U.S. patent application number 15/803369 was filed with the patent office on 2018-02-22 for methods and compositions for modifying sensorial perception.
The applicant listed for this patent is The Procter & Gamble Company. Invention is credited to Yakang Lin, Koti Tatachar Sreekrishna.
Application Number | 20180049960 15/803369 |
Document ID | / |
Family ID | 52815337 |
Filed Date | 2018-02-22 |
United States Patent
Application |
20180049960 |
Kind Code |
A1 |
Sreekrishna; Koti Tatachar ;
et al. |
February 22, 2018 |
Methods And Compositions For Modifying Sensorial Perception
Abstract
Compounds and methods for modifying sensory perception
associated with transient sensory receptors TRPA1, TRPV1, and
TRPA1V1. A method for screening compounds for modulation of TRPA1,
TRPV1, and/or TRPA1V1. Compositions comprising TRPA1V1 agonists or
antagonists, for modifying sensory perception of the
compositions.
Inventors: |
Sreekrishna; Koti Tatachar;
(Mason, OH) ; Lin; Yakang; (Liberty Township,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Procter & Gamble Company |
Cincinnati |
OH |
US |
|
|
Family ID: |
52815337 |
Appl. No.: |
15/803369 |
Filed: |
November 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15225852 |
Aug 2, 2016 |
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15803369 |
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14666394 |
Mar 24, 2015 |
9427415 |
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15225852 |
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61973566 |
Apr 1, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 8/33 20130101; A61K
31/11 20130101; G01N 33/6872 20130101; A61P 29/00 20180101; G01N
2500/10 20130101; A61Q 11/00 20130101; A61K 8/35 20130101; G01N
33/502 20130101; A61K 8/31 20130101; A61K 8/4973 20130101; G01N
33/5041 20130101 |
International
Class: |
A61K 8/35 20060101
A61K008/35; A61Q 11/00 20060101 A61Q011/00; A61K 8/49 20060101
A61K008/49; A61K 8/31 20060101 A61K008/31; G01N 33/50 20060101
G01N033/50; G01N 33/68 20060101 G01N033/68; A61K 31/11 20060101
A61K031/11; A61K 8/33 20060101 A61K008/33 |
Claims
1. A method of altering the sensory perception of burning,
irritation, or pain from exposure to an oral care composition, the
method comprising: incorporating a compound that modulates TRPA1V1
activity into an oral care composition; wherein the compound is
selected from the group consisting of ethyl vinyl ketone, vanillin
propylene glycol acetal, bisabolene, and combinations thereof;
wherein the compound is present in an amount effective to reduce
the activation of TRPA1V1 relative to the activation of at least
one of TRPA1 or TRPV1.
2. The method of claim 1, wherein the compound is ethyl vinyl
ketone.
3. The method of claim 1, wherein the compound is vanillin
propylene glycol acetal.
4. The method of claim 1, wherein the compound is bisabolene.
5. The method of claim 1, wherein the oral care composition is
selected from the group consisting of dentifrice, mouth rinse,
lozenge, chewable tablet, chewing gum, and combinations
thereof.
6. A method of altering the sensory perception of burning,
irritation, or pain from exposure to a personal care composition,
the method comprising: incorporating a compound that modulates
TRPA1V1 activity into a personal care composition; wherein the
compound is selected from the group consisting of ethyl vinyl
ketone, vanillin propylene glycol acetal, bisabolene,
trans,trans-2,4-undecadien-1-al, and combinations thereof; wherein
the personal care composition is selected from the group consisting
of hair coloring compositions, cough and cold compositions, shower
gels, lotions, moisturizers, sunscreens, shampoos, conditioners and
combinations thereof; wherein the compound is present in an amount
effective to reduce the activation of TRPA1V1 relative to the
activation of at least one of TRPA1 or TRPV1.
7. The method of claim 6, wherein the compound is ethyl vinyl
ketone.
8. The method of claim 6, wherein the compound is vanillin
propylene glycol acetal.
9. The method of claim 6, wherein the compound is bisabolene.
10. The method of claim 6, wherein the compound is
trans,trans-2,4-undecadien-1-al.
Description
FIELD OF THE INVENTION
[0001] The invention is directed to compounds, compositions, and
methods of altering sensory perception associated with TRPA1,
TRPV1, or TRPA1V1 receptor activation. The invention further
relates to methods for identifying and/or assessing the efficacy of
agonists or antagonists of TRPA1, TRPV1 or TRPA1V1 sensory
receptors.
BACKGROUND OF THE INVENTION
[0002] Transient receptor potential (TRP) ankyrin 1 (TRPA1) and
vanilloid 1 (TRPV1) receptors are implicated in the modulation of
cough and nociception. TRPA1 and TRPV1 have important roles in the
sensation of pain, temperature, inflammation and cough in animals
and man. TRPV1 is activated by warm temperatures (above 43.degree.
C.), protons and noxious chemicals such as capsaicin and
resiniferatoxin. TRPA1 is activated by cold temperatures (below
17.degree. C.), and a wide range of irritating and pain stimulating
chemicals such as acrolein (found in smoke), formalin, mustard oil
and allicin (found in onions and garlic) as well as cinnamaldehyde
(extracted from cinnamon). Occasionally, TRPA1 and/or TRPV1 respond
to compounds which are potentially beneficial, possibly creating a
sense of irritation, burning, or pain that can discourage the use
of beneficial products.
[0003] Functional TRP channels have been thought to be tetramers,
possibly either homo-tetramers or even hetero-tetramers. In vivo,
TRPA1 is known to be expressed in the same sensory neurons as TRPV1
and pharmacological interaction between the two receptors has been
established. Direct interaction resulting in hetero-tetramers
between these two channels has been demonstrated using transient
co-expression of the two receptors in CHO cells. Nonetheless, in
vitro models for irritation, burning, and pain have tended to focus
on TRPA1 or TRPV1 rather than TRPA1V1.
[0004] There remains a need to modify unpleasant sensations
associated with beneficial products.
SUMMARY OF THE INVENTION
[0005] In some aspects, the invention relates to a method for
screening compounds for modulation of TRPA1V1 activity. The method
may comprise providing a cell line stably co-expressing TRPA1 and
TRPV1, wherein the cell line may comprise TRPA1V1 hetero-tetramer
and at least one of TRPA1 homo-tetramer or TRPV1 homo-tetramer. The
method may comprise measuring the baseline calcium ion channel
activity of the cell line. The method may comprise exposing the
cell line to a test composition. The method may comprise measuring
the ion channel activity of the cell line after exposure to the
test compound.
[0006] In some aspects, the invention relates to a method for
screening compounds for modulation of TRPA1 or TRPV1 activity. The
method may comprise providing a cell line stably co-expressing
TRPA1 and TRPV1, wherein the cell line may comprise TRPA1V1
hetero-tetramer and at least one of TRPA1 homo-tetramer or TRPV1
homo-tetramer. The method may comprise measuring the baseline
calcium ion channel activity of the cell line. The method may
comprise exposing the cell line to a test composition. The method
may comprise measuring the ion channel activity of the cell line
after exposure to the test compound.
[0007] In some aspects, the invention relates to a method of
reducing the sensory perception of burning, irritation, or pain
from exposure to an oral care or topical composition. The method
may comprise providing an oral care or topical composition
comprising an agonist of TRPA1 and/or TRPV1. The method may
comprise incorporating into the oral care or topical composition a
TRPA1V1 modulator selected from the group consisting of isoamyl
3-(2-furan)propionate; ethyl 3-hydroxyoctanoate; geraniol;
5-oxodecanoic acid; vanillylacetone; isobutyl N-methyl
anthranilate; 2-ethylbutyric acid; decanoic acid; p-cresol;
1-ethylhexyl tiglate; ethyl vinyl ketone;
3,7-Dimethyl-7-octen-1-ol; Octyl 2-furoate; cis-3-nonen-1-ol;
piperine; gluconic acid, monopotassium salt; tartaric acid;
trans,trans-2,4-undecadien-1-al; 3,5,5-Trimethylhexanal;
(E)-2-Nonenal; Citronellol; 5-Phenyl-1-pentanol;
3-Methyl-5-propyl-2-cyclohexen-1-one; Benzaldehyde propylene glycol
acetal; Vanillin propylene glycol acetal; Rhodinol;
(E,E)-2,4-Dodecadienal; (E,E)-2,4-Decadienal; 2-Hexen-4-one;
Bisabolene; 10-Undecenal; 3-Methyl-1-phenyl-3-pentanol; and
combinations thereof. The TRPA1V1 modulator may be present in an
amount effective to reduce the activation of TRPA1V1 relative to
the activation of TRPA1 and/or TRPV1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A and 1B illustrates the appearance of cell cultures
associated with Example 1.
[0009] FIG. 2 is a graph showing fluorescence of cells under
different growth conditions, as described in Example 1.
DETAILED DESCRIPTION OF THE INVENTION
[0010] Despite the prior observation of hetero-tetramer receptors
comprising TRPA1 and TRPV1 subunits, previous efforts to study
irritation potential in vitro have focused on the TRPA1 and TRPV1
receptors, without regard to the TRPA1V1 hetero-tetramers. We have
found that there is value in using TRPV1 (SEQ ID NO: 1) and TRPA1
(SEQ ID NO: 2) co-expressing cells to study the irritation
potential of compounds because TRPA1V1 activation is not
predictable based on TRPA1 and/or TRPV1 activation. In addition to
possible down-modulation of TRPA1V1 by TRPA1 or TRPV1 activators,
in some cases there are synergistic increases in response across
the three receptor types, and, additionally, there are compounds
which can activate TRPA1V1 without activating either TRPA1 or
TRPV1. This is important for the practical application of irritancy
data, because TRPA1V1 activation is believed to be a better
predictor of irritancy, as TRPA1V1 hetero-tetramers are more common
in sensory nerves than TRPA1 or TRPV1 homo-tetramers.
[0011] TRPA1 and TRPV1 are co-expressed in many sensory cells,
including in sensory cells found across the exterior of the human
body, head-to-toe, and in the mucous membranes of the respiratory
and gastrointestinal tracts. TRPA1 and TRPV1 are believed to be
associated with irritancy triggered by a wide variety of stimuli,
including food, medications, environmental conditions, illnesses,
and topical treatments. In many cells where TRPA1 and TRPV1 are
both expressed, TRPA1V1 hetero-tetramers are also observed.
[0012] As used herein, "isolated compound" refers to a composition
that has been purified or synthesized such that a single chemical
entity predominates, excluding any solvent. In contrast, a "complex
extract," as used herein, refers to a composition comprising two or
more chemical entities, in addition to any solvent, and may
comprise dozens or hundreds of distinct chemical entities. A
complex extract may be characterized by its source material and
extraction process, rather than by its chemical composition.
[0013] As used herein, the terms "agonist" or "activator" refers to
a composition that activates a sensory receptor. Activation of the
sensory receptors TRPA1, TRPV1, and TRPA1V1 may be measured by
calcium flux, as described in Example 1, or suitable alternative
methods, including, without limitation, measurement of membrane
potential changes, cellular calcium imaging, electrophysiological
methods, or other methods for observing the activation of ion
channels. An agonist or activator generates at least 20%,
preferably at least 25% more receptor activity than a
physiologically relevant baseline. An exemplary baseline is
described in Example 1. In contrast, an "antagonist" refers to a
composition that decreases the activity of a sensory receptor. An
antagonist generates at least 20%, preferably at least 25% less
receptor activity than a physiologically relevant baseline.
[0014] As used herein, the term "modulator" refers to a composition
that can alter the activity of a sensory receptor, either by
increasing or decreasing activity of the sensory receptor relative
to a control condition.
[0015] 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.
[0016] All measurements referred to herein are made at 25.degree.
C. unless otherwise specified.
[0017] As used herein, the word "or" when used as a connector of
two or more elements is meant to include the elements individually
and in combination; for example X or Y, means X or Y or both.
[0018] 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 hair coloring
compositions, oral care compositions, after shave gels and creams,
pre-shave preparations, shaving gels, creams, or foams,
moisturizers and lotions, cough and cold compositions, leave-on
skin lotions and creams, shampoos, conditioners, shower gels, bar
soaps, toilet bars, antiperspirants, deodorants, depilatories,
lipsticks, foundations, mascara, sunless tanners and sunscreen
lotions.
[0019] By "oral care composition", as used herein, is meant a
product, which in the ordinary course of usage, is not
intentionally swallowed for purposes of systemic administration of
particular therapeutic agents, but is rather retained in the oral
cavity for a time sufficient to contact dental surfaces or oral
tissues. Examples of oral care compositions include dentifrice,
mouth rinse, mousse, foam, mouth spray, lozenge, chewable tablet,
chewing gum, tooth whitening strips, floss and floss coatings,
breath freshening dissolvable strips, or denture care or adhesive
product. The oral care composition may also be incorporated onto
strips or films for direct application or attachment to oral
surfaces.
[0020] 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.
[0021] As used herein, the term "topical", in reference to a
composition, includes any composition intended to be applied to the
skin, including the scalp, hair, or nails. Exemplary topical
compositions include lotions; moisturizers; sunscreens; perfumes;
color cosmetics, such as blush, foundation, or eye shadow; hair
treatments, including hair dyes, shampoos, conditioners, texture
modifiers such as relaxers, curling products, and treatments to
increase volume or shine, and styling aids, such as hair gel and
hairspray; nail polish or nail strengtheners; cuticle oil; soaps,
detergents, and body washes; compositions for shaving, including
shave gels or foams and after-shave treatments; toners; and
medicaments.
[0022] A "medicament", as used herein, is any agent that promotes
recovery from injury or illness, inclusive of pain relievers.
[0023] As used herein, "toxic" refers to a composition or substance
capable of damaging tissues under relevant conditions of exposure,
including duration of exposure, nature of exposure (e.g.,
respiratory, ingestion), and concentration of the composition or
substance.
[0024] As used herein, "gastrointestinal symptoms" refers to
undesired gastrointestinal events, including bloating, cramping,
vomiting, diarrhea, excessive gas production, with or without
associated tissue damage.
[0025] As used herein, "stably co-expressing" refers to a cell line
which produces a consistent, reproducible response to known
agonists of TRPV1 (capsaicin or suitable alternative) and TRPA1
(allylisothiocyanate or suitable alternative).
[0026] As used herein, the term "irritation", "burning", and "pain"
refer to undesirable sensory perceptions, noticeable to a human
subject able to report them or measurable by ion channel activation
or comparable analytical methodology. These sensations are
associated with, among other receptors, TRPA1 and TRPV1. The words
irritation, burning, and pain may variously be used by human
subjects to describe their perception of stimuli which are known to
activate TRPA1 and/or TRPV1.
[0027] Irritation, burning, and pain may be associated with stimuli
which are, at least in the short-term or at low concentrations,
safe. As an example, a hot surface may create a sensation of
burning or pain significant enough to trigger an involuntary
movement of the body away from the hot surface, even if the surface
is not, in fact, hot enough to cause tissue damage from indirect or
very brief contact. If the stimulus has benefits, such as cosmetic
or medical benefits, it may be desirable to reduce or entirely
override the perception of irritancy, burning, or pain associated
with the stimulus.
[0028] In some aspects, the invention relates to a method of
reducing the sensory perception of burning, irritation, or pain
from exposure to an oral care or topical composition. The method
may comprise providing an oral care or topical composition
comprising an agonist of TRPA1 and/or TRPV1. The method may
comprise incorporating into the oral care or topical composition a
TRPA1V1 modulator, particularly a modulator which decreases the
measurable activity of TRPA1V1. The TRPA1V1 modulator may be
selected from the group consisting of isoamyl
3-(2-furan)propionate; ethyl 3-hydroxyoctanoate; geraniol;
5-oxodecanoic acid; vanillylacetone; isobutyl N-methyl
anthranilate; 2-ethylbutyric acid; decanoic acid; p-cresol;
1-ethylhexyl tiglate; ethyl vinyl ketone;
3,7-Dimethyl-7-octen-1-ol; Octyl 2-furoate; cis-3-nonen-1-ol;
piperine; gluconic acid, monopotassium salt; tartaric acid;
trans,trans-2,4-undecadien-1-al; 3,5,5-Trimethylhexanal;
(E)-2-Nonenal; Citronellol; 5-Phenyl-1-pentanol;
3-Methyl-5-propyl-2-cyclohexen-1-one; Benzaldehyde propylene glycol
acetal; Vanillin propylene glycol acetal; Rhodinol;
(E,E)-2,4-Dodecadienal; (E,E)-2,4-Decadienal; 2-Hexen-4-one;
Bisabolene; 10-Undecenal; 3-Methyl-1-phenyl-3-pentanol; and
combinations thereof. The TRPA1V1 modulator may be present in the
composition in an amount effective to reduce the activation of
TRPA1V1 relative to the activation of TRPA1 and/or TRPV1. The
TRPA1V1 modulator may be trans,trans-2,4-undecadien-1-al.
[0029] The method may be used with oral compositions which are
ingestible or non-ingestible. Oral compositions of interest may
include rinses, foods, beverages, medicaments, dentifrices, and the
like. The method may be used with topical compositions. Exemplary
topical compositions include moisturizers; medicaments; toners;
depilatories; color cosmetics; compositions intended to treat the
hair or scalp, including shampoos, conditioners, hair dyes, styling
aids, and/or texture modifiers; and combinations thereof. In any of
these exemplary compositions, the TRPA1V1 modulator may be
trans,trans-2,4-undecadien-1-al.
[0030] In some aspects, the invention relates to a method for
isolating trigeminal response in a subject. The method may comprise
exposing the subject to a TRPA1 activator that does not modulate
TRPV1 or TRPA1V1. The method may comprise exposing the subject to a
TRPV1 activator that does not modulate TRPA1 or TRPA1V1 activity.
The method may comprise exposing the subject to a TRPA1V1 activator
that does not modulate TRPA1 or TRPV1 activity. By comparing the
responses to the TRPA1, TRPV1, and TRPA1V1 activators, it is
possible to isolate the trigeminal response. By studying the
trigeminal effect of individual compounds known to specifically
activate only one of the TRPA1, TRPV1, and TRPA1V1 receptors, it is
possible to isolate the trigeminal response specific to each of
those receptors.
[0031] An exemplary TRPA1 activator which does not modulate TRPV1
or TRPA1V1 is propylparaben. Suitable TRPV1 activators which do not
modulate TRPA1 or TRPA1V1 include, without limitation,
2,4,5-trimethyl-3-oxazoline; heptaldehyde; 2-ethylfuran;
desoxycholic acid; malic acid; 2-methylbutyl isovalerate, and
combinations thereof.
[0032] Just as irritation, burning, and pain may be associated with
stimuli which are safe, some stimuli which are dangerous may not be
associated with irritation, burning, or pain. Exemplary
compositions which may be useful but dangerous to ingest or touch
with bare skin include many household cleaners, or topical
medicaments that are not suitable for ingestion. If a composition
is dangerous if touched or ingested, it may be desirable to
increase the perception of irritancy, burning, or pain associated
with touching or ingesting the composition.
[0033] In some aspects, the invention relates to a method for
increasing the perception of burning, irritation, or pain
associated with topical exposure to a composition. The method may
comprise providing a composition for which it is desired to
discourage contact between the composition and the skin or scalp,
and incorporating into the composition a TRPA1V1 activator. The
TRPA1V1 activator may also activate TRPA1 or TRPV1. The TRPA1V1
activator may also activate TRPA1 and TRPV1. The composition may be
toxic. The composition may cause undesirable cosmetic changes in
the skin, such as discoloration. It is not necessary that the
composition provide a noticeable perception of burning, irritation,
or pain without the addition of the TRPA1V1 activator, although, in
some cases, the composition will provoke some discomfort which is
intensified by the addition of the TRPA1V1 activator.
[0034] In some aspects, the invention relates to a method for
increasing the perception of burning, irritation, or pain
associated with oral exposure to a composition. The method may
comprise providing a composition for which it is desired to
discourage contact between the composition and the mouth, mucous
membranes, or digestive tract, and incorporating into the
composition a TRPA1V1 activator. The TRPA1V1 activator may also
activate TRPA1 or TRPV1. The TRPA1V1 activator may also activate
TRPA1 and TRPV1. The composition may be toxic. The composition may
cause gastrointestinal symptoms if ingested. It is not necessary
that the composition provide a noticeable perception of burning,
irritation, or pain without the addition of the TRPA1V1 activator,
although, in some cases, the composition will provoke some
discomfort which is intensified by the addition of the TRPA1V1
activator.
[0035] In some aspects, the invention relates to a composition
comprising a TRPA1V1 antagonist. The composition may be an oral
care or topical composition. The composition may comprise an
agonist of TRPA1 and/or TRPV1. Counter intuitively, the TRPA1V1
antagonist may help reduce the perception of irritation, burning,
or pain from oral care or topical contact with the composition,
even if the particular antagonist used is an agonist for TRPA1
and/or TRPV1. In some aspects, the invention relates to a
composition comprising a TRPA1 agonist that down-modulates TRPV1
activity. In some aspects, the invention relates to a composition
comprising a TRPV1 agonist that down-modulates TRPA1 activity.
[0036] In some aspects, the invention relates to a composition
comprising a TRPA1V1 agonist. The composition may be an oral care
or topical composition. The composition may be toxic, or may cause
undesirable cosmetic changes in the skin, or may cause
gastrointestinal symptoms if ingested. The composition may comprise
an antagonist of TRPA1 and/or TRPV1. Counter intuitively, the
TRPA1V1 agonist may increase the perception of irritation, burning,
or pain from oral care or topical contact with the composition
(thereby discouraging prolonged contact or ingestion), even if the
particular agonist used is an antagonist for TRPA1 and/or TRPV1. In
some aspects, the invention relates to a composition comprising a
TRPA1 antagonist that activates TRPV1. In some aspects, the
invention relates to a composition comprising a TRPV1 agonist that
activates TRPA1.
[0037] In some aspects, the invention relates to a method for
screening compounds for modulation of TRPA1 activity. The method
may comprise providing cell lines stably co-expressing TRPA1,
TRPV1, and/or TRPA1V1. The method may comprise measuring the
baseline ion channel activity of the cell line. The baseline ion
channel activity is measured prior to exposure to any control
substance (e.g., known modulator with predictable effect) or test
composition. With respect to TRPA1, TRPV1, and TRPA1V1, ion channel
activity may be measured as calcium flux across a cell membrane.
Suitable alternatives to calcium flux measurements include
measurement of membrane potential changes, cellular calcium
imaging, electrophysiological methods, and other methods of
observing ion channel activation. The measurement of calcium flux
is well known and can be used with commercially available test
kits, such as the kit described in Example 1. TRPA1 activity can be
distinguished from TRPV1 and TRPA1V1 activity as known in the art.
As an example, by using a cell expressing only TRPA1 or TRPV1, it
is possible to screen for compounds that activate specifically
TRPA1 or TRPV1.
[0038] The method may comprise exposing the cell line to a test
composition. The test composition may be a substance with unknown
effect (if any) on the receptor of interest. The test composition
may be added to the cell culture in concentrations ranging in
molarity from 200 nM to 5 mM, more preferably from 350 nM to 1 mM,
more preferably from 100 .mu.M to 1 mM. If the test composition is
a complex extract or for any other reason cannot be conveniently
measured by molarity, the test composition may be added to the cell
culture in concentrations ranging from 0.0002% to 0.008%. It should
be understood that the test composition will be further diluted
upon addition to the cell culture. The exemplary ranges provided
are suggested for a 100 .mu.L aliquot of cell line suspended in
assay reagent. Significantly larger or smaller cell line aliquots
may require different test composition concentrations. The cell
line may be incubated with the test composition. The cell line may
be incubated with the test composition for 15-60 minutes, more
preferably, 20-40 minutes. The method may comprise measuring the
ion channel activity of the cell line after exposure to (or
incubation with) the test compound.
[0039] Screening compounds for modulation of TRPA1 activity using a
cell line stably co-expressing TRPA1, TRPV1, and TRPA1V1 may be
beneficial in identifying previously unappreciated interactions
between these receptors and/or their agonists. Similar benefits may
be obtained by screening compounds for modulation of TRPV1 or
TRPA1V1 activity using cell lines stably co-expressing TRPA1,
TRPV1, and/or TRPA1V1.
[0040] It is possible to compare the measured response to a test
composition to published, predicted, or previously obtained data.
In TRPV1-expressing cells, exposure to heat in excess of 30.degree.
C. may increase the basal activity of TRPV1, and may also be
associated with cell clumping and other signs of poor cell health.
FIG. 1A shows TRPV1-expressing cells incubated at 37.degree. C.
FIG. 1B shows TRPV1-expressing cells incubated at 25.degree. C.
Some agonists, such as capsaicin, may further exacerbate the
tendency for cells to clump at high temperature. Thus, it may be
desirable to pre-incubate TRPV1-expressing cells at a temperature
between 22.degree. C. and 30.degree. C. prior to exposing the cell
line to a control agonist and/or test composition. The cell line
may be pre-incubated between 22.degree. C. and 30.degree. C. for
15-60 minutes prior to exposing the cell line to the control
agonist and/or test composition, more preferably for 20-40
minutes.
[0041] It is possible to distinguish the ion channel activation
associated with one receptor versus another receptor or receptors
even in cell lines expressing two or more related receptors using
methods known in the art. For example, a parallel screening for
activation of related ion channels can be used to eliminate
positive activation results for compounds that activate multiple
types of ion channels. Another exemplary method is to test a
compound which appears to be an agonist for a specific receptor in
the presence of a known antagonist for that specific receptor, to
check for a reduction in response. More details about an exemplary
methodology are included in Zhang, Y.; Sreekrishna, K.; Lin, Y.;
Huang, L.; Eickhoff, D.; Degenhardt, D.; Xu T. Modulation of
Transient Receptor Potential (TRP) Channels by Chinese Herbal
Extracts. Phytother. Res. 2011, 25, 1666-1670.
Example 1
Materials
[0042] Allyl isothiocyanate (AITC), cinnamaldehyde, capsaicin, and
calcium ionophores (A23187 and ionomycin) were obtained from Sigma
Aldrich (St Louis, Mo.). All buffers, expression vectors,
antibiotics, calcium dyes (Fluo-3 AM and Fluo-4 AM), and other
reagents used were obtained from Life Technologies (Carlsbad,
Calif.). GRAS compound library was procured from Evotec (San
Francisco, Calif.).
TRPV1, TRPA1, TRPA1V1 and pcDNA3 Control Cells
[0043] Cells stably expressing human TRPV1 (sequence used is shown
in SEQ ID NO: 1), human TRPA1 (sequence used is shown in SEQ ID NO:
2), as well as cells that co-express both receptors (TRPA1V1) and
control (pcDNA3) cells have been described previously. Sadofsky, L.
R.; Campi, B.; Trevisani, M.; Compton, S. J.; Morice, A. H.
Transient receptor potential vanilloid-1-mediated calcium responses
are inhibited by the alkylamine antihistamines dexbrompheniramine
and chlorpheniramine Exp Lung Res. 2008, 34, 681-693. Mitchell, J.
E.; Campbell, A. P.; New, N. E.; Sadofsky, L. R.; Kastelik, J. A.;
Mulrennan, S. A.; Compton, S. J.; Morice, A. H. Expression and
characterization of the intracellular vanilloid receptor (TRPV1) in
bronchi from patients with chronic cough. Exp Lung Res. 2005, 31,
295-306. Sadofsky, L. R.; Sreekrishna, K.; Morice, A. H
Characterisation of a HEK293 cell line permanently co-expressing
the cough receptors Transient Receptor Potential Ankyrin 1 and
Vanilloid 1 (TRPA1 and TRPV1). The Sixth London International
Symposium on Cough: A translational approach, London 24-26 Jun.
2010 (Chung, K. F.; Widdicombe, J) [Abstracts/Pulmonary
Pharmacology and Therapeutics 2011, 24, e8].
Measurement of Intracellular Calcium for Activation of TRPA1V1,
TRPA1 and TRPV1 Cells by GRAS Compounds
[0044] TRPV1, TRPA1, TRPA1V1 and pcDNA3 cells 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 in a mammalian cell culture incubator at
33.degree. C. and 5% CO.sub.2. TRP Cells were detached with 8 mL of
PBS (without calcium or magnesium); for pcDNA3 cells, trypsin was
used for releasing the cells. The detached cells were spun at low
speed (800-900 rpm for 3 min) to pellet the cells. The PBS medium
was gently removed, and the cell pellet was re-suspended in 1 mL
growth medium; 12.5 .mu.g of Fluo-4 AM calcium dye dissolved in 5
.mu.L Pluronic F-127 (20% solution in DMSO), was added and
incubated for 30 min with gentle shaking at room temperature. The
cells were washed once with 45 mL assay buffer (1.times.HBSS, 20 mM
HEPES) by low speed centrifugation (800-900 rpm for 3 min) and
resuspended in 11 mL of the assay buffer in a reagent reservoir.
Aliquots of 100 .mu.L (approximately 5.times.10.sup.4 cells) were
dispensed in each well of the 96-well plate (BD Falcon micro-test
assay plate #353948). The plates were set at room temperature for
30 min. The plates were read in a FLIPR.sup.TETRA instrument
(Molecular Devices, Sunnyvale, Calif.) at .lamda..sub.ex 488 nm and
.lamda..sub.em 514 nm to record baseline fluorescence following
which 20 .mu.L of test material at 1 mM or 100 .mu.M final
concentration for pure compounds in a GRAS library or 0.004% for
extracts. For agonists, capsaicin 350 nM and AITC at 30 .mu.M; and
for controls, ionomycin 10 .mu.M and buffer alone) was added to
each well using the dispenser provided in the FLIPR. The data point
was recorded every 2 seconds for a total of 10 min. Data were
analyzed after baseline subtraction as described previously in
Smart, et al. Characterization using FLIPR of human vanilloid VR1
receptor pharmacology. Eur J Pharmacol 2001, 417, 51-58, and Zhang,
et al. Modulation of Transient Receptor Potential (TRP) Channels by
Chinese Herbal Extracts. Phytother. Res. 2011, 25, 1666-1670.
Activation of TRPA1V1, TRPA1 and TRPV1 Cells by GRAS Library
Compounds
[0045] We observed that during cultivation of cells for several
passages, TRPV1 cells cultivated at 37.degree. C. tended to form
clumps and were unhealthy presumably due to high basal activity of
TRPV1 (FIG. 1A), whereas cells grown at 25.degree. C. did not form
clumps and looked healthy (FIG. 1B). Pre-incubation of cells at
25.degree. C. for 30 minutes prior to addition of capsaicin gave
superior response as compared to cells pre-incubated at 33.degree.
C. or 37.degree. C. (FIG. 2). Cells pre-incubated at higher
temperatures had proportionally higher basal activity. Thus for
screening compounds we implemented the 33.degree. C. growth
temperature and 25.degree. C. pre-incubation conditions for all
three cell lines.
Agonist Hits Identified in the GRAS Compound Library Screen
[0046] Compounds that gave at least 20-25% of activity relative to
benchmark agonist against at least any one of A1, V1 or A1V1 were
considered an agonist. In all we identified 329 agonists out of
1620 compounds screened at 1 mM or 100 .mu.M final dose for pure
compounds or 0.004% or 0.0004% final concentration for extracts. Of
these agonists 67 activated all three receptors and in 41
instances, A1 or V1 had no obvious effect on A1V1 activity. By this
we mean that an A1 agonist gave similar A1V1 response (Table 1a).
Whereas in other 26 other instances, A1 and/or V1 impacted A1V1
response as noted in Table 1b. We found 137 compounds that
activated A1 and A1V1, but not V1 and had same or very similar
level of activity of both A1 and A1V1 (Table 1c), suggesting very
little or no impact of V1 on A1V1 activity in the majority of
instances. However, in 39 instances, V1 caused negative modulation
of A1V1 activity (Table 1d), whereas in 34 instances, V1 caused
positive modulation of A1V1 response (Table 1e). We also found 12
compounds that activated V1 and A1V1, but not A1 where V1 and A1V1
activities were comparable (Table 1f), suggesting that A1 did not
influence A1V1 activity in those cases. In 20 instances A1 enhanced
A1V1 response (Table 1g), whereas in 12 instances, A1 negatively
impacted A1V1 response (Table 1h). Interestingly, 6 compounds
activated only V1, one activated only A1, and one compound
activated both A1 and V1 but not A1V1 (Table 1i). These results
suggest that, although in majority of instances A1V1 response
correlates with A1 or V1 response, nevertheless in a significant
number of instances, the activity of A1V1 is modulated, positively
or negatively, by A1 or V1 in a compound specific manner.
Table 1.
[0047] Summary of GRAS compound library screen for activation of
TRPA1, TRPA1V1 and TRPV1 cells. Average value for activation from
three independent assay plates are presented as % of agonist value
(AITC, 30 .mu.M for TRPA1 and capsaicin, 350 nM for TRPV1. For
TRPA1V1, we used AITC, 30 .mu.M or capsaicin, 350 nM depending on
if the compound is an A1 agonist or a V1 agonist. For compounds
that activated both A1 and V1, we used AITC for normalization).
Variation among triplicates was within 10% of each other. Assayed
at 1 mM for pure compounds and 0.004% for extracts.
TABLE-US-00001 TABLE 1(a) Compounds that activated all three
receptors, with no apparent effect of V1 on A1V1. GRAS Compound at
1 mM or 100 .mu.M(*) A1 A1V1 V1 Allyl-trisulfide 83.45 89.08 32.85
Perillyl alcohol 82.00 73.28 20.37 Phenylpyruvic acid 68.03 74.05
32.49 Trans-2-Octen-1-ol 118.4 103.2 25.98 Ethyl
2-hydroxy-3-phenylpropanoate 95.60 90.76 29.18 Geranyl butyrate
76.94 76.26 28.86 Retinol palmitate 76.39 76.97 32.78 2-Butylfuran
68.54 62.77 20.19 Alpha-Amylcinnamyl alcohol 96.36 77.36 21.12
Pseudoionone 105.5 127.2 20.76 5-Octen-1-ol, (Z) 62.30 69.24 20.50
Helional 78.00 71.85 23.38 2-Methoxy-4-vinylphenol 74.81 81.90
20.66 2-(3-Phenylpropyl)pyridine 75.97 74.53 21.47 Farnesal 97.58
104.4 41.22 Beta-Cyclohomocitral 81.44 66.79 61.74 Saccharin 92.58
93.25 80.76 3-Methyl-2-Phenylbutyraldehyde 90.56 93.08 75.80
Gamma-Nonalactone 33.32 69.15 66.34 Vanillyl ethyl ether 38.00
59.64 67.57 Cyclohexaneaceticacid 20.60 61.28 56.63 Methyl
linolenate 108 104 31 Pyruvic caid cis-3-hexen-1-yl ester 45 40 21
4-Hydroxy-5-methyl-3-furanone 58 38 40.5 3-Octen-2-one 52.67 45.60
30.02 4-Hydroxybenzaldehyde* 80.4 163.2 123.1 Phloretin* 160.2
174.3 122.4 2-Napthlaenthiol * 66.3 85.2 120.3
2-Furfurylthio-3-methylpyrazine* 80.7 80.9 31.3 Benzothiazole* 51.3
74.4 33.4 Propyl 2-methyl-3-furyl disulfide 210.3 194.3 25
2,3,6-Trimethylphenol 90 91.4 74.8 o-Eugenol 128.4 158.3 64.5
2-Propylphenol 126.4 183.8 96.9 Isothiocyanic acid, benzyl ester
110.3 180.5 59.2 3-Mercapto-2-pentanone 92 72.6 59.2 Linoleic acid
169.7 170.4 24.9 1-Octen-3-one 118 98.9 63.2
1-Phenyl-1,2-propanedione 98.6 105.5 66.6 Butylparaben 104.7 98.1
79.1 2-Benzofurancarboxaldehyde 91 102.3 57.6
TABLE-US-00002 TABLE 1(b) Compounds that activated all three
receptors, with A1 and/or V1 having effect on A1V1. GRAS Compound
at 1 mM or 100 .mu.M (*) A1 A1V1 V1 Remarks Isoamyl
3-(2-furan)propionate 123.3 106.9 32.66 Negative effect of V1 Ethyl
3-hydroxyoctanoate 97.29 76.90 28.05 Negative effect of V1 Geraniol
82.52 63.93 42.16 Negative effect of V1 Thymol 159 105.7 105.8
Negative effect of V1 (E)-2-Nonenal 130 61.8 89 Negative effect of
V1 5-Oxodecanoic acid 28.57 39.34 53.38 Negative effect of A1
Vanillylacetone 22.26 37.75 60.05 Negative effect of A1
3,5,5-Trimethylhexanal 133 114.7 217.2 Negative effect of A1
Trans,trans-2,4-undecadien-1-al* 74 38.4 87.5 A1 and V1 have
negative effect Citronellol 102 34.4 106 A1 and V1 have negative
effect 5-Phenyl-1-pentanol 119 52.1 113.1 A1 and V1 have negative
effect 3-Phenylpropanol 51.43 64.94 32.05 A1 and V1 have a positive
effect 3-Methyl-5-propyl-2-cyclohexen-1- 69.00 101.0 59.22 A1 and
V1 have a one positive effect Benzaldehyde propylene glycol 48.03
78.33 34.38 A1 and V1 additive acetal Vanillin propylene glycol
acetal 76.21 94.49 57.97 A1 and V1 additive Piperonyl isobutyrate
61.03 82.33 30.00 A1 and V1 additive 4-Phenyl-2-butanol 58.83 81.29
37.89 A1 and V1 additive Methyl cyclohexanecarboxylate 45.50 85.17
69.51 A1 and V1 additive Piperitone 90.06 109.1 53.30 A1 and V1
additive Hedione 43.54 60.64 25.60 A1 and V1 additive Piperonyl
acetate 62.67 78.00 20.92 A1 and V1 additive Curcumin 95.3 171.6
43.2 A1 and V1 have a positive effect 2-Cyclopentylcyclopentanone
25 90.8 24 A1 and V1 have a positive effect Citral diethyl acetal
53 130.7 72 A1 and V1 have a positive effect 3-Mercaptobutanone
50.4 80.9 63.8 A1 and V1 have a positive effect Carvacrol 115 134.1
84.6 A1 and V1 have a positive effect
TABLE-US-00003 TABLE 1(c) Compounds that activate A1 and A1V1 with
similar level of Activation of A1 and A1V1. GRAS Compound at 1 mM
or 100 .mu.M (*) or at 0.004% for extract (**) A1 A1V1
2,4-Hexadienyl propionate 93.17 91.20 Methyl o-formamidobenzoate
75.42 75.64 (E,E)-2,6-Nonadienal 87.10 89.61 2-Furanmethanol,
5-methyl- 77.31 76.02 2,4-Hexadienol 78.00 78.68
Dihydro-beta-ionone 64.4 64.07 Isoeugenyl phenylacetate 59.08 58.40
Gum benzoin, Siam** 47.32 46.64 Rhodinyl acetate 89.06 89.69
Sandalwood oil** 92.67 94.94 Trivertal 35.43 36.46
3-Methyl-2-cyclohexen-1-one 94.48 94.76 Benzenemethanol 79.64 78.47
(+/-)-Alpha-Ionone 64.62 64.21 Amyl furoate 81.09 87.70
p-Hydroxyacetophenone 52.82 49.38 Methyl p-anisate 57.47 66.24
Ammonium carbonate 52.81 58.42 O-Tolyl isobutyrate 49.37 57.76
Allyl anthranilate 73.80 75.03 2-Octenoic acid 55.93 61.77
Methylparaben 62.00 66.33 Methyl dihydrojasmonate 54.44 59.58
Methyl sorbate 76.00 81.15 Sucrose 84.13 80.86 Methyl
2-methoxybenzoate 71.2 74.68 Eugenyl acetate 75.4 83.94
Veratraldehyde 73.8 69.76 Allyl phenylacetate 86.2 84.14
2-trans-6-cis-Nonadienal 77.3 81.22 4-Methoxybenzylacetone 62.3
71.16 Methyl N-methylanthranilate 83.62 86.81 Cinnamyl acetate
84.99 89.73 N,N-Dimethylanthranilic acid methyl ester 74.23 77.15
Valencene 61.97 66.32 Ethyl vanillin propylene glycol acetal 66.41
72.64 Rhodinyl isovalerate 54.92 48.29 Saffron extract (**) 86.70
92.32 Chicory extract(**) 40.72 45.18 L-Ascorbic acid, calcium salt
(2:1) 88.00 93.20 Maltol propionate 85.04 81.97 Carmine 66.21 61.54
(-)-Carvyl acetate 31.2 37 Basil oil(**) 73.84 67.89 Geranium oil
71.89 76.78 1-Methylpyrrole-2-carboxaldehyde 81.09 87.70
1-Cyclohexene-1-carboxaldehyde 45.88 48.37 Eugenol 79.00 91.29
3-Butylidenephthalide 119.3 115.4 (E,E)-2,4-Hexadienal 92.00 108.71
Bisabolol 68.05 73.53 Decanoic acid 65.12 73.45 2-Acetylnaphthalene
60.28 69.41 Isoamyl acetate 49.63 61.80 4-Pentenal 75.4 84.24
Rhodinyl propionate 75.56 86.44 Isoeugenyl acetate 57.6 68.34
(-)-Alpha-Bisabolol 71.26 83.53 Phenoxyethyl propionate 65.55 79.34
DL-Menthol 54.02 43.05 Trans-2-Pentenal 80.86 68.62 O-Methylbenzyl
acetate 87.22 77.24 Retinol 80.00 67.79 Phenethyl isothiocyanate
92.00 81.46 2,6-Nonadiene, 1,1-diethoxy-, (E,Z)- 89.30 74.31
Alpha-Isobutylphenethyl alcohol 73.00 58.62 Phenethyl
isothiocyanate 92.32 81.46 O-Methoxycinnamaldehyde 65.3 55.99
Methyl 4-phenylbutyrate 58.39 50.30 2-Chloroacetophenone* 93.6 88.8
Erucin* 74.2 78.9 Ethyl 2-mercaptopropionate* 88.3 70.2
Isothiocyanic acid 4-penten-1-yl ester* 51.6 47.3
3-(2-Furfuryl)-2-phenylpropenol* 81.3 72.4 1,2-Propanedithiol* 65.8
65.4 Citronellyl butyrate* 44.9 57.9 Skatole* 66.2 71.4 Linalyl
benzoate* 44.4 55.3 2,5-dimethyl-1,4-dithilane-2,5-diol* 77.5 87.2
Citronellyl propionate* 52.5 43.2 2-Methylundecanal* 54.9 57.3
Trans-2-dodecenal* 79.1 66 3-Mercapto-2-Pentanone* 103.4 100
Skatole 116.3 102.9 2,4-Octadienal 80.9 82.7 Methyl
2-methyl-3-furyl disulfide 52.6 52 3-Thiazoline,
4,5-dimethyl-2-isobutyl- 142.4 138.7 Benzoylanthranilic acid 145.9
125.6 cis-3-Hexenyl cis-3-hexenoate 106.6 97.4 2-Furylacetone 60.8
59.5 Citral 145.4 132.7 Cinnamyl alcohol 89.8 79.4 2-Furoic acid,
phenethyl ester 155 145 Benzyl propionate 103.4 90.7
2-Phenylpropionaldehyde 105.1 106.9 3-(Methylthio)hexyl acetate
97.9 87.6 2-Tridecenal 119 102 trans-2-Octanal 112 115 Theaspirane
99 84 Anisyl formate 89.5 78.2 3-mercaptopropanoate 42.7 41.8
Propyl 2-methyl-3-furyl disulfide 265.7 242.5
2,4-Dimethylbenzaldehyde 106.8 102.8 1,6-Hexanedithiol 68.6 54
1,2-Propanedithiol 66 73.3 Nonyl alcohol 71.1 69.4 (E)-2-Heptenal
112.4 96.6 Cinnamic acid 37.7 42.5 Myrtenal 95.8 111.3 Ethyl
p-anisate 91.2 93.1 cis-6-Nonenal 108 93 4-Hydroxybenzaldehyde 63
60.3 L-Perillaldehyde 127.7 138.2 alpha-Terpinyl formate 82.2 71
(E,E)-Hepta-2,4-dien-1-ol 145.6 133.3 2-Hexanoylfuran 118 109 Ethyl
benzoate 70 60 Ethyl 2-octenoate 127.7 120 2,6-Dimethylthiophenol
84.4 85.5 Benzyl alcohol, p,alpha-dimethyl- 77.2 71.8 beta-Ocimene
111.5 92 Benzyl butyrate 70 64.7 Melonal 52 57
3-Acetyl-2,5-dimethylfuran 73.5 61 Ethyl 2-benzylacetoacetate
(Ethyl 2- 98 115 acetyl-3-phenylpropionate) Salicylaldehyde 66.9
65.6 3-(2-Furyl)acrolein 68.4 66.5 Safranal 73 74.5 3-Butenyl
isothiocyanate 99.4 85.8 Myricitrin 70.5 83.2 Methyl
2-methyl-3-furyl disulfide 52.6 52 2,4-trans,trans-Undecadienal
86.4 85.4 2-Chloroacetophenone 91.4 103 2-Tridecenal 85.6 101
2,4-Decadienol 48.6 52.2 (2E)-Undecenal 59.6 48.8
alpha-Amylcinnamaldehyde 86.6 82.7
TABLE-US-00004 TABLE 1(d) Compounds that activate only A1 and A1V1,
with V1 having negative effect on A1V1 activity. V1 is considered
as having negative effect in instances where the A1V1 activity is
at least 20-25% lower than A1 value. GRAS Compound at 1 mM or 100
.mu.M(*) A1 A1V1 2-Ethylbutyric acid 67.73 34.87 Ethyl
5-oxodecanoate 51.02 28.24 p-Cresol 60.96 38.52 1-Ethylhexyl
Tiglate 50.60 31.87 Ethyl vinyl ketone 79.50 40.61 Rhodinol 116.27
78.75 Octyl 2-furoate 54.33 40.60 Cis-3-nonen-1-ol 55.00 41.22
Isobutyl N-methyl anthranilate 59.73 36.7
1-Phenyl-1,2-propanedione* 100.5 71.4 2-Tridecenal* 76.3 43.5
3-Mercapto-2-butanone* 68.9 53.3
4-(2-Methyl-3-furylthio)-5-nonanone* 52.5 34.1
(E,E)-2,4-Dodecadienal 158.8 55.4 Isopropyl phenylacetate 80.2 60.9
Methyl 2-nonynoate 100.7 76.1 2-mercaptopropanoate 101.5 54.3
(E,E)-2,4-Decadienal 142.3 46 Ethyl salicylate 41 28 Linalool 52.6
20 Hexyl nicotinate 72.9 54.4
1-(2,2,6-Trimethylcyclohex-2-en-1-yl)-1-penten-3-one 97.3 69.7
3-Penten-2-one 64.2 35 Acetaldehyde, phenethyl propyl acetal 86.4
62.2 beta-Caryophyllene 134.2 103.8 3,7-Dimethyl-7-octen-1-ol 93.7
70.7 2-Hexen-4-one 53 24.5 Bisabolene 77 46.2 10-Undecenal 61.8 28
3-(Methylthio)propionaldehyde 53.8 32.4 Furfuryl acetate 120 91.3
Ethyl hydrocinnamate 72.2 50.9 cis-3-Hexenyl isovalerate 71.7 51.6
3-Hepten-2-one 82 59 trans-2-Hexenyl butyrate 48.9 20 Propyl
benzoate 40.7 25.7 Furfurylthiol acetate 75.5 60 Phenoxyethyl
isobutyrate 81 53.9 dec-1-en-3-ol 90 69.5
TABLE-US-00005 TABLE 1(e) Compounds that activate only A1 and A1V1,
with V1 having positive effect on A1V1 activity. V1 is considered
as having positive effect in instances where the A1V1 activity is
at least 20-25% higher than A1 value. GRAS Compound at 1 mM or 100
.mu.M (*) or at 0.004% for extract (**) A1 A1V1 Eugenyl isovalerate
59.69 79.29 Benzoin 26.25 61.81 Prenyl thioacetate 47.27 68.99
Alpha Methyl-4-methoxycinnamaldehyde 67.06 85.80 Furan,
tetrahydro-2-(3-phenylpropyl)- 26.00 72.09 L-Piperitone 45 68.40
p-Methoxybenzyl butyrate 34 58.97 3-(Acetylmercapto)hexyl acetate
22.3 52.79 Trans-p-methoxy cinnamaldehyde 58.79 75.99
2',4'-Dimethylacetophenone 24.40 45.30 L-Menthyl acetate 44.88
60.47 Trans-p-methoxy cinnamaldehyde 58.40 75.99 Hexyl butyrate
43.2 55.89 4-Methylbenzyl acetate 58.94 88.05 Cinnamomum zeylanicum
bark oil** 50.33 65.88 Curcumin* 102.2 150.3 Butyl
4-hydroxybenzoate* 98.7 127.3 4-Ethoxybenzaldehyde 62 103 Citral
dimethyl acetal 101.6 137.2 6-Methoxyquinoline 57.8 130.5 Cyclamen
aldehyde 94.1 120.9 Allyl-alpha-ionone 52.6 89.7 Propane,
1-isothiocyanato-3-(methylthio)- 81.6 114.6 Isopropyl
isothiocyanate 41.8 71.6 Isothiocyanic acid 4-penten-1-yl ester
62.2 74.8 5-Methyl-2-phenyl-2-hexenal 60.5 89.8 2-Phenylpropyl
isobutyrate 67.4 102.3 3-Methylbutyl isothiocyanate 61.1 111.2
Methyl linoleate 31 81.7 2-Phenyl-2-butenal 65.8 82.8 2-Dodecenal
65.5 82.8 p-Menth-8-en-2-one 39.9 59.5 Cinnamaldehyde 51.2 77.3
Sorbic acid methyl ester 46.4 83.3
TABLE-US-00006 TABLE 1(f) Compounds that activate only V1 and A1V1,
with A1 having no impact on A1V1 activity GRAS Compound at 1 mM or
at 0.004% for extract A1V1 V1 Gamma-Undecalactone 57.8 64.1
Epsilon-Decalactone 52.9 47.7 4-Decanolide 35.8 40.9
3-Heptyldihydro-5-methylfuran-2(3H)-one 40.5 45.5 Tannins 69.4 52.7
Gamma-Octanolactone 70.2 72.9 3-(5-Methyl-2-furyl)butanal 91.6
103.4 2-Furanacrolein, alpha-phenyl- 66.2 55.7 p-tert-Butylphenol
108.9 110.5 Indole 42.9 53.3 Octanoic acid 83.4 73.7
3-Acetyl-2,5-dimethylthiophene 84.9 95.1
TABLE-US-00007 TABLE 1(g) Compounds that activate only V1 and A1V1,
with A1 having positive effect on A1V1 activity. A1 is considered
as having positive effect in instances where the A1V1 activity is
at least 20-25% higher than V1 value. Compound at 1 mM or at 0.004%
for extract A1V1 V1 Rosmarinus officinalis essential oils 50.7 37.7
Delta-tetradecalactone 61.7 33.0 Tolylaldehyde glyceryl acetyl
110.7 53.0 Isobutyle acetoacetate 81.9 54.1 Protocatechuic acid
121.05 60.6 Furfuryl methyl sulfide 111.6 50.1 Acetic acid, thio-
115.1 61.9 6-Pentyl-alpha-pyrone 95.2 66.1 m-Homosalicylaldehyde
140.7 56.5 Adipic acid 70.4 47.6 5-Nonanone,
4-[(2-methyl-3-furanyl)thio]- 64 40.6 Nonivamide 135.6 91.4
18beta-Glycyrrhizinic acid 162.7 100.9 1-Propene, 3,3'-thiobis-
108.4 47.3 alpha-Methylcinnamaldehyde 104.4 54.6
4-(p-Acetoxyphenyl)-2-butanone 91.8 58.9 4'-Methylacetophenone
136.6 55 5-Methyl-2-thiophenecarboxaldehyde 111.4 58.9
1,3-Dithiolane, 2-methyl- 119.9 66.2 4-Ethylbenzaldehyde 120.3
56.3
TABLE-US-00008 TABLE 1(h) Compounds that activate only V1 and A1V1,
with A1 having negative effect on A1V1 activity. A1 is considered
as having negative effect in instances where the A1V1 activity is
at least 20-25% lower than V1 value. Compound at 1 mM or 100 .mu.M
(**) A1V1 V1 Piperine 57.1 120.1 Gluconic acid, monopotassium salt
14.0 33.0 Tartaric acid 29.6 55.8 L-(+)-Tartaric acid* 38.4 60.4
Anisole* 34.7 48.3 2,4-Dodecadienal* 53.5 199.1 2,4-Dodecadienal*
53.5 199.1 2,5-Dimethylphenol 34.2 81.2 2,6-Dimethylphenol 45.8
72.1 2-Isopropylphenol 83.5 108.2 (+)-Neomenthol 44.8 68.8
3-Methyl-1-phenyl-3-pentanol 70.5 100.5
TABLE-US-00009 TABLE 1(i) Compounds that activate only A1, or A1
and V1, but not A1V1. Values less than 20-25% are considered
inactive. Compound at 1 mM or 100 .mu.M (*) A1 A1V1 V1
Propylparaben 93.2 2.1 1.6 Malic acid 1.9 2.4 67.5 2-Methylbutyl
isovalerate 1.7 2.1 75.3 2,4,5-Trimethyl-3-oxazoline* 2.1 4.1 100.7
Heptaldehyde* 4.3 2.4 65.3 2-Ethylfuran* 1.2 1.8 58.7 Desoxycholic
acid* 3.2 2.9 84.4 N-Vanillylnonanamide* 69 1.2 65.5
[0048] Among thousands of compounds screened, in the majority of
instances the response seen with TRPA1V1 cells is reflective of the
response predicted based on what is observed in cells expressing
only TRPA1 or TRPV1. Nonetheless, in a surprising number of
instances, there is a compound-dependent difference in the response
of TRPA1V1, which we hypothesize is due to modulation by TRPA1
and/or TRPV1. Because these key receptors are often co-expressed in
the same sensory cells in vivo, an in vitro system which provides
stable co-expression of TRPA1 and TRPV1, with formation of
hetero-tetramers, provides an advantage in being able to identify
extraordinary cases where the results of TRPA1 and TRPV1 studies do
not reflect possible interactions between the TRPA1, TRPV1, and
TRPA1V1 receptors. These interactions, in a significant number of
instances, do not give the predicted, additive result.
[0049] 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."
[0050] 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.
[0051] 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
212520DNAHomo 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 3360
* * * * *