Patent Publication Number: US-2018049960-A1

Title: Methods And Compositions For Modifying Sensorial Perception

Description:
FIELD OF THE INVENTION 
     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 
     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° C.), protons and noxious chemicals such as capsaicin and resiniferatoxin. TRPA1 is activated by cold temperatures (below 17° 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. 
     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. 
     There remains a need to modify unpleasant sensations associated with beneficial products. 
     SUMMARY OF THE INVENTION 
     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. 
     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. 
     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 
         FIGS. 1A and 1B  illustrates the appearance of cell cultures associated with Example 1. 
         FIG. 2  is a graph showing fluorescence of cells under different growth conditions, as described in Example 1. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     All measurements referred to herein are made at 25° C. unless otherwise specified. 
     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. 
     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. 
     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. 
     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. 
     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. 
     A “medicament”, as used herein, is any agent that promotes recovery from injury or illness, inclusive of pain relievers. 
     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. 
     As used herein, “gastrointestinal symptoms” refers to undesired gastrointestinal events, including bloating, cramping, vomiting, diarrhea, excessive gas production, with or without associated tissue damage. 
     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). 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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 μM 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 μL 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. 
     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. 
     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° 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° C.  FIG. 1B  shows TRPV1-expressing cells incubated at 25° 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° C. and 30° C. prior to exposing the cell line to a control agonist and/or test composition. The cell line may be pre-incubated between 22° C. and 30° 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. 
     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 
     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 
     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 
     TRPV1, TRPA1, TRPA1V1 and pcDNA3 cells were grown in 15 mL growth medium [high glucose DMEM (Dulbecco&#39;s modification of Eagle&#39;s medium) supplemented with 10% FBS (fetal bovine serum), 100 μg/mL penicillin/streptomycin, 100 μg/mL G418] in a 75 cm 2  flask for 3 days in a mammalian cell culture incubator at 33° C. and 5% CO 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 μg of Fluo-4 AM calcium dye dissolved in 5 μL 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×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 μL (approximately 5×10 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 TETRA  instrument (Molecular Devices, Sunnyvale, Calif.) at λ ex  488 nm and λ em  514 nm to record baseline fluorescence following which 20 μL of test material at 1 mM or 100 μM final concentration for pure compounds in a GRAS library or 0.004% for extracts. For agonists, capsaicin 350 nM and AITC at 30 μM; and for controls, ionomycin 10 μM 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 
     We observed that during cultivation of cells for several passages, TRPV1 cells cultivated at 37° C. tended to form clumps and were unhealthy presumably due to high basal activity of TRPV1 ( FIG. 1A ), whereas cells grown at 25° C. did not form clumps and looked healthy ( FIG. 1B ). Pre-incubation of cells at 25° C. for 30 minutes prior to addition of capsaicin gave superior response as compared to cells pre-incubated at 33° C. or 37° C. ( FIG. 2 ). Cells pre-incubated at higher temperatures had proportionally higher basal activity. Thus for screening compounds we implemented the 33° C. growth temperature and 25° C. pre-incubation conditions for all three cell lines. 
     Agonist Hits Identified in the GRAS Compound Library Screen 
     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 μM 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. 
     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 μM for TRPA1 and capsaicin, 350 nM for TRPV1. For TRPA1V1, we used AITC, 30 μM 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 1(a) 
               
             
            
               
                   
               
               
                 Compounds that activated all three receptors, 
               
               
                 with no apparent effect of V1 on A1V1. 
               
            
           
           
               
               
               
               
            
               
                 GRAS Compound at 1 mM or 100 μM(*) 
                 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 1(b) 
               
             
            
               
                   
               
               
                 Compounds that activated all three receptors, 
               
               
                 with A1 and/or V1 having effect on A1V1. 
               
            
           
           
               
               
               
               
               
            
               
                 GRAS Compound at 1 mM 
                   
                   
                   
                   
               
               
                 or 100 μM (*) 
                 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 1(c) 
               
             
            
               
                   
               
               
                 Compounds that activate A1 and A1V1 with similar 
               
               
                 level of Activation of A1 and A1V1. 
               
            
           
           
               
               
               
               
            
               
                   
                 GRAS Compound at 1 mM or 100 μM (*) 
                   
                   
               
               
                   
                 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 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 μM(*) 
                 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 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 μM (*) 
                   
                   
               
               
                   
                 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 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 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 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 μM (**) 
                 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 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 μM (*) 
                 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 
               
               
                   
                   
               
            
           
         
       
     
     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. 
     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.” 
     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. 
     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.