Transient Receptor Potential Channels As Therapeutic Targets

REVIEWS

Transient receptor potential channels as therapeutic targets

Magdalene M. Moran*, Michael Allen McAlexander‡, Tamás Bíró§ and Arpad Szallasi||¶ Abstract | Transient receptor potential (TRP) cation channels have been among the most aggressively pursued drug targets over the past few years. Although the initial focus of research was on TRP channels that are expressed by nociceptors, there has been an upsurge in the amount of research that implicates TRP channels in other areas of physiology and pathophysiology, including the skin, bladder and pulmonary systems. In addition, mutations in genes encoding TRP channels are the cause of several inherited diseases that affect a variety of systems including the renal, skeletal and nervous system. This Review focuses on recent developments in the TRP channel-related field, and highlights potential opportunities for therapeutic intervention.

Transient receptor potential (TRP) channels (BOX 1; FIG. 1) second messenger signalling cascades that are initiated are being ardently pursued as targets for drug discovery. by receptor activation, and some TRP channels function There are several factors that make TRP cation channels on intracellular membranes3. appealing as drug targets. First, although ion channels TRP channels are associated with several pathophysi- have been successful drug targets, achieving subtype- ological processes, which include (but are not limited to) selectivity has always been a major challenge, particu- pain, respiratory reflex hypersensitivity, cardiac hyper- larly with voltage-gated sodium and calcium channels. trophy and ischaemic cell death3. In addition, several As members of the TRP family of channels do not share gene association studies in humans have indicated that much homology with one another, the identification of single-nucleotide polymorphisms (SNPs) in the cod- subtype-selective compounds is likely to be more attain- ing regions and/or promoters of genes that encode TRP

*Hydra Biosciences, able. Second, TRP channels act as integrators of several channels are either associated with an increased risk of 790 Memorial Drive, well-described signalling systems, including those that multifactorial diseases or they appear to be causative Cambridge, Massachusetts are mediated by cell surface receptors (for example, factors in rare heritable conditions4. Interestingly, when 02139, USA. G protein-coupled receptors (GPCRs) and growth fac- these mutated TRP channels are expressed in recombi- ‡Neuronal Targets Discovery Performance Unit, tor receptors). Third, mutations in many of the genes nant systems, they generally display enhanced activity, GlaxoSmithKline that encode TRP channels are sufficient to cause disease which suggests that blockade of these channels may pro- Pharmaceuticals,King of in humans. vide therapeutic benefit. Prussia, Pennsylvania, USA. Pioneering research in the field of pain has established To date, target validation of TRP channels has largely § Department of Physiology, that a subset of TRP channels (those that are activated been generated via genetic studies; by comparison, the Research Center for Molecular Medicine, by temperatures; the so-called thermoTRP channels) identification of chemical modulators of TRP channels University of Debrecen, 4032 are capable of initiating sensory nerve impulses fol- is in its infancy. Several natural ligands (for example, Debrecen, Hungary. lowing the detection of chemical and thermal stimuli capsaicin and menthol) have provided valuable insights ||Department of Pathology, (reviewed in REFS 1,2). Although pain is currently the into the pharmacology of TRP channels (reviewed in Monmouth Medical Center, REFS 1,2,5 300 Second Avenue, most advanced TRP channel-related field, an increasing ). Although these molecules can be informa- Long Branch, number of gene deletion studies in animals and genetic tive when they are used as tools for compound screen- New Jersey 07740, USA. association studies in humans have demonstrated that ing, they rarely display the potency, selectivity and/or ¶Drexel University College the pathophysiological roles of TRP channels extend well the physical properties that are desirable in modern of Medicine, Philadelphia, beyond the sensory nervous system (reviewed in REF. 3). drug discovery programmes. However, despite these Pennsylvania 19102, USA. Correspondence to A.S. Indeed, even broadly classifying TRP channels as sensors obstacles several pharmaceutical companies have been e‑mail: [email protected] of environmental cues understates the diversity of their able to develop blockers of TRP cation channel subfam- doi:10.1038/nrd3456 function. In fact, many TRP channels are activated by ily V, member 1 (TRPV1; also known as the capsaicin

Pruritus receptor); these blockers have been sufficiently safe and developments in this rapidly evolving field, highlight Pruritus (also called an itch) is effective in preclinical studies to merit their testing in crucial advances and look ahead to the next steps in an unpleasant cutaneous clinical trials. Many of these trials are still underway elucidating the roles of TRP channels in neurology, der- sensation that is associated (TABLE 1; Supplementary information S1 (box)), but the matology, pulmonology, cardiology, urology, oncology with an urge to scratch. Various categories of pruritus have results of published trials have not been straightforward; and heritable diseases. been suggested, including rather, they have raised new questions regarding the role pruriceptive itch (which arises of TRPV1 in humans. TRP channels as analgesic targets from skin conditions), Despite these setbacks, we believe that the pace of The role of TRP channels is best understood in the pain neurogenic itch (which is drug discovery in the field of TRP channels has inten- area (FIG. 2). TRPV1 and TRPV3 antagonists have already caused by systemic disorders), (TABLE 1) neuropathic itch (which is due sified. The diversity in the expression, function and advanced to clinical trials , whereas TRPA1 to a primary neurological structure of TRP channels provides the opportunity antagonists (TABLE 2) are still in preclinical development. disorder) and psychogenic itch. to generate novel, selective chemical entities that can be tested in diverse clinical indications. Molecules that TRPV1. As the desensitization of nociceptive neurons to target TRPV3 have entered Phase I trials6 (see the Sanofi capsaicin has analgesic potential5, the cloning of the cap- website), and blockers of TRP cation channel subfamily saicin receptor, TRPV1 (REF. 9), has spurred considerable A, member 1 (TRPA1)6, TRP cation channel subfamily efforts in the pharmaceutical community to find TRPV1 M, member 8 (TRPM8)7 and TRPV4 (REF. 8) have been antagonists. However, side effects associated with the use efficacious in preclinical disease models and devoid of of TRPV1 antagonists have so far prevented any com- unexpected acute adverse effects that could limit their pounds from progressing beyond testing in Phase II tri- tolerability (TABLE 2). als. Particular concerns have surfaced around the effects Although results from clinical trials will serve as the of TRPV1 antagonism on the regulation of body tem- final arbiters of the utility of TRP channel modulators perature10 and in the detection of noxious heat (S. Eid, as therapeutics, current evidence indicates that these personal communication). channels contribute to the development and/or progression of the symptoms of many diseases (for example, TRPV1 and regulation of body temperature. neuropathic pain, overactive bladder, asthma, anxiety Trpv1‑null9,11 and -knockdown12 mice have an appar- disorders and pruritus) and that they are therapeutic ently normal body temperature, despite the fact that they targets that are amenable to blockade by small mole- prefer lower ambient temperatures13. These characteris- cules. In this Review, we summarize the state-of-the-art tics are also observed in rats in which TRPV1‑expressing

Box 1 | Introduction to TRP channels The transient receptor potential (TRP) cation channel superfamily is a diverse family of 28 cation channels that have varied physiological functions, including thermal sensation, chemosenation, magnesium transport and iron transport (reviewed in REF. 3). The TRP channel superfamily is classified into six related subfamilies: TRP cation channel subfamily C (canonical; TRPC), TRP cation channel subfamily V (vanilloid; TRPV), TRP cation channel subfamily M (melastatin; TRPM), TRP cation channel subfamily A (ankyrin; TRPA), TRP cation channel polycystin subfamily (TRPP) and TRP cation channel mucolipin subfamily (TRPML)3. The TRPML and TRPP subfamilies were named after the human diseases they are associated with (mucolipidosis and polycystic kidney disease, respectively). The founding member of the TRPM subfamily, TRPM1, was identified via comparative analysis of genes that distinguish benign nevi from malignant melanoma146. The TRPA subfamily has only one known member (TRPA1) and its name refers to the unusually high number of ankyrin repeats at the amino terminus of the channel protein (FIG. 1). Mammalian TRP channels that are most similar to the product of the Drosophila melanogaster Trp gene are referred to as TRPC proteins3. The TRPV subfamily was identified following expression cloning of TRPV1, which is the receptor for the prototypical irritant vanilloid, capsaicin9. Overall, few generalizations can be made about TRP channels. Most members of the TRP channel superfamily share a low level of structural similarity (FIG. 1), but some channels — such as TRPC3 and TRPC7, as well as TRPV5 and TRPV6 — are highly homologous to each other3. Most of the channels are predicted to have six transmembrane domains and large intracellular amino and carboxyl termini (FIG. 1). Many TRP channels form functional channels as homotetramers, although heteromultimerization is not uncommon3. The latter phenomenon may have important implications in drug discovery as it is crucial for understanding the endogenous subunit composition of the channels so that TRP channels can be appropriately targeted with a pharmacological agent. Consistent with their diverse structure, TRP channels also serve diverse functions. Although most members of the TRP channel superfamily are cation channels with limited selectivity for calcium, both calcium‑selective (such as TRPV5 and TRPV6) and sodium-selective (such as TRPM4 and TRPM5) members of the TRP channel subfamilies exist3. In addition, some TRP channels transport non-canonical cations such as iron (TRPML1) or magnesium (TRPV6). Temperature also exerts profound effects on several TRP channels. Although TRPV1 and TRPM8 have been clearly demonstrated to serve as sensors for changes in environmental temperature, many other TRP channels have temperature coefficients such that a change in temperature of 10°C has profound effects on channel activity3. These include TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5 and TRPA1. Data from animal models and human genetic studies have shown that TRP channel dysfunction (which is known as TRP channelopathy) can cause various pathological conditions, including an inherited pain syndrome, multiple kidney diseases and skeletal disorders (reviewed in REF. 4).

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Figure 1 | Diversity in structure among TRP channel families. The six transient receptor potential0CVWT (TRP)G4GX cationKGYU^&TWI&KUE familiesQX GT[ contain very different motifs in their amino and carboxyl termini. The TRP cation channel subfamily V (TRPV), TRP cation channel subfamily A (TRPA) and TRP cation channel subfamily C (TRPC) families have amino terminal ankyrin repeat (AnkR) domains that are not present in other TRP channel subfamilies. The TRP box, which is found in the TRPV, TRP cation channel subfamily M (TRPM) and TRPC families, is thought to be involved in gating. TRP cation channel polycystin subfamily (TRPP) and TRP cation channel mucolipin subfamily (TRPML) proteins both have endoplasmic reticulum (ER) retention domains that may be due to their functional localization on intracellular organelles. aa, amino acids; CIRB, calmodulin/inositol-1,4,5-tris-

phosphate (Ins(1,4,5)P3) receptor binding domain; NUDIX, nucleoside diphosphate-linked moiety X; PDZ, acronym for postsynaptic density protein 95 (PSD95), Drosophila disc large tumour suppressor (DLGA) and zonula occludens protein 1 (ZO1). Image is reproduced, with permission, from REF. 180 © (2003) Macmillan Publishers Ltd. All rights reserved.

neurons have been ablated by high-dose capsaicin (for example, ABT‑102 (REF. 16) and AZD1386 (REF. 17); treatment, which was administered when the rats were TABLE 1) are associated with hyperthermia, although the neonates5. Therefore, the discovery that some TRPV1 effects of these antagonists were not as pronounced as antagonists cause hyperthermia in preclinical studies the effects that were observed with AMG517. and humans was somewhat unexpected10. In rats, it was possible to eliminate hyperthermia Is the hyperthermic action of TRPV1 antagonists while preserving analgesic activity by differential block- separable from their analgesic action? Several studies ade of TRPV1 activation. Compounds (for example, have noted that treatment with antagonists that block AMG8562; see Supplementary information S2 (table)) the three primary TRPV1 activators (that is, capsaicin, that prevented the activation of rat TRPV1 by capsai- low pH and heat) in vitro results in transient hyper- cin, but not by low pH or heat, had no effect on body thermia in experimental animal models (reviewed in temperatures in the rat models; however, these com- REF. 14). The severity of this effect varies depending on pounds still caused hyperthermia in dogs18. How well the compound used but it is attenuated after several days this translates into clinical studies remains to be seen. of dosing15. In human volunteers, AMG517 (TABLE 1) Notably, PHE377 (which is currently in Phase Ib trials) caused a hyperthermic response that lasted for 1–4 days did not cause hyperthermia in rats or dogs, although it and raised body temperatures up to 40.2°C10. Other clin- did inhibit all three major modalities of TRPV1 activa- ical studies have also shown that TRPV1 antagonists tion (see the PharmEste website).

Table 1 | Therapeutic targeting of TRPV1 Compound Therapeutic indications Stage of development (status) Published data and press releases Agonists* ALGRX‑4975 Analgesia after total knee replacement Phase III trial (ongoing) See the Drugs.com website for further information surgery and bunionectomy on Anasevia’s Phase III trial results WN‑1001 Cluster headache, osteoarthritis Phase III trial (completed) ClinicalTrials.gov identifier: NCT00033839 NGX‑4010 (Qutenza; Postherpetic neuralgia Phase III trial (ongoing) See Drugs.com website for further information on Astellas Pharma/ post-hepatic FDA approval of Qutenza NeurogesX) Antagonists‡ ABT‑102 Pain associated with inflammation, Phase I trial ClinicalTrials.gov identifier: NCT00854659 tissue injury and ischaemia AMG‑517 Pain Phase Ib trial (terminated) REFS 11,21 AZD‑1386 Chronic nociceptive pain and GERD Phase II trial (terminated) REF. 19 DWP‑05195 Neuropathic pain Phase I trial (ongoing) Press release: 21 January 2009 (Daewoong Pharmaceutical website) GRC‑6211 Pain, migraine, urinary incontinence- Phase II osteoarthritis trial Press release: 24 October 2008 (Glenmark associated pain and osteoarthritis (suspended) Pharmaceuticals website) JTS‑653 Pain Phase II trial (ongoing) Clinical Development of Pharmaceuticals; 29 July 2010: Japan Tobacco website MK‑2295 Pain Phase II trial (completed) ClinicalTrials.gov identifier: NCT00387140 PHE377 Neuropathic pain Phase I trial (ongoing) See the PharmEste website for further information SB‑705498 Pain, migraine and rectal pain Phase II migraine and rectal ClinicalTrials.gov identifiers: NCT00269022; pain trial (terminated) NCT00731250 Phase II non-allergic intranasal rhinitis trial (ongoing) GERD, gastroesophageal reflux disease; TRPV1, transient receptor potential cation channel subfamily V, member 1. *These agonists have been reviewed in REF. 20. ‡See Supplementary information S1 (box) for further information.

TRPV1 antagonists and noxious heat perception in (Qutenza; NeurogesX) (TABLE 1) was recently approved humans. Clinical studies have confirmed the role of for the treatment of various pain conditions21. Injections TRPV1 as a noxious heat sensor in humans (S. Eid, of resiniferatoxin, an ultrapotent capsaicin analogue5 personal communication). Indeed, the threshold for (see Supplementary information S2 (table)), are being detecting painful heat was considerably elevated in evaluated as a so-called ‘molecular scalpel’ to achieve non-sensitized skin of healthy volunteers following long-term analgesia in patients with cancer who have oral administration of 400 mg of SB‑705498 per day chronic, intractable pain (ClinicalTrials.gov identifier: (Supplementary information S2 (table)), with sub- NCT00854659). A novel approach for minimizing the sequent studies reporting blunted heat perception in burning pain reaction at the application site, which is the healthy human subjects, which was not desensitized main adverse effect of capsaicin administration, is the after repeated dosing19. This effect could potentially activity-dependent targeting of TRPV1 using perma- cause scalding injuries during common activities such nently charged agonists that only permeate the core of as taking a hot shower or consuming hot food or bever- the TRPV1 channel when it is open22. Such agonists are ages. Indeed, some subjects taking MK‑2295 perceived expected to target (and subsequently desensitize) hyper- potentially harmful temperatures as innocuous (S. Eid, active TRPV1 and spare normal nociception. personal communication). In randomized clinical trials, similar findings were reported using ABT‑102 (which TRPA1. TRPA1 is a receptor for a range of environmental was administered at a dose of up to 4 mg twice a day) and irritants and oxidants23–29, and it has an important role in AZD1386 (which was administered at a single daily dose many preclinical models of pain6. TRPA1 is directly acti- of 95 mg)16,17. Notably, there were no other relevant safety vated by structurally diverse chemicals. These include: findings in these two trials and the investigators felt that cinnamaldehyde (which is present in cinnamon), allyl AZD1386 may have clinical potential in relieving pain isothiocyanate (which is found in mustard oil), allicin associated with gastroesophageal reflux disease17. (which is present in raw garlic), formalin (a chemical that is commonly used to induce experimental pain and TRPV1 agonists (capsaicin and resiniferatoxin) in the is also a hazardous respiratory irritant) and icilin, which clinic. Topical TRPV1 agonists (for example, capsaicin is a synthetic compound that produces a sensation of creams) have been used clinically for many years to alle- extreme cold (Supplementary information S2 (table); viate chronic painful conditions such as diabetic neurop- reviewed in REF. 6). When they are applied topically, athy20. An occlusive high-concentration capsaicin patch many of these compounds cause pain in humans. Several

Table 2 | Stage of development of drugs targeting TRP channels Channel Findings from in vitro Genetic deletion Studies linking Published Efficacy with Published clinical studies studies mutation to pharmacological pharmacological trial data disease modulators agents in vivo TRPV1 Acts as a capsaicin Decreased sensitivity None reported Discussed in Table 1 Desensitization See Supplementary receptor, and is to noxious heat, and to agonists; information S1 a heat-activated reduced thermal pharmacological (box) non-selective cation hyperalgesia in blockade by channel2 numerous pain models2 antagonists1,2,5 TRPV3 A heat-activated Effects on skin, heat None reported Agonists: incensole CCI, CFA GRC 15300 (details non-selective sensation44,50 acetate, 2‑APB (GRC 15300)49 not reported)6 cation channel that (nonspecific49) See the Sanofi is potentiated by Antagonists: website for further repeated activation6,49 GRC 15300 (no information structure available) TRPV4 A non-selective Increased bone Skeletal dysplasias Agonists: 4aPDD, Agonists cause None cation channel. Acts density114,115 altered including SMDK, GSK1016790A profound as an osmosensor urination brachyolmia type (REF. 69) circulatory that is activated by 3, hereditary Antagonist: collapse110 and metabolites of the motor and sensory HC‑067047 (REF. 67) antagonists arachidonic acid neuropathy have efficacy in pathway. Involved type 2C, bladder cystitis in osteoclast scapuloperoneal (HC‑067047; differentiation and and congenital REF. 67) bone resorption114,115 distal SMA116–120 TRPC3 A diacylglycerol- Ataxia and reduced None reported Antagonist: Reduced cardiac None activated non-selective mGluR signalling140 ethyl‑1-(4‑2,3,3- hypertrophy in cation channel that is trichloroacrylamide) mice a downstream target phenyl)‑5-

of multiple Gq-coupled (trifluoromethyl)- GPCRs3 1H-pyrazole‑4- carboxylate165 TRPC5 A non-selective Reduced anxiety None reported None None None cation channel that is behaviours potentiated by calcium and reduced

and Gq-coupled CCK4-invoked GPCR signalling, and currents in forms heteromultimers hippocampal slices141 with TRPC13 TRPML1 An intracellular Deletion in Drosophila Mucolipidosis None None None iron-permeable melanogaster results in type IV3,4 channel131 neurodegeneration*166 TRPM1 Cloned following a Mutation identified as Associated with None None None screen for genes that the potential cause of the complete mark melanoma cells146; heritable stationary form of human is a marker for melanoma night blindness in congenital progression146; small horses168 stationary night current, activated by blindness169–171 steroids and blocked by zinc167 TRPM2 An oxidant sensor Increased blood Downregulation Antagonist: None None that is potentiated by glucose levels in or mutation may N‑(p-amylcinnamoyl) increases in levels of Trpm2–/– mice172 be associated with anthranilic acid173 intracellular calcium, bipolar disorder cyclic ADP ribose, or hereditary hydrogen peroxide and deafness144 NADP172 TRPM6 An outwardly rectifying Genetic deletion is Inherited None None None channel, involved in lethal174; heterozygotes hypomagnesaemia4 magnesium transport4 show mild hypomagnesaemia174 TRPM7 An outwardly rectifying Knockout is lethal; Potential Antagonists: NDGA, None None channel. siRNAs targeted deletion susceptibility locus AA861 and MK886; reduce cell death in in T cells disrupts for Guamanian Antagonists: hippocampal neurons thymopoiesis without amyotrophic nafamostat mesylate‡ owing to oxygen and affecting magnesium lateral sclerosis activates TRPM7 glucose deprivation145 homeostasis175 and parkinsonism under physiological dementia176 conditions177

Table 2 cont. | Stage of development of drugs targeting TRP channels Channel Findings from in vitro Genetic deletion Studies linking Published Efficacy with Published clinical studies studies mutation to pharmacological pharmacological trial data disease modulators agents in vivo TRPM8 A cold- and Affects the sensation None reported Agonists: menthol, Agonists: wet dog Agonists: cutaneous menthol-activated of environmental icilin, WS‑12 and shakes; analgesia menthol (migraine); non-selective cation cold; mild pain WS‑5 (REF. 1) in CCI model of menthol patch channel (reviewed in phenotype52–54 Antagonists: chronic pain55 (mild to moderate REFS 1,2) benzimidazoles, Antagonists: muscle strain)78 AMTB (REF. 70) AMTB decreased Antagonists: D‑3263 volume-induced (ClinicalTrials. voiding events70 gov identifier: NCT00839631) TRPP2 A non-selective cation Pkd2–/– mice usually Mutation in None None None channel that forms die mid-gestation as a gene encoding heteromultimers with result of kidney cysts, polycystic kidney several other TRP oedema and placental disease 2 protein122 channels. Involved in abnormalities179 ciliary movement121,123 TRPA1 Channel activated by Reduced sensitivity Mutations cause Agonists: multiple Formalin, AITC, None reported reactive chemicals, to environmental familial episodic reactive chemicals cinnamaldehyde- potentiated by cold irritants; reduced pain syndrome43 (endogenous and flinch33–35; pain behaviours in exogenous) that CFA-cold38, numerous models; lead to sulfhydryl mechanical35; reduced responses modification of SNL-mechanical; in an allergic asthma cysteine reidues SNI-cold; model Antagonists: MIA-induced OA40 HC‑030031 (REF. 33), AP‑18 (REF. 34), A‑967079 (REFS 40,41) 2-APB, 2‑aminoethoxydiphenyl borate; 4aPDD, a-phorbol 12,13-didecanoate; AITC, allyl isothiocyanate; AMTB, N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]- N-(2-thienylmethyl)benzamide hydrochloride; CCI, chronic constriction injury; CCK4, cholecystokinin 4; CFA, complete Freund’s adjuvant; DAG, diacylglycerol;

GPCR, G protein-coupled receptor; IC50, half-maximal inhibitory concentration; mGluR, metabotropic glutamate receptor; MIA, monoiodoacetate; OA, osteoarthritis; SMA, spinal muscular atrophy; SMDK, spondylometaphyseal dysplasia, Koslowski type; SNI, spared nerve injury; SNL, spinal nerve ligation; TRPA1, transient receptor potential cation channel subfamily A, member 1; TRPC1, TRP cation channel subfamily C, member 1; TRPM1, TRP cation channel subfamily M, member 1; TRPML1, TRP cation channel mucolipin subfamily 1; TRPP2, TRP cation channel polycystin subfamily 2 (also known as PKD2); TRPV1, TRP cation channel ‡ subfamily V, member 1. *This is possibly owing to reduced clearance of macromolecules and apoptotic cells. IC50 varies with concentration of divalent cations.

endogenous compounds (for example, 4‑hydroxy- information S2 (table)), prevents the development of nonenal, A‑ and J‑series prostaglandins and hydrogen mechanical hyperalgesia in animal models of diabetes- peroxide) that are released following tissue damage and induced pain, which suggests that endogenously pro- inflammation also directly activate TRPA1 via covalent duced oxidant and inflammatory mediators may act via binding and induce pain behaviours in mice25,30,31. TRPA1 to contribute to diabetes-induced pain36. In addi- Key pronociceptive signalling pathways also indi- tion, animals that were treated with Chembridge‑5861528 rectly potentiate TRPA1 activity by increasing intracel- had reduced nerve damage in response to streptozotocin, lular calcium concentration. For example, bradykinin, which indicates that TRPA1 antagonists could be used for proteinase-activated receptor 2 and nerve growth factor disease modification in painful diabetic neuropathy36. In potentiate TRPA1 currents via their respective receptors rats, intrathecal TRPA1‑targeted antisense oligonucleo- (reviewed in REFS 1,6). Furthermore, mice with disrupted tides37 or TRPA1 antagonists can also dramatically reduce TRPA1 function fail to develop pain behaviour and ther- cold hypersensitivity after nerve injury or inflammation35. mal and mechanical hypersensitivity after intraplantar Cold temperatures are a considerably weaker activa- injection of bradykinin24,32, which provides evidence that tor of the TRPA1 channel38. However, cold temperatures this phenomenon is relevant in vivo. dramatically potentiate TRPA1 activity in the presence of Collectively, the above results led to efforts that aimed other agonists38. Furthermore, the selective TRPA1 antag- to identify TRPA1 antagonists as therapies for treating onist HC‑030031 reduces cold hypersensitivity in rodent pain6. Two moderately potent but highly selective TRPA1 models of inflammatory and neuropathic pain without antagonists, HC‑030031 (REF. 33) and AP‑18 (REF. 34), altering normal cold sensation in naive animals33,38,39. have subsequently been discovered (see Supplementary Similarly, A‑967079 — a compound that is structurally information S2 (table)). similar to AP‑18 (Supplementary information S2 (table)) In rodents, HC‑030031 reduces acute and chronic — reduces cold hypersensitivity after nerve injury without inflammatory pain, and reduces neuropathic pain with- affecting acute responses to environmental cold40. Thus, out having any apparent effect on motor coordination TRPA1 appears to selectively mediate cold hypersensitiv- or noxious cold detection33,35. A structurally related ity in pathological conditions in which other activators of compound, Chembridge‑5861528 (Supplementary the channel are also present.

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Figure 2 | TRP channels as nociceptors. Sensory neurons express multiple transient receptor0CVWT potentialG4GXKGYU (TRP)^&TWI&KUEQ channels. TRPXGT[ cation channel subfamily V, member 1 (TRPV1), TRPV3 and TRPV4 all respond to warming temperatures. Noxious heat activates TRPV2, but the physiological relevance of this is unclear. Acids are robust activators of TRPV1 and bases have emerged as activators of TRP cation channel subfamily A, member 1 (TRPA1). TRPA1 is a key chemoreceptor that responds to scores of reactive chemicals. At higher concentrations, some of these chemicals also activate TRPV1. TRP cation channel subfamily M, member 8 (TRPM8) serves as the key receptor for environmental cold, although TRPA1 also has a role in cold hyperalgesia. Activation of any of these TRP cation channels can trigger action potentials in the sensory neuron. Some of these channels, such as TRPV1, are also expressed in the spinal cord, where they seem to have an important role in the central nervous system as well. DRG, dorsal root ganglion. Image is reproduced, with permission, from REF. 181 © (2002) Macmillan Publishers Ltd. All rights reserved.

Although TRPA1 may have an analogous role in with diabetic neuropathy47. TRPV3 appears to be unique nociceptor mechanotransduction, whether TRPA1 is among TRP channels in that repeated stimulation of the mechanically activated per se remains to be determined. channel leads to sensitization that depends on the pres- 48 In rats, A‑967079 reduced the frequency of firing of wide- ence of calcium . Activation of Gq-coupled GPCRs — range dynamic neurons in lamina V of the spinal cord in including the histamine and bradykinin receptors — also response to noxious mechanical stimuli41. These data are potentiates TRPV3 activity45, thus allowing TRPV3 to consistent with data taken from an isolated rat skin-nerve serve as a convergence point for multiple pain pathways preparation using HC‑030031 and from mice lacking (reviewed in REF. 49). TRPV3 is activated in response to TRPA1, which suggests that TRPA1 is required for pro- temperatures in the range of 31–39 °C and to the chemicals longed neuronal firing in response to certain high-inten- camphor and 2‑aminoethoxydiphenyl borate (reviewed in sity mechanical stimuli42. REFS 1,49). Trpv3‑null mice had defects in thermal selec- In conclusion, preclinical data (and data from a tion behaviour in response to innocuous heat, and defects recent human genetic study43) highlight TRPA1 antago- in withdrawal behaviour in response to noxious heat50. nists as a promising new approach for the treatment of Taken together, these findings suggest an important role acute and chronic pain. The current status of several for TRPV3 in pain transduction. TRPV3 antagonists have drug development programmes is presented in TABLE 2. shown efficacy in preclinical neuropathic and inflammatory pain models (TABLE 2), and one molecule has entered TRPV3. Changes in the levels of expression of TRPV3, Phase I clinical trials (see the Sanofi website). which is usually highly expressed in the skin44,45, can occur in human disease states. For example, TRPV3 expres- TRPM8. In sensory ganglia, TRPM8 expression dis- sion is increased in painful breast tissue46 and decreased tinguishes a specific subpopulation of primary sensory in basal keratinocytes that are recovered from patients neurons that are cold-sensitive (reviewed in REFS 1,2).

The finding that the heat-sensitive TRPV1 and the cold- may also provide relief from some forms of pain. For sensitive TRPM8 are co-expressed in some neurons51 example, Trpm8–/– mice failed to develop cold allodynia underlines the hypothesis that individual neurons may after chronic constriction injury or injection of complete be able to sense ranges of both hot and cold temperatures. Freund’s adjuvant (CFA)54. Importantly, a TRPM8 antag- In vitro, TRPM8 is activated by cool temperatures in onist reduced cold allodynia in a chronic constriction the range of 10–23 °C, as well as the cooling agents men- injury model of chronic neuropathic pain, thus recapitu- thol (Supplementary information S2 (table)) and icilin lating the phenotype of genetic deletion56. (reviewed in REFS 1,2). Furthermore, TRPM8‑deficient The observation that both TRPM8 agonists and mice showed no preference for warm temperatures over TRPM8 antagonists may be useful for the treatment of cool temperatures, and they exhibited impaired cold pain highlights the importance of selecting appropriate avoidance behaviour, which highlights the essential role models to examine TRP channel modulators. of TRPM8 in the sensation of environmental cold52–54. Controversy surrounds the utility of TRPM8 as an TRP channels in bladder disorders analgesic target. The TRPM8 agonist menthol decreases Several TRP channels are expressed in the bladder — nociceptive responses in animal models of inflammatory in the urothelium, nerve endings and detrusor muscle and neuropathic pain55. However, TRPM8 antagonism (FIG. 3) — where they are thought to function as sensors of

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Figure 3 | Roles of TRP channels in bladder functions. The micturition reflex is mediated by transient receptor potential cation channel subfamily V, member 1 (TRPV1)-positive nerves and probably — albeit0CVWT G4Gto a XKGYUlesser^ degree&TWI&KUEQ — alsoXGT[ by TRP cation channel subfamily M, member 8 (TRPM8)-positive nerves. These same neurons convey nociceptive information (for example, bladder pain that is secondary to cystitis) to the central nervous system. TRPV4 is a key player in bladder function because it is present in both the urothelium and the detrusor muscle, and it is activated by stretch (bladder distension) and hypo-osmolar urine. The activator of TRPM8 in the bladder remains to be determined. The micturition reflex is under the control of a descending central nervous system pathway; when this pathway is disrupted (for example, as a result of spinal cord injury or multiple sclerosis), it becomes autonomous and partly driven by TRPV1. The existence of functional TRPV1 in urothelial cells remains controversial, as evidence for (REFS 64, 66) and against (REF. 65) the presence of a funtional channel has been presented. Collectively, these findings imply a therapeutic potential of TRPV1 antagonists (or TRPV1 desensitization) and/or TRPM8 agonists as therapies in painful bladder disorders (and in pain induced by benign prostatic hyperplasia) and in an overactive bladder. These findings also suggest that TRPV4 blockers could be useful in the management of an overactive bladder. DRG, dorsal root ganglion.

stretch and chemical irritation (reviewed in REFS 57,58). micturition pattern that is characterized by increased Intravesical administration of TRPV1 agonists has been intermicturation intervals and spotting behaviour due used in the management of the overactive bladder for to spontaneous (that is, not reflex-driven) contractions many years, largely on an empirical basis (reviewed in of the detrusor muscle66. In addition, Trpv4–/– mice show REF. 58). The recent recognition of disease state-related reduced urinary frequency and increased void volume changes in the expression of TRP channels has provided after bladder damage caused by intravesical administra- a new impetus to investigate the roles of these channels tion of cyclophosphamide67. Based on these findings, it in normal bladder function and dysfunction. has been postulated that TRPV4 plays a crucial part in the mechanosensory pathway in the bladder by detect- TRPV1. There is a dense network of nerve fibres that ing changes in intravesical pressure. Indeed, mechani- express TRPV1 in the suburothelium and muscular cal stretch has been shown to activate urothelial TRPV4 layer of the human renal pelvis, ureter, bladder and in vitro68. urethra57,58, but the existence of functional TRPV1 in Activation of TRPV4 that is located in the detrusor the urothelium and detrusor smooth muscle remains muscle can lead to muscle contractions. Accordingly, controversial59. The involvement of neuronal TRPV1 in the TRPV4 agonist GSK1016790A (Supplementary the micturition reflex is well established57,58. Indeed, rats information S2 (table)) causes direct contraction of in which TRPV1‑expressing nerves have been ablated the bladder, even in the absence of the urothelium69. by neonatal capsaicin treatment develop a dis- As predicted by data generated from Trpv4–/– mice, the tended bladder5. Trpv1–/– mice, however, show only TRPV4 antagonist HC‑067047 (Supplementary infor- spotty incontinence60. It is possible that another mation S2 (table)) improved bladder function in mice protein compensates for the lack of TRPV1 or that with cyclophosphamide-induced cystitis67. Acute dosing TRPV1‑containing, neuron-specific gene products of HC‑067047 did not alter water intake, core body tem- other than TRPV1 contribute to the phenotype that is perature, thermal selection behaviour, heart rate, loco- observed in these animals. motion or motor coordination in vivo. Taken together, In humans, desensitization therapy using intravesical these observations imply that TRPV4 antagonists could TRPV1 agonists (capsaicin or resiniferatoxin) is based be valuable in the management of an overactive bladder. on the concept that the C fibre-driven micturition reflex — which is inactive in adult life — reassumes control TRPM8. In the human bladder, TRPM8 is present in of micturition in the overactive bladder, in both neu- both urothelial and suburothelial myelinated nerve rogenic and non-neurogenic cases57,58. In patients with fibres, with increased expression in patients who have neurogenic detrusor overactivity disorders, intravesical bladder pain or idiopathic detrusor overactivity70. The administration of resiniferatoxin provides symptomatic TRPM8 agonist menthol evokes the micturition reflex relief by increasing bladder capacity and decreasing the in humans70. Conversely, the TRPM8 antagonist AMTB frequency of daily episodes of incontinence61. Moreover, (Supplementary information S2 (table)) decreases the intrathecal administration of resiniferatoxin blocks frequency of volume-induced bladder contractions in a detrusor overactivity in rats that have complete spinal rat model of painful bladder syndrome71. These findings cord transection62, and intravesical administration of imply that TRPM8 antagonists have therapeutic potential resiniferatoxin reduces the frequency of incontinence in the management of bladder disorders that are charac- episodes in patients with spinal cord injury (reviewed terized by pain and/or overactivity of the detrusor muscle. in REFS 61,63). The therapeutic value of TRPV1 antagonists in manag- TRP channels in the skin ing detrusor muscle overactivity is unclear, as no endoge- Populations of non-neuronal cells within the skin express nous agonist has been identified in the bladder of patients many different types of TRP channels (FIG. 4), which are who suffer from incontinence. By contrast, the pain and thought to be involved in various key cutaneous func- bladder hyperactivity that accompany interstitial cystitis tions including skin-derived pruritus, proliferation, dif- are thought to be amenable to therapy with TRPV1 antag- ferentiation, cancer and inflammatory processes (BOX 2). onists63. In a feline model of interstitial cystitis, abnor- Prurigo nodularis mally enhanced responses to capsaicin were detected TRPV1 as a key molecule in itch. TRPV1 is involved A skin condition that is after TRPV1 phosphorylation by protein kinase C64. in the development of skin-derived pruritus, which is characterized by itchy nodules (circumscribed, solid elevations In mice, genetic manipulation of the Trpv1 gene prevents thought to occur through itch-specific subpopulations of on the skin), which usually bladder reflex hyperactivity and spinal FOS overexpres- TRPV1‑expressing sensory afferent neurons (also known appear on the arms or legs. sion in experimental models of cystitis60. The TRPV1 as pruritoceptive neurons; reviewed in REFS 72,73). antagonist GRC‑6211 (Supplementary information S2 TRPV1 is also expressed in non-neuronal cell types of Pruritogens 74 Agents that induce itch by (table)) ameliorates micturition reflex activity in the human skin , and its expression is elevated in epidermal 65 75 stimulating pruritoceptive chronically inflamed bladder . Collectively, these find- keratinocytes of patients with prurigo nodularis . sensory afferent neurons. In ings imply a therapeutic value for TRPV1 antagonists in Certain endogenous signalling molecules that poten- the skin, they are synthesized the symptomatic treatment of interstitial cystitis. tiate TRPV1 activity (including acids, ATP, lipoxyge- by and released from multiple nase products, prostaglandins and histamine) are also non-neuronal cell types and pruritogens REFS 72,73 include histamine, acids, ATP, TRPV4. In the bladder, TRPV4 is predominantly potent (reviewed in ). It is probable prostaglandins and expressed in the urothelium, but it is also present in that on sensory neurons, histamine indirectly activates pro-inflammatory interleukins. the detrusor muscle57,59. Trpv4 –/– mice show an altered TRPV1 through histamine H1 receptor-dependent

synthesis of 12‑hydroperoxyeicosatetraenoic acid, hair follicles also inhibited hair growth91, these findings which is an endogenous activator of TRPV1 (reviewed imply a therapeutic potential for topical TRPV3 agonists in REF. 2). Consistent with this finding, genetic deletion and antagonists in the treatment of hirsutism and alo- of Trpv1 in mice substantially suppressed histamine- pecia, respectively. induced scratching behaviour76. In humans, TRPV1 TRPV3 is also involved in the development of skin mediates histamine-induced experimental itch77, as well inflammation. Stimulation of TRPV3 in cultured as pruritus in patients with seasonal allergic rhinitis78. By keratinocytes induced the release of pro-inflammatory contrast, histamine-independent itch — caused by chlo- mediators45. Importantly, DS‑Nh mice develop skin roquine or the endogenous pruritogen peptide BAM8‑22 alterations that are similar to those seen in human atopic (originally isolated from bovine adrenal medulla) — is dermatitis89. Moreover, Trpv3Gly573Ser transgenic mice that predominantly mediated by TRPA1 (REF. 79). express high levels of the mutated, constitutively active Paradoxically, the loss of TRPV1‑expressing neu- TRPV3 protein in epidermal keratinocytes also sponta- rons is also associated with excessive itch. Rats that neously develop an inflammatory condition that is simi- have had their capsaicin-sensitive neurons chemically lar to human atopic dermatitis90. As the skin-targeted ablated may develop skin ulcers as a result of extensive Trpv3 Gly573Ser transgenic mice also exhibit scratching scratching80. Mice lacking the vesicular glutamate trans- behaviour, these data suggest that TRPV3 channels may porter 2 — thereby ostensibly disabling glutamatergic function as important transducers of pro-inflammatory neurotransmission in the spinal dorsal horn, which is signals in the pathogenesis of various forms of dermatitis. the termination site of TRPV1‑positive primary afferent neurons — demonstrated enhanced scratching behav- TRP channels in the pulmonary system iour and reduced sensitivity to noxious heat81,82. Clearly, The mammalian respiratory tract is lined with a dense further neurophysiological studies in humans are plexus of sensory fibres, including those that express needed to dissect the multifaceted relationship between TRPA1 and TRPV1 (FIG. 5). Activation of this subset TRPV1‑positive primary afferent neurons and pruritus. of nerve fibres by irritant and/or inflammatory stimuli triggers multiple reflexes — such as sneezing, cough- Role of TRPV1 in the control of skin growth, skin cell ing, mucus secretion, bronchospasms and apnoea — survival and cutaneous inflammation. It has been sug- that limit ventilation and dilute and/or expel foreign gested that TRPV1 participates in the regulation of materials. Analogous to pain, in which inflammatory cutaneous growth and differentiation. TRPV1‑mediated mediators produce a hypersensitive response to vari- calcium influx in cultured human keratinocytes sup- ous stimuli, reflexes such as coughs are proposed to be Alopecia presses proliferation and promotes apoptosis83,84. In sensitized by mediators of inflammation and oxidant A type of pathological hair loss that mostly affects the scalp. addition, activation of TRPV1 by either capsaicin or stress, such that they become triggered by innocuous The most common forms of heat alters the formation of the epidermal permeability stimuli. Thus, although a distinct and unusual subpopu- alopecia are alopecia barrier in human skin in vivo85. lation of afferent fibres may be responsible for coughing universalis, alopecia areata and TRPV1 has also been suggested to regulate cutaneous reflexes92, airway nociceptors appear to exert a consider- alopecia androgenetica. Telogen Effluvium, which is inflammation. Capsaicin-induced activation of TRPV1 able amount of control over the sensitivity of this reflex. characterized by diffuse hair on human epidermal and hair follicle-derived keratino- shedding, is a form of alopecia. cytes in vitro results in the release of several pro-inflam- TRPV1. Capsaicin is a prototypical respiratory irritant matory cytokines83. In addition, as ultraviolet irradiation that causes noxious sensations as well as reflexes such Hirsutism upregulates TRPV1 expression in human skin86, TRPV1 as coughing, sneezing and fluid secretion when it is Excessive and increased hair growth (especially in women) that is expressed on keratinocytes is a specific media- applied to the human respiratory mucosa (reviewed in on regions of the body where tor of heat shock-induced and ultraviolet irradiation- REF. 5). Increased sensitivity to capsaicin aerosols occurs the occurrence of hair normally induced expression of matrix metalloproteinase 1 in several respiratory disorders of varying severity and is minimal or absent. (REF. 87), an enzyme that is implicated in skin inflam- aetiology (reviewed in REF. 93). Alterations in capsaicin-

Dermatitis mation and remodelling. Taken together, these findings induced coughing may be due to enhanced TRPV1 A universal term describing imply that topical TRPV1 modulators may be used in expression or activity in airway sensory neurons, altera- inflammation of the skin. It can the treatment of sunburn, acne vulgaris and alopecia or tions in the permeability of the epithelial barrier that be induced by various factors hirsutism. allows capsaicin to more readily access airway nocicep- such as allergens (allergic tor terminals and/or heightened responsiveness of the dermatitis), infections, eczema (atopic dermatitis) or external TRPV3. TRPV3, which is expressed at high levels by central nervous system to afferent inputs. Intriguingly, 88 compounds (contact keratinocytes , is crucial in promoting epidermal bar- treatment with the non-selective cyclooxygenase inhibi- dermatitis). rier formation and hair morphogenesis in mouse skin. tor indomethacin increases capsaicin-induced cough This probably occurs through the formation of a signal- thresholds in patients with asthma or chronic bronchitis Apnoea 94 Prolonged periods of time ling complex between TRPV3 and the epidermal growth but not in healthy subjects . These results offer com- 44 without respiratory flow. factor receptor . Consistent with this concept, genetic pelling evidence that inflammatory mediators that are Although humans can perform deletion of Trpv3 in mice caused hair abnormalities produced in diseased airways enhance the sensitivity of this manoeuvre voluntarily (by (that is, wavy hair coat and curly whiskers)44,50, whereas airway reflexes in a manner that can be at least partially holding their breath), reflex the constitutively active gain-of-function mutation reversed by pharmacological intervention. apnoeas can be induced in Gly573Ser human volunteers and animals Trpv3 resulted in a spontaneous hairless pheno- Therapy using intranasal capsaicin to desensitize 89,90 by irritant stimulation of the type in DS‑Nh mice (a mouse model of dermatitis) . TRPV1‑containing sensory neurons provides symp- respiratory tract. As the activation of TRPV3 in organ cultures of human tomatic relief in patients with rhinitis95; however, this

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Figure 4 | TRP channels in human skin. The role of transient receptor potential (TRP) channels0CVWT G4Gin theXKGYU skin^ is&TWI&KUEQ supportedX GT[ Hair cycle by strong evidence (shown using continuous arrows) indicating that TRP cation channel subfamily V, member 1 (TRPV1) is A life-long regeneration programme of the hair follicles expressed by various cell types (sebocytes, keratinocytes, sensory neurons and cells of the hair follicles). TRPV1 activation that can be divided into three is shown to induce heat sensation and the development of skin-derived pruritus, and suppress sebaceous lipid synthesis. phases: anagen (growth), Activation of TRPV1 and TRPV3 shifts the proliferation–differentiation balance of epidermal keratinocytes towards catagen (apoptosis-driven differentiation and, along with TRPV4 and TRPV6, TRPV1 and TRPV3 are involved in the regulation of epidermal barrier regression or involution) and formation. Moreover, activation of TRPV1 and TRPV3 either on epidermal or hair follicle-derived keratinocytes results telogen (resting or quiescence, in increased pro-inflammatory cytokine release, which suggests that these channels are key players in the in situ preparation for the next anagen immunoregulation of human skin. In addition, the hair cycle is regulated both directly (via stimulation of TRPV1 and phase). This cycle is controlled TRPV3) and indirectly (via TRPV1 activation that results in follicular growth factor production) by TRP channels. by promoters (for example, Preliminary findings (indicated using dashed arrows) suggest that TRP cation channel subfamily C, member 1 (TRPC1) insulin-like growth factor 1 and hepatocyte growth factor) and and TRPC4 are likely to have antitumour effects, whereas TRP cation channel subfamily M, member 7 (TRPM7) regulates inhibitors (for example, melanogenesis of melanocytes. In addition, TRP cation channel subfamily A, member 1 (TRPA1) and TRPM8 may have a interleukin‑1β, and synergistic effect with other TRP channels in the regulation of epidermal barrier formation and maturation (that is, transforming growth factor‑β2) differentiation) of keratinocytes.

Box 2 | Emerging functions of TRP channels in cutaneous biology In the human pilosebaceous unit, capsaicin-induced activation of transient receptor potential cation channel subfamily V, member 1 (TRPV1) suppresses hair shaft elongation and induces apoptosis-driven catagen regression84. In this study, TRPV1 activation was accompanied by a considerable alteration in the gene-expression profiles of the cells and modulation of Respiratory irritation intrafollicular production of cytokines and growth factors that control human hair growth in vivo84. Interestingly, compared A stereotypical reflex reduction to wild-type mice Trpv1 gene-deficient mice exhibited a significant delay in hair follicle cycling — that is, a delay in the in respiratory rate exhibited by onset of the catagen phase158. This supports the concept that TRPV1 may exert functions that are primarily small laboratory animals growth-inhibitory in mammalian skin epithelium. Moreover, stimulation of TRPV1 expressed in human sebaceous following irritant aerosol provocation. This behaviour gland-derived SZ95 sebocytes selectively inhibited lipid synthesis (sebum production) and altered the expression profiles 159 generally predicts the irritation of multiple genes that are involved in cellular lipid homeostasis . threshold for molecules in In addition to TRPV1 and TRPV3, other TRP channels (for example, TRP cation channel subfamily C, member 1 (TRPC1), humans. TRPC4, TRPV4, TRPV6, TRP cation channel subfamily A, member 1 (TRPA1) and TRP cation channel subfamily M, member 8 (TRPM8)) have been identified in skin keratinocytes, where they are probably involved in the differentiation — Pilosebaceous unit and malignant transformation — of keratinocytes and barrier formation. Intriguingly, several skin pathologies have Consists of the hair shaft, the altered TRP channel expression. For example, basal cell carcinoma cells lack both TRPC1 and TRPC4 expression160. hair follicle, the sebaceous Conversely, TRPC1 is overexpressed in the skin of patients with Darier’s disease161. TRPV4 seems to promote the gland and the erector pili development and maturation of intercellular junctions in the epidermis85, thus topical TRPV4 agonist preparations may muscle; causes the hair to stand up when it contracts. represent a novel approach in the treatment of acne vulgaris. Stimulation of TRPA1 affects the expression profile of genes that are involved in the control of keratinocyte proliferation and differentiation162. TRPV6 participates in the differ‑ Sebum entiation-stimulatory effects of 1,25‑dihydroxyvitamin D3 by increasing Ca2+ entry163. At present, it is unclear how specific A lipid-enriched, oily exocrine these effects are, as dramatic increases in calcium concentration are likely to affect the proliferation and differentiation of product of the sebaceous skin cells. Last, decreased and/or faulty TRPM7 production leads to impaired melanocytic differentiation164, which can glands that has various result in vitiligo. functions including waterproof barrier formation, antimicrobial 27–29,98 activity, transport and procedure is poorly tolerated and has therefore not to these irritants . Thus, emerging data from animal thermoregulation. been widely adopted. The explanation for the efficacy models largely indicate that TRPA1 is the sole effector of capsaicin desensitization therapy remains elusive. The of sensory neuron activation and respiratory irritation Basal cell carcinoma The most common type of beneficial effects of capsaicin may paradoxically extend reflexes that are triggered by acute exposure to a range malignant skin tumour, which beyond its actions on TRPV1, in that it may functionally of respiratory irritants. One noteworthy exception is develops from the basal cell inactivate the entire TRPV1‑expressing neuron5. Such nicotine, which activates sensory neurons by acting at layer of the epidermis. It rarely neurons express many other gene products in addition both TRPA1 and nicotinic acetylcholine receptors99. metastasizes, but without to TRPV1, including the related polymodal nocisensor Further clarification is required regarding the com- treatment it may cause substantial destruction by TRPA1 (see below). plex interplay between these two mechanisms in vivo. invading the deeper skin tissues. TRPA1 is necessary for increases in the Penh (enhanced TRPA1. Many of the irritants that activate TRPA1 are pause) respiratory measurement caused by intrana- Darier’s disease air pollutants that are produced by the combustion of sal nicotine99; however, similar provocations pro- A congenital skin condition that is characterized by materials (including tobacco products) that cause produce robust respiratory irritation in TRPA1‑deficient 28 dyskeratosis (abnormal nounced cutaneous, ocular and respiratory irritation in mice . keratinization of the epidermis) humans. Several classes of anaesthetic molecules — Although further studies will reveal the contribu- and the appearance of pruritic, including lidocaine, propofol, etomidate and volatile tion of TRPA1 to the overall respiratory pathology that greasy and scaly skin papules gaseous anaesthetics — also act as TRPA1 agonists96. is caused by chronic exposure to hazardous irritants in (circumscribed, solid elevations on the skin) and plaques Although these data raise the possibility that anaesthesia humans, existing data from mice already demonstrate (confluences of papules). may paradoxically increase postoperative pain, the more the role of TRPA1 in allergic airway inflammation. Mice immediate impact of these data is the identification of that either lacked TRPA1 or were pretreated with the Vitiligo TRPA1 as a possible mediator of the respiratory com- TRPA1 blocker HC‑030031 were protected from air- A skin disorder that is bronchoalveolar lavage characterized by plications of gaseous anaesthetics, which can include way inflammation (both in and in depigmentation of patches of coughing and laryngospasms. In support of this hypoth- lung tissue) and bronchial hyperreactivity in response skin. It develops as a result of esis, the TRPA1 blocker HC‑030031 has been shown to to acetylcholine. Transcription of the gene product for impaired functions or death of prevent desflurane-induced increases in airway resist- the gel-forming mucin, MUC5AC, was also reduced100. skin melanocytes, which can be ance in guinea pigs26. This suggests that HC‑030031 prevented gene transcrip- induced by various factors, such as autoimmune Additional hazardous irritants — which include iso- tion, although no measurement of the protein (that is, conditions, genetic factors, cyanates, ozone, chlorine and cigarette smoke extracts the gene product) was performed. Importantly, knock- oxidative stress and infections. — activate overexpressed TRPA1 and cause pulmo- ing out TRPV1 did not alter any of these parameters100, nary nociceptor activation, respiratory irritation and/ thus providing evidence that TRPA1 has a distinct role Penh (enhanced pause) A derived value that is or neurogenic inflammation in a TRPA1‑dependent in this model of allergic disease. In addition, TRPA1 has 25,27–29,97 supposed to characterize the manner . These molecules are broadly toxic, and been implicated in neurogenic inflammation of the ventilatory activity of freely exposure to them causes marked symptoms and injury airway caused by the acetaminophen (paracetamol) moving rodents in in human airways. Perhaps surprisingly, interruption metabolite N‑acetyl‑p-benzoquinone imine in sev- plethysmography chambers of TRPA1 function in rodents — via gene disruption eral species of small rodents101. These compelling data where airflow is measured. Interpretation of this or pharmacological blockade — nearly abolishes demonstrate that TRPA1 can contribute to both res- measurement is debated the activation of sensory neurons and/or respiratory piratory reflexes and inflammation in laboratory-based within the respiratory field. reflexes, including coughs produced by acute exposure animal models.

TRPC and TRPV channels in airway structural and lungs from these animals have marked deficits in arterial inflammatory cells. Several TRP channels are expressed oxygen saturation following airway instillation of saline in bronchial and/or vascular smooth muscle. TRP cat- to produce regional ventillatory failure102. Moreover, ion channel subfamily C (TRPC) has functional roles in a SNP (–254C→–254G) in the promoter region of the the pulmonary vasculature, particularly with regard to gene encoding TRPC6 has been linked to idiopathic responses to hypoxia. Regional alveolar hypoxia redirects pulmonary arterial hypertension103. Consistent with this blood flow to well-oxygenated areas. This reflex mecha- finding, pulmonary arterial smooth muscle cells from nism is impaired in TRPC6‑knockout mice, as ex vivo patients with idiopathic pulmonary arterial hypertension had considerably higher levels of the TRPC6 protein, higher resting levels of cytosolic Ca2+ and larger 8CICNUGPUQT[PGTXGVGTOKPCNU #KTYC[UOQQVJOWUENG 1‑oleyl‑2‑acetyl-sn-glycerol (OAG)-dependent cationic r642#CPF6428 r642%CPF6428 currents than samples from control subjects103. r(WPEVKQPUFGRQNCTK\CVKQPCEVKQP r(WPEVKQPUEQPUVTKEVKQP Smooth muscle cells in diseased airways typically dis- RQVGPVKCNHQTOCVKQPKPKVKCVKQPQH CKTȯQYQDUVTWEVKQP UGPUCVKQPUCPFTGȯGZGU play abnormalities in contraction and/or proliferation. 5GPUCVKQPU 8CIWUPGTXGU Although bronchodilators are efficacious therapies that r7TIGVQEQWIJ are capable of reversing airflow obstruction, treatments r%JGUVVKIJVPGUU that reduce contractility and pathological remodelling of r&[URPQGC CKTJWPIGT airway smooth muscle remain targets for active investi- %05FGRGPFGPVTGȯGZGU gation. Following incubation with the inflammatory r%JCPIGUKPTGURKTCVQT[ RCVVGTP cytokine tumour necrosis factor, human airway smooth r%QWIJ muscle cells had marked changes in Ca2+ homeostasis r2CTCU[ORCVJGVKETGȯGZ that are accompanied by increased expression of the CKTȯQYNKOKVCVKQP 104 r%JCPIGUKPCKTYC[DNQQFȯQY TRPC3 protein . Moreover, enhanced acetylcholine- induced elevations in cytosolic Ca2+ in human airway smooth muscle cells are blocked by RNA silencing of TRPC3. These data are consistent with studies in mice #KTYC[XCUEWNCVWTG with allergen-induced airway inflammation; in these r642%CPFQT642%! GZVTCCNXGQNCT 6428 CNXGQNCT studies TRPC3‑specific antibodies inhibited non-selec- r(WPEVKQPUXCUQEQPUVTKEVKQP tive cation conductances and restored resting membrane 642% KPETGCUGUKPGZVTC potentials of airway smooth muscle cells to values that CNXGQNCT 642%U CPF CNXGQNCT 6428 were more hyperpolarized and consistent with con- XCUEWNCT #NXGQNCTOCETQRJCIGU trols105. Compensatory elevation of TRPC3 expression r6428CPF6428 RGTOGCDKNKV[ –/– r(WPEVKQPUUVKOWNCVKQP occurs in Trpc6 mice, which may explain why these QHRJCIQE[VQUKU 6428 'PFQVJGNKWO mice have enhanced airway reactivity in response to a 405CPFQT415 muscarinic agonist, despite displaying reduced inflam- RTQFWEVKQP 6428 85/%U matory parameters in bronchoalveolar lavage fluid106. 2+ Figure 5 | Diverse roles of TRP channels in the pathophysiology of the mammalian TRPV4 has also been proposed to contribute to Ca 107 respiratory tract. Although transient receptor potential0CVWT (TRP)G4G channelsXKGYU^ generally&TWI&KUEQ XGT[ mobilization in airway smooth muscle . This function increase intracellular Ca2+ concentrations and/or depolarize membrane potentials, their of TRPV4 is one possible mechanism that may explain varied expression patterns and sensitivity to agonists result in considerable functional the genetic association between multiple SNPs in the diversity. For instance, in vagal sensory nerve terminals, noxious chemical and physical gene encoding TRPV4 and chronic obstructive pulmo- stimuli activate TRP cation channel subfamily A, member 1 (TRPA1) and TRP cation nary disease108, although the function of TRPV4 in the channel subfamily V, member 1 (TRPV1) to produce nerve activation, which initiates respiratory tract extends beyond airway smooth muscle reflexes and critically regulates sensations, as shown in the figure. In airway smooth responsiveness. Indeed, the TRPV4 gene containing the muscle cells, functional TRP cation channel subfamily C, member 3 (TRPC3) and TRPV4 chronic obstructive pulmonary disease-associated P19S channels have been identified and these are thought to contribute to constriction that leads to airflow obstruction, as these channels are Ca2+-permeable and Ca2+ is a SNP displays gain-of-function characteristics in human necessary mediator of smooth muscle constriction. TRPC6, which is present in airway airway epithelial cells, where the disease-associated SNP 2+ vascular smooth muscle cells (VSMCs), can mediate vessel constriction to reduce blood has been shown to increase Ca influx and secretion of flow. Several other TRPCs have been identified in endothelial cells of larger matrix metalloproteinase 1 in response to diesel exhaust (extra-alveolar) blood vessels within the lung, where their activation can increase fumes109. vascular permeability and cause fluid to leak into interstitial spaces between The most striking manifestation of TRPV4 biology in vessels. Activation of endothelial TRPV4 also increases vascular permeability, although the lung occurs in the alveolar septae. In isolated, per- this increase is specific to the small blood vessels that supply the alveoli of lungs. TRPV4 fused lungs in mice, activation of TRPV4 by elevated has also been identified on alveolar macrophages, where its activation triggers the vascular pressure110 or injurious high-pressure mechani- production of toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS). cal ventilation111 caused extravascular leakage of fluid, TRPV2 channels are also present on alveolar macrophages, as well as several other as reflected by increases in the filtration coefficient (K ) macrophage populations, where their activation stimulates phagocytosis of foreign f material. Although these data have largely been generated in laboratory animal species, of lung fluid via extravascular leakage. Consistent with clinical data already exist that support a role of TRPA1 and TRPV1 in respiratory sensory these findings, intravenous administration of the TRPV4 nerves, as acute exposure to agonists of either channel (notably, tear gases for TRPA1 and agonist GSK1016790A causes circulatory collapse that pepper sprays for TRPV1) can cause intense, incapacitating respiratory irritation in is characterized by the failure of the alveolar septal bar- humans. CNS, central nervous system. rier112. Although the same study also demonstrated that

Bronchoalveolar lavage GSK1016790A can produce dramatic deformation of TRP channels. Collectively however, these data suggest A procedure in which cultured human endothelial cells, it was recently dem- a potential value for TRPV4 modulators in the treatment inflammatory cells and other onstrated that TRPV4 that is expressed on alveolar mac- of heritable disorders, including skeletal dysplasias and materials within the airway rophages can have a crucial role in ventilator-induced sensory and motor neuropathies. lumen are collected via injury in isolated lungs of mice113. Although these data repeated washings. suggest that discerning the exact mechanism (or mecha- TRPP2. Mutations in the genes encoding two proteins, Alveoli nisms) of TRPV4‑mediated lung injury may be challeng- polycystic kidney disease 1 (PKD1) and TRP cation Distal regions of the lung in ing, they highlight the contribution of TRPV4 activation channel polycystin subfamily 2 (TRPP2; also known as which a thin barrier between to pulmonary oedema that is caused by aberrant vascu- PKD2), are causative factors in polycystic kidney dis- airspaces and capillaries allows for gas exchange. lar or airway pressures. ease and are responsible for ~85% and ~15% of cases, respectively121. The proteins encoded by these genes

Filtration coefficient (Kf) TRP channels and human genetic diseases are thought to interact with each other in vivo to form A measurement that is used to Mutations in at least six of the 28 members of the TRP a cation channel–receptor complex that is involved in reflect the permeability of the channel superfamily are associated with genetic dis- pressure sensing in the cilia122. The exact role of each pulmonary vasculature to fluid. eases in humans (reviewed in REFS 3,4). The diversity of protein is unclear, but the ratio of PKD1 expression to Autosomal dominant pathologies caused by TRP channel dysfunction high- TRPP2 expression seems to be crucial for normal pres- brachyolmia lights the range of roles TRP channels have in normal sure sensing123. These findings may help to explain A disorder that is typified by physiology and underscores the importance of calcium some of the cardiovascular abnormalities (for example, short stature, a short trunk and curved spine. signalling in various systems. Some of the rare muta- hypertension and aortic aneurysm) that are observed in tions may also point to a more general role for TRP polycystic kidney disease. In addition, they highlight the Spondylometaphyseal channels in these pathologies. In situations in which a challenge of restoring normal function in patients via a dysplasia gain-of-function mutation underlies the pathology of a TRPP2 modulator. A skeletal disorder that is TRP channelopathy, the potential utility of an antago- typified by short stature and abnormalities in the vertebrae nist is evident; however, TRP channel agonists could TRPC6. Mutations in TRPC6 also lead to kidney dys- and tubular bones. also have therapeutic benefits in situations in which function. A large family was analysed and it was discov- one allele is intact or the mutation reduces the probabil- ered that a single point mutation (P112Q) in TRPC6 was Charcot–Marie–Tooth ity of the channel being open but leaves TRP channel sufficient for causing focal segmental glomerular sclero- disease sis124 Also known as hereditary function intact. . Subsequently, additional mutations in TRPC6 that 125 motor and sensory neuropathy. led to nephrosis were identified , and there is increas- This disease, named after the TRPV4. Mutations in TRPV4 cause several divergent ing interest in TRPC6 as a potential target for the therapy three doctors who first heritable diseases that affect diverse systems. Genetic of acquired kidney diseases. When they are expressed identified it, is one of the most deletion of Trpv4 in mice leads to a substantial increase in heterologous systems, several of these mutated TRP common inherited neuropathies. Symptoms in bone mass and reduced bone loss owing to lack of channels show substantial increases in the amplitude 114 126 include weakness, motor weight bearing . TRPV4 also has an important role of the current and they implicate calcium handling atrophy and foot deformities. in bone resorption and osteoclast differentiation115. In in glomerular disease. One possible downstream effect line with this finding, more than 19 autosomal domi- of overactive TRPC6 is the increased activation of the Focal segmental glomerulosclerosis nant human mutations in TRPV4 are associated with nuclear factor of activated T cells (NFAT)–calcineurin 116 127 A disease that is typified by skeletal dysplasias . In two families, TRPV4 mutations signalling pathway . glomerular scarring, which (R616Q and V620I) that caused profound increases in results in proteinuria, oedema levels of current in heterologous systems led to autoso- TRPA1. Studies on a family have revealed an autoso- and the eventual need for mal dominant brachyolmia117. Other mutations in TRPV4 mal dominant mutation in the fourth transmembrane dialysis. lead to additional skeletal disorders of varying severity, domain of TRPA1 that underlies familial episodic pain Familial episodic pain including spondylometaphyseal dysplasia118. Both mild syndrome43: this is the first and as yet only example of syndrome and lethal forms of metatrophic dysplasia are also due a TRP channel mutation that is implicated in a human A rare disorder that is typified to mutations in TRPV4 that increase channel activity pain syndrome. The recombinantly expressed familial by periods of severe pain in the 116–118 trunk and upper body. in vitro . episodic pain syndrome mutant N855S TRPA1 carries Episodes are typically triggered Mutations in TRPV4 have also been linked to periph- more current than wild-type TRPA1 at negative poten- by cold temperatures and/or a eral neuropathies, including hereditary motor and tials, although maximal current responses to reactive low energy state brought about sensory neuropathy type 2C (also known as Charcot– chemical agonists appear to be unaltered43. Not only do by hunger or fatigue. Marie–Tooth disease type 2C), scapuloperoneal spinal these data highlight the relevance of the modulation of muscular atrophy and congenital distal spinal muscular TRPA1 at cold temperatures in humans but they also atrophy119,120. In five families, three separate missense suggest a possible connection between cellular energetics mutations were identified that altered arginine residues and TRPA1 function. in the amino terminal ankyrin repeat domains119,120. These mutations appear to be gain-of-function muta- TRPM6. Like the closely related TRPM7 channel, tions120, although they have also been characterized as TRPM6 is distinguished from other TRP channel family loss-of-function mutations that are potentially caused members because it has a large carboxyl terminal pro- by the decreased expression of functional channels119. tein kinase domain (reviewed in REF. 3). Loss-of-function These discrepancies need to be resolved by further stud- mutations in TRPM6 underlie inherited autosomal ies, as the cellular context in which the TRP channel is recessive hypomagnesaemia with secondary hypocalcae- expressed may influence the function of the mutated mia (reviewed in REF. 4). Several mutations, including

deletion of exons, point mutations, deletion mutations the strength of synaptic transmission in these regions. and premature stop codons can lead to the disease128–130. Future behavioural studies that directly compare selec- These findings raise the possibility that compounds that tive TRPV1 antagonists with differential central nervous increase TRPM6 activity could be useful in the treatment system penetrances are needed to elucidate the role of of both inherited and sporadic hypomagnesaemia. TRPV1 in brain function.

TRPML1. Mutations in TRP cation channel mucolipin TRPC3. TRPC3 is the most abundant TRP channel of subfamily 1 (TRPML1; also known as mucolipin 1) cause the TRPC subfamily in cerebellar Purkinje cells. Like mucolipidosis type IV. Subsequent studies on the func- TRPC6 and TRPC7, TRPC3 is activated by diacyl-

tion of TRPML1 have indicated that it is an intracellular glycerol, and Gq-coupled GPCRs are likely to be crucial protein that is localized to the endosome and lysosome, regulators of channel activity in vivo (reviewed in REF. 3). and is responsible for iron transport independently In addition, receptor tyrosine kinases activate TRPC3 of the divalent metal transporter 1 (REF. 131). Disease- in vitro and brain-derived neurotrophic factor-mediated associated mutations in TRPML1 compromise its ability survival of cerebellar granular cells depends on func- to transport iron, and this occurs in a manner that corre- tional TRPC3 expression138. lates well with disease severity131. These data suggest that Trpc3–/– mice show considerably attenuated inward molecules that improve the function of TRPML1 could currents in response to activation of metabotropic glu- be useful treatments for individuals with mucolipidosis tamate receptor 1 (mGluR1), which is a GPCR that type IV. However, screening for such compounds using is thought to be upstream of TRPC3; this finding is traditional fluorescent assays and electrophysiological consistent with the role of TRPC3 in the cerebellum. techniques is difficult given the intracellular localiza- Trpc3–/– mice also display impairments in walking behav- tion of the channel. iour, although — as predicted — the TRPC3‑knockout phenotype is less severe than the ataxia observed in TRP channels in the brain mGluR1–/– animals139. Many TRP channels are expressed by brain tissue; some A screen in chemically mutagenized mice also are expressed at high levels (for example, TRPC3 and identified a crucial role for TRPC3 in Purkinje cells. TRPC5), whereas others are expressed at low levels Moonwalker mice — mice with a T635A mutation in (for example, TRPV1). The function of these channels Trpc3 — showed progressive Purkinje cell degeneration remains to be elucidated, but there is evidence that sev- and ataxia140. In cerebellar slices, this mutation resulted eral TRP channels may contribute to neuronal excit- in a higher amplitude of currents in response to low ability and neurotransmitter-mediated signalling in concentration of an mGluR1 agonist140. Taken together, the brain. these data indicate that TRPC3 channels have an important role in synaptic transmission in cerebellar Purkinje TRPV1. There is controversy surrounding the expres- cells and in the survival of these cells. Identification of sion and role of TRPV1 in the brain. Some studies report selective pharmacological agents will be useful for deter- that TRPV1 is widely expressed throughout the whole mining whether TRPC3 has a broader role in ataxia. neuroaxis of the rat (albeit at much lower levels than in sensory neurons; reviewed in REF. 132). However, recent TRPC5. High levels of TRPC5 are expressed in the hip- research — that relies on a powerful combination of pocampus and amygdala141. Trpc5 –/– mice show less reporter mice, in situ hybridization, electrophysiological innate fear behaviour than their wild-type littermates recordings and calcium imaging — suggests that TRPV1 in an open-field test, an elevated plus maze test and a expression is restricted to very few regions of the brain, nose bumping assay141. In addition, their conditioned most notably the caudate nucleus of the hypothalmus133. fear memory is unaffected in a single fear conditioning Consequently, additional research will need to be car- paradigm141. These data are consistent with observations ried out to explain the differences that have been noted made in brain slice recordings in which Trpc5–/– mice in the behavioural responses between wild-type and showed normal membrane excitability and synaptic Trpv1–/– mice. function but reduced synaptic responses to activation Trpv1–/– mice adapt more easily than their wild-type of mGluRs and cholecystokinin (CCK) receptors141. littermates to aversive light, and they explore the open These data provide a potential link between TRPC5 arm of the elevated maze more freely; these results are and the CCK4 signalling pathway, the activation of indicative of a reduced unconditional fear response in which induces anxiety behaviours in rodents and Trpv1 –/– mice134. Furthermore, Trpv1 –/– mice exhibit humans. This suggests that TRPC5 antagonists might less freezing than wild-type mice in auditory fear con- be useful as anxiolytic agents. Additional work will be ditioning assays134. These findings imply that TRPV1 required to determine whether a pharmacological agent Mucolipidosis type IV antagonists have therapeutic potential as novel anxio- can recapitulate the effects of genetic deletion. Further A lysosomal storage disorder. lytic agents. Notably, TRPV1 is present in dopaminergic analysis of the role of TRPC5 in learning and memory Symptoms typically present neurons in the basal ganglia and it was speculated that is also warranted. during the first year of life and malfunction of TRPV1 may be involved in the patho- TRPC5 may also have an important role in the cal- affected individuals suffer from 135 142 psychomotor retardation, genesis of Parkinson’s disease . Some studies have also cium-mediated guidance of neuronal growth cones . ophthalmological reported on the presence of TRPV1 in the hippocam- Further in-depth studies on the brain architecture abnormalities and anaemia. pus136 and nucleus accumbens137; TRPV1 may modulate in developing Trpc5–/– mice and experiments that

examine receptor-activated neurite extension in brain negative Trpc6 transgene renders mice less susceptible slices should help to clarify the contribution of TRPC5 to hypertrophy153. Consequently, there is substantial to axonal pathfinding. interest in assessing the utility of TRPC6 antagonists in various models of cardiovascular disease. Owing to the TRPM2 and TRPM7. In the brain, TRPM2 is expressed dramatic upregulation of TRPC3 following genetic dele- by neurons and microglia. Consistent with the concept tion of TRPC6, selective TRPC6 antagonists are needed that TRPM2 functions as a redox sensor, Trpm2–/– mice to probe the utility of TRPC6 as a therapeutic target. are protected against various pathologies related to oxidative stress, including the focal ischaemia model of Metabolic disorders. Genetic deletion of TRPM5, a stroke143. The TRPM2 gene is also a candidate risk fac- known taste sensor, results in impaired glucose toler- tor gene for bipolar disorder144. Knockdown of TRPM7 ance in mice154. This phenotype may be due to the loss reduces cell death that is caused by oxygen- and/or glu- of high-frequency calcium oscillations in pancreatic cose deprivation in isolated neuronal cultures145, which β-cells155, although the effects of TRPM5 deletion can- implies that TRPM7 antagonists may have a role in the not be accounted for by simple changes in membrane treatment of stroke. potential. In animal models, inactivation of TRPV1 by genetic or pharmacological manipulation has been Other diseases shown to protect against the development of type 1 dia- In addition to the therapeutic areas described above, betes and improve glucose tolerance in type 2 diabetes TRP channels have been implicated in various other (reviewed in REF. 156). In Trpv1 –/– mice, both increased13 diseases. Although some are not reviewed here, we dis- and decreased157 body fat was reported, therefore the cuss the most noteworthy findings, in our opinion, for role of TRPV1 in the regulation of body weight remains therapeutic targeting. controversial.

Cancer. Several TRP channels have been linked to can- Conclusions cer, some as markers of biological behaviour (such as The recent expansion of research into TRP channels aggressive versus indolent phenotypes), whereas oth- has resulted in the identification of numerous potential ers could be putative therapeutic targets (reviewed in drug targets beyond TRPV1, and has elucidated roles REF. 146). TRPM8 is a prime example of a marker that is for TRP channels in diverse therapeutic areas includ- also a target. TRPM8 is overexpressed in prostate can- ing pain, pulmonary indications, oncology, neurology cer, and its level of expression correlates with tumour and genetic disorders. Interest is mounting as a result of severity147. At the same time, the TRPM8 agonist men- emerging data from animal models, human genetic dis- thol reduces the proliferation and viability of prostate orders and, in some cases, compounds entering clinical cancer cell lines148. Notably, the synthetic TRPM8 ago- trials. Indeed, at this early stage, with very limited clini- nist D‑3263, which reduces benign prostatic hyperplasia cal data available regarding the effects of small-molecule in rats, is in clinical trials (ClinicalTrials.gov identifier: blockade of a single TRP channel (TRPV1), it is decep- NCT00839631). The structure of this compound has not tively easy to speculate on the therapeutic potential — yet been published. As patients with benign prostatic or the potential to cause mechanism-based toxicological hyperplasia often have prostate cancer, they could con- liabilities — of TRP channel modifiers. Several questions ceivably benefit from TRPM8 agonist treatment, which remain unanswered, and are listed below. would both improve bladder function and reduce the risk of cancer. How predictive are the animal models that are used to test TRP channel modulators? Available data largely Cardiovascular diseases. TRPM4, which is activated by suggest that recombinant human TRP channels respond (but not permeable to) Ca2+ ions, regulates many aspects to the same sorts of stimuli as their laboratory animal of cardiovascular function (reviewed in REF. 3). Within the orthologues (for example, rat, mouse and human TRPM8 cerebral arteries of rats, antisense oligonucleotide deple- are all activated by menthol and cool temperatures). tion of TRPM4 reduces the potential depolarization of the Moreover, acute behavioural effects of known TRP chan- myocyte membrane and reduces myogenic vasoconstric- nel modulators tend to be similar between humans and tion caused by elevated intraluminal pressure149. TRPM4 laboratory animals (for example, topical capsaicin causes has also been implicated in human cardiac conduction nocifensive behaviour and swelling when it is applied to disorders150, as mutations in the channel that resulted in a rodent or human skin), even if the molecular identity net gain-of-function have been identified in several fami- of the protein (or proteins) underlying the response in lies that had a block of cardiac electrical conduction151. humans remains unconfirmed. Although most TRP Loss of TRPM4 expression is, however, not categorically channels have not been studied to this extent, in vitro cardioprotective as Trpm4–/– mice are hypertensive152. differences between mammalian orthologues to date are Mechanistic investigations have revealed that this is due generally quantitative (for example, differences in the to increased adrenal catecholamine release rather than potency of activating ligands) rather than qualitative. direct cardiac or vascular effects152. Overexpression of TRPC6 and calcineurin-NFAT Will target validation generated via genetic means be signalling are associated with angiotensin II‑induced predictive of blocker effects? Potent and selective small- cardiac hypertrophy, and the expression of a dominant molecule modulators of TRP channels are continuing

to emerge. These tools are vital for advancing the field, (such as alterations in thermosensation16,17 and/or ther- as acute blockade does not always mimic the protec- moregulation10,16,17) have been consistently observed, the tive and/or deleterious effects of genetic removal. magnitude of these effects has varied considerably owing Although these two methods often correlate well, a to factors that are only partially understood. number of notable exceptions have occurred. A prime example is TRPV1. Some TRPV1 antagonists cause Will an agonist or an antagonist be therapeutically use- hyperthermia, which has necessitated the withdrawal ful? The answer to this question is rarely as obvious as it of these compounds from clinical trials (reviewed in seems: in the pain area, both agonists and antagonists of REF. 14), yet Trpv1–/– mice have normal body tempera- TRPV1 are being evaluated in clinical trials, and the first tures9,11. Furthermore, the TRPV1 antagonist GRC‑6211 approved TRPV1 modulator is capsaicin, which robustly improves bladder capacity65; by contrast, Trpv1 –/– mice activates the channel (reviewed in REF. 5). Similar ques- show spotty incontinence60. Other examples include tions exist for TRPM8, as both agonists and antagonists TRPV4 (for example, the TRPV4 blocker HC‑067047 are being pursued for the treatment of pain (reviewed does not alter thermal selection behaviour or water in REF. 7). Clearly, the answer to this question will vary consumption in mice67) and TRPA1 (for example, block- depending on the nature of the disease state and the TRP ade but not knockout of TRPA1 inhibits CFA-induced channel (or channels) that are involved. mechanical hyperalgesia34). The answers to the above questions will help to determine the answer to the most pressing question in Will TRP channel modulators be safe and well toler- the TRP channel-related field: will blocking (or other- ated in humans? As with all novel targets, questions exist wise modulating) TRP channels ameliorate established regarding the potential mechanism-based toxicological human disease? This answer will only be obtained after liabilities of TRP channel modulators. Speculation in this long-term clinical studies have been carried out in area is rampant, owing at least in part to the paucity of patients. However, the broad and pronounced links to empirical evidence regarding the safety and tolerability pathophysiological processes that have been revealed by of modulators of any of these targets other than TRPV1. extensive preclinical target validation and human genetic Even in the case of TRPV1, in which several structur- studies — in addition to the relatively high chemical ally unrelated molecules have been dosed in patients and tractability of the TRP superfamily of ion channels — dose-limiting effects that appear to be mechanism-based provide strong cause for optimism.

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