MinireviewTRPM8 ion channel ligands for new therapeutic applications and as probes to study menthol pharmacology
Introduction
The monoterpenoid (−)-menthol, (1R,2S,5R)-2-isopropyl-5-methylcyclohexanol is a common and popular flavoring and cooling additive in several common household remedies (topical pain gels, throat lozenges, toothpaste), foods (gum, candies, teas) and in cigarettes regardless of whether they are labeled mentholated or non-mentholated (Giovino et al., 2004). Menthol as an additive in cigarettes has come under close scrutiny following the report from FDA's Tobacco Products Scientific Advisory Committee (TPSAC) (Samet et al., 2011) which suggests that menthol, as an additive in cigarettes and other tobacco products, may facilitate smoking behavior and promote the adverse effects of smoking on health. Mandated by the Family Smoking Prevention and Tobacco Control Act, this FDA committee studied and reported on the public health impact of menthol in cigarettes, particularly the use among children, African Americans, Hispanics, and other racial and ethnic minorities. The report highlights critical questions related to the effect of menthol in cigarettes and provides population-level evidence that can form the basis of pharmacological studies into the role of menthol in nicotine addiction.
Menthol is purported to decrease smoke irritation and yield a cooling sensation in the upper airway tract, enabling deeper inhalation of cigarette smoke. The TPSAC report suggests that these effects could possibly lead to the greater reinforcement of smoking behavior, lessen aversion to initial smoking among novice smokers, prevent successful quitting, and exacerbate the harmful effects of tobacco smoke toxins. Menthol has been used as a cigarette additive since the 1920s (Ferris Wayne and Connolly, 2004), and careful studies have shown that 2.5 to 3 mg of menthol is present in mentholated cigarettes (Benowitz et al., 2004, Celebucki et al., 2005). The TPSAC estimates that about 1250 mcg/L of menthol could be available in cigarette smoke for exerting pharmacological effects or for metabolism in the upper and lower airways. Menthol smokers absorb about 20% of menthol from cigarettes (Benowitz et al., 2004). Previous studies with known doses of menthol show that about 50% of a menthol dose is excreted in the urine as a glucuronide (Gelal et al., 1999). Menthol glucuronide was measurable in the urine in 82% of smokers of menthol cigarettes. Interestingly, 54% of smokers smoking non-mentholated cigarettes also showed measurable levels of menthol glucuronide (Benowitz et al., 2010). These findings bring up the possibility that the presence of menthol in cigarettes is pervasive, regardless of whether the cigarettes are marketed as mentholated or non-mentholated. Nevertheless, the use of ‘mentholated’ cigarettes has been determined to be higher among certain population groups, including African-Americans, Hispanics and in adolescents and first-time smokers, and these populations are at higher risks for the facilitatory effects of menthol on the adverse health impact of smoking. Several recent large clinical studies of mentholated and non-mentholated cigarette smokers have found that mentholated smokers have consistently lower abstinence rates, suggesting that additive menthol in cigarettes supports facilitation and maintenance of smoking behaviors (Gandhi et al., 2009, Gundersen et al., 2009, Okuyemi et al., 2007, Stahre et al., 2010).
However, it is still not understood whether menthol's effects on smoking behavior are just due to enhancement of the smoking experience or if it exerts pharmacological effects to modulate the reinforcing effects of nicotine or other addictive tobacco ingredients. The effect of menthol on other aspects of nicotine intake such as nicotine absorption, nicotine and carcinogen metabolism, cardiovascular and respiratory effects have also been studied but have not provided definitive answers that explain the facilitative effect of menthol on smoking behaviors.
Menthol is a nonselective agonist of the transient receptor potential melastatin 8 (TRPM8) receptor (McKemy et al., 2002, Peier et al., 2002) and exerts its cooling effect by activating the TRPM8 ion channel. TRPM8 stimulation is thought to yield an analgesic effect (Proudfoot et al., 2006), which may contribute to the counter-irritant properties of menthol (Willis et al., 2011). The transient receptor potential (TRP) cation channels are well-known for their involvement in sensory stimuli such as temperature and chemical irritation, as well as physical stimuli such as pain. Menthol is the natural ligand for TRPM8 (EC50 at hTRPM8 66.7 ± 3.3 μM) (McKemy et al., 2002) and its binding to TRPM8 produces a strong outward rectification of this ion channel (Hui et al., 2005). Menthol is not selective for TRPM8 and also acts at other TRP channels (Macpherson et al., 2006). It was shown to activate or inhibit the transient receptor potential ankyrin 1 (TRPA1) ion channel, depending on the concentration (Karashima et al., 2007). TRPA1 is a chemosensory TRP ion channel activated by irritants such as those present in tobacco smoke and vehicle exhaust (Andre et al., 2008, Bautista et al., 2006). Menthol is also an activator of the transient receptor potential vanilloid 3 (TRPV3) ion channel, a thermo-sensitive TRP ion channel activated by warmth (Macpherson et al., 2006, Vogt-Eisele et al., 2007).
Recently, menthol was shown to act as a broad-spectrum counter-irritant against cigarette smoke irritants such as acrolein (a reactive aldehyde), acetic acid and cyclohexanone (a volatile organic hydrocarbon, VOC) (Willis et al., 2011). Menthol's counter-irritant effects against acrolein-induced irritation appear to be a TRPM8-related phenomenon, as the effect diminishes upon administration of a selective TRPM8 antagonist, AMTB (vide infra) (Willis et al., 2011). This study suggests that by decreasing the effect of smoke irritants on sensory nerves, menthol may allow for increased inhalation and facilitate smoking behaviors. In addition to its well-recognized cooling effect in cigarettes, this counter-irritant effect further implicates menthol as a facilitator of smoking and nicotine addiction. However, whether menthol has direct effects on nicotine addiction is not really known, but such a line of research would be greatly facilitated by the availability of small-molecule ligands that bind to the known receptors of menthol, and have higher potency and specificity than menthol. For instance, the availability of selective TRPM8 antagonists could greatly help in understanding the role of TRPM8, menthol's cognate receptor, in its effect on nicotine addiction. Defined pharmacological studies with such tools would be very valuable in understanding the underlying interactions between the actions of nicotine and menthol, and if they contribute to increased smoking and addiction.
TRPM8 belongs to the transient receptor potential (TRP) cation channel superfamily, which is comprised of twenty-eight channels categorized into seven subfamilies: TRPC (TRP-Canonical), TRPV (TRP-Vanilloid), TRPM (TRP-Melastatin), TRPA (TRP-Ankyrin), TRPN (TRP-NompC), TRPP (Trp-Polycystin), TRPML (TRP-MucoLipin)(Venkatachalam and Montell, 2007). A subset of TRP channels termed the thermoTRP channels are able to start sensory nerve impulses after activation by thermal or chemical stimuli (Patapoutian et al., 2009); members of this subfamily include heat- activated TRPV1–4, TRPM2, TRPM4–5, and cold-activated TRPA1 and TRPM8. TRPM8 is a cation channel permeable to both monovalent and divalent cations, and exhibits a strong outward rectification (Hui et al., 2005). TRPM8 is activated by cold, chemical agents menthol and icilin, positive membrane potentials and the endogenous signaling lipid PIP2 (Brauchi et al., 2004, Liu and Qin, 2005, Rohacs et al., 2005, Voets et al., 2004); however, the mechanism by which these different stimuli result in activation of the receptor is unknown (Bandell et al., 2006). Menthol yields a cooling sensation by activating this thermally sensitive receptor; as a result, TRPM8 is often referred to as the ‘cold receptor’.
The TRPM8 gene was discovered by screening a prostate-specific subtracted cDNA library (Tsavaler et al., 2001). TRPM8 mRNA is found in prostate tissue, and is also found at elevated levels in tumors of the prostate and other organs such as the breast, colon, lung and skin (Tsavaler et al., 2001). TRPM8 mRNA is also expressed in Aδ- and C-fiber primary afferent neurons in rat, a subpopulation of dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons (Kobayashi et al., 2005). Localization of TRPM8 in this area, coupled to activation by cold temperatures (McKemy et al., 2002, Peier et al., 2002), led to the exploration of this ion channel as a target for cold hypersensitivity associated with inflammatory and neuropathic pain. Particularly noteworthy is the fact that TRPM8-expressing neurons are more abundant in the trigeminal ganglion (TG) than in the dorsal root ganglion (DRG), especially in the mandibular nerve region innervating the tongue (Kobayashi et al., 2005), which could be responsible for a rapid effect of menthol during cigarette smoking. Among other non-neuronal tissues, TRPM8 is found in the upper gut's nodose ganglion (Zhang et al., 2004), gastric fundus (Mustafa and Oriowo, 2005), vascular smooth muscle (Yang et al., 2006), liver (Henshall et al., 2003), bladder urothelium and the male genital tract (Stein et al., 2004).
The TRPM8 ion channel is a transmembrane protein of 1104 residues whose functional structure consists of a modular homotetramer channel (Latorre et al., 2007). Four transmembrane helices S1–S4 contain the voltage sensor module and the binding sites for chemical agonists menthol and icilin. TRPM8 shares > 40% homology with TRPM4/5 (Voets et al., 2004), and 42% similarity to TRPM2 (Peier et al., 2002).
Mutagenesis studies show two regions necessary for menthol's pharmacological effects: the S2 transmembrane domain and a region of the C-terminal domain called the TRP box (Bandell et al., 2006). A Y745 residue, in the S2 domain where menthol binds, was shown to be crucial for receptor sensitivity to menthol. Residues in the TRP box are required for coupling menthol binding to channel opening. At least one other binding site other than the menthol-binding domain possibly exists on TRPM8, as evidenced by differing sensitivities of several low-affinity antagonists to a TRPM8-Y745H mutant (Malkia et al., 2009).
A number of small-molecule compounds have been developed as high affinity ligands for the TRPM8 receptor and investigated for their therapeutic benefit in a variety of disease states such as prostate cancer, benign prostatic hyperplasia (BPH), urological disorders, and for the treatment of cold hypersensitivity associated with neuropathic and inflammatory pain. A menthol-based TRPM8 agonist D3263.HCl from Dendreon Corporation (see below) showed disease stabilization in patients with advanced prostate cancer in a small Phase I safety trial (Tolcher et al., 2010). However, there have been no reports of using TRPM8 agonists or antagonists to explore the role, if any, of this ion channel in the effect of menthol on nicotine addiction, possibly due to the lack of easy accessibility of such compounds for research use. As of the writing of this minireview, a small-molecule TRPM8 antagonist AMTB (see below) has been recently made available through a commercial supplier Tocris. TRPM8 ligands could be very useful as pharmacological tools to study the role of menthol in nicotine addiction and to investigate the suitability of menthol's cognate receptor, the TRPM8 ion channel, as a possible target for nicotine addiction pharmacotherapy.
Section snippets
TRPM8 agonists and antagonists
A range of chemical scaffolds have been reported in the patent and journal literature as possessing activity at TRPM8, the majority of which were discovered through high-throughput screening of proprietary in-house chemical libraries in several pharmaceutical companies. Both agonists and antagonists of TRPM8 have been investigated for various therapeutic applications as discussed below, however, none of these compounds have probed the pharmacological link of TRPM8 to nicotine addiction. A
Prostate cancer and benign prostatic hyperplasia (BPH)
Among the therapeutic indications investigated for TRPM8 ligands, only prostate cancer treatment and benign prostatic hyperplasia have been explored in human clinical trials, with the p-menthane-based agonist D3263 from Dendreon Corporation (Tolcher et al., 2010). D3263 was also effective in a model of BPH, alone or in combination with finasteride (used clinically for this indication), although this preclinical validation has yet to be investigated in human clinical trials. While the p-menthane
Conclusions
As evidenced in this review, many potent and selective TRPM8 agonists and antagonists have been developed mainly by the pharmaceutical industry. Several of these have also been optimized as preclinical drug candidates for various therapeutic indications discussed above. Lead optimization campaigns which have been reported in the literature, suggest tractable structure-activity relationships for improving drug-like suitability of TRPM8 ligands while maintaining the pharmacological and functional
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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The mechanisms of cold encoding
2022, Current Opinion in NeurobiologyCitation Excerpt :Appropriate cold receptor function is critical to survival, as it allows for the avoidance of harmful ambient conditions that could produce tissue damage, respiratory issues, or hypothermia. Furthermore, cold and cooling compounds, such as menthol, have been used for hundreds of years as both analgesics and antipruritics, highlighting the therapeutic potential of targeting these neurons [12–15]. In the twenty years since the initial cloning of TRPM8 [16,17], important progress has been made to further our knowledge of the molecules and mechanisms involved in cold processing; nevertheless, outstanding questions remain.
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2019, Neuroscience of Nicotine: Mechanisms and TreatmentAntidiabetogenic efficiency of menthol, improves glucose homeostasis and attenuates pancreatic β-cell apoptosis in streptozotocin–nicotinamide induced experimental rats through ameliorating glucose metabolic enzymes
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