Here we show that PBMC is a robust and selective TRPM8 antagonist. In vitro
, PBMC is the most potent TRPM8 antagonist reported to date and inhibits channel activation to both chemical and thermal stimuli. Using calcium microfluorimetry and whole-cell electrophysiology, we found that PBMC reduced TRPM8 activity in a dose-dependent manner. Indeed, we observed an IC50
concentration of less than 1 nM, a dosage approximately 100-fold lower than the most potent TRPM8 antagonist reported to date, CTPC 
. Thus, the two-orders-of-magnitude higher affinity of PBMC makes this compound a more amenable reagent in the study of TRPM8 channel function.
Importantly, and unlike other TRPM8 antagonists, we did not observe any cross reactivity with either TRPV1 or TRPA1, suggesting that PBMC is selective for TRPM8. However, these observations are not all inclusive of other cellular mechanisms, but application of PBMC to cultured TG neurons did not lead to any noticeable changes in cellular excitability, suggesting that PBMC does not have any appreciable off-target effects at the level of cultured sensory neurons. We found that PBMC exerts its antagonistic effect on TRPM8 by shifting the voltage-dependence of TRPM8 gating. This particular result, consistent with previous reports from our lab and others, suggests that many (if not all types) of functional regulation of TRPM8—whether by agonist, antagonist, or adaptive mechanisms—involves changes in voltage-dependent gating 
Emerging evidence suggests that TRPM8 plays a role in thermoregulation, both with the stimulation of skin afferents with chemical agonists 
or cooling 
. Here, we have confirmed that icilin, a chemical TRPM8 agonist more potent than menthol 
can also induce an increase in body temperature 
, an effect that is TRPM8-dependent 
, despite reports that icilin can also activate TRPA1 in vitro 
. Even though TRPM8-/-
mice do not respond to icilin, these animals retain the ability to mount a chemically-induced thermoregulatory response as we observed an identical effect in both wildtype and TRPM8-/-
mice in response to the TRPV1-agonist capsaicin. Therefore it appears that TRPM8-expressing afferents have the ability to affect thermoregulatory responses to both chemical and thermal stimuli, although the exact neurological mechanism remains to be explored.
Due to this evidence and recent reports of TRPV1 antagonists having undesired thermoregulatory effects 
, we were concerned that a TRPM8 antagonist would also affect thermoregulation. Indeed, when we administered PBMC at a dose of 20 mg/kg, we observed a profound hypothermic effect, with one mouse reaching body temperatures below the temperature range of the telemeter (<30°C), a temperature classified as deep hypothermia in humans 
. The pharmacokinetics of PBMC are as yet unknown, yet the hypothermic effect observed here lasted around four hours on average, and in thermoregulatory and behavioral experiments the effects were gone by less than one day after administration. Interestingly, halving the dose (10 mg/kg) almost completely abolished the hypothermic response, with core body temperatures dropping less than one degree—a surprising change in effect for such a small reduction in dose. Indeed, while this drop in core temperature was significantly different than vehicle injected control or TRPM8-/-
mice, it was not significant when compared to normal circadian changes in body temperature we observed in these mice. Thus, we suggest that the slight change in core temperature observed at the 10 mg/kg dose did not participate in the ability of PBMC to block acute cold sensation, as well as reduce injury-induced cold hypersensitivity.
It has been shown extensively that TRPM8 is required for cold sensation, particularly in the evaporative cooling assay 
. When a small volume of acetone is applied to the hindpaw of a mouse, it quickly evaporates and cools the skin down to temperatures as low 14–18°C 
, which is near the loose boundary of the transition from innocuous cool to cold pain 
. With 10 mg/kg PBMC, we observed a partial reduction in the normal acetone response score, demonstrating that by blocking TRPM8, this compound can alter cold thermosensation. These responses were further reduced with the highest concentration tested, 20 mg/kg, although the interpretation of these effects are complicated by the dramatic hypothermia produced at this dosage. It is important to note that the PBMC-treated scores did not drop to the level of TRPM8-/-
mice (), indicating partial blockade of the channel at this dose. Interestingly, we observed individual differences in the amplitude of the score reduction with 10 mg/kg PBMC under normal conditions, which may suggest that, at this low dose, individual variations in physiology may affect drug action. However, due to the thermoregulatory effects described above, we were limited in the amount of drug we could administer to the mice without potentially confounding thermosensory responses.
TRPM8 has also been implicated in the painful cold hypersensitivity that is a distressing symptom of inflammatory and neuropathic conditions, as well as platinum-based chemotherapy drugs 
. It would therefore be greatly beneficial to both chronic pain and chemotherapy patients to have a drug which could control such symptoms. Thus we tested whether PBMC could reduce the behavioral responses to evaporative cooling in models of inflammatory and neuropathic pain. In the CFA model of inflammatory pain and the CCI model of neuropathic pain, we saw a reduction in the response scores of mice treated with 10 mg/kg PBMC. Interestingly, both of these reduced scores remained higher than those seen at baseline or with TRPM8-/-
mice, again suggesting that at this dose PBMC only partially blocked TRPM8 function in vivo
. However, given that the aim of a good symptom-controlling drug would be to reduce the hypersensitivity to cold without abolishing normal thermosensation (e.g. numbness), this may not be a completely undesirable effect.
In contrast, when we examined oxaliplatin-treated animals given PBMC, we did not see a statistically significant reduction in response scores. It is puzzling that PBMC would be effective against one model of neuropathic pain (CCI) but not another. There are two probable explanations for this observation: First, it is possible that other mechanisms may also be involved in cold hypersensitivity in oxaliplatin-induced neuropathy and PBMC is ineffective against these mechanisms 
, although our and others' recent evidence suggests that TRPM8 plays a pivotal role in this pathology 
. Alternatively, it may be that the partial inhibition of TRPM8 we observe with PBMC prevents this compound from being effective in reducing the response scores in this pain model. Again, as we were constrained by the hypothermic side effect of a higher dose, we were unable to test if higher doses could provide some level of analgesia in oxaliplatin-induced cold hypersensitivity. Reformulation of the drug, if possible, may yield a compound that specifically targets sensory afferents without having the strong thermoregulatory effect observed here. Such a drug may bring much-needed relief to both chronic pain and chemotherapy patients experiencing symptoms of cold hypersensitivity. Nonetheless, our results show that PBMC is a potent and selective inhibitor of TRPM8, and that inhibition of this channel alters cold sensation, thermoregulation, and provides a modest level of relief in rodent models of injury-induced cold pain.