In the present study we demonstrate that continuous infusion of DHC, a TRPV1 receptor agonist, can induce a sustainable and clinically relevant mild hypothermia in rats, cynomologus monkeys, and in young cattle. In addition we show that also synthetic TRPV1 agonists such as MSK-195, olvanil, arvanil or rinvanil are able to produce hypothermia in rats. Similar, TRPV1 antagonists have been shown to induce hyperthermia in rats as well as in humans [28
], which collectively demonstrate the central role of TRPV1 channels in the regulation of body temperature [11
Based on death certificates, sudden cardiac arrest accounts for about 15% of all death in Western countries [29
] and about 330,000 per year in the United States alone [30
]. With the witness of a cardiac arrest and performance of CPR with return of spontaneous circulation (ROSC), mild therapeutic hypothermia has been shown to improve survival and neurological outcome [2
]. At present the only therapy available is mechanical/physical cooling, which is associated with side-effects including pneumonia, sepsis, and stress-related shivering [7
]. Moreover, it can be argued that mechanical cooling is connected to various practical issues that may affect the frequency of which the treatment is actually applied. Finally, the on-set of the use of mechanical cooling may take several hours following the ischemic insult [1
], which in turn may affect the outcome of the treatment. Taken together there is an un-met medical need for the development of drug-induced mild therapeutic hypothermia, which may lead to a safer, manageable, and instant treatment immediately following ROSC. In animals drug-induced hypothermia has previously been demonstrated by activation of a number of biological systems located in the brain including the CB1 receptor [31
], neurotensin receptor [33
], muscarine receptor [34
], and 5-HT-1A receptor [35
]. Here we focused our efforts on the TRPV1 receptor agonists and investigated the feasibility of a long-term infusion-based method for chemically induced hypothermia.
Previously it has been shown that single administration of capsaicin, DHC, and RTX [25
] can induce a short period of hypothermia in rats. In addition to the role in thermoregulation, the TRPV1 receptor is important in the sensation of pain and it contributes to the detection of noxious heat, which in turn may lead to effects on the cardiovascular and respiratory systems [36
]. In the current study focused on temperature regulation, we in general did not observe any major behavioral changes that may indicate any of these effects, except in calves during the first 1 to 2 hours of the infusion, where some discomfort was observed indicating a pain reaction. Our findings may be explained by relatively low dose levels tested, or the fact that DHC was administered by the intravenous route, which could cause a relative distribution of DHC to central receptors possibly less involved in nociception. On the other hand, intravenous infusion of the TRPV1 agonist capsaicin was previously shown to cause a hypertension and tachycardia in anesthetized dogs [37
]. Since the possible effect of DHC on the cardiovascular and system is central in understanding the therapeutic potential of the compound, the issue is currently a subject for specific studies. Notably, preliminary results demonstrate a dose-dependent cardiovascular effect including tachycardia and hypertension by continuous intravenous infusion of DHC [38
]. Also, future studies may address possible adverse effects of infusion of DHC on coagulopathy, hematological effects, liver enzymes, and fluid and electrolyte shifts.
Besides the ability of inducing hypothermia per se
to the relevant therapeutic level of 32-34°C, it is pivotal for a novel drug for the treatment of patients following ROSC after cardiac arrest, that mild therapeutic hypothermia can be sustained for an appropriate period of time. Thus, the current recommendation from ERC/AHA concerning the length of period of therapeutic hypothermia is 12-24 hours. Now, exposure of a TRPV1 agonist to the receptor may cause desensitization of the receptor [27
], and accordingly repeated dosing may lead to a less pronounced hypothermic response as observed in young non-naïve animals [39
]. In fact, very high exposure of a TRPV1 agonist may even lead to nerve fiber degeneration and subsequent re-innervation [40
]. Here we investigated the possibility of sustaining hypothermia by applying various TRPV1 agonists by a continuous intravenous infusion to adult rats. Interestingly, our data indicate that the role of desensitization vary among the heterogeneous class of TRPV1 agonists, as it has previously been indicated in vitro
in patch clamp studies [27
]. Thus, infusion of arvanil caused a rapid desensitization towards the hypothermic effect, which was also seen at lower dose of arvanil (data not shown), whereas infusion of olvanil or DHC did not. Interestingly, olvanil caused a prolonged hypothermia even after the infusion was stopped, whereas the hypothermic effect of DHC was short-lasting after stopping the infusion. It is currently not known what causes this differential desensitization effect, but it may relate to the binding of the agonist to the receptor and/or the associated permeability of the ion channel [19
]. Another possibility is that the observed difference in the pharmacological response of the various TRPV1 agonists is caused by different pharmacokinetic properties of the tested TRPV1 agonists.
From our initial screening of hypothermic properties DHC displayed a desirable profile showing a relevant and sustainable level of hypothermia as well as a rapid and controllable on- and off-set, and this candidate was therefore selected for further dose-response testing. An important component in the regulation of human body temperature is the ability to sweat, which causes heat loss via evaporative cooling. In contrast, in most animals including rats sweating does not contribute significantly to the body temperature homeostasis, but other mechanisms, such as panting, are important [42
]. Therefore we included dose-response studies in rats as well as in cynomologus monkeys, the latter animal which in contrast to rats possesses the ability to sweat [43
]. We observed that infusion of DHC caused a dose-dependent decrease in temperature in both rats and cynomologus monkeys. Notably, while the maximal response was at the same level, the sensitivity towards DHC was about 3-fold higher in the rats than in cynomologus monkeys. The reason for this is not clear, but may relate to the role of desensitization [39
], since the cynomologus monkeys were dosed on repeated occasions, whereas the rats were all naïve to DHC. It may also be explained by species differences in the molecular structure of the TRPV1 receptor [19
] or the components of thermoregulation [42
]. Finally, it may play a role that DHC was formulated in different vehicles in the two experiments, which may have resulted in a difference in the exposure of the compound. In this context it is of interest that when the cynomologus monkeys, yet naïve to DHC, were treated for the first time at the dose of 0.3 mg/kg/h, the response was still less pronounced than rats treated with 0.25 mg/kg/h. This may indicate that desensitization is not the only reason for the observed difference in the hypothermic response.
Both rats and monkeys are small animals compared to humans. In order to test the hypothermic properties of DHC in a large animal at the weight of a human being, we infused DHC to young cattle. We were able to sustain hypothermia at the relevant therapeutic level for more than 12 hours. It should be noted that despite a constant infusion of compound, there was a minor incline in body temperature during that last hours of the infusion. Also, it is important to notice that we saw a minor overshoot in the body temperature after discontinuation of the infusion. Likewise a minor overshooting was observed in the cynomologus monkeys following the highest dose (1.8 mg/kg/h) applied. The reason for this overshooting is not clear at present, and it is not known whether or not it may be prevented by a gradual discontinuation of the infusion or by the treatment of the fever with non-steroidal anti-inflammatory drugs or similar. Also, due to technical limitations here we report only peripheral body temperatures, whereas the brain temperature, was not assessed. These matters should be subject of further experimentation.
In rat models of ischemia a beneficial effect of mild hypothermia on neurological outcome has been demonstrated by infusion of the neurotensin receptor agonist NT77 [44
] or a cannabinod (CB) receptor agonist WIN 55,212-2 [45
]. However, the use of a CB1 agonist for inducing hypothermia may be connected with undesirable cardiac and respiratory side-effects [46
]. In the present study we demonstrate for the first time that a sustainable mild therapeutic hypothermia relevant for the human situation can be obtained in rodents and small and large non-rodents. Interestingly, feasibility studies in a rat model of cardiopulmonary resuscitation have demonstrated that infusion of DHC can also induce mild hypothermia in this model, and in a similar fashion to the one reported here [38