- Open Access
Positive allosteric modulation of TRPV1 as a novel analgesic mechanism
© Lebovitz et al.; licensee BioMed Central Ltd. 2012
Received: 6 July 2012
Accepted: 11 September 2012
Published: 21 September 2012
The prevalence of long-term opiate use in treating chronic non-cancer pain is increasing, and prescription opioid abuse and dependence are a major public health concern. To explore alternatives to opioid-based analgesia, the present study investigates a novel allosteric pharmacological approach operating through the cation channel TRPV1. This channel is highly expressed in subpopulations of primary afferent unmyelinated C- and lightly-myelinated Aδ-fibers that detect low and high rates of noxious heating, respectively, and it is also activated by vanilloid agonists and low pH. Sufficient doses of exogenous vanilloid agonists, such as capsaicin or resiniferatoxin, can inactivate/deactivate primary afferent endings due to calcium overload, and we hypothesized that positive allosteric modulation of agonist-activated TRPV1 could produce a selective, temporary inactivation of nociceptive nerve terminals in vivo. We previously identified MRS1477, a 1,4-dihydropyridine that potentiates vanilloid and pH activation of TRPV1 in vitro, but displays no detectable intrinsic agonist activity of its own. To study the in vivo effects of MRS1477, we injected the hind paws of rats with a non-deactivating dose of capsaicin, MRS1477, or the combination. An infrared diode laser was used to stimulate TRPV1-expressing nerve terminals and the latency and intensity of paw withdrawal responses were recorded. qRT-PCR and immunohistochemistry were performed on dorsal root ganglia to examine changes in gene expression and the cellular specificity of such changes following treatment.
Withdrawal responses of the capsaicin-only or MRS1477-only treated paws were not significantly different from the untreated, contralateral paws. However, rats treated with the combination of capsaicin and MRS1477 exhibited increased withdrawal latency and decreased response intensity consistent with agonist potentiation and inactivation or lesion of TRPV1-containing nerve terminals. The loss of nerve endings was manifested by an increase in levels of axotomy markers assessed by qRT-PCR and colocalization of ATF3 in TRPV1+ cells visualized via immunohistochemistry.
The present observations suggest a novel, non-narcotic, selective, long-lasting TRPV1-based approach for analgesia that may be effective in acute, persistent, or chronic pain disorders.
During the past decade, the use of opiates for chronic pain management has expanded from mainly postoperative and oncological patients to include those with chronic non-cancer pain. Consequently, the abuse of and dependence on prescription opioids has become an increasing public health concern [1–5]. Given the detrimental side effects and addiction potential of these medications, it is critical that we develop novel, non-opiate based pharmacological agents to treat chronic pain. One potential therapeutic avenue involves targeting TRPV1, a sodium/calcium ion channel highly expressed in a subpopulation of unmyelinated C- and lightly-myelinated Aδ-afferent primary afferent neurons [6–9]. This receptor responds to multifactorial inputs and is activated by temperature >43°C , protons , exogenous vanilloid ligands such as capsaicin and resiniferatoxin (RTX) , endogenous vanilloids such as NADA , the endocannibinoid anandamide [11, 12], and various fatty acids [13–15]. It is also sensitized by post-translational modifications through the actions of algesic molecules produced after tissue injury or during inflammation [16–20], and the plasma membrane expression of TRPV1 is greatly upregulated in small- to medium-diameter sensory neurons during inflammation [21, 22]. Extensive drug development has been directed at generating antagonists of the TRPV1 orthosteric capsaicin-binding site for acute and chronic pain. This research yielded many outstanding pharmacological agents in several chemical classes  that include, for example, ABT-102 , SB-705498 , AS1928370 , and AMG8562 . However, as a class, this group of antagonists encountered difficulties in transitioning to clinical utilization due to two main side effects. The first is an unpredictable level of hyperthermia; the propensity for which is variable among individual agents . The second is that TRPV1 antagonists potently block hot temperature sensation throughout the body and the loss of cutaneous sensation to noxious thermal stimuli can place these patients at risk for accidental burn injuries .
In an attempt to circumvent some of the issues with current TRPV1 antagonists, novel reversible or permanent interventional neuroablative therapies based on TRPV1 agonists are being explored [29, 30]. Administration of a vanilloid agonist, such as capsaicin or its ultrapotent analog RTX , can cause calcium-induced cytotoxicity and lead to a TRPV1-selective axonopathy that spares surrounding non-TRPV1-expressing somatosensory proprioceptive afferent and motor efferent nerve fibers. The potency and selectivity of vanilloid agonists for TRPV1 afferents has been demonstrated repeatedly in vivo [8, 32–40]. For example, ablation of TRPV1+ nerve terminals occurring after a single subcutaneous injection of RTX into the rat hind paw results in prolonged but reversible analgesia that can be detected for one to several weeks and induces up-regulation of molecular markers for axon damage/repair in neuronal perikarya of the dorsal root ganglia [8, 32]. We have also found that topical application of RTX onto the cornea results in temporary loss of the capsaicin eyewipe response in parallel with a loss of CGRP immunoreactive afferents in stromal fiber bundles . While the actions of peripherally administered RTX are localized and reversible, RTX given intrathecally produces a spatially broader effect over multiple dermatomes with permanent pain relief [33, 34, 36]. The therapeutic efficacy of intrathecal RTX is especially evident in veterinary canine patients with naturally-occurring osteosarcoma , and RTX delivered intrathecally to treat advanced cancer pain in humans is currently in a Phase I clinical trial (<http://clinicalstudies.info.nih.gov/detail/A_2009-D-0039.html>). At present, the only available FDA-approved agonist-based treatments contain capsaicin in an over-the-counter low dose (0.075%) topical cream and a high dose (8%) prescription cutaneous patch . Topical therapy is reported to be effective for post-herpetic neuralgia , HIV-associated distal sensory polyneuropathy , and painful diabetic neuropathy . Because TRPV1 agonists are painful initially upon application, in general, administration of a TRPV1 agonist necessitates pretreatment with a local anesthetic and, in the case of intrathecal administration, general anesthesia, which requires the patient to be either in the clinic or in the operating room. In addition, while capsaicin is effective when administered focally to treat Morton’s neuroma or osteoarthritis  and RTX is effective when administered intrathecally to treat osteosarcoma , they generally cannot be given systemically in large doses since they produce a decrease in core body temperature and may be cardiotoxic, although there is a much larger safety margin for RTX . Thus while some conditions may be amenable to local, interventional TRPV1 agonist administration, other pain conditions, where pain is more diffuse or delocalized, may not be appropriate candidates for treatment with localized therapies.
Male Sprague–Dawley rats (250–400g) were housed under a 12h light–dark cycle and allowed access to food and water ad libitum. The ambient temperature of the holding and testing rooms was ~22°C. Procedures were performed in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, and approved by the National Institute of Dental and Craniofacial Research (NIDCR) Animal Care and Use Committee. All efforts were made to minimize both animal numbers and distress within the experiments.
Drug solutions and administration
The drug vehicle for all experiments consisted of 7.5% Tween-80 (Sigma-Aldrich, P8074) and 0.05% ascorbic acid (Spectrum Chemical, AS105) in PBS, at pH 7.2. Resiniferatoxin from stock (“RTX”, custom purification, 100 μg/ml in vehicle) was further diluted in vehicle to 200 ng/100 μl for injection. A 20 mM stock solution of MRS1477 (FW = 389.51, ) was prepared in 100% DMSO (Sigma-Aldrich, D2650) and further diluted in vehicle to either 1 μg/100 μl (25 μM) or 2 μg/100 μl (50 μM), where specified. Capsaicin (“CAP”, Sigma-Aldrich, M2028) was prepared as a 100 mM stock solution in absolute ethanol, stored at −80°C, and was diluted directly into vehicle on the day of the experiments to 30 μg/100 μl. CAP-only injectates contained an equal amount of DMSO as those with MRS1477. All subcutaneous injections were made using a 29G×1/2", 3/10 cc insulin syringe (Terumo Medical Corp., Cat No. SS*30 M2913). The experimenter was blinded to the identity of the injectates in the various behavioral experiments.
Behavioral assessments were performed as reported previously . Briefly, unrestrained rats were placed on a clear glass platform under a small plastic cage (23×13×13 cm), which allowed them to move freely. Groups of rats were habituated to the testing apparatus for a minimum of three days by placing them under the cages on the glass platform and given both Aδ- and C-fiber laser stimuli. On the day of testing, rats were allowed to habituate for at least 10 min prior to thermal stimulation. Rats were tested prior to injection to establish a baseline, then at 2, 24, and 48 h post-injection. An infrared diode laser (Lasmed LLC, Mountain View, CA) was aimed at the ventral hind paw, with a perpendicular approach to the plantar skin. Aδ-fibers were preferentially stimulated with a short-pulse (100 msec) using a high-intensity small-diameter (1.6 mm) beam, and behavioral responses were assessed on a five-point scale: 0 = no response, 1 = orient to stimulus, 2 = orient with paw lift, 3 = lift paw, orient, and shake, and 4 = lift paw with lick . Only responses graded as 2 or above included a paw withdrawal. Aδ-fibers were stimulated with laser intensities ranging from 3.59 W/mm2 to 5.61 W/mm2. C-fibers were preferentially stimulated with a continuous, wide-diameter (5 mm), low-energy pulse (0.083 W/mm2) and paw withdrawal latency (sec) served as the primary endpoint. One cohort of rats (n = 4) receiving 2 μg MRS1477 + 30 μg CAP was tested repeatedly for 24 days to evaluate the time course for recovery of thermal sensitivity.
RT-PCR primer pairs and PCR product sizes
Comparisons of gene expression from ipsilateral and contralateral tissues were made by paired Student’s t-test. For other single comparisons between two different groups, unpaired t-tests were used. For multiple comparisons, one-way and two-way ANOVA tests were used with, respectively, Tukey’s post-hoc test and Bonferroni multiple comparisons. For ordinal response score data from behavioral ratings that were used to evaluate Aδ-fiber responses, the Mann–Whitney U test was used. Differences were considered significant where p < 0.05. Time versus drug effects for longitudinal behavioral data were analyzed using two-way ANOVA.
Rats used for immunohistochemistry were deeply anesthetized with sodium pentobarbital and transcardially perfused with cold PBS followed by 4% paraformaldehyde. The L4-L5-L6 dorsal root ganglia were harvested bilaterally, post-fixed in 4% paraformaldehyde for 2 h, cryopreserved in 20% sucrose, then mounted and frozen for sectioning on a cryostat.
All DRG tissues were sectioned at 10 μm and mounted directly onto slides and stored at −80°C until use. Prior to staining with antibodies, the sections were thawed at RT and allowed to air dry. The entire immunostaining procedure was carried out at RT. Sections were washed in HEPES Buffer (“HB”; see below) and fixed in 4% paraformaldehyde for 10 min, then washed in HB and counterfixed in absolute methanol for 5 min. Antigen retrieval was performed by incubation of sections in 1% SDS for 5 min and the tissues were then incubated in Background Buster solution (Innovex Biosciences, Cat No. NB306-50) for 20 min to block non-specific antibody binding. Sections were subsequently washed in HB and then incubated with primary antibodies of interest (see below) diluted in HB for 1 h in a humidified chamber. The unbound antibodies were washed away using HB and the sections were then incubated with appropriate fluorescently-labeled secondary antibodies (see below) for 1 h in a dark, humidified chamber. Finally, the sections were washed in HB to remove unbound antibodies, sealed with Immuno Mount (GeneTex, Cat No. GTX30928) and a coverslip, and were viewed the following day.
All sections were imaged using a fluorescence microscope (Axiovert 200 M; Carl Zeiss, Thornwood, NY) equipped with a 20× Plan-Apochromat objective (Carl Zeiss), a high-resolution cooled digital camera (ORCA-ER; Hamamatsu Photonics, Japan), a 100 W mercury-arc lamp light source and wavelengths selected with excitation/dichroic/emission filter sets (Semrock, Rochester, NY) optimized to detect the following fluorophores: DAPI, Alexa Fluor 488, and Alexa Fluor 546. Each labeling reaction was captured using appropriate fluorescence filter sets and the images individually digitized at 12-bit resolution using the Volocity image acquisition program (Improvision Inc., Lexington, MA). An appropriate color table was applied to each image to either match its emission spectrum or to set a distinguishing color balance. The pseudocolored images were then converted into TIFF files, exported to Adobe Photoshop, and overlaid as individual layers to display multi-colored merged composites.
HEPES Buffer (HB)
HB was made fresh prior to staining and kept at 4°C. Buffer was made in 1 liter batches and contained 145 mM NaCl, 5 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES, and 0.1% BSA. The solution was filter sterilized and the pH was adjusted to 7.3 with NaOH/HCl.
Polyclonal primary antibodies consisted of anti-TRPV1 (guinea pig, 1:100, Novus Biologicals, Cat No. NB300-122) and anti-ATF3 (rabbit, 1:200, Santa Cruz Biotechnology, Cat No. sc-188). Both primary antibodies were diluted with glycerol 1:1 prior to use to limit degradation following repeated freeze-thaw cycles. Fluorescently labeled secondary antibodies were purchased from Invitrogen and consisted of goat anti-guinea pig Alexa Fluor 488 (1:200, Cat No. A-11073) and goat anti-rabbit Alexa Fluor 546 (1:200, Cat No. A-11035). All antibodies were diluted from stock solutions to working concentrations with HB.
In order to evaluate the in vivo effects of MRS1477, we measured nociceptive responses to thermal stimuli in Aδ- and C-fibers after mid-plantar injections into rat hind paws. The CAP dose was determined empirically in pilot studies. To do this, we sought a dose that produced robust nocifensive responses from the rats upon acute injection (vocalization, paw shaking, paw licking, and erythema), but did not cause subsequent analgesia when tested with noxious thermal stimuli the next day. We found 30 μg of CAP in 100 μl to be effective for this purpose. The MRS1477 doses were based on our previous in vitro data, and we were primarily interested in finding a minimum effective dose when administered with 30 μg of CAP. Response scores (Aδ stimulus, 3.59-5.61 W/mm2) and paw withdrawal latencies (C-fiber stimulus, 0.083 W/mm2) were evaluated prior to and at 2, 24, and 48 h following subcutaneous injection of either MRS1477 alone, CAP alone, or the co-administration of MRS1477 and CAP. We chose the starting point of 2 h to minimize acute effects of the injection on response sensitivity and also to allow time for denervation to occur . A separate cohort of rats (n = 6) was used to compare MRS1477 alone to untreated contralateral paws, and the behavioral data were not found to be significantly different (not shown). Throughout the rest of the experiments, CAP alone and untreated contralateral paw data served as controls.
Paws treated with MRS1477 + CAP showed a loss of thermal sensitivity that persisted at 24 h (Figure 2C). The mean Aδ response score after 2 μg MRS1477 + 30 μg CAP (0.7 ± 0.1, n = 7) was significantly lower (p < 0.0001) than all other treatments; the mean response score was 2.4 ± 0.2 (n = 12) after 1 μg MRS1477 + 30 μg CAP and 2.4 ± 0.2 (n = 12) after CAP alone, which was not significantly different from the untreated contralateral paws (2.4 ± 0.2, n = 19). The mean C-fiber evoked paw withdrawal latency at 24 h (Figure 2D) after 2 μg MRS1477 + CAP (10.4 ± 0.6 s, n = 7) were significantly greater (p < 0.0001) than CAP alone (7.3 ± 0.3 s, n = 12), and compared to contralateral paws (7.8 ± 0.3 s, n = 19). The mean latency for 1 μg MRS1477 + CAP was significantly higher (p < 0.05) than CAP alone. At this time, the increase in latency produced by 2 μg MRS1477 + 30 μg CAP was not significantly different than that seen with 1 μg MRS1477 + 30 μg CAP (8.8 ± 0.6 s, n = 12).
Blunted nocifensive behavioral responses continued for 48 h and were now apparent with tests of Aδ fibers at both doses of MRS1477 (Figure 2E). The mean Aδ response scores after 1 μg MRS1477 + CAP (1.8 ± 0.2, n = 12) and 2 μg MRS1477 + CAP (1.5 ± 0.2, n = 7) were similar, but they were both significantly lower compared to CAP alone (3.2 ± 0.1, n = 12) and untreated contralateral paws (2.8 ± 0.1, n = 19) (p < 0.0001). At 48 h (Figure 2F), the mean C-fiber withdrawal latencies after 1 μg MRS1477 + CAP (9.2 ± 0.6 s, n = 12) and 2 μg MRS1477 + CAP (10.0 ± 0.6 s, n = 7) were both significantly greater (p < 0.01 and p < 0.0001, respectively) than CAP alone (7.4 ± 0.6 s, n = 12), and the untreated contralateral paws (7.0 ± 0.4 s, n = 19). A summary of the dose–response effect at 2, 24, and 48 h for the C-fiber stimuli is shown in Figure 2G. We combined CAP alone with the untreated contralateral paw data at each time point, since the CAP only injection had no effect. Again, the loss of acute thermal sensitivity after 2 μg MRS1477 + CAP is significant at all time points (p < 0.0001), while loss of thermal sensitivity in the 1 μg MRS1477 + CAP treatment group was significant at the 48 h test (p < 0.05). The persistent nature of the analgesia provided the initial indication that TRPV1-expressing afferent nerve endings are being ablated by the combined treatment.
The present data demonstrate the ability of MRS1477, a positive allosteric modulator of TRPV1, to potentiate the effect of capsaicin (CAP) in vivo resulting in a rapid peripheral nerve terminal inactivation and a significant, long-lasting yet reversible analgesia to cutaneous noxious thermal stimulation in rats. Neither MRS1477 nor capsaicin alone produced suppression of behavioral responses. Evidence for loss of the cutaneous nerve terminals was reinforced by parallel molecular studies of gene expression changes in DRG. The corresponding cellular specificity of the effect was examined by immunohistochemical double labeling for ATF3 (a nuclear stain) and TRPV1 (largely cytoplasmic). Intraplantar injection of the combination of MRS1477 and CAP produced colocalization of TRPV1 and ATF3 in neurons in lumbar DRG. These data indicate that potentiation of agonist-activated TRPV1 by a positive allosteric modulator can produce a localized nerve terminal inactivation in vivo that yields rapid and prolonged analgesia.
We previously demonstrated that MRS1477 exhibits no agonist activity at TRPV1, but potentiates the calcium influx induced by orthosteric vanilloid agonists and TRPV1 stimulation by protons or sensitization by phorbol ester [46, 47]. In the previous study, we also observed MRS1477 potentiation of intraperitoneal capsaicin-induced hypothermia . In the present study, we were able to induce analgesia through functional inactivation of peripheral primary afferent nerve terminals similar to the long-lasting effect of the potent TRPV1 agonist RTX, which served as our positive control . Inhibition of the response to noxious thermal stimuli was observed after administration of 2 μg MRS1477 plus 30 μg capsaicin, while 1 μg MRS1477 plus 30 μg capsaicin produced a less robust response, consistent with a dose-dependent relationship. Furthermore, the effect of the agonist-PAM combination was spatially discrete. When administered as a mid-plantar injection, the resulting analgesia occurred at the treated area and distally in the toes with relative sparing of the heel (see Figure 5). This localized analgesia in the mid-plantar and distal regions suggests that the nerve terminal inactivation is confined to the nerve endings in the immediate vicinity of the injection site and to axons projecting through it; an effect we have demonstrated previously with peripherally administered resiniferatoxin . We refer to the process involving nerve endings as “nerve terminal inactivation” or “deactivation,” rather than complete ablation, since the nerve endings can grow back and become active again [32, 55]. We also distinguish inactivation/deactivation from desensitization, the latter referring to the progressive decrement in response upon multiple, successive applications of a vanilloid agonist; desensitization is an endpoint frequently investigated in electrophysiological experiments. This inactivation process can form a new potential approach to analgesic pharmacology that is conditional and pain-state dependent .
When we tracked the recovery of Aδ-mediated thermal nociception, we observed substantial yet incomplete recovery by day 4, followed by a gradual, progressive return towards baseline over the next 20 days. With C-fiber stimulation, there was significant attenuation of nociceptive responses over the first 6 to 8 days and a convergence with control latency measurements by day 14. This slow return to baseline is consistent with an earlier study of the effects of RTX on cutaneous Aδ- and C-fiber TRPV1-expressing nerve terminals . These data support the notion that a TRPV1 PAM, in combination with an agonist in a non-deactivating dose, can produce a long-lasting analgesia that shares many of the behavioral and afferent fiber-type specific characteristics obtained by treatment with the potent, selective agonist RTX.
Gene upregulation of markers for neuronal damage in dorsal root ganglia also provides evidence that PAM treatment modifies the integrity of primary afferent nerve terminals. This was explored using two approaches: transcript amplification by qRT-PCR and immunohistochemical staining for relevant proteins and neuropeptides. Increased transcript levels of nerve damage biomarkers were measured in the dorsal root ganglia for the transcription factor ATF3  and the neuropeptides vasoactive intestinal peptide (VIP) [51–53], galanin (GAL) , and neuropeptide Y (NPY) . We previously demonstrated that these molecules can also be induced by injection of RTX into the hind paw; a result consistent with chemo-axotomy of the TRPV1-expressing nerve endings. By extension, we hypothesized that the PAM/agonist combination would produce a similar effect. Our qRT-PCR data demonstrated that MRS1477 plus CAP resulted in an induction of ATF3, VIP, NPY, and GAL transcripts. In contrast, neither CAP nor MRS1477 alone induced upregulation of any of the biomarkers except NPY and VIP, which were upregulated after CAP alone. This may be related to the low but detectable basal expression levels of NPY compared to the other genes, which are nearly undetectable at basal state. For VIP, while induction by CAP is higher than in the untreated contralateral DRG, treatment with MRS1477 + CAP produces both significantly greater VIP induction and behavioral effects compared to CAP alone.
We determined that the molecular axotomy was specific to TRPV1+ neurons by immunofluorescence double labeling using antibodies targeting TRPV1 and ATF3. Both the behavioral and molecular data suggest that MRS1477 is working on TRPV1-expressing afferent terminals. We reasoned that this could be further substantiated if the elevation in ATF3 was enriched in TRPV1-expressing neurons in the DRG. Figure 7 shows that in rats treated with MRS1477 and CAP, nuclear staining for ATF3 is found predominantly in TRPV1 expressing neurons: 93% (82/88) of ATF3 positive nuclei co-localized to TRPV1-stained neurons, and this percentage is similar following positive control injections of RTX (79%, 100/126). However not all TRPV1+ neurons expressed ATF3. Likely, these represent TRPV1+ neurons with axons terminating in the lower limb but not in the treated plantar surface of the hind paw. Extensive co-localization of axotomy-related molecular alterations to TRPV1+ neurons suggests a high degree of cellular specificity for MRS1477. At the same time, the rats did not exhibit autotomy behaviors that commonly accompany sciatic nerve transection [56–58]. These results are consistent with earlier studies on peripheral injections of RTX into the hind paw , perineurally [38, 55], or directly into rat or monkey trigeminal [33, 59]. These data suggest that our TRPV1 PAM is producing the expected on-target effects and does not produce unexpected sensory abnormalities.
Evidence from this experiment and our previous RTX studies [8, 32] suggest that TRPV1-expressing Aδ-fibers are more susceptible to long-term denervation. The greater structural complexity of lightly myelinated Aδ-fibers could account for the apparent disparity in functional recovery time compared to unmyelinated C-fibers . Also, the paranodal location of the plasma-membrane calcium ATPase , a major regulator of intracellular calcium levels in myelinated DRG neurons [62, 63], may accentuate calcium toxicity in Aδ-fibers. It is interesting to speculate whether differential efficacy at the two fiber types could form a useful strategy for treating pathological pain problems.
Allosteric modulation is an important approach to consider in therapeutic drug development . Currently, allosteric modulation of GABAA ligand-gated ion channels is a well-exploited mechanism for general anesthetics, benzodiazepine pre-anesthetic medications, and anxiolytics [65–67]. Agonist potentiation is also being explored to mitigate the side effects of poorly tolerated but otherwise effective analgesics acting on, for example, GABAB receptors  and nicotinic acetylcholine receptors . Conversely, negative allosteric modulation of glutamate receptors has been explored clinically for the treatment of migraine . Our studies investigate MRS1477 as a proof-of-concept molecule for allosteric potentiation of TRPV1. We have demonstrated that MRS1477 can effectively modulate CAP activation in vitro and in vivo, and while CAP is not a physiologically relevant agonist, our model serves as a framework for future testing of animal pain models. In pathological states there are several variables that can influence PAM efficacy. TRPV1 PAM actions are predicated on the existence and generation of endogenous TRPV1 activators (endovanilloids) that are adequately potent and present in sufficient concentrations at sites of tissue damage or inflammation to bind to the orthosteric vanilloid site; low pH also plays an activating role [71, 72]. A model for the interactions between TRPV1, PAMs, and endovanilloids is presented in Figure 1, which shows a diagram of four potential states of the receptor: closed, both sites unoccupied; closed with PAM site occupied; open with vanilloid site occupied; and open with both sites occupied. It may be that only certain pain conditions generate sufficient endovanilloids or protons to be permissive for positive allosteric modulation of TRPV1. In chronic pain states, nociceptive nerve endings remain active and do not transition into an inactive state without pharmacological intervention, since the endings have multiple mechanisms to cope with transmembrane calcium flux . Thus, despite a low pH and an endovanilloid-enriched environment, the added “push” from a PAM would be necessary to achieve nerve terminal inactivation .
Perhaps the greatest advantage of a TRPV1 PAM in terms of clinical pharmacology is the mechanism of calcium-mediated nerve terminal inactivation, which can potentially produce an effective, long-term analgesia. Even the most highly specific TRPV1 antagonist blocks just one ligand-gated ion channel, leaving numerous other TRPV1-independent receptors on the nerve endings available for stimulation by algesic mediators. In contrast, nerve terminal inactivation renders the fiber essentially insensitive to a variety of noxious stimuli. Thus, several practical and theoretical characteristics of a TRPV1 PAM offer the potential for a new, emerging class of analgesics.
EEL received funding from the Howard Hughes Medical Institute as an HHMI-NIH Research Scholar and from the Division of Intramural Research NIDCR, NIH. This work was supported by the Division of Intramural Research, NIDCR, NIH. NIH grant R03MH089480.
- Paulozzi LJ, Budnitz DS, Xi Y: Increasing deaths from opioid analgesics in the United States. Pharmacoepidemiol Drug Saf 2006, 15: 618–627. 10.1002/pds.1276View ArticlePubMedGoogle Scholar
- Edlund MJ, Martin BC, Devries A, Fan MY, Braden JB, Sullivan MD: Trends in use of opioids for chronic noncancer pain among individuals with mental health and substance use disorders: the TROUP study. Clin J Pain 2010, 26: 1–8. 10.1097/AJP.0b013e3181b99f35PubMed CentralView ArticlePubMedGoogle Scholar
- Boudreau D, Von Korff M, Rutter CM, Saunders K, Ray GT, Sullivan MD, Campbell CI, Merrill JO, Silverberg MJ, Banta-Green C, Weisner C: Trends in long-term opioid therapy for chronic non-cancer pain. Pharmacoepidemiol Drug Saf 2009, 18: 1166–1175. 10.1002/pds.1833PubMed CentralView ArticlePubMedGoogle Scholar
- Michna E, Jamison RN, Pham LD, Ross EL, Janfaza D, Nedeljkovic SS, Narang S, Palombi D, Wasan AD: Urine toxicology screening among chronic pain patients on opioid therapy: frequency and predictability of abnormal findings. Clin J Pain 2007, 23: 173–179. 10.1097/AJP.0b013e31802b4f95View ArticlePubMedGoogle Scholar
- Compton WM, Volkow ND: Major increases in opioid analgesic abuse in the United States: concerns and strategies. Drug Alcohol Depend 2006, 81: 103–107. 10.1016/j.drugalcdep.2005.05.009View ArticlePubMedGoogle Scholar
- Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D: Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000, 288: 306–313. 10.1126/science.288.5464.306View ArticlePubMedGoogle Scholar
- Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997, 389: 816–824. 10.1038/39807View ArticlePubMedGoogle Scholar
- Mitchell K, Bates BD, Keller JM, Lopez M, Scholl L, Navarro J, Madian N, Haspel G, Nemenov MI, Iadarola MJ: Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates. Molecular Pain 2010, 6: 94. 10.1186/1744-8069-6-94PubMed CentralView ArticlePubMedGoogle Scholar
- McQueen DS, Bond SM, Smith PJ, Balali-Mood K, Smart D: Cannabidiol lacks the vanilloid VR1-mediated vasorespiratory effects of capsaicin and anandamide in anaesthetised rats. Eur J Pharmacol 2004, 491: 181–189. 10.1016/j.ejphar.2004.03.045View ArticlePubMedGoogle Scholar
- Huang SM, Bisogno T, Trevisani M, Al-Hayani A, De Petrocellis L, Fezza F, Tognetto M, Petros TJ, Krey JF, Chu CJ, et al.: An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proc Natl Acad Sci U S A 2002, 99: 8400–8405. 10.1073/pnas.122196999PubMed CentralView ArticlePubMedGoogle Scholar
- Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB: The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol 2000, 129: 227–230. 10.1038/sj.bjp.0703050PubMed CentralView ArticlePubMedGoogle Scholar
- Olah Z, Karai L, Iadarola MJ: Anandamide activates vanilloid receptor 1 (VR1) at acidic pH in dorsal root ganglia neurons and cells ectopically expressing VR1. J Biol Chem 2001, 276: 31163–31170. 10.1074/jbc.M101607200View ArticlePubMedGoogle Scholar
- Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, Cho S, Min KH, Suh YG, Kim D, Oh U: Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances. Proc Natl Acad Sci U S A 2000, 97: 6155–6160. 10.1073/pnas.97.11.6155PubMed CentralView ArticlePubMedGoogle Scholar
- Premkumar LS, Ahern GP: Induction of vanilloid receptor channel activity by protein kinase C. Nature 2000, 408: 985–990. 10.1038/35050121View ArticlePubMedGoogle Scholar
- Matta JA, Miyares RL, Ahern GP: TRPV1 is a novel target for omega-3 polyunsaturated fatty acids. J Physiol 2007, 578: 397–411.PubMed CentralView ArticlePubMedGoogle Scholar
- Hong Y, Abbott FV: Behavioural effects of intraplantar injection of inflammatory mediators in the rat. Neuroscience 1994, 63: 827–836. 10.1016/0306-4522(94)90527-4View ArticlePubMedGoogle Scholar
- Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K, et al.: Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000, 405: 183–187. 10.1038/35012076View ArticlePubMedGoogle Scholar
- Yang HY, Mitchell K, Keller JM, Iadarola MJ: Peripheral inflammation increases Scya2 expression in sensory ganglia and cytokine and endothelial related gene expression in inflamed tissue. J Neurochem 2007, 103: 1628–1643. 10.1111/j.1471-4159.2007.04874.xView ArticlePubMedGoogle Scholar
- Maingret F, Coste B, Padilla F, Clerc N, Crest M, Korogod SM, Delmas P: Inflammatory mediators increase Nav1.9 current and excitability in nociceptors through a coincident detection mechanism. J Gen Physiol 2008, 131: 211–225. 10.1085/jgp.200709935PubMed CentralView ArticlePubMedGoogle Scholar
- Kessler W, Kirchhoff C, Reeh PW, Handwerker HO: Excitation of cutaneous afferent nerve endings in vitro by a combination of inflammatory mediators and conditioning effect of substance P. Experimental brain research Experimentelle Hirnforschung Experimentation cerebrale 1992, 91: 467–476.View ArticlePubMedGoogle Scholar
- Ji RR, Samad TA, Jin SX, Schmoll R, Woolf CJ: p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 2002, 36: 57–68. 10.1016/S0896-6273(02)00908-XView ArticlePubMedGoogle Scholar
- Yu L, Yang F, Luo H, Liu FY, Han JS, Xing GG, Wan Y: The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund’s adjuvant. Molecular Pain 2008, 4: 61. 10.1186/1744-8069-4-61PubMed CentralView ArticlePubMedGoogle Scholar
- Pal M, Angaru S, Kodimuthali A, Dhingra N: Vanilloid receptor antagonists: emerging class of novel anti-inflammatory agents for pain management. Curr Pharm Des 2009, 15: 1008–1026. 10.2174/138161209787581995View ArticlePubMedGoogle Scholar
- Gomtsyan A, Bayburt EK, Schmidt RG, Surowy CS, Honore P, Marsh KC, Hannick SM, McDonald HA, Wetter JM, Sullivan JP, et al.: Identification of (R)-1-(5-tert-butyl-2,3-dihydro-1 H-inden-1-yl)-3-(1 H-indazol-4-yl)urea (ABT-102) as a potent TRPV1 antagonist for pain management. J Med Chem 2008, 51: 392–395. 10.1021/jm701007gView ArticlePubMedGoogle Scholar
- Chizh BA, O’Donnell MB, Napolitano A, Wang J, Brooke AC, Aylott MC, Bullman JN, Gray EJ, Lai RY, Williams PM, Appleby JM: The effects of the TRPV1 antagonist SB-705498 on TRPV1 receptor-mediated activity and inflammatory hyperalgesia in humans. Pain 2007, 132: 132–141. 10.1016/j.pain.2007.06.006View ArticlePubMedGoogle Scholar
- Watabiki T, Kiso T, Tsukamoto M, Aoki T, Matsuoka N: Intrathecal administration of AS1928370, a transient receptor potential vanilloid 1 antagonist, attenuates mechanical allodynia in a mouse model of neuropathic pain. Biol Pharm Bull 2011, 34: 1105–1108. 10.1248/bpb.34.1105View ArticlePubMedGoogle Scholar
- Lehto SG, Tamir R, Deng H, Klionsky L, Kuang R, Le A, Lee D, Louis JC, Magal E, Manning BH, et al.: Antihyperalgesic effects of (R, E)-N-(2-hydroxy-2,3-dihydro-1 H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluorom ethyl)phenyl)-acrylamide (AMG8562), a novel transient receptor potential vanilloid type 1 modulator that does not cause hyperthermia in rats. J Pharmacol Exp Ther 2008, 326: 218–229. 10.1124/jpet.107.132233View ArticlePubMedGoogle Scholar
- Wong GY, Gavva NR: Therapeutic potential of vanilloid receptor TRPV1 agonists and antagonists as analgesics: Recent advances and setbacks. Brain Res Rev 2009, 60: 267–277. 10.1016/j.brainresrev.2008.12.006View ArticlePubMedGoogle Scholar
- Kissin I, Szallasi A: Therapeutic targeting of TRPV1 by resiniferatoxin, from preclinical studies to clinical trials. Current topics in medicinal chemistry 2011, 11: 2159–2170. 10.2174/156802611796904924View ArticlePubMedGoogle Scholar
- Iadarola MJ, Mannes AJ: The vanilloid agonist resiniferatoxin for interventional-based pain control. Current topics in medicinal chemistry 2011, 11: 2171–2179. 10.2174/156802611796904942PubMed CentralView ArticlePubMedGoogle Scholar
- Szallasi A, Blumberg PM: Resiniferatoxin, a phorbol-related diterpene, acts as an ultrapotent analog of capsaicin, the irritant constituent in red pepper. Neuroscience 1989, 30: 515–520. 10.1016/0306-4522(89)90269-8View ArticlePubMedGoogle Scholar
- Neubert JK, Karai L, Jun JH, Kim HS, Olah Z, Iadarola MJ: Peripherally induced resiniferatoxin analgesia. Pain 2003, 104: 219–228. 10.1016/S0304-3959(03)00009-5View ArticlePubMedGoogle Scholar
- Karai L, Brown DC, Mannes AJ, Connelly ST, Brown J, Gandal M, Wellisch OM, Neubert JK, Olah Z, Iadarola MJ: Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control. J Clin Investig 2004, 113: 1344–1352.PubMed CentralView ArticlePubMedGoogle Scholar
- Brown DC, Iadarola MJ, Perkowski SZ, Erin H, Shofer F, Laszlo KJ, Olah Z, Mannes AJ: Physiologic and antinociceptive effects of intrathecal resiniferatoxin in a canine bone cancer model. Anesthesiology 2005, 103: 1052–1059. 10.1097/00000542-200511000-00020View ArticlePubMedGoogle Scholar
- Backonja M, Wallace MS, Blonsky ER, Cutler BJ, Malan P Jr, Rauck R, Tobias J: NGX-4010, a high-concentration capsaicin patch, for the treatment of postherpetic neuralgia: a randomised, double-blind study. Lancet Neurol 2008, 7: 1106–1112. 10.1016/S1474-4422(08)70228-XView ArticlePubMedGoogle Scholar
- Jeffry JA, Yu SQ, Sikand P, Parihar A, Evans MS, Premkumar LS: Selective targeting of TRPV1 expressing sensory nerve terminals in the spinal cord for long lasting analgesia. PLoS One 2009, 4: e7021. 10.1371/journal.pone.0007021PubMed CentralView ArticlePubMedGoogle Scholar
- Kissin EY, Freitas CF, Kissin I: The effects of intraarticular resiniferatoxin in experimental knee-joint arthritis. Anesth Analg 2005, 101: 1433–1439. 10.1213/01.ANE.0000180998.29890.B0PubMed CentralView ArticlePubMedGoogle Scholar
- Kissin I, Davison N, Bradley EL Jr: Perineural resiniferatoxin prevents hyperalgesia in a rat model of postoperative pain. Anesth Analg 2005, 100: 774–780. table of contents 10.1213/01.ANE.0000143570.75908.7FView ArticlePubMedGoogle Scholar
- Robbins WR, Staats PS, Levine J, Fields HL, Allen RW, Campbell JN, Pappagallo M: Treatment of intractable pain with topical large-dose capsaicin: preliminary report. Anesth Analg 1998, 86: 579–583.PubMedGoogle Scholar
- Nolano M, Simone DA, Wendelschafer-Crabb G, Johnson T, Hazen E, Kennedy WR: Topical capsaicin in humans: parallel loss of epidermal nerve fibers and pain sensation. Pain 1999, 81: 135–145. 10.1016/S0304-3959(99)00007-XView ArticlePubMedGoogle Scholar
- Bates BD, Mitchell K, Keller JM, Chan CC, Swaim WD, Yaskovich R, Mannes AJ, Iadarola MJ: Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist. Pain 2010, 149: 522–528. 10.1016/j.pain.2010.03.024PubMed CentralView ArticlePubMedGoogle Scholar
- Irving GA, Backonja M, Rauck R, Webster LR, Tobias JK, Vanhove GF: NGX-4010, a capsaicin 8% dermal patch, administered alone or in combination with systemic neuropathic pain medications, reduces pain in patients with postherpetic neuralgia. The Clinical journal of pain 2012, 28: 101–107. 10.1097/AJP.0b013e318227403dView ArticlePubMedGoogle Scholar
- Simpson DM, Estanislao L, Brown SJ, Sampson J: An open-label pilot study of high-concentration capsaicin patch in painful HIV neuropathy. Journal of pain and symptom management 2008, 35: 299–306. 10.1016/j.jpainsymman.2007.04.015View ArticlePubMedGoogle Scholar
- Webster LR, Peppin JF, Murphy FT, Lu B, Tobias JK, Vanhove GF: Efficacy, safety, and tolerability of NGX-4010, capsaicin 8% patch, in an open-label study of patients with peripheral neuropathic pain. Diabetes Res Clin Pract 2011, 93: 187–197. 10.1016/j.diabres.2011.04.010View ArticlePubMedGoogle Scholar
- Remadevi R, Szallisi A: Adlea (ALGRX-4975), an injectable capsaicin (TRPV1 receptor agonist) formulation for longlasting pain relief. IDrugs: the investigational drugs journal 2008, 11: 120–132.PubMedGoogle Scholar
- Kaszas K, Keller JM, Coddou C, Mishra SK, Hoon MA, Stojilkovic S, Jacobson KA, Iadarola MJ: Small molecule positive allosteric modulation of TRPV1 activation by vanilloids and acidic pH. J Pharmacol Exp Ther 2012, 340: 152–160. 10.1124/jpet.111.183053PubMed CentralView ArticlePubMedGoogle Scholar
- Roh EJ, Keller JM, Olah Z, Iadarola MJ, Jacobson KA: Structure-activity relationships of 1,4-dihydropyridines that act as enhancers of the vanilloid receptor 1 (TRPV1). Bioorg Med Chem 2008, 16: 9349–9358. 10.1016/j.bmc.2008.08.048PubMed CentralView ArticlePubMedGoogle Scholar
- Tsujino H, Kondo E, Fukuoka T, Dai Y, Tokunaga A, Miki K, Yonenobu K, Ochi T, Noguchi K: Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: A novel neuronal marker of nerve injury. Mol Cell Neurosci 2000, 15: 170–182. 10.1006/mcne.1999.0814View ArticlePubMedGoogle Scholar
- Wakisaka S, Kajander KC, Bennett GJ: Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy. Neurosci Lett 1991, 124: 200–203. 10.1016/0304-3940(91)90093-9View ArticlePubMedGoogle Scholar
- Hokfelt T, Wiesenfeld-Hallin Z, Villar M, Melander T: Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci Lett 1987, 83: 217–220. 10.1016/0304-3940(87)90088-7View ArticlePubMedGoogle Scholar
- Shehab SA, Atkinson ME: Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons. Brain Research 1986, 372: 37–44. 10.1016/0006-8993(86)91456-3View ArticlePubMedGoogle Scholar
- Shehab SA, Atkinson ME: Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy. Experimental brain research Experimentelle Hirnforschung Experimentation cerebrale 1986, 62: 422–430.View ArticlePubMedGoogle Scholar
- Atkinson ME, Shehab SA: Peripheral axotomy of the rat mandibular trigeminal nerve leads to an increase in VIP and decrease of other primary afferent neuropeptides in the spinal trigeminal nucleus. Regul Pept 1986, 16: 69–81. 10.1016/0167-0115(86)90195-3View ArticlePubMedGoogle Scholar
- Toews AD, Barrett C, Morell P: Monocyte chemoattractant protein 1 is responsible for macrophage recruitment following injury to sciatic nerve. J Neurosci Res 1998, 53: 260–267. 10.1002/(SICI)1097-4547(19980715)53:2<260::AID-JNR15>3.0.CO;2-AView ArticlePubMedGoogle Scholar
- Neubert JK, Mannes AJ, Karai LJ, Jenkins AC, Zawatski L, Abu-Asab M, Iadarola MJ: Perineural resiniferatoxin selectively inhibits inflammatory hyperalgesia. Molecular pain 2008, 4: 3. 10.1186/1744-8069-4-3PubMed CentralView ArticlePubMedGoogle Scholar
- Wall PD, Devor M, Inbal R, Scadding JW, Schonfeld D, Seltzer Z, Tomkiewicz MM: Autotomy following peripheral nerve lesions: experimental anaesthesia dolorosa. Pain 1979, 7: 103–111. 10.1016/0304-3959(79)90002-2View ArticlePubMedGoogle Scholar
- Nissenbaum J, Devor M, Seltzer Z, Gebauer M, Michaelis M, Tal M, Dorfman R, Abitbul-Yarkoni M, Lu Y, Elahipanah T, et al.: Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2. Genome Res 2010, 20: 1180–1190. 10.1101/gr.104976.110PubMed CentralView ArticlePubMedGoogle Scholar
- Zeltser R, Beilin B, Zaslansky R, Seltzer Z: Comparison of autotomy behavior induced in rats by various clinically-used neurectomy methods. Pain 2000, 89: 19–24. 10.1016/S0304-3959(00)00342-0View ArticlePubMedGoogle Scholar
- Tender GC, Walbridge S, Olah Z, Karai L, Iadarola M, Oldfield EH, Lonser RR: Selective ablation of nociceptive neurons for elimination of hyperalgesia and neurogenic inflammation. J Neurosurg 2005, 102: 522–525. 10.3171/jns.2005.102.3.0522View ArticlePubMedGoogle Scholar
- Gaudet AD, Popovich PG, Ramer MS: Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. J Neuroinflammation 2011, 8: 110. 10.1186/1742-2094-8-110PubMed CentralView ArticlePubMedGoogle Scholar
- Mata M, Staple J, Fink DJ: Cytochemical localization of Ca2 + −ATPase activity in peripheral nerve. Brain research 1988, 445: 47–54. 10.1016/0006-8993(88)91072-4View ArticlePubMedGoogle Scholar
- Gover TD, Moreira TH, Kao JP, Weinreich D: Calcium regulation in individual peripheral sensory nerve terminals of the rat. J Physiol 2007, 578: 481–490.PubMed CentralView ArticlePubMedGoogle Scholar
- Gover TD, Moreira TH, Kao JP, Weinreich D: Calcium homeostasis in trigeminal ganglion cell bodies. Cell Calcium 2007, 41: 389–396. 10.1016/j.ceca.2006.08.014View ArticlePubMedGoogle Scholar
- Valant C, Robert Lane J, Sexton PM, Christopoulos A: The best of both worlds? Bitopic orthosteric/allosteric ligands of g protein-coupled receptors. Annu Rev Pharmacol Toxicol 2012, 52: 153–178. 10.1146/annurev-pharmtox-010611-134514View ArticlePubMedGoogle Scholar
- Rudolph U, Mohler H: Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annu Rev Pharmacol Toxicol 2004, 44: 475–498. 10.1146/annurev.pharmtox.44.101802.121429View ArticlePubMedGoogle Scholar
- Rudolph U, Knoflach F: Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes. Nat Rev Drug Discov 2011, 10: 685–697. 10.1038/nrd3502PubMed CentralView ArticlePubMedGoogle Scholar
- Saari TI, Uusi-Oukari M, Ahonen J, Olkkola KT: Enhancement of GABAergic activity: neuropharmacological effects of benzodiazepines and therapeutic use in anesthesiology. Pharmacol Rev 2011, 63: 243–267. 10.1124/pr.110.002717View ArticlePubMedGoogle Scholar
- Brusberg M, Ravnefjord A, Martinsson R, Larsson H, Martinez V, Lindstrom E: The GABA(B) receptor agonist, baclofen, and the positive allosteric modulator, CGP7930, inhibit visceral pain-related responses to colorectal distension in rats. Neuropharmacology 2009, 56: 362–367. 10.1016/j.neuropharm.2008.09.006View ArticlePubMedGoogle Scholar
- Lee CH, Zhu C, Malysz J, Campbell T, Shaughnessy T, Honore P, Polakowski J, Gopalakrishnan M: alpha4beta2 neuronal nicotinic receptor positive allosteric modulation: an approach for improving the therapeutic index of alpha4beta2 nAChR agonists in pain. Biochem Pharmacol 2011, 82: 959–966. 10.1016/j.bcp.2011.06.044View ArticlePubMedGoogle Scholar
- Marin JC, Goadsby PJ: Glutamatergic fine tuning with ADX-10059: a novel therapeutic approach for migraine? Expert opinion on investigational drugs 2010, 19: 555–561. 10.1517/13543781003691832View ArticlePubMedGoogle Scholar
- Jerman JC, Gray J, Brough SJ, Ooi L, Owen D, Davis JB, Smart D: Comparison of effects of anandamide at recombinant and endogenous rat vanilloid receptors. Br J Anaesth 2002, 89: 882–887. 10.1093/bja/aef281View ArticlePubMedGoogle Scholar
- Smart D, Jonsson KO, Vandevoorde S, Lambert DM, Fowler CJ: ‘Entourage’ effects of N-acyl ethanolamine’s at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism. Br J Pharmacol 2002, 136: 452–458. 10.1038/sj.bjp.0704732PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.