Abstract
GR79236 is a highly potent and selective adenosine A1 receptor agonist that has analgesic and anti-inflammatory actions in humans and animals. In animal models it inhibits trigeminal nerve firing and calcitonin gene-related peptide release which play a pivotal role in migraine pathophysiology. Thus GR79236 may have therapeutic potential in migraine. Although there are no validated human models of migraine, the trigeminal nociceptive pathways may be studied with a novel electrode to elicit nociception-specific blink reflex responses. Twelve healthy female volunteers were randomized in a double-blind, placebo-controlled, crossover trial to investigate the effect of GR79236 on trigeminal nociceptive pathways, as measured by the blink reflex. A secondary objective was to compare the use of two types of electrode, the standard (SE) and nociception-specific electrodes (NE), to investigate human trigeminal pharmacology. Blink reflexes were elicited with SE and NE before and 30 min after GR79236 (10 μg/kg i.v.) or placebo. The median area under the curve of repeated sweeps of the R2 component of the blink reflex was analysed using analysis of covariance with baseline as covariate. Using NE, GR79236 produced a non-significant reduction of the ipsilateral R2 compared with placebo (P = 0.097) and a significant reduction contralaterally (P = 0.008). No significant changes were observed using SE. There were no significant adverse events. The results suggest that NE is more sensitive than SE to detect pharmacological effects in the trigeminal nociceptive system. Furthermore, the adenosine A1 receptor agonist GR79236 inhibits trigeminal nociception in humans. These results support a possible therapeutic role for GR79236 in primary headache disorders.
Introduction
Treatment of migraine has been revolutionized by the discovery and development of selective 5-HT1B/D agonists, the triptans. However, the search continues for novel therapeutic agents that do not share the vasoconstrictor properties of triptans (1). 5-HT1B/D receptors transduce their effects via G-proteins of the Gi type. Agonists at other receptors that also activate Gi proteins may therefore have therapeutic potential in migraine without the peripheral vasoconstrictor action of triptans. One such receptor is the adenosine A1 receptor.
Adenosine and its agonists induce analgesia in humans and antinociception in animal models (2–4). For example, low-dose adenosine intravenous infusions reduce postoperative pain (5) and have pain-relieving properties in neuropathic pain (6). Adenosine acts via three known receptor types: A1, A2 and A3. The A1 receptor mediates antinociception, whereas the A2 and A3 receptors mediate pro-nociception. The A1 receptor mediates pre- and post-synaptic antinociceptive actions. Presynaptically, it inhibits cAMP to inhibit release of calcitonin gene-related peptide (CGRP), substance P and probably excitatory amino acids such as glutamate. Post-synaptically it produces hyperpolarization and thus inhibits the action of substance P and excitatory amino acids (7).
GR79236 (N-[(1S, trans)-2-hydroxycyclopentyl] adenosine) is a highly potent and selective A1-receptor agonist (8) that was originally developed for the treatment of Type 2 diabetes mellitus, as a cardioprotective agent and also for peripheral arterial occlusive diseases. It has also been shown to have antinociceptive and anti-inflammatory properties in animal models (9). In humans the compound has been investigated clinically for dental surgery pain (10). Evidence to support a potential role in the treatment of primary headache disorders has been gained from established animal models of trigeminal nociception. GR79236 inhibits the release of CGRP evoked by superior sagittal sinus (SSS) stimulation in the cat (11) and inhibits trigeminal nucleus firing in the cat (11) and rat (12). After intravenous administration it is widely distributed in all tissues except the central nervous system, and has a terminal half-life of 1–2 h.
Although animal models of trigeminal nociception have been successful in predicting anti-migraine effects, until recently there have been no good human models with which to study potential treatments. The recent development of a blink reflex electrode that specifically stimulates nociception-specific fibres (NE) (13) has allowed the non-invasive study of nociceptive transmission in the spinal trigeminal nucleus caudalis (TNC). Activation of TNC neurones is believed to play a critical role in central sensitization (14) and neurovascular positive feedback loop of migraine (15).
The aim of the present study was two-fold: first, to assess the response of GR79236 on human trigeminal nociception as measured by the blink reflex, and second, to compare the use of the standard electrode (SE) and NE in assessing changes in trigeminal pharmacology.
Methods
The study was approved by the Independent Ethics Committee of the Glaxo Wellcome Clinical Pharmacology Unit at Northwick Park Hospital, Harrow. Healthy female volunteers (n = 26) aged between 20 and 36 years were seen at a screening visit. All subjects gave informed consent. Twelve subjects with consistent blink reflex responses were recruited into the study. Individual pain sensation thresholds with NE were determined at the screening visit. None had a personal history of migraine (16) and all were using reliable methods of contraception. Subjects were excluded who had a history of cardiovascular or cerebrovascular disease, hypertension, Raynaud's syndrome, epilepsy, diabetes, impaired hepatic or renal function, alcohol or drug abuse, hypersensitivity to 5-HT1 agonists, heavy smoking, obesity, hepatitis B or C, or if taking any regular medication other than the oral contraceptive pill. Since pain perception and blink reflex activity may change through different phases of the menstrual cycle, we did not examine the volunteers in any one particular menstrual phase.
Stimulation and recording
All recordings were carried out between 8 and 11.30 a.m., by the same examiners, under identical conditions and using the same verbal instructions on each occasion in order to minimize any variation in the level of subject arousal. Blink reflexes were elicited with both SE and NE: SE is a commercial parallel assembly with two stimulation surfaces 25 mm apart, NE is a custom-built planar concentric electrode. The following stimulus parameters were used: monopolar square wave, duration 0.3 ms, interstimulus interval 12–18 s (pseudo-randomized); current intensity for nociception-specific stimulation 1.5 times individual pain threshold (mean 1.43 ± 0.06 mA), 25 sweeps; for standard stimulation 12–15 mA, six sweeps. EMG recordings were obtained from bilateral surface electrodes placed infraorbitally (different) and at the root of the nose (indifferent), acquisition bandwidth 1 Hz to 1 kHz; digitization: sampling rate 2.5 kHz, sweep length 150 ms. PC-based off-line analysis was performed with custom written software. The method is described in detail elsewhere (13).
Study design
In a randomized, double blind, cross-over design patients received either placebo or GR79236 with a 1-week wash-out period between treatments. GR79236 10 µg/kg or placebo was administered intravenously over 15 min with intermittent blood pressure and heart rate monitoring for 24 h after the start of dosing and continuous telemetric ECG monitoring. Blink reflexes were elicited with NE and SE at baseline and 30 min after the start of each drug dosing. Stimulating electrodes were positioned over the left supraorbital nerve.
Statistical analysis
The area under the curve (AUC) of the R2 component of both types of blink reflex was calculated from the rectified EMG. The median AUC at 30 min post start of drug dosing was modelled using analysis of covariance with subject, period and treatment as factors in the model, and the baseline median AUC as a dynamic covariate. Statistics were computed using SAS software package. The level of significance was set at P < 0.05.
Results
All 12 subjects completed both arms of the study.
Nociception-specific electrode blink reflex
Comparison of the two treatment groups demonstrated a greater reduction of the R2 response after GR79236 compared with placebo. Ipsilaterally there was a mean difference of 17% (AUC −567 µVms (95% confidence interval (CI) −1260 to 125.8)), P = 0.097 (Fig. 1). Contralaterally the mean difference was 20% (AUC −473 µVms (95% CI −786.6 to −158.7)), P = 0.008.
Reduction of R2 AUC elicited by nociceptive-specific electrode after GR79236 vs. placebo, mean difference and 95% confidence intervals (∗P < 0.05).
Standard electrode blink reflex
There was no significant difference (2.4%) of ipsilateral R2 response after GR79236 compared with placebo when blink reflexes were elicited with the SE (mean AUC reduction −152 µVms (95% CI −2038 to 1733)) (Fig. 2).
Reduction of R2 AUC elicited by SE after GR79236 vs. placebo, mean difference and 95% confidence interval. No significant effect was seen.
Adverse events
There were no significant adverse events after GR79236 or placebo. The electrodes were well tolerated. There were no significant changes in systolic or diastolic blood pressure or mean heart rate with GR79236 vs. placebo at any time point measured up to 24 h post dosing. There was an increase (compared with baseline) in the mean QTc interval at 5 min after GR79236 of 10.2 ± 23.4 ms, with a maximum recorded value of 476 ms (normal range < 460 ms). Likewise, there was a non-clinically significant increase (compared with baseline) in mean PR interval at 15 min of 13.8 ± 10.3 ms, but all recorded values for the 24 h were> 200 ms. No cardiac arrythmias were detected.
Discussion
The reduction of the bilateral R2 response elicited with nociception-specific electrode after the adenosine A1 receptor agonist GR79236 suggests that this compound inhibits trigeminal nociceptive pathways in humans. This supports the evidence for trigeminal nociceptive inhibition demonstrated in animal models where GR79236 attenuates trigeminal neurone firing in the TNC (11, 12) and suggests A1 receptor agonists may have acute anti-migraine actions.
The TNC is thought to play a pivotal role in the pathophysiology of primary headache disorders. In animal models of trigeminovascular nociception, stimulation of the superior sagittal sinus evokes trigeminal neuronal activation in the TNC (17, 18). The TNC has receptors that bind compounds with specific anti-migraine action; notably [3H]-dihydroergotamine in cat (19), and triptans in the cat and humans (20, 21). Additionally, the same anti-migraine drugs can inhibit activity of these TNC neurones (20, 22, 23) at clinically relevant doses. Adenosine A1-receptor agonists including GR79236 elicit a similar dose-dependent block of SSS-evoked trigeminal firing in animal models (11). The potential clinical relevance of these findings is highlighted by the localization of adenosine A1-receptor protein in human trigeminal ganglia (24).
Adenosine receptor agonists are effective in preclinical antinociceptive tests when administered systemically, intrathecally or centrally (7). Use of A1-receptor selective agonists and antagonists suggests that the A1 receptor is the predominant receptor subtype mediating these effects. A1-receptor agonists are highly effective against experimental inflammatory and neuropathic pain. For example, GR79236 causes a rapid reversal of sciatic ligature-induced allodynia (25) and inhibits inflammatory hyperalgesia (9) in rats. Adenosine itself has been reported to be effective following intravenous infusion in patients with peripheral neuropathic pain (6) and following intrathecal administration to neuropathic pain patients (7).
The blink reflex is a trigeminofacial brain stem reflex. Afferent fibres are activated by stimulation of the supraorbital nerve, a branch of the first division of the trigeminal nerve (V1). Efferent fibres pass through the facial nerve and the motor response can be quantified by surface EMG electrodes placed infraorbitally over the orbicularis oculi muscles. Three EMG components can be distinguished when the blink reflex is elicited with a SE: an oligosynaptic ipsilateral R1 (onset latency 11 ms), a polysynaptic bilateral medullary R2 (onset latency 33 ms), and a less well-defined R3. The R1 is mediated by non-nociceptive neurones only but the R2 is mediated by both nociceptive and non-nociceptive neurones. By contrast, the novel nociception-specific electrode (NE), by virtue of its concentric design, selectively depolarizes superficial nociceptive-specific fibres at low current intensities without recruiting deeper non-nociceptive (Aβ) fibres deeper in the skin. Thus, the NE blink reflex has no R1 component and the R2 nociceptive subcomponent response is abolished by topical lignocaine that anaesthetizes the superficial nociceptive (Aδ and C) fibres but not the deeper Aβ fibres (26). This technique may therefore have potential to assess trigeminal nociception in man non-invasively. In a recent study it was demonstrated that NE blink reflex responses are facilitated during acute migraine, suggesting central sensitization, an effect not detected with SE (27). Further studies should assess the effect of drugs known to be effective in migraine, such as the triptans, on the nociceptive blink reflex. Other trigemino-facial reflexes have been used to explore the action of centrally acting drugs, including the effect of triptans on the R3 component of the blink reflex in migraine (28), and that of opiates and benzodiazepines on the corneal reflex and the blink reflex (29), although the clinical relevance of these studies may be questioned as they did not specifically address the nociceptive trigeminal pathways.
Since NE directly depolarizes the trigeminal sensory axons, rather than stimulation via peripheral nociceptive receptors, the inhibitory effect of GR79236 on the blink reflex elicited with NE suggests that the compound must act directly at second-order trigeminal neurones in the TNC or indirectly via more rostral brain stem structures modulating trigeminal nociceptive pathways. This is in accordance with animal data of a reduction of trigeminal nerve firing with GR79236, reflecting a central mechanism of action. GR79236 may also act on peripheral terminals of the trigeminal nerve by inhibition of CGRP release (11). This effect could not have been observed in the present study because of direct neuronal depolarization by the electrode. Both central and peripheral effects are in keeping with the concept of prejunctional inhibitory adenosine A1 receptors being located on primary afferent neurones and causing inhibition of transmitter release.
The study was carried out only on female volunteers, as migraine is a predominately female condition and we wished to keep the study population as homogeneous as possible.
The inhibitory effect of GR79236 only reached statistical significance on the contralateral R2 blink reflex response but not ipsilaterally. This was due to the lower variation seen in the contralateral response (within subject SD = 340 µVms) compared with the ipsilateral response (within subject SD = 625 µVms). It is likely that a larger study would be required to demonstrate statistical significance for the ipsilateral response. The sample size in the current study was based on a pilot study of 16 men and women (age 22–42 years) with two sets of blink reflexes elicited with the NE 60 min apart: it was found that to detect a difference of 368 µVms (equivalent to a 20% change from baseline values) a sample size of 12 would be required for a power of 81% (α 0.05, two-tailed).
There are various factors that may affect the variability of the nociceptive blink reflex response. It is critically dependent on level of arousal of the subject and was controlled for as far as possible by performing the experiments under identical conditions. However, further studies should address methodological improvements such as varying the inter-stimulus interval, using differing stimulus intervals and changing the stimulation site to maintain arousal and reduce habituation. Circadian changes at the level of the brain stem may be important in the nociception-specific blink reflex, and to control for this we performed the tests at the same time of day.
The moderate size of the effect shown in this study is probably dose dependent. GR79236 was used at 10 µg/kg. A greater effect on trigeminal nociception may have been achieved by higher doses. In animal studies doses up to 100 µg/kg have shown dose-dependant inhibition (up to 80% reduction) of the probability of trigeminal nerve firing after SSS stimulation. However, these higher doses of A1-receptor agonists may cause bradycardia and adverse CNS effects in animals (30). Nevertheless, these effects are modest and only found with high doses. Prolongation of the PR and QTc intervals is a class effect of adenosine A1-receptor agonists and may limit higher doses (> 20 µg/kg) being used in humans. No significant changes in the ECG parameters or blood pressure were seen in the present study. Furthermore, GR79236 (10 µg/kg i.v.) has no effect on resting meningeal artery diameter in rats (31). This is in contrast to the effects of triptans which cause cranial vasoconstriction in a range of species, including cat and humans, in addition to inhibiting trigeminal nerve firing. Further clinical studies of GR79236 in migraine will show whether this narrow therapeutic window limits the clinical effectiveness of the drug.
Only the nociceptive-specific electrode but not the standard electrode showed a change in the blink reflex response with GR79236. Thus the NE seems to be a sensitive, specific and therefore a more useful tool to investigate trigeminal nociceptive pharmacology than SE.
The present study demonstrates that the nociception-specific electrode is non-invasive, well tolerated, can be applied repeatedly over time and can be used to study trigeminal nociceptive pharmacology in both healthy volunteers and headache patients. Additionally, the current study adds to the body of evidence that adenosine A1-receptor agonists, such as GR79236, inhibit trigeminal nociceptive pathways in humans as well as in animals. By inhibiting trigeminal nociception, adenosine A1-receptor agonists may be effective as novel therapeutic agents in migraine and other primary headache disorders.
Footnotes
Acknowledgements
The authors thank Paul Hammond for his technical assistance, Melanie Talbot for advice on statistics, and Professor Pat Humphrey (GIAP, Cambridge) and Dr Malcolm Thomas (ex-Director of Clinical Pharmacology at GlaxoSmithKline) for many stimulating discussions concerning this study.