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Anesthesiology:
doi: 10.1097/ALN.0b013e318195b51d
Pain Medicine

Clonidine and Dexmedetomidine Produce Antinociceptive Synergy in Mouse Spinal Cord

Fairbanks, Carolyn A. Ph.D.*; Kitto, Kelley F.†; Nguyen, H Oanh B.S.‡; Stone, Laura S. Ph.D.§; Wilcox, George L. Ph.D.∥

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Abstract

Background: Synergy between drugs manifests with increased potency and/or efficacy of the combination relative to either agonist given alone. Synergy is typically observed between drugs of different classes, as is the case with the α-adrenergic–opioid receptor synergy often observed in preclinical studies. However, rare studies report synergy between agonists of the same class. The current study examined the analgesic interaction between two intrathecally injected α2-adrenergic receptor (AR) agonists previously thought to act at the same receptor subtype when given spinally.
Methods: Mice were given clonidine, dexmedetomidine, or the combination spinally to evaluate the interaction between these two agonists. The ED50 values were calculated, and the interactions were tested by isobolographic analysis. The rotarod test was performed in the same mice after the completion of analgesic assessment to assess motor or sedative effects. These experiments were performed in outbred mice as well as in mice with mutant α2AARs, α2CAR knockout mice, or wild-type controls. Finally, analgesic cross-tolerance between clonidine and dexmedetomidine was evaluated.
Results: Clonidine and dexmedetomidine interacted synergistically in all lines except the α2CAR knockout line, implicating α2CARs in the interaction. In addition, clonidine and dexmedetomidine did not show analgesic cross-tolerance in the outbred strain, suggesting that the two drugs have distinct mechanisms of action.
Conclusions: The current study introduces a new synergistic agonist pair, clonidine–dexmedetomidine. These two drugs seem to require the α2AAR for spinal analgesia when given separately; when delivered as a combination, the resultant synergistic interaction requires the α2CAR as well.
SYNERGISTIC drug interactions result in enhanced potency and/or efficacy when one agent is given together with another. Therapeutic application of synergistic combinations carries the expectation of efficacy at reduced doses and, theoretically, reduced side effects. Although the mechanisms underlying synergistic interactions are not well understood, synergy is thought to result from simultaneous action of the two agents at two distinct sites, such as a common receptor located at disparate anatomical sites or distinct receptors coresiding at a common anatomical location. Examples of well-described synergistic agonist pairs include selective agonists of the μ- and δ-opioid receptor subtypes as well as either of those subtypes combined with agonists targeting the α2-adrenergic receptors (ARs).
The analgesic and anesthetic properties of α2AR-selective agonists have been known for decades. Development of clinical applications of these agonists remains an area of interest, particularly as adjuvants for pain management and as anesthetic-sparing agents.1 In contrast to the opioid receptor–selective agonists, definition of the pharmacologic profile of each α2AR agonist has been limited because of poor ligand selectivity across the three α2AR subtypes: α2AAR, α2BAR, and α2CAR.2 The α2AR subtypes are differentially expressed in specific regions of the central nervous system. For example, in the spinal cord, α2AARs seem to be principally of primary afferent neuron origin, whereas α2CARs seem to be expressed primarily on neurons intrinsic to the spinal cord.3 The evidence for α2BAR expression in spinal cord nerve terminals and intrinsic spinal neurons is not conclusive. Activation of both α2AARs4,5 and α2CARs6 has been reported to result in antinociception. Therefore, it is reasonable to propose that concurrent participation of α2AARs and α2CARs could result in analgesic synergy. Support for a positive interaction between α2AARs4,5 and α2CARs is provided in a previous report that evaluated interactions between two α2-adrenergic agonists7 that were thought to act at different α2AR subtypes based on differences in the pharmacology of their antagonist sensitivity. To approach this question systematically, we have initiated a broad evaluation of several α2AR agonist combinations in mouse lines deficient in α2AAR or α2CAR function. As part of this larger program, the current study evaluated the interaction between intrathecally administered clonidine and dexmedetomidine. Previous studies of α2AAR mutant mice have been interpreted to indicate that the potency and/or efficacy of both of these agonists is primarily dependent on α2AAR activation, particularly when administered intrathecally. Because of this prevailing view, we did not expect that coadministration of clonidine with dexmedetomidine would result in a synergistic analgesic interaction. Our observations indicate, however, that this combination produces definitive and replicable synergistic analgesia in several separate strains of mice: CD-1 Institute of Cancer Research (ICR) outbred mice, mice deficient in the α2AAR or the α2CAR subtype, and their wild-type (WT) controls. Further, the potential for cross-tolerance between the agonists was assessed after chronic intrathecal delivery of either agonist. Finally, the interaction between clonidine and dexmedetomidine on a measure of sedation and motor coordination (accelerating rotarod) was also evaluated.
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Methods and Materials

Animals
Experimental subjects were 20- to 25-g male ICR mice (Harlan, Madison, WI) or 15- to 20-g male and female mice (sex matched) with either a mixed C57BL/6-129/Sv genetic background (α2AAR-WT or α2AAR-D79N) or a pure C57BL/6 background (α2CAR-WT or α2CAR-knockout [KO]). Animals were maintained on a 12-h light/dark cycle and had unlimited access to food and water. The α2AAR-D79N mutant mice had been generated by hit-and-run gene targeting as previously described8 on a hybrid C57BL/6-129/Sv background. WT animals of the same mixed background were used as controls (α2AAR-WT). The α2CAR-KO mice were developed at Stanford University, Palo Alto, California,9 and were purchased from Jackson Labs, Bar Harbor, Maine, after 17 generations of backcrossing to C57BL/6 background. C57BL/6 mice pair-bred within our facility were used as WT controls (α2CAR-WT). Breeding pairs were established, and pups were weaned between 2 and 3 weeks of age. Within each experiment, animals were age- and sex-matched across groups. Animals were used no more than twice. In each case, a rest period of at least 1 week was used, and the animals were randomized across treatment groups. Although the use of transgenic or KO mice may result in compensatory changes, we chose to use these mouse lines because we have extensively characterized their spinal neuropharmacology,4–6 and they have been widely used by other groups with interest in α2AR-mediated antinociception and antihypertensive effects (for review, see Kable et al.10). Therefore, the results presented in this study are directly comparable to the previous literature. These experiments were approved by the Institutional Animal Care and Use Committee of the University of Minnesota, Minneapolis, Minnesota. Subjects were housed in groups of four in 25 × 48 × 15-cm plastic cages in a temperature- and humidity-controlled environment, were maintained on a 12-h light/dark cycle, and had free access to food and water.
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Chemicals
Clonidine HCl (2-[2,6-dichloroaniline]-2-imidazoline) and substance P (SP) were purchased from Sigma Chemical Co. (St. Louis, MO). SP was dissolved in acidified saline. Zeneca (Wilmington, DE) donated the dexmedetomidine [1-(S)-4-[1-(2,3-dimethylphenyl) ethyl]1H- imidazole]. Clonidine and dexmedetomidine were dissolved in 0.9% saline. All drugs were administered intrathecally by direct lumbar puncture in a 5-μl volume in conscious mice.11
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Nociceptive Assay
Equation (Uncited)
Equation (Uncited)
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Nociceptive responsiveness was tested in the SP nociceptive test. The SP assay is a sensitive indicator of milder analgesics.12 SP (10–20 ng) was injected intrathecally to produce approximately 40–60 behaviors (scratches and bites directed to the hindquarters) in the first minute after injection. The dose of SP required to produce this number of behaviors was confirmed with each new experiment. Coadministration of opioid or adrenergic analgesics dose-dependently inhibits those behaviors.13 To test the ability of dexmedetomidine and clonidine to inhibit SP-induced behavior, the drugs were coadministered with SP and inhibition was expressed as a percent of the mean response of the control group (determined with each new experiment) according to the following equation:
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Sedation/Motor Impairment Assay
In the same mice that received SP stimulation, doses of clonidine, dexmedetomidine, and their combination were tested for impairment of rotarod performance. In such experiments, the animals were trained the day before experimentation to walk 300 s on the accelerating rotarod, typically requiring three trials to learn the behavior. The following day, the drugs were administered with SP. After completion of the 1-min SP-evoked scratching and biting analysis, the mice were run on the rotarod test.
Equation (Uncited)
Equation (Uncited)
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Motor impairment or sedation was expressed as inhibition of the subjects’ ability to remain on the accelerating rotarod; baseline latencies to fall were typically at or near the cutoff of 300 s. Percent inhibition was expressed as a percent of the baseline latency of each mouse (determined prior to each new experiment) according to the following equation:
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Dose–Response Analysis
Individual dose and/or time points are expressed as mean and SEM. ED50 values and confidence limits were calculated according to the graded dose–response method of Tallarida and Murray14 on the linear portion of each dose–response curve. Statistical comparisons of potencies are based on the confidence limits of the ED50 values. A minimum of three doses were used for each drug or combination of drugs. A minimum of 50% was set for a drug to be classified as efficacious.
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Isobolographic Analysis
Fig. 1
Fig. 1
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Table 1
Table 1
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Dose–response curves were constructed for each agonist administered alone; the ED50 values were calculated and used to determine the potency ratio between the agonists (e.g., fig. 1A). This ratio was then maintained when both agonists were administered in combination, a third dose–response curve was constructed, and an experimentally derived combination ED50 was calculated. To test for interactions between agonists, the ED50 values and SE for all dose–response curves were arithmetically arranged around the ED50 value using the equation (ln10 × ED50) × (SE of log ED50).15 Isobolographic analysis (the standard for the evaluation of drug interactions14,15) necessitates this manipulation. When testing an interaction between two drugs, a theoretical additive ED50 value is calculated for the combination based on the dose–response curves of each drug administered separately. This theoretical value is then compared by a t test with the observed experimental ED50 value of the combination. These values are based on the total dose of both drugs. An interaction is considered synergistic if the experimental ED50 is significantly less (P < 0.05) than the calculated theoretical additive ED50 value.
Visualization of drug interactions can be facilitated and enhanced by graphical representation of isobolographic analysis. This representation depicts the ED50 of each agent as the x- or y-intercept. For example, figure 1B presents the ED50 of clonidine as the y-intercept and the ED50 of dexmedetomidine as the x-intercept. The line connecting these two points depicts the dose combinations expected to yield 50% efficacy if the interaction is purely additive and is called the theoretical additive line. The theoretical additive ED50 and its confidence interval are determined mathematically and plotted spanning this line. The observed ED50 for the combination is plotted at the corresponding x,y coordinates along with its 95% confidence interval for comparison to the theoretical additive ED50. All dose–response and isobolographic analyses were performed with the FlashCalc 4.5.3 pharmacologic statistics software package16,17 generously supplied by Michael Ossipov, Ph.D. (Professor, University of Arizona, Tucson, Arizona).
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Chronic Clonidine or Dexmedetomidine Tolerance Induction
To induce spinal clonidine or dexmedetomidine tolerance, clonidine or dexmedetomidine (10 nmol in 5 μl) was delivered intrathecally once on experimental day 1 and twice daily on experimental days 2 and 3. Repeated injections were separated by at least 8 h. A separate group of mice received an equal number of injections of saline as a control group. On experimental day 4, full dose–response curves were constructed for each agonist in each pretreatment group. The antinociceptive potencies (ED50 values) of clonidine and dexmedetomidine to inhibit SP-evoked behaviors were compared between mice pretreated with saline or clonidine or dexmedetomidine.
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Results

Clonidine–Dexmedetomidine Analgesic Synergy
Clonidine Produces Analgesic Synergy with Dexmedetomidine in ICR Mice.
We first determined the potency of each agonist to inhibit SP-evoked behavior in ICR mice. As expected, clonidine and dexmedetomidine inhibited the behavior with comparable potency and efficacy (fig. 1A). The calculated ED50 values of these dose–response curves formed the basis for the equieffective dose ratios used in the respective combinations (table 1). Coadministration of clonidine with dexmedetomidine resulted in combination dose–response curves shifted approximately 700-fold to the left compared with each agonist given separately (fig. 1 and table 1). The isobologram in figure 1B illustrates that the ED50 value of the observed combination differs significantly from the calculated theoretical additive ED50 value, indicating a synergistic interaction (fig. 1B and table 1; Student t test, P < 0.05). This experiment was replicated in a separate group of mice with comparable outcomes (synergism). The robust synergistic interaction of the clonidine–dexmedetomidine combination suggests a second spinal site of action for one of the two agonists.
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Clonidine–Dexmedetomidine Coadministration in α2AAR-WT Mice.
Fig. 2
Fig. 2
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The objectives for testing the clonidine–dexmedetomidine combination in α2AAR-WT and α2AAR-D79N mice were (1) to determine whether the synergistic interaction was observable across mouse strains and (2) to determine whether the combination demonstrated any efficacy in mice lacking α2AAR. Because clonidine consistently demonstrates no efficacy in α2AAR-D79N mice and dexmedetomidine is only efficacious at high doses, the expectation was that the combination would not yield significant efficacy in those mice; nonetheless, it was important to test the possibility that the combination resulted in a significantly different pharmacologic profile than either agonist alone. We first determined the potency of each agonist to inhibit SP-evoked behavior in α2AAR-WT mice. As expected, clonidine and dexmedetomidine inhibited the behavior with comparable potency and efficacy (fig. 2A). Coadministration of clonidine with dexmedetomidine resulted in combination dose–response curves shifted approximately sevenfold to the left compared with each agonist given separately (fig. 2A and table 1). The isobologram in figure 2B illustrates that the ED50 value of the observed combination differs significantly from the calculated theoretical additive ED50 value, indicating a synergistic interaction (fig. 2B and table 1; Student t test, P < 0.05). The synergistic interaction of the clonidine–dexmedetomidine combination in α2AAR-WT mice confirms that the observation was not unique to ICR mice. Although the magnitude of synergism is significantly different (100-fold) across these two strains, the observation of significant synergy for this combination is consistent. This difference also profiles the importance of evaluating combinations across multiple strains. Consistent with our previous reports, neither clonidine nor dexmedetomidine demonstrates antinociceptive efficacy in the α2AAR-D79N mice when given either alone or as a 1:1 combination, even at relatively high doses (10, 30, and 100 nmol, intrathecally; fig. 2C).
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Mechanism of Clonidine–Dexmedetomidine Analgesic Synergism
Clonidine and Dexmedetomidine Do Not Evoke Chronic Analgesic Cross-Tolerance.
Fig. 3
Fig. 3
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Table 2
Table 2
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The observation of synergy between clonidine and dexmedetomidine suggests that a receptor other than the α2AAR is involved in the interaction. In situations where two agonists act primarily at the same receptor, chronic administration of one agonist usually elicits cross-tolerance to the other.18 Conversely, in cases where two agonists act at different receptor sites, chronic exposure to one agonist typically does not invoke chronic tolerance to the other (e.g., μ-opioid receptor (MOP), δ-opioid receptor (DOP)19,20), although minor cross-tolerance is sometimes observed, perhaps because of changes in convergent downstream signaling pathways (e.g., MOP–α2AAR20–22). Therefore, to evaluate whether clonidine and dexmedetomidine may act on the same or different receptors, we conducted an evaluation of analgesic tolerance to clonidine or dexmedetomidine after repeated chronic exposure to spinally administered clonidine (fig. 3A) or dexmedetomidine (fig. 3B) in ICR mice. Whereas 3-day spinal pretreatment with clonidine significantly reduced the potency of “probe” doses of clonidine (16-fold tolerance), the analgesic dose–response curve for dexmedetomidine remained largely unchanged (fig. 3A and table 2). Similarly, 3-day spinal pretreatment with dexmedetomidine significantly reduced the potency of “probe” doses of dexmedetomidine (21-fold tolerance), but the analgesic response to clonidine was not significantly altered (fig. 3B and table 2). This lack of cross-tolerance suggests that, despite their apparent shared reliance on spinal α2AARs when given separately, clonidine-evoked or dexmedetomidine-evoked antinociception requires participation of a second distinct receptor.
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Clonidine Produces Analgesic Synergy with Dexmedetomidine in C57Bl/6 but Not α2CAR-KO Mice.
Fig. 4
Fig. 4
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A logical candidate for the second receptor is the α2CAR, given its localization in spinal cord and previous studies illustrating that α2CAR activation can result in antinociception.6,23 Therefore, we tested for clonidine–dexmedetomidine synergy in α2CAR-KO mice and their WT controls (C57BL/6 mice). The clonidine–dexmedetomidine combination demonstrated significant analgesic synergy in α2CAR-WT mice (figs. 4A and B and table 1), as was the case in ICR and α2AAR-WT mice. In contrast to the lack of efficacy observed in the α2AAR-D79N mice (fig. 2C), the analgesic potency of clonidine and dexmedetomidine decreased only twofold to threefold (though significantly) in α2CAR-KO mice relative to that in α2CAR-WT mice. These data indicate that, when given separately, neither agonist demonstrates an absolute requirement for the α2CAR (in contrast to that seen in α2AAR mutant mice), but that the α2CAR may participate in the full antinociceptive potential of the two agonists. However, despite this moderate KO effect on the individual dose–response curves of clonidine and dexmedetomidine, the synergistic interaction of their combination was clearly absent in the α2CAR-KO mice (figs 4C and D and table 1). The potency of the clonidine–dexmedetomidine combination was not altered relative to that of either agonist given alone; the combination ED50 value was significantly higher than that of the theoretical additive ED50 value. This result suggests that the clonidine–dexmedetomidine synergistic interaction requires the presence of α2CARs and that in the absence of α2CARs the two drugs may act at the same receptor, presumably the α2AAR.
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Clonidine–Dexmedetomidine Interactions in Other Assays
Clonidine–Dexmedetomidine Interactions in the Rotarod Assay of Sedation and Motor Impairment.
Fig. 5
Fig. 5
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In addition to their analgesic effects, α2AR agonists affect multiple physiologic systems, including the central nervous system (sedation, cardiovascular effects, addiction and withdrawal responses). In the current study, the rotarod test, which has been previously used as a measure of both sedation and motor impairment,24 was used to assess the sedative and/or motoric effects of the agonists or their combination immediately after SP nociceptive testing. In outbred ICR mice (fig. 5A), clonidine and dexmedetomidine each produced a mild reduction in rotarod performance at the highest dose tested (10 nmol); higher doses were not tested. The clonidine–dexmedetomidine combination reduced rotarod performance only 30% at the highest combination dose (1 nmol of each drug) tested, which produced approximately 90% antinociception (fig. 1A); potentiation was evident at 0.01 and 1 nmol. We distinguish this interaction in rotarod from the synergistic analgesic interaction by referring to the former as potentiation. In α2AAR-WT mice (fig. 5B), clonidine reduced rotarod performance approximately 70% at the highest dose tested (10 nmol), whereas dexmedetomidine produced only partial reduction (approximately 50%). The combination showed a moderate (< 10-fold) but significant increase in the potency of each agonist when coadministered, the interaction of which was statistically synergistic (isobole not shown). In α2CAR-WT mice (fig. 5C), both clonidine and dexmedetomidine inhibited rotarod performance at approximately 10-fold lower potency relative to inhibition of SP behavior. Further, the clonidine–dexmedetomidine combination demonstrates substantially increased potency (approximately 100-fold relative to each given alone) for reduction of rotarod performance. Isobolographic analysis confirmed a significant synergistic interaction (isobole not shown). Figure 5D reflects a minimal effect (< 25%) in α2AAR-D79N mutant mice (consistent with the lack of analgesic effect); figure 5E also shows moderate (< 50%) rotarod impairment in α2CAR-KO mice. In summary, using the rotarod assay as a model of sedation and/or motor impairment, the clonidine–dexmedetomidine combination resulted in differential pharmacologic outcomes across the three lines of mice tested in terms of relative potency and efficacy. Specifically, whereas the combination significantly impaired rotarod performance in the C57Bl/6 line, it impaired motor performance only moderately in the ICR line; the effect in the α2AAR-WT line was intermediate. These results that differ across mouse lines contrast with the concurrent antinociceptive measures in that the antinociceptive potency and efficacy and synergism were consistent (albeit of differing magnitude) across all WT mouse lines. Further analysis of other potential side effects of the combination in mice and other species will be needed to determine the utility of the clonidine–dexmedetomidine combination in pain management or anesthesia.
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Discussion

The current study reveals that two spinally active α2-adrenergic analgesics, clonidine and dexmedetomidine, interact synergistically in the production of antinociception in mice. These two agonists have previously been thought to act primarily on α2AARs to exert their various physiologic effects.10,25 Because clonidine requires α2AAR5 and the analgesic potency of dexmedetomidine is dramatically reduced in mice in the absence of functional α2AARs,4 the observation of synergism was an unexpected and novel finding. Upon further investigation in the current study, the participation of a second target, likely to be α2CARs, has become apparent. The concept of α2CAR as a synergistic partner with α2AARs is supported by previous anatomical3 and pharmacologic7 evidence.
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Synergistic Analgesic Pairs
Historically, synergistic analgesic partners have implicated the activation of two distinct receptors or receptor subtypes. Opioid receptor pairs with synergistic interactions include MOP–DOP and MOP–KOP26; both pairings involve agonists acting at separate receptor subtypes in the same G protein–coupled receptor family (opioid). Others have demonstrated synergy between agonists that activate receptors in different G protein–coupled receptor families: Examples include MOP and α2AR agonists,27,28 DOP and α2AAR agonists,4,13 and DOP and α2CAR agonists.4 Studies evaluating interactions between agonists acting on the same opioid receptor subtype have reported only additive interactions.29
One previous report studied the interactions between two α2AR agonists, dexmedetomidine and ST91.7 The rationale for assessing that combination for synergy derived from observations that, whereas dexmedetomidine had been largely thought to activate α2AAR, ST91 seemed to be independent of α2AARs. These assertions did not derive from binding studies because the affinities of these ligands do not differ appreciably among α2AR subtypes. Rather, the proposed selectivity was derived from pharmacologic studies using antagonists with differential affinity for the three receptor subtypes.30,31 The selectivity of dexmedetomidine was subsequently validated by studies using genetically altered mice,32 but ST91 did not show substantial dependence on either α2A or α2CARs in genetically altered mice.32 However, the observation that synergy was detected between dexmedetomidine and ST91 is consistent with the participation of two distinct receptor subtypes. The distinct localizations of α2AAR (thought to be restricted to the spinal terminals of primary afferent neurons) and α2CAR (thought to be restricted to spinal neurons)3 in spinal cord positions this pair to operate in such a synergistic manner.
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Clonidine–Dexmedetomidine Analgesic Synergy
The total lack of clonidine efficacy in α2AAR functional knockout mice suggested that clonidine acts only at α2AARs to produce antinociception.5 Although the potency of dexmedetomidine was dramatically reduced in the same mice, dexmedetomidine retained analgesic efficacy, albeit at thousandfold higher doses.4 This distinction between clonidine and dexmedetomidine leaves open the possibility that the latter acts on another AR, such as the α2CAR. It is also conceivable that clonidine acts on α2CAR with an effect below the threshold of detection in our nociceptive assay. These possibilities in turn suggested that clonidine–dexmedetomidine synergy may result from the participation of both α2AARs and α2CARs. Two experimental tests of this hypothesis yielded concurrent results. First, cross-tolerance did not occur between clonidine and dexmedetomidine, indicating that the two agonists act at different receptors when given as a combination. Second, clonidine–dexmedetomidine synergy was not observed in α2CAR-KO mice but did occur in WT mice. Therefore, whereas clonidine and dexmedetomidine given separately by the intrathecal route seem to rely primarily on activation of α2AAR, their spinal synergistic interaction requires the recruitment of α2CAR as well. Activity at both receptors is consistent with competition binding studies, where both agonists bind with comparable affinity to both receptors.2,33 However, competition binding studies are incongruent with functional assays (e.g., guanosine 5′-O-[γ-thio]triphosphate binding) in transfected cell lines where dexmedetomidine has shown a rank order preference for α2BAR > α2CAR > α2AAR and clonidine was a partial agonist at α2BAR > α2AAR and inactive at α2CAR.34 It is clear that in vitro binding or functional studies may not model the in vivo condition adequately. Furthermore, the participation of α2CAR may not be at the level of direct agonist–receptor interaction but rather could represent an indirect contribution within a more complex pathway. The current study indicates that the efficacy of single agonists delivered spinally may not adequately predict the efficacy, potency, or mechanism of combined agonists given spinally.
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Interaction Studies of Sedation and Motor Impairment
Assessing the analgesic utility of the clonidine–dexmedetomidine combination warrants determination of the effects of the combination on at least one non–analgesic-dependent measure. Accordingly, sedation and motor impairment were assessed using the accelerating rotarod test immediately after antinociceptive testing. Unlike the antinociceptive measure, the sedative efficacy of the agonists and their combination varied across the strains studied. The individual agonists produced moderate (< 50%) sedation in the outbred ICR strain, intermediate effects in the α2AAR-WT (mixed strain: C57BL/6-129sv), and pronounced sedation in the α2CAR-WT (inbred strain: C57BL/6). Interestingly, the individual agonists produced minimal sedation in the two mutant lines of mice, α2AAR-D79N and α2CAR-KO, indicating that both receptor subtypes contribute to the sedative effects. The clonidine–dexmedetomidine combination showed a small sedative effect at lower doses in ICR mice, synergistic sedation in both α2AAR-WT and α2CAR-WT mice, and minimal to no sedation in both α2AAR-D79N and α2CAR-KO mice. Interestingly, a previous study of α2AAR-D79N heterozygous mice revealed a clear difference between the antihypertensive and sedative effects of dexmedetomidine; dexmedetomidine’s cardiovascular effects were fully manifest in heterozygous α2AAR-D79N mice, whereas its sedative effect was absent.35 The authors attributed this difference in response to a different receptor occupancy requirement for decreasing blood pressure versus sedation. They postulated that partial α2AAR agonists might provide that separation of effect in WT mice, and in fact observed a similar separation of effects in WT mice with the partial agonist moxonidine. Conceivably, the separation of analgesia and sedation in the outbred ICR strain results from a similar partial agonist character of the clonidine–dexmedetomidine combination. Dexmedetomidine is considered a full agonist at both α2A and α2CARs, whereas clonidine is considered a partial agonist at both.2 We speculate that the relation between receptor occupancy and sedation could be a strain-dependent effect and account for the difference in sedative effects in the strains studied; however, further testing is required to address this hypothesis. Further study is needed to refine the combination to optimize clinical outcomes for either analgesia with moderate sedation or improved sedative/anesthetic efficacy, depending on the target therapeutic application.
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Clinical Relevance
Clinical application of interdrug synergy between G protein–coupled receptor agonists carries the potential for reduced dose and side effect profiles of drug combinations compared with the drugs given alone. There is an expectation that the dose reduction enabled by a synergistic interaction might reduce side effects. The utility of clonidine as a monotherapy36–41 or combined with spinal opioids42,43 and/or local anesthetics has been studied for decades.44,45 Although the primary clinical use of dexmedetomidine has been as a sedative and anesthetic agent,46,47 the combination of intrathecal dexmedetomidine with bupivacaine has recently been shown to be effective for analgesic control, comparing favorably with the combination of intrathecal clonidine and bupivacaine.48 Further, a recent case report documents the use of intrathecal dexmedetomidine combined with morphine to restore analgesic control in a morphine-tolerant cancer patient.49 Therefore, both clonidine and dexmedetomidine produce antinociception when given intrathecally both in animal models4,5,50–52 and humans.36,48,49
However, before clinical application of any single agent or novel combination of spinal analgesics, the conduct of preclinical animal neurotoxicity studies53 and controlled clinical trials to establish safety of the singly delivered agents54 and the synergistic combinations (a requisite separate study from that of the singly delivered agents)55 is imperative.56 The importance of neurotoxicity evaluation of potential neuraxial therapeutics cannot be overemphasized.57,58 Whereas the safety profile of intrathecally delivered clonidine has been previously established,53 the neurotoxicity of intrathecally delivered dexmedetomidine is largely unknown. A recent evaluation59 of toxicity of epidurally delivered commercial dexmedetomidine formulation in rabbits found white matter demyelination in the spinal cord, potentially attributable to the pH (4.5–7.0) of the current formulation. For the novel combination of clonidine–dexmedetomidine to be considered useful for application, substantially more work would be needed.54 A further consideration is that the anatomical organization of α2ARs subtypes, although well defined in rodents, has not been evaluated in human spinal cord. Differences between species in receptor subtype expression pattern in the spinal cord could ultimately account for differences in agonist combination interactions. Isobolographic analysis of a combination (fentanyl–clonidine) well established to be synergistic in rodents did not demonstrate statistically significant synergism in one clinical evaluation42; the reason for the difference between rodents and humans is not clear. Regardless of these considerations, the current study reveals an unexpected interaction between two α2AR agonists and suggests further evaluation of other α2AR agonists as potentially useful synergistic partners.
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Conclusion

Application of interdrug synergy between G protein–coupled receptor agonists carries the potential for reduced dose and side effect profiles of drug combinations compared with the drugs given alone. The potential of such positive interactions encourages the continued search for novel useful combinations. The opportunities of therapeutic application of α2AR agonists either as single agents or as combinations (particularly with opioids and local anesthetics) continues to expand with recent clinical studies.1 In the current study, spinally coadministered clonidine and dexmedetomidine demonstrated a replicable and consistent synergistic interaction that was not predicted by previous pharmacologic studies of the agonists in genetic KO mice. The application of isobolographic analysis to this unexpected combination in genetic KO mice revealed an interaction between α2AAR and α2CAR that would be otherwise difficult to identify.60 Therefore, the combination of these two agonists or other coactivators of this α2A–α2CAR pair may have utility in pain management and sedative anesthesia.
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References

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