Pain is one of the most common reasons for patients to seek medical advice, although modern pharmacological treatments are limited. Selective activation of peripheral receptors has the important advantage of providing effective analgesia without side effects typically associated with centrally acting drugs. However, in clinical practice most pain treatment strategies are based on systemic administration of conventional centrally penetrating substances. It is well established that stimulation of central opioid receptors results in analgesia by modulating nociceptive information; however, both opioid receptors and endogenous opioids can also be widely detected throughout the peripheral nervous system and in peripheral tissues1,2 Typically, the application of conventional opioid receptor agonists in small, systemically inactive doses directly into injured peripheral tissues or the administration of opioids with limited access to the central nervous system has been used efficiently in animal and human studies as well.2–6 Furthermore, centrally penetrating mu-, delta-, and kappa-receptor agonists, when administered systemically, produce a considerable part of antinociception through peripheral opioid receptors.5 It is also well known that the organism can express very effective antinociception in different circumstances by releasing various endogenous ligands. These substances have potentially advantageous features, and their synthesizing and breakdown enzymes are available in the body; therefore, they have short half-lives and lower toxicity.7 On the other hand, some of the endogenous ligands exhibit lower specificity and affinity for their receptors compared with exogenous drugs, and/or they exert their effects at several types of receptors in different parts of the body. Therefore, the net effect depends on the localization of the ligands/receptors, and on which receptors, and where they will be influenced by a ligand. Accordingly, their efficacy might significantly differ from synthetic drugs. With the discovery of the two endogenous tetrapeptides, endomorphin-1 (EM1) and endomorphin-2, highly specific μ-opioid receptor agonists have been identified.8 It is an open question whether EMs are real endogenous opioid neurotransmitters/modulators, because their synthesis has not been clarified. However, several studies have identified EMs in the different parts of the organism, and their metabolizing enzymes have also been shown. Furthermore, some data suggest that EMs can be synthesized from dipeptides and not from a large propeptide.9 A huge amount of data proved the antinociceptive potential of these tetrapeptides at both spinal and supraspinal levels.10 The antinociceptive effects of endogenous ligands at the peripheral level are not well known. Only some data suggest that endogenous opioids can produce effective antinociception peripherally,6 and even fewer data are available on endomorphins.11,12 Some data suggest the role of EM1 in the control of inflammatory processes at the joint level13–17; however, data are not available on its antinociceptive potential in this model. Therefore, the first goal was to determine the dose-dependent antinociceptive potency of intraarticularly administered EM1 in the carrageenan-induced inflammatory pain model.
Glutamate is a major excitatory amino acid neurotransmitter acting on metabotropic and ionotropic glutamate receptors. Several studies indicate that glutamate and its ionotropic receptors, including the N-methyl-d-Aspartate (NMDA) receptors, play a role in peripheral nociceptive transmission.18 Thus, both animal and human studies suggest that NMDA antagonists produce antinociception after local administration,19–21 whereas others found them to be ineffective.22 Kynurenic acid (KYNA) is an endogenous excitatory amino acid antagonist with preferential activity at the NMDA receptors and is also a noncompetitive antagonist at alpha7 nicotinic receptor (nAChR).23,24 It is produced endogenously both centrally and peripherally.25 Our earlier data have revealed that intrathecally administered KYNA produced antinociception, but the effective dose caused motor impairment as well.26 Only a few studies suggest the role of KYNA in the periphery. Thus, it has been detected in synovial fluid collected from knee joints of rheumatoid arthritic patients, and it inhibited the proliferation of synoviocytes in vitro.27 The second goal of this study was to determine the effect of intraarticularly administered KYNA on inflammation-induced allodynia.
A good possibility for overcoming the problems of endogenous ligands (lower specificity and affinity and plateau effect) is to use their combinations,28,29 and several studies proved synergistic interactions between drugs acting at different receptors.30,31 The interaction of endogenous ligands at the peripheral level is largely unknown. Spinally coadministered EM1 and KYNA produced enhanced antinociception,26 and their coadministration might also be beneficial at the peripheral level. Therefore, the third goal of our study was to examine the interaction of these ligands at the joint level.
After institutional ethical approval had been obtained (Institutional Animal Care Committee of the Faculty of Medicine at the University of Szeged), male Wistar rats (Charles River strain, Bioplan, Budapest, Hungary; 247 ± 2.2 g; n = 7-12/group) were housed in groups of 5-6 per cage, with free access to food and water, and with a natural light/dark cycle. Animals suffering and the number of animals per group were kept at a minimum.
The following drugs were administered: λ-carrageenan, EM-1 (formula weight: FW = 611), KYNA (FW = 189), and naltrexone (NTX; FW = 378) (Sigma-Aldrich, Budapest, Hungary). Carrageenan, EM1, and NTX were dissolved in physiological saline, whereas KYNA was dissolved in 0.1 M NaOH. The excess NAOH was back titrated with 0.1 M HCl to a neutral pH, and the volume was adjusted with physiological saline. The solutions were freshly prepared on the day of experiment. Physiological saline was used as control.
Inflammation was produced by injecting carrageenan (300 μg/20 μL) into the tibiotarsal joint of the right hind leg as previously described.32 Briefly, all treatments were given to gently restrained conscious animals, using a 27-gauge needle, without anesthesia so as to exclude any drug interaction. These injections did not elicit any sign of major distress.
To determine the changes in the size of the inflamed joint, we measured the anteroposterior and mediolateral diameter of the paw at the level of ankle joint with a digital caliper. The cross-section area was calculated with the formula a × b × π, where a and b are the radius in the two aspects.
Behavioral Nociceptive Testing
The threshold for withdrawal from mechanical stimulation to the plantar aspect of the hindpaws was determined with a logarithmic series of calibrated von Frey monofilaments (SenseLab—Aesthesiometer, Somedic, Sweden). Before baseline testing, each rat was habituated to a testing box with a wire-mesh grid floor for at least 15 min. Von Frey filaments (bending force ranging from 0.064 to 110 g) were applied in ascending order using a single, steady 1-2 s application perpendicularly through the grid floor to the plantar surface of the right hindpaw of each rat until a paw withdrawal occurred.33 The lowest force producing a withdrawal response was considered the threshold. Only robust and immediate withdrawal responses from the stimulus were considered.
After baseline determination of joint diameter and mechanical paw withdrawal threshold (precarrageenan baseline value at −180 min), carrageenan was injected. These measurements were obtained again 3 h after carrageenan injection (postcarrageenan baseline values at 0 min). EM1 (30, 100, and 200 μg; 50, 150, and 300 nmol), KYNA (30, 100, 200, and 400 μg; 160, 530, and 1060 nmol), and their combinations in a fixed-dose ratio: 1:1 (30-30, 100-100, and 200-200 μg) were given into the inflamed joint (20 μL), and mechanical sensitivity was defined at 10, 20, 30, 45, 60, and 75 min after the drug administrations. The control group received physiological saline. At the end of the experiment, the joint diameters were measured again. To reveal the role of opioid receptor activation by EM1, a group of animals was pretreated with NTX (a well-known antagonist on μ-opioid receptors; 4 mg/kg subcutaneously) 20 min before 200-μg EM-1 administration.
Data are presented as means ± sem. Paw withdrawal latencies on the inflamed side were transformed to % maximum possible effect (%MPE) by using the following formula:
%MPE = ([observed force − postcarrageenan baseline force]/[precarrageenan baseline force − postcarrageenan baseline force]) × 100.
Therefore, 100% MPE means perfect relief of allodynia (equivalent to precarrageenan baseline value, which generally means the maximum value 110 g), whereas 0% MPE means that the observed force is equivalent to the postcarrageenan baseline value. Thus, these two baseline values are not shown in the figures. Because treatments generally produced their effects between 30 and 60 min, their mean values on the inflamed side were used for dose-effect curves and linear regression analysis (a common method for the determination of dose-response effects in in vivo studies). The 50% effective dose (ED50) was defined as the dose that yielded 50% MPE. Because a lower level of the effect might also be important for therapeutic practice, we also determined 30% effective dose (ED30), which means about a 10 times increase in the pain threshold compared with the postcarrageenan baseline value. The ED30 and ED50 values with 95% confidence intervals (CIs) were calculated by linear regression. Data sets were examined by one-way and two-way analyses of variance (ANOVA). The significance of differences between experimental and control values was calculated using the Fisher’s LSD test for post hoc comparison (P value <0.05 was considered significant).
Three hours after the injection of carrageenan into the right ankle, there was a significant (P < 0.01) increase in joint cross-section compared with preinjection control levels (from 46 ± 0.7 mm2 to 82 ± 0.9 mm2). This conspicuous increase in joint diameter was a result of edema formation, confirming that carrageenan treatment resulted in an inflammatory reaction. None of the treatments influenced the degree of edema (data are not shown).
The basal mechanical withdrawal threshold was 105 ± 1.0 g and 107 ± 0.8 g for the left and right sides, respectively, i.e., about 90% of the animals did not withdraw their paws at the cutoff value. Carrageenan caused a significant decrease in the paw withdrawal threshold on the inflamed side (0.36 ± 0.054 g), but it did not have a significant influence on the noninflamed side (106 ± 1.0 g). None of the treatments changed the mechanosensitivity on the normal side; therefore, results were analyzed only on the inflamed paws.
EM1 produced dose-dependent antinociceptive effect, which developed relatively slowly (Fig. 1A). ANOVA with repeated measurements showed significant effects of treatment (F3,30 = 6.9, P < 0.005), time (F5,150 = 8.5, P < 0.001), and interaction (F15,150 = 5.4, P < 0.001). Thus, 30-μg EM1 was ineffective, whereas 200 μg caused a prolonged effect, which was about 80% MPE at the 30th and 45th min, leading to nearly perfect relief of allodynia. The ED30 and ED50 values were 112 μg (CI: 80-146) and 167 μg (CI: 135-220), respectively. NTX pretreatment alone did not influence the pain threshold (data are not shown) but prevented the antiallodynic effect of EM1 (200 μg) (Fig. 1A).
KYNA by itself also caused a dose-dependent antiallodynic effect, which developed 30 min after the injection. Only the highest dose produced prolonged antinociception and almost total relief of allodynia (Fig. 1B). ANOVA proved significant effects of treatment (F4,40 = 16.1, P < 0.001), time (F5,200 = 10.5, P < 0.001), and interaction (F20,200 = 3.9, P < 0.001). Its potency was lower compared with EM, i.e., the ED30 and ED50 values were 204 μg (CI: 160-251) and 330 μg (CI: 280-407), respectively.
Regarding the interaction of these ligands, coadministration of 30-30 μg EM1 and KYNA did not produce any antiallodynic effect (Fig. 2A). Concerning the coadministration of 100-100 μg, ANOVA revealed significant effects of treatment (F3,34 = 4.2, P < 0.05), time (F5,170 = 9.4, P < 0.001), and interaction (F15,170 = 2.4, P < 0.005). Post hoc comparison revealed that this combination produced an increased antinociception at some time points compared with vehicle, KYNA, and EM1 (Fig. 2B). EM + KYNA 200-200 μg produced long lasting antinociception compared with the single treatment (Fig. 2C). ANOVA showed significant effects of treatment (F3,31 = 11.6, P < 0.001), time (F5,155 = 14.0, P < 0.001), and interaction (F15,155 = 4.4, P < 0.001).
Because the ratio of the ED50 values of EM/KYNA was about 2, the doses of the combinations were calculated in this proportion.34 The dose-response curve of the cocktail is between the EM and KYNA lines (Fig. 3). The ED30 and ED50 values were 141 μg [CI: 83-182] and 231 μg [CI: 190-293], respectively, which did not differ significantly from the theoretically additive values (ED30 and ED50 145 μg [CI: 68-237] and 220 μg [CI: 144-230], respectively).35
We did not examine or quantify motor behavior systematically, but the animals’ behaviors were observed, and there were no signs of altered behavior immobility, flaccidity, excitation, or motor weakness). Therefore, in contrast to intrathecally administered KYNA, the local administration of this ligand did not produce local anesthetic effects.26
This study has shown that the intraarticularly administered endogenous μ-opioid receptor agonist EM1 and the NMDA receptor antagonist KYNA dose-dependently decreased mechanical allodynia without signs of systemic side effects. The coadministration of these ligands produced additive interaction, thus a decreased dose of each drug can lead to effective antinociception. Furthermore, we did not find any changes on the normal side. Therefore, we may exclude systemic antinociceptive effects of these ligands.
Locally released opioid peptides at the site of injury inhibit the inflammatory response and reduce the pain associated with it.6 Only a few studies supported the beneficial effects of EM1 at the peripheral level. Thus, intraplantar administration of 20-160-μg EM1 dose-dependently decreased neuropathic pain,11 whereas Labuz et al.12 found that EM1 was effective in lower doses (0.3-1.25 μg) in the inflammatory pain model. The differences in the potency might be due to the alterations in the pain models and/or the applied pain tests (von Frey versus paw pressure test). However, morphine also has much lower potency peripherally than at the spinal level.36 Furthermore, the effect of EM1 might have been decreased by endopeptidases, because synovial fibroblasts are a rich source of these enzymes.37–39 The effects of EM1 developed relatively slowly compared with other studies,11,12,16 which might have been due to the differences in the routes of administration, the pain models, and/or the investigated variables (vascular reactivity versus pain threshold). Regarding the effects of EM1 on joint pain, one study has detected decreased afferent nerve activity in response to noxious hyperrotation of the joint in anesthetized rats after EM1 administration.14 EM1 (60 μg) applied intraarterially close to the knee joint, caused up to a 75% reduction in joint afferent nerve activity, but its effect was lost during chronic inflammation. Considering the action mechanism of EM1, it is suggested that the activation of μ-opioid receptors by EM1 (because it was reversible by μ-opioid antagonist) can inhibit the release of pain-producing substances (e.g., Substance P) from primary sensory neurons.40
Regarding the action mechanism of KYNA, this study does not provide direct evidence for specific receptor involvement in mediating the efficacy of KYNA, but data from receptor binding studies performed in other laboratories do allow us to suggest potential receptor mechanisms, which may be involved. Several peripheral NMDA receptors also play a significant role in sensory processing at the peripheral level.18,41 The possibility that glutamate may be released by neuronal endings in the inflamed knee joint has been demonstrated in rats, and its contribution to the hyperalgesic events initiated during the development of joint inflammation has strongly been supported through the activation of NMDA on primary sensory neurons.41,42 Because KYNA produces its effects mainly by the inhibition of NMDA receptors, we therefore suppose that primarily this effect might have led to its effectivity in this model. However, KYNAs antinociceptive potency and the side effects could not have been predicted from the earlier results. We found that KYNA had lower potency compared with EM1, but in contrast to our former results at the spinal level,26 the intraarticular administration of KYNA did not produce any motor impairment.
The inhibition of alpha7 nAChRs by KYNA also could have a role in its effects.25 It has been found that nAChRs play a role in modulating pain transmission both centrally and peripherally; however, the results are controversial.43,44 Stimulation of neuronal nAChR excites or sensitizes peripheral sensory nerve fibers, but it has also been reported to mediate cholinergic antinociception.45,46 There is controversy about the localization of alpha7 nAChR at the periphery as well, because Haberberger et al.47 found these receptors on all nociceptive neurons, whereas Lang et al.48 could not detect it. Although the deficiency in this receptor did not influence pain sensitivity,49 one study suggests that activation of alpha7 nAChR may elicit antinociceptive effects with a peripheral mechanism in an inflammatory pain model.50 Therefore, the results of inhibition of alpha7 nAChR are uncertain in the periphery, but it might be involved in the effects of KYNA. A study has shown that GPR35, a previously orphan G-protein coupled receptor (GPR), functions as a receptor for the KYNA.51 KYNA elicits calcium mobilization and IP3 production in a GPR35-dependent manner in the presence of Gqi/o chimeric G proteins and also induces the internalization of GPR35. Expression analysis indicates that GPR35 is predominantly detected in immune cells and in the gastrointestinal tract, but it has also been found in the dorsal root ganglion on small-diameter to medium-diameter neurons.52 The results suggest that GPR35 may modulate nociception, and continued study of this receptor will provide additional insight into the role of KYNA in pain perception. However, no in vivo data are available regarding the role of GPR35 in the effects of KYNA, and until now no specific antagonists have been developed. Regardless of the mechanism of action, the results clearly show that KYNA has antihyperalgesic potency at the peripheral level. Therefore, KYNA can influence several systems, which might be involved in the effects of KYNA on pain threshold.
An important technique used to decrease the side effects is the use of a combination of several drugs in low doses that produce the same therapeutic effects as a single drug administered in a higher dose. In this respect, there is a growing body of experimental data, which indicates that NMDA antagonists enhance the analgesic effect of opiates and may block or reduce the development of tolerance after long-term opiate administration.53–57 Controversially, there have been some reports that the NMDA antagonist MK-801 neither has an antinociceptive effect of its own nor does it alter that of morphine,53 whereas other data indicate a simple additive interaction of MK-801 with morphine in the carrageenan-induced inflammatory pain model.58 Some studies have investigated the interactions of EM-1 with different drugs to improve its efficacy at the central level.59–62 Our earlier studies suggest that the antinociceptive effect of EM-1 is enhanced by S(+)-ketamine and KYNA at the spinal level,26,63 whereas this study showed that the type of the interaction at the peripheral level was additive. Because both opioid and glutamate receptors are present on the primary sensory neurons at the periphery, the coactivation and antagonism of these receptors could have a beneficial effect on the inhibition of pain sensation at doses which do not cause side effects. The explanation for the additive interaction at the periphery might be the differences in the number and/or density of these receptors compared with the spinal level; however, there are no data supporting this theory. Because the ratio of the administered drugs can also influence the type of interactions,35 another ratio of these drugs may produce another type of interaction.
The acute administration of EM1 and/or KYNA did not influence the degree of edema in our study. However, it cannot be excluded that their administration would produce not only antinociception but also an antiinflammatory effect, and this may contribute to their antinociceptive effects.13,27,64 Thus, opioid receptors have been demonstrated on primary sensory neurons and immune cells,6,40 and locally released opioid peptides (including EM1) inhibit the inflammatory response at the site of injury.6,13,15 Similarly, KYNA can also control the inflammatory process by inhibition of the proliferation of synoviocytes.27
The reported data indicate that EM1 is an effective antinociceptive drug after intraarticular administration. Similar to other NMDA receptor antagonists, KYNA has a limited therapeutic range and may therefore not be particularly useful alone as an analgesic for the treatment of clinical pain. The interaction of the endogenous substances, which act at different receptor types, might be beneficial in attenuating carrageenan-induced mechanical allodynia. These results suggest an important direction for the development of pain strategies that focus on the coadministration of different endogenous ligands at the peripheral level.
The authors thank Agnes Tandari and Zita Petrovszki for technical assistance.
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