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1.  Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities 
Bioorganic & medicinal chemistry letters  2013;23(17):10.1016/j.bmcl.2013.06.065.
The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr1-DAla2-Gly3-Phe4-Gly5-Trp6-O-[3′,5′-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.
doi:10.1016/j.bmcl.2013.06.065
PMCID: PMC3810412  PMID: 23899615
bifunctional compounds; opioid receptor agonists; neutokinin-1 receptor antagonists; Truncation of peptide sequence; NMR structure
2.  Synthesis and biological evaluation of new opioid agonist and neurokinin-1 antagonist bivalent ligands 
Bioorganic & medicinal chemistry  2011;19(20):6135-6142.
Newly designed bivalent ligands—opioid agonist/NK1-antagonists have been synthesized. The synthesis of new starting materials—carboxy-derivatives of Fentanyl (1a–1c) was developed. These products have been transformed to ‘isoimidium perchlorates’ (2a–c). The new isoimidium perchlorates have been successfully implemented in nucleophilic addition reactions, with L-tryptophan 3,5-bis(trifluoromethyl)benzyl ester to give the target compounds—amides (3a–c). Perchlorates (2a–c) successfully undergo reactions with other nucleophiles such as alcohols, amines or hydrazines. The obtained compound 3b exhibited μ-opioid agonist activity and NK1-antagonist activity and may serve as a useful lead compound for the further design of a new series of opioid agonist/NK1-antagonist compounds.
doi:10.1016/j.bmc.2011.08.027
PMCID: PMC4137774  PMID: 21925887
Analgesic; Bivalent ligands; μ-Opioids; NK1 antagonist; Fentanyl
3.  Descending Facilitation Maintains Long-term Spontaneous Neuropathic Pain 
Neuropathic pain is frequently characterized by spontaneous pain (i.e. pain at rest) and in some cases, cold and touch-induced allodynia. Mechanisms underlying the chronicity of neuropathic pain are not well understood. Rats received spinal nerve ligation (SNL) and were monitored for tactile and thermal thresholds. While heat hypersensitivity returned to baseline levels within approximately 35-40 days tactile hypersensitivity was still present at 580 days after SNL. Tactile hypersensitivity at post-SNL day 60 (D60) was reversed by microinjection of (a) lidocaine or (b) a CCK2 receptor antagonist into the rostral ventromedial medulla (RVM) or (c) dorsolateral funiculus (DLF) lesion. RVM lidocaine at D60 or spinal ondansetron, a 5HT3 antagonist, at post-SNL day 42 produced conditioned place preference (CPP) selectively in SNL treated rats, suggesting long-lasting spontaneous pain. Touch-induced FOS was increased in the spinal dorsal horn of SNL rats at D60 and prevented by prior DLF lesion suggesting that long-lasting tactile hypersensitivity depends upon spinal sensitization, which is mediated in part, by descending facilitation, in spite of resolution of heat hypersensitivity.
Perspective
These data suggest that spontaneous pain is present for an extended period of time and, consistent with likely actions of clinically effective drugs, is maintained by descending facilitation.
doi:10.1016/j.jpain.2013.02.011
PMCID: PMC4028636  PMID: 23602267
nerve injury; neuropathic pain; spontaneous pain; central sensitization; RVM; spinal cord
4.  Discovery of Amphipathic Dynorphin A Analogues to Inhibit the Neuroexcitatory Effects of Dynorphin A through Bradykinin Receptors in the Spinal Cord 
We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but allowed us to discover a potential neuroexcitatory target. Well known BR ligands, BK, DALKD, and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg7]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn A’s biological activity, is not necessary, and that a type I β-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naïve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.
doi:10.1021/ja501677q
PMCID: PMC4021566  PMID: 24742335
pathological chronic pain states; hyperalgesia; dynorphin A; bradykinin receptor recognition; non-opioid; amphipathic pharmacophore
5.  Chiral Effect of a Phe Residue in Position 3 of the Dmt1-l(or d)-Tic2 Analogues on Opioid Functional Activities 
ACS Medicinal Chemistry Letters  2013;4(7):656-659.
In this letter, we describe a structure–activity relationships study, specifically related to the chirality of third amino acid residue in our H-Dmt-l(or d)-Tic analogues, of which C-terminus is attached to a piperidinyl moiety. Observed selectivities and functional activities of these analogues demonstrated that the chiralities of the second and third position residues are crucial for determining whether these ligands act as antagonists or agonists at the δ opioid receptor, but not at the μ opioid receptor.
doi:10.1021/ml400115n
PMCID: PMC3956057  PMID: 24648867
Dmt-Tic; opioid functional activities; structure−activity relationship; δ opioid receptor; chirality
6.  Capturing the aversive state of cephalic pain preclinically 
Annals of neurology  2013;74(2):257-265.
Objective
Preclinical evaluation of headache by behavioral assessment of reward from pain relief.
Methods
Inflammatory mediators (IM) or control solutions were applied to the rat dura mater to elicit a presumed state of cephalic pain. Hindpaw incision was used in separate groups of animals to model non- cephalic post-surgical pain. Drugs were given systemically or microinjected within the rostral ventromedial medulla (RVM), nucleus accumbens (NAc) or rostral anterior cingulate cortex (rACC). Peripheral nerve block (PNB) was produced at the level of the popliteal fossa and behavior was assessed using evoked sensory stimuli or conditioned place preference (CPP). Immunohistochemistry and brain microdialysis measurements were performed.
Results
Dural IM produced long-lasting generalized cutaneous allodynia (CA). RVM lidocaine produced CPP, increased NAc c-FOS and dopamine (DA) release selectively in rats receiving dural IM; CPP was blocked by intra-NAc α-flupenthixol, a dopaminergic antagonist. Intravenous αCGRP(8-37) produced CPP and elicited NAc dopamine release selectively in rats with dural IM. Prior lesion of the rACC or treatment with systemic sumatriptan, or αCGRP(8-37) abolished RVM lidocaine-induced CPP in IM-treated rats. Sumatriptan treatment blocked NAc dopamine release in IM treated rats receiving RVM lidocaine. Systemic sumatriptan did not alter pain-relief induced CPP in rats with incisional injury.
Interpretation
Cephalic pain was unmasked in rats by assessment of motivated behavior to seek relief. Relief of pain activates the dopaminergic reward pathway to elicit negative reinforcement of behavior. Medications clinically effective for migraine headache selectively elicit relief of ongoing cephalic, but not post-surgical, non-cephalic pain. These studies provide a platform for exploring migraine pathophysiology and for the discovery of new headache therapies.
doi:10.1002/ana.23922
PMCID: PMC3830648  PMID: 23686557
7.  Reversal of Inflammatory and Non-Inflammatory Visceral Pain by Central or Peripheral Actions of Sumatriptan 
Gastroenterology  2008;135(4):1369-1378.
Background and Aims
Sumatriptan is used specifically to relieve headache pain. The possible efficacy of sumatriptan was investigated in two models of visceral pain.
Methods
Pancreatic inflammation was induced by intravenous injection of dibutyltin dichloride. Non-inflammatory irritable bowel syndrome was induced by intracolonic instillation of sodium butyrate. The effects of systemic sumatriptan on referred hypersensitivity were tested in both models. Effects of sumatriptan within the rostral ventromedial medulla (RVM), a site of descending modulation of visceral pain, was determined by (a) testing the effects of RVM administration of 5HT1B/D antagonists on systemic sumatriptan action and (b) determining whether RVM application of sumatriptan reproduced the actions of systemic drug administration.
Results
Systemic sumatriptan elicited a dose- and time-related blockade of referred hypersensitivity in both models that was blocked by systemic administration of either 5HT1B or 5HT1D antagonists. Sumatriptan administered into the RVM similarly produced dose- and time-related blockade of referred hypersensitivity in both visceral pain models. This was blocked by local microinjection of the 5HT1B antagonist, but not the 5HT1D antagonist. Microinjection of 5HT1B or 5HT1D antagonists into the RVM did not block the effects of systemic sumatriptan.
Conclusions
Our findings suggest that sumatriptan suppresses either inflammatory or non-inflammatory visceral pain, most likely through peripheral 5HT1B/1D receptors. Actions at 5HT1B receptors within the RVM offer an additional potential site of action for the modulation of visceral pain by triptans. These studies offer new insights into the development of strategies which may improve therapy of visceral pain conditions using already available medications.
doi:10.1053/j.gastro.2008.06.085
PMCID: PMC4028637  PMID: 18694754
8.  Opioid-induced latent sensitization in a model of non-inflammatory viscerosomatic hypersensitivity 
Brain research  2010;1358:64-70.
Exposure to opioids can induce a state of “latent sensitization” characterized by long-lasting enhanced responses to subsequent cutaneous injury. Here, we explored the possibility that prior treatment with morphine could induce a state of latent sensitization to visceral pain conditions. Following butyrate enemas to induce non-inflammatory visceral pain, acute morphine administration produced dose-related inhibition of referred viscerosomatic hypersensitivity. Treatment with morphine for a period of six days resulted in a persistent hyperalgesia that resolved many days after termination of drug administration. In morphine pre-exposed rats, butyrate-induced referred hypersensitivity was enhanced and extended in duration. No differences were observed in the morphine dose-response curve in suppression of acute nociception (i.e., the hot-plate assay) when morphine pre-exposed rats were compared to naïve rats indicating that opioid antinociceptive tolerance was not present. However, the morphine dose-response curve to suppress evoked viscerosomaticl hypersensitivity was displaced to the right by approximately 4-fold in morphine pre-exposed rats. Induction of viscerosomativ hypersensitivity resulted in an increased labeling of CGRP-, but not substance P-positive cells in the lumbar dorsal root ganglia; increased labeling was not affected by prior exposure to morphine. The data indicate that a period of morphine exposure can induce a state of “latent-sensitization” to subsequent visceral pain characterized by extended duration of hypersensitivity. This condition likely reflects enhanced visceral “pain” intensity as a consequence of persistent pronociceptive adaptive changes.
doi:10.1016/j.brainres.2010.08.032
PMCID: PMC4028661  PMID: 20727859
IBS; opioid-induced latent sensitization
9.  Nerve Injury-induced Tactile Allodynia Is Present In The Absence Of FOS Labeling In Retrogradelly Labeled Post-Synaptic Dorsal Column Neurons 
Pain  2006;129(0):143-154.
The dorsal column pathway consists of direct projections from primary afferents and of ascending fibers of the post-synaptic dorsal column (PSDC) cells. This pathway mediates touch but may also mediate allodynia after nerve injury. The role of PSDC neurons in nerve injury-induced mechanical allodynia is unknown. Repetitive gentle, tactile stimulus or noxious pinch was applied to the ipsilateral hindpaw of rats with spinal nerve ligation (SNL) or sham surgery that had previously received tetramethylrhodamine dextran in the ipsilateral n. gracilis. Both touch and noxious stimuli produced marked increases in FOS expression in other cells throughout all laminae of the ipsilateral dorsal horn after nerve injury. However, virtually none of the identified PSDC cells expressed FOS immunofluorescence in response to repetitive touch or pinch in either the nerve-injured or sham groups. In contrast, labeled PSDC cells expressed FOS in response to ureter ligation and labeled spinothalamic tract (STT) cells expressed FOS in response to noxious pinch. Identified PSDC neurons from either sham-operated or SNL rats did not express immunoreactivity to substance P, CGRP, NPY, PKCg, MOR, the NK1 and the NPY-Y1 receptor. Retrogradely labeled DRG cells of nerve injured rats were large diameter neurons, which expressed NPY, but no detectable CGRP or substance P. Spinal nerve injury sensitizes neurons in the spinal dorsal horn to repetitive light touch but PSDC neurons apparently do not participate in touch-evoked allodynia. Sensitization of these non-PSDC neurons may result in activation of projections integral to the spinal/supraspinal processing of enhanced pain states and of descending facilitation, thus priming the central nervous system to interpret tactile stimuli as being aversive.
doi:10.1016/j.pain.2006.10.009
PMCID: PMC4028680  PMID: 17156921
PSDC cells; spinal cord; neuropathic pain; FOS expression
10.  Evaluation of reward from pain relief 
The human experience of pain is multidimensional and comprises sensory, affective, and cognitive dimensions. Preclinical assessment of pain has been largely focused on the sensory features that contribute to nociception. The affective (aversive) qualities of pain are clinically significant but have received relatively less mechanistic investigation in preclinical models. Recently, operant behaviors such as conditioned place preference, avoidance, escape from noxious stimulus, and analgesic drug self-administration have been used in rodents to evaluate affective aspects of pain. An important advance of such operant behaviors is that these approaches may allow the detection and mechanistic investigation of spontaneous neuropathic or ongoing inflammatory/nociceptive (i.e., nonevoked) pain that is otherwise difficult to assess in nonverbal animals. Operant measures may allow the identification of mechanisms that contribute differentially to reflexive hypersensitivity or to pain affect and may inform the decision to progress novel mechanisms to clinical trials for pain therapy. Additionally, operant behaviors may allow investigation of the poorly understood mechanisms and neural circuits underlying motivational aspects of pain and the reward of pain relief.
doi:10.1111/nyas.12095
PMCID: PMC4028681  PMID: 23496247
operant behavior; conditioned place preference; spontaneous pain; pain affect; dopamine; mesolimbic reward circuit
11.  SPINAL NK-1 RECEPTOR EXPRESSING NEURONS MEDIATE OPIOID-INDUCED HYPERALGESIA AND ANTINOCICEPTIVE TOLERANCE VIA ACTIVATION OF DESCENDING PATHWAYS 
Pain  2006;129(0):35-45.
Opioids can induce hyperalgesia in humans and in animals. Mechanisms of opiate-induced hyperalgesia and possibly of spinal antinociceptive tolerance may be linked to pronociceptive adaptations occurring at multiple levels of the nervous system including activation of descending facilitatory influences from the brainstem, spinal neuroplasticity, and changes in primary afferent fibers. Here, the role of NK-1 receptor-expressing cells in the spinal dorsal horn in morphine-induced hyperalgesia and spinal antinociceptive tolerance was assessed by ablating these cells with intrathecal injection of SP-saporin (SP-SAP). Ablation of NK-1 receptor expressing cells prevented (a) morphine-induced thermal and mechanical hypersensitivity, (b) increased touch-evoked spinal FOS expression, (c) upregulation of spinal dynorphin content and (d) the rightward displacement of the spinal morphine antinociceptive dose-response curve (i.e., tolerance). Morphine-induced hyperalgesia and antinociceptive tolerance were also blocked by spinal administration of ondansetron, a serotonergic receptor antagonist. Thus, NK-1 receptor expressing neurons play a critical role in sustained morphine-induced neuroplastic changes which underlie spinal excitability reflected as thermal and tactile hypersensitivity to peripheral stimuli, and to reduced antinociceptive actions of spinal morphine (i.e., antinociceptive tolerance). Ablation of these cells likely eliminates the ascending limb of a spinal-bulbospinal loop that engages descending facilitation and elicits subsequent spinal neuroplasticity. The data may provide a basis for understanding mechanisms of prolonged pain which can occur in the absence of tissue injury.
doi:10.1016/j.pain.2006.09.033
PMCID: PMC4028682  PMID: 17123731
Opioid-induced hyperalgesia; descending facilitation; spinal plasticity; spinal tolerance; NK-1 receptors; projection cells
12.  Descending Facilitatory Pathways From the Rostroventromedial Medulla Mediate Naloxone-precipitated Withdrawal in Morphine-Dependent Rats 
Opioids produce analgesic effects and extended use can produce physical dependence in both humans and animals. Dependence to opiates can be demonstrated by either termination of drug administration or through precipitation of the withdrawal syndrome by opiate antagonists. Key features of the opiate withdrawal syndrome include hyperalgesia, anxiety and autonomic signs such as diarrhea. The rostral ventromedial medulla (RVM) plays an important role in the modulation of pain and for this reason, may influence withdrawal-induced hyperalgesia. The mechanisms that drive opiate withdrawal-induced hyperalgesia have not been elucidated. Here, rats made dependent upon morphine received naloxone to precipitate withdrawal. RVM microinjection of lidocaine, kynurenic acid (excitatory amino acid antagonist) or YM022 (CCK2 receptor antagonist) blocked withdrawal-induced hyperalgesia. Additionally, these treatments reduced both somatic and autonomic signs of naloxone-induced withdrawal. Spinal application of ondansetron, a 5HT3 receptor antagonist thought to ultimately be engaged by descending pain facilitatory drive, also blocked hyperalgesia and somatic and autonomic features of the withdrawal syndrome. These results indicate that the RVM plays a critical role in mediating components of opioid withdrawal that may contribute to opioid dependence.
Perspective
Manipulations targeting these descending pathways from the RVM may diminish the consequences of prolonged opioid administration-induced dependence and be useful adjunct strategies in reducing the risk of opioid addiction.
doi:10.1016/j.jpain.2010.12.007
PMCID: PMC4028695  PMID: 21354865
naloxone-induced withdrawal; hyperalgesia; RVM; descending facilitation; morphine dependence
13.  Opioid and noradrenergic contributions of tapentadol in experimental neuropathic pain 
Neuroscience letters  2013;562:91-96.
Tapentadol is a dual action molecule with mu opioid agonist and norepinephrine (NE) reuptake blocking activity that has recently been introduced for the treatment of moderate to severe pain. The effects of intraperitoneal (i.p.) morphine (10 mg/kg), tapentadol (10 or 30 mg/kg) or duloxetine (30 mg/kg), a norepinephrine/serotonin (NE/5HT) reuptake inhibitor, were evaluated in male, Sprague-Dawley rats with spinal nerve ligation (SNL) or sham surgery. Additionally, the effects of these drugs on spinal cerebrospinal fluid (CSF) NE levels were quantified. Response thresholds to von Frey filament stimulation decreased significantly from baseline in SNL, but not sham, operated rats. Duloxetine, tapentadol and morphine produced significant and time-related reversal of tactile hypersensitivity. Duloxetine significantly increased spinal CSF NE levels in both sham and SNL rats and no significant differences were observed in these groups. Tapentadol (10 mg/kg) produced a significant increase in spinal NE levels in SNL, but not in sham, rats. At the higher dose (30 mg/kg), tapentadol produced a significant increase in spinal CSF NE levels in both SNL and sham groups; however, spinal NE levels were elevated for an extended period in the SNL rats. This could be detected 30 min following tapentadol (30 mg/kg) in both sham and SNL groups. Surprisingly, while the dose of morphine studied reversed tactile hypersensitivity in nerve-injured rats, CSF NE levels were significantly reduced in both sham- and SNL rats. The data suggest that tapentadol elicits enhanced elevation in spinal NE levels in a model of experimental neuropathic pain offering a mechanistic correlate to observed clinical efficacy in this pain state.
doi:10.1016/j.neulet.2013.08.017
PMCID: PMC4018237  PMID: 23969300
Tapentadol; Spinal norepinephrine; Opioid; Noradrenergic; Norepinephrine reuptake inhibition
14.  Chiral Effect of a Phe Residue in Position 3 of the Dmt1-L(or D)-Tic2 Analogues on Opioid Functional Activities 
ACS medicinal chemistry letters  2013;4(7):656-659.
In this letter, we describe a structure–activity relationships study, specifically related to the chirality of third amino acid residue in our H-Dmt-L(or D)-Tic analogues, of which C-terminus is attached to a piperidinyl moiety. Observed selectivities and functional activities of these analogues demonstrated that the chiralities of the second and third position residues are crucial for determining whether these ligands act as antagonists or agonists at the δ opioid receptor, but not at the μ opioid receptor.
doi:10.1021/ml400115n
PMCID: PMC3956057  PMID: 24648867
Dmt-Tic; opioid functional activities; structure–activity relationship; δ opioid receptor; chirality
15.  Effect of anchoring 4-anilidopiperidines to opioid peptides 
We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.
doi:10.1016/j.bmcl.2013.03.065
PMCID: PMC3942532  PMID: 23623418
Opioid peptide; Dynorphine analog; Bivalent ligand; Fentanyl; Analgesic
16.  Biological Active Analogues of the Opioid Peptide Biphalin: Mixed α/β3-Peptides 
Journal of medicinal chemistry  2013;56(8):3419-3423.
Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-β3 amino acids. The derivative 1 containing hβ3 Phe in place of Phe showed good μ- and δ-receptor affinities ( Kiδ=0.72nM;Kiμ=1.1nM) and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma.
doi:10.1021/jm301456c
PMCID: PMC3942533  PMID: 23547584
17.  Update on Medication-overuse Headache 
Medication-overuse headache (MOH) is a syndrome that can develop in migraineurs after overuse of antimigraine drugs, including opiates and triptans especially. MOH manifests as increased frequency and intensity of migraine attacks and enhanced sensitivity to stimuli that elicit migraine episodes. Although the mechanisms underlying MOH remain unknown, it is hypothesized that repeated use of antimigraine drugs could elicit increased headache attacks as a consequence of neuronal plasticity that may increase responsiveness to migraine triggers. Preclinical studies show that exposure to either opiates or triptans can induce pronociceptive neuroadaptive changes in the orofacial division of the trigeminal ganglia that persist even after discontinuation of the drug treatment. Additionally, medications can elicit increased descending facilitatory influences that may amplify evoked inputs from trigeminal afferents leading to behavioral hypersensitivity reminiscent of cutaneous allodynia observed clinically. Importantly, enhanced descending facilitation may manifest as an inhibition of diffuse noxious inhibitory control. Persistent, pronociceptive adaptations in nociceptors as well as within descending modulatory pathways thus may jointly contribute to the development of MOH.
doi:10.1007/s11916-010-0155-1
PMCID: PMC3780979  PMID: 21058047
Migraine; Medication-overuse headache; Opiates; Triptans; Neuroadaptive changes
18.  Evaluation of cutaneous allodynia following induction of cortical spreading depression in freely moving rats 
Background
Cortical spreading depression (CSD) is a wave of depolarization followed by depression of bioelectrical activity that slowly propagates through the cortex. CSD is believed to be the underlying mechanism of aura in migraine; however, whether CSD can elicit pain associated with migraine headache is unclear.
Methods
Awake, freely moving rats were monitored for both CSD events and behavioral responses resulting from dural-cortical pinprick and/or KCl injection to the occipital cortex.
Results
We observed tactile allodynia of the face and hindpaws, as well as enhanced Fos expression within the trigeminal nucleus caudalis (TNC) following CSD induced by KCl injection into the cortex, but not by pinprick. Application of KCl onto the dura elicited cutaneous allodynia and increased Fos staining in the TNC but did not elicit CSD events.
Conclusions
These data suggest that sustained activation of trigeminal afferents that may be required to establish cutaneous allodynia may not occur following CSD events in normal animals.
doi:10.1177/0333102411410609
PMCID: PMC3780988  PMID: 21700643
CSD; migraine; nucleus caudalis; trigeminal; allodynia; fos
19.  ACTIVATION OF TRPA1 ON DURAL AFFERENTS: A POTENTIAL MECHANISM OF HEADACHE PAIN 
Pain  2012;153(9):1949-1958.
Activation of transient receptor potential ankyrin-1 (TRPA1) on meningeal nerve endings has been suggested to contribute to environmental irritant-induced headache but this channel may also contribute to other forms of headache such as migraine. The preclinical studies described here examined functional expression of TRPA1 on dural afferents and investigated whether activation of TRPA1 contributes to headache-like behaviors. Whole-cell patch-clamp recordings were performed in vitro using two TRPA1 agonists, mustard oil (MO) and the environmental irritant umbellulone (UMB), on dural-projecting trigeminal ganglion neurons. Application of MO and UMB to dural afferents produced TRPA1-like currents in approximately 42% and 38% of cells, respectively. Using an established in vivo behavioral model of migraine-related allodynia, dural application of MO and UMB produced robust time-related tactile facial and hindpaw allodynia that was attenuated by pretreatment with the TRPA1 antagonist HC-030031. Additionally, MO or UMB were applied to the dura and exploratory activity was monitored for 30 minutes using an automated open-field activity chamber. Dural MO and UMB decreased the number of vertical rearing episodes and the time spent rearing in comparison to vehicle treated animals. This change in activity was prevented in rats pretreated with HC-030031 as well as sumatriptan, a clinically effective anti-migraine agent. These data indicate that TRPA1 is expressed on a substantial fraction of dural afferents and activation of meningeal TRPA1 produces behaviors consistent with those seen in patients during migraine attacks. Further, they suggest that activation of meningeal TRPA1 via endogenous or exogenous mechanisms can lead to afferent signaling and headache.
doi:10.1016/j.pain.2012.06.012
PMCID: PMC3413768  PMID: 22809691
migraine; TRPA1; mustard oil; umbellulone; allodynia; headache; dura
20.  Contribution of PKMζdependent and independent amplification to components of experimental neuropathic pain 
Pain  2012;153(6):1263-1273.
Injuries can induce adaptations in pain processing that result in amplification of signaling. One mechanism may be analogous to long-term potentiation (LTP) and involve the atypical protein kinase C, PKMζ The possible contribution of PKMζ-dependent, and independent amplification mechanisms to experimental neuropathic pain was explored in rats with spinal nerve ligation (SNL) injury. SNL increasedp-PKMζin the rostral anterior cingulate cortex (rACC) a site that mediates, in part, the unpleasant aspects of pain. Inhibition of PKMζ within the rACC by a single administration of ζ-pseudosubstrate inhibitory peptide (ZIP)reversedSNL-induced aversivenesswithin24 hrswhereasNMDA receptor blockade with MK-801 had no effects. The SNL-induced aversive state (reflecting “spontaneous” pain),was re-established in a time-dependent manner, with full recovery observed 7 days post-ZIP administration. Neither rACC ZIPnor MK-801altered evoked responses. In contrast, spinal ZIP or MK-801, but not scrambled peptide,transiently reversedevoked hypersensitivity but had no effect on nerve-injury induced spontaneous pain.PKMζ phosphorylation was not altered by SNL in the spinal dorsal horn.These data suggest thatamplification mechanisms contribute to different aspects of neuropathic pain at different levels of the neuraxis. Thus, PKMζ-dependent amplification contributes to nerve-injury induced aversivenesswithin the rACC. Moreover, unlike mechanisms maintaining memory, the consequences of PKMζ inhibition within the rACC are not permanent in neuropathic pain, possibly reflecting the re-establishment of amplification mechanisms by ongoing activity of injured nerves. In the spinal cord, however, both PKMζ-dependent and independent mechanisms contribute to amplification of evoked responses, but apparently not spontaneous pain.
doi:10.1016/j.pain.2012.03.006
PMCID: PMC3358498  PMID: 22482911
rACC; spinal cord; PKMζ; ZIP; aversiveness; spontaneous pain; neuropathic pain
21.  Negative reinforcement reveals non-evoked ongoing pain in mice with tissue or nerve injury 
The Journal of Pain  2012;13(6):598-607.
Patients with chronic pain experience spontaneous or ongoing pain as well as enhanced sensitivity to evoked stimuli. Spontaneous or ongoing pain is rarely evaluated in preclinical studies. In fact, it remains controversial whether ongoing or spontaneous pain even develops in mice after tissue or nerve injury. This study tested a hypothesis that negative reinforcement can be used to unmask the presence of pain in mice with tissue or nerve injury. We found that spinal administration of clonidine or lidocaine did not elicit CPP in uninjured or sham-operated mice. However, these agents produced CPP in mice with chronic inflammation induced by complete Freund’s adjuvant (CFA) or following L5/L6 spinal nerve ligation (SNL). These data indicate the presence of non-evoked (i.e., stimulus-independent) ongoing pain in mice with chronic inflammation (CFA) or following nerve injury (SNL). In addition, this study validates the use of negative reinforcement to unmask non-evoked ongoing pain in mice. Given the existence of a large collection of transgenic and knockout mice, our data show the application of this approach to elucidate molecular mechanisms underlying non-evoked pain and to contribute to drug discovery for pain.
Perspective
We demonstrated the presence of non-evoked ongoing pain in mice with chronic inflammation or following nerve injury. The study also validates the use of negative reinforcement to unmask non-evoked pain in mice. We propose to apply this approach to identify molecular mechanisms and effective drugs for chronic pain.
doi:10.1016/j.jpain.2012.03.011
PMCID: PMC3367085  PMID: 22609247
spontaneous pain; conditioned place preference; neuropathic pain; inflammatory pain; negative reinforcement
22.  Afferent Drive Elicits Ongoing Pain in a Model of Advanced Osteoarthritis 
Pain  2012;153(4):924-933.
Osteoarthritis (OA) is a chronic condition characterized by pain during joint movement. Additionally, patients with advanced disease experience pain at rest (i.e., ongoing pain)that is generally resistant to non-steroidal anti-inflammatory drugs (NSAIDs). Injection of monosodium iodoacetate (MIA) into the intra-articular space of the rodent knee is a well-established model of OA that elicits weight-bearing asymmetry and referred tactile and thermal hypersensitivity. Whether ongoing pain is present in this model is unknown. Additionally, the possible relationship of ongoing pain to MIA dose is not known. MIA produced weight asymmetry, joint osteolysis, and cartilage erosion across a range of doses (1, 3, and 4.8 mg). However, only rats treated with the highest dose of MIA showed conditioned place preference to a context paired with intra-articular lidocaine, indicating relief from ongoing pain. Diclofenac blocked the MIA-induced weight asymmetry but failed to block MIA-induced ongoing pain. Systemic AMG9810, a TRPV1 antagonist, effectively blocked thermal hypersensitivity, but failed to block high dose MIA-induced weight asymmetry or ongoing pain. Additionally, systemic or intra-articular HC030031, a TRPA1 antagonist, failed to block high dose MIA-induced weight asymmetry or ongoing pain. Our studies suggest that a high dose of intra-articular MIA induces ongoing pain originating from the site of injury that is dependent on afferent fiber activity but apparently independent of TRPV1 or TRPA1 activation. Identification of mechanisms driving ongoing pain may enable development of improved treatments for patients with severe OA pain and diminish the need for joint replacement surgery.
doi:10.1016/j.pain.2012.01.022
PMCID: PMC3313555  PMID: 22387095
23.  Supraspinal peroxynitrite modulates pain signaling by suppressing the endogenous opioid pathway 
Peroxynitrite (PN, ONOO−) is a potent oxidant and nitrating agent that contributes to pain through peripheral and spinal mechanisms, but its supraspinal role is unknown. We present evidence here that PN in the rostral ventromedial medulla (RVM) is essential for descending nociceptive modulation in rats during inflammatory and neuropathic pain through PN-mediated suppression of opioid signaling. Carrageenan-induced thermal hyperalgesia was associated with increased 3-nitrotyrosine (NT), a PN biomarker, in the RVM. Furthermore, intra-RVM microinjections of the PN decomposition catalyst (PNDC), Fe(III)-5,10,15,20-tetrakis(N-methyl-pyridinium-4-yl)porphyrin (FeTMPyP5+) dose-dependently reversed this thermal hyperalgesia. These effects of FeTMPyP5+ were abrogated by intra-RVM naloxone, implicating potential interplay between PN and opioids. In support, we identified NT co-localization with the endogenous opioid, enkephalin (ENK), in the RVM during thermal hyperalgesia, suggesting potential in situ interactions. To address the functional significance of such interactions, we exposed methionine-enkephalin (MENK) to PN and identified the major metabolite, 3-nitrotyrosine-methionine-sulfoxide (NSO-MENK), using liquid chromatography-mass spectrometry (LCMS). Next, we isolated, purified, and tested NSO-MENK for opioid receptor binding affinity and analgesic effects. Compared to MENK, this NSO-MENK metabolite lacked appreciable binding affinity for δ, µ, and κ opioid receptors. Intrathecal injection of NSO-MENK in rats did not evoke antinociception suggesting that PN-mediated chemical modifications of ENK suppress opioid signaling. When extended to chronic pain, intra-RVM FeTMPyP5+ produced naloxone-sensitive reversal of mechanical allodynia in rats following chronic constriction injury (CCI) of the sciatic nerve. Collectively, our data reveal the central role of PN in RVM descending facilitation during inflammatory and neuropathic pain potentially through anti-opioid activity.
doi:10.1523/JNEUROSCI.6345-11.2012
PMCID: PMC3511865  PMID: 22875915
Peroxynitrite; rostral ventromedial medulla (RVM); enkephalin; superoxide; descending modulation; descending facilitation; chronic pain; oxidative stress; nitrative stress; post-translational modification
24.  Activation of Descending Pain Facilitatory Pathways from the Rostral Ventromedial Medulla by Cholecystokinin Elicits Release of PGE2 in the Spinal Cord 
Pain  2011;153(1):86-94.
Cholecystokinin (CCK) has been suggested to be both pro-nociceptive and anti-opioid by actions on pain modulatory cells within the RVM. One consequence of activation of RVM CCK2 receptors may be enhanced spinal nociceptive transmission but how this might occur, especially in states of pathological pain is unknown. Here, in vivo microdialysis was used to demonstrate that levels of RVM CCK increased by approximately 2-fold following ligation of L5/L6 spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. Additionally, RVM CCK elicited a time-related increase in PGE2 measured in cerebrospinal fluid from the lumbar spinal cord. The peak increase in spinal PGE2 was approximately 5-fold and was observed at approximately 80-min post-RVM CCK, a time coincident with maximal RVM CCK-induced mechanical hypersensitivity. Spinal administration of naproxen, a non-selective COX-inhibitor, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. RVM-CCK also resulted in a 2-fold increase in spinal 5-HIAA, a 5-HT metabolite, as compared to controls, and mechanical hypersensitivity that was attenuated by spinal application of ondansetron, a 5-HT3 antagonist. The present studies suggest that chronic nerve injury can result in activation of descending facilitatory mechanisms that may promote hyperalgesia via ultimate release of PGE2 and 5-HT in the spinal cord.
doi:10.1016/j.pain.2011.09.021
PMCID: PMC3245767  PMID: 22030324
rostral ventromedial medulla; descending facilitation; cholecystokinin; serotonin; PGE2; microdialysis; tactile hypersensitivity; nerve injury
25.  Improving Metabolic Stability By Glycosylation: Bifunctional Peptide Derivatives That Are Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists 
Journal of medicinal chemistry  2009;52(16):5164-5175.
In order to obtain a metabolically more stable analgesic peptide derivative, O-β-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3’,5’-Bzl(CF3)2) which was a previously reported bifunctional compound with delta/mu opioid agonist and neurokinin-1 receptor antagonist activities, and with a half life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3’,5’-Bzl(CF3)2) was found to have effective bifunctional activities with a well-defined conformation with two β-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 ± 9 % of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.
doi:10.1021/jm900473p
PMCID: PMC3521585  PMID: 20560643
bifunctional peptide derivatives; glycopeptides; analgesics; opioid induced tolerance; opioid receptor agonist; neurokinin-1 receptor antagonist; conformation-activity relationships; NMR structure; DPC micelles

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