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author:("vander, Todd")
1.  Blockade of non-opioid excitatory effects of spinal dynorphin A at bradykinin receptors 
Dynorphin A (Dyn A) is an endogenous opioid ligand that possesses neuroinhibitory (antinociceptive) effects via μ, δ, and κ opioid receptors. However, under chronic pain conditions, up-regulated spinal Dyn A can also interact with bradykinin receptors (BRs) to promote hyperalgesia through a neuroexcitatory(pronociceptive) effect. These excitatory effects cannot be blocked by an opioid antagonist, and thus are non-opioid in nature. On the basis of the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin(BK) and kallidin (KD), Dyn A's interaction with BRs could not be predicted, and provided an opportunity to identify a novel potential neuroexcitatory target. Systematic structure-activity relationship (SAR) studies discovered a minimum pharmacophore of Dyn A, [des-Arg7]-Dyn A-(4-11) LYS1044 for antagonist activity at the BRs, along with insights into the key structural features for BRs recognition, i.e., amphipathicity. The des-Tyr fragment of dynorphin does not bind to opioid receptors. Intrathecal administration of des-Tyr dynorphin produces hyperalgesia reminiscent of behaviors seen in peripheral n europathic pain models and at higher doses, neurotoxicity. Our lead ligand LYS1044 negatively modulated Dyn A-(2-13)-induced neuroexcitatory effects in naïve animals and blocked mechanical hypersensitivity and thermal hyperalgesia in a dose-dependent manner in animals with experimental neuropathic pain. Based on these results, ligand LYS1044 might prevent abnormal pain states by blocking the neuroexcitatory effects of increased levels of Dyn A that are seen in experimental models of neuropathic pain and that likely promote excitation mediated by BRs in the spinal cord.
PMCID: PMC4515361  PMID: 26221618
2.  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 it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg10, Leu9]-kallidin (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 ( 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.
PMCID: PMC4021566  PMID: 24742335
3.  Novel fentanyl-based dual μ/δ-opioid agonists for the treatment of acute and chronic pain 
Life sciences  2013;93(0):1010-1016.
Approximately one third of the adult U.S. population suffers from some type of on-going, chronic pain annually, and many more will have some type of acute pain associated with trauma or surgery. First-line therapies for moderate to severe pain include prescriptions for common mu opioid receptor agonists such as morphine and its various derivatives. The epidemic use, misuse and diversion of prescription opioids has highlighted just one of the adverse effects of mu opioid analgesics. Alternative approaches include novel opioids that target delta or kappa opioid receptors, or compounds that interact with two or more of the opioid receptors.
Here we report the pharmacology of a newly synthesized bifunctional opioid agonist (RV-Jim-C3) derived from combined structures of fentanyl and enkephalin in rodents. RV-Jim-C3 has high affinity binding to both mu and delta opioid receptors.
Main Methods
Mice and rats were used to test RV-Jim-C3 in a tailflick test with and without opioid selective antagonist for antinociception. RV-Jim-C3 was tested for anti-inflammatory and antihypersensitivity effects in a model of formalin-induced flinching and spinal nerve ligation. To rule out motor impairment, rotarod was tested in rats.
Key findings
RV-Jim-C3 demonstrates potent-efficacious activity in several in vivo pain models including inflammatory pain, antihyperalgesia and antiallodynic with no significant motor impairment.
This is the first report of a fentanyl-based structure with delta and mu opioid receptor activity that exhibits outstanding antinociceptive efficacy in neuropathic pain, reducing the propensity of unwanted side effects driven by current therapies that are unifunctional mu opioid agonists.
PMCID: PMC3962292  PMID: 24084045
chronic pain; allodynia; hyperalgesia; inflammatory; mice; rat; fentanyl; mu opioid; delta opioid; spinal nerve ligation; formalin flinch; naloxone
4.  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.
PMCID: PMC3810412  PMID: 23899615
bifunctional compounds; opioid receptor agonists; neutokinin-1 receptor antagonists; Truncation of peptide sequence; NMR structure
5.  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.
PMCID: PMC4137774  PMID: 21925887
Analgesic; Bivalent ligands; μ-Opioids; NK1 antagonist; Fentanyl
6.  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 ( 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.
PMCID: PMC4021566  PMID: 24742335
pathological chronic pain states; hyperalgesia; dynorphin A; bradykinin receptor recognition; non-opioid; amphipathic pharmacophore
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.
PMCID: PMC4028682  PMID: 17123731
Opioid-induced hyperalgesia; descending facilitation; spinal plasticity; spinal tolerance; NK-1 receptors; projection cells
8.  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.
PMCID: PMC3942532  PMID: 23623418
Opioid peptide; Dynorphine analog; Bivalent ligand; Fentanyl; Analgesic
9.  Evaluation of cutaneous allodynia following induction of cortical spreading depression in freely moving rats 
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.
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.
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.
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.
PMCID: PMC3780988  PMID: 21700643
CSD; migraine; nucleus caudalis; trigeminal; allodynia; fos
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.
PMCID: PMC3413768  PMID: 22809691
migraine; TRPA1; mustard oil; umbellulone; allodynia; headache; dura
11.  Tachykinin NK1 receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation 
European journal of pharmacology  2012;684(1-3):64-70.
Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK1 (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK1 receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK1 receptor antagonist (N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 μg/injection) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in morphine-withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK1 antagonist pharmacophor yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK1 receptor antagonists may provide a novel paradigm for long-term pain management.
PMCID: PMC3565540  PMID: 22724132
opioid-induced hyperalgesia; spinal glia; Tachykinin NK1 receptor; Tachykinin NK1 receptor antagonist
12.  Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist 
Journal of Neuroimmunology  2012;244(1-2):23-31.
Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine–mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1β, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.
PMCID: PMC3298577  PMID: 22285397
morphine; spinal glia; CB2 agonist; hyperalgesia; allodynia; pain sensitization
13.  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.
PMCID: PMC3245767  PMID: 22030324
rostral ventromedial medulla; descending facilitation; cholecystokinin; serotonin; PGE2; microdialysis; tactile hypersensitivity; nerve injury
14.  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.
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
15.  Engagement of Descending Inhibition from the Rostral Ventromedial Medulla Protects Against Chronic Neuropathic Pain 
Pain  2011;152(12):2701-2709.
A puzzling observation is why peripheral nerve injury results in chronic pain in some, but not all, patients. We explored potential mechanisms that may prevent the expression of chronic pain. Sprague-Dawley (SD) or Holtzman (HZ) rats showed no differences in baseline sensory thresholds or responses to inflammatory stimuli. However, spinal nerve ligation (SNL)-induced tactile allodynia occurred in approximately 85% of SD and 50% of HZ rats, respectively. No apparent differences were observed in a survey of DRG or spinal “neuropathic markers” following SNL regardless of allodynic phenotype. SNL-induced allodynia was reversed by administration of lidocaine within the rostral ventromedial medulla (RVM), a site that integrates descending pain modulation via pain inhibitory (i.e., OFF) and excitatory (i.e., ON) cells. However, in SD or HZ rats with SNL but without allodynia, RVM lidocaine precipitated allodynia. Additionally, RVM lidocaine produced conditioned place preference in allodynic SD or HZ rats but conditioned place aversion in non-allodynic HZ rats. Similarly, RVM U69,593 (kappa opioid agonist) or blockade of spinal α2 adrenergic receptors precipitated allodynia in previously non-allodynic HZ rats with SNL. All rats showed an equivalent first phase formalin responses. However, HZ rats had reduced second phase formalin behaviors along with fewer RVM OFF cell pauses and RVM ON cell bursts. Thus, expression of nerve-injury induced pain may ultimately depend on descending modulation. Engagement of descending inhibition protects in the transition from acute to chronic pain. These unexpected findings might provide a mechanistic explanation for medications that engage descending inhibition or mimic its consequences.
PMCID: PMC3222148  PMID: 21745713
16.  Unmasking the tonic-aversive state in neuropathic pain 
Nature neuroscience  2009;12(11):1364-1366.
Tonic pain has been difficult to demonstrate in animals. Because relief of pain is rewarding, analgesic agents that are not rewarding in the absence of pain should become rewarding only when there is ongoing pain. We used conditioned place preference to concomitantly determine the presence of tonic pain in rats and the efficacy of agents that relieve it. This provides a new approach for investigating tonic pain in animals and for evaluating the analgesic effects of drugs.
PMCID: PMC3427725  PMID: 19783992
17.  Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury 
Nature neuroscience  2008;11(4):488-496.
Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.
PMCID: PMC3417340  PMID: 18344995
18.  TRPV1 Receptor in Expression of Opioid-induced hyperalgesia 
The Journal of Pain  2008;10(3):243-252.
Opiates are currently the mainstay for treatment of moderate to severe pain. However, prolonged administration of opiates has been reported to elicit hyperalgesia in animals and examples of opiate-induced hyperalgesia have been reported in humans as well. In spite of the potential clinical significance of such opiate-induced actions, the mechanisms of opiate-induced hypersensitivity remain unknown. The TRPV1 receptor, a molecular sensor of noxious heat, acts as an integrator of multiple forms of noxious stimuli and plays an important role in the development of inflammation-induced hyperalgesia. As animals treated with opiates show thermal hyperalgesia, we examined the possible role of TRPV1 receptors in the development of morphine-induced hyperalgesia using TRPV1 wild-type (WT) and knock-out (KO) mice and with administration of a TRPV1 antagonist in mice and rats. Administration of morphine by subcutaneous implantation of morphine pellets elicited both thermal and tactile hypersensitivity in TRPV1 WT mice, but not in TRPV1 KO mice. Moreover, oral administration of a TRPV1 antagonist reversed both thermal and tactile hypersensitivity induced by sustained morphine administration in mice and rats. Immunohistochemical analyses indicate that sustained morphine administration modestly increases TRPV1 labeling in the dorsal root ganglia (DRG). In addition, sustained morphine increased flinching and plasma extravasation after peripheral stimulation with capsaicin, suggesting an increase in TRPV1 receptor function in the periphery in morphine treated animals. Collectively our data indicate that the TRPV1 receptor is an essential peripheral mechanism in expression of morphine-induced hyperalgesia.
Opioid-induced hyperalgesia possibly limits the usefulness of opioids, emphasizing the value of alternative methods of pain control. We demonstrate that TRPV1 channels play an important role in peripheral mechanisms of opioid-induced hyperalgesia. Such information may lead to the discovery of analgesics lacking such adaptations and improving treatment of chronic pain.
PMCID: PMC3385996  PMID: 18774343
19.  Novel peptide ligands with dual acting pharmacophores designed for the pathophysiology of neuropathic pain 
Brain research  2011;1395:1-11.
The conventional design of high affinity drugs targeted to a single molecule has not resulted in clinically useful therapies for pain relief. Recent reviews have suggested that newly designed analgesic drugs should incorporate multiple targets. The distributions of cholecystokinin (CCK) and CCK receptors in the central nervous system (CNS) overlap significantly with endogenous opioid systems and can be dually targeted. CCK has been shown to act as an endogenous “anti-analgesic” peptide and neuropathic pain conditions promote endogenous CCK release in CNS regions of pain modulation. Administration of CCK into nuclei of the rostral ventromedial medulla induces pronociceptive behaviors in rats. RSA 504 and RSA 601 are novel bifunctional compounds developed to target neuropathic pain by simultaneously acting as agonists at two distinct opioid receptors and antagonizing CCK receptors in the CNS. RSA 504 and RSA 601 demonstrate agonist activity in vitro and antihypersensitivity to mechanical and thermal stimuli in vivo using the spinal nerve ligation model of neuropathic pain. Intrathecal administration of RSA 504 and RSA 601 did not demonstrate antinociceptive tolerance over 7 days of administration and did not display motor impairment or sedation using a rotarod. These are the first behavioral studies that demonstrate how multi-targeted molecule design can address the pathology of neuropathic pain. These compounds with δ and μ opioid agonist activity and CCK antagonist activity within one molecule offer a novel approach with efficacy for neuropathic pain while lacking the side effects typically caused by conventional opioid therapies.
PMCID: PMC3105124  PMID: 21550594
neuropathic pain; spinal nerve ligation; cholecystokinin; opioids
20.  Discovery of a Potent and Efficacious Peptide Derivative for δ/μ Opioid Agonist/Neurokinin 1 Antagonist Activity with a 2′, 6′-Dimethyl-L-Tyrosine: In Vitro, In Vivo and NMR-Based Structural Studies 
Journal of medicinal chemistry  2011;54(7):2029-2038.
Multivalent ligands with delta/mu opioid agonist and NK1 antagonist activities have shown promising analgesic potency without detectable sign of toxicities, including motor skill impairment and opioid-induced tolerance. To improve their biological activities and metabolic stability, structural optimization was performed on our peptide-derived lead compounds by introducing 2′, 6′-Dimethyl-L-Tyrosine (Dmt) instead of Tyr at the first position. The compound 7 (Dmt-D-Ala-Gly-Phe-MetPro-Leu-Trp-NH-[3′,5′-(CF3)2-Bzl]) showed improved multivalent bioactivities compared to those of the lead compounds, had more than 6 h half-life in rat plasma, and significant antinociceptive efficacy in vivo. The NMR structural analysis suggested that Dmt1 incorporation in compound 7 induces the structured conformation in the opioid pharmacophore (N-terminus), and simultaneously shifts the orientation of the NK1 pharmacophore (C-terminus), consistent with its affinities and activities at both opioid and NK1 receptors. These results indicate that compound 7 is a valuable research tool to seek a novel analgesic drug.
PMCID: PMC3090346  PMID: 21366266
analgesics; bifunctional peptide derivatives; 2′, 6′-Dimethyl-L-Tyrosine; DPC micelle; opioid induced tolerance; opioid receptor agonist; neurokinin-1 receptor antagonist; NMR structure
21.  Development of potent μ and δ opioid agonists with high lipophilicity 
Journal of medicinal chemistry  2010;54(1):382-386.
An SAR study on the Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-yl propionamide moiety at C-terminal was performed, and has resulted in highly potent ligands at μ and δ opioid receptors. In general, ligands with the substitution of D-Nle2 and halogenation of the aromatic ring of Phe4 showed highly increased opioid activities. Ligand 6 with good biological activities in vitro demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay.
PMCID: PMC3136578  PMID: 21128594
22.  Sustained morphine treatment augments prostaglandin E2-evoked Calcitonin Gene-Related Peptide release from primary sensory neurons in a PKA- dependent manner 
European journal of pharmacology  2010;648(1-3):95-101.
Tissue damage leads to pain sensitization due to peripheral and central release of excitatory mediators such as prostaglandin E2 (PGE2). PGE2 sensitizes spinal pain neurotransmitter such as calcitonin gene-related peptide (CGRP) release via activation of cyclic AMP (cAMP)/Protein kinase A (PKA) -dependent signaling mechanisms. Our previous data demonstrates that sustained morphine pretreatment sensitizes adenylyl cyclase(s) (AC) toward the direct stimulator, forskolin, in cultured primary sensory neurons (AC superactivation). In the present work we investigated the hypothesis that morphine pretreatment also sensitizes ACs toward Gs protein-coupled excitatory modulators (such as PGE2), leading to augmented PKA-dependent CGRP release from PGE2-stimulated primary sensory dorsal root ganglion (DRG) neurons. Our results show that sustained morphine treatment potentiated PGE2-mediated cAMP formation and augmented PGE2-evoked CGRP release from cultured primary sensory neurons in a PKA-dependent manner. Our data suggests that attenuation of AC-superactivation in primary sensory neurons may prevent the development of opioid-induced hyperalgesia.
PMCID: PMC2955884  PMID: 20826131
Morphine; PGE2; PKA; CGRP release; DRG neurons; opioid-induced hyperalgesia; Gs protein signaling
23.  Inhibition of p38-MAPK signaling pathway attenuates breast cancer induced bone pain and disease progression in a murine model of cancer-induced bone pain 
Molecular Pain  2011;7:81.
Mechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs.
In cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden.
Blockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.
PMCID: PMC3212934  PMID: 22014040
24.  Biological and Conformational Evaluation of Bifunctional Compounds for Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists Possessing Two Penicillamines 
Journal of medicinal chemistry  2010;53(15):5491-5501.
Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, which have anti-opioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[D-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3′,5′-(CF3)2-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.
PMCID: PMC2943425  PMID: 20617791
bifunctional compound; opioid receptor agonists; neutokinin-1 receptor antagonists; NMR structure; membrane-compound interaction
25.  A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss 
Life sciences  2010;86(17-18):646-653.
Cannabinoid CB2 agonists have been shown to alleviate behavioral signs of inflammatory and neuropathic pain in animal models. AM1241, a CB2 agonist, does not demonstrate central nervous system side-effects seen with CB1 agonists such as hypothermia and catalepsy. Metastatic bone cancer causes severe pain in patients and is treated with analgesics such as opiates. Recent reports suggest that sustained opiates can produce paradoxical hyperalgesic actions and enhance bone destruction in a murine model of bone cancer. In contrast, CB2 selective agonists have been shown to reduce bone loss associated with a model of osteoporosis. Here we tested whether a CB2 agonist administered over a 7 day period inhibits bone cancer-induced pain as well as attenuates cancer-induced bone degradation.
Main Methods
A murine bone cancer model was used in which osteolytic sarcoma cells were injected into the intramedullary space of the distal end of the femur. Behavioral and radiographic image analysis was performed at days 7, 10 and 14 after injection of tumor cells into the femur.
Key Findings
Osteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 acutely or for 7 days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures.
These findings suggest a novel therapy for cancer-induced bone pain, bone loss and bone fracture while lacking many unwanted side effects seen with current treatments for bone cancer pain.
PMCID: PMC2871326  PMID: 20176037
CB2 agonists; AM1241; osteolytic sarcoma; bone cancer pain

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