Development of opioid analgesic tolerance in rat to extended-release buprenorphine formulated for laboratory subjects.

Buprenorphine in an extended-release formulation intended for use in laboratory subjects is frequently administered to rats to provide extended analgesia without repeated handling. While levels of buprenorphine may persist in serum once extended-release buprenorphine has been introduced, exposure to opioids can cause opioid tolerance or opioid-induced hypersensitivity. This work examined the analgesic duration and efficacy of a single administration of extended-release buprenorphine intended for use in laboratory subjects in models of inflammatory pain and post-operative pain and the development of opioid tolerance in rat. After subcutaneous administration of 1 mg/kg extended-release buprenorphine, analgesic efficacy did not persist for the expected 72 hours. No changes were observed in mechanical thresholds in the hindpaws that were contralateral to the injury, suggesting a lack of centrally mediated opioid-induced hypersensitivity. To determine whether opioid tolerance arose acutely after one exposure to extended-release buprenorphine, we conducted the warm water tail flick assay; on Day 1 we administered either saline or extended-release buprenorphine (1 mg/kg) and on Day 3 we quantified the standard buprenorphine dose-response curve (0.1-3 mg/kg). Rats previously given extended-release buprenorphine displayed decreased analgesic responses after administration of standard buprenorphine as compared to the robust efficacy of standard buprenorphine in control subjects. Males appeared to show evidence of acute opioid tolerance, while females previously exposed to opioid did not demonstrate a decreased response at the doses examined. Taken together, these results suggest that opioid tolerance arises quickly in male rats after exposure to the extended-release formulation of buprenorphine. This tolerance may account for the brief period of antinociception observed.

Biodistribution of Agmatine to Brain and Spinal Cord after Systemic Delivery.

Agmatine, an endogenous polyamine, has been shown to reduce chronic pain behaviors in animal models and in patients. This reduction is due to inhibition of the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR) in the central nervous system (CNS). The mechanism of action requires central activity, but the extent to which agmatine crosses biologic barriers such as the blood-brain barrier (BBB) and intestinal epithelium is incompletely understood. Determination of agmatine distribution is limited by analytical protocols with low sensitivity and/or inefficient preparation. This study validated a novel bioanalytical protocol using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for quantification of agmatine in rat biologic matrices. These protocols were then used to determine the plasma pharmacokinetics of agmatine and the extent of distribution to the CNS. Precision and accuracy of the protocol met US Food and Drug Administration (FDA) standards in surrogate matrix as well as in corrected concentrations in appropriate matrices. The protocol also adequately withstood stability and dilution conditions. Upon application of this protocol to pharmacokinetic study, intravenous agmatine showed a half-life in plasma ranging between 18.9 and 14.9 minutes. Oral administration led to a prolonged plasma half-life (74.4-117 minutes), suggesting flip-flop kinetics, with bioavailability determined to be 29%-35%. Intravenous administration led to a rapid increase in agmatine concentration in brain but a delayed distribution and lower concentrations in spinal cord. However, half-life of agmatine in both tissues is substantially longer than in plasma. These data suggest that agmatine adequately crosses biologic barriers in rat and that brain and spinal cord pharmacokinetics can be functionally distinct. SIGNIFICANCE STATEMENT: Agmatine has been shown to be an effective nonopioid therapy for chronic pain, a significantly unmet medical necessity. Here, using a novel bioanalytical protocol for quantification of agmatine, we present the plasma pharmacokinetics and the first report of agmatine oral bioavailability as well as variable pharmacokinetics across different central nervous system tissues. These data provide a distributional rationale for the pharmacological effects of agmatine as well as new evidence for kinetic differences between brain and spinal cord.

Long-term reversal of chronic pain behavior in rodents through elevation of spinal agmatine.

Chronic pain remains a significant burden worldwide, and treatments are often limited by safety or efficacy. The decarboxylated form of L-arginine, agmatine, antagonizes N-methyl-d-aspartate receptors, inhibits nitric oxide synthase, and reverses behavioral neuroplasticity. We hypothesized that expressing the proposed synthetic enzyme for agmatine in the sensory pathway could reduce chronic pain without motor deficits. Intrathecal delivery of an adeno-associated viral (AAV) vector carrying the gene for arginine decarboxylase (ADC) prevented the development of chronic neuropathic pain as induced by spared nerve injury in mice and rats and persistently reversed established hypersensitivity 266 days post-injury. Spinal long-term potentiation was inhibited by both exogenous agmatine and AAV-human ADC (hADC) vector pre-treatment but was enhanced in rats treated with anti-agmatine immunoneutralizing antibodies. These data suggest that endogenous agmatine modulates the neuroplasticity associated with chronic pain. Development of approaches to access this inhibitory control of neuroplasticity associated with chronic pain may yield important non-opioid pain-relieving options.

Adeno-associated virus-mediated gene transfer of arginine decarboxylase to the central nervous system prevents opioid analgesic tolerance.

Agmatine, a decarboxylated form of L-arginine, prevents opioid analgesic tolerance, dependence, and self-administration when given by both central and systemic routes of administration. Endogenous agmatine has been previously detected in the central nervous system. The presence of a biochemical pathway for agmatine synthesis offers the opportunity for site-specific overexpression of the presumptive synthetic enzyme for local therapeutic effects. In the present study, we evaluated the development of opioid analgesic tolerance in ICR-CD1 mice pre-treated with either vehicle control or intrathecally delivered adeno-associated viral vectors (AAV) carrying the gene for human arginine decarboxylase (hADC). Vehicle-treated or AAV-hADC-treated mice were each further divided into two groups which received repeated delivery over three days of either saline or systemically-delivered morphine intended to induce opioid analgesic tolerance. Morphine analgesic dose-response curves were constructed in all subjects on day four using the warm water tail flick assay as the dependent measure. We observed that pre-treatment with AAV-hADC prevented the development of analgesic tolerance to morphine. Peripheral and central nervous system tissues were collected and analyzed for presence of hADC mRNA. In a similar experiment, AAV-hADC pre-treatment prevented the development of analgesic tolerance to a high dose of the opioid neuropeptide endomorphin-2. Intrathecal delivery of anti-agmatine IgG (but not normal IgG) reversed the inhibition of endomorphin-2 analgesic tolerance in AAV-hADC-treated mice. To summarize, we report here the effects of AAV-mediated gene transfer of human ADC (hADC) in models of opioid-induced analgesic tolerance. This study suggests that gene therapy may contribute to reducing opioid analgesic tolerance.

Central Nervous System Distribution of an Opioid Agonist Combination with Synergistic Activity.

Novel combinations of specific opioid agonists like loperamide and oxymorphindole targeting the µ- and δ-opioid receptors, respectively, have shown increased potency with minimized opioid-associated risks. However, whether their interaction is pharmacokinetic or pharmacodynamic in nature has not been determined. This study quantitatively determined whether these drugs have a pharmacokinetic interaction that alters systemic disposition or central nervous system (CNS) distribution. We performed intravenous and oral in vivo pharmacokinetic assessments of both drugs after discrete dosing and administration in combination to determine whether the combination had any effect on systemic pharmacokinetic parameters or CNS exposure. Drugs were administered at 5 or 10 mg/kg i.v. or 30 mg/kg orally to institute for cancer research (ICR) mice and 5 mg/kg i.v. to Friend leukemia virus strain B mice of the following genotypes: wild-type, breast cancer resistance protein (Bcrp-/- ) (Bcrp knockout), Mdr1a/b-/- [P-glycoprotein (P-gp) knockout], and Bcrp-/- Mdr1a/b-/- (triple knockout). In the combination, clearance of oxymorphindole (OMI) was reduced by approximately half, and the plasma area under the concentration-time curve (AUC) increased. Consequently, brain and spinal cord AUCs for OMI in the combination also increased proportionately. Both loperamide and OMI are P-gp substrates, but administration of the two drugs in combination does not alter efflux transport at the CNS barriers. Because OMI alone shows appreciable brain penetration but little therapeutic efficacy on its own, and because loperamide's CNS distribution is unchanged in the combination, the mechanism of action for the increased potency of the combination is most likely pharmacodynamic and most likely occurs at receptors in the peripheral nervous system. This combination has favorable characteristics for future development. SIGNIFICANCE STATEMENT: Opioids have yet to be replaced as the most effective treatments for moderate-to-severe pain and chronic pain, but their side effects are dangerous. Combinations of opioids with peripheral activity, such as loperamide and oxymorphindole, would be valuable in that they are effective at much lower doses and have reduced risks for dangerous side effects because the µ-opioid receptor agonist is largely excluded from the CNS.

Expression of and correlational patterns among neuroinflammatory, neuropeptide, and neuroendocrine molecules from cerebrospinal fluid in cerebral palsy.

BACKGROUND: The underlying pathogenesis of cerebral palsy (CP) remains poorly understood. The possibility of an early inflammatory response after acute insult is of increasing interest. Patterns of inflammatory and related biomarkers are emerging as potential early diagnostic markers for understanding the etiologic diversity of CP. Their presence has been investigated in plasma and umbilical cord blood but not in cerebrospinal fluid (CSF). METHODS: A clinical CP sample was recruited using a single-time point cross-sectional design to collect CSF at point-of-care during a standard-of-care surgical procedure (intrathecal pump implant). Patient demographic and clinical characteristics were sourced from medical chart audit. RESULTS: Significant (p ≤ 0.001) associations were found among neuroinflammatory, neuroendocrine, and nociceptive analytes with association patterns varying by birth status (term, preterm, extremely preterm). When between birth-group correlations were compared directly, there was a significant difference between preterm and extremely preterm birth subgroups for the correlation between tumour necrosis factor alpha (TNFα) and substance P. CONCLUSION: This investigation shows that CSF can be used to study proteins in CP patients. Differences in inter-correlational patterns among analytes varying by birth status underscores the importance of considering birth status in relation to possible mechanistic differences as indicated by biomarker signatures. Future work should be oriented toward prognostic and predictive validity to continue to parse the heterogeneity of CP's presentation, pathophysiology, and response to treatment.

Biodistribution of Adeno-Associated Virus Serotype 5 Viral Vectors Following Intrathecal Injection.

The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.

Agmatine requires GluN2B-containing NMDA receptors to inhibit the development of neuropathic pain.

A decarboxylated form of L-arginine, agmatine, preferentially antagonizes NMDArs containing Glun2B subunits within the spinal cord and lacks motor side effects commonly associated with non-subunit-selective NMDAr antagonism, namely sedation and motor impairment. Spinally delivered agmatine has been previously shown to reduce the development of tactile hypersensitivity arising from spinal nerve ligation. The present study interrogated the dependence of agmatine's alleviation of neuropathic pain (spared nerve injury (SNI) model) on GluN2B-containing NMDArs. SNI-induced hypersensitivity was induced in mice with significant reduction of levels of spinal GluN2B subunit of the NMDAr and their floxed controls. Agmatine reduced development of SNI-induced tactile hypersensitivity in controls but had no effect in subjects with reduced levels of GluN2B subunits. Ifenprodil, a known GluN2B-subunit-selective antagonist, similarly reduced tactile hypersensitivity in controls but not in the GluN2B-deficient mice. In contrast, MK-801, an NMDA receptor channel blocker, reduced hypersensitivity in both control and GluN2B-deficient mice, consistent with a pharmacological pattern expected from a NMDAr antagonist that does not have preference for GluN2B subtypes. Additionally, we observed that spinally delivered agmatine, ifenprodil and MK-801 inhibited nociceptive behaviors following intrathecal delivery of NMDA in control mice. By contrast, in GluN2B-deficient mice, MK-801 reduced NMDA-evoked nociceptive behaviors, but agmatine had a blunted effect and ifenprodil had no effect. These results demonstrate that agmatine requires the GluN2B subunit of the NMDA receptor for inhibitory pharmacological actions in pre-clinical models of NMDA receptor-dependent hypersensitivity.

FBNTI, a DOR-Selective Antagonist That Allosterically Activates MOR within a MOR-DOR Heteromer.

This report describes the unique pharmacological profile of FBNTI, a potent DOR antagonist that acts as a MOR agonist via an allosteric mechanism. Binding of FBNTI to opioid receptors expressed in HEK 293 cells revealed a 190-fold greater affinity for DOR (Ki = 0.84 nM) over MOR (Ki = 160 nM). In mice, intrathecal FBNTI produced potent antinociception (ED50 = 46.9 pmol/mouse), which was antagonized by selective MOR antagonists (CTOP, ß-FNA). Autoantagonism of the MOR agonism by FBNTI was observed above the ED75 dose, suggesting antagonism of activated MOR. That FBNTI is devoid of agonism in DOR knockout mice is consistent with allosteric activation of the MOR protomer via FBNTI bound to within a MOR-DOR heteromer. This proposed mechanism is supported by calcium mobilization assays, which indicate that FBNTI selectively activates the MOR-DOR heteromer and functionally antagonizes the MOR protomer at >ED75. The unprecedented mode of MOR activation by FBNTI may be responsible for the lack of tolerance after intrathecal (i.t.) administration. FBNTI was highly effective upon topical administration to the ipsolateral hind paw in the Hargreaves assay (EC50 = 0.17 ± 0.08 µM) and without significant contralateral activity, suggesting a lack of systemic exposure.

Topical Application of Loperamide/Oxymorphindole, Mu and Delta Opioid Receptor Agonists, Reduces Sensitization of C-fiber Nociceptors that Possess NaV1.8.

It was recently shown that local injection, systemic administration or topical application of the peripherally-restricted mu-opioid receptor (MOR) agonist loperamide (Lo) and the delta-opioid receptor (DOR) agonist oxymorphindole (OMI) synergized to produce highly potent anti-hyperalgesia that was dependent on both MOR and DOR located in the periphery. We assessed peripheral mechanisms by which this Lo/OMI combination produces analgesia in mice expressing the light-sensitive protein channelrhodopsin2 (ChR2) in neurons that express NaV1.8 voltage-gated sodium channels. These mice (NaV1.8-ChR2+) enabled us to selectively target and record electrophysiological activity from these neurons (the majority of which are nociceptive) using blue light stimulation of the hind paw. We assessed the effect of Lo/OMI on nociceptor activity in both naïve mice and mice treated with complete Freund's adjuvant (CFA) to induce chronic inflammation of the hind paw. Teased fiber recording of tibial nerve fibers innervating the plantar hind paw revealed that the Lo/OMI combination reduced responses to light stimulation in naïve mice and attenuated spontaneous activity (SA) as well as responses to light and mechanical stimuli in CFA-treated mice. These results show that Lo/OMI reduces activity of C-fiber nociceptors that express NaV1.8 and corroborate recent behavioral studies demonstrating the potent analgesic effects of this drug combination. Because of its peripheral site of action, Lo/OMI might produce effective analgesia without the side effects associated with activation of opioid receptors in the central nervous system.

Sustained-release buprenorphine induces acute opioid tolerance in the mouse.

Sustained-release buprenorphine is widely used in mice with the intention of providing long-lasting analgesia. Statements about duration of therapeutic efficacy are based on persistence of serum buprenorphine levels over a minimum threshold, but behavioral data demonstrating sustained efficacy is not established. Additionally, chronic opioid exposure can induce tolerance and/or hyperalgesia; mice receiving sustained-release buprenorphine have not been evaluated for these effects. This study assessed clinical efficacy and duration of sustained-release buprenorphine in inflammatory, post-operative, and cancer pain; and screened for centrally-mediated opioid-induced hyperalgesia as well as opioid tolerance. At 1-2 mg/kg sustained-release buprenorphine, statistically significant analgesic efficacy occurred only at time points up to 2 h. These animals showed no changes in von Frey thresholds on the contralateral side, i.e. no centrally-mediated opioid hyperalgesia. To establish whether acute onset opioid tolerance resulted from a single sustained-release buprenorphine administration, we used the tail flick assay, exposing mice to sustained-release buprenorphine or saline on Day 1 and buprenorphine on Day 2. We measured duration and efficacy of 1 mg/kg buprenorphine after 1 mg/kg sustained-release buprenorphine, and also quantified a dose-response curve of buprenorphine (0.1-3 mg/kg) after 2 mg/kg sustained-release buprenorphine. Compared to control animals, mice previously exposed to sustained-release buprenorphine showed diminished analgesic response to buprenorphine; the resultant dose-response curve showed decreased efficacy. Pretreatment with naloxone, an opioid receptor antagonist, blocked sustained-release buprenorphine analgesic action. The short duration of antinociception following administration of sustained-release buprenorphine in mice is caused by the rapid development of tolerance.

Evidence for neurogenic inflammation in lichen planopilaris and frontal fibrosing alopecia pathogenic mechanism.

Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are lymphocytic scarring alopecias affecting primarily the scalp. Although both diseases may share some clinical and histopathological features, in the last decade, FFA has become an "epidemic" particularly in Europe, North and South America with unique clinical manifestations compared to LPP, thus, raising the idea that this disease may have a different pathogenesis. Symptoms such as scalp burning, pruritus or pain are usually present in both diseases, suggesting a possible role for nerves and neuropeptides in the pathogenesis of both diseases. Based on some previous studies, neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), have been associated with lipid metabolism and many chronic inflammatory disorders. In this study, we asked if these neuropeptides are associated with LPP and FFA scalp lesions. Alteration in the expression of SP and CGRP in affected and unaffected scalp skin from patients with both diseases was found with examination of sections using immunohistochemical techniques and confocal microscopy. We then quantitatively assessed and compared SP and CGRP expression from control, LPP and FFA scalp biopsies. Although LPP and FFA share similar histopathologic findings, opposite results were found in affected and unaffected scalp in the ELISA tests, suggesting that these diseases may have different pathogenic mechanisms. We also found presence of histopathological inflammation irrespective of evident clinical lesions, which raises the possibility that both diseases may be more generalized processes affecting the scalp.

The Study of Pain in Rats and Mice.

Pain is a clinical syndrome arising from a variety of etiologies in a heterogeneous population, which makes successfully treating the individual patient difficult. Organizations and governments recognize the need for tailored and specific therapies, which drives pain research. This review summarizes the different types of pain assessments currently being used and the various rodent models that have been developed to recapitulate the human pain condition.

Combination of a δ-opioid Receptor Agonist and Loperamide Produces Peripherally-mediated Analgesic Synergy in Mice.

BACKGROUND: The long-term use of opioids for analgesia carries significant risk for tolerance, addiction, and diversion. These adverse effects are largely mediated by µ-opioid receptors in the central nervous system. Based on the authors' previous observation that morphine and δ-opioid receptor agonists synergize in spinal cord in a protein kinase Cε-dependent manner, they predicted that this µ-opioid receptor-δ-opioid receptor synergy would take place in the central terminals of nociceptive afferent fibers and generalize to their peripheral terminals. Therefore, the authors hypothesized that loperamide, a highly efficacious µ-opioid receptor agonist that is excluded from the central nervous system, and oxymorphindole, a δ-opioid receptor agonist that was shown to synergize with morphine spinally, would synergistically reverse complete Freund's adjuvant-induced hyperalgesia. METHODS: Using the Hargreaves assay for thermal nociception, the von Frey assay for mechanical nociception and the complete Freund's adjuvant-induced model of inflammatory pain, we tested the antinociceptive and antihyperalgesic effect of loperamide, oxymorphindole, or the loperamide-oxymorphindole combination. Animals (Institute for Cancer Research [ICR] CD1 strain mice; n = 511) received drug by systemic injection, intraplantar injection to the injured paw, or a transdermal solution on the injured paw. Dose-response curves for each route of administration and each nociceptive test were generated, and analgesic synergy was assessed by isobolographic analysis. RESULTS: In naïve animals, the loperamide-oxymorphindole combination ED50 value was 10 times lower than the theoretical additive ED50 value whether given systemically or locally. In inflamed animals, the combination was 150 times more potent systemically, and 84 times more potent locally. All combinations showed statistically significant synergy when compared to the theoretical additive values, as verified by isobolographic analysis. The antihyperalgesia was ablated by a peripherally-restricted opioid antagonist. CONCLUSIONS: From these data we conclude that the loperamide-oxymorphindole combination synergistically reverses complete Freund's adjuvant-induced inflammatory hyperalgesia. The authors also conclude that this interaction is mediated by opioid receptors located in the peripheral nervous system.

Interleukin-8 as a therapeutic target for chronic low back pain: Upregulation in human cerebrospinal fluid and pre-clinical validation with chronic reparixin in the SPARC-null mouse model.

BACKGROUND: Low back pain (LBP) is the leading global cause of disability and is associated with intervertebral disc degeneration (DD) in some individuals. However, many adults have DD without LBP. Understanding why DD is painful in some and not others may unmask novel therapies for chronic LBP. The objectives of this study were to a) identify factors in human cerebrospinal fluid (CSF) associated with chronic LBP and b) examine their therapeutic utility in a proof-of-concept pre-clinical study. METHODS: Pain-free human subjects without DD, pain-free human subjects with DD, and patients with chronic LBP linked to DD were recruited and lumbar MRIs, pain and disability levels were obtained. CSF was collected and analyzed by multiplex cytokine assay. Interleukin-8 (IL-8) expression was confirmed by ELISA in CSF and in intervertebral discs. The SPARC-null mouse model of progressive, age-dependent DD and chronic LBP was used for pre-clinical validation. Male SPARC-null and control mice received systemic Reparixin, a CXCR1/2 (receptors for IL-8 and murine analogues) inhibitor, for 8 weeks. Behavioral signs of axial discomfort and radiating pain were assessed. Following completion of the study, discs were excised and cultured, and conditioned media was evaluated with a protein array. FINDINGS: IL-8 was elevated in CSF of chronic LBP patients with DD compared to pain-free subjects with or without DD. Chronic inhibition with reparixin alleviated low back pain behaviors and attenuated disc inflammation in SPARC-null mice. INTERPRETATION: These studies suggest that the IL-8 signaling pathway is a viable therapy for chronic LBP. FUND: Supported by NIH, MMF, CIHR and FRQS.

AAV-Mediated Gene Delivery to the Spinal Cord by Intrathecal Injection.

Gene therapy targeting the spinal cord is an important tool for analyzing mechanisms of nervous system diseases and the development of gene therapies. Analogous to a lumbar puncture in humans, the rodent spinal cord can be accessed through an efficient, noninvasive injection. Here we describe a method for AAV-mediated gene transfer to cells of the spinal cord by intrathecal injection of small quantities of AAV vector.

Detailed Method for Intrathecal Delivery of Gene Therapeutics by Direct Lumbar Puncture in Mice.

Delivery of viral vectors directly into the central nervous system (CNS) has emerged as an important tool for the refinement of gene therapy. Intrathecal delivery by direct lumbar puncture in conscious rodents offers a minimally invasive approach that avoids tissue damage and/or destruction. Here we describe delivery of small quantities of viral vector product to the intrathecal space of rodents via direct lumbar puncture aided by a catheter.

Agmatine preferentially antagonizes GluN2B-containing N-methyl-d-aspartate receptors in spinal cord.

The role of the N-methyl-d-aspartate receptor (NMDAr) as a contributor to maladaptive neuroplasticity underlying the maintenance of chronic pain is well established. Agmatine, an NMDAr antagonist, has been shown to reverse tactile hypersensitivity in rodent models of neuropathic pain while lacking the side effects characteristic of global NMDAr antagonism, including sedation and motor impairment, indicating a likely subunit specificity of agmatine's NMDAr inhibition. The present study assessed whether agmatine inhibits subunit-specific NMDAr-mediated current in the dorsal horn of mouse spinal cord slices. We isolated NMDAr-mediated excitatory postsynaptic currents (EPSCs) in small lamina II dorsal horn neurons evoked by optogenetic stimulation of Nav1.8-containing nociceptive afferents. We determined that agmatine abbreviated the amplitude, duration, and decay constant of NMDAr-mediated EPSCs similarly to the application of the GluN2B antagonist ifenprodil. In addition, we developed a site-specific knockdown of the GluN2B subunit of the NMDAr. We assessed whether agmatine and ifenprodil were able to inhibit NMDAr-mediated current in the spinal cord dorsal horn of mice lacking the GluN2B subunit of the NMDAr by analysis of electrically evoked EPSCs. In control mouse spinal cord, agmatine and ifenprodil both inhibited amplitude and accelerated the decay kinetics. However, agmatine and ifenprodil failed to attenuate the decay kinetics of NMDAr-mediated EPSCs in the GluN2B-knockdown mouse spinal cord. The present study indicates that agmatine preferentially antagonizes GluN2B-containing NMDArs in mouse dorsal horn neurons. NEW & NOTEWORTHY Our study is the first to report that agmatine preferentially antagonizes the GluN2B receptor subunit of the N-methyl-d-aspartate (NMDA) receptor in spinal cord. The preferential targeting of GluN2B receptor is consistent with the pharmacological profile of agmatine in that it reduces chronic pain without the motor side effects commonly seen with non-subunit-selective NMDA receptor antagonists.

Defining and Managing Pain in Stroke and Traumatic Brain Injury Research.

Neurologic conditions such as stroke and traumatic brain injury are challenging conditions to study in humans. Animal models are necessary to uncover disease processes and develop novel therapies. When attempting to model these or other neurologic diseases, the accompanying anesthesia and analgesia create variables that are not part of the onset of the clinical disease in the human population but are critical components of the postinjury care both in humans and animals. To maximize model validity, researchers must consider whether the disease process or a novel therapy is being studied. Damage to the neurons of the brain or the spinal cord is not painful at the neural tissue itself, but alterations to nociceptive signaling along the pain pathway can induce chronic pain. In addition, trauma or surgery leading to the event is associated with damage to peripheral tissue. Inflammation is inextricably associated with tissue injury. Inflammation is known to evoke nociception in the periphery and drive long-term changes to neurons in the CNS. Analgesics and anesthetics alter these responses yet are required as part of humane animal care. Careful planning for effective drug administration consistent with the standard of care for humans and equivalent animal care is required.

Bivalent ligand that activates mu opioid receptor and antagonizes mGluR5 receptor reduces neuropathic pain in mice.

The mu opioid receptor (MOR) and metabotropic glutamate receptor 5 (mGluR5) are well-established pharmacological targets in the management of chronic pain. Both receptors are expressed in the spinal cord. MMG22, a bivalent ligand containing 2 pharmacophores separated by 22 atoms, which simultaneously activates MOR and antagonizes mGluR5, has been shown to produce potent reversal of tactile hypersensitivity in rodent models of lipopolysaccharide (LPS)-and bone cancer-induced chronic pain. This study assessed whether intrathecal MMG22 also is effective in reducing pain of neuropathic origin. Furthermore, we theorized that MMG22 should reduce hyperalgesia in nerve-injured mice in a manner consistent with a synergistic interaction between MOR and mGluR5. Several weeks after spared nerve injury, tactile hypersensitivity was reversed in mice by the intrathecal injection of MMG22 (0.01-10 nmol) but also by its shorter spacer analog, MMG10, with similar potency. The potencies of the bivalent ligands were 10- to 14-fold higher than those of the compounds upon which the bivalent structure was based, the MOR agonist oxymorphone and the mGluR5 antagonist MPEP. Coadministration of oxymorphone and MPEP demonstrated analgesic synergism, an interaction confirmed by isobolographic analysis. This study indicates that in the spared nerve injury-induced model of neuropathic pain, the 2 pharmacophores of the bivalent ligands MMG22 and MMG10 target MOR and mGluR5 as separate receptor monomers. The observed increase in the potency of MMG22 and MMG10, compared with oxymorphone and MPEP, may reflect the synergistic interaction of the 2 pharmacophores of the bivalent ligand acting at their respective separate receptor monomers.