RESUMO
Aberrations in spinal glycinergic signaling are a feature of pain chronification. Normalizing these changes by inhibiting glycine transporter (GlyT)-2 is a promising treatment strategy. However, existing GlyT2 inhibitors (e.g., ORG25543) are limited by narrow therapeutic windows and severe dose-limiting side effects, such as convulsions, and are therefore poor candidates for clinical development. Here, intraperitoneally administered oleoyl-D-lysine, a lipid-based GlyT2 inhibitor, was characterized in mouse models of acute (hot plate), inflammatory (complete Freund's adjuvant), and chronic neuropathic (chronic constriction injury) pain. Side effects were also assessed on a numerical rating score, convulsions score, for motor incoordination (rotarod), and for respiratory depression (whole body plethysmography). Oleoyl-D-lysine produced near complete antiallodynia for chronic neuropathic pain, but no antiallodynia/analgesia in inflammatory or acute pain. No side effects were seen at the peak analgesic dose, 30 mg/kg. Mild side effects were observed at the highest dose, 100 mg/kg, on the numerical rating score, but no convulsions. These results contrasted markedly with ORG25543, which reached less than 50% reduction in allodynia score only at the lethal/near-lethal dose of 50 mg/kg. At this dose, ORG25543 caused maximal side effects on the numerical rating score and severe convulsions. Oleoyl-D-lysine (30 mg/kg) did not cause any respiratory depression, a problematic side effect of opiates. These results show the safe and effective reversal of neuropathic pain in mice by oleoyl-D-lysine and provide evidence for a distinct role of glycine in chronic pain over acute or short-term pain conditions. SIGNIFICANCE STATEMENT: Partially inhibiting glycine transporter (GlyT)-2 can alleviate chronic pain by restoring lost glycinergic function. Novel lipid-based GlyT2 inhibitor ol-D-lys is safe and effective in alleviating neuropathic pain, but not inflammatory or acute pain. Clinical application of GlyT2 inhibitors may be better suited to chronic neuropathic pain over other pain aetiologies.
Assuntos
Dor Aguda , Dor Crônica , Neuralgia , Insuficiência Respiratória , Animais , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Glicina , Hiperalgesia/tratamento farmacológico , Lipídeos , Lisina/farmacologia , Lisina/uso terapêutico , Masculino , Camundongos , Neuralgia/tratamento farmacológico , Insuficiência Respiratória/induzido quimicamente , Insuficiência Respiratória/tratamento farmacológicoRESUMO
An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.
Assuntos
Analgésicos Opioides/farmacologia , Proteínas Fúngicas/farmacologia , Oligopeptídeos/farmacologia , Penicillium/química , Receptores Opioides mu/agonistas , Analgésicos Opioides/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas Fúngicas/química , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Oligopeptídeos/química , Ligação Proteica , Receptores Opioides mu/química , Receptores Opioides mu/metabolismoRESUMO
Changes in ion channel function and expression are characteristic of neuropathic pain. Voltage-gated calcium channels (VGCCs) are integral for neurotransmission and membrane excitability, but relatively little is known about changes in their expression after nerve injury. In this study, we investigate whether peripheral nerve ligation is followed by changes in the density and proportion of high-voltage-activated (HVA) VGCC current subtypes in dorsal root ganglion (DRG) neurons, the contribution of presynaptic N-type calcium channels in evoked excitatory postsynaptic currents (EPSCs) recorded from dorsal horn neurons in the spinal cord, and the changes in expression of mRNA encoding VGCC subunits in DRG neurons. Using C57BL/6 mice [8- to 11-wk-old males (n = 91)] for partial sciatic nerve ligation or sham surgery, we performed whole cell patch-clamp recordings on isolated DRG neurons and dorsal horn neurons and measured the expression of all VGCC subunits with RT-PCR in DRG neurons. After nerve injury, the density of P/Q-type current was reduced overall in DRG neurons. There was an increase in the percentage of N-type and a decrease in that of P/Q-type current in medium- to large-diameter neurons. No changes were found in the contribution of presynaptic N-type calcium channels in evoked EPSCs recorded from dorsal horn neurons. The α2δ-1 subunit was upregulated by 1.7-fold and γ-3, γ-2, and ß-4 subunits were all downregulated 1.7-fold in injured neurons compared with sham-operated neurons. This comprehensive characterization of HVA VGCC subtypes in mouse DRG neurons after nerve injury revealed changes in N- and P/Q-type current proportions only in medium- to large-diameter neurons.
Assuntos
Adaptação Fisiológica , Canais de Cálcio/metabolismo , Potenciais Pós-Sinápticos Excitadores , Gânglios Espinais/metabolismo , Neurônios Aferentes/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Animais , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Aferentes/classificação , Neurônios Aferentes/fisiologia , Especificidade de Órgãos , Traumatismos dos Nervos Periféricos/fisiopatologiaRESUMO
BACKGROUND: The current pharmacological treatments for chronic pain are limited. The first analgesic drug approved for clinical use in decades that has a novel molecular target is the synthetic version of a naturally occurring conotoxin. Several conotoxins that target ion channels have progressed to clinical trials for the relief of pain. Vc1.1 and RgIA are analgesic α-conotoxins that target α9-subunit-containing nicotinic acetylcholine receptors (α9-nAChR) as well as GABAB receptor mechanisms. However, the evidence for the involvement of α9-nAChRs in pain is controversial. In the present study, the role of the α9-nAChR in pain was assessed using a battery of behavioural pain tests and pain models in α9-nAChR knockout (KO) mice. RESULTS: α9-nAChR KO mice showed normal responses to acute noxious thermal and mechanical stimuli, and developed normal chronic cold and mechanical allodynia in inflammatory and nerve injury pain models. However, KO animals developed mechanical hyperalgesia to a lesser extent than their wild type (WT) counterparts in both inflammatory and neuropathic pain models. Chronic neuropathic pain is sustained in WT mice for at least 21 days post injury, while KO mice show significant recovery by 14 days post injury. KO sham mice were also resistant to the repeated-measures effect of the noxious pain test that caused a gradual onset of mild mechanical hyperalgesia in WT sham animals. CONCLUSIONS: The α9-nAChR is not involved in acute pain perception or chronic thermal or mechanical allodynia or thermal hyperalgesia but does contribute to the intensity and duration of chronic mechanical hyperalgesia, suggesting that pain-relieving actions of antagonists that target this site may be restricted to high threshold mechanosensation. The α9-nAChR appears to be a valid target for pharmacological compounds that alleviate long-term mechanical hyperalgesia and may be of use as a prophylactic drug to prevent the development of some symptoms of chronic pain.
Assuntos
Hiperalgesia/genética , Limiar da Dor/fisiologia , Receptores Nicotínicos/metabolismo , Animais , Dor Crônica/complicações , Dor Crônica/genética , Temperatura Baixa , Constrição , Modelos Animais de Doenças , Adjuvante de Freund/toxicidade , Lateralidade Funcional/genética , Temperatura Alta/efeitos adversos , Hiperalgesia/etiologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Knockout , Neuralgia/complicações , Medição da Dor , Receptores Nicotínicos/deficiência , Fatores de Tempo , TatoRESUMO
BACKGROUND: Antagonists of N-type voltage-gated calcium channels (VGCC), Ca(v)2.2, can manage severe chronic pain with intrathecal use and may be effective systemically. A series of novel ω-conotoxins that selectively inhibit N-type VGCCs was isolated from Conus catus. In the present study, the potency and reversibility of ω-conotoxins CVID, CVIE and CVIF to inhibit N-type calcium currents were investigated in mouse isolated dorsal root ganglion (DRG) neurons. The systemic potency of each ω-conotoxin to reverse signs of mouse chronic inflammatory pain was also compared. RESULTS: In DRG neurons, the rank order of potency to inhibit N-type calcium currents was CVIE > CVIF > CVID. After subcutaneous administration, CVID and CVIE, but not CVIF, partially reversed impaired weight bearing in mice injected with Freund's complete adjuvant (CFA) three days prior to testing. No side-effects associated with systemic administration of ω-conotoxins were observed. CONCLUSIONS: The present study indicates a potential for CVID and CVIE to be developed as systemically active analgesics with no accompanying neurological side-effects.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo N/metabolismo , Dor/tratamento farmacológico , ômega-Conotoxinas/uso terapêutico , Analgésicos/administração & dosagem , Analgésicos/uso terapêutico , Animais , Bloqueadores dos Canais de Cálcio/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor/metabolismo , Técnicas de Patch-Clamp , ômega-Conotoxinas/administração & dosagemRESUMO
Dysfunction at glutamatergic synapses has been proposed as a mechanism in the development of neuropathic pain. Here we sought to determine whether peripheral nerve injury-induced neuropathic pain results in functional changes to primary afferent synapses. Signs of neuropathic pain as well as an induction of glial fibrillary acidic protein in immunostained spinal cord sections 4 days after partial ligation of the sciatic nerve indicated the induction of neuropathic pain. We found that following nerve injury, no discernable change to kinetics of dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) or N-methyl-d-aspartate receptor (NMDAR)-mediated evoked excitatory postsynaptic currents (eEPSCs) could be observed in dorsal horn (lamina I/II) neurons compared with those of naïve mice. However, we did find that nerve injury was accompanied by slowed decay of the early phase of eEPSCs in the presence of glutamate transporter inhibition by the competitive nontransportable inhibitor dl-threo-ß-benzyloxyaspartic acid (TBOA). Concomitantly, expression patterns for the two major glutamate transporters in the spinal cord, excitatory amino acid transporters (EAAT) 1 and EAAT2, were found to be reduced at this time (4 days postinjury). We then sought to directly determine whether nerve injury results in glutamate spillover to NMDARs at dorsal horn synapses. By employing the use-dependent NMDAR blocker (±)MK-801 to block subsynaptic receptors, we found that although TBOA-induced spillover to extrasynaptic receptors trended to increased activation of these receptors after nerve injury, this was not significant compared with naïve mice. Together, these results suggest the development of neuropathic pain involves subtle changes to glutamate transporter expression and function that could contribute to neuropathic pain during excessive synaptic activity.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Regulação da Expressão Gênica/fisiologia , Ciática/metabolismo , Ciática/patologia , Medula Espinal/metabolismo , Análise de Variância , Animais , Área Sob a Curva , Ácido Aspártico/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
Animal models of human pain conditions allow for detailed interrogation of known and hypothesized mechanisms of pain physiology in awake, behaving organisms. The importance of the glycinergic system for pain modulation is well known; however, manipulation of this system to treat and alleviate pain has not yet reached the sophistication required for the clinic. Here, we review the current literature on what animal behavioral studies have allowed us to elucidate about glycinergic pain modulation, and the progress toward clinical treatments so far. First, we outline the animal pain models that have been used, such as nerve injury models for neuropathic pain, chemogenic pain models for acute and inflammatory pain, and other models that mimic painful human pathologies such as diabetic neuropathy. We then discuss the genetic approaches to animal models that have identified the crucial glycinergic machinery involved in neuropathic and inflammatory pain. Specifically, two glycine receptor (GlyR) subtypes, GlyRα1(ß) and GlyRα3(ß), and the two glycine transporters (GlyT), GlyT1 and GlyT2. Finally, we review the different pharmacological approaches to manipulating the glycinergic system for pain management in animal models, such as partial vs. full agonism, reversibility, and multi-target approaches. We discuss the benefits and pitfalls of using animal models in drug development broadly, as well as the progress of glycinergic treatments from preclinical to clinical trials.
RESUMO
The midbrain ventral tegmental area (VTA) contains neurons largely with either a dopaminergic (DAergic) or GABAergic phenotype. Physiological and pharmacological properties of DAergic neurons have been determined using tyrosine hydroxylase (TH) immunohistochemistry but many properties overlap with non-DAergic neurons presumed to be GABAergic. This study examined properties of GABAergic neurons, non-GABAergic neurons and TH-immunopositive neurons in VTA of GAD67-GFP knock-in mice. Ninety-eight per cent of VTA neurons were either GAD-GFP or TH positive,with the latter being five times more abundant. During cell-attached patch-clamp recordings, GAD-GFP neurons fired brief action potentials that could be completely distinguished from those of non-GFP neurons. Pharmacologically, the µ-opioid agonist DAMGO inhibited firing of action potentials in 92% of GAD-GFP neurons but had no effect in non-GFP neurons. By contrast, dopamine invariably inhibited action potentials in non-GFP neurons but only did so in 8% of GAD-GFP neurons. During whole-cell recordings, the narrower width of action potential in GAD-GFP neurons was also evident but there was considerable overlap with non-GFP neurons. GAD-GFP neurons invariably failed to exhibit the potassium-mediated slow depolarizing potential during injection of positive current that was present in all non-GFP neurons. Under voltage-clamp the cationic current, I(h), was found in both types of neurons with considerable overlap in both amplitude and kinetics. These distinct cellular properties may thus be used to confidently discriminate GABAergic and DAergic neurons in VTA during in vitro electrophysiological recordings.
Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios GABAérgicos/citologia , Área Tegmentar Ventral/citologia , Potenciais de Ação/efeitos dos fármacos , Animais , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Eletrofisiologia/métodos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Neurônios GABAérgicos/metabolismo , Técnicas de Introdução de Genes/métodos , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Camundongos , Técnicas de Patch-Clamp/métodos , Potássio/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
Inhibitors that target the glycine transporter 2, GlyT2, show promise as analgesics, but may be limited by their toxicity through complete or irreversible binding. Acyl-glycine inhibitors, however, are selective for GlyT2 and have been shown to provide analgesia in animal models of pain with minimal side effects, but are comparatively weak GlyT2 inhibitors. Here, we modify the simple acyl-glycine by synthesizing lipid analogues with a range of amino acid head groups in both l- and d-configurations, to produce nanomolar affinity, selective GlyT2 inhibitors. The potent inhibitor oleoyl-d-lysine (33) is also resistant to degradation in both human and rat plasma and liver microsomes, and is rapidly absorbed following an intraperitoneal injection to rats and readily crosses the blood-brain barrier. We demonstrate that 33 provides greater analgesia at lower doses, and does not possess the severe side effects of the very slowly reversible GlyT2 inhibitor, ORG25543 (2).
Assuntos
Aminoácidos/uso terapêutico , Analgésicos/uso terapêutico , Dor Crônica/prevenção & controle , Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Aminoácidos/química , Aminoácidos/farmacocinética , Animais , Barreira Hematoencefálica , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Meia-Vida , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The α9α10-subtype of nicotinic acetylcholine receptor (nAChR) has recently garnered interest in biomedicine and is being pursued as an analgesic target. However, the receptor exhibits diverse tissue distribution, the function of which is known to varying degrees, and targeting this receptor for clinical treatments without a broad understanding of its function may have adverse consequences. The α9α10-nAChR is expressed in the adrenal and pituitary glands, suggesting a potential role in the stress response, but little is known about its function in this tissue. Here we determined a role for the α9α10-nAChR in behavioural and physiological stress responses, by comparing the stress- and affect-related phenotypes of wildtype and α9-nAChR knockout mice. Naïve knockout mice exhibited largely normal behaviour on standard tests of affective behaviour. However, after sub-chronic restraint stress knockout mice showed significantly decreased stress-induced arousal and increased anxiety-like behaviour when compared to wildtype animals. Physiologically, corticosterone responses were muted in knockout mice after an acute stressor, but exaggerated in response to the same stressor after undergoing sub-chronic stress. Behavioural profiling of the α9-nAChR knockout mice in the home-cage revealed that circadian patterns of activity were altered when compared to wildtype controls. Furthermore, knockout mice showed altered responses to a period of reward discounting, resulting in anhedonia-like behaviour in a sucrose preference test where WT mice continued to seek reward. These experiments uncover a novel role for the α9α10-nAChR in mounting a normal stress response and in the regulation of affective- and reward-related behaviour, and suggest that pursuing the receptor for clinical treatments may not be as straightforward as has been suggested.
Assuntos
Afeto/fisiologia , Receptores Nicotínicos/deficiência , Recompensa , Estresse Psicológico/metabolismo , Anedonia/fisiologia , Animais , Ansiedade/metabolismo , Nível de Alerta/fisiologia , Ritmo Circadiano/fisiologia , Corticosterona/sangue , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Receptores Nicotínicos/genéticaRESUMO
The development of neuropathic pain involves persistent changes in signalling within pain pathways. Reduced inhibitory signalling in the spinal cord following nerve-injury has been used to explain sensory signs of neuropathic pain but specific circuits that lose inhibitory input have not been identified. This study shows a specific population of spinal cord interneurons, radial neurons, lose glycinergic inhibitory input in a rat partial sciatic nerve ligation (PNL) model of neuropathic pain. Radial neurons are excitatory neurons located in lamina II of the dorsal horn, and are readily identified by their morphology. The amplitude of electrically-evoked glycinergic inhibitory post-synaptic currents (eIPSCs) was greatly reduced in radial neurons following nerve-injury associated with increased paired-pulse ratio. There was also a reduction in frequency of spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSC) in radial neurons without significantly affecting mIPSC amplitude. A subtype selective receptor antagonist and western blots established reversion to expression of the immature glycine receptor subunit GlyRα2 in radial neurons after PNL, consistent with slowed decay times of IPSCs. This study has important implications as it identifies a glycinergic synaptic connection in a specific population of dorsal horn neurons where loss of inhibitory signalling may contribute to signs of neuropathic pain.
Assuntos
Neuralgia/metabolismo , Células do Corno Posterior/metabolismo , Receptores de Glicina/metabolismo , Nervo Isquiático/metabolismo , Transmissão Sináptica , Animais , Modelos Animais de Doenças , Masculino , Neuralgia/patologia , Células do Corno Posterior/patologia , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/patologiaRESUMO
The α9α10-nicotinic acetylcholine receptor (nAChR) has been implicated in pain and has been proposed to be a novel target for analgesics. However, the evidence to support the involvement of the α9α10-nAChR in pain is conflicted. This receptor was first implicated in pain with the characterisation of conotoxin Vc1.1, which is highly selective for α9α10-nAChRs and is an efficacious analgesic in chronic pain models with restorative capacities and no reported side effects. Numerous other analgesic conotoxin and non-conotoxin molecules have been subsequently characterised that also inhibit α9α10-nAChRs. However, there is evidence that α9α10-nAChR inhibition is neither necessary nor sufficient for analgesia. α9α10-nAChR-inhibiting analogues of Vc1.1 have no analgesic effects. Genetically-modified α9-nAChR knockout mice have a phenotype that is markedly different from the analgesic profile of Vc1.1 and similar conotoxins, suggesting that the conotoxin effects are largely independent of α9α10-nAChRs. Furthermore, an alternative mechanism of analgesia by Vc1.1 and other similar conotoxins involving non-canonical coupling of GABAB receptors to voltage-gated calcium channels is known. Additional incongruities regarding α9α10-nAChRs in analgesia are discussed. A more comprehensive characterisation of the role of α9α10-nAChRs in pain is crucial for understanding the analgesic action of conotoxins and for improved drug design.