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1.
Pain ; 159(11): 2277-2284, 2018 Nov.
Article in English | MEDLINE | ID: mdl-29994988

ABSTRACT

Recently, (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP), a newly designed µ-opioid receptor (MOR) agonist with a low pKa, has been shown to produce injury-restricted analgesia in models of inflammatory and postoperative pain, without exhibiting typical opioid side effects. Here, we investigated MOR binding of NFEPP in brain and dorsal root ganglia, pH in injured tissues, and the analgesic efficacy of NFEPP compared with fentanyl in a chronic constriction injury model of neuropathic pain, and in the acetic acid-induced abdominal writhing assay in rats. Binding experiments revealed significantly lower affinity of NFEPP compared with fentanyl at pH 7.4. In vivo, pH significantly dropped both at injured nerves after chronic constriction injury and in the abdominal cavity after acetic acid administration. Intravenous NFEPP as well as fentanyl dose-dependently diminished neuropathy-induced mechanical and heat hypersensitivity, and acetic acid-induced abdominal constrictions. In both models, NFEPP-induced analgesia was fully reversed by naloxone methiodide, a peripherally restricted opioid receptor antagonist, injected at the nerve injury site or into the abdominal cavity. Our results indicate that NFEPP exerts peripheral opioid receptor-mediated analgesia exclusively in damaged tissue in models of neuropathic and abdominal pain.


Subject(s)
Abdominal Pain/drug therapy , Neuralgia/drug therapy , Piperidines/therapeutic use , Receptors, Opioid, mu/agonists , Animals , Brain/cytology , Cell Membrane/drug effects , Disease Models, Animal , Ganglia, Spinal/cytology , Hydrogen-Ion Concentration , Hyperalgesia/drug therapy , Male , Pain Measurement/drug effects , Pain Threshold/drug effects , Protein Binding/drug effects , Rats , Rats, Wistar , Receptors, Opioid, mu/metabolism , Statistics, Nonparametric
2.
Sci Rep ; 8(1): 8965, 2018 06 12.
Article in English | MEDLINE | ID: mdl-29895890

ABSTRACT

Novel pain killers without adverse effects are urgently needed. Opioids induce central and intestinal side effects such as respiratory depression, sedation, addiction, and constipation. We have recently shown that a newly designed agonist with a reduced acid dissociation constant (pKa) abolished pain by selectively activating peripheral µ-opioid receptors (MOR) in inflamed (acidic) tissues without eliciting side effects. Here, we extended this concept in that pKa reduction to 7.22 was achieved by placing a fluorine atom at the ethylidene bridge in the parental molecule fentanyl. The new compound (FF3) showed pH-sensitive MOR affinity, [35S]-GTPγS binding, and G protein dissociation by fluorescence resonance energy transfer. It produced injury-restricted analgesia in rat models of inflammatory, postoperative, abdominal, and neuropathic pain. At high dosages, FF3 induced sedation, motor disturbance, reward, constipation, and respiratory depression. These results support our hypothesis that a ligand's pKa should be close to the pH of injured tissue to obtain analgesia without side effects.


Subject(s)
Analgesics , Drug Design , Neuralgia/drug therapy , Pain, Postoperative/drug therapy , Receptors, Opioid, mu/agonists , Analgesics/chemical synthesis , Analgesics/chemistry , Analgesics/pharmacology , Animals , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Male , Molecular Structure , Muramidase , Neuralgia/genetics , Neuralgia/metabolism , Neuralgia/pathology , Pain, Postoperative/genetics , Pain, Postoperative/metabolism , Pain, Postoperative/pathology , Peptide Fragments , Rats , Rats, Wistar , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism
3.
Front Pharmacol ; 9: 1478, 2018.
Article in English | MEDLINE | ID: mdl-30618766

ABSTRACT

Neuropathic pain often arises from damage to peripheral nerves and is difficult to treat. Activation of opioid receptors in peripheral sensory neurons is devoid of respiratory depression, sedation, nausea, and addiction mediated in the brain, and ameliorates neuropathic pain in animal models. Mechanisms of peripheral opioid analgesia have therefore gained interest, but the role of G protein-coupled inwardly rectifying potassium (Kir3) channels, important regulators of neuronal excitability, remains unclear. Whereas functional Kir3 channels have been detected in dorsal root ganglion (DRG) neurons in rats, some studies question their contribution to opioid analgesia in inflammatory pain models in mice. However, neuropathic pain can be diminished by activation of peripheral opioid receptors in mouse models. Therefore, here we investigated effects of the selective µ-opioid receptor (MOR) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) on potassium conductance in DRG neurons upon a chronic constriction injury (CCI) of the sciatic nerve in mice. For verification, we also tested human embryonic kidney (HEK) 293 cells transfected with MOR and Kir3.2. Using patch clamp, we recorded currents at -80 mV and applied voltage ramps in high extracellular potassium concentrations, which are a highly sensitive measures of Kir3 channel activity. We found a significantly higher rate of HEK cells responding with potassium channel blocker barium-sensitive inward current (233 ± 51 pA) to DAMGO application in transfected than in untransfected group, which confirms successful recordings of inward currents through Kir3.2 channels. Interestingly, DAMGO induced similar inward currents (178 ± 36-207 ± 56 pA) in 15-20% of recorded DRG neurons from naïve mice and in 4-27% of DRG neurons from mice exposed to CCI, measured in voltage clamp or voltage ramp modes. DAMGO-induced currents in naïve and CCI groups were reversed by barium and a more selective Kir3 channel blocker tertiapin-Q. These data indicate the coupling of Kir3 channels with MOR in mouse peripheral sensory neuron cell bodies, which was unchanged after CCI. A comparative analysis of opioid-induced potassium conductance at the axonal injury site and peripheral terminals of DRG neurons could clarify the role of Kir3 channel-MOR interactions in peripheral nerve injury and opioid analgesia.

4.
Elife ; 62017 07 04.
Article in English | MEDLINE | ID: mdl-28673386

ABSTRACT

Novel painkillers are urgently needed. The activation of opioid receptors in peripheral inflamed tissue can reduce pain without central adverse effects such as sedation, apnoea, or addiction. Here, we use an unprecedented strategy and report the synthesis and analgesic efficacy of the standard opioid morphine covalently attached to hyperbranched polyglycerol (PG-M) by a cleavable linker. With its high-molecular weight and hydrophilicity, this conjugate is designed to selectively release morphine in injured tissue and to prevent blood-brain barrier permeation. In contrast to conventional morphine, intravenous PG-M exclusively activated peripheral opioid receptors to produce analgesia in inflamed rat paws without major side effects such as sedation or constipation. Concentrations of morphine in the brain, blood, paw tissue, and in vitro confirmed the selective release of morphine in the inflamed milieu. Thus, PG-M may serve as prototype of a peripherally restricted opioid formulation designed to forego central and intestinal side effects.


Subject(s)
Analgesia/methods , Analgesics/pharmacology , Analgesics/pharmacokinetics , Glycerol/pharmacology , Glycerol/pharmacokinetics , Morphine/pharmacology , Morphine/pharmacokinetics , Polymers/pharmacology , Polymers/pharmacokinetics , Analgesics/chemistry , Animal Structures/chemistry , Animals , Glycerol/chemistry , Hydrophobic and Hydrophilic Interactions , Molecular Weight , Morphine/chemistry , Polymers/chemistry , Rats
5.
ACS Chem Neurosci ; 8(8): 1638-1640, 2017 08 16.
Article in English | MEDLINE | ID: mdl-28603962

ABSTRACT

Conventional opioids mediate analgesia as well as severe adverse effects via G-protein coupled opioid receptors (OR) in both inflamed (peripheral injured tissue) and healthy (brain, intestinal wall) environments. To exclude side effects, OR activation can be selectively achieved in damaged tissue by lowering the pKa of an opioid ligand to the acidic pH of inflammation. As a result, protonation of the ligand and consequent OR binding and activation of G-proteins is pH- and injury-specific. A novel compound (NFEPP) demonstrates the feasibility of this approach and displays blockade of pain transmission only at the peripheral site of injury, but with lack of central and gastrointestinal adverse effects. These findings suggest disease-specific receptor activation as a new strategy in drug design.


Subject(s)
Analgesics, Opioid/adverse effects , Analgesics, Opioid/therapeutic use , Receptors, Opioid/metabolism , Analgesics, Opioid/pharmacology , Animals , Drug Design , Humans
6.
Biotechnol Bioeng ; 114(10): 2328-2338, 2017 10.
Article in English | MEDLINE | ID: mdl-28574582

ABSTRACT

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein-coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell-free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell-free system, performed within a short time and in a cost-effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect-based cell-free system. Moreover, we have chosen the µ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell-free synthesized MOR in comparison to MOR expressed in a human cell line by "one-point" radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328-2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.


Subject(s)
Cell Fractionation/methods , Genetic Enhancement/methods , Insecta/metabolism , Protein Engineering/methods , Receptors, G-Protein-Coupled/biosynthesis , Animals , Cell-Free System/chemistry , Cell-Free System/metabolism , HEK293 Cells , Humans , Insecta/chemistry , Receptors, G-Protein-Coupled/chemistry
7.
Expert Opin Investig Drugs ; 26(2): 155-160, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28001096

ABSTRACT

INTRODUCTION: Opioids are widely used to treat severe pain. Most clinically used opioids activate µ-opioid receptors (MOR). Their ligands induce potent analgesia but also adverse effects. The δ-opioid receptor (DOR) is another member of the opioid receptor family that has been under intense investigation with the aim to avoid MOR-induced side effects. Areas covered: This article reviews DOR ligands which appeared to be promising after preclinical evaluation. A literature search using Pubmed, Cochrane library, ClinicalTrials.gov, EudraCT, AdisInsight database and EBSCO Online Library was conducted. Out of numerous newly synthesized molecules, only few candidates entered phase I and/or II clinical investigation. The publicly accessible results are presented here. Expert opinion: Many compounds showed potent DOR-specific pain inhibition in preclinical studies. ADL5859 and ADL5747 entered clinical trials and successfully passed phase I. However, in phase II studies the primary endpoint (pain reduction) was not met and further investigation was terminated. A third compound, NP2, is in phase II clinical evaluation and results are pending. These findings suggest a potential of DOR ligands according to preclinical studies. Further clinical research and secondary analysis of unpublished data is needed to identify molecules which are useful in humans.


Subject(s)
Analgesics, Opioid/therapeutic use , Pain/drug therapy , Receptors, Opioid, delta/agonists , Analgesics, Opioid/adverse effects , Analgesics, Opioid/pharmacology , Animals , Benzamides/pharmacology , Benzamides/therapeutic use , Benzopyrans/pharmacology , Benzopyrans/therapeutic use , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Drug Design , Humans , Pain/physiopathology , Spiro Compounds/pharmacology , Spiro Compounds/therapeutic use
8.
Neuropharmacology ; 101: 330-40, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26453963

ABSTRACT

Targeting peripheral neuropathic pain at its origin may prevent the development of hypersensitivity. Recently we showed this can be mediated by opioid receptors at the injured nerve trunk. Here, we searched for the most relevant peripheral site to block transient receptor potential vanilloid 1 (TRPV1), and investigated analgesic interactions between TRPV1 and opioids in neuropathy. In a chronic constriction injury (CCI) of the sciatic nerve in mice, we assessed the effects of µ-, δ- and κ-opioid receptor agonists and TRPV1 antagonist (SB366791) injected at the CCI site or into the injured nerve-innervated paw on spontaneous paw lifting, heat and mechanical sensitivity. We also examined TRPV1 expression in total membrane and plasma membrane fractions from nerves and paws. We found that opioids and SB366791 co-injected in per se nonanalgesic doses at the CCI site or into the paw diminished heat and mechanical sensitivity. SB366791 alone dose-dependently alleviated heat and mechanical sensitivity. TRPV1 blockade in the paw was more effective than at the CCI site. None of the treatments diminished spontaneous paw lifting. TRPV1 expression analysis suggests that the levels of functional TRPV1 do not critically determine the TRPV1 antagonist-mediated analgesia. Together, the identification of the primary action site in damaged nerves is crucial for effective pain control. Contrary to opioids, the TRPV1 blockade in the injured nerve peripheral terminals, rather than at the nerve trunk, appears promising against heat pain. Opioid/TRPV1 antagonist combinations at both locations partially reduced neuropathy-triggered heat and mechanical pain.


Subject(s)
Analgesics, Opioid/therapeutic use , Neuralgia/drug therapy , TRPV Cation Channels/metabolism , Analysis of Variance , Anilides/therapeutic use , Animals , Arabidopsis Proteins , Cinnamates/therapeutic use , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Combinations , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Gene Expression Regulation/drug effects , Hyperalgesia/complications , Hyperalgesia/drug therapy , Male , Mice , Mice, Inbred C57BL , Nociception/drug effects , Nuclear Proteins , Pain Measurement/drug effects , Pain Threshold/drug effects , TRPV Cation Channels/genetics , Time Factors
9.
Methods Mol Biol ; 1230: 187-96, 2015.
Article in English | MEDLINE | ID: mdl-25293326

ABSTRACT

As the activation of opioid receptors leads to the modulation of potassium and calcium channels, the ion imaging represents an attractive method to analyze the function of the receptors. Here, we describe the imaging of potassium using the FluxOR™ potassium ion channel assay, and of calcium using Fura-2 acetoxymethyl ester. Specifically, we (1) characterize the activation of the G-protein-coupled inwardly rectifying potassium 2 channel by agonists of µ- and δ-opioid receptors with the aid of the FluxOR™ assay in cultured mouse dorsal root ganglion neurons, and (2) describe calcium imaging protocols to measure capsaicin-induced transient receptor potential vanilloid 1 channel activity during opioid withdrawal in transfected human embryonic kidney 293 cells.


Subject(s)
Calcium/analysis , Potassium/analysis , Receptors, Opioid/isolation & purification , Animals , Calcium Channels/chemistry , Ganglia, Spinal/metabolism , Humans , Mice , Molecular Biology/methods , Neurons/chemistry , Neurons/metabolism , Potassium Channels/chemistry , Receptors, Opioid/genetics
10.
Methods Mol Biol ; 1230: 197-211, 2015.
Article in English | MEDLINE | ID: mdl-25293327

ABSTRACT

The patch clamp is a valuable electrophysiological technique, which allows the study of single or multiple ion channels in cells, and it is particularly useful in testing the excitable cells such as neurons. Activation of neuronal opioid receptors results in the modulation of various ion channels, which enables to examine the receptors' action with the patch clamp. In this chapter, we analyze the activation of the G-protein-coupled inwardly rectifying potassium channel 2 by opioids, and the capsaicin-induced transient receptor potential vanilloid 1 channel currents during opioid withdrawal, using the whole cell patch clamp in transfected human embryonic kidney 293 cells as well as in mouse and rat primary dorsal root ganglion neurons.


Subject(s)
Analgesics, Opioid/administration & dosage , Electrophysiology/methods , Patch-Clamp Techniques/methods , Receptors, Opioid/metabolism , Animals , Capsaicin/administration & dosage , Ganglia, Spinal/metabolism , HEK293 Cells , Humans , Membrane Potentials/drug effects , Mice , Potassium Channels/metabolism , Rats , Receptors, Opioid/isolation & purification , TRPV Cation Channels/metabolism
11.
Mol Pharmacol ; 85(2): 335-44, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24275229

ABSTRACT

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ligand-gated cation channel responding to noxious heat, protons, and chemicals such as capsaicin. TRPV1 is expressed in sensory neurons and plays a critical role in pain associated with tissue injury, inflammation, and nerve lesions. Transient receptor potential ankyrin 1 (TRPA1) is coexpressed with TRPV1. It is activated by compounds that cause a burning sensation (e.g., mustard oil) and, indirectly, by components of the inflammatory milieu eliciting nociceptor excitation and pain hypersensitivity. Previous studies indicate an interaction of TRPV1 and TRPA1 signaling pathways. Here we sought to examine the molecular mechanisms underlying such interactions in nociceptive neurons. We first excluded physical interactions of both channels using radioligand binding studies. By microfluorimetry, electrophysiological experiments, cAMP measurements, and site-directed mutagenesis we found a sensitization of TRPV1 after TRPA1 stimulation with mustard oil in a calcium and cAMP/protein kinase A (PKA)-dependent manner. TRPA1 stimulation enhanced TRPV1 phosphorylation via the putative PKA phosphorylation site serine 116. We also detected calcium-sensitive increased TRPV1 activity after TRPA1 activation in dorsal root ganglion neurons. The inhibition of TRPA1 by HC-030031 (1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide) after its initial stimulation (and the calcium-insensitive TRPA1 mutant D477A) still showed increased capsaicin-induced TRPV1 activity. This excludes a calcium-induced additive TRPA1 current after TRPV1 stimulation. Our study shows sensitization of TRPV1 via activation of TRPA1, which involves adenylyl cyclase, increased cAMP, subsequent translocation and activation of PKA, and phosphorylation of TRPV1 at PKA phosphorylation residues. This suggests that cross-sensitization of TRP channels contributes to enhanced pain sensitivity in inflamed tissues.


Subject(s)
Calcium Channels/physiology , Nerve Tissue Proteins/physiology , TRPV Cation Channels/physiology , Transient Receptor Potential Channels/physiology , Adenylyl Cyclases/physiology , Calcium/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , HEK293 Cells , Humans , Phosphorylation , TRPA1 Cation Channel
12.
Pain ; 154(4): 598-608, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23398938

ABSTRACT

Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via µ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia.


Subject(s)
Analgesics, Opioid/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Sensory Receptor Cells/drug effects , TRPV Cation Channels/metabolism , Animals , Calcium/metabolism , Capsaicin/adverse effects , Cells, Cultured , Cyclic AMP/metabolism , Disease Models, Animal , Diterpenes/pharmacokinetics , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacokinetics , Enzyme Inhibitors/pharmacology , Fentanyl/pharmacology , Ganglia, Spinal/cytology , Humans , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Male , Membrane Potentials/drug effects , Membrane Potentials/genetics , Morphine/pharmacology , Mutagenesis, Site-Directed , Protein Binding/drug effects , Rats , Receptors, Opioid, mu/metabolism , Sensory Receptor Cells/metabolism , Substance Withdrawal Syndrome/metabolism , TRPV Cation Channels/genetics , Tritium/pharmacokinetics
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