From opium to analgesic tests: An introduction to the functioning and studying of the opioid system
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1 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP C-13/1/KONV From opium to analgesic tests: An introduction to the functioning and studying of the opioid system Ferenc Zádor Laboratory of Opioid Research Institute of Biochemistry
2 Introduction: Why study the opioid system?
3 Introduction: Why study the opioid system? Nutt et al., 2007, Lancet
4 Introduction: Why study the opioid system?
5 Introduction: The OxyContin story
6 Part I. What are opioids? Opium Opiates or opioids? Part II. How do they work? GPCRs in general Opioid receptors Endogenous opioids Part III. What do they do? Pain pathways Opioids and analgesia Opioids and addiction Opioids in medicine Part IV. What is the future? Improving the safety Future applications Biochemical assays Analgesic tests
7 Part I. What are opioids? Opium Opiates or opioids? Part II. How do they work? GPCRs in general Opioid receptors Endogenous opioids Part III. What do they do? Pain pathways Opioids and analgesia Opioids and addiction Opioids in medicine Part IV. What is the future? Improving the safety safety Future applications Biochemical assays Analgesic tests
8 Part I: Opium, opiates, opioids János Kabay Morphine Codeine Thebaine Fentanyl Met-enkephalin Heroin Naloxone
9 Part I. What are opioids? Opium Opiates or opioids? Part II. How do they work? GPCRs in general Opioid receptors Endogenous opioids Part III. What do they do? Pain pathways Opioids and analgesia Opioids and addiction Opioids in medicine Part IV. What is the future? Improving safety Future applications Biochemical assays Analgesic tests
10 Part II: G-protein coupled receptors in general ECL2 Extracellular space G α β 2 AR TM1 ~50 Å 90 G β TM5 H8 G γ BI G α G γ G β N ECL1 ECL2 ECL3 BI ICL3 ICL1 ICL2 8 C α β γ Lipid anchors Brian Kobilka Robert Lefkowitz
11 Part II: G-protein coupled receptors in general Venkatakrishnan et al., 2013, Nature
12 Part II: G-protein coupled receptors in general Venkatakrishnan et al., 2013, Nature
13 Part II: G-protein coupled receptors in general Agonist G α G β G γ GTP GDP Secondary messenger/effector protein
14 Part II: Opioid receptor signaling Opioid agonist Opioid agonist N Extracellular space N Extracellular space Adenylate cyclase Adenylate cyclase - GTP + α β γ C β ATP camp α β γ C α β ATP camp GDP Periaqeductal gray (midbrain) Substantia gelatinosa (spinal cord) Brainstem (respiration) Enteric neurons Biological response (%) Basal activity Agonist Log drug concentration
15 Part II: Opioid receptor signaling Opioid agonist N Extracellular space K+ Ca Adenylate cyclase β γ C α β ATP camp K+ Ca Analgesia Constipation (ACh) Respiratory depression
16 Part II: Opioid receptor signaling Opioid antagonist Opioid agonist N Opioid agonist Extracellular space N Opioid antagonist Extracellular space Adenylate cyclase Adenylate cyclase GTP α β γ C β ATP camp GTP α β γ C β ATP camp GDP GDP Biological response (%) Agonist Basal activity Antagonist Log drug concentration
17 Part II: Opioid receptor types μ (MOPr) δ (DOPr) κ (KOPr) NOPr Manglik et al., 2012 Granier et al., 2012 Wu et al., 2012 Thompson et al., 2012
18 Part II: Endogenous opioids
19 Part II: Endogenous opioids Morphine Met-enkephalin
20 Part II: Short summary Opioid receptors belong to the large GPCR superfamily GPCRs share several structural and functional similarities GPCRs have second messenger systems which forwards the signal of the bound ligand Opioid receptors overall inhibit the release of several neurotransmitters (GABA, serotonin) Three types of opioid receptors (classically), with overlaping functionalities Endogenous opioid peptides mainly act as hormones and share certain structural motifs with exogenous ligands
21 Part I. What are opioids? Opium Opiates or opioids? Part II. How do they work? GPCRs in general Opioid receptors Endogenous opioids Part III. What do they do? Pain pathways Opioids and analgesia Opioids and addiction Opioids in medicine Part IV. What is the future? Improving safety Future applications Biochemical assays Analgesic tests
22 Part III: The pain pathways
23 Part III: The opioid system and analgesia
24 Part III: Currently available classic opioid analgesics
25 Part III: Opioid use disorder
26 Part III: Opioid withdrawal and overdose symptoms
27 Part III: The reward system and addiction Prefrontal cortex Corpus callosum Pons Medulla oblongata Dopamine (3,4-dihydroxyphenethylamine) Nucleus accumbens Hippocampus Ventral tegmentum (ventral tegmental area) Cerebellum Substantia nigra
28 Part III: The reward system and opioids VTA NA Soma GABA receptor GABA Opioid receptor (mu type) Morphine/heroin VTA NA Soma GABA receptor - GABA Opioid receptor (mu type) Morphine/heroin
29 Part III: The traces of tolerance and addiction in the brain NA NA
30 Part III: The traces of tolerance and addiction in the brain
31 Part III: The consequences of addiction and its medication
32 Part III: Opioids for diarrhea, constipation and cough Loperamide Methylnaltrexone
33 Part III: Short summary The opioid system reduces pain signals both in the spinalcord and in the brain Opioid receptor activation results GABA, serotonin or noradrenaline neurotransmitter release inhibition in the descending pain pathways Opioid medications can cause serious side effects (opioid use disorder) Opioids can be highly addictive and can cause serious withdrawal symptoms The opioid system strongly interacts with the reward system and increases dopamine levels Medications targeting the opioid system can help to overcome withdrawal symptoms and addiction Opioids for diarrhea, constipation and as cough suppressants, due to the presence of the opioid system in the GI tract and respiratory control system
34 Part I. What are opioids? Opium Opiates or opioids? Part II. How do they work? GPCRs in general Opioid receptors Endogenous opioids Part III. What do they do? Pain pathways Opioids and analgesia Opioids and addiction Opioids in medicine Part IV. What is the future? Improving the safety Future applications Biochemical assays Analgesic tests
35 Part IV: Approaches to overcome the side-effects MOR binding and agonist activity Antinociceptive activity Limited BBB penetrance Limited side-effects Morphine-6-O-sulfate 14-O-methylmorphine-6-O-sulfate Lackó et al. Curr Med Chem, 2012 Morphine 14-O-methylmorphine Zádor et al. Eu J Pharmacol, 2017 MOR binding, selectivity and agonist activity Antinociceptive activity Promising results in neurophatic pain Dr. Mahmoud Al-Khrasani Department of Pharmacology and Pharmacotherapy, Semmelweis University Budapest, Hungary
36 Part IV: Approaches to overcome the side-effects Buprenorphine + naloxone Oxycodone + naloxone Analgesic, modified-release Significantly reduces constipation μ opioid receptor agonist AND norepinephrine reuptake inhibitor Tapentadol
37 Part IV: Approaches to overcome the side-effects Mollica et al. Chem Biol Drug, 2014 Opioid receptor agonist AND N-type voltage dependent Ca 2+ channel blocker Dr. Adriano Mollica University of Chieti-Pescara, Italy Zádor & Wollemann, Pharmacol Res, 2015
38 Part IV: Future plans Targeting endogenous opioid degrading enzymes (enkephalinase inhibitors) Appetite supression by opioid antagonists Treating anxiety and depression Against alcoholism
39 Part IV: How to study the opioid system 1 Binding affinity, selectivity and capacity with radiolabeled ligands N Extracellular space Adenylate cyclase - 3 Agonist activity with labeled camp GTP GDP α 2 β γ C β α ATP camp Agonist activity with radiolabeled GTP 4 Smooth muscle contraction inhibition Acethylcholine release inihibition Isolated mouse vasa deferentia, rodent intestines, guinea pig illeum Ligand characterization
40 Part IV: How to study the opioid system 5 Antinociception Tail-flick test Plantar test Hot-plate test Thermal pain Paw pressure test Mechanical pain
41 Part IV: Short summary Introducing new chemical groups to the opioid structures to improve the safety profile of opioids Combination therapy, multitarget compounds are also promising Further therapeutic applications are in developement We can study opioid receptors at the ligand binding and signaling level We can study thermal, mechanical or other pain types with animal behavior tests
42 Final summary 1 There are plant derived, half-synthetic and fully synthetic opioids, which mimic the molecular signitures of the endogenous opioids Opioid receptors are GPCRs, transferring the extracellular first messenger signal molecules (opioid ligands) through a molecular switch (G-protein) to second messenger systems (e.g. adenylate cyclase) Opioid ligands can bind to and activate opioid receptors (agonists) or bind to the receptor without triggering the signalling pathway (antagonists), the latter also blocks receptor activity Opioids reduce pain both in the brain and in the spinal cord by inhibiting the singals in the pain pathways through reducing the release of certain neurotransmitters (e.g. GABA, serotinin) Opioids are highly addictive, they strongly interact with the reward system and increase dopamine levels The opioid system can be targeted for many therapuetic applications apart from analgesia (e.g. addiction, bowel movements, cough etc.) The opioid system can be studied in multiple levels with biochemical assays and animal behavior tests
43 Contact information Ferenc Zádor Ph.D. Room: 233, 135 Phone: 580 Edina Szűcs Room: 235b, 135 Laboratory of Opioid Research Sándor Benyhe D.Sc. Head of research group Ferenc Ötvös Ph.D. Senior research associate Anna Erdei Ph.D. student Zsuzsa Benyhe Laboratory assistant
44 THANK YOU FOR YOUR ATTENTION! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practiceoriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMOP C-13/1/KONV project.
From opium to analgesic tests: An introduction to the functioning and studying of the opioid system
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