ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT. financed by the program

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keretében finanszírozott ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the

1 TÁMOP C-13/1/KONV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: NOVEMBER 2. / NOVEMBER 2, 2017 Helyszín / Place: MTA SZBK BIOFIZIKAI INTÉZET, TANÁCSTEREM / LECTURE ROOM, INST. OF BIOPHYSICS, BIOLOGICAL RESEARCH CENTRE SZEGED, TEMESVÁRI KRT. 62. Az előadás címe / Title of the presentation: FROM OPIUM TO ANALGESIC TESTS: AN INTRODUCTION TO THE FUNCTIONING AND STUDYING OF THE OPIOID SYSTEM Előadó / Speaker: FERENC ZÁDOR Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB

Practice-oriented, student-friendly modernization of the biomedical education for

functioning and studying of the opioid system Ferenc Zádor Laboratory of Opioid

2 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 Introduction: Why study the opioid system? Introduction: Why study the opioid system? Nutt et al., 2007, Lancet 1

3 Introduction: Why study the opioid system? Introduction: The OxyContin story 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 2

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?

4 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 the safety Future applications Biochemical assays Analgesic tests Part I: Opium, opiates, opioids János Kabay Morphine Codeine Thebaine Fentanyl Met-enkephalin Heroin Naloxone 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 3

Part II: G-protein coupled receptors in general

H8 G BI 167107 G G G N ECL1 ECL2 ECL3 BI-167107 1

5 Part II: G-protein coupled receptors in general ECL2 Extracellular space G 2 AR TM1 ~50 Å 90 G TM5 H8 G BI G G G N ECL1 ECL2 ECL3 BI ICL3 Brian Kobilka ICL1 ICL2 8 C β Lipid anchors 2012 Robert Lefkowitz Part II: G-protein coupled receptors in general Venkatakrishnan et al., 2013, Nature Part II: G-protein coupled receptors in general Venkatakrishnan et al., 2013, Nature 4

Part II: G-protein coupled receptors in general Agonist G G β G GTP GDP Secondary messenger/effector protein Part II: Opioid receptor signaling Opioid agonist Opioid agonist N Extracellular space N

6 Part II: G-protein coupled receptors in general Agonist G G β G GTP GDP Secondary messenger/effector protein Part II: Opioid receptor signaling Opioid agonist Opioid agonist N Extracellular space N Extracellular space Adenylate cyclase Adenylate cyclase GTP + α β C β ATP camp α β C β α ATP camp GDP Periaqeductal gray (midbrain) Substantia gelatinosa (spinal cord) Brainstem (respiration) Enteric neurons Biological response (%) 200 Agonist 150 Basal activity Log drug concentration Part II: Opioid receptor signaling Opioid agonist N Extracellular space K+ Ca Adenylate cyclase + β C β α ATP camp K+ Ca Analgesia Constipation (ACh) Respiratory depression 5

Part II: Opioid receptor signaling Opioid antagonist Opioid agonist N 1 2 3 4 5 6 7 Extracellular space Adenylate cyclase N Opioid agonist Extracellular Opioid antagonist space 1 2 3 4 5 6 7

7 Part II: Opioid receptor signaling Opioid antagonist Opioid agonist N Extracellular space Adenylate cyclase N Opioid agonist Extracellular Opioid antagonist space Adenylate cyclase GTP α β C β ATP camp GTP α β C β ATP camp GDP GDP Biological response (%) Agonist Basal activity Antagonist Log drug concentration 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 Part II: Endogenous opioids 6

Part II: Endogenous opioids Met-enkephalin Morphine Part II: Short summary Opioid receptors belong to the large GPCR superfamily GPCRs share several structural and functional similarities GPCRs have

(classically), with overlaping functionalities Endogenous opioid peptides mainly act as hormones and share certain structural motifs with exogenous ligands Part I. What are opioids?

8 Part II: Endogenous opioids Met-enkephalin Morphine 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 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 7

9 Part III: The pain pathways Part III: The opioid system and analgesia Part III: Currently available classic opioid analgesics 8

10 Part III: Opioid use disorder Part III: Opioid withdrawal and overdose symptoms Part III: The reward system and addiction Prefrontal cortex Corpus callosum Pons Medulla oblongata Dopamine (3,4-dihydroxyphenethylamine) Nucleus accumbens Hippocampus Ventral tegmentum Cerebellum (ventral tegmental area) Substantia nigra 9

11 Part III: The reward system and opioids VTA NA Soma GABA receptor GABA Opioid receptor (mu type) Morphine/heroi n NA Soma GABA receptor VTA GABA Opioid receptor (mu type) Morphine/heroi n Part III: The traces of tolerance and addiction in the brain NA NA Part III: The traces of tolerance and addiction in the brain 10

Part III: The consequences of addiction and its medication Part III: Opioids for diarrhea, constipation and cough Loperamide Methylnaltrexone Part III: Short summary The opioid system reduces pain

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

12 Part III: The consequences of addiction and its medication Part III: Opioids for diarrhea, constipation and cough Loperamide Methylnaltrexone 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 11

13 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 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 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 12

Part IV: Approaches to overcome the side-effects Mollica et al.

Adriano Mollica University of Chieti-Pescara, Italy Zádor & Wollemann, Pharmacol Res, 2015 Part IV: Future

antagonists Treating anxiety and depression Against alcoholism Part IV: How to study the opioid system N 1

GDP Adenylate cyclase C β α β α ATP camp 2 Agonist activity with radiolabeled GTP Smooth muscle contraction

14 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 Part IV: Future plans Targeting endogenous opioid degrading enzymes (enkephalinase inhibitors) Appetite supression by opioid antagonists Treating anxiety and depression Against alcoholism Part IV: How to study the opioid system N 1 Binding affinity, selectivity and capacity with radiolabeled ligands Extracellular space GTP GDP Adenylate cyclase C β α β α ATP camp 2 Agonist activity with radiolabeled GTP Smooth muscle contraction inhibition 3 Agonist activity with labeled camp Acethylcholine release inihibition Isolated mouse vasa deferentia, rodent intestines, guinea pig illeum Ligand characterization 13

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 Part IV: Short summary Introducing new chemical

study opioid receptors at the ligand binding and signaling level We can study thermal, mechanical or other pain types with animal behavior tests Final summary 1 2 3 4 5 6 7 There are plant derived,

15 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 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 Final summary 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 14

16 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 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. 15

From opium to analgesic tests: An introduction to the functioning and studying of the opioid system

From opium to analgesic tests: An introduction to the functioning and studying of the opioid system Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP-4.1.1.C-13/1/KONV-2014-0001 From