Program & Abstracts Table of Contents

Home , Norhydrocodone

International Narcotics Research Conference

Kansas City, MO USA July 15 – 20, 2012

INRC 2012 Committees 3

Acknowledgements 4

General Information 5

INRC Program 6

Poster Titles Monday, July 16 11 Wednesday, July 18 17

Program Abstracts Oral Presentations 22 Poster Presentations 47

Index 72

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Program Committee INRC Executive Committee

Chair Officers Thomas Prisinzano (USA) President: John Traynor Past-President: Lakshmi Devi Members Treasurer: Jean Bidlack Jane Aldrich (USA) Treasurer-Elect: Lawrence Toll Michael Bruchas (USA) Information: Craig Stevens Carmine Coscia (USA) Shuanglin Hao (USA) Members Christopher McCurdy (USA) Susan Ingram (USA) Elyssa Margolis (USA) Jay McLaughlin (USA) Jay McLaughlin (USA) Louis Gendron (Canada) Scott Runyon (USA) Ingrid Nylander (Sweden) Stephen Husbands (UK) Local Organizing Committee Graeme Milligan (UK) Thomas Prisinzano Lih-Chu Chiou (Taiwan) Jane Aldrich Elena Bagley (Australia) Jeffery Aubé Shiro Kishioka (Japan) Michael Bruchas Carmine Coscia Jean Bidlack (INRC Treasurer)

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Acknowledgements

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General Information

Badges Every registered participant will receive a name badge that must be worn to gain access to the scientific sessions and meals/coffee breaks onsite.

Registration Desk The registration desk will be located on the Mezzanine floor adjacent to the Ballroom on Sunday from 4:00 – 7:00 PM and Monday – Thursday 7:30 – 8:30 AM.

Meals Breakfast, coffee/snack breaks and lunches will be provided for all registered participants for the duration of the conference at the Sheraton Kansas City Hotel at Crown Center. For dinner you can experience Kansas City’s fantastic restaurants, some of which are located within walking distance of the Sheraton. Please check out our restaurant listings provided with the conference bag and use the concierge at the Sheraton Hotel for ideas and reservations.

Social Program Sunday, July 15 7 – 9 PM Opening Reception, Sheraton Kansas City Hotel

Tuesday, July 17 5 – 7 PM Data Blitz, Arthur Stillwell Room at Union Station

Wednesday, July Free time in the afternoon 18 Thursday, July 19 8 – 11 PM Banquet, Sheraton Kansas City Hotel

Visitor Information and Transportation Nicknamed “the Paris of the Plains,” Kansas City is particularly known for its jazz and its barbecue. Located in the southern part of downtown, the hotel is connected by an enclosed walkway to the sprawling Crown Center, which comprises dozens of shops, restaurants, theaters, and businesses. It is also home to the headquarters of Hallmark Cards, Inc., as well as Kaleidoscope, Hallmark’s creative workshop for children. Numerous restaurants and attractions are within walking distance, and downtown’s government and financial district is about 10 blocks away. On weekdays, the hotel operates complimentary shuttle service that can take you to government buildings, corporate offices, restaurants, and other attractions within 4 miles of the hotel.

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INRC 2012 Program

Sunday, July 15 4:00 – 7:00 PM Registration Sheraton Kansas City

7:00 – 9:00 PM Welcome Reception Sheraton Kansas City

Monday, July 16 7:00 – 8:15 AM Breakfast

8:15 – 8:30 Welcome and Announcements John Traynor, INRC President Jean Bidlack, INRC Treasurer Thomas Prisinzano, INRC Chair

8:30 – 9:30 Plenary Lecture (P1) Marc Caron, Duke University INTEGRATED APPROACHES TO UNDERSTAND THE ACTIONS OF GPCRS

9:30 – 12:30 Symposium #1 – Neuropeptidergic Targets for Addiction: Chemistry and Biology Organizer and Chair: Scott Runyon, Research Triangle Institute

9:30 – 9:55 Michael Bruchas, Washington University THE ORL1 – NOCICEPTIN SYSTEM: RECEPTOR TRAFFICKING, SIGNALING AND BEHAVIOR

9:55 – 10:15 Coffee Break

10:15 – 10:40 Jeffrey Aubé, University of Kansas NEW PROBES FOR THE KAPPA OPIOID RECEPTOR: SCREENING, CHEMISTRY, AND PHARMACOLOGY

10:40 – 11:05 Rainer Reinscheid, University of California at Irvine NEUROPEPTIDE S INFLUENCES MEMORY MECHANISMS THAT MAY AFFECT REINFORCEMENT LEARNING

11:05 – 11:40 Scott Runyon, Research Triangle Institute THE SEARCH FOR NEUROPEPTIDE S RECEPTOR LIGANDS AS TREATMENTS FOR SUBSTANCE ABUSE

11:40 – 12:05 Stewart Clark, SUNY Buffalo THE UROTENSIN II RECEPTOR IS EXPRESSED PRESYNAPTICALLY IN THE VTA BY CHOLINERGIC MESOPONTINE NEURONS

12:05 – 12:30 Yanan Zhang, Research Triangle Institute OREXIN RECEPTOR LIGANDS: IDENTIFICATION OF NEW TOOLS TO STUDY DRUG ABUSE

12:30 – 1:30 Lunch

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1:30 – 4:00 Symposium #2 – Glial Role in Opioid Analgesia, Tolerance and Dependence Organizers and Chairs: Shuanglin Hao, University of Miami, FL and Linda Watkins, University of Colorado at Boulder

1:30 – 2:00 Shuanglin Hao, University of Miami, FL ANTI-INFLAMMATORY CYTOKINES MEDIATED BY HSV VECTOR REDUCES MORPHINE TOLERANCE AND PHYSICAL WITHDRAWAL

2:00 – 2:30 Linda Watkins, University of Colorado at Boulder THE “TOLL” OF GLIAL ACTIVATION BY OPIOIDS: ENHANCING THE CLINICAL EFFICACY OF OPIOIDS BY BLOCKING TOLL-LIKE RECEPTOR 4 WITH THE NON-OPIOID (+)-ISOMERS OF NALOXONE AND NALTREXONE

2:30 – 2:45 Coffee Break

2:45 – 3:15 Daniela Salvemini, Saint Louis University TARGETING CERAMIDE AS A NOVEL THERAPEUTIC APPROACH IN OPIATE-INDUCED HYPERALGESIA AND ANTINOCICEPTIVE TOLERANCE

3:15 – 3:30 Discussion

4:00 – 6:00 Poster Session #1

Tuesday, July 17 7:00 – 8:15 AM Breakfast

8:30 – 9:30 Founder’s Lecture F. Ivy Carroll, Research Triangle Institute DESIGN AND DEVELOPMENT OF OPIOID RECEPTOR ANTAGONISTS FOR SUBSTANCE ABUSE RESEARCH

9:30 – 12:00 Symposium #3 – Opioid Peptides in Motivational Circuits Organizer and Chair: Elyssa Margolis, Ernest Gallo Clinic & Research Center/UCSF

9:30 – 9:55 Leslie Sombers, North Carolina State University THE VOLTAMMETRIC DETECTION AND CHARACTERIZATION OF MET- ENKEPHALIN IN BRAIN TISSUE

9:55 – 10:15 Coffee Break

10:15 – 10:40 Robert Kennedy, University of Michigan DEVELOPMENT AND APPLICATION OF CAPILLARY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY METHODS FOR MONITORING ENDOGENOUS OPIOID PEPTIDES IN VIVO

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10:40 – 11:05 Steve Laviolette, University of Western Ontario CORTICAL AND SUB-CORTICAL INTERACTIONS CONTROLLING MEMORY FORMATION DURING THE OPIATE ADDICTION PROCESS

11:05 – 11:40 Elyssa Margolis, Ernest Gallo Clinic & Research Center/UCSF MU OPIOID RECEPTOR ACTIONS IN THE VENTRAL TEGMENTAL AREA; THE JURY IS STILL OUT

11:40 – 12:00 Shanna Resendez, University of Michigan OPIOID REGULATION OF PAIR BOND FORMATION AND MAINTENANCE

12:00 – 1:30 Lunch

1:30 – 4:00 Symposium #4 – Young Investigators Organizer and Chair: Thomas Prisinzano, University of Kansas

1:30 – 2:00 Susan Mercer, Lipscomb University INVESTIGATION OF STERICALLY HINDERED MEPERIDINE ANALOGS AT P-GLYCOPROTEIN AND CYP3A4

2:00 – 2:30 Emily Jutkiewicz, University of Michigan THE EFFECTS OF DELTA-OPIOID RECEPTOR LIGANDS ON THE CONDITIONED STIMULI ASSOCIATED WITH COCAINE

2:30 – 2:45 Coffee Break

2:45 – 3:15 Grégory Scherrer, Columbia University THE CELLULAR LOGIC AND MOLECULAR MECHANISMS OF CONTROL OF CUTANEOUS MECHANOSENSATION BY OPIOID RECEPTORS

3:15 – 3:45 Sylvia Fitting, Virginia Commonwealth University MORPHINE AND HIV-1 TAT-INDUCED SUBLETHAL INJURY IS CAUSED BY A DISRUPTION IN ION HOMEOSTASIS AND CAN BE ATTENUATED BY NMDAR & AMPAR BLOCKADE IN THE STRIATUM

3:45 – 4:00 Discussion

5:00 – 7:00 Data Blitz Arthur Stillwell Room, Union Station

Wednesday, July 18 7:00 – 8:15 AM Breakfast

8:30 – 9:30 Plenary Lecture (P2) Kenner Rice, National Institute on Drug Abuse and National Institute on Alcoholism and Alcohol Abuse MEDICINAL CHEMISTRY IN DRUG ABUSE RESEARCH AT THE NIH – LOOKING BACK

9:30 – 12:30 Symposium #5 – Opioid/Cannabinoid Interactions Organizer and Chair: Jean Bidlack, University of Rochester

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9:30 – 10:00 Dana Selley, Virginia Commonwealth University CB1 CANNABINOID AND MU OPIOID RECEPTORS INTERACT IN CNS SIGNALING, ANALGESIA AND DRUG DEPENDENCE

10:00 – 10:15 Coffee Break

10:15 – 10:45 Ken Mackie, Indiana University RECEPTORS GONE WILD: CONSEQUENCES OF CB2 CANNABINOID RECEPTOR FUNCTIONAL SELECTIVITY

10:45 – 11:15 Sandy Welch, Virginia Commonwealth University CANNABINOIDS, OPIOIDS AND SPHINGOSINE-1-PHOSPHATE1 (S1P1) RECEPTOR INTERACTIONS IN ANTINOCICEPTION

11:15 – 11:45 Fernando Berrendero, University Pompeu Fabra, PRBB INVOLVEMENT OF THE ENDOCANNABINOID SYSTEM IN DRUG ADDICTION

11:45 – 12:00 Somnath Mukhopadhyay, North Carolina Central University TUNING CANNABINOID RECEPTOR SIGNALING BY RGS (REGULATORS OF G-PROTEIN SIGNALING) PROTEINS: NEW PLAYER IN CANNABINOID RECEPTOR SIGNALOSOME

12:00 – 12:15 Li Wei Tung, National Taiwan University OREXIN A INCREASES DOPAMINERGIC ACTIVITY VIA OX1 RECEPTOR- INITIATED ENDOCANNABINOID RETROGRADE DISINHIBITION IN THE VENTRAL TEGMENTAL AREA -A NOVEL ADDICTION MECHANISM

12:15 – 12:30 Tamara Vasiljevik, University of Kansas FURTHER ELUCIDATION OF THE SAR OF K2/SPICE AMINOALKYLINDOLES

12:30 – 2:30 Lunch and Poster Session #2

Thursday, July 19 7:00 – 8:15 AM Breakfast

8:30 – 9:30 Plenary Lecture (P3) Bryan Roth, University of North Carolina at Chapel Hill NEW APPROACHES FOR DECONSTRUCTING DRUG ACTIONS

9:30 – 12:00 Symposium #6 – Medicinal Chemistry of Opioid Peptides Organizer and Chair: Jane Aldrich, University of Kansas

9:30 – 10:00 Jane Aldrich, University of Kansas NOVEL KAPPA OPIOID RECEPTOR PEPTIDE LIGANDS AS POTENTIAL TREATMENTS FOR DRUG ABUSE

10:00 – 10:15 Coffee Break

10:15 – 10:45 Steve Ballet, Vrije Universiteit Brussel DESIGN AND STRUCTURE-ACTIVITY RELATIONSHIPS OF COMPACT, CONFORMATIONALLY CONSTRAINED, AND BIFUNCTIONAL OPIOID

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AGONIST-NEUROKININ 1 RECEPTOR ANTAGONISTS

10:45 – 11:15 Victor Hruby, University of Arizona NEW APPROACHES TO THE DEVELOPMENT OF OPIOID MULTIVALENT LIGANDS WITHOUT TOXICITIES, TOLERANCE OR DEPENDENCE FOR ACUTE AND NEUROPATHIC PAIN

11:15 – 11:45 Henry Mosberg, University of Michigan STRUCTURE-BASED DESIGN OF MIXED EFFICACY, BIFUNCTIONAL OPIOIDS

11:45 – 12:00 Discussion

12:00 – 1:30 Lunch

1:30 – 4:00 Symposium #7 – Opioid Ligand-Directed Signaling Organizer and Chair: Jay McLaughlin, Torrey Pines Institute for Molecular Studies

1:30 – 2:00 Ping-Yee Law, University of Minnesota IN VIVO CONSEQUENCES OF OPIOID AGONIST-DEPENDENT PATHWAY-SELECTIVE SIGNALING

2:00 – 2:30 Charles Chavkin, University of Washington LIGAND DIRECTED SIGNALING AT KAPPA OPIOID RECEPTORS

2:30 – 3:00 Amynah Pradhan, University of California at Los Angeles ARRESTIN MEDIATED FUNCTIONAL SELECTIVITY AT THE DELTA OPIOID RECEPTOR

3:00 – 3:15 Coffee Break

3:15 – 3:30 Jean Bidlack, University of Rochester LONG-ACTING KAPPA OPIOID ANTAGONISTS DO NOT ACTIVATE C-JUN N-TERMINAL KINASE

3:30 – 3:45 William Clarke, University of Texas Health Science Center at San Antonio KOR AGONIST FUNCTIONAL SELECTIVITY IN PERIPHERAL SENSORY NEURONS IN VIVO AND EX VIVO

3:45 – 4:00 Erin Bobeck, Washington State University DIFFERENCES IN ANTINOCICEPTION AND TOLERANCE DUE TO LIGAND-BIASED SIGNALING AT THE MU OPIOID RECEPTOR

4:00 – 4:15 John Traynor, University of Michigan MODULATION OF DELTA OPIOID RECEPTOR SIGNALING BY REGULATOR OF G PROTEIN SIGNALING PROTEIN 4 IN RODENT BRAIN

4:30 – 5:30 INRC Business Meeting Sheraton Kansas City

7:00 – 8:00 Pre-dinner reception Westin Kansas City at Crown Center

8:00 – 11:00 Banquet Dinner Westin Kansas City at Crown Center

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Poster Session #1 – Monday Afternoon (4:00 – 6:00 pm)

Behavior 1. BUPRENORPHINE/NALTREXONE: AN EFFECTIVE COMBINATION THERAPY APPROACH TO RELAPSE PREVENTION. S.M. Husbands, S. Cordery, A. Taverner, R. H. Guy, B. Delgado-Charro, C.P. Bailey Department of Pharmacy and Pharmacology, University of Bath, UK.

2. KOR AGONIST FUNCTIONAL SELECTIVITY IN PERIPHERAL SENSORY NEURONS IN VIVO AND EX VIVO. R.J. Jamshidi, B.A. McGuire, L.C. Sullivan, W.P. Clarke, K.A. Berg. Dept. of Pharmacol., University of Texas Health Science Center at San Antonio, San Antonio, TX.

3. DISTINCT KAPPA OPIOID RECEPTOR (KOR) ANTAGONISTS SUPPRESS ERK ½ MAP KINASE SIGNALING CORRELATING WITH ANTIDEPRESSANT-LIKE ACTIVITY. J.P. McLaughlin (1,2), M.R. Hoot (1), K. Rasakham (2) (1) Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA, (2) Northeastern University, Dept. of Psychology, Boston, MA USA

4. INVOLVEMENT OF PITUITARY POMC T-BOX TRANSCRIPTION FACTOR T-PIT IN COCAINE CONDITIONED PLACE PREFERENCE AND HYPOTHALAMIC-PITUITARY ADRENAL AXIS RESPONSIVITY IN MICE. S Chen(1), Y Zhou(1), K Niikura(1), A Ho(1), J Drouin(2), MJ Kreek(1) (1)The Rockefeller University, NY, USA, (2)IRCM, QC, Canada

5. PARENTING WITH PILLS: THE DUAL IDENTITIES OF PRESCRIPTION OPIOID ANALGESICS IN MIDWESTERN RURAL COMMUNITIES. K. Bensley, MSc. (1) Department of Anthropology, University College London, London, UK

6. EFFECTS OF THE WIDELY AVAILABLE KAPPA-OPIOID RECEPTOR (KOP-R) AGONIST HALLUCINOGEN, SALVINORIN A ON THE HPA AXIS: TRANSLATIONAL STUDIES. Eduardo R. Butelman (1), Thomas E. Prisinzano (2), Mary Jeanne Kreek (1) (1)Laboratory on the Biology of Addictive Diseases, The Rockefeller University, New York, NY 10065, USA, (2) Department of Medicinal Chemistry, School of Pharmacy, University of Kansas, Lawrence KS 66045, USA

7. ELECTROACUPUNCTURE ATTENUATES PRURITOGENS-EVOKED SCRATCHING. Y.- H. Chen (1), H.-Y. Yang (2) and J.-G. Lin (2) (1)Grad. Inst. Acupuncture Science, (2) Sch. of Chinese Medicine, China Medical University, Taichung, Taiwan.

8. LONG TERM CHANGES IN MOTIVATION DURING AVERSIVE CONDITIONS FOLLOWING MORPHINE WITHDRAWAL ARE PREDICTED BY ALTERED NMDAR1 SPLICING IN THE AMYGDALA. Ethan M. Anderson (2) and Robert M. Caudle (1,2). (1) Department of Oral Surgery, University of Florida, Gainesville, FL, United States (2) Department of Neuroscience University of Florida College of Medicine, Gainesville, FL

9. MORPHINE PRODUCES INVERTED DOSE-DEPENDENT REWARD IN NEUROPATHIC RATS. CM Cahill(1), L Xue(1), S LeCour(1), C Magnussen(1), S Metcalfe(2), P Grenier(1), MC Olmstead(2) Depts of (1)Biomed and Mol Sci and (2)Psychology, Queen’s Univ, Kingston ON.

10. A ROLE FOR NORADRENERGIC SYSTEMS IN KAPPA-OPIOID DEPENDENT REINSTATEMENT OF COCAINE PLACE PREFERENCE. R. Al-Hasani (1,2), A.M. Foshage (1), J.G. McCall (1,2,3), M.R. Bruchas (1,2,4) (1) Department of Anesthesiology (2)

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Department of Neurobiology (3) Program in Neuroscience, (4) Pain Center Washington University in St. Louis, St. Louis MO, USA

11. EFFECTS OF MU, DELTA AND KAPPA OPIOID RECEPTOR AGONISTS ON PAIN- STIMULATED VS. PAIN-DEPRESSED BEHAVIORS IN RATS. A. Altarifi and S. Negus; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA

12. GALPHAO PROTEIN PLAYS A ROLE IN MORPHINE-INDUCED LOCOMOTOR SENSITIZATION. J. Lamberts (1), J. Gateley (1), J. Borlagdan (1), M. VandenBerg (1), J. Traynor (1, 2) (1) Department of Pharmacology, (2) Substance Abuse Research Center, University of Michigan, Ann Arbor, MI, USA

13. COMPARISON OF PERIPHERAL AND CENTRAL ANTINOCICEPTIVE EFFECTS OF BIPHALIN AND MORPHINE IN A MOUSE MODEL OF CANCER PAIN. AA Lesniak(1), M Bochynska-Czyz(1), M Sacharczuk(2), Andrzej W. Lipkowski(1) (1)Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, (2)Institute of Genetics and Animal Breeding Polish Academy of Sciences, Postepu 1 05-552 Wolka Kossowska, Poland

14. TOWARD A PHARMACOGENETICS APPROACH TO INDIVIDUALIZED METHADONE MAINTENANCE TREATMENT FOR OPIOID ADDICTION. Orna Levran (1), Einat Peles (2), Matthew Randesi (1), Jurg Ott (1,3), Miriam Adelson (2), Mary Jeanne Kreek (1). (1)Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY, USA, (2)Dr. Miriam and Sheldon G. Adelson Clinic for Drug Abuse, Treatment and Research, Elias Sourasky Medical Center, Tel Aviv, Israel , (3)Institute of Psychology CAS, Beijing, China.

15. ZYKLOPHIN, THE SHORT-ACTING KAPPA OPIOID ANTAGONIST, INDUCES SCRATCHING IN MICE. K.M. DiMattio(1), T.V. Yakovleva(2), J.V. Aldrich(2), L.Y. Liu- Chen(1), A. Cowan(1) (1)Center for Substance Abuse Research and Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA; (2)Department of Medicinal Chemistry, University of Kansas, Lawrence, KS

16. KAPPA OPIOID RECEPTOR-MEDIATED DISRUPTION OF NOVEL OBJECT RECOGNITION: RELEVANCE FOR PSYCHOSTIMULANT TREATMENT. J.J. Paris (1), K.J. Reilley (1), J.P. McLaughlin (1) (1)Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA

17. THE INTERACTION OF SOME ANTIDEPRESSANT DRUGS WITH ACUTE VS. CHRONIC METHADONE IN MICE. Schreiber, S (1), Hostovsky, A (2), Barak, Y (2), Rubovitch, V (2), and Pick, CG (2) (1)Department of Psychiatry, Tel Aviv Sourasky Medical Center and Tel Aviv University Sackler Faculty of Medicine; (2)Department of Anatomy, Tel-Aviv University Sackler Faculty of Medicine, Tel Aviv, Israel

18. DELTA OPIOID RECEPTOR AGONISTS INHIBIT MIGRAINE-ASSOCIATED HYPERALGESIA, AVERSIVE STATE, AND CORTICAL SPREADING DEPRESSION. Monique L Smith (1,2,3), Jekaterina Zyuzin (2), Andrew Charles (2,3), and Amynah A Pradhan (1,3). (1) Semel Institute for Neuropsychiatry & Human Behavior, University of California Los Angeles, (2) Headache Research and Treatment Program, Department of Neurology, David Geffen School of Medicine at UCLA, (3) Shirley and Stephan Hatos Center for Neuropharmacology, UCLA.

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19. ASSOCIATION OF POLYMORPHISMS OF THE DELTA OPIOID RECEPTOR AND PROENKEPHALIN GENES WITH THE SEVERITY OF HIV INFECTION AND RESPONSE TO HIV TREATMENT. Dmitri Proudnikov (1), Matthew Randesi (1), Howard Crystal (2), Jurg Ott (3, 4), Ann Ho (1) and Mary Jeanne Kreek (1) (1) Lab. Biol. Addict. Diseases, Rockefeller Univ., New York, NY, USA (2) Dep. Pathol., SUNY Downst. Med. Center, Brooklyn, NY (3) Lab. Statist. Genet., Rockefeller Univ., New York, NY, USA (4) Inst. Psychol. CAS, Beijing, China

20. NORMAL HEALTHY WOMEN BEARING THE MINOR ALLELE A OF MC2R GENE (ACTHR) -184G>A HAVE DECREASED BASAL CORTISOL LEVELS COMPARED TO MALES, WHICH ALL INCREASE TO EQUAL LEVELS AFTER ACTH CHALLENGE. B. Ray, D. Proudnikov, S. Chen, E. Ducat, A. Ho, M.J. Kreek, Laboratory of the Biology of Addictive Diseases, Rockefeller University, New York, NY

21. THE ANTIDEPRESSANT-LIKE AND ANTINOCICEPTIVE EFFECTS OF KNT-127, THE NOVEL DELTA OPIOID RECEPTOR AGONIST, IN MICE. Akiyoshi Saitoh (1), Azusa Sugiyama (1,2), Toru Nemoto (3), Hideaki Fujii (3), Keiji Wada (4), Jun-Ichiro Oka (2), Hiroshi Nagase (3), Mitsuhiko Yamada (1) (1)Dept. of Neuropsychopharmacology, NIMH, NCNP, Tokyo, Japan; (2) Lab. Pharmacol., Fac. Pharm. Sci., Tokyo Univ. Sci., Chiba, Japan; (3) Laboratory of Med. Chemistry, School of Pharmacy, Kitasato Univ., Tokyo, Japan; (4)Dpartment of Degenerative Neurological Diseases, NIN, NCNP, Tokyo, Japan

22. LIPOXYGENASE-MEDIATED REGULATION OF DELTA OPIOID RECEPTORS IN PERIPHERAL SENSORY NEURONS. L.C. Sullivan, K.A. Berg, and W.P. Clarke Dept. of Pharmacology, University of Texas Health Science Center at San Antonio, TX, USA

23. THE DELTA-OPIOID RECEPTOR CONTRIBUTES TO MORPHINE TOLERANCE IN AN ANIMAL MODEL OF CHRONIC INFLAMMATORY PAIN. L. Hipólito (1), H. Beaudry (2), A. Gupta (3), L. Devi (3), L. Gendron (2), J.A. Morón Concepción (1). (1) Dept. of Anesthesiology, Columbia University Medical Center, New York, USA (2) Dept. of Physiology and Biophysics, Université de Sherbrooke, Sherbrooke, Canada, (3) Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, USA.

24. EFFECTS OF ADOLESCENT OXYCODONE SELF-ADMINISTRATION ON OXYCODONE- INDUCED CONDITIONED PLACE PREFERENCE AND LOCOMOTOR ACTIVITY IN C57BL/6J MICE. Yong Zhang, Eduardo R. Butelman, Ann Ho, Mary Jeanne Kreek, The Rockefeller University, New York, NY 10065

25. POSSIBLE INVOLVEMENT OF ENDOGENOUS OPIOID SYSTEM LOCATED IN THE DOWNSTREAM OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR IN NICOTINE- INDUCED ANTINOCICEPTION IN MICE. S. Kishioka, N. Wakida, K. Ueno, F. Saika, C. Yamamoto, Y. Kobayashi, N. Kiguchi Department of Pharmacology, Wakayama Medical University, Wakayama, Japan

26. SADOWSKI MOUSE STRAINS OF HIGH AND LOW STRESS INDUCED ANALGESIA AS A MODEL OF BLOOD BRAIN BARRIER PERMEABILITY STUDIES OF OPIOID PEPTIDES AND ALKALOIDS. AW Lipkowski(1), A Kosson(1), AA Lesniak(1), M Sacharczuk (2), G Tóth(3), J Molnár(3), IA. Krizbai(3), I Wilhelm(3) (1)Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, (2)Institute of Genetics and Animal Breeding Polish Academy of Sciences, Postepu 1 05-552 Wolka Kossowska, Poland, (3)Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary

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27. UNIDIRECTIONAL CROSS ACTIVATION OF GRPR BY MOR1D UNCOUPLES OPIOID- INDUCED ANALGESIA AND OPIOID-INDUCED ITCH. X.Y. Liu (1), Z.C. Liu (1), Y.G. Sun (1), M. Ross (1), S. Kim (1), Q.F. Li (1), H.H. Loh (2), Z.F. Chen (1) (1) Dep. of Anesthesiology, Center for the Study of Itch, Washington Univ., St Louis, USA (2) Dep. of Pharmaco., Univ. of Minnesota, Minneapolis, USA

28. A NOVEL MECHANISM FOR ACUPUNCTURE ANALGESIA: OREXIN 1 RECEPTOR- MEDIATED ENDOCANNABINOID RETROGRADE DISINHIBITION IN THE PERIAQUEDUCTAL GRAY. L.-C. Chiou (1, 2), Y.-H. Chen (3), H.-J. Lee (1) and Y.-T. Wu (3). (1) Dept. Pharmacology, (2) Grad. Inst. Brain and Mind Sciences, Coll. Medicine, National Taiwan University, Taipei, Taiwan; (3)Grad. Inst. Acupuncture Science, China Medical University, Taichung, Taiwan.

29. MODULATION OF MORPHINE TOLERANCE BY GLIAL RECEPTOR TOLL-LIKE RECEPTOR 4 IN THE MIDBRAIN PERIAQUEDUCTAL GRAY OF MALE RATS. Lori N. Eidson & Anne Z. Murphy Neuroscience Institute, Georgia State University, Atlanta, GA

30. COUPLING BETWEEN OPIOID RECEPTORS AND POTASSIUM CHANNELS IN PAIN REDUCTION: RELEVANCE FOR SPECIES DIFFERENCES AND EFFICACY. D. Nockemann (1), D. Labuz (1), C. Stein (1) and P.A. Heppenstall (2) (1)Dep. Anesthesiology, Freie Universität, Campus Benjamin Franklin, Charité, Berlin, Germany; (2)Mouse Biology Unit, EMBL Monterotondo, Italy

31. SPINAL MU OPIOID RECEPTORS REGULATE BOTH HEAT AND MECHANICAL PAIN IN RATS. A. Normandin(1), P. Luccarini(2), J-L. Molat(2), L. Gendron (1) and R. Dallel(2) (1) Département de Physiologie et Biophysique, Université de Sherbrooke, Québec, Canada; (2) Inserm/UdA U1107, Université d’Auvergne, Clermont-Ferrand, France

32. INHIBITION OF ANTINOCICEPTION BY THE MU OPIOID PARTIAL AGONIST BU08073. L. Toll (1), T.V. Khroyan (2), W.E. Polgar (2), G. Cami-Kobeci (3), S. Husbands (3). (1) Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA; (2) SRI International, Menlo Park, CA, USA; (3) University of Bath, Bath, UK.

33. EFFECTS OF SINGLE-PROLONGED STRESS ON NOCICEPTIVE SENSITIVITY AND THE NOCICEPTIN/ORPHANIN FQ (N/OFQ) SYSTEM IN SPINAL CORD OF RATS. Y. Zhang (1), P.R. Gandhi (1), K.M. Standifer (1, 2). (1)Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, (2)Department of Cell Biology and Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA

34. INVOLVEMENT OF THE LONG-CHAIN FATTY ACID RECEPTOR GPR40 AS A PAIN CONTROL SYSTEM. Kazuo Nakamoto (1), Takashi Nishinaka (1), Kengo Matsumoto (1), Naoya Sato (1), Kasuya Fumiyo (2), Mitsumasa Mankura (3), Yutaka Koyama (4) and Shogo Tokuyama (1) (1) Department of Clinical Pharmacy, Kobe Gakuin University, School of Pharmaceutical Sciences, (2) Biochemical Toxicology Laboratory, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, (3) Ikeda Tohka Industries Co., Ltd., (4) Laboratory of Pharmacology, Faculty of Pharmacy, Osaka Ohtani University

35. OPTOGENETIC DISSECTION OF THE LOCUS COERULEUS IN NEGATIVE AFFECTIVE BEHAVIOR AND NOCICEPTION. J.G. McCall (1,2,3,4), B. Sebastian (2), C.P. Ford (5), M.R. Bruchas (2,3,4) (1) Neurosciences Program (DBBS), (2) Dept. of Anesthesiology, (3) Anatomy & Neurobiology, (4) Pain Center, Washington University, St. Louis, MO, USA, (5) Dept. of Physiology & Biophysics, Department of Neurosciences, Case Western Reserve University, Cleveland, OH, USA

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36. MICROGLIAL ACTIVATION IS ESSENTIAL FOR OPIOID BUT NOT PSYCHOSTIMULANT REWARD IN DRUG DEPENDENT STATES. AMW. Taylor (1, 2), P. Vayssiere (1), C. Evans (1), CM. Cahill (2) (1) UCLA, Los Angeles, CA, USA; (2) Queens University, Kingston, ON, Canada

Chemistry 37. PHARMACOKINETICS OF 7-HYDROXYMITRAGYNINE, A µ-OPIOID RECEPTOR AGONIST, IN MALE SPRAGUE DAWLEY RATS. B.A. Avery (1), P.K. Vuppala (1), S. Jamalapuram (1), E.B. Furr (2), C.R. McCurdy (2,3). Departments of (1)Pharmaceutics, (2)Medicinal Chemistry, and (3) Pharmacology, University of Mississippi, MS 38677

38. PHENETHYL ORVINOLS: SAR AT OPIOID AND NOP RECEPTORS. Gerta Cami-Kobeci (1), Willma E. Polgar (2), Taline V. Khroyan (2), Lawrence Toll (2), Stephen M. Husbands (1). (1)Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, UK and (2)SRI International, Menlo Park, USA.

39. IDENTIFICATION OF OPIOID PEPTIDES IN THE SPINAL CORD OF SNI MALE MICE USING NANO LC-MS/MS. A. Carlsson (1), E. Brolin (1), H. Watanabe (1), H. Wadensten (2), P. Andrén (2), F. Nyberg (1). Uppsala University, Department of Pharmaceutical Biosciences (1) Division of Biological Research on Drug Dependence (2) Division of Medical Mass Spectrometry, Uppsala, Sweden

40. MIXTURE LINKAGE ANALYSIS: FUNCTIONAL ACTIVITY VALIDATION COMPARING SRU BIND TECHNOLOGY AND CAMP INHIBITION VIA MU OPIOID RECEPTORS. Margaret E. Cazares, Jaime A. Misler, and Colette T. Dooley. Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida

41. SYNTHESIS AND EVALUATION OF ARYL-NALOXAMIDE OPIATE ANALGESICS TARGETING TRUNCATED EXON 11-ASSOCIATED MOR-1 SPLICE VARIANTS. Susruta Majumdar (1), Joan Subrath (1), Valerie Le Rouzic (1), Lisa Polikar (1), Maxim Burgman (1), Kuni Nagakura (1), Julie Ocampo (1), Nathan Haselton (1), Anna R. Pasternak (1), Steven G. Grinnell (1), Ying-Xian Pan (1) and Gavril W. Pasternak (1). (1)Department of Neurology and Molecular Pharmacology and Chemistry Program Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA

42. PHARMACOLOGICAL CHARACTERIZATION OF SYNTHETIC MITRAGYNINE ANALOGS. Jessica V. Mankus (1), Lisa L. Wilson (2), Edward B. Furr (1), Marco Arribas (1), Christophe Mesangeau (1), Stephen J. Cutler (1) and Christopher R. McCurdy (1,2). Departments of (1)Medicinal Chemistry, and (2)Department of Pharmacology, University of Mississippi, University, MS 38677

43. ENDOMORPHIN ANALOGS SHOW REDUCED ABUSE POTENTIAL RELATIVE TO MORPHINE. M.R. Nilges (1), X. Zhang (2), J.E. Zadina (1,2,3,4). (1) Neuroscience Program, (2) Dept of Medicine and (3) Pharmacology, Tulane University, and (4) SE LA Veterans Health Care System, New Orleans, LA 70112

44. OPIOID AFFINITY, SELECTIVITY AND ANTINOCICEPTIVE ACTIVITY OF THIAZOLE- CONTAINING CYCLIZED ENKEPHALIN ANALOGS. SO Eans (1), ML Ganno (1), C Dooley (1), A Nefzi (1), JP McLaughlin (1) (1)Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA

45. EVALUATION OF CHRONIC RETENTION OF NOR-BNI IN MOUSE BRAIN OVER WEEKS AFTER ADMINISTRATION USING LC-MS/MS. K. A. Patkar, M. R. Hoot, S. O. Eans, K. J.

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Reilley and J. P. McLaughlin. Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987

46. INSULIN DEGRADING ENZYME MEDIATED PROTEOLYSIS OF EXTRACELLULAR NEUROPEPTIDES: DYNORPHINS, CRF, AND GALANIN. Brian Reed (1,2), Brian T. Chait (2), Mary Jeanne Kreek (1) (1) Lab. Biology Addictive Diseases, Rockefeller Univ., New York, NY, USA (2) Lab. Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, NY, USA

47. DEVELOPMENT AND IN VITRO CHARACTERIZATION OF NOVEL MOR AGONISTS DISPLAYING BIAS AGAINST βARRESTIN2 RECRUITMENT. N. C. Ross (1,2), J.M. Streicher (1), T. Bannister (2), L. M. Bohn (1) (1) Departments of (1) Molecular Therapeutics and Neuroscience and (2) Medicinal Chemistry, The Scripps Research Institute, Jupiter, FL, USA

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Poster Session #2 – Wednesday Afternoon (12:30 – 2:30 pm)

Biology 1. ACTIN CYTOSKELETON DISRUPTION MODULATES MU OPIOID RECEPTOR INTERNALIZATION AND DESENSITIZATION IN LOCUS COERULEUS NEURONS. S. Arttamangkul and J.T. Williams, Vollum Institute, Oregon Health & Science University, Portland, Oregon, USA

2. OPIOID PEPTIDES INDUCE LONG-TERM DEPRESSION AT GLUTAMATERGIC SYNAPSES IN THE DORSAL STRIATUM. Brady K. Atwood and David M. Lovinger National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, USA

3. INHIBITION OF CDK5 IMPAIRS MORPHINE- AND INFLAMMATION-INDUCED FUNCTIONAL EMERGENCE OF THE DELTA OPIOID RECEPTOR IN RATS. Andrée-Ann Mercier-Blais, Hélène Beaudry and Louis Gendron, Université de Sherbrooke, Canada

4. TNF-ALPHA-INDUCED MOR UP-REGULATION IN HUMAN GLIOBLASTOMA CELLS IS COUPLED TO A LOSS OF MOR-ACTIVATED SIGNALING TO INTRACELLULAR EFFECTORS. A. Bedini, G. Vincelli, M. Baiula and S. Spampinato Dept. of Pharmacology, University of Bologna – Irnerio 48, 40126 Bologna – Italy

5. AGONIST-INDUCED IMPRINTING OF THE MU OPIOID RECEPTOR VISUALIZED BY FLUORESCENT OPIOID LIGANDS. William T. Birdsong(1), Seksiri Arttamangkul(1), Mary J. Clark(2), Kenner Rice(3), John Traynor (2), Mark von Zastrow(4), John T. Williams(1) (1)Vollum Institute, Oregon Health & Science University, Portland, OR, USA. (2)University of Michigan, Ann Arbor, MI, USA; (3)National Institute on Drug Abuse, Bethesda, MD, USA; (4)University of California San Francisco, San Francisco, CA, USA

6. REGIONAL MRNA EXPRESSION OF GABA RECEPTOR SUBUNITS IN BRAINS OF C57BL/6J AND 129P3/J MICE: STRAIN AND HEROIN EFFECTS. M. Buonora, S.D. Schlussman, Y. Zhang, A. Ho and M.J. Kreek, The Laboratory on the Biology of Addictive Diseases, The Rockefeller University, New York, USA

7. DEVELOPMENT OF A REAL-TIME, FLUORESCENCE BASED ASSAY OF MU-OPIOID RECEPTOR MEDIATED INHIBITION OF ADENYLYL CYCLASE ACTIVITY IN CHINESE HAMSTER OVARY CELLS. Alisa Knapman, Mark Connor Australian School of Advanced Medicine, 2 Technology Place, Macquarie University, NSW, Australia, 2109

8. DIFFERENTIAL EXPRESSION AND HIV-1 REGULATION OF MU-OPIOID RECEPTOR SPLICE VARIANTS ACROSS HUMAN CENTRAL NERVOUS SYSTEM CELL TYPES. S.M. Dever (1), R. Xu (2), S. Fitting (1), P.E. Knapp (1,2,3), K.F. Hauser (1,3) (1)Dept. of Pharmacol. and Toxicol., (2)Dept. of Anat. and Neurobiol., (3)Inst. for Drug and Alcohol Studies, Virginia Commonwealth Univ. Sch. of Medicine, Richmond, Virginia, USA

9. CHARACTERIZATION OF NON-CLASSICAL OPIOID ACTIVITY AT TOLL-LIKE RECEPTOR 4. S. Dodson, R. Castoro, S. Das, R. Davis, and C. Stevens. Department of Pharmacology and Physiology, Oklahoma State University-Center for Health Sciences, Tulsa, OK, USA

10. THE ROLE OF NMDA RECEPTORS IN THE MECHANISMS UNDERLYING MORPHINE- INDUCED ALTERATIONS IN SYNAPTIC PLASTICITY. A.K. Fakira, G.S. Portugal, B. Carusillo, Z. Melyan* and J.A. Morón Concepción* Dept. of Anesthesiology, College of Physicians and Surgeons, Columbia University Medical Center, New York, United States

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11. G PROTEIN-COUPLED RECEPTOR HETEROMERIZATION: A ROLE IN ALLOSTERIC MODULATION OF OPIOID RECEPTOR ACTIVITY IN HEALTH AND DISEASE. Ivone Gomes (1), Achla Gupta (1) and Lakshmi A. Devi (1,2) (1) Departments of Pharmacology & Systems Therapeutics and (2) Neuroscience, Mount Sinai School of Medicine, New York, NY, USA, 10029

12. GENERATION AND CHARACTERIZATION OF G PROTEIN-COUPLED RECEPTOR HETEROMERS. Achla Gupta (1), Ivone Gomes (1), and Lakshmi A. Devi (1,2) (1)Departments of Pharmacology & Systems Therapeutics and (2)Neuroscience, Mount Sinai School of Medicine, New York, NY, USA, 10029

13. ENDOMORPHIN-2 IS AN ARRESTIN-BIASED AGONIST BECAUSE IT INDUCES GREATER MOPR PHOSPHORYLATION THAN PREDICTED FROM ITS EFFICACY FOR G PROTEIN COUPLING. G. Rivero (1), J. Llorente (1), J. McPherson (1), A.E. Cooke (1), S.J. Mundell (1), C.A. McArdle (1), E.M. Rosethorne (2), S.J. Charlton (2), C. Krasel (3), C.P. Bailey (4), G. Henderson (1), E. Kelly (1) (1) School of Physiology and Pharmacology, Univ of Bristol, UK, (2) Novartis, Horsham UK (3) Univ of Marburg, Germany, (4) Dept of Pharmacy and Pharmacology, Univ of Bath, UK

14. DESENSITIZATION OF MOPR: HOMOLOGOUS OR HETEROLOGOUS? J. Llorente (1), J. Lowe (2), E. Tsisanova (1), E. Kelly (1), G. Henderson (1), C.P. Bailey (2), (1) School of Physiology and Pharmacology, University of Bristol, UK, (2) University of Bath, UK

15. REVERSAL OF TOLERANCE TO MORPHINE BY ETHANOL: ROLE OF PKC INHIBITION. S. Withey (1), J. Llorente (1), C.P. Bailey (2), E. Kelly (1), G. Henderson (1) (1) School of Physiology and Pharmacology, University of Bristol, UK, (2) Dept of Pharmacy and Pharmacology, University of Bath, UK

16. TRAFFICKING OF MOPR FOLLOWING CHRONIC MORPHINE TREATMENT. A.E. Cooke (1), S.J. Mundell (1), G. Henderson (1), E. Kelly (1) (1) School of Physiology and Pharmacology, University of Bristol, Bristol UK

17. CHARACTERIZATION OF A NATURALLY OCCURRING VARIANT OF MOPR, L83I. A.E. Cooke (1), S.J. Mundell (1), G. Henderson (1), E. Kelly (1) (1) School of Physiology and Pharmacology, University of Bristol, Bristol UK

18. PATHOLOGIC CHANGES IN MURINE AND HUMAN NEURONS INDUCED BY HIV-1SF162: INTERACTIONS WITH MORPHINE. Masvekar RR (1), Zou S (1), Hauser KF (2), and Knapp PE (1,2). (1) Depts. Anatomy & Neurobiology, (2) Pharmacology & Toxicology, Virginia Commonwealth Univ., Richmond, VA

19. MORPHINE DESENSITIZATION AND LONG-TERM TOLERANCE ARE DISTINGUISHED BY PKC AND PHOSPHATASE ACTIVITY. ES Levitt and JT Williams, Vollum Institute, Oregon Health & Science University, Portland, OR

20. ANTICANCER PROPERTIES OF PEPTIDE OPIOID AGONIST – TACHYKININ ANTAGONIST CHIMERAS. J Matalinska (1), M Bochynska-Czyz(1), H. Skurzak (2), S Markowicz(2), AW Lipkowski(1) (1)Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, (2) Maria Sklodowska-Curie Oncological Research Centre, Warsaw, Poland

21. VTA INTERNEURONS ARE NOT THE MAJOR OPIOID-SENSITIVE GABA INPUT TO DOPAMINE NEURONS. Aya Matsui and John T. Williams Vollum Institute, Oregon Health and Science University, Portland, OR, USA

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22. MECHANISM OF OPIOID MODULATION OF THROMBOSPONDIN LEVELS IN ASTROCYTES. E. Phamduong, N.R. Crews, A.L. Leinweber, P. Kappera, M. Rathore, C. J. Coscia E.A. Doisy Dept. of Biochem. and Mol. Biol., St. Louis Univ. School of Med, St. Louis, MO, 63104 USA

23. INHIBITION/DELETION OF CCR5 BLOCKS INTERACTIVE MORPHINE AND TAT NEURODEGENERATION. E.M. Podhaizer (1), Y. Zhang (2), P.E. Knapp (1,3), K.F. Hauser (1). (1) Depts. of Pharmacol. & Toxicol., (2) Med. Chem., (3) Anat. & Neurobiol., Virginia Commonwealth Univ., Richmond, VA

24. NORBNI INACTIVATES KOR EXPRESSED IN TRANSFECTED HEK393 AND NG108 CELLS THROUGH THE C JUN KINASE MECHANISM. Selena Schattauer, Haripriya Shankar, Mayumi Miyataki, Charles Chavkin, Department of Pharmacology, University of Washington, Seattle, WA 98195

25. OPIOID RECEPTOR LIKE-1 (ORL1) INTRACELLULAR TRAFFICKING AND JNK SIGNALING REQUIRES C-TERMINAL SERINE 363. N. Zhang (1,4), E.R. Siuda (1,2), W. Planer (1), L. Stickler (1), M. Baird (1,5), T. Palmer (1), R. Al-Hasani (1,3), M.R. Bruchas (1,2,3) (1)Dept. of Anesthesiology, (2)Program in Neuroscience, (3)Dept. of Anatomy/Neurobiology, (4) HHMI SURF Fellow, (5) AMGEN Scholar Washington University in St. Louis, St. Louis MO, USA

26. OPIOID AND HIV-1 ASSOCIATED NEURODEGENERATION: IMPLICATIONS FOR P2X4 RECEPTOR INVOLVEMENT. Sorrell, M.E. (1), Zou, S. (2), Knapp, P.E. (1, 2), and Hauser, K.F. (1); (1) Dept. Pharmacol. & Toxicol., (2) Dept. Anat. & Neurobiol., Virginia Commonwealth Univ. Coll. of Medicine, Richmond, VA, USA.

27. EVOLUTIONARY DIFFERENCES OF OPIOID RECEPTORS ARE REFLECTED IN THEIR PHARMACOLOGICAL PROFILES. E. Vardy (1), C.W. Stevens (2), B.L. Roth (1). (1) Department of Pharmacology, Program in Neuroscience and Division of Chemical Biology and Medicinal Chemistry, and National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina Chapel Hill Medical School, Chapel Hill, NC 27514, USA; (2) Department of Pharmacology and Physiology, Oklahoma State University-Center for Health Sciences, College of Osteopathic Medicine, Tulsa, OK 74107, USA

28. SORTILIN REGULATES CELL SURFACE EXPRESSION AND TRAFFICKING OF THE HUMAN KAPPA OPIOID RECEPTOR (HKOPR). Y-J. Wang, C. Chen, J. Li, L.-Y. Liu-Chen. Ctr Sub Abuse Res, Temple Univ, Phila, PA, USA

29. AQUAPORIN 4 DEFICIENCY ATTENUATES OPIOID DEPENDENCE THROUGH THE SUPPRESSION OF GLUTAMATE TRANSPORTER-1 DOWN-REGULATION AND THE MAINTENANCE OF GLUTAMATE HOMEOSTASIS. N. Wu, H.T. Yan, X.Q. Lu, R.B. Su, J.Q. Zheng, J. Li Beijing Institute of Pharmacology and Toxicology, Beijing, China

30. REGION-SPECIFIC ALTERATIONS IN EXPRESSION OF EPHA4, EPHB2 AND OPRK1 RECEPTORS IN POSTMORTEM BRAIN OF SUBJECTS INFECTED WITH HIV COMPARED TO CONTROLS. V. Yuferov (1), A. Ho (1), S. Morgello (2), M.J. Kreek (1). (1) Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY, USA, (2) Pathology and Neuroscience, Mount Sinai Medical Center, New York, NY, USA

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Chemistry 31. DISCOVERY OF NEW KAPPA OPIOID RECEPTOR AGONIST AND ANTAGONIST CHEMOTYPES THROUGH SCREENING A NONPROPRIETARY COMPOUND COLLECTION AND SAR EXPANSION. Kevin J. Frankowski(1), Michael P. Hedrick(2), Palak Gosalia(2), Kelin Li(1), Shenghua Shi(2), Partha Ghosh(1), David Whipple(1), Thomas E. Prisinzano(1), Frank J. Schoenen(1), Ying Su(2), S. Vasile(2), Eduard Sergienko(2), Wilson Gray(2), Santosh Hariharan(2), Loribelle Milan(2), Susanne Heynen-Genel(2), Arianna Mangravita-Novo(3), Michael Vicchiarelli(3), Layton H. Smith(3), John M. Streicher(4), Marc G. Caron(5), Lawrence S. Barak(5), Laura M. Bohn(4), Thomas D.Y. Chung(2), Jeffrey Aubé(1) (1)University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, KS 66047; (2)Conrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; (3)Conrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL 32827; (4)Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458; (5)Department of Cell Biology, Duke University, Durham, NC 27710

32. NOVEL ANALOGS OF THE KAPPA OPIOID RECEPTOR LIGAND CJ-15,208 WITH POTENTIAL FOR DRUG DEVELOPMENT. S. N. Senadheera (1), S. O. Eans (2), N. C. Ross (2), J. P. McLaughlin (2), T. F. Murray (3), J. V. Aldrich (1) (1)Dept. of Medicinal Chemistry, The Univ. of Kansas, Lawrence, KS, USA; (2)Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA; (3)Dept. of Pharmacol., Creighton Univ. Sch. of Med., Omaha, NE, USA

33. PHARMACOLOGICAL EVALUATION OF NOVEL OPIOID LIGANDS: A STUDY TO DETERMINE THE EFFECTS OF MIXED MU/DELTA ACTIVITY IN VITRO AND IN VIVO. Jason R. Healy (1), Padmavani Bezawada (2), Matthew Metcalf (2), Chris Cunningham (2), Sucheta Kudrimoti (2), Andrew Coop (2), Rae R. Matsumoto (1) (1)West Virginia University School of Pharmacy, Morgantown, WV; (2)University of Maryland School of Pharmacy, Baltimore, MD

34. DESIGN AND SYNTHESIS OF AMIDE AND AMINE CONTAINING LIGANDS RELATED TO BUPRENORPHINE. Emma M. Turner (1), Juan Pablo Cueva (1), Mary J. Clark (2), John R. Traynor (2), Stephen M. Husbands (1). (1)Department of Pharmacy and Pharmacology, University of Bath, Bath, UK; (2)Department of Pharmacology, University of Michigan, Ann Arbor, USA

35. ANALYSIS OF HYDROCODONE, HYDROMORPHONE AND NORHYDROCODONE IN PLASMA USING LIQUID CHROMATOGRAPHY - TANDEM MASS SPECTROMETRY (LC/MS/MS). Sandra Valtier, Robert Mueck, Toni Vargas, and Vikhyat S. Bebarta, 59th Medical Wing, Lackland AFB, TX.

36. HYDROCODONE METABOLISM FOLLOWING SINGE DOSE ADMINISTRATION. Sandra Valtier, Robert Mueck, Toni Vargas, and Vikhyat S. Bebarta, 59th Medical Wing, Lackland AFB, TX.

37. PHYSICOCHEMICAL AND PHARMACOKINETIC PROPERTIES OF NOVEL MACROCYLIC PEPTIDE KAPPA OPIOID RECEPTOR LIGANDS. A. Joshi (1), T. Khaliq (1), S. Senadheera (1), A. Mukhopadhyay (1), S. M. Lunte (2), J. V. Aldrich (1) Dept of (1)Medicinal Chem. and (2) Pharmaceutical Chem., Univ. of Kansas, Lawrence, KS, USA

38. MIXTURE LINKAGE ANALYSIS: EFFICIENCY OF IDENTIFICATION OF DISTINCT LIGAND FAMILIES FROM POSITIONAL SCANNING COMBINATORIAL LIBRARIES.

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Jaime A. Misler, Margaret E. Cazares, Tina L. Yates and Colette T. Dooley. Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida

39. EXPLORING POTENTIAL INTERACTION MODES OF BASIC AND NON-BASIC AGONISTS WITH THE KAPPA-OPIOID RECEPTOR. E. Vardy (1), P. D. Mosier (2), K. J. Frankowski (3), J. Aubé (3), R. B. Westkaemper (2), B. L. Roth (1) (1) Dept. of Pharmacol., Univ. of North Carolina, Chapel Hill, North Carolina, 27599, USA, (2) Dept. of Med. Chem., Virginia Commonwealth Univ., Richmond, Virginia, 23298, USA, (3) Dept. of Med. Chem., Univ. of Kansas, Lawrence, Kansas, 66045, USA.

40. MIXED EFFICACY AND IMPROVED DRUGABILITY: WORKING TOWARD A BETTER OPIOID ANALGESIC. Jessica P. Anand, Katarzyna Sobczyk-Kojiro, Lauren C. Purington, Larisa Yeomans, Aubrie A. Harland, Vanessa R. Porter, Aaron M. Bender, Emily M. Jutkiewicz, John R. Traynor, and Henry I. Mosberg Departments of Medicinal Chemistry and Pharmacology, University of Michigan, Ann Arbor MI, USA

41. INSUFFLATION OF PERIPHERALLY-RESTRICTED KOR AGONISTS PRODUCES RAPID ANTINOCICEPTION IN MULTIPLE PAIN MODELS WITHOUT OPIOID LIABILITIES. M.R. Hoot (1), S.O. Eans (1), K.J. Reilley (1), M.L. Ganno (1), M. Guilianotti (1), R.A. Houghten (1), A. Leone-Bay (2), J.P. McLaughlin (1). (1)Torrey Pines Inst. Molecular Studies, Port St. Lucie, FL 34987 (2) MannKind Corp., Danbury, CT 06810

42. REDUCED TOLERANCE DEVELOPMENT TO ANTINOCICEPTION WITH BIFUNCTIONAL NOP/MU OPIOID AGONISTS. NT Zaveri (1), TV Khroyan (2), L Toll (3), WE Polgar (2), VB Journigan (1), Dennis Yasuda (1). (1) Astraea Therapeutics, Mountain View, CA (2) SRI International, Menlo Park, CA (3) Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL.

43. OPPOSING ROLES OF MOP AND NOP ON LOCOMOTOR ACTIVITY IS REVEALED BY THE USE OF A SERIES OF BIFUNCTIONAL NOP/MOP AGONISTS. N. Zaveri (1), A. Hamid (3), A. Tseng (2), P. Marquez (2,3), V. Journigan (2), K. Lutfy (2,3). (1)Astraea Therapeutics, Mountain View, CA; (2)Western University of Health Sciences, Pomona, CA; (3)Charles Drew University of Medicine and Sciences, Los Angeles, CA

44. THE SYNTHESIS OF N-ALKYL-OCTAHYDROISOQUINOLIN-1-ONE-8-CARBOXYLIC ACIDS UNDER MILD, NEARLY-NEAT CONDITIONS. S. R. Slauson (1), P. Ghosh (1), and J. Aubé (1). (1)Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66047

45. MOLECULAR DYNAMICS SIMULATIONS OF A HOMOLOGY MODEL OF THE ACTIVE STATE OF THE NOCICEPTIN RECEPTOR NOP REVEAL NEW INSIGHTS INTO AGONIST BINDING AND ACTIVATION. NT Zaveri (1) and PR Daga (1). (1) Astraea Therapeutics, LLC. Mountain View, CA.

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Oral Presentations

22 INRC 2012 Kansas City, Missouri

(P1) Plenary Lecture #1

INTEGRATED APPROACHES TO UNDERSTAND THE ACTIONS OF GPCRS Marc G. Caron, Department of Cell Biology, Medicine and Neurobiology, Duke University Medical Center, Durham, NC.

G protein-coupled receptors (GPCR-7TM) mediate numerous physiological responses and are the targets of many therapies. Classically, GPCR responses are mediated through G protein activation. However, evidence indicates that the processes of phosphorylation of GPCRs by receptor kinases (GRK) and binding of β-arrestins normally associated with desensitization of signaling can support G protein-independent modes of signaling. Interestingly, β-arrestin- dependent signaling is slower but more sustained than G protein-dependent signaling and agonists and antagonists can selectively discriminate between these modes of signaling thus creating the opportunity for new therapeutic targets. In the brain, dopamine (DA) is implicated in the control of locomotion, cognition, emotion and affect as well as the rewarding effects of drugs and natural stimuli. Mice lacking the β-arrestin2 gene show markedly diminished responses to DA receptor stimulation consequent to psychostimulant exposure. This unexpected effect is presumably associated with the ability of the DA D2 receptor to engage an Akt/GSK3 signaling pathway via the scaffolding of a β-arrestin2/Akt/PP2A/GSK3 complex (Beaulieu et al., 2008; O’Brien et al., 2011). This D2 receptor-mediated complex is a target for clinically effective antipsychotics. In order to further validate the physiological role of this signaling pathway, we have used genetic approaches like the selective deletion of the downstream GSK3β gene in D2R, but not in D1R, expressing medium spiny neurons (MSN) to confirm that it mimics antipsychotic actions and recapitulates loss of D2 receptor responsiveness. In collaboration with colleagues at UNC Chapel Hill we have developed novel aripiprazole-based D2R/β-arrestin2 functionally selective ligands that show antipsychotic activity with fewer side effects in animal models (Allen et al., 2011). In addition, we have developed mutant D2Rs that can selectively signal either through the G protein- or β-arrestin2-dependent pathways. Reconstitution of these D2R mutants in vivo into MSNs lacking the endogenous D2R receptor should facilitate elucidation of the cellular, molecular, and behavioral responses associated with each mode of D2R signaling. Similar considerations can be examined in the context of the actions of L-DOPA as a therapy for Parkinson’s disease. Thus, our genetic approaches should provide a further validation of DA receptor functional selectivity and the identification downstream cellular events. Moreover, the functional selectivity of GPCR signaling and the possibility to identify functionally selective ligands for each signaling pathway may provide new approaches for more selective therapies for the many conditions associated with deregulation of GPCR signaling.

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Symposium #1 - Neuropeptidergic Targets for Addiction: Chemistry and Biology Organizer and Chair: Scott Runyon, Research Triangle Institute

THE ORL1 – NOCICEPTIN SYSTEM: RECEPTOR TRAFFICKING, SIGNALING AND BEHAVIOR N. Zhang (1), Mat Arujo (1,2), W. Planer (1), E. Siuda (1,2), L. Stickler (1), Tiffani Palmer (1), R. Al-Hasani (1), M.R. Bruchas (1,3,4). (1)Department of Anesthesiology, Washington University, St. Louis, MO, USA; (2)Program in Neuroscience, Washington University-St. Louis; (3)Department of Anatomy-Neurobiology, Washington University-St. Louis, St. Louis MO; (4)Washington University Pain Center

Opioid Receptor Like-1 (also called OFQ/N or NOP1) is the newest member of the opioid receptor family and least understood. ORL1 plays a key role in pain modulation, opiate tolerance, and responses to stress, reward, and anxiety. It has been reported that c-terminal phosphorylation at serine (S), threonine (T), and tyrosine (Y) residues are required for mu and kappa opioid receptor internalization, desensitization, arrestin recruitment, and mitogen- activated protein kinase (MAPK) signaling. To determine the molecular mechanisms that mediate ORL1-mediated internalization, desensitization, and MAPK signaling we mutated the putative c-terminal phosphorylation residues of human ORL1-YFP. Using wild type and mutant YFP-tagged ORL1 receptors expressed in HEK293 cells we visualized and compared Nociceptin (Noci) and SCH221510-induced (SCH) ORL1 internalization. ORL1 rapidly internalized within 15 to 30 minutes following Noci or SCH treatment. In contrast, c-terminal mutation of ORL1 significantly attenuated agonist-induced internalization of ORL1. Inhibition of calcium influx responses were desensitized in ORL1 wild type but not mutant receptors. In addition, we determined the time course and concentration-dependence of ORL1 mediated MAPK phosphorylation. C-terminal mutants of ORL1 showed markedly different MAPK signaling properties. We also measured ORL1-dependent trafficking and MAPK in primary dorsal root ganglion cells and found similar results. Finally using ORL1 knockout mice and selective antagonists we examined the role of ORL1 in anxiety-like behaviors and cocaine place preference. Together, these data implicate the c-terminus and GRK/arrestin in ORL1 signaling, and support the growing body of data for arrestin-mediated GPCR signal transduction and behavioral function.

NEW PROBES FOR THE KAPPA OPIOID RECEPTOR: SCREENING, CHEMISTRY, AND PHARMACOLOGY Kevin Frankowski (1), John Streicher (2), Stephen Slausen (1), Michael Cameron (2), Thomas Prisinzano (1), Laura Bohn (2), and Jeffrey Aubé (2). (1)University of Kansas Specialized Chemistry Center, Lawrence, KS and (2) Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL

One goal of our laboratories is to provide probes to explore the kappa opioid rcceptor (KOR) and its possible role in addiction. Screening of chemical libraries carried out in collaboration with the Psychoactive Drug Screening Program and the Molecular Libraries Probe Production Centers Network has resulted in the identification of five chemotypes that selectively bind to the KOR. The results of this work will be described along with investigations into the pharmacological action of selected compounds.

NEUROPEPTIDE S INFLUENCES MEMORY MECHANISMS THAT MAY AFFECT REINFORCEMENT LEARNING Rainer K. Reinscheid (1), Celia Garau (1), Wei Si (1). (1)Department of Pharmaceutical Sciences, University of California Irvine, Irvine, CA 92697, USA

Early research on Neuropeptide S (NPS) identified arousal-promoting and anxiolytic-like effects as two prominent physiological functions. We also showed that activation of NPS receptors

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(NPSR) can facilitate fear extinction in mice. NPSR transcripts are prominently expressed in brain structures involved in learning and memory. Accordingly, we recently demonstrated that post-training central NPS administrations dose dependently enhanced memory consolidation in mice, while administration of a synthetic NPSR antagonist (SHA 68) attenuated NPS-induced memory enhancement. In contrast, NPSR knockout mice displayed deficits in aversive and neutral types of memory, suggesting that activity of the endogenous NPS system is critically involved during memory formation. Other reports demonstrated significant effects of NPS on reinstatement of drug-seeking behaviors. Based on these observations we set out to investigate whether NSP signaling might also contribute to memory mechanisms underlying reward learning that occurs during drug abuse. NSPR-deficient mice were tested for acquisition and extinction of reinforced behaviors. Our results suggest that NPS signaling might affect memory mechanisms required for formation of reward memories and that drugs targeting NPSR might be useful therapeutics to prevent relapse.

THE SEARCH FOR NEUROPEPTIDE S RECEPTOR LIGANDS AS TREATMENTS FOR SUBSTANCE ABUSE Scott P. Runyon (1), Rainer Reinscheid (2), Yanan Zhang (1), Carla Hassler (1), Danni Harris (1), Nicolas Goeders (3), and Chris Schmoutz (3). (1)Organic and Medicinal Chemistry; Research Triangle Institute, Research Triangle Park. NC; (2)Department of Pharmaceutical Sciences; UC Irvine, Irvine CA; (3)Department of Pharmacology, Toxicology & Neuroscience; LSU Health Sciences Center

The coupling of putative neurotransmitters with orphan receptors using reverse pharmacology has led to the identification of several important ligand-receptor associations that are beginning to demonstrate novel pharmacology in vivo. In particular, this technique has led to the pairing of neuropeptide S (NPS) with its cognate G-protein coupled receptor (NPSR). Neuropeptide S is a 20-amino acid peptide that functions as an agonist through activation of its cognate GPCR receptor system. Modulation of the Neuropeptide S (NPS) receptor has been associated with a variety of disease states including anxiety, panic disorder, narcolepsy, PTSD, and importantly substance abuse. Our program has been focused on developing novel entities based on the bicyclic piperazine scaffold (SHA-68) that suffers from poor drug-like properties. We have now defined a comprehensive NPS antagonist pharmacophore using traditional medicinal chemistry and computational/scaffold hopping approaches. The compounds developed in our laboratory are highly potent at blocking the NPS receptor in vitro and are also active in models of cocaine self-administration, stress induced, cue induced, and drug induced reinstatement. Further modification of these analogs will be of significant value in determining the comprehensive role of NPS in modulating substance abuse behaviors. Support: Grant MH087826 and MH081247

THE UROTENSIN II RECEPTOR IS EXPRESSED PRESYNAPTICALLY IN THE VTA BY CHOLINERGIC MESOPONTINE NEURONS Stewart D. Clark, Department of Pharmacology and Toxicology. University of Buffalo SUNY. Buffalo, NY.

In the last decade there has been the discovery of a number of neuropeptides which have greatly increased our understanding of brain function. We have previously shown that the mRNA of the receptor for the novel neuropeptide urotensin II (UII) is expressed by mesopontine cholinergic neurons (laterodorsal tegmentum, LDTg ; pedunculopontine tegmentum, PPTg). It was also found that areas known to be axonal projection sites of these mesopontine tegmental neurons, which had no detectable UII receptor (UIIR) mRNA, express the UIIR protein (receptor binding of 125I-UII; lateral septal nucleus dorsal (LSD), medial habenular nucleus (MHb), ventral tegmental area (VTA)). Subsequently, we have shown that UII microinjection into the VTA produces a dose-dependent, sustained dopamine efflux in the nucleus accumbens (NAc). Vis- à-vis, the UIIR-mediated release of dopamine in the NAc is similar to which is seen after

INRC 2012 Kansas City, Missouri 25 administration of drugs of abuse or acquisition of natural ‘rewarding’ stimuli. Moreover, preliminary data suggest that the UIIR agonists mediate reward- like behaviors such as conditioned place preference. Together, these data suggest UIIR may have a role in regulating goal-oriented behaviors. Our working hypothesis is that UIIR is expressed presynaptically by cholinergic mesopontine neurons, and that UIIR activation facilitates release of neurotransmitter(s) from these terminals that subsequently excite VTA dopaminergic neurons. However, due to the detection and resolution limitations of in situ hybridization and receptor binding, the presynaptic localization of the UIIR has not been firmly established. Therefore, to demonstrate that UIIR expressed in the VTA, and other areas, are due to presynaptic expression by the LDTg & PPTg neurons, we have used a combination of 125I-UII in situ autoradiography receptor binding and a novel fusion toxin (UII-diphtheria). Bilateral stereotaxic injection of the toxin into both the LDTg & PPTg selectively killed the UIIR expressing neurons. Consequently, this abolished 125I-UII receptor binding in the VTA while leaving the binding in the LSD and MHb intact. These findings suggest that the UIIR expression in VTA, but not in the LSD or MHb, is due to mesopontine innervation. Funding: NIDA R00DA024754

OREXIN RECEPTOR LIGANDS: IDENTIFICATION OF NEW TOOLS TO STUDY DRUG ABUSE Yanan Zhang, Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park. NC.

Orexin-expressing neurons, although small in number and highly constrained in the lateral hypothalamus, have extensive projections throughout the central nervous system (CNS) and affect a variety of physiological functions including sleep/wakefulness, feeding behaviors, and reward and motivation. Animal studies implicated that the orexins, and the orexin-1 receptor in particular, are involved in drug reinforcement, conditioned reinforcement and drug reward. Drug development targeting the orexin system, however, has thus far primarily focused on selective OX2 antagonists and/or dual OX1/OX2 antagonists for sleep disorders such as insomnia. Only a small number of OX1 selective antagonists have been described and SB-334867 represents the primary pharmacological entity in evaluating the physiological role of the OX1 receptor in vivo. Despite its high selectivity, SB-334867 has undesirable properties such as limited bioavailability and a relatively short half-life. Our efforts in developing OX1 selective ligands with improved drug-likeness and pharmacological profiles have focused on two structure classes – diaryl ureas based on SB334867 and tetrahydroisoquinolines based on the dual OX1/OX2 antagonist ACT-078573 (almorexant). Various modifications have been performed on these scaffolds and some of the synthesized analogs showed low nanomolar potency at and excellent selectivity for the OX1 receptor. Select compounds were tested in animal models of drug abuse including morphine induced behavioral sensitization and conditioned place preference. Continued effort along this venue may eventually lead to novel and druggable OX1 selective ligands for the treatment of drug abuse. Support: Grant DA026582, DA032837

Symposium #2 – Glial Role in Opioid Analgesia, Tolerance and Dependence Organizers and Chairs: Shuanglin Hao, University of Miami, FL and Linda Watkins, University of Colorado at Boulder

ANTI-INFLAMMATORY CYTOKINES MEDIATED BY HSV VECTOR REDUCES MORPHINE TOLERANCE AND PHYSICAL WITHDRAWAL S. Hao, Department of Anesthesiology, University of Miami Miller school of Medicine, Miami, FL

Clinical utility of opiate analgesics is often hampered by the development of tolerance/dependence. Glia is highly heterogeneous within the CNS; glial activation during chronic neuroinflammatory diseases may demonstrate regional differences in expression levels of proteins in the nervous system. Chronic administration of systemic or intrathecal morphine 26 INRC 2012 Kansas City, Missouri activates glia cells and upregulates proinflammatory cytokines, such as, tumor necrosis factor alpha (TNFα). TNF soluble receptor may prevent TNFα from binding TNF receptors in the cell surface to neutralize the effect of TNFα. Interleukin 4(IL-4) is one of anti-inflammatory cytokines. We have reported that replication-defective Herpes Simplex Virus (HSV) vector expressing p55TNF soluble receptor or IL-4 reduces neuropathic pain induced by peripheral nerve injury or spinal cord damage. Here, we report that the effect of HSV vector expressing p55TNF soluble receptor/IL-4 on the development of morphine tolerance and/or physical withdrawal. p55TNF soluble receptor/IL-4 mediated by HSV delayed the development of morphine tolerance and suppressed physical withdrawal behavior response, and reversed the increase in proinflammatory molecules. The studies suggested that proinflammatory cytokines were involved in the development of morphine tolerance/physical withdrawal, and that gene therapy expressing anti-inflammatory cytokines may provide a novel approach to treating morphine tolerance/physical withdrawal. Supported by NIH DA20078, DA026734, DA025527, NS066792.

THE “TOLL” OF GLIAL ACTIVATION BY OPIOIDS: ENHANCING THE CLINICAL EFFICACY OF OPIOIDS BY BLOCKING TOLL-LIKE RECEPTOR 4 WITH THE NON-OPIOID (+)- ISOMERS OF NALOXONE AND NALTREXONE L.R. Watkins, Dept. of Psychology & Neuroscience, Univ. of CO-Boulder, Boulder, CO

It has been known for several decades that opioids can alter the function of glial cells and endothelial cells. However, it is only within the past 5 years that it has become clear that opioids affect CNS non-neuronal cells through a key activation receptor called toll-like receptor 4 (TLR4). This is in keeping with the recent recognition of TLR4 as an innate immune receptor whose functions include not only the recognition of pathogens and alarmins (signals of host cell stress and damage), but also the recognition of xenobiotics, that is, chemicals not expected to be present within an organism. The activation of TLR4 by such diverse chemical structures sets it apart from every neurotransmitter receptor classically studied in the opioid literature. The study of the role of TLR4 in modulating the effects of opoiods arose as a natural extension of studies over the past 20 years documenting that glia (astrocytes and microglia) are activated under conditions of neuropathic pain and that such glial activation powerfully contributes to such nerve injury-induced pain through the release of proinflammatory, neuroexcitatory substances. It recently became clear that this pain-enhancing effect of glial activation was occurring, in part, through nerve damage-induced release of alarmins, causing activation of TLR4. As, by then, glial activation had been implicated in the development of morphine tolerance, and many parallelisms exist in mechanisms underlying neuropathic pain and morphine tolerance, this link led to our investigations of the role of TLR4 in various opioid effects. The results were striking. Blockade of TLR4 enhanced the utility of opioids for pain control; removing the opioid-induced, TLR4-mediated neuroexcitation increased the potency and duration of morphine analgesia while decreasing tolerance, dependence, reward, opioid-induced constipation, and likely opioid- induced itch. The study of the role of TLR4 in modulating opioid actions was made possible through the discovery of non-opioid (+)-naloxone and (+)-naltrexone as blood brain barrier permeable, selective TLR4 inhibitors. Characterization of these non-opioid, unnatural isomers via assays of 68 potential receptors, enzymes, second messengers, and biogenic amine transporters failed to find significant non-TLR4 activity for either compound. Hence these are the best characterized blood-brain barrier permeable TLR4 antagonists to date, opening up the ability to study the impact of TLR4 on a wide array of opioid actions classically believed to occur solely through opioid receptors.

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TARGETING CERAMIDE AS A NOVEL THERAPEUTIC APPROACH IN OPIATE-INDUCED HYPERALGESIA AND ANTINOCICEPTIVE TOLERANCE Daniela Salvemini, Dept of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St Louis, MO

Opiates, exemplified by morphine, are the most effective analgesics for treating acute and severe pain, but their use is limited by the development of hyperalgesia and analgesic tolerance necessating escalating doses to achieve equivalent pain relief. This complex pathophysiological cycle represents a critical barrier to the quality of life of patients due to the development of many side-effects and fear of addiction. The causative mechanisms remain poorly understood, and consequently, there are currently no target-directed therapeutic approaches. A better understanding of the underlying biochemical pathways is of great significance and clinical importance. Moreover, in order to develop novel therapeutic approaches new targets for potential intervention must be identified. To this end, our recent findings implicating the ceramide metabolic pathway as a critical determinant in the development of morphine hyperalgesia and antinociceptive tolerance define viable target for intervention. The development of morphine-induced hyperlagesia and anitinociceptive was associated with increased formation of ceramide and S1P the end-product of ceramide metabolism in spinal cord glial cells. Pharmacological inhibition of ceramide (with inhibitors of the de novo pathway or the sphingomyelin pathway), or S1P (with sphingosine kinase 1 and 2 inhibitors) formation or of S1P’s action (with S1P receptor antagonists) prevented hyperalgesia/antinociceptive tolerance. Our results further revealed that ceramide and S1P contribute to the development of hyperlagesia and anitinociceptive tolerance through increased formation of glial-related proinflammatory cytokines, in particular TNF-a, IL-1b, and IL6, which are known modulators of neuronal excitability, and 2) peroxynitrite-mediated post-translational nitration and inactivation of glial-related enzymes (glutamine synthetase and the glutamate transporter, GLT-1) known to play critical roles in glutamate neurotransmission. Our findings establish a varied platform approach of pharmacological manipulation that can intercept the ceramide metabolic pathway at many levels to pre-empt opioid-induced hyperalgesia and tolerance. Inhibitors of the ceramide metabolic pathway may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

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INRC 2012 Founder’s Lecture

DESIGN AND DEVELOPMENT OF OPIOID RECEPTOR ANTAGONISTS FOR SUBSTANCE ABUSE RESEARCH F. Ivy Carroll, Research Triangle Institute

Opiates, cocaine, methamphetamine, nicotine, and alcohol abuse have been and still remain major public health problems. This address will highlight our studies directed toward the design and development of opioid receptor antagonists as pharmacological and clinical research tools for use in obtaining a better understanding of substance abuse disorders. The main focus will be on the development of the selective kappa opioid receptor antagonist JDTic. Studies leading to selective mu and delta opioid receptor antagonists and delta inverse agonists will also be presented. All studies presented have been supported by the National Institute on Drug Abuse (NIDA).

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Symposium #3 – Opioid Peptides in Motivational Circuits Organizer and Chair: Elyssa Margolis, Ernest Gallo Clinic & Research Center/UCSF

THE VOLTAMMETRIC DETECTION AND CHARACTERIZATION OF MET-ENKEPHALIN IN BRAIN TISSUE Leslie A. Sombers, Department of Chemistry, North Carolina State University, Raleigh, NC

Endogenous opioid function has been implicated in specific aspects of reward in models of addiction. In order to understand the precise molecular mechanisms involved, an analytical method is required for real-time detection of endogenous opioids in living brain tissue. Background-subtracted fast-scan cyclic voltammetry coupled with carbon-fiber microelectrodes has proven to be sensitive and selective for detecting rapidly fluctuating neurochemicals in vivo. However, many challenges exist for applying this approach to the detection of neuropeptides. We have developed and characterized a novel voltammetric waveform for the detection of tyrosine-containing neuropeptide molecules, such as met-enkephalin (mENK). We have established that the main contributor to the electrochemical signal is tyrosine, and that conventional waveforms provide poor peak resolution and allow fouling of the electrode surface. With our analyte-specific waveform we have selectively distinguished mENK from common endogenous interferents, such as dopamine and pH shifts, and have detected exogenous mENK in living brain tissue. This work provides a foundation for real-time measurements of endogenous mENK, and is broadly applicable to the voltammetric detection of a variety of tyrosine-containing neuropeptides. Sponsored by NC State University, Raleigh, NC.

DEVELOPMENT AND APPLICATION OF CAPILLARY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY METHODS FOR MONITORING ENDOGENOUS OPIOID PEPTIDES IN VIVO Robert T. Kennedy, Omar Mabrouk, Alex Delifictano, Kent Berridge. Department of Chemistry, University of Michigan, Ann Arbor, MI

Microdialysis has been a valuable method for monitoring low molecular neurotransmitters in vivo. Measurement of neuropeptides has proven more difficult because of their low concentrations and the many similar forms of peptide that result from metabolism by peptidases. We have found that capillary LC-MS has the sensitivity and specificity to allow monitoring of several opioid peptides in vivo. In particular, we have developed methodology to simultaneously measure met-enkephalin, leu-enkephalin, dynorphin, and b-endorphin. Using this method we have uncovered reciprocal interactions of dopamine and opioids in the globus pallidus using pharmacological manipulations. We have also demonstrated that enkephalins are released in select regions of the striatum during feeding on palatable foods (M&Ms). Similarly, injection of opioids into the same region induces excess feeding. These results demonstrate that the method can be used to track both behavioral related changes and pharmacological changes in neuropeptide release in vivo. Sponsored by the NIH, award number R37 EB003320, and the University of Michigan Substance Abuse Research Center.

CORTICAL AND SUB-CORTICAL INTERACTIONS CONTROLLING MEMORY FORMATION DURING THE OPIATE ADDICTION PROCESS Steven Laviolette, Dept. of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, University of Western Ontario, Ontario, Canada.

Addiction to both illicit and prescription opiate-class drugs is on the rise. From a motivational perspective, opiates, such as morphine and heroin serve as potently rewarding stimuli that serve as powerful associative memory cues. Indeed, the ability of opiate-related memories to trigger drug craving is one of the most critical factors underlying opiate addiction relapse and compulsive abuse. Two neural regions in particular, the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) are involved importantly in the formation and recall 30 INRC 2012 Kansas City, Missouri of memories related to the opiate addiction process. These neural regions are functionally linked to the ventral tegmental area (VTA) and nucleus accumbens (NAc), and modulate activity within the mesolimbic dopamine system. Within this neural circuitry, signaling through dopamine and glutamate receptors is critical for the processing of opiate related learning and memory plasticity. Using a combination of multi-unit and single-unit neuronal recordings in awake behaving rodents combined with behavioural pharmacological approaches, we are investigating how glutamatergic and dopaminergic pathways influence the functional relationships between these cortical and sub-cortical regions during the encoding, recall and extinction of opiate- related associative memories.

MU OPIOID RECEPTOR ACTIONS IN THE VENTRAL TEGMENTAL AREA; THE JURY IS STILL OUT Elyssa B. Margolis, Ernest Gallo Clinic and Research Center, Emeryville, & University of California, San Francisco, CA

Mu opioid receptors (MORs) in the ventral tegmental area (VTA) are required for morphine reward, however the critical synaptic and local circuit actions of MOR agonists on VTA output the lead to positive reinforcement are uncertain. The canonical model of VTA MOR reward asserts that MOR in the VTA produces reward by inhibiting GABA neurotransmission, thereby disinhibiting dopamine neurons. However, intra-VTA MOR reward can be dopamine independent, and there are several other actions of MOR in the VTA that could contribute to reward. For instance, many VTA neurons, including dopamine neurons, are post-synaptically inhibited by MOR agonist DAMGO. Further, MOR effects sort by projection target. The fact that MOR acts on multiple circuits that include both dopamine and non-dopamine VTA neurons raises the intriguing possibility that MOR signaling regulates a distributed set of VTA outputs that act in concert to produce a reinforcement or reward signal. The effort depicted was supported by DA-030529-01 and the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco.

OPIOID REGULATION OF PAIR BOND FORMATION AND MAINTENANCE Shanna Resendez (1), Mackenzie Dome (2), Brandon Aragona (1,2) (1) Program in Neuroscience, University of Michigan, Ann Arbor, (2) Department of Psychology, University of Michigan, Ann Arbor

The socially monogamous prairie vole is an ideal animal model for studying the neurobiology of social attachment. Prairie voles form enduring pair bonds that begin with the development of an initial preference for their mating partner and is associated with affiliative social interactions. However, following the initial formation of the bond, there is a reduction in general affiliative behavior and an enhancement in aversive social encounters, such as selective aggression toward novel conspecifics. Here, we demonstrate that the initial formation and maintenance of the pair bond are regulated by opioid receptors that encode either positive reward (Mu-opioid receptors; MOR) or aversion (kappa-opioid receptors; KOR). We first used test of partner preference to demonstrate that MORs within the dorsal medial, but not ventral, nucleus accumbens (NAc) shell are important for initial pair bond formation. We next show that activation of KORs, but not MORs, within the NAc mediates selective aggression that is characteristic of an established pair bond. Together, these data demonstrate that opioids within the NAc that encode positive reward are important for initial pair bond formation while those that encode aversion are important for pair bond maintenance. We next extended our findings by examining interactions between the opioid system and dopamine system that has previously been identified as important for pair bonding. Specifically, activation of D1-like receptors within the NAc mediates selective aggression and stimulation of these receptors increase dynorphin, the endogenous ligand of KORs. We therefore hypothesized that D1-mediated aggression occurs through interactions with KORs. Indeed, activation of KORs within the NAc prevented reductions in selective aggression that normally occur through blockade of D1-like receptors.

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These data suggest that activation of KORs within the NAc directly drives aversive social interactions necessary for pair bond maintenance. This work was supported by NSF grant 0953106 to BA.

Symposium #4 – Young Investigators Organizer and Chair: Thomas Prisinzano, University of Kansas

INVESTIGATION OF STERICALLY HINDERED MEPERIDINE ANALOGS AT P- GLYCOPROTEIN AND CYP3A4 Susan L. Mercer (1,2), (1)Department of Pharmaceutical Sciences, Lipscomb University College of Pharmacy, Nashville, TN 37204 and (2)Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232

Chronic clinical pain remains poorly treated. The use of mu opioid analgesics such as morphine can treat the pain, but the rapid development of tolerance to the analgesic effects necessitates ever increasing doses to be administered. Extensive research has investigated the development of central tolerance focused at the cellular and receptor levels; however, recent studies suggest that a systems level approach including metabolism and efflux transporters is involved. The efflux transporter P-glycoprotein (P-gp) is present at the BBB, and actively pumps morphine out of the CNS. Animals tolerant to morphine display up-regulation of P-gp and therefore lower levels of morphine in the brain compared to naïve animals, demonstrating that P-gp contributes to central tolerance. P-gp substrates are generally characterized as lipophilic compounds containing a basic nitrogen(s) and are also commonly CYP3A4 substrates. Most opioids have these characteristics and therefore most clinically used opioids have P-gp activity. Meperidine is an exception in that it does not have P-gp activity although it is a lipophilic CYP3A4 substrate with a basic nitrogen. However, meperidine is not an optimal pain treatment due to low potency and toxic metabolite formation. CYP3A4 N-demethylates meperidine into normeperidine, a toxic metabolite, which leads to convulsions and potential death in accumulation. Our hypothesis is that meperidine can be optimized by introducing steric hindrance to the piperidine ring at the 2- and 6-positions to eliminate toxic metabolite formation. Preventing CYP3A4 metabolism will eliminate toxic metabolite formation and allow meperidine to be administered over a longer period of time. The synthesis of the 2,6-dimethyl and 2,2,6,6- tetramethyl analogs will be presented, along with biological analyses including opioid binding, P- gp activity, and CYP3A4 metabolism. The ideal meperidine analog maintains opioid activity, lacks P-gp interaction, and is not metabolized by CYP3A4, potentially leading to a novel opioid lacking tolerance development for use in the treatment of chronic, severe pain.

THE EFFECTS OF DELTA-OPIOID RECEPTOR LIGANDS ON THE CONDITIONED STIMULI ASSOCIATED WITH COCAINE Emily M. Jutkiewicz, Department of Pharmacology, University of Michigan University of Michigan Substance Abuse Research Center, University of Michigan

Data from pharmacological and genetic studies suggest that the delta opioid receptor system modifies addiction-related behaviors and neurocircuitry. For example, delta opioid receptors influence ethanol consumption and the reinforcing effects of ethanol (for review see Mendez and Morales-Mulia, 2008). Delta opioid receptor activation also increases cocaine-seeking in self-administration and conditioned place preference paradigms (Simmons and Self, 2009; Kotlinska et al., 2010) and increases amphetamine-stimulated dopamine efflux (Bosse et al., 2008). On their own, delta opioid agonists have stimulant-like effects, but nonpeptidic delta opioid agonists typically fail to promote or maintain self-administration behavior in animal models. Considering delta opioid agonists influence drugs of abuse without having primary reinforcing effects, this study investigated the effects of delta-opioid ligands on the conditioned reinforcing effects of cocaine in rats. The delta opioid agonist SNC80 either failed to maintain 32 INRC 2012 Kansas City, Missouri responding or maintained low levels of responding in naïve rats. In rats with a history of cocaine self-administration, non-contingent SNC80 administration enhanced operant responding maintained by stimuli previously associated with cocaine, but did not alter responding in the absence of the cocaine-paired stimuli or on the inactive manipulandum. The delta opioid antagonist naltrindole decreased responding for cocaine-paired stimuli in the absence of cocaine. Overall, these data suggest that the DOR system may modulate the reinforcing effectiveness of conditioned stimuli associated with drugs of abuse. These studies were supported by the Small Grants Program from the University of Michigan Substance Abuse Research Center.

THE CELLULAR LOGIC AND MOLECULAR MECHANISMS OF CONTROL OF CUTANEOUS MECHANOSENSATION BY OPIOID RECEPTORS Grégory Scherrer, Department of Physiology & Cellular Biophysics, Columbia University

Sensory neurons of the dorsal root ganglia (DRG) are heterogenous in order to respond to a variety of environmental stimuli and transmit somatosensory information to spinal neurons. Opioids can relieve pain by altering activity in spinal neuronal circuits, but the precise cellular and molecular mechanisms of spinal opioid analgesia remain poorly understood. Here we used a combination of mouse genetics, neuroanatomical and electrophysiological approaches to investigate the organization of spinal opioidergic circuits that regulate somatosensation. We found that mu opioid receptor (MOR) expression is restricted to populations of C and Adelta pain fibers (nociceptors) that express CGRP and TRPV1, and depend on TrkA signaling for their survival. By contrast, the delta opioid receptor (DOR) is predominantly expressed by both rapidly and slowly adapting populations of Abeta touch fibers (low-threshold mechanoreceptors, LTMRs) coexpressing the neurotrophin receptors TrkC and Ret. Furthermore, calcium imaging in primary DRG culture and electrophysiological recordings in spinal cord slices confirmed the Abeta LTMR identity of DOR-expressing DRG neurons and showed that DOR agonists depress Abeta fiber input to lamina III spinal neurons. Finally, we have identified a discrete population of CGRP-expressing Adelta nociceptors that coexpresses MOR and DOR. In these neurons, which have been implicated in the detection of noxious mechanical stimuli, MOR and DOR are colocalized at the plasma membrane in basal conditions. Together our data provide new insights into the organization of the opioid system in sensory neurons and the logic with which opioid receptors regulate integration and transmission of pain and touch information in the spinal cord.

MORPHINE AND HIV-1 TAT-INDUCED SUBLETHAL INJURY IS CAUSED BY A DISRUPTION IN ION HOMEOSTASIS AND CAN BE ATTENUATED BY NMDAR & AMPAR BLOCKADE IN THE STRIATUM S. Fitting (1), S. Zou (2), P. E. Knapp (2), K. F. Hauser (1). Dept. of (1)Pharmacol. & Toxicology and (2)Anat. and Neurobiol., Virginia Commonwealth Univ. - MCV Campus, Richmond, VA

Synaptodendritic damage and accompanying synaptic losses are correlated with neurobehavioral deficits seen in chronic HIV-1 infection and opioid abuse. Parallel in vivo (GFAP-driven, doxycycline-inducible, transgenic Tat mice) and in vitro (primary striatal neuron culture) studies examined the mechanisms underlying synaptodendritic injury and neurobehavioral defects caused by Tat ± morphine exposure. Our findings indicated that Tat and/or morphine uniquely disrupt neuronal synaptic organization (as assessed in Golgi- impregnations and by electron microscopy); however, combined opioid and Tat induction caused synergistic dendritic pathology at 7-10 days following continuous exposure to both. Pathology included the formation of bead-like varicosities and/or eventual fragmentation of spiny neuron dendrites in Golgi impregnations (Fitting et al., Am J Pathol, 2010). In vitro studies revealed, within a 10 min time window, nearly identical dendritic swellings as seen in vivo. Tat + 2+ caused significant focal increases in [Na ]i and [Ca ]i along the dendrites within the first 2 min, but returned to baseline levels after the initial elevation. In contrast, Tat ± morphine treatment

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+ 2+ caused significant sustained increases in [Na ]i and [Ca ]i, as well as corresponding increases in focal dendritic swellings. Importantly, Tat ± morphine-induced initial losses in ion homeostasis 2+ and increased [Ca ]i production were attenuated by the AMPA receptor antagonist, CNQX, + 2+ whereas the later sustained increases in [Na ]i and [Ca ]i depended more on NMDAR. The Tat 2+ and morphine-driven excitotoxic, glutamatergic dysregulation of [Ca ]i homeostasis appears to be an important event in opioid-HIV-1 Tat synergistic interactions, and is likely to be critical in underlying the loss of synaptic connectivity. The studies outlined herein will contribute to better therapeutic approaches toward allaying the CNS consequences of opioid abuse-HIV-1 comorbidity. Support: NIDA DA018633, DA027374.

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(P2) Plenary Lecture #2

MEDICINAL CHEMISTRY IN DRUG ABUSE RESEARCH AT THE NIH – LOOKING BACK Kenner Rice, National Institute on Drug Abuse and National Institute on Alcohol Abuse and Alcoholism

The present research program in our laboratory is one of the oldest continuous programs at NIH. It was originated in 1929 by the National Research Council as part of the first coordinated program to develop improved strong analgesics and better understand the complex pharmacology of opioids. The program has been under the sequential leadership of Drs. Lyndon Small, Nathan Eddy, Everette May, Arnold Brossi and the present author since its inception. Dedicated efforts in organic and medicinal chemistry during this work have provided many novel drugs and research tools that enabled advances in the pharmacology of narcotic analgesics, their antagonists, and other drugs of abuse. Early studies demonstrated the proof of principle of the original program hypothesis that specific chemical modification of the morphine molecule could separate the beneficial and detrimental effects of morphine. Later work afforded tools for PET imaging of opioid receptors, purification of the delta opioid receptor to homogeneity seven years prior to cloning, introduction of conformationally restrained 5-phenylmorphans as probes of opioid receptor structure and function, and the development of the NIH Opiate Total Synthesis. The latter renders the natural opiate enantiomers available independent of the opium poppy and thus offers opium poppy eradication as a strategy to combat heroin abuse. It also provides unnatural opiates as valuable research tools and as potential agents for the treatment of drug abuse via functional antagonism of Toll-like 4 receptors. This presentation will highlight the role of organic and medicinal chemistry in these and other discoveries emanating from the NIH program.

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Symposium #5 – Opioid/Cannabinoid Interactions Organizer and Chair: Jean Bidlack, University of Rochester

CB1 CANNABINOID AND MU OPIOID RECEPTORS INTERACT IN CNS SIGNALING, ANALGESIA AND DRUG DEPENDENCE Dana E. Selley (1), Sandra P. Welch (1), Divya Ramesh (1), Benjamin F. Cravatt (2), Aron H. Lichtman (1) and Laura J. Sim-Selley (1). (1)Dept. Pharmacology & Toxicology, Virginia Commonwealth University., Richmond, VA; (2)Dept. Chemical Physiology, The Scripps Research Institute, La Jolla, CA

CB1 cannabinoid (CB1R) and mu opioid receptors (MOR) mediate the psychoactive effects of marijuana and opiates such as morphine, respectively. These receptors are co-distributed in CNS regions that mediate reward, appetite, motor activity, analgesia, stress responses and cognition, and are co-localized in some neurons in regions including striatum and spinal cord. Both receptors are coupled mainly to Gi/o-proteins, and mediate inhibition of adenylyl cyclase and Ca2+ channels, and stimulate K+ channels and several kinases. Agonists of either receptor can produce tolerance and dependence with chronic administration. CB1Rs are generally more sensitive to agonist-induced desensitization and downregulation, whereas similar adaptation of MORs exhibits lower magnitude and a more restricted regional distribution. Co-administration of low doses of CB1R and MOR agonists produces synergistic antinociception, and avoids tolerance and receptor adaptation. Chronic administration of CB1R or MOR agonists induces the transcription factor DFosB in the striatum, which is associated with enhanced rewarding effects of opioids. Transgenic expression of DFosB in nucleus accumbens enhances MOR but not CB1R signaling. Chronic CB1R agonist treatment similarly enhances MOR signaling in this region, suggesting that cannabinoids might cross-sensitize reward pathways to opioids. A new approach to target CB1Rs indirectly with fewer side effects is to block endocannabinoid catabolism. We found that inhibition of fatty acid amide hydrolase or monoacylglycerol lipase increases specific endocannabinoid levels, produces antinociception, and reduces opioid withdrawal symptoms in morphine-dependent mice. Together these results suggest that the endogenous cannabinoid system can serve as a target for drug development to treat pain and opioid dependence with minimal adverse effects. We thank Dr. Eric J. Nestler for DFosB transgenic mice. This work was supported by grants from NIH/NIDA.

RECEPTORS GONE WILD: CONSEQUENCES OF CB2 CANNABINOID RECEPTOR FUNCTIONAL SELECTIVITY Brady Atwood, Alex Straiker, Jim Wager-Miller, and Ken Mackie. The Gill Center and the Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47405

Classically, responses mediated through ligands acting at G protein-coupled receptors (GPCR’s) have been quantified by potency and one-dimensional efficacy. Recent studies examining a range of GPCR’s have revealed that this conceptual framework is overly simplistic, and efficacies for a particular signaling pathway may vary widely among different ligands; a phenomenon termed functional selectivity. When examining the signaling of a range of CB2 ligands, we have found striking functional selectivity. Most remarkable was that the aminoalkylindoles (including WIN55,212-2, AM1241, and JWH015) failed to internalize CB2 receptors, or inhibit voltage-dependent calcium channels (VDCC’s), despite activation of ERK1/2, recruitment of beta-arrestin2 to the membrane, and inhibition of adenylyl cyclase. Other CB2 ligands from a variety of chemical classes also displayed a range of functional selectivity. Interestingly, CB2 inverse agonists also displayed functional selectivity, whereby SR144258 both activated VDCC’s and promoted cell surface accumulation of CB2, while AM630 on inhibited VDCC’s, but had no effect on the density of cell surface CB2 receptors. Functional selectivity may offer a path to develop more specific therapeutic agents. For example, if the pathways mediating a desired outcome can be identified, it might be possible to synthesize ligands that only activate those pathways. (However, it must be kept in mind that by activating 36 INRC 2012 Kansas City, Missouri only those pathways, the functional selective ligand is blocking activation of the non-preferred pathways by endogenous ligands.) In ongoing studies we are correlating the particular signaling pathways activated by CB2 agonists and their efficacy in various preclinical models. Supported by NIH grants DA021696, DA011322, and DA009158

CANNABINOIDS, OPIOIDS AND SPHINGOSINE-1-PHOSPHATE1 (S1P1) RECEPTOR INTERACTIONS IN ANTINOCICEPTION Sandra P. Welch, Dana E. Selley, Laura J. Sim-Selley Dept. of Pharmacology/Tox., Virginia Commonwealth Univ., Richmond, VA

A novel class of compounds that bind to sphingosine-1 phosphate (S1P) receptors is strikingly similar to cannabinoids (CBs) in their activity and are localized with both CB and opioid receptors in the central nervous system in pain pathways. Such evidence led us to evaluate the potential interaction of the three diverse receptor systems in the modulation of antinociception. The antinociceptive effects of morphine are attenuated by the S1P1/3 antagonist VPC44116 in mice. In addition, in sphingosine kinase2 knockout mice (lacking S1P), both morphine- and THC-induced antinociceptive efficacy is significantly reduced. Thus, the antinociceptive effects of both morphine and THC are dependent upon phosphorylation of sphingosine to S1P. The antinociceptive effects of both morphine and THC are enhanced by the administration of subactive doses of the S1P analog FTY720 (fingolimod), S1P, and the S1P1 receptor- selective drug, CYM- 5442. Conversely, the antinociceptive effects of FTY720 are enhanced in FAAH knockout mice, but are non-CB1, non-CB2 mediated and are not altered in THC-tolerant mice. The effects of several S1P analogs, including FTY720, produce antinociception blocked by the opioid non-selective (naloxone) or kappa (nor-binaltorphimine) antagonists, but are not cross- tolerant to morphine. Using transgenic mice, deletion of CB1- or mu opioid receptors fails to alter FTY720-induced antinociception. Although S1P and FTY720 have been shown to interact with CB receptors in vitro (Paugh et al., 2006), morphine-induced G-protein activation via [35S] GTPgS autoradiography in several brain regions is not attenuated in FTY720-tolerant mice. Our data indicate that the three receptor systems are highly dependent upon one another in the production of antinociception. Subsequently, the potential use of S1P agonists as adjuncts to opioid or cannabinoid analgesics might be a potential new therapeutic target for such drugs. The Presidential Research Incentive Grant (VCU) and NIDA RO3DA-05274 supported this work.

INVOLVEMENT OF THE ENDOCANNABINOID SYSTEM IN DRUG ADDICTION Fernando Berrendero, Laboratory of Neuropharmacology, Department of Experimental and Health Sciences, University Pompeu Fabra, PRBB, C/ Doctor Aiguader 88, 08003 Barcelona, Spain

A growing body of evidence indicates that the endogenous cannabinoid system is involved in the common neurobiological substrate underlying drug addiction which is characterized by the compulsive use of drugs in spite of their adverse consequences, loss of control over drug-taking and relapse even after long periods of drug abstinence. CB1 cannabinoid receptors and endocannabinoids are distributed in the mesolimbic system and modulate dopaminergic activity within these reward circuits. Thus, this system participates in the primary rewarding effects of cannabinoids, nicotine, alcohol and opioids probably through the release of endocannabinoids in the ventral tegmental area. In addition, recent studies also point to a role of CB2 cannabinoid receptors in the rewarding and psychomotor stimulating-effects of cocaine. Drug-induced alterations in the endocannabinoid system may also contribute to the processes driving to relapse to drug-seeking behavior. Based on these data, pharmacological manipulation of the endocannabinoid system could constitute a new therapeutic strategy for addictive disorders.

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TUNING CANNABINOID RECEPTOR SIGNALING BY RGS (REGULATORS OF G-PROTEIN SIGNALING) PROTEINS: NEW PLAYER IN CANNABINOID RECEPTOR SIGNALOSOME Somnath Mukhopadhyay (1,2), Vladimir Poltoratsky (1), Genevieve LaRoche (2), Shailendra Devkota (1), Patrick GiGuere (2), and David Siderovski (2) (1)Neuroscience & Cancer Research Program Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA; (2)Department of Pharmacology, UNC-Chapel Hill School of Medicine, Chapel Hill NC 27599

CB1 and CB2 cannabinoid receptors belong to Gi protein-coupled receptor superfamily. RGS proteins are classically known for turning off Gi and Gq-coupled receptor signaling. Activation of CB1 and CB2 cannabinoid receptors elicits a plethora of cellular and physiological responses acting through different Gi-proteins. Although agonist-directed differential signaling via preferential coupling with Gi subtypes is known for cannabinoid receptors but very little is known about how these signals are regulated in cannabinoid receptor signalosomes. In the current work we for the first time to define the role of specific RGS-proteins in the regulation of CB1 and CB2 receptor signaling. Previous results from our laboratory and others have showed that stimulation of CB1 and CB2 receptors produce nitric oxide via the activation of nitric oxide synthase (nNOS or eNOS depending on the cell type) in a pertussis-toxin sensitive manner. In the current study using real-time PCR we first screened for native expression of individual RGS proteins in CB1 and CB2 receptor expressing neuronal and endothelial cells lines respectively. We then selectively knock down (using SiRNA) individual RGS proteins in these cell lines and measured cannabinoid receptor-mediated cAMP and nitric oxide production in these RGS- knock down cell lines to determine the specificity of RGS proteins for intercepting CB1 vs. CB2 receptor signaling. Our findings showed that a) coupling profiles of CB1/CB2 receptor with RGS proteins are different and b) RGS proteins differentially regulate (inhibit or activate) CB1 and/or CB2 receptor-mediated signaling depending on the cell types and composition of CB/CB2R- RGS signalosomes. These results showed for the first time that RGS protein regulate CB1/CB2 receptor signaling and can be targeted to selectively dissect cannabinoid receptor-mediated signals. Acknowledgement: This work is supported by NIH grant AA 19765 and CA156735 to SM and NIH grant GM082892 to DS.

OREXIN A INCREASES DOPAMINERGIC ACTIVITY VIA OX1 RECEPTOR-INITIATED ENDOCANNABINOID RETROGRADE DISINHIBITION IN THE VENTRAL TEGMENTAL AREA -A NOVEL ADDICTION MECHANISM L.-W. Tung (1) and L.-C. Chiou (2, 3). (1)Grad. Inst. and (2) Dept. Pharmacology, (3) Grad. Inst. Brain and Mind Sciences, Coll. Medicine, National Taiwan University, Taipei, Taiwan.

The orexin system, consisting of orexin A and B and their receptors, OX1R and OX2R, is implicated in the regulation of motivation, feeding, and adaptive behaviors. Orexin neurons project widely throughout the brain, including the ventral tegmental area (VTA), an important reward processing site where dopamine neuron plasticity can be altered by addictive substances. However, the role of orexin signaling in dopamine neural plasticity is poorly understood. Our recent study revealed a novel analgesic mechanism in the periaqueductal gray: Activation of postsynaptic OX1R, a Gq-protein coupled receptor, results in synthesis of 2- arachydonoylglycerol (2-AG) via phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic pathway. 2-AG, an endocannabinoid, produces retrograde inhibition of GABA release (disinhibition) by activating presynaptic cannabinoid 1 receptors (CB1R).[1] Here, we validate if this OX1R-PLC-DAGL-2-AG retrograde disinhibition cascade also exists in VTA dopamine neurons and contributes to addiction mechanism by examining the effect of orexin A on GABAergic inhibitory postsynaptic currents (IPSCs) in dopamine neurons of rat VTA slices. We found orexin A (100 nM) did depress IPSCs in VTA dopamine neurons. This IPSC depressant effect of orexin A was reversed by AM 251 (3 microM), a CB1R antagonist, and THL (10 microM), a 2-AG synthesizing enzyme (DAGL) inhibitor. These results suggest that orexin A

38 INRC 2012 Kansas City, Missouri also inhibits GABAergic transmission (disinhibition) in VTA dopamine neurons via a retrograde signaling mediated by 2-AG, an endocannabinoid. This endocannabinoid-mediated retrograde disinhibiton mechanism in VTA dopamine neurons may explain the contribution of the orexin system in addictive behavior. Supported by NSC 100-2325-B002-050 [1]Ho YC et al. J Neurosci 31:14600 (2011)

FURTHER ELUCIDATION OF THE SAR OF K2/SPICE AMINOALKYLINDOLES Tamara Vasiljevik (1), Lisa K. Brents (2), Paul L. Prather (2), William E. Fantegrossi (2), and Thomas E. Prisinzano (1). (1)Department of Medicinal Chemistry, School of Pharmacy, The University of Kansas, Lawrence, Kansas, and (2)Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas

In early 2011, five synthetic cannabinoids, JWH-018, JWH-073, JWH-200, CP-47,497, and cannabicyclohexanol, were emergency scheduled as Schedule I substances by the U.S. Drug Enforcement Administration (DEA). However, despite the ban of these compounds, their use has greatly increased with more than 7,000 calls to the Poison Control Center in 2011, which is more than double that of the previous year. Additionally, according to a 2011 study by the National Institute on Drug Abuse, synthetic cannabinoids are the third most frequently abused drug among high school seniors. Despite their increased popularity very little is known regarding the pharmacology, toxicology, and metabolism of these synthetic cannabinoids other than their ability to induce psychoactive effects by activating one of the most abundant G-protein coupled receptors in the central nervous system, the cannabinoid type 1 receptor (CB1R). JWH-018 and JWH-073 are the most prevalent aminoalkylindoles (AAIs) found in the incense blend known as K2/Spice. These AAIs were originally synthesized by Dr. John Huffman for the purposes of studying the endocannabinoid system and the structure-activity relationships (SAR) of the cannabinoid receptors. It was recently reported that a mono-hydroxylated metabolite of JWH-073 exhibited neutral antagonist activity at the CB1R. Disease states that involve increased endocannabinoid signaling could be potentially treated with the use of a CB1R neutral antagonist, which will in effect attenuate the endocannabinoid system. One of these medical conditions is overweight/obesity. Herein, we report the design, synthesis and biological results of indole derivatives resembling the JWH scaffold in efforts of identifying a potential appetite suppressant.

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(P3) Plenary Lecture #3

NEW APPROACHES FOR DECONSTRUCTING DRUG ACTIONS Bryan L. Roth, Department of Pharmacology and Division of Chemical Biology and Medicinal Chemistry and NIMH Psychoactive Drug Screening Program, University of North Carolina Chapel Hill Medical School, Chapel Hill, NC 27514

Pharmacology is the science of drug action and elucidating the molecular, cellular and organismal substrates responsible for the effects of drugs has long been the province of pharmacologists. In this talk I will describe new approaches my lab has pioneered to deconstruct drug action and will highlight recent breakthroughs we have achieved in the area of opioid receptor pharmacology. I will use salvinorin A and κ-opioid receptors as a proof-of- concept. I will first describe new computational approaches (Keiser et al, Nature, 2009; Laggoner et al, Nature Chem Biol, in press) which allow for near druggable genome-wide identification of novel drug targets. I will then debut a novel genome-wide physical screening platform which facilitates the massively parallel screening of the entire non-olfactory human GPCR-ome and show how using this platform we have identified new orphan and non-orphan GPCRs as targets for opioid and non-opioid ligands. I will also demonstrate how recent atomic- level insights into the structures of opioid receptors in complex with their ligands will accelerate the structure-based design of novel opioid ligands. Finally, I will show how a recent chemical- genetic approach my lab has developed can be used to deconstruct circuits responsible for the actions of opioid ligands like salvinorin A.

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Symposium #6 – Medicinal Chemistry of Opioid Peptides Organizer and Chair: Jane Aldrich, University of Kansas

NOVEL KAPPA OPIOID RECEPTOR PEPTIDE LIGANDS AS POTENTIAL TREATMENTS FOR DRUG ABUSE J.V. Aldrich (1), S.N. Senadheera (1), K. Patkar (2), S.O. Eans (2), J.P. McLaughlin (2) (1) Dept. of Med. Chem., Univ. of Kansas, Lawrence, KS, USA, (2) Torrey Pines Inst. for Molecular Studies, Port St. Lucie, FL, USA

Previous studies have demonstrated that ligands with both agonist and antagonist activity at kappa opioid receptors (KOR) have potential as treatments of drug abuse, affecting different stages of drug seeking behavior, from blocking acute drug self administration to preventing reinstatement of drug seeking after extinction. We are exploring a series of novel cyclic peptides with KOR activity as potential treatments for drug abuse. These cyclic peptides should show stability to proteases, and therefore we expected them to remain active following systemic, including oral, administration. The activity profile of these cyclic peptides at KOR and mu opioid receptors (MOR) in vivo was found to depend on the identity and stereochemistry of amino acid residues in the peptides. Thus one stereoisomer of the lead peptide displayed exclusively KOR antagonist activity while the lead peptide exhibited a profile of mixed opioid agonism/KOR antagonism. Moreover, alanine analogs of the lead structures exhibited significant changes in efficacy, agonist selectivity and/or agonist potency. The lead peptide exhibiting solely KOR antagonist activity is orally active, antagonizing both KOR agonist-induced antinociception in the 55o warm water tail withdrawal assay and diuresis. Preliminary pharmacokinetic data demonstrates that this peptide penetrates the blood-brain barrier to antagonize KOR in the CNS, and this peptide blocks stress-induced reinstatement of cocaine seeking behavior after oral administration. The lead peptide exhibiting mixed opioid agonist/KOR antagonist activity is also orally active and exhibits a unique profile in blocking both drug and stress-induced reinstatement of cocaine seeking behavior. These orally active cyclic peptides represent promising leads for drug development. Research supported by grants R01 DA018832 and R01 DA023924.

CONFORMATIONALLY CONSTRAINED AMINO ACIDS IN THE DESIGN OF OPIOID AND BIFUNCTIONAL OPIOID – NK1 RECEPTOR LIGANDS K. Guillemyn (1), I. Van den Eynde (1), A. Keresztes (2), E. Varga (2), F. Porreca (2), N.N. Chung (3), C. Lemieux (3), P. W. Schiller (3), D. Tourwé (1), S. Ballet (1). (1) Dept. of Chemistry, Vrije Universiteit Brussel, Brussels, Belgium; (2) Dept. of Pharmacol., Univ. Arizona, Tucson, U.S.A.; (3) Dept. Chem. Biol. & Pept. Res., Clinical Research Institute of Montreal,Montreal, Canada.

Earlier research demonstrated that bifunctional peptides possessing both NK1R antagonist properties and opioid agonist potency show advantages over current analgesic drugs, such as a potent analgesic effect in both acute and neuropathic pain states, suppression of the development of tolerance and the presentation of antiallodynic and antihyperalgesic effects.[1-3] Different types of constrained aromatic amino acids 1 to 4 were prepared and derivatized to prepare new neurokinin 1 receptor (NK1R) antagonists. This allowed the identification of three novel NK1R antagonists that were able to counteract the agonist of substance P, the natural ligand of the neurokinin receptor. Subsequent identification of the most potent antagonist 7, allowed the design of a chimeric structure 6 possessing the desired dual opioid-NK1 activity.[4] Bifunctional ligand 6 (NK1 Ki = 0.5 nM, pA2 = 7.8; MOR Ki= 0.4 nM) was obtained, after framework combination with an opioid tetrapeptide analogue 5, and possessed a high degree of overlap between both pharmacophores.[4] Efforts in the synthesis and derivatization of scaffolds 1 to 4, in addition to the design and preparation of dual ligands of type 6, will be discussed.

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Refs: 1) Bonney, I.M. et al. Eur. J. Pharmacol. 2004, 488, 91-99. 2) Hruby, V.J. J. Org. Chem. 2009, 74, 9245-9264. 3) Yamamoto T.et al. J. Med. Chem. 2010, 53, 5491-5501. 4) Ballet, S. et al. J. Med. Chem. 2011, 54, 2467-2476. Funding: SB, DT and PWS thank the Research Foundation-Flanders, the Ministère du Développement Economique, de l'Innovation et de l'Exportation (MDEIE) du Québec and the Canadian Institutes of Health Research (grant MOP-89716) for financial support.

Figure 1. Constrained amino acid scaffolds 1 to 4 and combination of the NK1R antagonist 7 and opioid agonist 5 into dual ligand 6.

NEW APPROACHES TO THE DEVELOPMENT OF OPIOID MULTIVALENT LIGANDS WITHOUT TOXICITIES, TOLERANCE OR DEPENDENCE FOR ACUTE AND NEUROPATHIC PAIN Victor J. Hruby, Department of Chemistry and Biochemistry and Department of Medical Pharmacology, University of Arizona, Tucson, Arizona 85721 USA

Despite many advances in our understanding of the underlying mechanism associated with acute and prolonged pain, our treatment of pain, especially prolonged and neuropathic pain is still primitive and woefully inadequate. During the past decade or so, we came to the conclusion that most current approaches to the development of drugs for pain, especially prolonged and neuropathic pain, were inappropriate. Our own work and analysis of the literature suggested a new multivalent approach was needed, based on the fact that treatment of pain with mu opioids and other analgesics leads to changes in the expressed genome in pain pathways that in turn leads to the development of tolerance, dependency and many other toxicities. Our approach has been to design and examine multivalent ligands with a variety of biological profiles consistent with eliminating undesirable side effects of current drugs for pain. In this talk, we will discuss the multidisciplinary approach we have taken with the design and development of multivalent ligands with mu/delta opioid agonist activities and NK-1 receptor antagonist activities, and novel peptide-nonpeptide ligands with highly potent delta/mu opioid agonist activities and unexpected and novel biological activity profiles. Most of these ligands cross the blood brain barrier as a result of their physical/chemical properties. Supported in part by grants from the U.S. Public Health Service, National Institutes of Health, and National Institute of Drug Abuse.

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MOR/DOR MIXED FUNCTION OPIOIDS – ONE DOES NOT SIMPLY WALK INTO IT Henry I. Mosberg, Katarzyna Sobczyk-Kojiro, Lauren C. Purington, Jessica P. Anand, Larisa Yeomans, Aubrie A. Harland, Vanessa R. Porter, Emily M. Jutkiewicz, and John R. Traynor. Departments of Medicinal Chemistry and Pharmacology, University of Michigan, Ann Arbor MI, USA

The observation that antagonists at the delta opioid receptor (DOR) can mitigate some adverse effects of mu opioid receptor (MOR) agonists – such as development of tolerance and dependence – has motivated several groups to pursue bifunctional MOR agonist/DOR antagonist ligands. Using a combination of structure-based design and serendipity we have developed several peptide ligands with similar affinity toward MOR and DOR that function as MOR agonists and DOR antagonists. Members of this series possess advantages over previously reported examples of MOR agonist/DOR antagonist ligands, but, like most peptides, these analogs poorly penetrate the blood brain barrier. Consequently, our focus has turned to improving the bioavailability of potential MOR/DOR ligands. The two towers of our approach consist of: 1. Modifying our lead MOR/DOR peptides to improve membrane penetration, while preserving their pharmacological profile and 2. Transferring the key MOR/DOR pharmacophore elements to more drug-like peptidomimetic scaffolds. Successes, failures, and observations from these parallel approaches toward the development of opioid analgesics with reduced tolerance and dependence liability will be described. Supported by NIDA grant DA003910 and Fellowship support from NIDA Training Grants DA007281 (JPA) and DA007267 (LY).

Symposium #7 – Opioid Ligand-Directed Signaling Organizer and Chair: Jay McLaughlin, Torrey Pines Institute

IN VIVO CONSEQUENCES OF OPIOID AGONIST-DEPENDENT PATHWAY-SELECTIVE SIGNALING Hui Zheng (1,2), Lei Zhang (1), and Ping-Yee Law (1). (1)Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455 and (2)Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

Using MAPK activation as a readout, we have demonstrated that morphine activation of ERK1/2 was PKCe-dependent, while fentanyl or etorphine activation of ERK1/2 was ßArrestin2- dependent. Apparently, the pathway selected by the agonist was affected by the receptor phosphorylation state. Such pathway selectivity was observed also in the rapid desensitization of OPRM1 and also with the adenylyl cyclase (AC) superactivation during prolonged agonist exposure. Whether the pathway-selected signaling will have in vivo consequences, especially in the responses to repeated exposure to the drugs is being investigated in our group. In the current talk, we will present studies in which the pathway-selected by morphine and fentanyl to activate ERK1/2 can affect the extinction rate of the drug-related contextual memory due to the two drugs’ differential regulation of the miRNA190 and transcription factor NeuroD activities. We will also present studies to demonstrate that the pathway selectivity in receptor desensitization and AC superactivation has correlates in opioid tolerance development and withdrawal. Hence, it is possible to differentially modulate the responses to repeated exposures to agonists by regulating the activities of the components within pathways differentially used by the agonists for signaling.

LIGAND DIRECTED SIGNALING AT KAPPA OPIOID RECEPTORS Selena S. Schattauer and Charles Chavkin, Univ. of Washington, Seattle

Prior work established that signal transduction events initiated by different ligand binding to the kappa opioid receptor (KOR) can be strikingly different. Both neutral competitive antagonists and collateral agonists that inactivate KOR by initiating a JNK-1 dependent cascade have been INRC 2012 Kansas City, Missouri 43 distinguished. Kappa ligands that activate Gbg-dependent signaling to increase K+ and inhibit Ca++ conductances result in analgesia, whereas the dysphoric effects of kappa agonists are caused by a different pathway involving G-protein receptor kinase (GRK) and arrestin. Based on these distinctions, we hypothesize that a partial KOR agonist that does not efficiently activate arrestin dependent signaling may produce analgesia without dysphoria. No KOR-selective partial agonists are currently available and the preclinical assessment of partial agonists is complicated by structural differences between rodent (r) and human (h) KOR at the GRK phosphorylation sites required for arrestin recruitment. In the present study we compared the signaling events initiated by the prototypical partial kappa agonist, pentazocine in hKOR and rKOR. We found that pentazocine was significantly more potent in activating p38-MAPK in hKOR than rKOR in transfected HEK293 cells. In contrast, pentazocine was equally potent in arrestin-independent activation of ERK1/2 in hKOR and rKOR. There was no potency difference in U50,488 activation of ERK1/2 or p38 MAPK. We confirmed that pentazocine was a partial agonist at both receptors for both signaling pathways. hKOR lacks the Ser369 phosphorylation site required for GRK/arrestin dependent p38 activation, but mutation of Ser358 to alanine in hKOR blocked p38 activation. Although pentazocine is reported to produce dysphoria in humans, its lower efficacy at p38 activation of rKOR suggests that it may be unlikely to produce aversion in rodents. Consistent with this prediction, pentazocine (10 mg/kg i.p.) produced analgesia and a MOR-dependent place preference but did not produce KOR-dependent aversion. This difference has important implications in screening for partial KOR agonists with ligand directed signaling properties.

ARRESTIN MEDIATED FUNCTIONAL SELECTIVITY AT THE DELTA OPIOID RECEPTOR Amynah Pradhan, Semel Institute for Neuropsychiatry & Human Behavior, and Shirley and Stephan Hatos Center for Neuropharmacology University of California Los Angeles

G protein coupled receptors exist in multiple conformations, and agonists can stabilize different active states. These specific ligand-induced receptor conformations result in distinct receptor- effector complexes, which initiate diverse signaling and trafficking events. This concept, referred to as ligand-directed signaling or functional selectivity, has important biological and therapeutic implications. Delta opioid receptors are currently being developed for the treatment of chronic pain and emotional disorders. Convergent evidence from a number of in vitro and in vivo studies has revealed functional selectivity at the delta opioid receptor. Specifically, we compared agonists with similar binding and analgesic properties, but high-(SNC80) or low- (ARM390) internalization potencies. We found that following chronic administration two distinct forms of tolerance were observed depending on the internalization property of the agonist. SNC80 produced a generalized tolerance resulting in receptor downregulation throughout the central and peripheral nervous systems. In contrast, ARM390 produced an analgesic specific tolerance at the level of the dorsal root ganglia. We recently examined the effects of SNC80 and ARM390 in β-arrestin 1 and β-arrestin 2 knockout mice. The behavioral effects of SNC80, but not ARM390, were potentiated in b-arrestin 1 knockout mice, and these mice did not show receptor desensitization to this particular agonist. In contrast, in β-arrestin 2 knockout mice, the effects of SNC80 were unaffected, whereas the properties of ARM390 were adversely affected. These results show that high and low internalizing delta opioid receptor agonists differentially recruited b-arrestins, resulting in divergent biological effects. Research supported by NIH NIDA grant #DA031243 and DA05010, and the Shirly and Stefan Hatos Research Foundation. As well as BK was also supported by the CNRS, INSERM, the Université de Strasbourg, INSERM- FRSQ, FRM and the ANR grant IMOP

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LONG-ACTING KAPPA OPIOID ANTAGONISTS DO NOT ACTIVATE C-JUN N-TERMINAL KINASE J.M. Bidlack and B.I. Knapp, Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY USA

Using Western blot analysis, long-acting kappa opioid receptor (KOR) antagonists have been reported to activate c-Jun N-terminal kinase (JNK), resulting in phosphorylated JNK (pJNK) in HEK293 cells expressing the rat KOR (Bruchas et al., 2007). The current study used a cell- based ELISA to characterize total JNK and pJNK levels mediated by the human KOR. Chinese hamster ovary (CHO) cells stably expressing the human KOR were used in an ELISA to determine if agonists, partial agonists, and antagonists altered the levels of JNK and pJNK. Time-course experiments showed that within 5 min after incubating KOR-CHO cells with the KOR agonist U50,488, pJNK levels were increased by 130%. Maximal activation was maintained for 40 min and gradually decreased to basal levels by 5 hr after treatment. Titration experiments with KOR agonists, U50,488 and dynorphin peptides, showed an increase pJNK levels with low nM EC50 values. U50,488 had an EC50 value of 0.51 ± 0.18 nM. U50,488 did not increase pJNK levels in native CHO cells, indicating a KOR-mediated JNK activation. Total JNK levels did not change under any conditions. The KOR partial agonist diprenorphine had an EC50 value of 0.092 nM and an Emax value of 74% in increasing pJNK levels. The long-acting antagonists, nor-BNI, GNTI, and JDTic at concentrations up to 10 mM did not increase pJNK levels, but they inhibited agonist-induced increases in pJNK levels. In conclusion, U50,488 was a very potent stimulator of pJNK levels, having an EC50 value of 0.51 nM. In contrast, the previous study reported an EC50 value of 1240 nM for U50,488 (Bruchas et al., 2007). The current study did not observe any JNK activation by long-acting KOR antagonists, suggesting that the long duration of action of these antagonists is not mediated by JNK activation. (Supported by the Paul Stark Professorship and the Margo Cleveland Fund.)

KOR AGONIST FUNCTIONAL SELECTIVITY IN PERIPHERAL SENSORY NEURONS IN VIVO AND EX VIVO R.J. Jamshidi, B.A. McGuire, L.C. Sullivan, W.P. Clarke, K.A. Berg. Dept. of Pharmacol., University of Texas Health Science Center at San Antonio, San Antonio, TX

Functional selectivity is a term used to describe the ability of drugs to differentially activate signaling cascades coupled to a single receptor subtype. Here we studied the functional selectivity of two KOR agonists, U50488 and Salvinorin A (Sal A), measuring activation of Extracellular Signal-Regulated Kinase (ERK) and inhibition of PGE2-stimulated cAMP accumulation in primary sensory neuron cultures, and inhibition of PGE2-stimulated thermal allodynia following intraplantar (i.pl.) injection in the rat hindpaw. Ex vivo, both agonists inhibited PGE2-stimulated cAMP accumulation and increased ERK activity. However, although U50488- stimulated ERK activity was sensitive to pre-treatment with PTx (i.e., Gi-protein mediated), Sal A-stimulated ERK activity was PTx insensitive (i.e., Gi-protein independent). In vivo, U50488 and Sal A reduced PGE2-induced thermal allodynia; however for both agonists, the dose- response curve for anti-allodynia was an inverted U-shape. Consistent with reports of increased ERK activity leading to pro-nociception, the downward phase of the inverted U-shaped curve for U50488 was reversed by a MEK inhibitor, U0126, which prevents activation of ERK. However, the MEK inhibitor did not affect the inverted U-shaped curve for Sal A. Since previous studies have shown that activation of c-Jun N-terminal kinase (JNK) disrupts KOR signaling, we examined the effect of JNK inhibition on Sal A-mediated thermal anti-allodynia. In the presence of the JNK inhibitor, SP600125, the downward phase of the inverted U- shaped curve for Sal A was partially reversed. These data indicate that KOR agonists, U50488 and Sal A, differentially activate ERK and possibly JNK, and suggest differences in the functional selectivity profile of these KOR agonists in peripheral sensory neurons. Understanding mechanisms by which KOR agonist efficacy in the periphery is regulated may lead to improved pharmacotherapy for

INRC 2012 Kansas City, Missouri 45 treatment of pain with reduced CNS-mediated adverse effects. Supported by DA026619 and DA024865

DIFFERENCES IN ANTINOCICEPTION AND TOLERANCE DUE TO LIGAND-BIASED SIGNALING AT THE MU-OPIOID RECEPTOR E. N. Bobeck (1), D. M. Fitzgibbon (1), S. L. Ingram (2), & M. M. Morgan (1). (1) Dept. of Psychology, Washington State University, Vancouver, WA, (2) Dept. of Neurological Surgery, Oregon Health & Science University, Portland, OR

A variety of downstream signaling proteins from the mu-opioid receptor (MOP) have recently been shown to be activated in a ligand-biased manner. One known difference is that extracellular regulated kinase ½ (ERK1/2) is activated in a beta-arrestin dependent manner following fentanyl and DAMGO, but not morphine, administration. Our recent data determined that blockade of ERK1/2 potentiates tolerance to morphine within the ventrolateral periaqueductal gray (vlPAG), but other studies have found mixed results on tolerance to morphine administration in other areas. The role of ERK1/2 in antinociception and tolerance to fentanyl and DAMGO administration in the vlPAG has not been assessed. Rats were pretreated with a MEK inhibitor that prevents ERK1/2 activation (U0126—100ng/0.5microL) 20 min prior to microinjection of fentanyl or DAMGO into the vlPAG. ERK1/2 inhibition had no effect on fentanyl antinociception. However, a single or repeated microinjections of U0126 caused a decrease in subsequent DAMGO potency (i.e., decreased antinociception). Tolerance was then induced by two days of twice daily microinjections of fentanyl or DAMGO resulting in a rightward shift in the dose-response curve on Day 3. Daily co-administration with U0126 reversed tolerance to DAMGO, but not fentanyl. Taken together, these data show that ERK1/2 activation modulates antinociception produced by DAMGO microinjections into the vlPAG, but does not play a role in fentanyl-induced antinociception. This study adds to the growing body of evidence that different signaling pathways contribute to antinociception and tolerance to different MOP agonists. Funding by NIH grant DA 015498. Morphine sulfate was a gift from NIDA.

MODULATION OF DELTA OPIOID RECEPTOR SIGNALING BY REGULATOR OF G PROTEIN SIGNALING PROTEIN 4 IN RODENT BRAIN Q. Wang, E. M. Jutkiewicz, Y. Zhao, J.T. Lamberts, and J.R. Traynor. Department of Pharmacology and Substance Abuse Research Center, University of Michigan, Ann Arbor, USA

Regulator of G protein signaling (RGS) proteins act as GTPase accelerating proteins (GAPs) for GTP-bound Galpha subunits and so negatively regulate downstream signaling. Previously we have shown that RGS4 is a GAP for delta opioid receptor (DOR) but not mu-opioid receptor (MOR) signaling in SH-SY5Y cells. RGS4 protein is abundantly expressed in the striatum. To confirm a role for RGS4 in modulating DOR signaling in the striatum, we investigated the ability of the DOR agonist SNC80 to stimulate MAPK activation in tissue from RGS4 knockout mice compared with tissue from their wild-type littermates. SNC80 (1.0 µM) treatment of striatal slices from wild-type mice stimulated ERK phosphorylation by 92 % over basal levels. In contrast, the level of ERK phosphorylation in striatal tissue from RGS4 knockout mice was much more robust, giving 225 % stimulation over basal levels. We have also shown in SH-SY5Y cells that chronic morphine causes a marked down regulation of RGS4 and this results in altered DOR signaling. Chronic treatment of rats with morphine (i.p. with increasing doses ramping from 10-100 mg/kg, twice daily for 14 days) decreased RGS4 protein level in the striatum to half the amount of that seen in rats treated with saline vehicle. These findings indicate a role for RGS4 in modulating DOR signaling in rodent brain and suggest that morphine-induced alterations in RGS4 protein levels may lead to enhanced DOR signaling that could help to maintain cell homeostasis in the morphine-dependent state. Supported by DA04087 and MH083754.

46 INRC 2012 Kansas City, Missouri

Poster Presentations

INRC 2012 Kansas City, Missouri 47

M1 Buprenorphine/naltrexone: an effective combination therapy M2 KOR Agonist Functional Selectivity in Peripheral Sensory approach to relapse prevention Neurons In Vivo and Ex Vivo S.M. Husbands, S. Cordery, A. Taverner, R. H. Guy, B. Delgado-Charro, C.P. R.J. Jamshidi, B.A. McGuire, L.C. Sullivan, W.P. Clarke, K.A. Berg. Dept. Bailey Department of Pharmacy and Pharmacology, University of Bath, UK. of Pharmacol., University of Texas Health Science Center at San Antonio, San Antonio, TX. There is currently no medication licensed for treatment of cocaine dependence, and whilst there are treatments available for opioid dependence, no single Functional selectivity is a term used to describe the ability of drugs to treatment is effective for everyone. As many users of crack cocaine are also differentially activate signaling cascades coupled to a single receptor dependent on heroin, a relapse prevention medication that was effective in the subtype. Here we studied the functional selectivity of two KOR agonists, polydrug user population would be a notable step forward. Encouraging U50488 and Salvinorin A (Sal A), measuring activation of Extracellular results have been observed in two clinical trials using a buprenorphine (BUP) Signal-Regulated Kinase (ERK) and inhibition of PGE2-stimulated cAMP and naltrexone (NTX) combination therapy (Rothman et al, 2000; Gerra et al, accumulation in primary sensory neuron cultures, and inhibition of PGE2- 2006); significant reduction of both heroin and cocaine use was demonstrated. stimulated thermal allodynia following intraplantar (i.pl.) injection in the However, the reduction in cocaine use could simply be a consequence of rat hindpaw. Ex vivo, both agonists inhibited PGE2-stimulated cAMP reduction in heroin use (these two drugs of abuse are used to complement each accumulation and increased ERK activity. However, although U50488- other). Therefore, we wanted to assess the effect of BUP/NTX on cocaine- stimulated ERK activity was sensitive to pre-treatment with PTx (i.e., Gi- seeking in a controlled experiment. We used male adult Sprague Dawley rats protein mediated), Sal A-stimulated ERK activity was PTx insensitive (i.e., in the conditioned place preference (CPP) method to establish an animal model Gi-protein independent). In vivo, U50488 and Sal A reduced PGE2- of relapse to drug-seeking and to measure the ability of a BUP/NTX induced thermal allodynia; however for both agonists, the dose-response combination to block drug-primed reinstatement to cocaine seeking and, for curve for anti-allodynia was an inverted U-shape. Consistent with reports comparison, morphine seeking. 1.0 mg/kg NTX was found to block the mu of increased ERK activity leading to pro-nociception, the downward phase agonism of 0.3 mg/kg BUP, in both a tail withdrawal assay and the CPP assay, of the inverted U-shaped curve for U50488 was reversed by a MEK indicating that this combination was not rewarding and was suitable for use in inhibitor, U0126, which prevents activation of ERK. However, the MEK the reinstatement assays. As expected, the combination of BUP/NTX inhibited inhibitor did not affect the inverted U-shaped curve for Sal A. Since morphine-primed reinstatement of morphine CPP. But furthermore, the previous studies have shown that activation of c-Jun N-terminal kinase combination of BUP/NTX completely abolished cocaine-primed reinstatement (JNK) disrupts KOR signaling, we examined the effect of JNK inhibition of cocaine CPP. Thus, behavioral data generated using the CPP-extinction- on Sal A-mediated thermal anti-allodynia. In the presence of the JNK reinstatement method showed clearly that BUP/NTX reduced reinstatement to inhibitor, SP600125, the downward phase of the inverted U- shaped curve drug-seeking following a drug prime, in both cocaine-and morphine- for Sal A was partially reversed. These data indicate that KOR agonists, conditioned rats. Unexpectedly, these data suggest that the effect may even be U50488 and Sal A, differentially activate ERK and possibly JNK, and more dramatic in the cocaine-conditioned rats providing further evidence that suggest differences in the functional selectivity profile of these KOR this combination may become an effective pharmacological treatment for agonists in peripheral sensory neurons. Understanding mechanisms by cocaine abuse. Supported by MRC Grant no. 90121. which KOR agonist efficacy in the periphery is regulated may lead to improved pharmacotherapy for treatment of pain with reduced CNS- mediated adverse effects. Supported by DA026619 and DA024865

M3 Distinct Kappa Opioid Receptor (KOR) Antagonists Suppress ERK ½ M4 Involvement of Pituitary POMC T-box Transcription Factor T-Pit MAP Kinase Signaling Correlating with Antidepressant-like Activity in Cocaine Conditioned Place Preference and Hypothalamic-Pituitary J.P. McLaughlin (1,2), M.R. Hoot (1), K. Rasakham (2) (1) Torrey Pines Adrenal Axis Responsivity in Mice Institute for Molecular Studies, Port St. Lucie, FL, USA, (2) Northeastern S Chen(1), Y Zhou(1), K Niikura(1), A Ho(1), J Drouin(2), MJ Kreek(1) University, Dept. of Psychology, Boston, MA USA (1)The Rockefeller University, NY, USA, (2)IRCM, QC, Canada

The potential for distinct receptor ligands to exert pathway-selective signaling, Disruption to the stress responsive hypothalamic-pituitary adrenal (HPA) termed ligand-directed signaling, may further distinguish the activity of related axis predicts relapse to and amount of cocaine use. However, the role of ligands. As recent reports suggest that some KOR antagonists possess pituitary pro-opiomelanocortin (POMC) in mediating cocaine-induced signaling properties that cannot be attributed to partial agonist effects, we reward and drug-seeking behaviors has not been investigated. The present examined if nor-BNI and four other structurally distinct KOR antagonists studies used a conditioned place preference (CPP) model to determine (JDTic, arodyn, zyklophin and naloxone) could alter ERK1/2 MAP kinase whether deletion of the pituitary cell-specific POMC transcription T-box activity in KOR-GFP/HEK293 or untransfected HEK293 cells. Single factor (Tpit) (resulting in pituitary POMC deficiency) alters: 1) cocaine- incubations with each ligand (0.1 µM-10 µM) were followed by Western Blot induced reward; 2) CPP reinstatement induced by cocaine priming and by analysis of total ERK1/2 and phosphorylated ERK 1/2 (pERK 1/2) levels. Yohimbine (YOH) stress; and 3) HPA hormonal responses to acute cocaine Surprisingly, the antagonists demonstrated differential suppression of ERK1/2 administration. Cocaine place conditioning over 2 weeks (30 kinase signaling. Incubation with nor-BNI, JDTic or arodyn produced a min/sessions/day at 0, 5, or 15 mg/kg cocaine, i.p.) in Tpit heterozygotes concentration-dependent suppression of ERK1/2 activity, with a maximal (+/-) and wild-type (+/+) mice was given to males (n=4-7) and females reduction of pERK 1/2 labeling of approximately 50% after incubation with 10 (n=5-7). It was followed by CPP testing and 3 weeks of extinction. Cocaine µM nor-BNI that was significantly different from vehicle control, and lasted up (15 mg/kg) priming-induced reinstatement of CPP was tested on week 4 and to 24 h post drug incubation. In contrast, zyklophin and naloxone were YOH-(1 or 2.5 mg/kg) induced reinstatements on week 5. In a separate without effect. The effects of nor-BNI were KOR receptor-mediated, as no study, plasma corticosterone (CORT) was measured 30 min. after a single 5 change in pERK 1/2 labeling was observed in non-transfected HEK293 cells, mg/kg cocaine or saline injection in both genotypes and genders (n=5-7). At and the effect was abolished by co-incubation with naloxone. We further high dose cocaine (15 mg/kg), both genotypes and genders showed hypothesized the distinct signaling properties by these ligands may produce equivalent levels of cocaine-induced CPP. However, at low dose cocaine (5 different behavioral consequences in vivo. Initial behavioral testing with mg/kg), only Tpit +/- mice developed CPP. In the reinstatement tests, C57BL/6J mice in a forced swimming test found that only those KOR cocaine priming (15 mg/kg) reinstated CPP in both genotypes and YOH at antagonists suppressing ERK1/2 MAP kinase activity in vitro produced 2.5 mg/kg only reinstated cocaine-induced CPP in the Tpit +/- mice, all with antidepressant-like activity. In conclusion, these results suggest KOR- no gender difference. Finally, acute cocaine at 5 mg/kg elevated plasma selective antagonists demonstrate significant differences in ligand-directed CORT levels in males with no genotype difference. Basal CORT levels in signaling which may mediate changes in mood behaviors, potentially offering females were higher than those in males and cocaine did not cause further new therapeutic approaches for treatment. This work was supported by R03 plasma CORT elevations in either genotype. These data suggest that 016415 from NIDA and funds from the State of Florida to JPM. pituitary POMC (leading to ACTH) may be involved in gender differences in HPA responsivity. Support: NIH-NIDA P60-05130(MJK), NCIC (JD)

48 INRC 2012 Kansas City, Missouri

M5 Parenting with Pills: The dual identities of prescription opioid M6 Effects of the widely available kappa-opioid receptor (KOP-r) analgesics in Midwestern rural communities agonist hallucinogen, salvinorin A on the HPA axis: Translational K. Bensley, MSc. (1) Department of Anthropology, University College studies London, London, UK Eduardo R. Butelman (1), Thomas E. Prisinzano (2), Mary Jeanne Kreek (1) (1)Laboratory on the Biology of Addictive Diseases, The Rockefeller This study tries to better understand the perceived role of prescription opioid University, New York, NY 10065, USA, (2) Department of Medicinal analgesics in rural West Michigan, USA. By better understanding the social Chemistry, School of Pharmacy, University of Kansas, Lawrence KS contexts within which its dual roles are understood, opioid analgesic abuse 66045, USA and pain mistreatment can be dealt with better by health professionals. There were 19 participants interviewed in this study in the Summer of 2011, from Exogenous KOP-r agonists and endogenous KOP-r ligands (the four communities in West Michigan. From this study it emerged that dynorphins) have robust effects in the regulation of CNS functions, participants were using the words “medication” and “drug” to represent including neuropsychiatric-like states (e.g., depression or anxiety, different semantic meanings of opioid analgesics: participants were dysphoria and psychotomimesis), as well as drug addiction. KOP-r agonists identifying these substances as separate based on their role as a healing also have prominent neuroendocrine effects (e.g., prolactin release), and are substance (treatment for pain) or a harmful substance (opioid analgesic also known to activate the HPA axis. The widely available hallucinogen abuse). This allowed rural participants to think of their communities as salvinorin A (from the plant Salvia divinorum) is a high efficacy selective socially and geographically isolated and able to downplay their own substance KOP-r agonist. Salvinorin A has prominent KOP-r mediated behavioral abuse risk on a community level. The presences of opioids can be understood effects, and also causes prolactin release. We investigated the effects of i.v. as medicines in their communities, and drugs in other communities. This bolus salvinorin A (0.0032-0.032 mg/kg; n=3-4) on release of ACTH in understanding of opioid analgesics questions “War on Drugs” dualistic rhesus macaques, a translationally valuable model of KOP-r function, due approach to drug abuse. In order to effectively address opioid analgesic abuse to genetic similarities in the KOP-r gene between macaques and humans. and treat pain effectively, this dichotomy must be questioned and a Salvinorin A caused a dose-dependent increase in ACTH levels, with a compromise must be reached. peak effect at the earliest time point after administration (5 min). These effects of salvinorin A largely declined by 30 min after administration, consistent with the fast onset and short duration of action of salvinorin A in humans and non-human primates. For comparison, the synthetic selective KOP-r agonist U69,593 (0.01-0.032 mg/kg) produced relatively sustained effects on ACTH for at least 30 min, and also tended to produce greater maximal effects that salvinorin A. Funding by National Institutes of Health / National Institute on Drug Abuse grants DA018151 (TEP) and DA05130 (MJK) is gratefully acknowledged.

M7 Electroacupuncture attenuates pruritogens-evoked scratching M8 Long term changes in motivation during aversive conditions Y.-H. Chen (1), H.-Y. Yang (2) and J.-G. Lin (2) (1)Grad. Inst. Acupuncture following morphine withdrawal are predicted by altered NMDAR1 Science, (2) Sch. of Chinese Medicine, China Medical University, Taichung, splicing in the amygdala. Taiwan. Ethan M. Anderson (2) and Robert M. Caudle (1,2). (1) Department of Oral Surgery, University of Florida, Gainesville, FL, United States (2) The aims of the present study were as follows: (1) to investigate the influence Department of Neuroscience University of Florida College of Medicine, of electroacupuncture (EA) on compulsive scratching in mice elicited by the Gainesville, FL subcutaneous administration (behind the neck) of 5’-guanidinonaltrindole (GNTI), a κ opioid antagonist or of compound 48/80, a common pruritogen The NR1 subunit of the NMDA receptor can be alternatively spliced by the that rapidly releases inflammatory mediators such as histamine from mast cells insertion or removal of the N1, C1, C2, or C2’ regions. Morphine in connective tissue and skin; and (2) to determine the effects of EA on dependence and withdrawal were previously demonstrated to alter these suppressing c-fos expression provoked by subcutaneously injected pruritogens. variants as well as altering motivational and anxiety/stress behaviors in rats. We discovered that the application of EA to the Hegu (LI4) and Quchi (LI11) We tested the hypothesis that NR1 splicing would predict changes in these acupoints at 2 Hz, but not at 100 Hz, attenuated GNTI (0.3 mg/kg and 0.6 behaviors during an extended withdrawal period of two months. Motivation mg/kg)-evoked scratching. EA at 100 Hz, but not at 2 Hz, attenuated was measured using the operant orofacial assay at non-aversive compound 48/80 (5 mg/kg)-evoked scratching. Moreover, [D-Ala2, N-Me- temperatures while anxiety and stress were measured by examining this Phe4, Gly5-ol]-enkephalin acetate (DAMGO), a well-established selective µ- behavior at aversive temperatures. Altered NR1 splice variant expression opioid receptor agonist, and trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2- (1- levels were observed in the amygdala in a subset of rats after two months of pyrrolidinyl) cyclohexyl] benzeneacetamide hydrochloride hydrate (U- morphine withdrawal. This subset was associated with changes in 50488H), a selective κ opioid receptor agonist, both attenuated GNTI-evoked motivational behavior and/or hypersensitivity to adverse conditions. The scratching behavior. U-50488H, but not DAMGO, attenuated scratching changes in NR1 splicing may reflect long term, and possibly life long, evoked by compound 48/80. GNTI and compound 48/80 provoked c-fos alterations in the withdrawn population. Funding for this project was expression on the lateral side of the superficial layer of the dorsal horn in the provided by the NIDA. DA030044 cervical spinal cord. The application of 2 Hz EA to LI4 and LI11 decreased the number of c-fos positive nuclei induced by GNTI. In addition, the application of 100 Hz EA to LI4 and LI11 decreased the number of c-fos positive nuclei induced by compound 48/80. It is concluded that EA attenuates scratching behavior induced by GNTI and compound 48/80 in mice, in a frequency- dependent manner. The µ and κ opioid systems are involved, at least in part, in the anti-pruritic effects of EA.

INRC 2012 Kansas City, Missouri 49

M9 Morphine produces inverted dose-dependent reward in neuropathic M10 A role for noradrenergic systems in kappa-opioid dependent rats reinstatement of cocaine place preference CM Cahill(1), L Xue(1), S LeCour(1), C Magnussen(1), S Metcalfe(2), P R. Al-Hasani (1,2), A.M. Foshage (1), J.G. McCall (1,2,3), M.R. Bruchas Grenier(1), MC Olmstead(2) Depts of (1)Biomed and Mol Sci and (1,2,4) (1) Department of Anesthesiology (2) Department of Neurobiology (2)Psychology, Queen’s Univ, Kingston ON. (3) Program in Neuroscience, (4) Pain Center Washington University in St. Louis, St. Louis MO, USA In addition to the obvious sensory disturbances that accompany neuropathic (NP) pain, this condition is associated with an important affective state. Previous reports have demonstrated that stress causes dynorphin release, Clinical and basic science studies demonstrate that opioids are less effective in activating kappa-opioid receptors (KOR) in monoamine circuits resulting in alleviating NP pain compared to other painful conditions. In the present study, dysphoria-like behavioral responses, potentiation of cocaine conditioned we used a three compartment conditioned place preference (CPP) paradigm to place preference (CPP) and reinstatement of drug seeking. Activation of investigate rewarding properties of analgesics in NP pain. Neuropathy was noradrenergic (NA) receptor systems have also been implicated in similar induced by chronic constriction of the common sciatic nerve. Rats underwent behaviors. Together, these reports suggest a possible link between the surgery (or not) 6 days prior to habituation, and conditioning to drug and noradrenergic and dynorphin/kappa opioid circuits. The anatomical and vehicle over 8 days using an unbiased, balanced CPP paradigm. Intrathecal behavioral implications of this putative interaction have not been (i.t.) administration of the alpha 2 adrenergic receptor agonist, clonidine demonstrated. We investigated the role of NA circuits in KOR-dependent (13ug) or the delta opioid receptor agonist, deltorphin (30ug) produced a CPP stress-induced reinstatement of CPP. CPP was used to measure U50488 in NP, but not sham animals. Consistent with the literature, systemic (KOR agonist)-mediated reinstatement of cocaine seeking, in the presence administration of morphine dose-dependently produced reward in pain-naive and absence of the beta-adrenergic receptor (beta-AR) antagonist animals. However, the dose response curve was inverted in NP animals, propranolol, or the alpha2-agonist clonidine in male wild-type C57BL/6 where low dose morphine (2mg/kg) produced a greater CPP than higher doses mice. We found that injection of propranolol or clonidine prior to injection of morphine (8mg/kg). We then determined whether administration of an of U50,488, significantly potentiated reinstatement of CPP. Preinjection of analgesic on post-condition day would affect the expression of morphine the beta1 selective antagonist (Betoxolol) also caused potentiation of reward. I.t. clonidine and deltorphin strengthened the preference for the reinstatement. Recent data suggest that dynorphin containing neuronal morphine-paired compartment in pain-naïve animals, however, clonidine, but projections terminate within the locus coeruleus (LC), the primary source not deltorphin, caused the animals to significantly avoid the morphine-paired of norepinephrine in the forebrain. We report that phospho-ERK (pERK) is compartment in NP animals. These results indicate that the rewarding effects coexpressed in tyrosine hydroxylase (TH) cells in the LC following of clonidine and deltorphin as well as low dose morphine are attributable to U50,488 injection, further supporting KOR expression in the LC. We also removal of the tonic aversive nature of pain. However, higher dose morphine found that the potentiation of kappa-opioid dependent reinstatement by may exacerbate the negative affect associated with nerve injury. We speculate propranolol is blocked following injection of the KOR antagonist NorBNI that clonidine unmasks an aversive effect of morphine present in NP, but not into the LC where pERK is also absent, confirming localized KOR- pain-naïve animals. inactivation. Together, these findings suggest that there may be a causal connection between dynorphin/KOR circuitry and NA systems and that beta-ARs may act to tonically-inhibit KOR-dependent behaviors. Supported by NIDA, R00DA025182 (MRB)

M11 Effects of mu, delta and kappa opioid receptor agonists on pain- M12 Galphao protein plays a role in morphine-induced locomotor stimulated vs. pain-depressed behaviors in rats sensitization A. Altarifi and S. Negus; Department of Pharmacology and Toxicology, J. Lamberts (1), J. Gateley (1), J. Borlagdan (1), M. VandenBerg (1), J. Virginia Commonwealth University, Richmond, VA Traynor (1, 2) (1) Department of Pharmacology, (2) Substance Abuse Research Center, University of Michigan, Ann Arbor, MI, USA Pain is a significant health problem. Pain is associated with stimulation of some behaviors (e.g. withdrawal reflexes) and depression of others (e.g. Opioids such as morphine are known to cause behavioral sensitization in feeding, locomotion and positively reinforced operant responding). Preclinical rodents. This is commonly manifested as a sustained enhancement of the contributions to analgesic drug development have relied almost exclusively on locomotor response following chronic, intermittent drug treatment. measures of pain-stimulated behaviors, but drug effects on these measures However, the cellular mechanisms that underlie the development of have not reliably predicted human analgesic efficacy. Pain-depressed morphine-induced locomotor sensitization are not well understood. behaviors are used to diagnose pain in human and veterinary medicine, and Morphine elicits its behavioral effects by activating the mu-opioid receptor, assays that incorporate measures of pain-depressed behavior may improve which in turn stimulates the Galphai/o subunit of heterotrimeric G proteins predictive validity of preclinical drug evaluation. To evaluate this hypothesis, – most notably Galphao. To evaluate the role of Galphao in morphine mu, delta and kappa opioid receptor agonists were evaluated in sensitization, locomotor activity was measured in mice heterozygous for a complementary assays of pain-stimulated and pain-depressed behavior. Galphao knockout allele (Galphao (+/-) mice), in comparison with wild Intraperitoneal injection of dilute lactic acid (1.8% in 1 ml/kg) served as a type littermates. Mice were treated with either saline (i.p.) or cumulative common noxious stimulus to stimulate a stretching response or depress doses of morphine (10-320 mg/kg, i.p.) and locomotor activity was intracranial self-stimulation (ICSS) in separate groups of male Sprague- recorded in 5 min intervals for a total of 150 min; this procedure was Dawley rats. Mu and delta opioid agonists dose-dependently blocked both repeated one-weekly for three weeks. As a biochemical measure, G protein acid-stimulated stretching and acid-induced depression of ICSS. Mu but not activation was evaluated in separate groups of mice in striata collected in delta opioid agonists produced significant stimulation of ICSS in the absence week three. In wild type mice, exposure to morphine in week one was of the noxious stimulus, suggesting that antinociceptive mu-agonist doses may sufficient to produce locomotor sensitization in week two, and sensitization also produce abuse-related effects in the absence of pain. Kappa agonists dose- was maintained during the third week of morphine exposure. Moreover, dependently blocked acid-stimulated stretching but only exacerbated acid- this enhancement of morphine-stimulated locomotor activity was induced depression of ICSS at doses that also decreased control ICSS in the associated with an approximately 3-fold increase in the potency of absence of a noxious stimulus. The failure of kappa agonists to block acid- morphine to stimulate G protein activity in striatal homogenates. In induced depression of ICSS agrees with the poor analgesic efficacy of kappa contrast, Galphao (+/-) mice did not exhibit locomotor sensitization until agonists in humans. These results suggest that a preclinical assay of acid- week three, and this was not associated with changes in G protein depressed ICSS can dissociate effects of mu, delta and kappa opioid agonists. activation in the striatum. Together, these results demonstrate that Galphao Supported by R01 NS070715 and Jordan University of Science and plays a role in the development and/or expression of locomotor Technology sensitization to morphine and that this involves an enhancement of G protein signaling. This work was supported by DA04087 to JT; JL was supported by training grants GM077667 and DA007267.

50 INRC 2012 Kansas City, Missouri

M13 Comparison of peripheral and central antinociceptive effects of M14 Toward a pharmacogenetics approach to individualized biphalin and morphine in a mouse model of cancer pain methadone maintenance treatment for opioid addiction AA Lesniak(1), M Bochynska-Czyz(1), M Sacharczuk(2), Andrzej W. Orna Levran (1), Einat Peles (2), Matthew Randesi (1), Jurg Ott (1,3), Lipkowski(1) (1)Mossakowski Medical Research Centre Polish Academy of Miriam Adelson (2), Mary Jeanne Kreek (1). (1)Laboratory of the Biology Sciences, Pawinskiego 5, 02-106 Warsaw, (2)Institute of Genetics and Animal of Addictive Diseases, The Rockefeller University, New York, NY, USA, Breeding Polish Academy of Sciences, Postepu 1 05-552 Wolka Kossowska, (2)Dr. Miriam and Sheldon G. Adelson Clinic for Drug Abuse, Treatment Poland and Research, Elias Sourasky Medical Center, Tel Aviv, Israel , (3)Institute of Psychology CAS, Beijing, China. It is generally accepted that classical opioids exert their antinociceptive effect mainly when binding to opioid receptors located in the central nervous system. Individual methadone dosage optimization is one of the key factors in However, a growing body of evidence points to the relevance of peripheral effective methadone maintenance treatment (MMT) for opioid addiction, opioid receptors in periphery pathology, including cancer. A cancer is very since methadone has large inter-individual variability in response. This often the cause of pain resulting from peripheral metastasis. The peripheral study was designed to identify genetic factors that may modulate component of antinociception induced by a dimeric enkephalin analog-biphalin methadone dose requirements and to confirm previous finding in a larger showing limited blood-brain-barrier permeability may prove important in sample. We have recently shown that specific variants in ABCB1, CYP2B6 cancer pain therapy. An additional advantage of biphalin is a possibility to treat and NGFB are associated with methadone dose requirements, in an Israeli pain symptoms with reduction of side-effects - a result of the central action of sample. Other groups have indicated association with OPRM1, BDNF, some other opioid analgesics e.g morphine. We examined the peripheral and MYOCD, and CRY1 and DRD2 variants. To expand and validate these central analgesic effect of biphalin in a murine skin cancer pain model studies, we have analyzed 105 variants in 14 genes (ABCB1, CYP3A4, developed by an intraplantar inoculation of B16T0 melanoma cells. Animals CYP2D6 and CYP2B6, POMC, OPRM1, NGFB, BDNF, NTRK1, NTRK2, developed robust thermal hypersensitivity in the tumor-bearing paw compared DRD2, ARRB2, MYOCD, and CRY1) in a hypothesis-driven approach. The to PBS-injected individuals. Biphalin produced stronger analgesia in the expanded sample includes well-characterized unrelated former heroin tumor-bearing paw than morphine upon a comparable central effect. Our addicts in stable MMT that were admitted to the Adelson Clinic in Israel results suggest that biphalin analgesia manifested in the periphery is linked to a since 1993 (n=221). The daily methadone dose range was 10-280 mg less effective transport through the blood-brain barrier. We speculate that the (normal distribution, mean 140±50 mg). Fifteen HIV+ patients were centrally effective dose of biphalin equipotent to morphine simultaneously excluded. Structure analysis of 161 ancestry informative markers (AIMs) produces analgesia via peripheral opioid receptors. Thus, biphalin may become revealed a European and/or Middle Eastern ancestry for the majority of the useful in cancer pain treatment as an alternative drug executing a local as well patients. Nine SNPs in CYP2B6, ABCB1, OPRM1, BDNF and NTRK2 as a central analgesic response with limited undesirable side-effects. This work (BDNF receptor) showed significant association (nominal p<0.05) with has been partially supported by EU Normolife – 6FP Project-037733 methadone dose requirements by quantitative trait association and/or extreme value analysis (≤120, ≥170). The study confirmed that carriers of two T alleles of the functional SNP CYP2B6 516G>T (Q172H, rs3745274) require lower methadone doses than patients with other genotypes. It also suggests new variants including variants in intron 3 of OPRM1. Support: NIDA-P60-05130, NCRR-UL1RR024143 & The Adelson Medical Research Foundation.

M15 Zyklophin, the short-acting kappa opioid antagonist, induces M16 Kappa opioid receptor-mediated disruption of novel object scratching in mice recognition: Relevance for psychostimulant treatment K.M. DiMattio(1), T.V. Yakovleva(2), J.V. Aldrich(2), L.Y. Liu-Chen(1), A. J.J. Paris (1), K.J. Reilley (1), J.P. McLaughlin (1) (1)Torrey Pines Cowan(1) (1)Center for Substance Abuse Research and Department of Institute for Molecular Studies, Port St. Lucie, FL, USA Pharmacology, Temple University School of Medicine, Philadelphia, PA; (2)Department of Medicinal Chemistry, University of Kansas, Lawrence, KS Kappa opioid receptor (KOR) agonists are potentially valuable as therapeutics for the treatment of psychostimulant reward as they suppress We have previously shown that 5΄-GNTI, the long-lasting kappa opioid dopamine signaling in reward circuitry to repress drug-seeking behavior. receptor antagonist, causes dose-dependent immediate frenzied scratching in However, KOR agonists are also associated with sedation and cognitive Swiss-Webster mice that is not blocked by pretreatment with dysfunction. We examined the extent to which learning and memory norbinaltorphimine (norBNI), the kappa antagonist, or by naloxone, the non- disruption or hypolocomotion underlie KOR agonists ’ role in selective opioid antagonist, and persists in KOR -/- mice. The incidence of counteracting the rewarding effects of psychostimulants. C57BL/6J mice this scratching is lowered in a dose-dependent manner by pretreating the mice were pretreated with vehicle (saline, 0.9%), the KOR agonist U50,488, or with nalfurafine, the kappa opioid agonist. In the current study, we found that the peripherally restricted KOR agonist D-Phe-D-Phe-D-lle-D-Arg-NH2 zyklophin, the short-acting tetrapeptide kappa opioid antagonist, also elicited (ffir-NH2), through central (i.c.v.) or peripheral (i.p.) routes of scratching behavior when injected subcutaneously in the nape of the neck in administration. Locomotor activity was assessed via activity monitoring mice. Zyklophin induced hindleg scratching of the neck between +3 and +15 chambers and rotorod. Cognitive performance was assessed in a novel min in a dose-dependent manner at doses of 0.1, 0.3 and 1 mg/kg. Pretreating object recognition task. Prolonged hypolocomotion was observed following mice with norBNI (20 mg/kg, i.p.) at 18-20 hr before challenge with zyklophin administration of 1.0 and 10.0, but not 0.3 mg/kg U50,488. Central, but not (0.3 mg/kg) did not markedly affect scratching. Additionally, KOR -/- mice peripheral, administration of ffir-NH2 (a KOR agonist that does not cross given our standard dose of 0.3 mg/kg of zyklophin did not display decreased the blood-brain barrier) also reduced motor behavior. Systemic scratching when compared to wild-type animals. The absence of kappa pretreatment with the low dose of U50,488 (0.3 mg/kg, i.p.) significantly receptors was confirmed with ex vivo radioligand binding using [3H]-U69,593. impaired performance in the novel object recognition task. Likewise, ffir- To examine the role of substance P in zyklophin-induced scratching, we NH2 significantly reduced novel object recognition after central (i.c.v.), but injected mice with the selective neurokinin 1 receptor antagonist, RP 67580 (5 not peripheral (i.p.), administration. U50,488- and ffir-NH2 mediated and 10 mg/kg, i.p.), 1 hr before zyklophin. Neither dose markedly influenced deficits in novel object recognition were prevented by pretreatment with the peptide-induced scratching. Taken together, our data suggest that the KOR antagonists. Cocaine-induced conditioned place preference was presence of kappa receptors is not required for the excessive scratching caused subsequently assessed and was reduced by pretreatment with U50,488 (0.3 by zyklophin and 5΄-GNTI. These chemically diverse kappa antagonists mg/kg, i.p.). Together, these results suggest that the activation of centrally- appear to act at other targets to elicit scratching (and potentially the sensation located kappa opioid receptors may induce cognitive and mnemonic of itch). disruption independent of hypolocomotor effects which may contribute to (Supported by DA17302 and PA Department of Health grants) the KOR-mediated suppression of psychostimulant reward. Funded by: NIMH (MH085607), NIDA (DA031370), and the Florida Executive Office of the Governor’s Office of Tourism, Trade, and Economic Development.

INRC 2012 Kansas City, Missouri 51

M17 The interaction of some antidepressant drugs with acute vs. chronic M18 Delta Opioid Receptor Agonists Inhibit Migraine-Associated methadone in mice Hyperalgesia, Aversive State, and Cortical Spreading Depression Schreiber, S (1), Hostovsky, A (2), Barak, Y (2), Rubovitch, V (2), and Pick, Monique L Smith (1,2,3), Jekaterina Zyuzin (2), Andrew Charles (2,3), and CG (2) (1)Department of Psychiatry, Tel Aviv Sourasky Medical Center and Amynah A Pradhan (1,3). (1) Semel Institute for Neuropsychiatry & Tel Aviv University Sackler Faculty of Medicine; (2)Department of Anatomy, Human Behavior, University of California Los Angeles, (2) Headache Tel-Aviv University Sackler Faculty of Medicine, Tel Aviv, Israel Research and Treatment Program, Department of Neurology David Geffen School of Medicine at UCLA, (3) Shirley and Stephan Hatos Depression is highly prevalent among both chronic-pain patients and Center for Neuropharmacology, UCLA. methadone maintenance treatment (MMT) patients. However, controversy about effective treatment for patients with either version of the both disorders Migraine is an extraordinarily common brain disorder for which therapeutic persists, more so regarding the ‘right’ antidepressants to be used in MMT options continue to be limited, in part because of the lack of reliable pre- depressed patients. In the model of depressed pain patients, the impact of low, clinical models. The aim of this study was to further develop acute and sub-threshold doses of 4 antidepressant drugs with different mechanisms of chronic migraine models, and to investigate the effects of delta opioid action (fluvoxamine, escitalopram, reboxetine, venlafaxine) on the receptor agonists on basic mechanisms of migraine within these models. antinociceptive properties of a single dose of methadone was evaluated in The nitric oxide donor, nitroglycerin, is a consistent migraine trigger in mice using the hotplate assay. Following injection, methadone elicited humans, and was shown to evoke both acute mechanical and thermal analgesia in a dose-dependent manner. Fluvoxamine given at a fix sub- hyperalgesia in mice. Chronic intermittent administration of nitroglycerin threshold dose induced a synergistic effect (at the 1mg/kg methadone point the resulted in a progressive and sustained hyperalgesia, as well as conditioned group with fluvoxamine showed an analgesic effects of 60% while without place aversion. Two different delta opioid receptor agonists, SNC80 and fluvoxamine it was only 10% analgesia; P<0.05). Escitalopram, reboxetine ARM390, significantly reduced nitroglycerin-evoked hyperalgesia. SNC80 and venlafaxine given separately at a fix sub threshold dose induced no also abolished nitroglycerin-induced conditioned place aversion, suggesting interaction. Possible clinical implications of these findings are that while that delta opioid receptor activation may also alleviate the negative escitalopram, reboxetine and venlafaxine do not affect methadone’s emotional state associated with migraine. In addition, we found that SNC80 antinociception in mice and are safe to be given together with methadone, significantly attenuated cortical spreading depression, a model of migraine fluvoxamine notably augments methadone induced antinociceptive effects and aura that is considered useful in predicting the efficacy of migraine should be avoided in this population due to the risk of inducing opiate- preventive therapies. These data show that delta opioid receptor agonists overdose. In the model of depressed MMT patients, the impact of low, sub- modulate multiple basic mechanisms associated with migraine, indicating threshold doses of 2 antidepressant drugs with different mechanisms of action that delta opioid receptors are a promising therapeutic target for this (escitalopram, desipramine) on the antinociceptive properties of chronic disorder. This research was supported by NIH-NIDA Grants DA05010 and treatment with methadone was evaluated in mice using the hotplate assay. K99DA031243, the Migraine Research Foundation, and the Shirley and Following two weeks of methadone administration (through an implanted Stefan Hatos Research Foundation. mini-pump, i.p. injection of escitalopram did not elicit any analgesic effect. The study with desipramine is underway, and possible clinical implications remain to be determined later.

M19 Association of Polymorphisms of the Delta Opioid Receptor and M20 Normal healthy women bearing the minor allele A of MC2R gene Proenkephalin Genes with the Severity of HIV Infection and Response to (ACTHR) -184G>A have decreased basal cortisol levels compared to HIV Treatment males, which all increase to equal levels after ACTH challenge. Dmitri Proudnikov (1), Matthew Randesi (1), Howard Crystal (2), Jurg Ott (3, B. Ray, D. Proudnikov, S. Chen, E. Ducat, A. Ho, M.J. Kreek, Laboratory 4), Ann Ho (1) and Mary Jeanne Kreek (1) (1) Lab. Biol. Addict. Diseases, of the Biology of Addictive Diseases, Rockefeller University, New York, Rockefeller Univ., New York, NY, USA (2) Dep. Pathol., SUNY Downst. NY Med. Center, Brooklyn, NY (3) Lab. Statist. Genet., Rockefeller Univ., New York, NY, USA (4) Inst. Psychol. CAS, Beijing, China We have hypothesized that dysregulation of the hypothalamic-pituitary- adrenal axis is central to drug addiction. Cortisol provides negative Delta opioid receptors (OPRD1) expressed in lymphocytes affect T cell feedback on the hypothalamic production of corticotropin releasing proliferation and interleukin-2 production. Cross-desensitization between hormone and on the anterior pituitary production of proopiomelanocortin OPRD1 and chemokine receptors involved in HIV infection has been reported. which yields beta-endorphin and ACTH. The melanocortin receptor type 2 We examined whether genetic variants of OPRD1 and its ligands, the (MC2R, ACTH receptor) is the only melanocortin receptor that activates enkephalins derived from proenkephalin (PENK), influence HIV status and production of cortisol in the adrenal cortex. The MC2R gene, located in response to therapy. We examined 11 variants of OPRD1 and 8 variants of chromosome 18, has two exons. We evaluated the influence of -184G>A, PENK with change of viral load and CD4 count between admission to the rs2186944 in exon 2, on cortisol response to placebo and to ACTH Women’s Interagency HIV Study, before and after initiation of HAART, in challenge. Of 63 normal healthy volunteers with no addiction history 1270 subjects including African Americans, Hispanics and Caucasians. In recruited from the NYC area who consented to neuroendocrine studies and OPRD1 before HAART, a genotype effect of 1462A>G (rs4654327) on CD4 to genetic testing, there were 4 males and 4 females with the genotype - count change was found in Caucasians; genotype effects of IVS1+634G>A 184GA and 1 male and 1 female with AA. Their cortisol response to (rs2236861), IVS1+22487T>C (rs2236857), IVS1+30437T>C (rs3766951), placebo and to ACTH challenge was compared to 10 males and 10 females IVS1+37091A>G and 921T>C (rs2234918) on viral load change were found homozygous for the prototype allele (184GG). Subjects were admitted to in Hispanics. After HAART, genotype effects of IVS1+634G>A (rs2236861), Rockefeller University Hospital and normal saline 10 ml or cosyntropin (a IVS1+22487T>C (rs2236857), IVS1+27016T>G (rs2298896), synthetic derivative of ACTH) 750 mcg was administered intravenously on IVS1+30437T>C (rs3766951) and IVS1+37091A>G (rs12749204) on the 2 consecutive days at 10 AM. Cortisol levels were drawn on each subject CD4 count change were found in African Americans; genotype effects of beginning at 9:50 AM (-10), then at 0 (immediately preceding 921T>C (rs2234918) and 1462A>G (rs4654327) on change of CD4 count administration of study medication), 10, 20, 30, 40, 50, 60, 75, 90, 120, were found in Hispanics. Also, after HAART, a genotype effect of 240, and 360 minute time points. Analysis of variance of cortisol area under IVS1+30437T>C (rs3766951) on viral load change was found in Caucasians. the curve (0-90 min) in the placebo condition showed that while there was In PENK, a genotype effect of -23C>G (rs2078280) on CD4 count change was no significant main effect of genotype or of gender, females with the minor found in African Americans before HAART. Also in PENK, genotypes - allele A of MC2R -184G>A have lower cortisol levels than males (p<0.02, 1946A>G (rs2609997) and -1522C>T (rs2609998) were associated with HIV planned comparison). Cortisol levels after ACTH challenge were increased status in Hispanics. Support: NIH-NIDA P60-DA05130 (MJK), NIH-NIMH in both males and females to equal levels. Support: NIH-NIDA P60- MH79880 (MJK), NIH-NIMH MH076537 (HC) and NSFC from the Chinese DA05130 (M.J.K.), The Adelson Medical Research Foundation and Gov. 30730057 (JO). NIH/NCRR-CTSA UL1-RR024143 (B.C.)

52 INRC 2012 Kansas City, Missouri

M21 The antidepressant-like and antinociceptive effects of KNT-127, the M22 Lipoxygenase-mediated regulation of delta opioid receptors in novel delta opioid receptor agonist, in mice. peripheral sensory neurons Akiyoshi Saitoh (1), Azusa Sugiyama (1,2), Toru Nemoto (3), Hideaki Fujii L.C. Sullivan, K.A. Berg, and W.P. Clarke Dept. of Pharmacology, (3), Keiji Wada (4), Jun-Ichiro Oka (2), Hiroshi Nagase (3), Mitsuhiko University of Texas Health Science Center at San Antonio, TX, USA Yamada (1) (1)Dept. of Neuropsychopharmacology, NIMH, NCNP, Tokyo, Japan; (2) Lab. Pharmacol., Fac. Pharm. Sci., Tokyo Univ. Sci., Chiba, Japan; Peripheral administration of delta opioid receptor (DOR) agonists rarely (3) Laboratory of Med. Chemistry, School of Pharmacy, Kitasato Univ., produces analgesic responses in normal (uninflamed) tissue. We have Tokyo, Japan; (4)Dpartment of Degenerative Neurological Diseases, NIN, found that the DOR system in peripheral sensory neurons can become NCNP, Tokyo, Japan responsive (functionally competent) upon brief pre-treatment with inflammatory mediators such as bradykinin (BK) or arachidonic acid (AA). Objective: We previously reported that the delta opioid receptor (DOP) In cultures of peripheral sensory neurons from adult rat trigeminal agonists SNC80 and TAN-67 produce potent antidepressant-like and ganglion, the DOR agonist, DPDPE, does not inhibit PGE2-stimulated antinociceptive effects in rodents. However, SNC80 produced convulsive adenylyl cyclase activity unless cells are pre-treated (15 min) with BK or effects. Recently, we succeeded in synthesizing a novel DOP agonist called AA. Following 15 min pre-treatment, the DOR system remains functionally KNT-127. The present study examined the convulsive, antidepressant-like, competent for only 30-60 min. Here we examined if DOR functional and antinociceptive effects of KNT-127 in mice. competence could be re-induced by subsequent BK and/or AA applications. Methods: Male ICR mice weighing 30-40g were used. Convulsive effects on DPDPE (100 nM)–mediated inhibition of PGE2-stimulated cAMP mice were measured for 20 min after the subcutaneously drug treatment. The accumulation was measured in primary cultures of trigeminal ganglion antidepressant-like effect was evaluated using the forced swim test. The treated (15 min) with BK (10 uM) or exogenous AA (50 uM), 60 min after antinociceptive effects were evaluated using the acetic acid-abdominal initial BK or AA pre-treatment (15 min). DOR-mediated responses could constriction (writhing) test and the formalin test. KNT-127 was administered be re-established after initial BK, but not AA, pre-treatment suggesting that 30 min before the test session. Naltrindole (NTI), 7-benzylidenenaltrexone the DOR system became “refractory” to re-induction of functional and naltriben were administered 30 min before KNT-127 treatment. competence following the initial AA pre-treatment. This refractory Results: In contrast to SNC80, KNT-127 produced no convulsions at doses to response was blocked by inhibition of the lipoxygenase (LOX) pathway. 100 mg/kg. In mice subjected to the forced swim test, KNT-127 (1 mg/kg, Pre-treatment with the LOX inhibitors NGDA (10 uM) or DPE (10 uM) s.c.) significantly decreased the duration of immobility and increased the allowed for re-induction of DOR functional competence by BK 60 min duration. These behavioral changes were similar to that observed for the after initial AA treatment, whereas inhibition of the cytochrome P450 tricyclic antidepressant imipramine (6 mg/kg). The antidepressant-like effect pathway (with ODYA, 10 uM) had no effect. Overall these studies suggest of KNT-127 in mice was antagonized by pretreatment with NTI, a selective distinct AA-dependent pathways can regulate DOR function. In addition to

DOP antagonist, or naltriben, a putative DOP2 subtype antagonist. In addition, cyclooxygenase-dependent induction of competence, the current data KNT-127 (3 mg/kg, s.c.) significantly reduced the number of acetic acid- indicate a novel LOX-dependent pathway is involved in the loss of induced abdominal constrictions and the duration of licking time, respectively, functional competence of DOR. Thus inhibition of LOX, combined with a in mice subjected to the writhing test and the formalin test. These competence-inducing stimulus may promote initiation and maintenance of antinociceptive effects were antagonized by pretreatment with either NTI or 7- peripheral DOR-mediated analgesia. Supported by DA024865 and NIDCR benzylidenenaltrexone, a putative DOP1 subtype antagonist. grant DE14318 for the COSTAR Program. Conclusion: We propose that KNT-127 should be considered as a candidate compound for the development of DOP-based antidepressants and/or analgesics that lack convulsive effects.

M23 The delta-opioid receptor contributes to morphine tolerance in an M24 Effects of adolescent oxycodone self-administration on animal model of chronic inflammatory pain oxycodone-induced conditioned place preference and locomotor L. Hipólito (1), H. Beaudry (2), A. Gupta (3), L. Devi (3), L. Gendron (2), J.A. activity in C57BL/6J mice. Morón Concepción (1). (1) Dept. of Anesthesiology, Columbia University Yong Zhang, Eduardo R. Butelman, Ann Ho, Mary Jeanne Kreek Medical Center, New York, USA (2) Dept. of Physiology and Biophysics, The Rockefeller University, New York, NY 10065 Université de Sherbrooke, Sherbrooke, Canada, (3) Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, Illicit use of opioid analgesics (e.g., oxycodone) in adolescence is a New York, USA. pressing public health issue. Objective: Our goal was to determine whether adolescent oxycodone self-administration affects subsequent oxycodone- Opioid use in chronic inflammatory pain is limited by the development of tolerance induced conditioned place preference (CPP) and locomotor activity in and unwelcomed side effects. Although the topic remains controversial, functional mice. Methods: A group of adolescent mice (4 weeks old, n=6) and a and physical interactions between the mu- (MOP) and the delta- (DOP) opioid group of adult mice (10 weeks old, n=6) underwent surgery during which a receptors have been recently reported. Indeed, recent studies suggest that catheter was implanted into their jugular veins. Mice were given five days interactions between the MOP and DOP may play an important role in morphine- to recover from the surgery and were placed in the self-administration induced behavioral adaptations. In fact, blockade of the DOP or disruption of chambers for acquisition of oxycodone self administration. Mice self MOP/DOP heteromers was shown to enhance morphine-induced analgesia. Here, administered oxycodone (0.25mg/kg/infusion) 2hrs daily for 14 days. we investigated the regulation of DOP and the MOP/DOP heteromer during the Fifteen days after the last oxycodone self-administration session, mice development of morphine tolerance in a rat model of chronic inflammatory pain. We find an increase in the expression of DOP at the postsynaptic density fraction in underwent a 10-day CPP (3 mg/kg) training procedure. Results: Mice that the dorsal horn of the spinal cord in morphine tolerant rats and this effect is even had self-administered oxycodone during adolescence spent more time in more robust in the presence of chronic inflammation. In addition, ELISA studies the oxycodone-paired chamber than those that had self-administered using a selective MOP/DOP heteromer antibody show an increase in the levels of oxycodone during adulthood. Oxycodone increased locomotor activity the MOP/DOP heteromer in the dorsal horn in morphine tolerant rats; interestingly significantly in both age groups compared with saline with sensitization this effect is even more dramatic during chronic inflammatory pain. Thus, our data developed only in the younger group. Conclusion: Adolescent oxycodone show that the increase in the levels of postsynaptic DOP correlates with an increase self-administration leads to long-term alterations of the rewarding effect of in the DOP/MOP heteromer formation. Furthermore, behavioral analyses show that oxycodone in adulthood. pretreatment (s.c. or i.t.) with the DOP antagonist, naltrindole, attenuates the Support: This work was supported by NIH-NIDA 1R01DA029147-01A1 development of morphine tolerance. Overall, our data show that the DOP play an (YZ) and NIH-NIDA DA05130 (MJK) important role during morphine tolerance, an effect further amplified in the presence of chronic inflammation. We also show that the increase in DOP levels is associated with an increase in the levels of the MOP-DOP heteromer in the spinal cord. Altogether, our findings highlight the DOP as a key player in the development of morphine tolerance during chronic inflammation. Further studies are needed to analyze whether the above observed changes in DOP levels and MOP/DOP abundance are also observed in other components of the opioid-sensitive pain- modulating circuit and to define the exact consequences of this phenomenon. This work was supported by Departmental start up funds to JAMC and by a CIHR grant to LG. INRC 2012 Kansas City, Missouri 53

M25 Possible involvement of endogenous opioid system located in the M26 Sadowski mouse strains of high and low stress induced analgesia downstream of alpha7 nicotinic acetylcholine receptor in nicotine- as a model of blood brain barrier permeability studies of opioid induced antinociception in mice peptides and alkaloids. S. Kishioka, N. Wakida, K. Ueno, F. Saika, C. Yamamoto, Y. Kobayashi, N. AW Lipkowski(1), A Kosson(1), AA Lesniak(1), M Sacharczuk (2), G Kiguchi Department of Pharmacology, Wakayama Medical University, Tóth(3), J Molnár(3), IA. Krizbai(3), I Wilhelm(3) (1)Mossakowski Wakayama, Japan Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02- 106 Warsaw, (2)Institute of Genetics and Animal Breeding Polish Nicotine is a neuronal stimulant and produces various pharmacological effects Academy of Sciences, Postepu 1 05-552 Wolka Kossowska, Poland, through the activation of nicotinic acetylcholine receptor (nAChR). And it is (3)Biological Research Centre, Hungarian Academy of Sciences, Szeged, well-known that alpha7 and alpha4beta2 nAChRs are expressed in the central Hungary nervous system. In this study, we clarified the nAChR subtypes involved in nicotine-induced antinociception and activation of the hypothalamo-pituitary- The blood-brain barrier (BBB) forms filtering system between peripheral adrenal axis (i.e., the elevation of serum corticosterone; SCS) in ICR mice, circulating blood and central nervous system. Pathological leaking of BBB and predicted the existence of endogenous opioid system located in the is probably responsible for various dysfunctions and diseases. Up to now, downstream of nAChR subtype. good natural animal model of differences of BBB permeability is not Antinociceptive effect was evaluated by tail-pinch test. SCS level was known. Over twenty years ago Sadowski et al separated two lines of mouse, quantified by fluorometric assay. RT-PCR and immunohistochemical assays one with high sensitivity (HA) and the other with low (LA) sensitivity to were performed according to standard procedures. Nicotine (s.c. and i.t.)- stress induced analgesia (SIA). We propose that distinct leaking of BBB is induced antinociception was inhibited by both mecamylamine (MEC) and responsible for difference in SIA of “Sadowski mouse” model. Recently, naloxone (NLX). Conversely, nicotine (s.c.)-induced increase in SCS level we presented some structural evidences for this hypothesis [1]. The was prevented by MEC, but not NLX. Nicotine-induced antinociception was “Sadowski mouse” could be very useful model of BBB permeability role suppressed by repeated morphine treatment, while SCS increase was not. The and significance in physiological and pharmacological studies. This preproenkephalin mRNA expression in spinal cord was significantly increased communication is focusing on distinct effects of opioid peptides and 2 hrs after nicotine (s.c.), which was inhibited by MEC. On the other hand, alkaloids in HA and LA Sadowski mouse. nicotine had no effect on the expression of preprodynorphin and preproopiomelanocortin mRNA in the spinal cord. Moreover, nicotine- [1] Gajkowska B., Kosson A., Sacharczuk M., Kosson P., Lipkowski, AW. induced antinociception was suppressed by dihydro-beta-erythroidine Blood-brain barrier permeability differentiates Sadowski mouse lines of (DHbetaE; an antagonist for the alpha4beta2 nAChR) and methyllycaconitine high and low stress-induced analgesia. Electron microscopy analysis. Folia (MLA; an antagonist for the alpha7 nAChR). The nicotine-induced SCS Neuropathol 2011; 49: 311-318 increase was suppressed by DHbetaE, but not MLA. These results suggest that alpha4beta2 nAChR subtype involves in both nicotine-induced antinocieption and SCS elevation, while alpha7 nAChR participates only in antinociception. And the endogenous enkephalinergic opioid system may be localized on the downstream of alpha7 nAChR, but not alpha4beta2. This work was supported by a grant from the Smoking Research Foundation.

M27 Unidirectional Cross Activation of GRPR by MOR1D Uncouples M28 A novel mechanism for acupuncture analgesia: Orexin 1 receptor- Opioid-Induced Analgesia and Opioid-Induced Itch mediated endocannabinoid retrograde disinhibition in the X.Y. Liu (1), Z.C. Liu (1), Y.G. Sun (1), M. Ross (1), S. Kim (1), Q.F. Li (1), periaqueductal gray. H.H. Loh (2), Z.F. Chen (1) (1) Dep. of Anesthesiology, Center for the Study L.-C. Chiou (1, 2), Y.-H. Chen (3), H.-J. Lee (1) and Y.-T. Wu (3). (1) of Itch, Washington Univ., St Louis, USA (2) Dep. of Pharmaco., Univ. of Dept. Pharmacology, (2) Grad. Inst. Brain and Mind Sciences, Coll. Minnesota, Minneapolis, USA Medicine, National Taiwan University, Taipei, Taiwan; (3)Grad. Inst. Acupuncture Science, China Medical University, Taichung, Taiwan. Spinal opioid-induced itch, a prevalent side effect of pain management, has long been considered to arise as a result of pain inhibition. We report that Previously, we revealed a novel analgesic mechanism mediated by orexin 1 morphine-induced scratching (MIS) is abolished in mice lacking either receptor (OX1R) in the ventrolateral periaqueductal gray (vlPAG). gastrin-releasing peptide receptor (GRPR), an itch-specific receptor in the Activation of postsynaptic OX1R, a Gq-protein coupled receptor, results in spinal cord or the mu opioid receptor (MOR). Exon mapping studies show synthesis of 2-arachydonoylglycerol (2-AG) via phospholipase C (PLC)- that MOR1D is essential for MIS, whereas MOR1 is important for morphine- diacylglycerol lipase (DAGL) enzymatic pathway. 2-AG, an induced analgesia (MIA) with no cross-activity present. MOR1D and GRPR endocannabinoid, produces retrograde inhibition of GABA release expression overlaps in lamina I of the spinal cord, which can form (disinhibition) by activating presynaptic cannabinoid 1 receptors (CB1R) in heterodimers, co-internalize and activate phospholipase C (PLC) and inositol the vlPAG, leading to analgesia via activating the descending pain 1,4, 5-trisphosphate (IP3)-mediated Ca2+ signaling upon morphine activation inhibitory pathway.1 Interestingly, it has been reported that in heterologous system. By contrast, GRP is unable to induce electroacupuncture at the PC5 (Jianshi)-PC6 (Neiguan) acupoints induced a heterodimerization and co-internalization of MOR1D and GRPR. Thus, these CB1R-mediated inhibition of GABA release in the vlPAG. We, therefore, results suggest a unidirectional cross-activation of GRPR signaling by validated a hypothesis that EA at PC5-PC6 acupoints induces analgesia by MOR1D, and demonstrate that opioid-induced itch is an active process and releasing endogenous orexins through this OX1R-PLC-DAGL-2-AG thereby uncouples MIS and MIA. The uncoupling of MIA and MIS retrograde disinhibition mechanism in the vlPAG. EA-induced analgesia underscores the necessity of elucidating the function of individual MOR was accessed by the hot-plate response in mice receiving electrical isoforms which may promise novel pain therapy without side effects. stimulation (2 Hz, 2 mA, 0.15 ms) for 20 min at PC5-PC6 acupoints under isofluorane anesthesia. Indeed, the antinociceptive effect induced by EA at PC5-PC6 acupoints of mice was prevented by either i.p. injection or intra- vlPAG (i.pag.) microinjection of the antagonist of OX1R (SB-334867, 15 mg/kg, i.p., or 15 nmol, i.pag.) or CB1R (AM 251, 1.1 mg/kg, i.p., or 30 nmol, i.pag.), but not by naloxone (1 mg/kg, i.p.). Mice receiving 20 min EA had more c-Fos containing orexin neurons in the lateral hypothalamus than control mice. It is suggested that EA activates hypothalamic orexin neurons and releases orexins which induce analgesia via a novel mechanism mediated by OX1R-initiated 2-AG retrograde disinhibition in the vlPAG. (Supported by NSC 100-2325-B002-050) 1Ho YC et al. J Neurosci 31:14600 (2011).

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M29 Modulation of Morphine Tolerance by Glial Receptor Toll-like M30 Coupling between opioid receptors and potassium channels in Receptor 4 in the Midbrain Periaqueductal Gray of Male Rats pain reduction: relevance for species differences and efficacy Lori N. Eidson & Anne Z. Murphy Neuroscience Institute, Georgia State D. Nockemann (1), D. Labuz (1), C. Stein (1) and P.A. Heppenstall (2) University, Atlanta, GA (1)Dep. Anesthesiology, Freie Universität, Campus Benjamin Franklin, Charité, Berlin, Germany; (2)Mouse Biology Unit, EMBL Monterotondo, Morphine is amongst the most effective analgesics available for the treatment Italy of chronic pain. However, prolonged morphine treatment results in the development of analgesic tolerance. Recent evidence indicates that morphine Opioid treatment of pain is limited by adverse side effects on the central activates CNS glia via the innate immune receptor Toll-like receptor 4 nervous system (CNS). A better understanding of the mechanisms (TLR4), and that glial cell activity opposes morphine analgesia. While the underlying opioid analgesia mediated outside the CNS would help to midbrain periaqueductal gray (PAG) is an essential neural substrate mediating develop opioids acting at peripheral sites of tissue injury without centrally the development of morphine tolerance, no studies have assessed the role of mediated unwanted effects. In the CNS activation of opioid receptors PAG-glia and Toll-like receptor 4 (TLR4) in this phenomenon. Here we test inhibits voltage-gated calcium (Ca2+)-channels and activates G protein- the hypothesis that PAG glia mediate the development of morphine tolerance coupled inwardly rectifying potassium (GIRK) channels through binding of via TLR4. Our data suggest that PAG TLR4 mediates the development of G-protein Gβγ subunits. Both events reduce membrane excitability. morphine tolerance as antagonizing PAG TLR4 while co-administering However, studies on GIRK channel expression and function in peripheral subcutaneous morphine dose-dependently prevents morphine tolerance neurons are scarce and have produced conflicting results. Here we report development. Similarly, intra-PAG microinjections of TLR4 agonists lead to that GIRK channels in sensory dorsal root ganglion (DRG) neurons are tolerance to subsequent doses of morphine. These experiments could provide crucially involved in the generation of opioid antinociception. We found novel and crucial information about the mechanism by which central nervous very low mRNA and protein expression of different GIRK subunits in DRG system glia regulate morphine tolerance generally, and could provide a of naïve mice. However, expression of mRNA and protein of GIRK-1 and - potential therapeutic target for the enhancement of analgesic efficacy in the 2 was prominent in DRG of naïve rats. We detected GIRK currents after clinical treatment of chronic pain specifically. mu-opioid receptor activation by DAMGO in rat but not in mouse DRG neurons. We generated transgenic mice expressing a Flag-GIRK2 construct selectively in DRG neurons by use of a Nav1.8 promoter. DAMGO evoked GIRK currents in DRG neurons isolated from these Nav1.8-GIRK2 mice but not from wildtype littermates. We assessed how expression of GIRK2 affects nociceptive behavior by measuring hindpaw withdrawal in animals with painful hindpaw inflammation. Local injection of DAMGO abolished

the thermal and mechanical hypersensitivity in Nav1.8-GIRK2 mice but not in wildtype littermates. Our data show that GIRK channels in DRG neurons are crucial for generation of peripheral opioid antinociception and that these molecular mechanisms may explain species differences. These findings should be considered in drug development and in basic and clinical pain research.

M31 Spinal mu opioid receptors regulate both heat and mechanical pain M32 Inhibition of Antinociception by the Mu Opioid Partial Agonist in rats BU08073 A. Normandin(1), P. Luccarini(2), J-L. Molat(2), L. Gendron (1) and R. L. Toll(1), T.V. Khroyan(2), W.E. Polgar(2), G. Cami-Kobeci(3), S. Dallel(2) (1) Département de Physiologie et Biophysique, Université de Husbands(3). (1) Torrey Pines Institute for Molecular Studies, Port St. Sherbrooke, Québec, Canada; (2) Inserm/UdA U1107, Université Lucie, FL, USA; (2) SRI International, Menlo Park, CA, USA; (3) d’Auvergne, Clermont-Ferrand, France University of Bath, Bath, UK.

It was recently suggested that mu opioid receptors (MOPR) are present in only BU08073 is a buprenorphine analog that binds with high affinity to all a subset of unmyelinated nociceptors that express the neuropeptide substance opioid receptors, including NOP receptors, with Ki values of less than 10 P (SP) and the noxious thermal transducer TRPV1. Such a specific expression nM at each site. This compound has very low efficacy (less than 10% of MOPR in peptidergic C-fiber primary afferents suggests that spinally- relative to full agonists) at delta, kappa, and NOP receptors. At mu delivered MOPR agonists should differentially regulate heat and mechanical receptors BU08073 has moderate efficacy but high potency (EC50 2.9 nM, pain. To test this hypothesis, we assessed the effect of directly activating % stimulation 31.9%), having both higher efficacy and potency than spinal MOPR by intrathecal application of the MOPR agonist, DAMGO (5 buprenorphine. Surprisingly, acute injection of BU08073 does not increase µg), on both diffuse noxious inhibitory controls (DNIC) and spinal NK1 tail flick latency in mice. Despite moderate efficacy at mu receptors, receptor (NK1R) internalization triggered by heat and mechanical noxious BU08073 blocks analgesic activity of both morphine (mu-mediated) and stimulation of the hindpaw. We recorded from wide-dynamic range neurons in U50-488 (kappa-mediated). However, the kinetics of inhibition of opiate- the spinal trigeminal nucleus of rats. Trains of 105 electrical shocks were mediated analgesia are unusual. BU08073 is basically inactive at 1 h. 6 h delivered to the excitatory cutaneous receptive field at 3 times the threshold following an injection of BU08073 (3 mg/kg) both morphine and U50,488- for C-fibers activation. To trigger DNIC, either heat (immersion in a 49°C induced analgesia are blocked. A single injection of BU08073 still blocks waterbath) or mechanical (pressure 300 g) noxious stimulation was applied to antinociception for 3 days at the kappa receptor, and up to 6 days at the mu the hindpaw between the 36th and 60th stimuli. During the control periods, receptor. BU08073 was also tested for anxiolytic or anxiogenic activity inhibitions produced by heat and mechanical noxious stimulations were 87 ± using the elevated zero-maze. BU08073 alone decreased the time spent in 5%, and 58 ± 9%, respectively. I.t. DAMGO reduced heat and mechanical the open quadrant, indicating potential anxiogenic activity. Surprisingly, a stimulation-induced DNIC by 63 ± 9% and 57 ± 9%, respectively. Such single injection of BU08073, given as a pretreatment, induced anxiolytic reductions were reversed by administration of the non-selective opioid activity for up to 10 days when mice were tested one hour after receptor antagonist, naloxone (0.4 mg/kg i.v.). Because spinal SP is known to administration of the anxiogenic compound U50-488. The reason this be involved in activation of DNIC, we examined the effect of i.t. DAMGO on compound has no antinociceptive activity, in the tail flick assay, despite superficial laminae NK1R internalization induced by heat and mechanical greater efficacy at mu receptors than buprenorphine and other partial noxious stimulation of the hindpaw. DAMGO reduced heat (37.3 ± 4.4% of agonists is unclear. The mechanism by which two anxiogenic compounds neurons with internalized NK1R vs 72.3 ± 3.8% for saline-treated rats) and can become anxiolytic is also unclear. However, both of these phenomena mechanical (33.0 ± 7.0% vs 65.6 ± 5.2%) stimulation-evoked NK1R probably have to do with the slow onset of action and unusual kinetics of internalization compared to saline-treated rats. These results indicate that BU08073. Supported by NIH grants R01DA023281 to LT and activation of spinal MOPR can efficiently relieve both heat- and mechanical- R01DA020469 to SH. induced pain. This work was supported by Inserm, the NSERC and a training award from the QPRN. INRC 2012 Kansas City, Missouri 55

M33 Effects of Single-Prolonged Stress on Nociceptive sensitivity and the M34 Involvement of the long-chain fatty acid receptor GPR40 as a pain Nociceptin/Orphanin FQ (N/OFQ) System in Spinal Cord of Rats control system Y. Zhang (1), P.R. Gandhi (1) , K.M. Standifer (1, 2). (1)Department of Kazuo Nakamoto (1), Takashi Nishinaka (1), Kengo Matsumoto (1), Naoya Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Sato (1), Kasuya Fumiyo (2), Mitsumasa Mankura (3), Yutaka Koyama (4) Health Sciences Center, Oklahoma City, OK 73117, (2)Department of Cell and Shogo Tokuyama (1) (1) Department of Clinical Pharmacy, Kobe Biology and Oklahoma Center for Neuroscience, University of Oklahoma Gakuin University, School of Pharmaceutical Sciences, (2) Biochemical Health Sciences Center, Oklahoma City, OK 73117, USA Toxicology Laboratory, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, (3) Ikeda Tohka Industries Co., Ltd., (4) Laboratory of Single-prolonged stress (SPS) is an established animal model for post- Pharmacology, Faculty of Pharmacy, Osaka Ohtani University traumatic stress disorder (PTSD). Clinical studies indicate that post-traumatic stress disorder (PTSD) frequently shares co-morbidity with chronic pain. GPR 40 has been reported to activate by a long-chain fatty acids, such as Although acute stress-induced antinociception is well documented in animals, docosahexaenoic acid (DHA) and arachidonic acid. However, reports the effect of PTSD-like stress on pain sensitivity is not. We hypothesized that studying the role and functions of GPR40 in the brain are lacking. In the SPS would increase the nociceptive sensitivity of rats. Indeed, SPS induced present study, we focused on the relationship between pain regulation and mechanical and thermal allodynia within 3-5 days after initiation of SPS, and GPR40, and investigated the involvement of GPR40 in the brain on DHA- lasted 28-35 days. SPS also induced anxiety-like behavior 9 days after SPS induced antinociceptive effects. Additionally, we examined about the and decreased levels of circulating corticosterone 28 days post SPS; simulating involvement of brain GPR40 protein expression during chronic pain by PTSD-like symptoms. Nociceptin/orphanin FQ (N/OFQ), an endogenous using complete Freund's adjuvant (CFA)-induced inflammatory model mice. ligand for the N/OFQ peptide (NOP) receptor, modulates various biological Expression of GPR40 protein was a widely observed in the brain as well as functions in the central nervous system that are affected by PTSD, including the spinal cord. Intracerebroventricular (i.c.v.), but not intrathecal (i.t.) nociceptive sensitivity, stress and anxiety, learning and memory. Examination injection of DHA (25 and 50 mg/mouse) and GW9508 (a GPR40- and of N/OFQ levels in CSF, serum and spinal cord dorsal horn (SC) revealed that GPR120-selective agonist; 0.1 and 1.0 mg/mouse) significantly reduced N/OFQ levels were increased in CSF and serum by day 9 and 14 after formalin-induced pain behavior. These effects were inhibited by initiation of SPS, respectively. No changes in N/OFQ levels in SC were noted pretreatment with the m opioid receptor antagonist β-funaltrexamine (β- at any time point. Real-time PCR indicated NOP receptor mRNA was FNA), naltrindole (d opioid receptor antagonist) and anti-b-endorphin increased 2.7-fold at day 28. NOP receptor activity was measured as antiserum. On the other hand, in CFA-induced inflammatory model mice, stimulation of 35S-GTPγS binding by N/OFQ at 9, 14 and 28 days after SPS GPR40 expression in the medulla oblongata and hypothalamus were exposure. NOP receptor efficacy in SC of SPS rats was significantly increased significantly increased compared to sham group. On the other side, there is at day 14 and 28, but not at day 9. Our results indicate that the N/OFQ-NOP no change in the cortex. These findings suggest that DHA-induced receptor system dynamically responds to SPS and pain, suggesting that N/OFQ antinociception via b-endorphin release may be mediated (at least in part) may play an important role in the plasticity of nociceptive sensitivity that through GPR40 signaling in the supraspinal area. Furthermore, brain GPR40 occurs following PTSD. This study was supported by the Department of the may be an important role in pain control system. Supported by grants-in-aid Army DMRDP W81XWH-11-2-0077. from grants-in-aid for Scientific Research (C) (24592364) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

M35 Optogenetic Dissection of the Locus Coeruleus in Negative Affective M36 Microglial activation is essential for opioid but not Behavior and Nociception psychostimulant reward in drug dependent states J.G. McCall (1,2,3,4), B. Sebastian (2), C.P. Ford (5), M.R. Bruchas (2,3,4) AMW. Taylor (1, 2), P. Vayssiere (1), C. Evans (1), CM. Cahill (2) (1) (1) Neurosciences Program (DBBS), (2) Dept. of Anesthesiology, (3) UCLA, Los Angeles, CA, USA; (2) Queens University, Kingston, ON, Anatomy & Neurobiology, (4) Pain Center, Washington University, St. Louis, Canada MO, USA, (5) Dept. of Physiology & Biophysics, Department of Neurosciences, Case Western Reserve University, Cleveland, OH, USA Chronic exposure to opioids and cocaine results in glial activation in the brain. In this study, we explored the role of activated microglia in opioid The locus coeruleus (LC) and its projections are the primary source of and cocaine reward in acute and chronically exposed mice. Mice were norepinephrine for the mammalian forebrain. The LC is a target for conditioned in a balanced, unbiased conditioned place preference (CPP) corticotropin-releasing factor (CRF), opioid neuropeptide containing neurons, apparatus to morphine (10mg/kg, i.p.) or cocaine (10mg/kg, i.p.) over 8 and is highly enriched with all four opioid receptor types. LC tonic firing days. The day following conditioning, mice were placed in the CPP boxes, increases during stress and this increase is thought to be controlled by opioid drug-free, and the time spent in the drug-paired chamber was measured. and CRF activity. Therefore, we asked if specific modulation of LC neuronal Before conditioning, mice were made either opioid-dependent by chronic firing could lead to anxiety-like, aversive, and antinociceptive behaviors. morphine treatment (10, 20, 30, 40mg/kg, i.p. Q12h) or cocaine dependent Genetically targeting channelrhodopsin-2 to only LC noradrenergic (LC/NA) (30mg/kg, i.p. Q12h) for 2 days. Mice not made drug dependent (naïve) neurons, we used optogenetic light stimulation to specifically increase LC/NA received saline injections over the same time course. To determine the neuronal activity. We report an increase in tyrosine hydroxylase and c-fos co- effect of glial inhibition on opioid or cocaine CPP, mice received daily immunoreactivity in the LC following light stimulation. We then examined injections of vehicle or the glial inhibitor minocycline (30 mg/kg, i.p. the effect of increasing tonic LC/NA firing on anxiety- and aversion-like Q12h). Injections were at least 3 hours before conditioning and in the home behaviors using a conditioned place aversion paradigm. We found that cage, so there was no association of minocycline injections with increasing tonic firing of LC/NA neurons in a context dependent manner, conditioning. Morphine and cocaine naïve and dependent mice exhibited consistent with CRF release in the LC, induces a subsequent aversion to the CPP. Minocycline had no effect on cocaine preference in either cocaine- stimulated context. Furthermore, increased LC/NA firing results in decreased naïve or dependent mice. Similarly, morphine preference was unaffected by open arm time in the elevated zero maze, an assay of anxiety-like behavior. In chronic minocycline treatment in opiate-naïve mice. However, minocycline addition to its role in negative affective behaviors, the LC also sends blocked morphine CPP in opioid-dependent mice. This effect was specific descending projections to the spinal cord to modulate nociception in response to the rewarding properties of morphine, as minocycline did not block to stress. We demonstrated that activation of LC neurons causes a decrease in morphine-induced locomotor sensitization. Both chronic cocaine and thermal nociception in the Hargreaves Test. These results are consistent with morphine, but not acute treatment, caused glial activation in reward areas opioid release in the LC. These data suggest that increasing the firing rate of of the brain that was prevented by minocycline. These results suggest there LC/NA neurons can modulate both affective and antinociceptive behaviors. is a shift in opioid reward processing in dependent states mediated by Supported by NIDA, DA025182 (MRB), DA026417 (CPF), and DBBS activation of microglia. Cocaine reward in dependent mice did not display (JGM). reliance on microglial activation, suggesting differences in opioid and psychostimulant reward processing. Funding CIHR, CRC, NIH Grant DA05010, The Shirley and Stephan Hatos Foundation.

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M37 Pharmacokinetics of 7-hydroxymitragynine, a µ-opioid receptor M38 Phenethyl orvinols: SAR at opioid and NOP receptors agonist, in male Sprague Dawley rats Gerta Cami-Kobeci1, Willma E. Polgar2, Taline V Khroyan2, Lawrence B.A. Avery (1), P.K. Vuppala(1), S. Jamalapuram(1), E.B. Furr (2), C.R. Toll2, Stephen M. Husbands1 McCurdy(2,3) Departments of (1)Pharmaceutics, (2)Medicinal Chemistry, and 1Department of Pharmacy and Pharmacology, University of Bath, Bath, (3) Pharmacology, University of Mississippi, MS 38677 BA2 7AY, UK and 2SRI International, Menlo Park, USA.

7-hydroxymitragynine (7-OHMG) is a minor constituent in the plant material of Mitragyna speciosa. The leaves of this herb have long been used in It has been shown that buprenorphine as an opiate agent, posses mu opioid Thailand for its opium- and coca-like effects, and as a replacement for opium. (MOP) receptor partial agonist activity, alongside kappa/delta (KOP/DOP) In 2005, Horie et al. studied the opioid agonistic effects of the constituents of receptor antagonist activity. However significant behavioural features of Mitragyna speciosa using in vitro assays. The results suggested that the opioid buprenorphine have been postulated to be mediated by activation of effect is mostly based on the activity of 7-OHMG. Having a novel structural nociceptin/ orphanin FQ peptide (NOP) receptors, despite moderate scaffold for opioid receptor affinity and activity, 7-OHMG promotes further affinity and low efficacy at these receptors. A ligand with a similar profile investigation of it as a novel lead compound for opioid studies. We but increased NOP receptor activity would be of significant interest as a investigated the pharmacokinetic characteristics and bioavailability of 7- drug abuse medication and analgesic. Previously we reported that OHMG in rats after a 4 mg/kg IV and 20 mg/kg oral dose. A validated BU08028, a close analogue of buprenorphine had the desired profile in bioanalytical method was used for the analysis of rat plasma samples. After an vitro. In order to improve the profile that we were looking for, we focused

IV dose of 7-OHMG, a mean maximum plasma concentration (Cmax) was on the phenethylorvinol derivative, which was amongst the most potent found to be 3.0 µg/mL. The plasma concentration of 7-OHMG decreased antinociceptive agents in the orvinol series. Using this strategy we rapidly, and was eliminated from plasma with a terminal half-life (T1/2) of designed and synthesised a number of compounds with differently approximately 22.9 min. The volume of distribution (Vd) and clearance (CL) substituted phenethyl groups at C20 and replacement of 17-Me with 17- were found to be 1595.8 mL/kg and 44.2 mL/min/kg, respectively. After oral CPM. In binding assays all compounds within this series have high affinity administration, the mean Cmax of 2.8 µg/mL was observed at 25.8 min. The 7- at each of the opioid receptors and good affinity (low nM) at NOP OHMG was distributed extensively in the body with a Vd of 3451.2 mL/kg. receptors. The ligands were antagonists or low efficacy partial agonist at 35 The CL and T1/2 were found to be 91.4 mL/min/kg and 7.1 min, respectively. each of the receptors in the [ S]GTPgammaS binding assay. The further The absolute oral bioavailability was found to be 52%. The project described evaluation of these ligands, including initial in vivo evaluation will be was supported by grant number P20RR021929 from the National Center for presented. This work was supported by NIDA grants DA020469 (SMH) Research Resources. The content is solely the responsibility of the authors and and DA023281 (LT). does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. This investigation was conducted in a facility constructed with support from research facilities improvement program C06 RR-14503-01 from the National Center for Research Resources.

M39 Identification of opioid peptides in the spinal cord of SNI male mice M40 Mixture Linkage Analysis: Functional activity validation using nano LC-MS/MS comparing SRU BIND technology and cAMP inhibition via mu opioid A. Carlsson (1), E. Brolin (1), H. Watanabe (1), H. Wadensten (2), P. Andrén receptors (2), F. Nyberg (1). Uppsala University, Department of Pharmaceutical Margaret E. Cazares, Jaime A. Misler, and Colette T. Dooley. Torrey Pines Biosciences (1) Division of Biological Research on Drug Dependence (2) Institute for Molecular Studies, Port St Lucie, Florida Division of Medical Mass Spectrometry, Uppsala, Sweden Positional scanning deconvolution is a very powerful method for the Chronic pain is known to affect 10-30% of the population and the identification of active compounds from mixture based combinatorial identification of pain biomarkers is of great importance in the search for new libraries. However, in certain cases the number of individual compounds treatment regimens as well as diagnostic tools. The aim of this study was to required for synthesis is prohibitive. Furthermore, while some active investigate the presence of opioid peptides in the spinal cord in an animal peptides are identified many other compounds synthesized are found to be model of chronic pain, Spared Nerve Injury (SNI). In addition, it was aimed to inactive. A new analysis for mixture libraries is described wherein the investigate the levels of opioid peptides in the spinal cord using nano LC- physical mixture of two mixtures (a dimixture) indicates whether the amino MS/MS, comparing these compounds in nerve-injured mice with those in acids defined are likely to be present on the same active peptide. This sham operated and control mice. NMRI mice were anesthetized with isofluran analysis is based on mixture behavior obeying the harmonic mean, whereby and surgery was performed on the left sciatic nerve. Animals were kept for 6 the most active component(s) of a mixture drives the greatest proportion of weeks after surgery and the appearance and persistence of cold and tactile the activity. After active ligand families are determined based on receptor allodynia was investigated using the acetone test and von Frey test, binding assays, their functionality, i.e., agonism/antagonism, needs to be respectively. After decapitation, lumbar and sacral parts of the spinal cord established. Mu opioid receptors are Gi-protein-coupled and, therefore, were collected and divided: dorsal and ventral divided into left and right. The inhibit cyclic adenosine monophosphate (cAMP) production within the prepared samples were analyzed by nano LC-MS/MS. Quantification of cell. Current technologies provide a system to induce cAMP synthesis opioid peptides was done with Selected Reaction Monitoring. Identification of within the cell and then monitor potential inhibition by the addition of peptides was performed through database search using X!Tandem as search ligands. Here, we present a new technology (BIND) that uses a specialized engine. Several opioid peptides were identified in variuos samples, among plate reader to project white light onto plates with optical biosensors and them were Met-enkephalin, a fragment of dynorphin A and nociceptin. measure differences in the reflected light in response to changes in cell Results indicated that the levels of most peptides analyzed were affected in mass. The different G-proteins have been previously characterized by the the SNI mice compared to control and sham animals. For instance, the company; therefore, we can use the Gi-protein profile to validate its detectable opioid peptide structures Met-enkephalin, Leu-enkephalin and activation at the mu opioid receptor. We utilized this system in tandem dynorphin A(10-17) as well as nociceptin displayed an increase in the right with cAMP using three cell lines: those that endogenously express mu dorsal horn of the SNI mice compared to controls. In the ventral spinal cord opioid receptors, SH-SY5Y, HEK293 that overexpress cloned mu opioid dynorphin A(10-17) was found to decrease in the SNI mice. The significance receptors, and HEK293 without the receptor expression as a counter screen of these observations and the relevance of the present approach will be to rule out false positive activity. discussed. This work was supported by the Berzelii Technology Centre for Neurodiagnostics

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M41 Synthesis and evaluation of aryl-naloxamide opiate analgesics M42 Pharmacological Characterization of Synthetic Mitragynine targeting truncated exon 11-associated MOR-1 splice variants Analogs Susruta Majumdar (1), Joan Subrath (1), Valerie Le Rouzic (1), Lisa Polikar Jessica V. Mankus (1), Lisa L. Wilson (2), Edward B. Furr (1), Marco (1), Maxim Burgman (1), Kuni Nagakura (1), Julie Ocampo (1), Nathan Arribas (1), Christophe Mesangeau (1), Stephen J. Cutler (1) and Haselton (1), Anna R. Pasternak (1), Steven G. Grinnell (1), Ying-Xian Pan Christopher R. McCurdy (1,2) Departments of (1)Medicinal Chemistry, and (1) and Gavril W. Pasternak (1) (1)Department of Neurology and Molecular (2)Department of Pharmacology, University of Mississippi, University, MS Pharmacology and Chemistry Program Memorial Sloan-Kettering Cancer 38677 Center, New York, NY 10065, USA. Mitragynine is the major alkaloid extracted from the leaves of the Thai 3-Iodobenzoylnaltrexamide (IBNtxA) is a potent analgesic lacking many side- traditional herb, Mitragyna speciosa Korthals (Rubiaceae). Extracts of this effects of traditional opiates by acting through a novel receptor target plant have been used as substitutes for opium. Yet, mitragynine does not composed, in part, of exon 11-associated truncated six transmembrane domain possess the structural features of classic opiate ligands. In an attempt to MOR-1 (6TM/E11) splice variants. To better understand structure-activity further explore this unique feature of the natural product, synthetic analogs aspects of this drug target, a number of 4,5-epoxymorphinan analogs were were made. Recent work by N. Farah Idayu et al. has shown that synthesized with substitutions at the 6, 17 and 3 positions. Results show the mitragynine has antidepressant properties in forced swim test and tail importance of 1) a free 3-phenolic group, 2) a phenyl ring at the 6 position, 3) suspension assays. However, to date no anxiolytic effects have been shown an iodine at the 3’or 4’ position of the phenyl ring and 4) an N-allyl or N-c- for mitragynine or any synthetic analogs. The primary objective of this propylmethyl group to maintain high 6TM/E11 affinity and activity. The N- work is to test the analogs for their anxiolytic and antinociceptive effects. methyl compounds showed markedly diminished affinity for the 6TM/E11 The antinociceptive effects of the analogs were tested intraperitoneal (i.p.) target. The N-allyl and c-propylmethyl groups show similar affinity towards at various doses utilizing tail-flick and hot plate assays. In addition to the 6TM/E11, but the allyl analog displayed lower delta opioid receptor testing the antinociceptive effects of the analogs, the anxiolytic effects were affinity. 3-Iodobenzoylnaloxamide (3) was 10-fold more potent an analgesic also tested i.p. at various doses using the elevated plus maze assay. The than morphine and elicited no respiratory depression or physical dependence antinociceptive effect of intraperitoneal (i.p.) injection of mitragynine (10- and only limited inhibition of gastrointestinal transit. Thus, the aryl- 30 mg/kg) was investigated as compared to morphine (5-10 mg/kg) using naloxamide scaffold can generate potent analgesics acting through the the tail-flick and hotplate tests. In both tests, mitragynine exerted a dose- 6TM/E11 sites with advantageous side-effect profile and greater selectivity. dependent antinociceptive activity that was maximal at 30 min but was less This work was supported, in part, by research grants from the National potent than morphine. Such effects were completely abolished by the Institute on Drug Abuse (DA06241, DA02165, and DA07242) to GWP, a opioid receptor antagonist naloxone (10 mg/kg, i.p.). In the end Core Grant from the National Cancer Institute to MSKCC (CA08748) as well characterization of the anxiolytic effects of mitragynine and synthetic as the Technology Development Fund of Memorial Sloan-Kettering Cancer analogs allows for greater opportunities to develop a potentially new class Center. of anti-anxiety agents. Funding provided by Grant Number 5P20RR021929 from the National Center for Research Resources. Content sole responsibility of authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

M43 Endomorphin analogs show reduced abuse potential relative to M44 Opioid affinity, selectivity and antinociceptive activity of thiazole- morphine containing cyclized enkephalin analogs M.R. Nilges (1), X. Zhang (2), J.E. Zadina (1,2,3,4). (1) Neuroscience SO Eans (1), ML Ganno (1), C Dooley (1), A Nefzi (1), JP McLaughlin (1) Program, (2) Dept of Medicine and (3) Pharmacology, Tulane University, and (1)Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA (4) SE LA Veterans Health Care System, New Orleans, LA 70112 We theorized cyclized enkephalin analogs would retain opioid receptor Morphine is the gold standard treatment for moderate to severe pain, but side selectivity and activity while increasing oral bioavailability and resistance effects including abuse liability limit its usefulness. Analgesic effects of to metabolism. Different thioalkylation approaches were utilized for the morphine occur via agonism of mu opioid receptors (MOR). The discovery of formation of the 1924-series cyclic peptide library from 10 enkephalin endogenous MOR agonists called endomorphins (EM-1 and EM-2) led to the analogs, while the 1936-series library was constructed from cyclized development of several metabolically stable EM analogs that are currently enkephalin of varying ring sizes using the new developed Hantzsch-based being studied in our lab. These EM analogs produce antinociceptive responses macrocyclization strategy. In radioligand competition binding assays, the equal to, or greater than morphine. Repeated pairing of an environment with compounds demonstrated modest affinity (submicromolar) but high drug administration produced a place preference for morphine, but not for equi- selectivity for the delta opioid receptor. Antinociceptive activity of each antinociceptive doses of EM analogs. A complementary test of abuse liability compound (1 nmol, i.c.v.) was assessed in vivo with the use of C57BL/6J was used in the present study; morphine and EM analogs were tested for mice in the 55oC warm-water tail-withdrawal test. Cyclic compounds intravenous self-administration under short- and long-term access sessions to 1924-10, 1924-21 and 1924-29 produced antinociception equivalent to or characterize pain, reward, and extinction patterns. Under short-term (40 greater than morphine alone, with ED50 (and 95% confidence interval) min/dose/day) sessions, rats dose-dependently self-administered morphine, but values of 0.39 (0.20-0.77), 1.26 (0.61-2.68), 0.45 (0.19-1.11) nmol i.c.v., not analogs. Vehicle substitution for morphine, but not analogs, produced respectively. Likewise, cyclic enkephalin analogs 1936-1, 1936-7 and extinction bursting, consistent with active drug seeking behavior. 1936-9 all proved significantly potent, with ED50 values of 0.32 (0.06- Antinociception after self-administration was similar for morphine and EM 1.69), 0.40 (0.20-0.88), 0.75 (0.34-1.69) nmol i.c.v., respectively. After analogs, suggesting a separation of analgesia from reward. Under long-term oral administration (10 mg/kg p.o.), all but 1936-7 produced time- (12 hours/day for 7 days) access sessions, rats lever-pressed for morphine to a dependent antinociception significantly greater than vehicle. The greater extent than for vehicle or equal doses of EM analogs. Within-group antinociception produced by each compound was antagonized by comparisons showed that rats pressed the active lever more than the inactive pretreatment with opioid receptor antagonist naloxone (6 µg i.c.v., -20 “non-drug” lever for morphine, but not EM analogs. When the ratio of presses min), and opioid receptor selectivity was confirmed in vivo by pretreating to infusions was raised to fixed ratio 3 and 5, requiring greater effort per mice with either β-FNA (5 mg/kg, i.p., -24 h), nor-BNI (10 mg/kg, i.p., -24 infusion, rats self-infusing morphine, but not analogs, significantly increased h) or naltrindole (20 mg/kg i.p., -20 min) prior to testing with each their rate of responding. Our data provide a thorough evaluation of EM analog compound (10 nmol, i.c.v.). Comparison of the cyclized enkephalin reward properties in 3 paradigms. We conclude that EM analogs show reduced analogs after central (10 nmol i.c.v.) administration to morphine-induced abuse potential relative to the current gold standard of morphine. Funding: VA, respiratory depression and hyperlocomotion will be discussed. DOD, and ONR. Funded by: NIDA (R03 DA025850 to AN and R01 DA031370), and the Florida Governor’s Office of Tourism, Trade, and Economic Development.

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M45 Evaluation of chronic retention of nor-BNI in mouse brain over M46 Insulin Degrading Enzyme Mediated Proteolysis of Extracellular weeks after administration using LC-MS/MS Neuropeptides: Dynorphins, CRF, and Galanin K. A. Patkar, M. R. Hoot, S. O. Eans, K. J. Reilley and J. P. McLaughlin. Brian Reed (1,2), Brian T. Chait (2), Mary Jeanne Kreek (1) (1) Lab. Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987. Biology Addictive Diseases, Rockefeller Univ., New York, NY, USA (2) Lab. Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller We utilized liquid chromatography-mass spectrometry (LC-MS/MS) to University, New York, NY, USA evaluate the presence of the kappa opioid receptor (KOR) selective antagonist nor-BNI in C57BL/6J mouse brain over time following a single administration, The termination of neuropeptide neurotransmission, in contrast to most determining if nor-BNI was detectable in brain during its prolonged duration of classical small molecule neurotransmitters, require extracellular breakdown activity in vivo. In the mouse 55 ºC warm-water tail-withdrawal assay, a single to terminate signaling. Proteases present in the extracellular fluid include administration of nor-BNI (10 mg/kg, i.p.) antagonized KOR agonist-induced “enkephalinase” (neprilysin), which cleaves Met- and Leu-enkephalin, as antinociception up to 14 days. Mice brains harvested at various times over 21 well as insulin degrading enzyme (IDE), which we have previously shown d post single administration (10 mg/kg, i.p.) were analyzed for the presence of to be responsible for the extracellular biotransformation of beta-endorphin. nor-BNI using LC-MS/MS; nor-BNI was detected in the brain samples up to IDE is also responsible for the breakdown of insulin as well as beta- 21 d. Subsequent administration of nor-BNI at doses corresponding to the amyloid peptides, and and is being actively pursued as a therapeutic target amounts found in the brain tissue (3-20 pmol/mg) of the pretreated mice for the treatment of Alzheimer’s Disease. We report here on investigations, significantly antagonized the antinociceptive effect of U50,488 in a dose- in vitro, of the breakdown of other neuropeptides known to be important to dependent manner. The dose (1.35 nmol, i.c.v.) corresponding to the lowest reward and/or stress responsivity pathways. We investigated the amount of nor-BNI detected in brain on day 21 did not antagonize U50,488- breakdown of CRF, ACTH, dynorphin A (1-17), dynorphin B, galanin, induced antinociception, consistent with the absence of U50,488 antagonism Met-Enk-Arg-Phe, PACAP-38, and Neuropeptide Y, as well as beta- observed in vivo at this time point after pre-treatment. We also evaluated the endorphin and insulin-B-chain(oxidized) as controls, both in the wash fluid distribution of nor-BNI in brain regions with low and high KOR expression of dissected brain tissue from rats, as well as using purified recombinant and in the brain homogenates from the nor-BNI pretreated KOR knockout IDE. The enzyme is capable of cleaving several of the peptides, including mice. No significant differences were observed in the levels of nor-BNI CRF, dynorphin A (1-17), dynorphin B, galanin, and PACAP-38, with no detected in brain regions of wild type mice (WT) regardless of KOR activity in cleaving ACTH, Neuropeptide Y, or Met-Enk-Arg-Phe. Similar expression over 21 d post treatment or in KOR-knockout brain homogenates profiles of cleavage were obtained in the wash fluid obtained from compared to that of WT 7 d post single administration. Although the physical dissected striatal tissue, although other enxymes were also present, as presence of nor-BNI in the mouse brain paralleled it’s in vivo pharmacological expected. IDE-like activity was also observed in the hypothalamus and profile, its presence in the low KOR expression brain region of WT and in the hippocampus, and is thus likely responsible for the extracellular KOR-knockout mouse brain suggests that the chronic retention may be a due degradation of neurpeptide substrates throughout the brain. Funded by to non-specific binding of nor-BNI to the brain tissue, and not exclusively due NIH-NIDA grant P60-DA05130-25 (M.J.K.) and NIH-NCRR grant to a non-equilibrium interaction with the KOR. (Supported by the State of RR00862 (B.T.C.). Florida.)

M47 Development and in vitro characterization of novel MOR agonists displaying bias against βarrestin2 recruitment N. C. Ross (1,2), J.M. Streicher (1), T. Bannister (2), L. M. Bohn (1) (1) Departments of (1) Molecular Therapeutics and Neuroscience and (2) Medicinal Chemistry, The Scripps Research Institute, Jupiter, FL, USA

Mu opioid receptor (MOR) agonists are the most widely used analgesics for the relief of severe and acute pain. However, their clinical use is limited due to their adverse side effects. Opioids produce most of their physiological effects by activating mu opioid receptors (MOR). As a G protein coupled receptor, the MOR has been shown to interact with and be regulated by the intracellular scaffolding and regulatory protein βarrestin2. Previously, we have shown that βarrestin2 knockout mice that have been treated with morphine display enhanced and prolonged antinociception with reduced antinociceptive tolerance, physical dependence, constipation and respiratory suppression. These studies suggest that MOR agonists that do not induce βarrestin2 interactions with the receptor may provide pain relief without the development of these side effects. The goal of our current work is to identify novel MOR ligands that stimulate MOR-mediated signaling, yet are biased against promoting βarrestin2 recruitment. We have developed a diverse library of compounds, which we have screened for activity at MOR receptors. Cell lines stably expressing MOR were used to screen compounds for their ability to induce G protein coupling and ERK phosphorylation. Recruitment of βarrestin2 was assessed using the DiscoveRx PathHunter assay as well as confocal microscopy. In addition to identifying ligand bias between G protein coupling and βarrestin2 recruitment, we have also identified bias between G protein coupling and ERK activiation. Here we present early data arising from these new scaffolds. The identification of MOR agonists which are functionally selective against βarrestin2 recruitment may allow for the development of more effective antinociceptive agents with reduced side effects, as well as pharmacological probes to further elucidate the role of βarrestin2 in MOR mediated physiological responses. (Supported by NIDA grant R01 DA03307301.)

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W1 Actin cytoskeleton disruption modulates mu opioid receptor W2 Opioid peptides induce long-term depression at glutamatergic internalization and desensitization in locus coeruleus neurons. synapses in the dorsal striatum S. Arttamangkul and J.T. Williams, Vollum Institute, Oregon Health & Brady K. Atwood and David M. Lovinger National Institute on Alcohol Science University, Portland, Oregon, USA Abuse and Alcoholism, Rockville, MD, USA

The actin cytoskeleton acts to support cellular structure, but also plays many The dorsal striatum is a brain region that is critically involved in action roles in GPCR signaling regulation. The actin network beneath plasma selection and habit formation and as such plays an important role in membrane has been shown to confine mu opioid receptors and have an effect addiction to drugs of abuse. Opioid peptides and their receptors are on lateral diffusion properties. The present study used transgenic-expressing abundantly expressed in this brain region. Many components of this Flag-mu opioid receptors (FMORs) from slices of locus coeruleus neurons to endogenous opioid system in the striatum are modulated by drugs of abuse, study [Met5]-enkephalin(ME)-induced FMOR internalization when actin suggesting that the opioid system is possibly a critical component of the polymerization was disrupted by latrunculin B (Lat-B). The amount of machinery involved in addiction. There is a paucity of data concerning internalized FMOR caused by ME (30 µM, 10 min) increased in the Lat-B opioid roles in long-term synaptic plasticity in the dorsal striatum. Data is treated cells as compared to control. ME (30 µM) still induced FMOR also lacking for what effects in vivo exposure to opiates will have on desensitization as measured by the decline in membrane hyperpolarization corticostriatal synaptic plasticity in this brain region. We utilized whole- during perfusion for 10 minutes. Recovery from desensitization was delayed. cell patch clamp electrophysiological recordings of medium spiny neurons The effect of Lat-B on lateral diffusion of FMOR on the membranes of in the dorsal striatum in a brain slice preparation to determine whether HEK293 cells was studied by line-scanning fluorescence correlation opioids could produce long-lasting changes in synaptic plasticity at spectroscopy. In Lat-B treated cells, FMOR diffused at a faster rate than corticostriatal synapses. We confirmed what has been shown previously by control. ME (1µM) slowed down the lateral mobility while morphine (1µM) others that bath application of either mu or delta opioid receptor agonists did not change the diffusion rate even in the present of Lat-B. These inhibit glutamatergic neurotransmission in the dorsal striatum. However preliminary results suggest that the actin cytoskeleton may play a role in the we also discovered that this inhibition is long-lasting and is able to be regulation of MOR. blocked, but not reversed by opioid receptor antagonists, suggesting that this is opioid-induced long-term depression (LTD). We also found that intra-striatal stimulation coupled with a brief application of peptidase inhibitors also produced LTD that could be blocked by opioid receptor antagonists. Determining the identity of the endogenous opioid(s) that mediate this LTD is an ongoing effort. We are also in the process of determining what effects both acute and chronic exposures to opiates in vivo have on opioid-LTD. These data demonstrate a novel form of long- lasting synaptic plasticity in the dorsal striatum that is induced by at least a brief in vitro exposure to opioid peptides.

W3 Inhibition of Cdk5 impairs morphine- and inflammation-induced W4 TNF-alpha-induced MOR up-regulation in human glioblastoma functional emergence of the delta opioid receptor in rats cells is coupled to a loss of MOR-activated signaling to intracellular Andrée-Ann Mercier-Blais, Hélène Beaudry and Louis Gendron, Université effectors de Sherbrooke, Canada A. Bedini, G. Vincelli, M. Baiula and S. Spampinato Dept. of Pharmacology, University of Bologna – Irnerio 48, 40126 Bologna – Italy Our group has previously shown that chronic morphine (escalating doses; 5, 8, 10, 15 mg/kg for 48h) and CFA-induced inflammation increase the membrane Glial cells act as neuronal support cells and immune cells in the nervous expression of DOPR as well as the analgesic efficacy of its agonists. Among system and actively participate to pain transmission. Opiates are among the other cellular mechanisms, receptor phosphorylation is believed to be strongest pain relievers, although they show poor efficacy in some chronic important for the regulation of DOPR trafficking. More specifically, pain states and antinociceptive tolerance can be developed. phosphorylation of Thr161 of DOPR by Cdk5 was recently shown to be Activation of spinal glia is involved in both opioid-resistant chronic pain and necessary for the formation of the MOPR/DOPR dimer, an event possibly opioid tolerance: once activated, glia release pro-inflammatory mediators, as involved in DOPR trafficking. The present study therefore aimed to determine TNF-alpha, that increase neuronal excitability, sustain glial activation and the role of Cdk5 in the functional emergence of DOPR. The role of this kinase contribute to impair opioid efficacy. Opioids themselves may induce glial was studied using Roscovitine as a selective inhibitor. We first evaluated the activation through their non-stereoselective interaction with Toll-like effect of intrathecal Roscovitine (1-30 µg) on DOPR-mediated receptor 4 (TLR4). In this study we investigated any effect elicited by TNF- antinociception in animals treated with morphine for 48h. In rats treated for alpha (0.1-10 ng/ml; 24-72 h) on MOR expression and intracellular signaling 48h with morphine, we found that Roscovitine (1 injection every 12h for 48h) in U-87 MG human glioblastoma, a cell line commonly adopted as a model dose-dependently reduced the antinoniceptive effect of deltorphine in the tail of glial cells. We found that morphine and DAMGO significantly inhibited flick test. In order to verify whether Cdk5 inhibition affects the regulation of adenylyl cyclase and induced ERK1/2 phosphorylation. Furthermore, this deltorphin II’s analgesic potency, increasing doses of this DOPR agonist were latter effect was still present in glioblastoma cells whose TLR4 expression then injected intrathecally to animals pretreated or not with 30 µg had been knocked-down by siRNA. Interestingly, MOR expression in U-87 Roscovitine. We observed that Roscovitine shifted deltorphin II’s ED50 from MG cells was significantly up-regulated upon prolonged exposure to TNF- 9.8 µg to 26.4 µg. DOPR-mediated analgesia was also significantly reduced in alpha (10ng/ml; 72h); however, under these conditions, neither morphine nor the CFA model of inflammation. Finally, the fact that DAMGO-mediated DAMGO could induce ERK1/2 phosphorylation or inhibit adenylyl cyclase. spinal analgesia was not modified by Roscovitine treatment suggests that the Our findings show that morphine and DAMGO trigger MOR-mediated, inhibition of DOPR functions are not due to impaired MOPR signalling. TLR4 independent, signaling pathways in untreated human glial cells; a Together, our results demonstrate that functional emergence of DOPR induced prolonged exposure to TNF-alpha, albeit significantly up-regulating MOR either by chronic morphine or inflammation involves Cdk5 activity. Further expression, leads to a loss of MOR-activated signaling to intracellular studies are required to understand how Cdk5 is activated by morphine and effectors, thus suggesting that prolonged pro-inflammatory stimuli may CFA-induced inflammation. Supported by CIHR, FRQS and NSERC determine adaptive changes in MOR sensitization. Such alterations occurring in glial cells may affect opioid efficacy and substantially contribute to both chronic pain maintenance and tolerance development. This research was funded by University of Bologna (RFO granted projects).

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W5 Agonist-Induced Imprinting of the mu Opioid Receptor Visualized by W6 Regional mRNA expression of GABA receptor subunits in brains of Fluorescent Opioid Ligands. C57BL/6J and 129P3/J mice: Strain and heroin effects. William T. Birdsong(1), Seksiri Arttamangkul(1), Mary J. Clark(2), Kenner M. Buonora, S.D. Schlussman, Y. Zhang, A. Ho and M.J. Kreek Rice(3), John Traynor (2), Mark von Zastrow(4), John T. Williams(1) The Laboratory on the Biology of Addictive Diseases, The Rockefeller (1)Vollum Institute, Oregon Health & Science University, Portland, OR, USA. University, New York, USA (2)University of Michigan, Ann Arbor, MI, USA; (3)National Institute on Drug Abuse, Bethesda, MD, USA; (4)University of California San Francisco, We have shown differences in heroin-induced conditioned place preference San Francisco, CA, USA in C57BL/6J (C57) and 129P3/J (129) mice as well as strain differences in the regional expression of several receptor and peptide mRNAs. We here Prolonged exposure to high-efficacy agonists results in desensitization of the extend our studies by examining the effects of heroin on levels of mRNA of mu opioid receptor. Desensitized receptors are thought to be unable to couple GABA receptor subunits in the brains of these same mice. GABA is the to G-proteins, preventing downstream signaling. The effect of desensitization main inhibitory neurotransmitter in the CNS, binding to ionotropic GABAA on ligand-receptor interactions is not well characterized. In the current study, receptors and G-protein coupled GABAB receptors. Using real time PCR, confocal imaging is used to determine whether desensitizing conditions cause dose and strain-related differences were observed in GABA receptors’ a change in agonist-receptor interactions. Using rapid solution exchange, subunit mRNA levels in the nucleus accumbens (NAc), caudate putamen binding kinetics of fluorescently labeled opioid agonist – dermorphin (CPu), and the SN/VTA. Compared to C57 animals, 129 mice had Alexa594 (derm A594) – to mu opioid receptors were found to be altered by significantly higher levels of GABAA1 mRNA and GABAB2 mRNA in the conditions which are known to cause receptor desensitization. In contrast, NAc. Levels of GABAB2 mRNA were also higher in the CPu of 129 binding of a fluorescent antagonist, naltrexamine Alexa 594, was unaffected compared to C57 mice. In contrast, levels of GABAA1 mRNA were lower in by similar agonist pre-treatment. Desensitizing conditions increased the the CPu of 129 compared to C57 mice. In the NAc of the 129 mice only, affinity of derm A594 for the receptor in a long-lived manner following dose-dependent differences in mRNA expression were observed. 129 mice agonist treatment that was only partially reversed by extended washing. Thus, receiving the highest dose (20mg/kg) of heroin had higher levels of opioid receptors were imprinted with a memory of prior agonist exposure that GABAA1 mRNA compared to mice receiving saline or the lowest doses affected how the receptor subsequently interacted with a fluorescent agonist (1.25mg/kg, 2.5mg/kg). In addition, 129 mice receiving the lowest doses of but not a fluorescent antagonist. Supported by DA08163, DA026617 (JTW) heroin had lower levels of GABAB2 mRNA compared to mice receiving and DA007262-18 (WTB) saline. Strain and dose differences were not observed in levels of GABAA2 mRNA in any region examined. In the SN/VTA, dose effects were observed in mRNA expression of GABAA1 within both strains of mice. Mice receiving 20 mg/kg of heroin had higher levels of GABAA1 mRNA in the SN/VTA compared to mice receiving saline or the two lowest doses (1.25mg/kg, 2.5mg/kg). Similarly, mice receiving 10mg/kg of heroin had higher levels of GABAA1 compared to mice receiving saline or 1.25mg/kg, with no strain difference. Thus, the GABAergic system may underlie behavioral differences in these strains of mice. Support: NIH-NIDA (DA05130), the Arcadia Charitable Trust and The Carson Family Charitable Trust to MJK.

W7 Development of a real-time, fluorescence based assay of mu-opioid W8 Differential expression and HIV-1 regulation of mu-opioid receptor mediated inhibition of adenylyl cyclase activity in Chinese receptor splice variants across human central nervous system cell types hamster ovary cells S.M. Dever (1), R. Xu (2), S. Fitting (1), P.E. Knapp (1,2,3), K.F. Hauser Alisa Knapman, Mark Connor Australian School of Advanced Medicine, 2 (1,3) (1)Dept. of Pharmacol. and Toxicol., (2)Dept. of Anat. and Technology Place, Macquarie University, NSW, Australia, 2109 Neurobiol., (3)Inst. for Drug and Alcohol Studies, Virginia Commonwealth Univ. Sch. of Medicine, Richmond, Virginia, USA Activation of the mu-opioid receptor (MOR) leads to inhibition of adenylyl cyclase (AC) and reduction of cAMP levels. Modulation of AC activity is Evidence suggests that opiate drugs, through actions at the mu-opioid frequently used as an assay for measuring opioid ligand potency and efficacy. receptor (MOR), can exacerbate the central nervous system (CNS) Most currently used AC assays are single time point, requiring multiple consequences of HIV-1 infection. MOR is known to undergo extensive reagents and/or cell lysis and lengthy development times. We have developed alternative splicing; however, despite this knowledge, the functional a real-time, fluorescence based assay of MOR-mediated AC inhibition in significance of MOR variants, as well as how splice variants other than intact Chinese hamster ovary (CHO) cells. CHOK1 cells stably expressing MOR-1 might differentially regulate HIV-1 pathogenesis in the CNS, or human MOR were grown in 96-well microplates and membrane potential was elsewhere, is uncertain. We found that although MOR-1A mRNA was measured using the Molecular Devices FLIPR membrane potential dye in a detected in astroglia, microglia and neurons, MOR-1 and MOR-1X were Flexstation 3. Dye emission intensity increases on membrane depolarization, only found in astroglia. The expression of the various forms of MOR along and decreases upon hyperpolarization. Application of forskolin hyperpolarized with the chimeric G protein qi5 in HEK-293T cells resulted in differences

CHO-MOR cells with a pEC50 of 7.3 ± 0.05 and a maximum reduction in in calcium-driven NFAT signaling with morphine treatment, and fluorescence of 53 ± 1 %. The hyperpolarization induced by forskolin differences were detected in other signaling cascades such as MAPK/ERK (300nM) was inhibited in a dose-dependent manner by DAMGO (Emax 57% using luciferase-driven response elements. These results suggest that unique

± 3%, pEC50 7.8 ± 0.1 and morphine (Emax 58% ± 8.7%, pEC50 7.1 ± 0.3). patterns of MOR variant expression underlie functional differences in The effects of opioids were prevented by treatment with pertussis toxin MOR-effector coupling and signaling among neural cell types. Moreover, (200ng/mL overnight), and blocked by naloxone. The forskolin the results unexpectedly reveal that MOR-1 may not be the predominant hyperpolarization was inhibited by H89, a PKA inhibitor, and mimicked by MOR variant expressed by some CNS cell types; rather, splice variants 100uM SP-8-CPT-cAMPs, a direct PKA activator. The forskolin besides MOR-1, with possible differing functions, may contribute to the hyperpolarization was not blocked by K+ channel inhibitors TEA (10mM), 4- diversity of MOR functions in the CNS. To explore the relationship of AP (300uM), glibenclamide (10uM), VU-591 (100uM) and charybdotoxin MOR-1 and other MOR variants to neuroAIDS further, MOR variant (100nM). However increasing extracellular K+ from 2.5mM to 30mM and transcript levels were measured in the brains of HIV infected subjects. We 75mM reduced the forskolin-induced (1uM) change in fluorescence from 42% found a significant association between MOR-1 expression levels with to 17% and 2%, respectively, suggesting the involvement of a K+ channel. neurocognitive impairment and encephalitis. Collectively, our findings This assay is a novel method for rapid, no-wash, real-time measurement of AC suggest that there are unique differences in the profile of MOR variant inhibition by MOR ligands in intact CHO cells, which may be suitable for expression among CNS cell types, and that the expression levels of MOR HTS. Supported by the NH&MRC of Australia. variants are differentially regulated by HIV-1. Support: NIH DA018633, DA028741, DA007027, and the National NeuroAIDS Tissue Consortium.

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W9 Characterization of non-classical opioid activity at Toll-Like W10 The role of NMDA receptors in the mechanisms underlying Receptor 4 morphine-induced alterations in synaptic plasticity. S. Dodson, R. Castoro, S. Das, R. Davis, and C. Stevens. Department of A.K. Fakira, G.S. Portugal, B. Carusillo, Z. Melyan* and J.A. Morón Pharmacology and Physiology, Oklahoma State University-Center for Health Concepción* Dept. of Anesthesiology, College of Physicians and Sciences, Tulsa, OK, USA Surgeons, Columbia University Medical Center, New York, United States *Authors contributed equally to this work. Classical opioids signal through a stereoselective G-protein coupled receptor. The research presented here illustrates novel, opioid interaction at Toll-Like Previous studies in our laboratory demonstrated that escalating doses of Receptor 4 (TLR4). TLR4 is a receptor that upon stimulation by morphine paired with a novel context produced robust locomotor lipopolysaccharide (LPS) signals to NF kappa B to initiate transcription of sensitization and impaired long-term potentiation (LTP) in the pro-inflammatory cytokines. We have shown that in the HEK Blue-4 cell line, hippocampus. Whereas behavioral sensitization was attenuated by mutating a Human Embryonic Kidney 293 cell line stably transfected to overexpress Ca2+-permeable AMPA receptors in the hippocampus, LTP remained TLR4 and the necessary accessory proteins to TLR4, MD-2 and CD14, opioid impaired. Ca2+-permeable NMDA receptors (NMDAR), composed of agonists such as morphine and fentanyl inhibit LPS signaling of TLR4 through NR2A, NR2B and NR1 subunits, are negatively regulated by Ca2+-activated the NF kappa B pathway. Opioid antagonists such as naltrexone and beta- small-conductance potassium channels (SK channels) and modulation of funaltrexamine (beta-FNA) also inhibit LPS induced TLR4 signaling. A this feedback loop can affect LTP. In this study we examined the role of general order of potency comparing these drugs was found to be: beta-FNA > NMDAR and SK2 channels in the mechanisms underlying morphine- fentanyl > morphine > naltrexone. The next set of studies was decided induced alterations in synaptic plasticity. Mice displaying behavioral primarily on the fact that astrocytes are the predominant cell type found in the sensitization to escalating doses of morphine (5, 8, 10 and 15 mg/kg) after brain. However, most immunological studies focus on peripheral immunity receiving a morphine challenge (5mg/kg; Mor + Mor) one week later and growing evidence exists that the central nervous system (CNS) actively demonstrated increased NR2A subunit trafficking to the hippocampal participates in the immune response too. Therefore, we sought to investigate postsynaptic density. Whole cell patch-clamp recordings from CA1 the effects of opioids on Normal Human Astrocytes (NHA). It was found that pyramidal cells showed that sensitivity of EPSCs to NR2A subunit blocker beta-FNA inhibited tumor necrosis factor-alpha (TNF-alpha) interleukin-1- NVP was increased suggesting an increase in NR2A containing NMDAR. beta (IL-1beta) induced CXCL10 protein expression in NHA cells. It was Moreover, sensitivity to NR2B blocker Ro 25-6981 was decreased in subsequently found that beta-FNA also inhibits TNF-alpha induced increases morphine treated animals suggesting morphine-induced changes in in CXCL10 mRNA. Taken together, the data presented here indicates that NMDAR composition. In addition to alterations in NMDARs, expression opioid agonists and antagonists both inhibit TLR4 signaling in both the HEK of SK2 channels was increased in the hippocampus of Mor + Mor mice. Blue-4 and the NHA cell lines. This research helps to explain part of the Furthermore, bath application of SK channel blocker apamin produced a immunomodulatory actions in the CNS that have been attributed to opioid use higher increase of NMDAR-EPSCs in Mor + Mor group compared to and/or abuse. We are grateful to Kelly McCracken and Daniel Buck for their saline-treated mice (Sal + Mor), indicating enhanced activation of SK- technical assistance and the support given in part by NIH grants NS 062664 channel–NMDAR feedback loop in morphine treated animals. Finally, we (RLD) and an Oklahoma Center for the Advancement of Science and found that apamin application can restore the ability of CA1 cells to Technology (OCAST) contract HR10-031 (CWS). produce LTP. Our data suggest that morphine-induced changes in NMDAR composition can affect SK channel–NMDAR loop leading to down-regulation of LTP. Therefore, we provide an evidence for a new key player to the mechanism of impaired synaptic plasticity after morphine-induced sensitization. This work was supported by NIH grant R01 DA025036 to JMC.

W11 G protein-coupled Receptor Heteromerization: A Role in Allosteric W12 Generation and Characterization of G Protein-coupled Receptor Modulation of Opioid Receptor Activity in Health and Disease Heteromers Ivone Gomes (1), Achla Gupta (1) and Lakshmi A. Devi (1,2) (1) Departments Achla Gupta (1), Ivone Gomes (1), and Lakshmi A. Devi (1,2) of Pharmacology & Systems Therapeutics and (2) Neuroscience, Mount Sinai (1)Departments of Pharmacology & Systems Therapeutics and School of Medicine, New York, NY, USA, 10029 (2)Neuroscience, Mount Sinai School of Medicine, New York, NY, USA, 10029 Recent advances in G protein-coupled receptor (GPCR) pharmacology have demonstrated that different ligands binding to the same receptor can activate G protein-coupled receptors (GPCRs) are important molecular targets in discrete sets of downstream effectors, a phenomenon known as “ligand- drug discovery. These receptors play a pivotal role in physiological directed signal specificity”, which is currently being explored for drug signaling pathways and are targeted by nearly 50% of currently available development due to its potential therapeutic advantage. Emerging studies drugs. Mounting evidence suggests that GPCRs, including opioid suggest that GPCR responses can also be modulated by interactions with other receptors, form dimers and various studies have shown that dimerization is GPCRs. Association between GPCR types leads to the formation of necessary for receptor maturation, signaling and trafficking. However, the complexes, or GPCR heteromers, with distinct and unique signaling physiological implications of dimerization in vivo have not been well properties. Some of these heteromers activate discrete sets of signaling explored since detection of GPCR dimers in endogenous systems has been molecules upon activation by the same ligand, a phenomenon termed a challenging task. One exciting new approach to this challenge is the “heteromer-directed signaling specificity”. This has been shown to be generation of antibodies against specific GPCR dimers. We have recently involved in the physiological role of receptors, and in some cases, in disease- generated antibodies to several GPCR heteromer pairs and used them as specific dysregulation of a receptor effect. We used opioid receptors as a tools for characterization of heteromer-specific function, as reagents for model, and demonstrated allosteric modulation of their activity by their purification, tissue localization and regulation in vivo and as probes heteromerization; this serves as a novel concept for the design of new ligands for mapping their functional domains. Currently we are exploring the and/or ligand combinations that target heteromeric receptor entities and possibility of developing such antibodies to block heteromer activities in differentially influence their activity. This provides a unique strategy to select vivo. These heteromer-specific antibodies represent novel tools for the receptor-specific responses and is likely to be useful for achieving specific exploration and manipulation of GPCR dimer pharmacology. Supported by beneficial therapeutic effects. Supported by NIH grants, DA 08863 and NIH grants, DA 08863 and DA019521 (to L.A.D.). DA019521 (to L.A.D.).

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W13 Endomorphin-2 is an arrestin-biased agonist because it induces W14 Desensitization of MOPr: homologous or heterologous? greater MOPr phosphorylation than predicted from its efficacy for G J. Llorente (1), J. Lowe (2), E. Tsisanova (1), E. Kelly (1), G. Henderson protein coupling (1), C.P. Bailey (2), (1) School of Physiology and Pharmacology, G. Rivero (1), J. Llorente (1), J. McPherson (1), A.E. Cooke (1), S.J. Mundell University of Bristol, UK, (2) University of Bath, UK (1), C.A. McArdle (1), E.M. Rosethorne (2), S.J. Charlton (2), C. Krasel (3), C.P. Bailey (4), G. Henderson (1), E. Kelly (1) (1) School of Physiology and There is controversy over whether MOPr desensitization is homologous or Pharmacology, Univ of Bristol, UK, (2) Novartis, Horsham UK (3) Univ of heterologous and over the mechanisms underlying such desensitization Marburg, Germany, (4) Dept of Pharmacy and Pharmacology, Univ of Bath, (Raveh et al., 2010, Cell 143:750). In different cell types desensitization UK has been reported to involve MOPr phosphorylation by various kinases as well as perturbation of the MOPr effector pathway by GRK sequestration Our previous results (McPherson et al., 2010, Mol Pharm 78:756) suggested of G protein betagamma subunits or ion channel modulation. In this study that the endomorphins show bias towards arrestin recruitment. Here we have brainstem slices were prepared from young (<20 day old) and relatively investigated the signalling of endomorphin-2 in more detail. We firstly mature (>150g) rats and whole cell voltage clamp recordings of K+ currents analysed our previous data according to the method described by Rajagopal et were made from LC neurones. In slices from mature rats rapid MOPr al (2011, Mol Pharm 80:367) in order to quantify ligand bias; this indicated desensitization induced by DAMGO was largely homologous in that during that endomorphin-2 is significantly biased towards arrestin (bias factor - a 15 min exposure to 10 uM DAMGO the evoked current decreased by 57 0.81±0.18; p<0.05), whilst morphine was not biased (bias factor -0.02±0.05). ± 4 % whereas the amplitude of the response to a subsequent application We next determined the efficacy of endomorphin-2 and other agonists for of a submaximal concentration of 5 uM noradrenaline was decreased by 375 Ser phosphorylation of MOPr, by quantification of HEK293 cell imaging only 14 ± 6 % following DAMGO exposure. Given that MOPrs and alpha2- using an antiphosphoSer375 antibody in an INCell Analyser. This indicated that adrenoceptors couple through G proteins to the same set of K+ channels it the efficacy of endomorphin-2-induced phosphorylation was as great as that of is unlikely that in mature neurones MOPr desensitization involves G DAMGO, and much higher than that of morphine. Analysis of MOPr protein subunit betagamma sequestration or ion channel modulation. In internalization data also indicated that endomorphin-2 has greater efficacy than contrast, in slices from immature animals, MOPr desensitization was morphine. Finally, the ability of endomorphin-2 to induce desensitization of observed to be heterologous in that the 10 uM DAMGO evoked current inwardly rectifying K+ channels in neurones of the rat LC was examined. The decreased by 60± 4 % over 15 min and the response to a subsequent rate of desensitization induced by endomorphin-2 was faster than that of application of 5 uM noradrenaline was decreased by 58 ± 6 %. Western DAMGO and other agonists examined; the t0.5 of desensitization for blotting on extracts of rat brain demonstrated that GRK2 expression was endomorphin-2 was 1.6 min [1.5-1.6] and that for DAMGO 3.0 min [2.8-3.2] higher in immature than mature rat brain in a number of regions examined (CI in square brackets). These data indicate that endomorphin-2 is an arrestin including cortex, striatum, hippocampus and LC. These observations are biased ligand which has distinctive properties at MOPr in brain. The arrestin compatible with the hypothesis that in LC neurones from immature animals bias may be to the ability of endomorphin-2 to induce greater MOPr heterologous desensitization could result from GRK2 sequestration of free phosphorylation than expected from its efficacy to induce coupling to G G protein betagamma subunits but that in mature animals, with lower protein. Supported by grants from the MRC UK (G0600943) and NIDA GRK2 levels, desensitization is largely homologous. (DA020836), and a Fellowship from the Basque Govt to G.R. Supported by project grants from the MRC UK (G0600943) and NIDA (DA020836).

W15 Reversal of tolerance to morphine by ethanol: role of PKC inhibition. W16 Trafficking of MOPr following chronic morphine treatment S. Withey (1), J. Llorente (1), C.P. Bailey (2), E. Kelly (1), G. Henderson (1) A.E. Cooke (1), S.J. Mundell (1), G. Henderson (1), E. Kelly (1) (1) (1) School of Physiology and Pharmacology, University of Bristol, UK, (2) School of Physiology and Pharmacology, University of Bristol, Bristol UK Dept of Pharmacy and Pharmacology, University of Bath, UK Sustained agonist activation of MOPr rapidly initiates regulatory events Ethanol is detected in approximately 50% of heroin related overdose deaths. including desentization and internalization, that are thought to contribute to Here we examined whether ethanol can reduce tolerance to opioids, thus the development of tolerance. Here we studied the molecular mechanisms increasing the risk of overdose. PKC has been implicated in MOPr that mediate the trafficking of the MOPr following chronic (72h) desensitisation and cellular tolerance to morphine (Bailey et al., 2009, Eur J morphine treatment. Using ELISA to quantify internalization in HEK 293 Neurosci 29:307). To investigate the interaction between ethanol and cellular cells stably expressing HA-tagged MOPr, the extent of DAMGO-induced tolerance at the level of MOPr we measured MOPr function in rat brain internalization was significantly reduced in cells that had been exposed to neurones and examined whether ethanol inhibited PKC activity. Whole cell 1 uM morphine for 72h in comparison to control cells (14.6 ± 1.7% voltage clamp recordings of K+ currents were made from individual LC internalization versus 25.2 ± 2.5% internalization, respectively; P<0.05). neurones in rat brain slices, which were prepared from rats pretreated with This effect was due to chronic morphine as following exposure to 1 uM morphine for 3 days prior to death and maintained in morphine (1 uM). When morphine for only 24h the observed decrease in DAMGO-induced LC neurones were exposed to a low concentration of 20 mM ethanol for 10 internalization was absent. Furthermore, in cells that have been exposed to min prior to and during the morphine challenge, cellular tolerance was reversed morphine chronically, the MOPr does not appear to recycle normally, only (maximum morphine response as a % of maximum response to noradrenaline 0.8 ± 10.4 % of the DAMGO-internalized receptor had recycled back after was: naïve, 103 ± 7; naïve + 20 mM ethanol, 110. ± 7; morphine treated, 68 ± 30 min in the presence of naltrexone (1 uM), whereas in control cells, 49.4 2.5*; morphine treated + 20 mM ethanol, 110 ± 8.5)(*p<0.01 compared to ± 8.4 % of the receptor was found to recycle back to the plasma membrane naïve). As PKCalpha has been implicated in morphine tolerance in LC at 30 min (P< 0.01). Chronic morphine treatment did not alter the neurones we investigated the effects of ethanol on PKCalpha activity. A expression of arrestins, dynamin or GRK2 in the cells, but there was a PepTag assay kit was used to measure PKC activity in vitro. 100 mM ethanol substantial decrease (40.1±7.2%) in MOPr. Immunofluorescence produced a 16.8 ± 2.4 % and 21.0 ± 2.6 % decrease in PKCalpha activity in microscopy indicated that chronic morphine treatment induced lysosomal vesicles containing 4% and 8% Diacylglycerol (DAG) respectively (p<0.001). colocalization of MOPr with LysoTracker Red. Thus after chronic However, 20 mM ethanol did not reduce PKCalpha activity. These data morphine exposure, DAMGO induces significantly less internalization of demonstrate that ethanol reverses opioid cellular tolerance. Although MOPr, whilst the receptor also appears to be unable to recycle to the cell PKCalpha is involved in cellular morphine tolerance we cannot conclude that surface. Supported by a BPS AJ Clark Studentship to A.E.C. ethanol reverses tolerance by inhibiting PKCalpha as ethanol only inhibited kinase activity at a high concentration. The interaction between ethanol and PKCalpha appears to be dependent on the concentration of DAG present. Supported by project grants from the MRC UK (G0600943) and NIDA (DA020836), and a BBSRC Studentship to S.W.

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W17 Characterization of a naturally occurring variant of MOPr, L83I W18 Pathologic changes in murine and human neurons induced by A.E. Cooke (1), S.J. Mundell (1), G. Henderson (1), E. Kelly (1) (1) School of HIV-1SF162: interactions with morphine. Physiology and Pharmacology, University of Bristol, Bristol UK Masvekar RR (1), Zou S (1), Hauser KF (2), and Knapp PE (1,2). (1) Depts. Anatomy & Neurobiology, (2) Pharmacology & Toxicology, A recently identified naturally occurring variant (L83I) has been observed to Virginia Commonwealth Univ., Richmond, VA undergo significant internalization in response to morphine (Ravindranathan et al., 2009, PNAS USA. 106:10811). In the current study we examined the Microglia are principal targets of HIV infection in the CNS. HIV trafficking and signalling of WT-MOPr in comparison with that of the L83I infected/activated glial cells release viral and cellular toxins that drive variant expressed in HEK293 cells. Using ELISA, DAMGO induced secondary toxicity in neurons and other cells. Using murine models, we internalization of both the WT-MOPr and the L83I variant (33.9 ± 3.0% and have shown that many lethal and sublethal effects of HIV-1 Tat on neurons 36.6 ± 4.3% respectively). In contrast, morphine induced significant are enhanced by co-exposure to morphine, an opiate that preferentially acts internalization of the L83I variant but had little effect on the WT-MOPr (27.2 through mu-opioid receptors. This mimics the co-morbid effects of opiates ± 4.1% and 1.6 ± 7.8% respectively). Inhibition of dynamin function with and HIV observed in human patients. Many studies related to effects of dynasore (40 uM) inhibited the DAMGO-induced internalization of both the HIV ± opiates have focused on neuron death mediated by particular HIV WT-MOPr and the L83I variant. Furthermore, dynasore also inhibited the proteins (e.g. Tat, gp120). To more closely model the disease process, we morphine-induced internalization of the L83I variant. Expression of DNM tested whether opiate exposure potentiated neurotoxic effects of supernatant GRK2 attenuated the internalization of the L83I variant in response to both from HIV-infected monocytes. HIVSF162, an R5 tropic strain, was DAMGO and morphine, in addition to inhibiting the internalization of the WT- propagated in U937 human leukemic monocyte lymphoma cells. MOPr in response to DAMGO. Following immunoprecipitation of MOPr, Extracellular p24 levels were measured by ELISA. Supernatant was added DAMGO induced substantial phosphorylation of Ser375 in both the WT-MOPr to primary murine striatal neurons plated 6 d previously, in the presence or and the L83I variant, as assessed with an antiphosphoserine375 antibody; absence of 500 nM morphine ± 1.5 mM naloxone. Lethal and sublethal morphine induced far less phosphorylation of this residue, but morphine- effects on neurons were assessed in populations, and by computer-assisted induced phosphorylation was the same for WT-MOPr and L83I. Unlike with digital imaging of individual cells for at least 48 h. At specified intervals, DAMGO, we were unable to detect co-immunoprecipitation of arrestins with cells were fixed and immunostained for MAP-2 (neurite marker) and the morphine-activated WT-MOPr or the L83I variant. Investigations of cAMP TUNEL (apoptotic marker). Neurotoxicity was assessed by percent + + signalling revealed no significant change in the EC50 of either DAMGO or TUNEL /MAP-2 neurons, loss of neuritic arborization, and MTT analysis. morphine to inhibit cAMP formation when compared to values obtained for Supernatant from HIV infected monocytes caused dose dependent toxicity the WT-MOPr. These results show that unlike the WT-MOPr, the L83I variant over a range of p24 levels (< 500 pg/ml), affecting survival, primary rapidly internalizes in response to morphine in a GRK- and dynamin- process length, and complexity of neuritic fields. Significant interactions dependent manner. The enhanced internalization of L83I in response to with morphine were noted, especially at lower HIV levels. Importantly, morphine is not due to increased phosphorylation of Serine375, increased ability treatment with HIV alone caused reversible arrest of neurite outgrowth; in of MOPr to interact with arrestins, or an increased ability to signal via G- neurons co-exposed to HIV and morphine this reversibility was proteins. Supported by a BPS AJ Clark Studentship to AEC significantly reduced. Studies in progress show similar HIV and morphine effects in human primary neurons and those differentiated from human cell lines. Support: DA019398; DA024461.

W19 Morphine desensitization and long-term tolerance are distinguished W20 Anticancer properties of peptide opioid agonist – tachykinin by PKC and phosphatase activity antagonist chimeras ES Levitt and JT Williams, Vollum Institute, Oregon Health & Science J Matalinska (1), M Bochynska-Czyz(1), H. Skurzak (2), S Markowicz(2), University, Portland, OR AW Lipkowski(1) (1)Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, (2) Maria Mu opioid receptor desensitization is considered an initial step in the Sklodowska-Curie Oncological Research Centre, Warsaw, Poland development of tolerance. Morphine produces robust tolerance, but minimal acute desensitization. Rather, morphine desensitization develops over several Malignant cells overexpress various GPCRs as well as release of hours. Given this prolonged time-course, this study was designed to test the neuropeptides. Both of these phenomena are important factors of hypothesis that desensitization is indeed present in rats that have been treated cancerogenesis that result with an increase of cell proliferation and with morphine continuously for several days and can be distinguished from migration. Tachykinin receptors are one example of such phenomena. cellular tolerance by time-course of recovery and mechanism. Morphine- Recently it has been successfully shown that peptidomimetic tachykinin mediated activation of G protein-coupled inwardly-rectifying potassium antagonists (aprepitant) may reduce various cancer cell proliferation. Our conductance was measured using voltage-clamp recordings from locus group has been involved in development of new medicines for treatment of coeruleus neurons in brain slices from naïve or morphine-treated rats. Cellular cancer pain. Opioid agonist-tachykinin antagonist chimeras were one of the tolerance was observed as a decrease in morphine efficacy in slices from most interesting targets. Under this direction several new peptidomimetic morphine-treated rats. This tolerance was long-lasting and persisted for up to 6 compounds have been synthesized that expressed high analgesic potency. hours after morphine was washed from the slice. An additional reduction in These compounds were further studied in vitro for their influence on morphine-mediated current was observed when slices from morphine-treated proliferation of various human cancer cell lines. The compounds expressed rats were continuously maintained in morphine. This reduction represents antiproliferative activity in the range comparable to aprepitant. In addition, desensitization that developed in the animal and recovered within an hour after compounds showed synergic effects with colchicines, reference anticancer morphine was washed from the slice. The addition of ser/thr phosphatase compound interacting with tubulins. In conclusion we showed that opioid inhibitors calyculin A or okadaic acid in the morphine-free wash revealed that agonist-tachykinin antagonist chimeras could be applied as new analgesics recovery from desensitization, but not long-lasting tolerance, was facilitated by for treatment cancer pain. The cancer cells antiproliferative properties of protein phosphatase 1 (PP1) activity. Furthermore, desensitization, but not these compounds are positive adjuvant effects in chemotherapeutic cancer tolerance, could be reversed by protein kinase C (PKC) inhibitors, but not an treatment. This work has been partially supported by EU Normolife – 6FP inhibitor of c-Jun N-terminal kinase. Therefore, morphine treatment leads to Project-037733 both long-lasting cellular tolerance and short-term desensitization, which are differentially dependent on PP1 and PKC activity and combine to result in a substantial decrease in morphine effectiveness. Supported by DA08163 (JTW) and DA33036 (ESL).

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W21 VTA interneurons are not the major opioid-sensitive GABA input to W22 Mechanism of opioid modulation of thrombospondin levels in dopamine neurons astrocytes Aya Matsui and John T. Williams Vollum Institute, Oregon Health and E. Phamduong, N.R. Crews, A.L. Leinweber, P. Kappera, M. Rathore, C. J. Science University, Portland, OR, USA Coscia E.A. Doisy Dept. of Biochem. and Mol. Biol., St. Louis Univ. School of Med, St. Louis, MO, 63104 USA Opioids increase dopamine release in the brain through inhibition of GABA input to dopamine neurons in the midbrain. The mechanism of disinhibition Astrocytes are dynamic partners with neurons in synaptogenesis as they was originally believed to result from inhibition of VTA interneurons. release critical gliotransmitters to regulate synaptic function and synapse Recently, the rostromedial tegmental nucleus (RMTg), also known as the tail maintenance. Gliotransmitters such as thrombospondins (TSPs) 1 and 2 are of the ventral tegmental area (tVTA), has been shown to send a dense opioid- secreted from astrocytes in developing and injured brain. TSP1 and 2 have sensitive GABA input to dopamine neurons. The strong inhibition of GABA individual functions in brain and display a marked divergence in coding innervation from RMTg increased dopamine neuron firing rates. The opioid sequence despite conserved exon/intron organization. We will present sensitivity of GABA inputs from VTA interneurons has not been directly evidence to suggest the modulation of TSP1 by chronic mu opioids tested. The present study utilized an optogenetic approach to address if involves crosstalk between three different classes of receptors and the dopamine neurons received opioid-sensitive GABA inputs from VTA mediation of ERK/MAPK in rat immortalized cortical astrocytes. Using interneurons. Channelrhodopsin2 (ChR2) was expressed using AAV virus in selective antagonists, inhibitors and prototypic ligands, we implicated the both dopamine neurons and interneurons in the rat midbrain. Whole-cell mu opioid receptor, EGFR and TGFbetaR. TGFbeta1 stimulates TSP1 voltage clamp recordings were performed in sagittal brain slice from dopamine expression via EGFR and ERK phosphorylation. Therefore, our studies neurons. Focal photo activation of ChR2 successfully evoked GABA-A IPSCs indicate that EGFR is a signaling hub for opioid and TGFbeta1 modulated in dopamine neurons. GABA-A IPSC from VTA interneurons were decreased TSP1 expression. In prior studies, TGFbeta 1 and serum induced TSP1 in amplitude by Met-enkephalin (1 µM) to 82.7±4.2 % of control, whereas protein expression in astrocytes. TSP2 cellular levels are not stimulated by RMTg GABA input decreased to 25.4±4.7% of control. This study TGFbeta 1 under the same conditions or earlier time periods. In in vivo demonstrates that VTA interneurons that express mu-opioid receptors were studies, gene profiling data revealed that chronic opiates down regulate less sensitive to opioids than RMTg neurons. The functional connection from both TSP1 and TSP2 expression in discrete brain regions. TSP1 and TSP2 the RMTg to the VTA mediated by a dense, opioid-sensitive GABA protein expression were down regulated at 8 days of opioid treatment of innervation is the key structure in the mu-opioid receptor dependent regulation astrocytes under the same conditions that we found morphine attenuates of dopamine neurons. Supported by DA08163 (JTW). both neurite outgrowth and synapse formation in co-cultures of astrocytes and neurons. Therefore, chronic morphine may deter synaptogenesis via TSP1 and TSP2. Since synaptic plasticity is one of the major cellular adaptations that ensue upon chronic opioid exposure, astrocytic TSP1/2 may play a role in opioid tolerance, withdrawal and dependence. Supported by NIDA grant R01DA-05412.

W23 Inhibition/deletion of CCR5 blocks interactive morphine and Tat W24 norBNI inactivates KOR expressed in transfected HEK393 and neurodegeneration NG108 cells through the c Jun Kinase mechanism. E.M. Podhaizer (1), Y. Zhang (2), P.E. Knapp (1,3), K.F. Hauser (1). (1) Selena Schattauer, Haripriya Shankar, Mayumi Miyataki, Charles Chavkin Depts. of Pharmacol. & Toxicol., (2) Med. Chem., (3) Anat. & Neurobiol., Department of Pharmacology, University of Washington, Seattle, WA Virginia Commonwealth Univ., Richmond, VA. 98195

Previous studies have shown that dual morphine & Tat treatment produce a Opioid receptor inactivation has been shown to result from ligand directed synergistic increase in neurotoxicity. We chose to examine CCR5’s role in activation of JNK. Long lasting KOR antagonists activate JNK in mouse this effect due to its interactions with the mu-opioid receptor, and its ligand’s striatum and spinal cord and in transfected cells following receptor involvement in glial activation in response to morphine & Tat treatment. We binding. The selective activation of JNK1 by long lasting KOR hypothesized that blockade/deletion of CCR5 would prevent morphine and antagonists has been shown to result in receptor inactivation in vivo Tat interactive neurodegeneration. A CCR5 antagonist, maraviroc (MVC), through unknown mechanisms. We sought to determine if norBNI also was used first to assess CCR5’s role in morphine & Tat toxicity in neuron- causes JNK mediated KOR inactivation in transfected cells. HEK293 and mixed glia co-cultures. While MVC had no effect on morphine or Tat toxicity differentiated NG108 cells stably expressing KOR were treated 24hr with alone, it blocked the enhanced effect of the combined treatment. To vehicle, naloxone, or norBNI. Following antagonist treatment, cells were compliment this study, use of CCR5 knockout (KO) glia with wild-type (WT) either immediately stimulated with U50,488 (T0), or washed extensively neurons delayed the onset of neurotoxicity in morphine, Tat, or dual-treated and then maintained 24 hr in drug free media prior to stimulation with cells and the overall toxicity of the combined treatment was reduced. When U50,488 (T24). KOR activation was measured by immunoblot analysis of WT glia were cultured with CCR5 KO neurons, morphine & Tat toxicity was ERK phosphorylation following 5 min U50,488 treatment. In both significantly reduced, while interestingly, morphine toxicity was enhanced. HEK293 and differentiated NG108, norBNI treatment (1uM or 10uM) However, deletion of CCR5 in both neurons and glia showed significant blocked U50,488 stimulated pERK at both T0 and T24, whereas 10 uM toxicity under basal conditions and suggests that CCR5, per se, is naloxone treatment blocked U50,488 stimulated pERK at T0 but not T24. neuroprotective, while there was no treatment effect. To determine if the glial This shows that in these cell systems, similar to in vivo mouse models, component of the CCR5 response could be due to beta-chemokine release, norBNI but not naloxone, causes a long lasting inactivation of KOR that CCR5’s endogenous ligands, MIP-1alpha, MIP-1beta, and RANTES were persists after drug has been washed out. Pretreatment with the JNK applied to co-cultured cells between 0.1 pg/mL and 1.0 micrograms/mL. inhibitor SP600125 (100nM) prevented the block of U50,488 stimulated While the highest concentrations did show significant neurotoxicity, pERK by norBNI at T24, but not T0. These results show that norBNI physiologically relevant levels did not produce an effect, suggesting that binding to KOR results in both competitive antagonism at TO and JNK- elevated levels of CCR5 agonists were not responsible for enhanced morphine mediated inactivation of KOR in transfected HEK293 and differentiated & Tat toxicity. A follow-up experiment confirmed that cytokine/chemokine NG108. These cell models may be useful in probing the mechanism of release was not the primary effect, as secretion of RANTES and MCP-1 was JNK mediated opioid receptor inactivation. Supported by DA11672 and only modestly reduced with MVC pretreatment. These results suggest that DA20570 CCR5, in both neurons and glia, contributes to interactive morphine & Tat neurotoxicity through an alternative mechanism.

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W25 Opioid Receptor Like-1 (ORL1) intracellular trafficking and JNK W26 Opioid and HIV-1 Associated Neurodegeneration: Implications signaling requires c-terminal serine 363 for P2X4 Receptor Involvement N. Zhang* (1,4), E.R. Siuda* (1,2), W. Planer (1), L. Stickler (1), M. Baird Sorrell, M.E. (1), Zou, S. (2), Knapp, P.E. (1, 2), and Hauser, K.F. (1); (1) (1,5), T. Palmer (1), R. Al-Hasani (1,3), M.R. Bruchas (1,2,3) (1)Dept. of Dept. Pharmacol. & Toxicol., (2) Dept. Anat. & Neurobiol., Virginia Anesthesiology, (2)Program in Neuroscience, (3)Dept. of Commonwealth Univ. Coll. of Medicine, Richmond, VA, USA. Anatomy/Neurobiology, (4) HHMI SURF Fellow, (5) AMGEN Scholar Washington University in St. Louis, St. Louis MO, USA HIV-1-associated neurocognitive disorders (HAND) are seen in about 40% of AIDS patients and individuals who abuse opiates can have an increased Opioid Receptor Like-1 (ORL1) is the least understood opioid receptor and incidence of HAND. Microglia play a major role in HIV plays a key role in pain modulation, opiate tolerance, and responsivity to neuropathogenesis. Their activation produces inflammatory molecules that stress and anxiety. It has been reported that c-terminal phosphorylation at can lead to neuronal injury and death. Extracellular ATP, which activates serine (S), threonine (T), and tyrosine (Y) residues are required for mu and P2X receptors, can increase microglial activation and neuronal injury. kappa opioid receptor internalization, desensitization, arrestin recruitment, Morphine has also been reported to increase microglial motility via P2X4 and mitogen-activated protein kinase phosphorylation (pMAPK). To signaling (Horvath & DeLeo, J. Neurosci., 2009). To test the hypothesis determine the molecular mechanisms that mediate ORL1 internalization, that HIV-1 and opioid-induced neurotoxicity are mediated via purinergic desensitization, and pMAPK we mutated c-terminal residues of human ORL1- signaling, co-cultures of primary neurons and mixed glia from mouse YFP. We generated alanine mutations at T362/S363, S363, T362/S363/T365, striatum were treated for 72 h with combinations of Tat, morphine, and and S337/346/351 in the ORL1 c-terminus. Using wild type and mutant YFP- TNP-ATP, a non-selective P2X antagonist. Tat and morphine interactive tagged ORL1 receptors expressed in HEK293 cells, we visualized and neurotoxicity was reversible by treatment with TNP-ATP. Next we compared Nociceptin (Noci)-induced ORL1 internalization. ORL1 investigated sub-lethal neuronal injury resulting from treatment with Tat + internalized within 15 to 30 minutes following Noci treatment. In contrast, morphine. At 72 h, dendritic length was significantly decreased by Tat + S363A of ORL1 significantly blocked internalization. In addition, we morphine; however, dendritic pruning was prevented by treatment with determined the time course and concentration-dependence of ORL1-mediated TNP-ATP. Other results show Tat and morphine significantly increased 2+ 2+ 2+ pMAPK. Noci caused robust increases in pMAPK cascades: pERK and intracellular Ca ([Ca ]i)levels, which can lead to excitoxicity. [Ca ]i pJNK. ORL1-induced pERK was rapid and peaked at 10 minutes following increases were preventable by TNP-ATP pretreatment. To investigate agonist exposure while pJNK peaked at 30 minutes. C-terminal serine 363 P2X4 receptor involvement, selective antagonists against the P2X1, P2X3, mutants of ORL1 showed markedly reduced pJNK MAPK phosphorylation. and P2X7 receptor subtypes were also screened. The findings showed that We also measured ORL1-dependent MAPK in primary cultures of dorsal root these receptor subtypes were not involved in Tat + morphine neurotoxicity ganglion cells and found similar results. Further, we determined that ORL1 further supporting the hypothesis that P2X4 receptors are involved. internalization is blocked via arrestin3 and GRK3 shRNAs, and that ORL1 Finally, human P2X microarray data from HIV infected and non-infected S363A internalization and pJNK can be rescued via expression of a individuals [courtesy National NeuroAIDS Tissue Consortium (NNTC)], dominant+ arrestin 3. Together, these data implicate GRK/arrestin in ORL1 suggest that P2X4 is significantly altered in neuroAIDS and that the P2X4 MAPK signaling, and highlight the potential for the development of receptor may be a novel therapeutic target in the prevention of HAND. functionally selective ORL1-ligands. (funded by NIH/NIDA-R00DA025182- Support: NIH DA018633, DA028741, DA007027, and the NNTC. MRB)

W27 Evolutionary differences of opioid receptors are reflected in their W28 Sortilin regulates cell surface expression and trafficking of the pharmacological profiles human kappa opioid receptor (hKOPR) E. Vardy (1), C.W. Stevens (2), B.L. Roth (1). (1) Department of Y-J. Wang, C. Chen, J. Li, L.-Y. Liu-Chen. Ctr Sub Abuse Res, Temple Pharmacology, Program in Neuroscience and Division of Chemical Biology Univ, Phila, PA, USA and Medicinal Chemistry, and National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina Chapel Sortilin is one of five members of the Vps 10p-domain family of sorting Hill Medical School, Chapel Hill, NC 27514, USA; (2) Department of receptors and functions in intracellular trafficking and signaling. We have Pharmacology and Physiology, Oklahoma State University-Center for Health found by proteomic analyses that sortilin is one of the proteins

Sciences, College of Osteopathic Medicine, Tulsa, OK 74107, USA immunoprecipitated with KOPR from the extract of N2A-HA-hKOPR cells. Expression of sortilin siRNA in N2A-HA-hKOPR cells reduced The evolutionary differences between mammalian and amphibian opioid sortilin by ~90% and decreased fully glycosylated hKOPR (55kd) receptors (ORs) are examined here by a direct comparison of the ligand expression by 28% as determined by immunoblotting, and cell surface induced Gi coupling signaling profiles of mammalian and amphibian opioid receptor expression decreased by 35% when measured by flow cytometry receptors. Agonist induced Gi response was determined for amphibian (Rana analysis and [3H]diprenorphine binding. In contrast, expression of sortilin pipiens) opioid receptors (rpMOR, rpDOR,rpKOR and rpORL) and the cDNA increased fully glycosylated hKOPR expression. Neither sortilin human opioid receptors (hMOR, hDOR, hKOR and hORL) in transiently expression nor knockdown affected % of total receptor present on cell transfected human embryonic kidney cells (HEK293T). Identifying the surface. In addition, sortilin siRNA significantly decreased U50,488H differences in response profile is an important step in understanding the (10µM, 30min)-induced internalization compared to control siRNA. These effects of evolutionary pressure on the sensitivities of these receptors. The results indicate that sortilin interacts with hKOPR and regulates cell rank of opioid ligand selectivity was mostly consistent with previously surface KOPR expression and trafficking. Supported by DA17302 characterized type-selectivity. However, most of the opioid ligands tested had significant differences in affinity between rpORs and hORs. In most cases type-selective ligands had much lower affinities for the amphibian receptors than the human receptors with the nociceptin receptor standing out with equivalent affinities between the human and the amphibian receptor. This work provide information on how the molecular differences generated by evolution affect the pharmacological profile of opioid receptors and can be used to help guide structure – function analysis of agonist activation of opioid receptors. Supported by RO1DA017204 to BLR

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W29 Aquaporin 4 deficiency attenuates opioid dependence through the W30 Region-specific alterations in expression of EPHA4, EPHB2 and suppression of glutamate transporter-1 down-regulation and the OPRK1 receptors in postmortem brain of subjects infected with HIV maintenance of glutamate homeostasis compared to controls N. Wu, H.T. Yan, X.Q. Lu, R.B. Su, J.Q. Zheng*, J. Li* Beijing Institute of V. Yuferov (1), A. Ho (1), S. Morgello (2), M.J. Kreek (1). (1) Laboratory Pharmacology and Toxicology, Beijing, China of the Biology of Addictive Diseases, The Rockefeller University, New York, NY, USA, (2) Pathology and Neuroscience, Mount Sinai Medical Repeated exposure to abused substances produces adaptations in glutamate Center, New York, NY, USA neurotransmission that result in addictive behaviors such as withdrawal, craving and relapse. Therefore, preventing the disruption of glutamate Neurocognitive disorders persist in many individuals infected with HIV homeostasis or re-establishing glutamate homeostasis is a novel strategy for (HIV+). Earlier studies showed this neurocognitive impairment was the treatment of addiction. Glutamate transporters are primarily responsible for strongly associated with reduced synaptic complexity. We examined clearing extracellular glutamate and thereby maintaining glutamate alterations in expression of several ephrin genes that are involved in homeostasis. Our previous work demonstrated that aquaporin 4 (AQP4) synapse formation, in the caudate and anterior cingulate in postmortem deficiency attenuated morphine dependence, but the mechanisms are unclear. brain of HIV+ subjects compared to HIV-negative (HIV-) subjects, along Given the recent evidence that AQP4 might form a functional complex with with expression of OPRK1, and neuronal and astroglial/macroglial markers. glutamate transporter-1 (GLT-1), the present study focused on whether AQP4 Postmortem brain tissues of HIV-infected and control subjects were participates in the modulation of GLT-1 and glutamate homeostasis in obtained from the Manhattan HIV Brain Bank (The Mount Sinai Medical morphine-dependent mice. We found that AQP4 deficiency reduced either Center, NY). Quantification of mRNA levels of OPRK1, basal GLT-1 expression or the ability of GLT-1 to clear extracellular astroglial/macroglial markers (GFAP, CD163, CD68), neuronal marker glutamate in the prefrontal cortex, striatum, hippocampus and cerebellum. SNAP25, and ephrin genes (EPHA4 and EPHB2 receptors, and ligands Similarly, AQP4 knockout prevented the down-regulation of GLT-1 EFNB1 and EFNB2) was carried out using SYBR Green RT-PCR. Copy expression and function induced by repeated morphine treatment. AQP4 number of cDNA transcripts of each gene was expressed normalized to knockout abolished both increases and decreases in the extracellular glutamate GAPDH cDNA. Normalized values of copy numbers of mRNA of each levels in the prefrontal cortex during repeated morphine treatment and gene were expressed as natural log. Levels of EPHA4 and EFNB2 mRNA naloxone-precipitated withdrawal, indicating that glutamate homeostasis was in the caudate, and EPHB2 mRNA in anterior cingulate were significantly maintained. Collectively, these results suggest that AQP4 deficiency lower in HIV+ compared to HIV- subjects (p<0.002, p<0.02, p<0.05, suppresses the down-regulation of GLT-1 and the disruption of glutamate respectively). In contrast, OPRK1 mRNA level was significantly higher in homeostasis caused by repeated exposure to morphine, pointing to a strategy the anterior cingulate in HIV+ subjects (p<0.005). Correlation analysis for maintaining glutamate homeostasis and thereby treating addiction. This showed strikingly coordinated expression of the genes studied in both brain work was supported by grants from the National Basic Research Program of regions of HIV+ subjects, but not in HIV- subjects. There was significant China (2009CB522008) and the Beijing Nature Science Foundations (No. correlation of EPHA4 mRNA levels with expression of OPRK1 and 7113162 and No. 7102124). SNAP25 in the caudate of HIV+ subjects. Reduction in ephrin receptors EPHA4 and EPHB2, and the ephrin ligand EFNB2, and the elevation of OPRK1 mRNA levels in brain may contribute to the development of the neurocognitive disorder associated with HIV. Support: NIH NIDA-P60- 05130 (MJK), NIMH-U01-MH083501 (SM)

W31 Discovery of New Kappa Opioid Receptor Agonist and Antagonist W32 Novel analogs of the kappa opioid receptor ligand CJ-15,208 with Chemotypes through screening a Nonproprietary Compound Collection potential for drug development and SAR Expansion S. N. Senadheera (1), S. O. Eans (2), N. C. Ross (2), J. P. McLaughlin (2), Kevin J. Frankowski(1), Michael P. Hedrick(2), Palak Gosalia(2), Kelin Li(1), T. F. Murray (3), J. V. Aldrich (1) (1)Dept. of Medicinal Chemistry, The Shenghua Shi(2), Partha Ghosh(1), David Whipple(1), Thomas E. Univ. of Kansas, Lawrence, KS, USA; (2)Torrey Pines Institute for Prisinzano(1), Frank J. Schoenen(1), Ying Su(2), S. Vasile(2), Eduard Molecular Studies, Port St. Lucie, FL, USA; (3)Dept. of Pharmacol., Sergienko(2), Wilson Gray(2), Santosh Hariharan(2), Loribelle Milan(2), Creighton Univ. Sch. of Med., Omaha, NE, USA Susanne Heynen-Genel(2), Arianna Mangravita-Novo(3), Michael Vicchiarelli(3), Layton H. Smith(3), John M. Streicher(4), Marc G. Caron(5), Recently kappa opioid receptor (KOR) antagonists have demonstrated Lawrence S. Barak(5), Laura M. Bohn(4), Thomas D.Y. Chung(2), Jeffrey therapeutic potential for the treatment of drug abuse. Macrocyclic Aubé(1) (1)University of Kansas Specialized Chemistry Center, University of tetrapeptides are promising candidates for development because of their Kansas, Lawrence, KS 66047; (2)Conrad Prebys Center for Chemical low molecular weight, expected metabolic stability in vivo and potential Genomics at Sanford-Burnham Medical Research Institute, La Jolla, CA oral bioavailability. The macrocyclic tetrapeptide CJ-15,208 (cyclo[L-Phe- 92037; (3)Conrad Prebys Center for Chemical Genomics at Sanford-Burnham D-Pro-L-Phe-Trp]) is a natural product that was reported to be a novel Medical Research Institute at Lake Nona, Orlando, FL 32827; (4)Department KOR antagonist (Saito et al., J. Antiobiot. 2002, 55, 847). We used of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida modifications to our initial synthetic protocol (Ross et al., Tetrahedron 33458; (5)Department of Cell Biology, Duke University, Durham, NC 27710 Lett. 2010, 51, 5020) to prepare larger quantities of the synthetically challenging CJ-15,208 Trp isomers and their alanine scan analogs for The high throughput screening (HTS) of the NIH compound collection using a detailed pharmacological evaluation in vivo. As expected, both isomers ß-arrestin recruitment assay platform has identified several new chemotypes exhibited activity after oral administration. The D-Trp macrocyclic for the modulation of the kappa opioid receptor (KOR). Here we describe the tetrapeptide exhibited potent KOR selective antagonism in vivo in mice and successful optimization of two agonist and two antagonist chemotypes through prevented stress-induced reinstatement of extinguished cocaine-seeking a brief, collaborative SAR campaign. The resulting KOR probe molecules behavior (Ross et al., Br. J. Pharmacol. 2012, 165, 1097). The Ala scan were profiled against a set of 44 GPCR and other molecular targets and analogs of the promising D-Trp isomer, which exhibit a range of affinities characterized in a basic set of PK assays. The four chemotypes presented for opioid receptors (Dolle et al., Bioorg. Med. Chem. Lett. 2009, 19, complement the existing arsenal of KOR ligands through their modular, easily 3647), showed distinct pharmacological profiles in vivo in the 55 °C warm constructed, and, in most cases achiral structures. The development of such water tail-withdrawal assay, surprisingly exhibiting either mixed readily-modified, KOR-selective scaffolds provides new molecular tools for agonist/antagonist activity or exclusive agonist activity. One analog that the investigation of KOR modulation pathways. This work was supported by shows mixed agonist/ KOR antagonist activity in the antinociceptive assay grants 5U54HG005033 (John Reed, PI) and 5U54HG005031 (JA) from the prevented stress-induced, but not cocaine-induced, reinstatement in mice, Molecular Libraries Initiative, and 1X01DA026208 (LSB) and similar to the parent peptide. These novel macrocyclic tetrapeptides are 5U01DA022950-03 (MC) from the National Institute on Drug Abuse. promising candidates for the development of potential peptide KOR Receptor profiling was provided by the Psychoactive Drug Screening Program therapeutic ligands. Research supported by NIDA grants R01 DA018832 at the University of North Carolina, Chapel Hill (Contract # HHSN-271-2008- and R01 DA023924. 00025-C (NIMH PDSP)).

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W33 Pharmacological Evaluation of Novel Opioid Ligands: A Study to W34 Design and Synthesis of Amide and Amine Containing Ligands Determine the Effects of Mixed Mu/Delta Activity in vitro and in vivo Related to Buprenorphine Jason R. Healy (1), Padmavani Bezawada (2), Matthew Metcalf (2), Chris Emma M. Turner (1), Juan Pablo Cueva (1), Mary J. Clark (2), John R. Cunningham (2), Sucheta Kudrimoti (2), Andrew Coop (2), Rae R. Traynor (2), Stephen M. Husbands (1). (1)Department of Pharmacy and Matsumoto (1) (1)West Virginia University School of Pharmacy, Pharmacology, University of Bath, Bath, UK; (2)Department of Morgantown, WV; (2)University of Maryland School of Pharmacy, Pharmacology, University of Michigan, Ann Arbor, USA Baltimore, MD In the search for new opioid ligands for the treatment of opiate abuse, The three opioid receptor subtypes mu, delta and kappa have long been buprenorphine is the most noteworthy recent addition to our armour. In associated with analgesia. Traditional opioid analgesics exert their effects addition to its activity as a mu opioid (MOP) receptor partial agonist, through mu receptors located in the CNS. Recent studies suggest that the buprenorphine is a kappa/delta (KOP/DOP) receptor antagonist and more development of opioid analgesics displaying dual properties of mu agonism recently profiled as a partial agonist at the nociceptin/orphanin FQ (NOP) and delta antagonism could be of benefit by retaining potent analgesic receptor. In 2004 the structurally related amide-containing ligand TAN- properties while reducing the development of tolerance with chronic 821 was shown both in in vitro and in vivo assays to be an antagonist of all administration. Novel ligands incorporating features of potent mu agonism three classical opioid receptors, MOP, KOP and DOP, and described as an and low efficacy at delta were synthesized and tested for their binding affinity agonist of the putative epsilon opioid receptor. Our interest in ligands at each receptor subtype. Specifically, UMB 425 and 426 display high affinity similar to buprenorphine and in amides closely related to TAN-821 at the mu receptor (Ki = 3.2 ± 0.14, 33 ± 3.1 nM) moderate affinity at the delta prompted us to investigate a series of amide and amine containing ligands. receptor (Ki = 208 ± 18, 121 ± 7.2 nM), with moderate to low affinity at the Synthesis and retesting of TAN-821, and some of its close analogues 35 kappa receptor (Ki = 212 ± 22, 1804 ± 444 nM). In vitro [ S]GTPγS suggests this series of amides have high efficacy at KOP (89-110% in the functional assay results indicate that UMB 425 and 426 behave as partial GTPgammaS assay), as well as moderate to low MOP efficacy (10-71%). agonists at the mu receptor (ED50 = 35 ± 3.7, 342 ± 94 nM; %Emax = 73 ± 7.3, This was coupled with low efficacy at NOP (-5-10%), a profile in marked 79 ± 7.3), whilst having competitive antagonistic properties at the delta contrast to the original publication. The series of ligands has now been receptor (pA2 = 6.12(-0.91), 6.31(-1.08)). In vivo studies in mice show that extended to include alkyl and aryl-based amide compounds, some of which UMB 425 displays potent analgesic activity for both the hot plate (ED50 = have improved NOP efficacy, but retain high efficacy at KOP and low to 2.73 mg/kg) and tail-flick (ED50 = 6.85 mg/kg) assays. In addition, naloxone moderate efficacy at MOP. Further evaluation of these compounds will be (1 mg/kg, i.p.; t = -30 min) pretreatment significantly attenuates the analgesic presented. This work was supported by NIDA grant DA07315 (SMH). effects induced by an acute ED90 of UMB 425 (15 mg/kg, s.c.) treatment (p<0.001 for each assay, respectively). Additional in vivo studies are underway to determine the level of chronic tolerance developed by UMB 425. This project is supported by the National Institute on Drug Abuse (DA- 13583); we also thank Larry Toll (Torrey Pines Institute for Molecular Studies) for providing stably transfected CHO cells overexpressing each of the human opioid receptor subtypes.

W35 Analysis of Hydrocodone, Hydromorphone and Norhydrocodone in W36 Hydrocodone Metabolism Following Singe Dose Administration Plasma using Liquid Chromatography - Tandem Mass Spectrometry Sandra Valtier, Robert Mueck, Toni Vargas, and Vikhyat S. Bebarta, (LC/MS/MS) 59th Medical Wing, Lackland AFB, TX. Sandra Valtier, Robert Mueck, Toni Vargas, and Vikhyat S. Bebarta, 59th Medical Wing, Lackland AFB, TX. Abuse of prescription opioids for non-medical use has been on the rise over the past decade. The most commonly abused opioid is hydrocodone, a The goal of this study is to present a validated liquid chromatography tandem frequently prescribed pain medication metabolized by the body to mass spectrometry (LC/MS/MS) method for quantitative analysis of hydromorphone, norhydrocodone and other minor metabolites. hydrocodone (HC) and its metabolites, hydromorphone (HM) and Quantitative analysis of HC and its primary metabolites, hydromorphone norhydrocodone (NHC) in plasma. HC, a semi-synthetic opioid analgesic (HM) and norhydrocodone (NHC), can aid in monitoring pain management, used for moderate and severe pain relief, is metabolized to its major distinguishing prescribed from unauthorized drug use, and reduce drug metabolite, HM, and to a lesser extent to minor metabolite, NHC. To diversion. This study describes the metabolism profile of hydrocodone, effectively monitor and evaluate metabolism profiles, a sensitive and specific hydromorphone and norhydrocodone in plasma following a single dose (10 test is needed to ensure that the drug and its metabolites can be measured to mg) administration of hydrocodone to human subjects (n = 12). Blood the lowest detectable amount. Standards spiked with concentrations of HC, samples were collected in lithium heparin tubes prior to administration of HM and NHC ranging from 0.25 - 100 ng/mL were prepared in opioid the drug and at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 72, 96 and 168 hours negative plasma. The LC gradient mobile phase consisted of (A) 0.1% formic following drug administration. Blood collected at 72, 96 and 168 hours acid and (B) acetonitrile; flow rate was set at 0.5 mL/minute. The internal were collected as part of a bigger protocol involving proteomic analysis. standard solution contained 1µg/mL HC-D6, HM-D6 and NHC-D3 in Plasma was then separated from the sample and stored at ≤ 20ºC prior to methanol. A 500 µL aliquot of standard was mixed with 25 µL of internal analysis. Following solid phase extraction, a validated LC/MS/MS method standard solution. Solid phase extraction (SPE) was performed, followed by was used for analysis of the drug and its metabolites. Peak concentrations injection on the LC/MS/MS system. The mass spectrometer was set in ESI of hydrocodone were found at 0.5 to 5.0 hours post-dose and were in the positive mode and analysis was performed using two multiple reaction range of 12.20 to 31.70 ng/mL. Hydromorphone peak concentrations were monitoring (MRM) transitions per analyte. The ion transitions monitored were found at 1.0 to 8.0 hours post-dose and ranged from 1.13 to 3.53 ng/mL. m/z 300.2→199.1 and 300.2→171.0 for HC; m/z 286.1→185.0 and For norhydrocodone, peak concentrations were found at 0.5 to 6.0 hours 286.1→157.0 for HM; m/z 286.2→199.1 and 286.2→241.1 for NHC; m/z post-dose and ranged from 2.40 to 7.40 ng/mL. Hydrocodone was detected 306.2→174.0 for HC-D6, 292.2→185.2 for HM-D6 and m/z 289.0→202.0 for for up to 24 hours in 10 of 12 subjects. Hydromorphone was last detected NHC-D3. The linear range was determined for this procedure by analysis on 6 for up to 10 to 24 hours post dose and norhydrocodone was last detected for different runs on concentrations ranging from 1 to 100 ng/mL of each analyte up to 11 to 24 hours post dose. Following the metabolism and excretion prepared in plasma. The linear range was shown to be 1 to 100 ng/mL for HC, profile of an individual with a sensitive and specific drug test can help in HM and NHC with r value > 0.99 for all compounds. The limit of detection determining time since last dose and expected peak concentrations. Funding (LOD) was 0.25 ng/mL and the limit of quantitation (LOQ) was 1.0 ng/mL for source: Department of Defense (DOD) Congressionally Directed Medical all analytes. The method yielded good precision with RSDs of < 6.3% at 10 Research Programs (CDRMP) Grant Award and 25 ng/mL HC, HM, and NHC. Funding source: Department of Defense (DOD) Congressionally Directed Medical Research Programs (CDRMP) Grant Award. 68 INRC 2012 Kansas City, Missouri

W37 Physicochemical and pharmacokinetic properties of novel W38 Mixture Linkage Analysis: Efficiency of Identification of Distinct macrocylic peptide kappa opioid receptor ligands Ligand Families from Positional Scanning Combinatorial Libraries A. Joshi (1), T. Khaliq (1), S. Senadheera (1), A. Mukhopadhyay (1), S. M. Jaime A. Misler, Margaret E. Cazares, Tina L. Yates and Colette T. Lunte (2), J. V. Aldrich (1) Dept of (1)Medicinal Chem. and (2) Dooley. Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida Pharmaceutical Chem., Univ. of Kansas, Lawrence, KS, USA Positional scanning deconvolution is a very powerful method for the We are interested in metabolically stable peptidic ligands for kappa opioid identification of active compounds from mixture based combinatorial receptors (KOR) as potential treatments for drug abuse and pain. The natural libraries. However, in certain cases the number of individual compounds product CJ 15,208, a macrocyclic tetrapeptide (cyclo[L-Phe-D-Pro-L-Phe- required for synthesis is prohibitive. Furthermore, while some active Trp]), was reported to exhibit KOR antagonism in vitro (Saito et al., J. peptides are identified many the other compounds synthesized are found to Antibiot. 2002, 55, 847). The L- and D-Trp isomers of this peptide exhibit be inactive. A new analysis for mixture libraries is described wherein the different pharmacological profiles in vivo after intracerebroventricular physical mixture of two mixtures (a dimixture) indicates whether the amino administration to mice (Ross et al., Br. J. Pharmacol. 2012, 165, 1097), and acids defined are likely to be present on the same active peptide. This both exhibit activity following oral administration. Therefore both isomers analysis is based on mixture behavior obeying the harmonic mean, whereby serve as lead compounds for further exploration. In order to enhance the oral the most active component(s) of a mixture drives the greatest proportion of activity of these macrocyclic peptides we are exploring the physicochemical the activity. In Mixture Linkage Analysis, activity of a mixture with a and pharmacokinetic properties of these two lead compounds. Because of single active component is halved if an equal volume of second mixture their hydrophobicity we have examined various solubilizing agents, including lacking activity is added; however addition of a second mixture having an organic cosolvents, cyclodextrins and surfactants that are compatible with in identical active component will result in activity retention. In this case vitro and in vivo studies, to solubilize these compounds. We have studied the study we determined whether mixture linkage analysis is capable of bidirectional permeability of these compounds in the Caco-2 cell monolayer identifying distinct active peptide sequences (families) from a complex model for intestinal absorption using liquid chromatography-tandem mass mixture based library (over 5 million peptides). The library used was from spectrometry (LC-MS/MS) to quantify the compounds. The interaction of the a previously published study where novel peptides for the mu opioid compounds with recombinant P-glycoprotein is also being evaluated directly receptors were identified from three sublibraries OOXXXX-NH2, using an enzymatic assay. In addition the stability of the compounds in blood XXOOXX-NH2 and XXXXOO-NH2. Distinct peptide sequences were and liver homogenate is being determined, along with protein binding. The identified in our first study and this knowledge was used to determine the results of these studies will be presented. This evaluation of the efficiency of using mixture linkage analysis physicochemical and pharmacokinetic properties of these lead compounds is important in their development for in vivo studies and as potential therapeutic agents. Research supported by NIDA grant R01 DA023924.

W39 Exploring potential interaction modes of basic and non-basic W40 Mixed Efficacy and Improved Drugability: Working Toward A agonists with the kappa-opioid receptor Better Opioid Analgesic E. Vardy (1), P. D. Mosier (2), K. J. Frankowski (3), J. Aubé (3), R. B. Jessica P. Anand, Katarzyna Sobczyk-Kojiro, Lauren C. Purington, Larisa Westkaemper (2), B. L. Roth (1) (1) Dept. of Pharmacol., Univ. of North Yeomans, Aubrie A. Harland, Vanessa R. Porter, Aaron M. Bender, Emily Carolina, Chapel Hill, North Carolina, 27599, USA, (2) Dept. of Med. Chem., M. Jutkiewicz, John R. Traynor, and Henry I. Mosberg Departments of Virginia Commonwealth Univ., Richmond, Virginia, 23298, USA, (3) Dept. Medicinal Chemistry and Pharmacology, University of Michigan, Ann of Med. Chem., Univ. of Kansas, Lawrence, Kansas, 66045, USA. Arbor MI, USA

The recently-solved crystal structures of the kappa-, mu-, and It has been recognized that the simultaneous modulation of multiple targets nociceptin/orphanin FQ-opioid receptors (KOP, MOP, and NOP respectively) may generate a more desirable drug profile, in some cases even reducing the represent a major advance in our understanding of the structural similarities development of negative side effects. This concept has been illustrated in and differences among the members of this pharmaceutically relevant subclass the field of opioid analgesics, where the co-administration of a mu opioid of rhodopsin-like G protein-coupled receptors (GPCRs) and their interactions receptor (MOR) agonist with a delta opioid receptor (DOR) antagonist gives with antagonists. Here we report putative initial interaction modes of the all the expected analgesia of a MOR agonist, but reduced tolerance and structurally diverse KOP receptor-selective agonists N-naphthoyl-beta- dependence liabilities. In this vein we are exploring mixed efficacy ligands naltrexamine (NNTA), U69593, salvinorin A and dynorphin A (1–13) with the where the MOR agonist activity is combined with DOR antagonism in the human KOP receptor. Experimentally-guided automated docking routines same molecule to ease pharmacokinetic and patient compliance issues. were used to derive the putative binding modes for the agonists. Functional Peptides provide a unique opportunity for the development of data for each agonist at the wild-type KOP receptor and 27 KOP receptor multifunctional ligands as the density of chemical information allows for mutants was determined using a cellular cAMP response assay. These fine-tuning of receptor affinity and efficacy for multiple targets. mutations clearly exhibited a differential effect on the agonists’ signaling Unfortunately, peptides are often seen as “un-druggable”, with poor blood ability. Notably, mutation of the conserved transmembrane helix 3 aspartate brain barrier penetration and rapid enzymatic degradation. Our goal is to D138(3.32) to asparagine dramatically increased the IC50 value of the basic develop a lead peptide with the desired binding and efficacy profile and amine-containing agonists while significantly reducing the IC50 value for the then, using the SAR information from our peptide series, develop a more non-basic amine-containing agonists. By comparing the agonists’ putative “druggable” ligand. Our approach has two main arms; first, we are trying to binding modes and incorporating current knowledge about the activation of improve the bioavailability and membrane permeability of peptide leads rhodopsin-like GPCRs, hypotheses were generated that explain their through modifications such as glycosylation. Second, we are translating the individual mechanisms of action. Support: NIH-NIMH MH-R01DA017204 key binding elements of our peptide leads to a small molecule scaffold (BLR). which more closely resembles a classical drug molecule to improve activity in vivo. Examples of both approaches will be described. Supported by NIDA grant DA003910 and Fellowship support from NIDA Training Grants DA007281 (JPA) and DA007267 (LY).

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W41 Insufflation of peripherally-restricted KOR agonists produces rapid W42 Reduced Tolerance Development to Antinociception with antinociception in multiple pain models without opioid liabilities Bifunctional NOP/Mu Opioid Agonists M.R. Hoot (1), S.O. Eans (1), K.J. Reilley (1), M.L. Ganno (1), M. Guilianotti NT Zaveri (1), TV Khroyan (2), L Toll (3), WE Polgar (2), VB Journigan (1), R.A. Houghten (1), A. Leone-Bay (2), J.P. McLaughlin (1). (1)Torrey (1), Dennis Yasuda (1). (1) Astraea Therapeutics, Mountain View, CA (2) Pines Inst. Molecular Studies, Port St. Lucie, FL 34987 (2) MannKind Corp., SRI International, Menlo Park, CA (3) Torrey Pines Institute for Molecular Danbury, CT 06810 Studies, Port St. Lucie, FL.

Two agonists previously identified from a library of 6,250,000 tetrapeptides, Several studies have shown that tolerance develops to the antinociceptive ffir-NH2 and ff(nle)r-NH2, demonstrated affinity (IC50 = ~2 nM) and effects of morphine during chronic use, necessitating increases in dosage, selectivity for the kappa opioid receptor (KOR). In the 55°C warm water tail followed by other undesirable consequences. Nociceptin/orphaninFQ withdrawal test both compounds dose-dependently increased tail withdrawal (N/OFQ) via its cognate receptor NOP modulates several opioid-mediated latency following i.p., i.c.v., i.v., per os (p.o.) or i.v. administration. actions, particularly in reward and nociceptive pathways. Activation of the Antinociception produced by each tetrapeptide (ffir-NH2 ED50=0.53 (0.08- NOP receptor system has been shown to reduce tolerance development to 2.50) mg/kg, i.p.; ff(nle)r-NH2 ED50=0.25 (0.05-1.08) mg/kg) was 9.9- and opioid-mediated antinociception. N/OFQ, administered 21.0-times more potent than morphine, respectively. Pretreatment with the intracerebroventricularly, decreases tolerance development to morphine- opioid antagonist, naloxone methiodide, confirmed the peripherally-restricted induced antinociception in rats. We have hypothesized that bifunctional activity of the tetrapeptides. Insufflation (i.n.s.) of ffir-NH2 or ff(nle)r-NH2 NOP/mu agonists could lead to non-addicting analgesics with a decreased formulated as Technosphere® dry powders for oral inhalation, resulted in side-effect profile (i.e. reward, tolerance development). We have previously maximal antinociception in the tail withdrawal assay 5 minutes post demonstrated that the bifunctional NOP/mu receptor partial agonist administration at doses as low as 0.13µg. Pharmacokinetic examination SR16435, produces dose-dependent antinociception but exhibits decreased following ff(nle)r-NH2-Technosphere after i.v. (60 nmol) or i.n.s. (1.25 µg) tolerance development to its antinociceptive effects when administered over administration found blood levels consistent with the duration of nine days, compared to morphine. In the present study, we further examined antinociception, but tetrapeptide was not found in perfused brain tissue. whether tolerance would develop to antinociception produced by two other Further analysis of i.n.s. ff(nle)r-NH2-Technosphere on thermal hyperalgesia bifunctional NOP/mu agonists, SR14150 (now AT-200) and AT-203, using induced by chronic constriction injury demonstrated a dose-dependent return the mouse tail-flick assay. The results showed that tolerance developed to to baseline withdrawal latencies. Insufflation (0.125 mg) of ffir-NH2- or morphine antinociception (3 mg/kg) by the fourth injection, whereas ff(nle)r-NH2–Technosphere did not produce any significant differences from tolerance did not develop to the antinociception produced by AT-200 or vehicle in measures of respiration, locomotion or place conditioning, although AT-203. When the NOP antagonist SB612111 was given as a pretreatment i.n.s. morphine-Technosphere produced significant respiratory depression, prior to daily injections of AT-200 and AT-203, tolerance to hyperlocomotion and conditioned place preference. Overall, these results antinociception developed at a similar rate as that observed with morphine. suggest that i.n.s. administration of ffir-NH2 and ff(nle)r-NH2 is capable of These latter findings confirm that the inhibition of tolerance development to producing rapid and potent antinociception with fewer side effects. (Supported antinociception is mediated by the NOP agonist activity of AT-200 and AT- by NIDA (DA031370) and the State of Florida.) 203. These results support the therapeutic application of such bifunctional NOP/mu agonists as non tolerance-developing analgesics. This work was supported by grants DA14026 and DA027811.

W43 Opposing roles of MOP and NOP on locomotor activity is revealed W44 The Synthesis of N-Alkyl-octahydroisoquinolin-1-one-8- by the use of a series of bifunctional NOP/MOP agonists carboxylic Acids Under Mild, Nearly-Neat Conditions N. Zaveri (1), A. Hamid (3), A. Tseng (2), P. Marquez (2,3), V. Journigan (2), S. R. Slauson, P. Ghosh, and J. Aubé K. Lutfy (2,3). (1)Astraea Therapeutics, Mountain View, CA; (2)Western Department of Medicinal Chemistry, University of Kansas, Lawrence, University of Health Sciences, Pomona, CA; (3)Charles Drew University of Kansas 66047 Medicine and Sciences, Los Angeles, CA A new procedure for the synthesis of N-substituted-octahydroisoquinolin-1- Morphine and related mu opioid (MOP) agonists have been shown to augment one-8-carboxylic acids under mild, nearly-neat conditions is reported. neurotransmission along the mesolimbic dopaminergic neurons and induce When amine-tethered dienes were reacted with maleic anhydride at 0°C to locomotor stimulation. On the other hand, orphanin FQ/nociceptin, the room temperature for 20 minutes, the resulting isoquinolone carboxylic endogenous ligand of the opioid receptor-like receptor (ORL1, also known as acids were obtained in moderate to good yields. Mechanistic investigation NOP), has been demonstrated to negatively regulate the function of these by NMR monitoring revealed an initial acylation step followed by the neurons and induce locomotor suppression. We recently synthesized a series of intramolecular Diels-Alder reaction. Compared to previous methods, the bifunctional NOP/MOP compounds with varying efficacies at the NOP and new protocol allows quick, easy scale-up by eliminating the requirement of MOP receptors, based on the hypothesis that these compounds would produce microwave batch chemistry as well as significantly reducing the quantity of potent analgesia with limited rewarding effects and thus less abuse liability, halogenated solvent utilized. since NOP agonists have been shown to reduce the rewarding actions of This work was supported by grant R01DA031927 from the National morphine and other addictive drugs. Measurement of locomotor activity in Institute on Drug Abuse. rodents has been used as an indirect measure of reward and function of the mesolimbic dopaminergic neurons. Thus, we assessed the contribution of NOP and MOP in the motor effects of two bifunctional NOP/MOP ligands, in mice lacking MOP and their wild-type controls. AT-201, which is a partial agonist at NOP and MOP, induced a MOP-mediated motor stimulatory effect in wild- type mice, but suppressed motor activity in MOP knockout mice. AT-203, which is a full agonist at NOP, elicited motor suppressant effects in mice of both genotypes. These results suggest that the efficacy of each ligand at NOP mediates its overall motor suppressant effect in wild-type mice and the MOP efficacy determines their motor stimulatory effect. Therefore, a motor suppressant effect is observed following administration of bifunctional compounds possessing full agonist activity at the NOP receptor and partial efficacy at MOP. On the other hand, a motor stimulatory effect is observed after administration of a drug with partial agonist activity at both receptors. Supported by R01DA016682 (KL); R24 DA017298 (KL); R01DA014026 (NZ) and R01DA027811 (NZ)

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W45 Molecular Dynamics Simulations of a Homology Model of the Active State of the Nociceptin Receptor NOP Reveal New Insights into Agonist Binding and Activation. Zaveri NT and Daga PR. Astraea Therapeutics, LLC. Mountain View, CA.

The nociceptin receptor NOP, like other members of the opioid receptor family, is a G-protein coupled receptor (GPCR), and shares about 60% homology with the opioid receptors. However, none of the endogenous or synthetic opiates have high affinity for NOP, while the endogenous NOP ligand, nociceptin/orphanin FQ (N/OFQ) does not bind to opioid receptors. Site-directed mutagenesis studies suggest that the functional architecture and activation mechanism of NOP is distinct from that of the other opioid receptors. While the crystal structure of the NOP receptor GPCR has not yet been resolved, as of the writing of this abstract, previously reported homology models of the NOP receptor have been based on the antagonist-bound ‘inactive-state’ GPCR crystal structures, and give limited information on the binding of ‘agonists’ and the activation-associated receptor changes that occur upon agonist binding. We report here the first ‘active-state’ homology model of the NOP receptor, based on the opsin GPCR crystal structure. An inactive- state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP receptor activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches consistent with GPCR activation. Docking of selective nonpeptide NOP agonists into the active-state model reveals active site residues in NOP that play a role in the high selectivity of such agonists for NOP over other opioid receptors. Docking the shortest active fragment of N/OFQ (residues 1–13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues 8–13 of N/OFQ. NOP agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks during receptor activation and the involvement of the EL2 loop residues in binding and possibly activation. Support: Grant DA14026-07S1.

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INRC Index A Chung, N. N. 41 Adelson, M. 51 Chung, T. D. Y. 67 Aldrich, J. V. 41, 51, 67, 69 Clark, M. J. 61, 68 Al-Hasani, R. 24, 50, 66 Clark, S. 25 Altarifi, A. 50 Clarke, W. P. 45, 48, 53 Anand, J. P. 43, 69 Connor, M. 61 Anderson, E. M. 49 Cooke, A. E. 63, 64 Andrén, P. 57 Coop, A. 68 Aragona, B. 31 Cordery, S. 48 Arribas, M. 58 Coscia, C. J. 65 Arttamangkul, S. 60, 61 Cowan, A. 51 Arujo, N, 24 Cravatt, B. F. 36 Atwood, B. K. 36, 60 Crews, N. R. 65 Aubé, J. 24, 67, 69, 70 Crystal, H. 52 Avery, B.A. 57 Cueva, J. P. 68 Cunningham, C. 68 B Cutler, S. J. 58 Bailey, C. P. 48, 63 Baird, M. 66 D Baiula, M. 60 Dallel, R. 55 Ballet, S. 41 Das, S. 62 Bannister, T. 59 Davis, R. 62 Barak, L. S. 67 Delgado-Charro, B. 48 Barak, Y. 52 Delifictano, A. 30 Beaudry, H. 53, 60 Dever, S. M. 61 Bebarta, V. S. 68 Devi, L. A. 53, 62 Bedini, A. 60 Devkota, S. 38 Bender, A. M. 69 DiMattio, K. M. 51 Bensley, K. 49 Dodson, S. 62 Berg, K. A. 45, 48, 53 Doisy, E. A. 65 Berrendero, F. 37 Dome, M. 31 Berridge, K. 30 Dooley, C. T. 57, 58, 69 Bezawada, P. 68 Draga, P. R. 71 Bidlack, J. M. 45 Drouin, J. 48 Birdsong, W. T. 61 Ducat, E. 52 Bobeck, E. 46 Bochynska-Czyz, M. 51, 64 E Bohn, L. M. 24, 59, 67 Eans, S. O. 41, 58, 59, 67, 70 Borlagdan, J. 50 Eidson, L. N. 55 Brents, L. K. 39 Evans, C. 56 Brolin, E. 57 Bruchas, M. R. 24, 50, 56, 66 F Buonora, M. 61 Fakira, A. K. 62 Burgman, M. 58 Fantegrossi, W. E. 39 Butelman, E. R. 49, 53 Fitting, S. 33, 61 Fitzgibbon, D. M. 46 C Ford, C. P. 56 Cahill, C. M. 50, 56 Foshage, A. M. 50 Cameron, M. 24 Frankowski, K. J. 24, 67, 69 Cami-Kobeci, G. 55, 57 Fujii, H. 53 Carlsson, A. 57 Fumiyo, K. 56 Caron, M. G. 23, 67 Furr, E. B. 57, 58 Carroll, F. I. 29 Carusillo, B. 62 G Castoro, R. 62 Gandhi, P. R. 56 Caudle, R. M. 49 Ganno, M. L. 58, 70 Cazares, M. E. 57, 69 Garau, C. 24 Chait, B. T. 59 Gateley, J. 50 Charles, A. 52 Gendron, L. 53, 55, 60 Charlton, S. J. 63 Ghosh, P. 67, 70 Chavkin, C. 43, 65 GiGuere, P. 38 Chen, C. 66 Goeders, N. 25 Chen, S. 48, 52 Gomes, I. 62 Chen, Y.-H. 49, 54 Gosalia, P. 67 Chen, Z. F. 54 Gray, W. 67 Chiou, L.-C. 38, 54 Grenier, P. 50 72 INRC 2012 Kansas City, Missouri INRC Index Grinnell, S. G. 58 Leinweber, A. L. 65 Guilianotti, M. 70 Lemieux, C. 41 Guillemyn, K. 41 Leone-Bay, A. 70 Gupta, A. 53, 62 Lesniak, A. A. 51, 54 Guy, R. H. 48 Levitt, E. S. 64 Levran. O. 51 H Li, J. 66, 67

Hamid, A. 70 Li, K. 67 Hao, S. 26 Li, Q. F. 54 Hariharan, S. 67 Lichtman, A. H. 36 Harland, A. A. 43, 69 Lin, J.-G. 49 Harris, D. 25 Lipkowski, A. W. 51, 54, 64 Haselton, N. 58 Liu, X. Y. 54 Hassler, C. 25 Liu, Z. C. 54 Hauser, K. F. 33, 61, 64, 65, 66 Liu-Chen, L.-Y. 51, 66 Healy, J. R. 68 Llorente, J. 63 Hedrick, M. P. 67 Loh, H. H. 54 Henderson, G. 63, 64 Lovinger, D. M. 60 Heppenstall, P. A. 55 Lowe, J. 63 Heynen-Genel, S. 67 Lu, X. Q. 67 Hipólito, L. 53 Luccarini, P. 55 Ho, A. 48, 52, 53, 61, 67 Lunte, S. M. 69 Hoot, M. R. 48, 59, 70 Lutfy, K. 70 Hostovsky, A. 52 Houghten, R. A. 70 M Hruby, V. J. 42 Mabrouk, O. 30 Husbands, S. M. 48, 55, 57, 68 Mackie, K. 36 Magnussen, C. 50 IJ Majumdar, S. 58 Ingram, S. L. 46 Mangravita-Novo, A. 67 Jamalapuram, S. 57 Mankura, M. 56 Jamshidi, R. J. 45, 48 Mankus, J. V. 58 Joshi, A. 69 Margolis, E. 31 Journigan, V. B. 70 Markowicz, S. 64 Jutkiewicz, E. M. 32, 43, 46, 69 Marquez, P. 70 Masvekar, R. R. 64 K Matalinska, J. 64 Kappera, P. 65 Matsui, A. 65 Kelly, E. 63, 64 Matsumoto, K. 56 Kennedy, R. 30 Matsumoto, R. R. 68 Keresztes, A. 41 McArdle, C. A. 63 Khaliq, T. 69 McCall, J. G. 50, 56 Khroyan, T. V. 55, 57, 70 McCurdy, C. R. 57, 58 Kiguchi, N. 54 McGuire, B. A. 45, 48 Kim, S. 54 McLaughlin, J. P. 41, 48, 51, 58, 59, 67, 70 Kishioka, S. 54 McPherson, J. 63 Knapman, A. 61 Melyan, Z. 62 Knapp, B. I. 45 Mercer, S. 32 Knapp, P. E. 33, 61, 64, 65, 66 Mercier-Blais, A.-A. 60 Kobayashi, Y. 54 Mesangeau, C. 58 Kosson, A. 54 Metcalf, M. 68 Koyama, Y. 56 Metcalfe, S. 50 Krasel, C. 63 Milan, L. 67 Kreek M. J. 48, 49, 51, 52, 53, 59, 61, 67 Misler, J. A. 57, 69 Krizbai, I. A. 54 Miyataki, M. 65 Kudrimoti, S. 68 Molat, J-L. 55 Molnár, J 54 L Morgan, M. M. 46 Labuz, D. 55 Morgello, S. 67 Lamberts, J. 46, 50 Morón Concepción, J. A. 53, 62 LaRoche, G. 38 Mosberg, H. I. 43, 69 Laviolette, S. 30 Mosier, P. D. 69 Law, P.-Y. 43 Mueck, R. 68 Le Rouzic, V. 58 Mukhopadhyay, A. 69 LeCour, S. 50 Mukhopadhyay, S. 38 Lee, H.-J. 54 Mundell, S. J. 63, 64 INRC 2012 Kansas City, Missouri 73 INRC Index Murphy, A. Z. 55 Murray, T. F. 67 S Sacharczuk, M. 51, 54 N Saika, F. 54 Nagakura, K. 58 Saitoh, A. 53 Nagase, H. 53 Salvemini, D. 27 Nakamoto, K. 56 Sato, N. 56 Nefzi, A. 58 Schattauer, S. 43, 65 Negus, S. 50 Scherrer, G. 33 Nemoto, T. 53 Schiller, P. W. 41 Niikura, K. 48 Schlussman, S. D. 61 Nilges, M.R. 58 Schmoutz, C. 25 Nishinaka, T. 56 Schoenen, F. J. 67 Nockemann, D. 55 Schreiber, S. 52 Normandin, A. 55 Sebastian, B. 56 Nyberg, F. 57 Selley, D. 36, 37 Senadheera, S. N. 41, 67, 69 O Sergienko, E. 67 Ocampo, J. 58 Shankar, H. 65 Oka, J.-I. 53 Shi, S. 67 Olmstead, M. C. 50 Si, W. 24 Ott, J. 51, 52 Siderovski, D. 38 Sim-Selley, L. J. 36, 37 P Siuda, E. R. 66 Palmer, T. 24, 66 Skurzak, H. 64 Pan, Y.-X. 58 Slausen, S. 24, 70 Paris, J. J. 51 Smith, L. H. 67 Pasternak, A. R. 58 Smith, M. L. 52 Pasternak, G. W. 58 Sobczyk-Kojiro, K. 43, 69 Patkar, K. A. 41, 59 Sombers, L. 30 Peles, E. 51 Sorrell, M. E. 66 Phamduong, E. 65 Spampinato, S. 60 Pick, C. G. 52 Standifer, K. M. 56 Planer, W. 24, 66 Stein, C. 55 Podhaizer, E. M. 65 Stevens, C. W. 62, 66 Polgar, W. E. 55, 57, 70 Stickler, L. 24, 66 Polikar, L. 58 Straiker, A. 36 Poltoratsky, V. 38 Streicher, J. M. 24, 59, 67 Porreca, F. 41 Su, R. B. 67 Porter, V. R. 43, 69 Su, Y. 67 Portugal, G. S. 62 Subrath, J. 58 Pradhan, A. A. 44, 52 Sugiyama, A. 53 Prather, P. L. 39 Sullivan, L. C. 45, 48, 53 Prisinzano, T. E. 24, 39, 49, 67 Sun, Y. G. 54 Proudnikov, D. 52 Purington, L. C. 43, 69 T Taverner, A. 48 R Taylor, A. M. W. 56 Ramesh, D. 36 Tokuyama, S. 56 Randesi, M. 51, 52 Toll, L. 55, 57, 70 Rasakham, K. 48 Tóth, G. 54 Rathore, M. 65 Tourwé, D. 41 Ray, B. 52 Traynor, J. R. 43, 46, 50, 61, 68, 69 Reed, B. 59 Tseng, A. 70 Reilley, K. J. 51, 59, 70 Tsisanova, E. 63 Reinscheid, R. 24, 25 Tung, L.-W. 38 Resendez, S. 31 Turner, E. M. 68 Rice, K. C. 35, 91 Rivero, G. 63 UV Rosethorne, E. M. 63 Ueno, K. 54 Ross, M. 54 Valtier, S. 68 Ross, N. C. 59, 67 Van den Eynde, I. 41 Roth, B. L. 40, 66, 69 VandenBerg, M. 50 Rubovitch, V. 52 Vardy, E. 66, 69 Runyon, S. 25 Varga, E. 41 Vargas, T. 68 74 INRC 2012 Kansas City, Missouri INRC Index Vasile, S. 67 Vasiljevik, T. 39 Vayssiere, P. 56 Vicchiarelli, M. 67 Vincelli, G. 60 von Zastrow, M. 61 Vuppala, P. K. 57

WX Wada, K. 53 Wadensten, H. 57 Wager-Miller, J. 36 Wakida, N. 54 Wang, Q. 46 Wang, Y.-J. 66 Watanabe, H. 57 Watkins, L. 27 Welch, S. 36, 37 Westkaemper, R. B. 69 Whipple, D. 67 Wilhelm, I 54 Williams, J. T. 60, 61, 64, 65 Wilson, L. L. 58 Withey, S. 63 Wu, N. 67 Wu, Y.-T. 54 Xu, R. 61 Xue, L. 50

Y Yakovleva, T. V. 51 Yamada, M. 53 Yamamoto, C. 54 Yan, H. T. 67 Yang, H.-Y. 49 Yasuda, D. 70 Yates, T. L. 69 Yeomans, L. 43, 69 Yuferov, V. 67

Z Zadina, J.E. 58 Zaveri, N. T. 70, 71 Zhang, L. 43 Zhang, N. 24, 66 Zhang, X. 58 Zhang, Y. 25, 26, 53, 56, 61, 65 Zhao, Y. 46 Zheng, H. 43 Zheng, J. Q. 67 Zhou, Y. 48 Zou, S, 33, 64, 66 Zyuzin, J. 52

INRC 2012 Kansas City, Missouri 75 2013 INRC Meeting: Cairns, Australia

July 14-18th

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