New Approaches in the Development of Opioids and Other Analgesics. Thomas E. Prisinzano, PhD Department of Medicinal Chemistry School of Pharmacy

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1 New Approaches in the Development of pioids and ther Analgesics Thomas E. Prisinzano, PhD Department of Medicinal Chemistry School of Pharmacy

2 Disclosures The University of Iowa Research Foundation has filed an issued patent and several patent applications related to the material presented. U. S. Patent #7,728,001 issued June 1, TEP is a co-founder and consultant for Mencuro Therapeutics, Inc. TEP has received compensation for reviewing grants from the National Institutes of Health and Department of Defense in the past year 2

3 utline Background Selected Chemistry Selected Pharmacology Future Directions Conclusions 3

4 verview of pioid Receptor Pharmacology Receptors Cellular Effects Systemic Effects MP GIRK Acute Chronic DP Ca 2+ Pain Inhibition Tolerance KP NT Release Reward Dependence NP camp CH 3 N H N N CH 3 N CH3 H H 4

5 Life Cycle of an pioid Receptor A Morphine Methadone Fentanyl A Desensitization -arrestin GRK Resensitization Internalization 5

6 Summary of b-arrestin-2 K Responses to Morphine Enhanced and prolonged morphine antinociception (Hot plate and Tail Flick) Loss of morphine antinociceptive tolerance in hot plate (tail flick: significantly attenuated No difference in physical dependence (WT & K mice exhibit same withdrawal symptoms) Reduced respiratory depression and constipation 1. Bohn LM, Lefkowitz RJ, Gainetdinov RR, Peppel K, Caron MG, Lin FT Science 1999;286: Bohn LM, Gainetdinov RR, Lin FT, Lefkowitz RJ, Caron MG Nature 2000;408: Bohn LM, Lefkowitz RJ, Caron MG J. Neurosci. 2002;22: Bohn LM, Gainetdinov RR, Sotnikova TD, Medvedev I, Lefkowitz RJ, Dykstra LA, Caron MG. J. Neurosci. 2003;23: Raehal KM, Walker JK, Bohn LM. J. Pharmacol. Exp. Ther. 2005;314:

7 b-arrestin-2 (barr2) as a Drug Target ne approach to enhance morphine analgesia and decrease morphine tolerance would be to inhibit barr2 directly No known inhibitors Lack of receptor selectivity An alternative approach is to develop agonists that do not induce MP - barr2 interactions or subsequent receptor internalization Identify and develop promising scaffolds as analgesics 7

8 Design Hypothesis Novel pioids A Morphine Methadone Fentanyl A Desensitization -arrestin GRK Tolerance Constipation Respiratory Failure Signaling Resensitization Internalization Antinociception 8

9 Approach Salvinorin A is a unique kappa opioid receptor (KP) agonist that promotes 40-fold less efficient KP internalization compared to U50,488H but is antinociceptive in vivo Suggests that salvinorin A binding is conducive to G-protein signaling but resistant to internalization-mediated regulation Potential to develop analogues with altered regulation Develop MP agonists derived from salvinorin A 9

10 Pharmacological Evaluation of Herkinorin H H H 1. Na2C3, MeH 2. BzCl, NEt3, DMAP, CH2Cl2 H H H C 2 Me C 2 Me KP Ki = 1.9 ± 0.2 nmdp Ki = 5,790 ± 980 nm MP Ki = partial inhibition KP EC50 = 40 ± 10 (Emax = 120)TF ED50 = 1.98 mg/kg/scppq ED50 = 0.59 mg/kg/schp ED50 = Inactive at 1, 28% at 10 and inactive at 30 mg/kg/sc KP Ki = 90 ± 2 nm DP Ki = 1,170 ± 60 nm MP Ki = 12 ± 1 nm KP EC50 = 1320 ± 150 (Emax = 140) MP EC50 = 500 ± 140 (Emax = 130) Harding WW, Tidgewell K, Byrd N, Cobb H, Dersch CM, Butelman ER, Rothman RB, Prisinzano TE. J Med Chem. 2005;48: Tidgewell K, Groer C, Harding WW, Lozama A, Schmidt M, Marquam AL, Hiemstra J, Dersch CM, Partilla JS, Rothman RB, Bohn LM, Prisinzano TE. J Med Chem. 2008;51:

11 Immunofluorescence (% Control) Immunofluorescence of Cell Surface Expression M Herkinorin 1 M DAMG Time (min) Immunofluorescence of cell surface MP expression reveals that DAMG, but not herkinorin, leads to a loss of cell surface expression following 2 hours of drug treatment. Data are normalized to the control in which no agonists were added. Non-specific secondary antibody interactions were subtracted from each point. Data are the average of 4 samples/timepoint. Two-way ANVA analysis reveals that the curves differ (P<0.001) and that the DAMG treated cells display less surface receptors at each time point as determined by Bonferoni post-hoc analysis (p<0.001 at each time point). Groer CE, Tidgewell K, Moyer RA, Harding WW, Rothman RB, Prisinzano TE, Bohn LM. Mol Pharmacol. 2007;71:

12 Herkinorin does not induce arr2-gfp translocation in HEK293 cells overexpression GRK2. Cells were transiently transfected with haemagglutinin (HA-N-terminus) tagged mr (10 µg cdna) and arr2-gfp (2 µg cdna) and GRK2 (10 µg cdna). Herkinorin treatment for 5 or 10 or 30 minutes does not lead to arr2-gfp recruitment to the receptor. However, morphine treatment of the same cells treated for 30 minutes with herkinorin promotes arr2-gfp translocation indicating that the cells do express the receptor as well as the GRK2. Multiple cells were viewed following two separate transfections; shown are representative cells. Laura Bohn 12

13 Prolactin ( ng/ml) Herkinorin 0.32 mg/kg alone +Quaternary naloxone 3.2 mg/kg PT +Nalmefene 0.01 mg/kg PT Q/N Time (min) Herkinorin was partially blocked by a relatively low dose of nalmefene (0.01 mg/kg) and was blocked by the peripherally selective antagonist, quaternary naloxone. Butelman ER, Rus S, Simpson DS, Wolf AKH, Prisinzano TE, Kreek MJ. J Pharmacol Exp Ther. 2008;327:

14 In vivo Evaluation of Antinociception g Sprague Dawley Rats Acute Formalin Test 100 L injection of 1.25% formalin or drug into right hindpaw 250 L injection of antagonists into back of neck Vehicle of saline or DMA Count the number of flinches and separate into 5 minute bins Chronic Formalin Test Dosed for 4 days alternating dorsal and plantar surface and tested on the 5 th day Generate tolerance using 75 mg/kg of morphine delivered by minipump 14

15 Number of Flinches per 5 min A. Vehicle B. C. Herkinorin 10 mg/kg 120 Herkinorin 10 + Naloxone 10 Morphine 10 mg/kg vehicle Vehicle Day 1 Vehicle Day 5 Herkinorin Day 1 Herkinorin Day Vehicle Herkinorin Morphine tolerant + Herkinorin Morphine tolerant + Morphine morphine Time (minutes) Time (minutes) Time (minutes) Herkinorin effects are partially reversed by Naloxone The same dose of Herkinorin remains effective after 5 days of treatment Herkinorin is effective in Morphine tolerant rats Lamb K, Tidgewell K, Simpson DS, Bohn LM, Prisinzano TE. Drug Alcohol Depend. 2012;121:

16 A New Era Has Begun Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis WI, Kobilka BK, Granier S. Nature Mar 21. doi: /nature

17 Conclusions Illustrates the ability of selecting or designing novel agents that differentially activate regulation pathways of a single receptor Herkinorin is a novel chemotype for MP receptors that possesses unique pharmacological properties Active in rats and nonhuman primates Appears to be have reduced tolerance Natural products are excellent leads for opioid drug discovery Structure-based drug design for MP receptors is now possible 17

18 NIDA Richard B. Rothman Christina M. Dersch Heng Xu John S. Partilla Xiaoying Wang John M. Rutherford Research Triangle Institute Hernán A. Navarro Keith Warner Brian Gilmour Holden Laboratories Kenneth G. Holden Acknowledgments Rockefeller University Eduardo R. Butelman Mary Jean Kreek Johns Hopkins University Roland R. Griffiths Matthew W. Johnson Amy K. Goodwin Universidad Nacional Autónoma de México Baldomero Esquivel Alfredo rtega Scripps Florida Laura M. Bohn Robert Moyer Kirsten Raehal Chad Groer Victoria University of Wellington Bronwyn Kivell Susan Schenk Università degli Studi di Palermo Guiseppe Savona Gianfranco Fontana CSIC, Madrid Benjamin Rodríguez 18

19 Acknowledgments 2 Current Members Michael Caspers Katherine Prevatt-Smith Andrew Riley Tamara Vasiljevik Mark Madhavan Marci Seuferling Molecular Structures Group Todd D. Williams Justin T. Douglas Victor W. Day Financial Support R01DA R01DA018151S1 T32GM