new approaches in the development of opioids and other...
TRANSCRIPT
Thomas E. Prisinzano, PhD
Department of Medicinal Chemistry
School of Pharmacy
New Approaches in the Development of
Opioids and Other Analgesics
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, 2010.
• 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
Outline
Background
Selected Chemistry
Selected Pharmacology
Future Directions
Conclusions
3
Receptors Cellular Effects Systemic Effects
MOP ↑ GIRK Acute Chronic
DOP ↓ Ca2+ Pain Inhibition Tolerance
KOP ↓ NT Release Reward Dependence
NOP ↓ cAMP
Overview of Opioid Receptor Pharmacology
O
N
OH
H
CH3
HO
N
N
O
O
NCH3
CH3
4
Life Cycle of an Opioid Receptor
5
Desensitization
GRK
-arrestin
A A
Internalization
Morphine
Methadone
Fentanyl
Resensitization
Summary of b-Arrestin-2 KO 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 & KO 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;286:2495-2498.
2. Bohn LM, Gainetdinov RR, Lin FT, Lefkowitz RJ, Caron MG Nature 2000;408:720-723.
3. Bohn LM, Lefkowitz RJ, Caron MG J. Neurosci. 2002;22:10494-10500.
4. Bohn LM, Gainetdinov RR, Sotnikova TD, Medvedev IO, Lefkowitz RJ, Dykstra LA, Caron MG. J. Neurosci.
2003;23:10265-10273.
5. Raehal KM, Walker JK, Bohn LM. J. Pharmacol. Exp. Ther. 2005;314:1195-1201.
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b-Arrestin-2 (barr2) as a Drug Target
• One 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 MOP - barr2
interactions or subsequent receptor internalization
– Identify and develop promising scaffolds as analgesics
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Design Hypothesis
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Desensitization
GRK
-arrestin
A A
Internalization
Signaling
Antinociception
Morphine
Methadone
Fentanyl
Resensitization
Tolerance
Constipation
Respiratory Failure
Novel
Opioids
Approach
• Salvinorin A is a unique kappa opioid receptor (KOP) agonist that promotes 40-fold
less efficient KOP 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 MOP agonists derived from salvinorin A
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Pharmacological Evaluation of Herkinorin
1. Na2CO3, MeOH
2. BzCl, NEt3, DMAP, CH2Cl2
KOP Ki = 1.9 ± 0.2 nMDOP Ki = 5,790 ± 980 nM
MOP Ki = partial inhibition
KOP 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
KOP Ki = 90 ± 2 nM
DOP Ki = 1,170 ± 60 nM
MOP Ki = 12 ± 1 nM
KOP EC50 = 1320 ± 150 (Emax = 140)
MOP 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;48:4765-4771.
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;51:2421-2431.
O
O
O
CO2Me
HH
H
O
O
O O
O
O
CO2Me
HH
H
O
O
O
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Immunofluorescence of cell surface MOP
expression reveals that DAMGO, 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/time-
point. Two-way ANOVA analysis reveals that the
curves differ (P<0.001) and that the DAMGO
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;71:549-557.
Immunofluorescence of Cell Surface Expression
0 30 60 90 12040
60
80
100
120
1 M DAMGO
10 M Herkinorin
Time (min)
Imm
un
ofl
uo
rescen
ce
(% C
on
tro
l)
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Herkinorin does not induce arr2-GFP translocation in HEK293 cells
overexpression GRK2. Cells were transiently transfected with haemagglutinin
(HA-N-terminus) tagged mOR (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
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;327:154-160.
Q/N 5 15 30 60 90 120
0
100
200
300
400
+Nalmefene 0.01 mg/kg PT
Herkinorin 0.32 mg/kg alone
+Quaternary naloxone 3.2 mg/kg PT
Time (min)
Pro
lacti
n (
ng
/ml)
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In vivo Evaluation of Antinociception
• 250 – 275 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 5th day
– Generate tolerance using 75 mg/kg of morphine delivered by minipump
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Lamb K, Tidgewell K, Simpson DS, Bohn LM, Prisinzano TE. Drug Alcohol Depend. 2012;121:181-188.
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
0 10 20 30 40 50 60
0
20
40
60
80
100
120
Herkinorin
Morphine tolerant + Herkinorin
Vehicle
Morphine tolerant + Morphine
Time (minutes)
A. B. C.
0 10 20 30 40 50 60
0
20
40
60
80
100
120
Herkinorin 10 + Naloxone 10
Morphine 10 mg/kg
Vehicle
Herkinorin 10 mg/kg
Time (minutes)
Nu
mb
er
of
Flin
ch
es p
er
5 m
in
0 10 20 30 40 50 60
0
20
40
60
80
100
120
Vehicle Day 1
Herkinorin Day 1
Vehicle Day 5
Herkinorin Day 5
Time (minutes)
vehicle
morphine
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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. 2012 Mar 21. doi: 10.1038/nature10954.
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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 MOP 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 MOP receptors is now possible
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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
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 Ortega
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
Acknowledgments
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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
Acknowledgments – 2
R01DA018151
R01DA018151S1
T32GM008545
Financial Support
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