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.

6

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

7

Design Hypothesis

8

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

9

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

10

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

15

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.

16

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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