The promises of opioids: Future and presentGavril W. Pasternak, MD PhD
Anne Burnett Tandy Chair of Neurology
Laboratory Hear, Molecular Pharmacology Program
Memorial Sloan-Kettering Cancer CenterandProfessor of Pharmacology, Neurology & Neuroscience and Psychiatry
Weill Cornell Medical College

Dedication
E. Leong Way(1915-2017)
This talk is dedicated to Eddie Way

Eddie provided a foundation for opioid pharmacology
His work and insights have brought us to where we are today

Pain“And only YOU can hear this whistle?”

The study of pain is difficult due to its
subjective nature and theunpredictable contributions of genetics

Opioid analgesia
Perception is a corticaland subcortical process

Activation ofPeripheral
Nociceptors

Opioid are active:

Descending
Modulatory
Pathway

Ascending
Pathway• Spinally• Supraspinally• Peripherally

There is synergy among sites:• Spinal/supraspinal• Systemic/spinal

Synapse in dorsal horn andascend through neo- andpaleospinothalamic pathways.

4Complexity of opioid analgesia
Genetic backgrounds impact potency
100

Morphine 5 mg/kg

80
60
40
20CXBK

Swiss Webster

C57/+HSC57/bgj

CD-1

0Balb/c

Analgesia (% of mice)

Genetic backgrounds impact selectivity

Mouse Strain

5

Complexity of opioid analgesia
Genetic backgrounds impact potency

Genetic backgrounds impact selectivity

100*P <0.001

75

50250*

CD-1
CXBK

6

Why Mu Opioids Differ?

•Different pharmacokinetics•

Different metabolic profile•

Differential function activation of the receptor (Biased Signaling)•

Differential activation of subtypes of receptors

Defining Opioid Receptors
Opioid receptors were originally defined by ligands.
• 1965: ‘Opioid’ defined by sensitivity to naloxone
Subtypes of opioid receptors were defined by agonists:• 1967: Receptor Dualism: M & N: morphine & nalorphine• 1976: Mu and kappa: morphine and ketocyclazocine• 1977: Delta: Enkephalins• 1983: Kappa: U50,488H
Subsequent studies utilized antagonists:• 1980: Mu: β-Funaltrexamine (β-FNA)• 1980: Mu subtypes: naloxazone/naloxonazine• 1987: Kappa: norBinaltorphimine (norBNI)• 1988: Delta: naltrindole
After the cloning of the mu, delta and kappa receptor genes,
subtypes have been defined molecularly at the genetic level

G-Protein Coupled Receptors• Traditional GPCR have 7 transmembrane (TM) domains

Extracellular• Splicing, both 5’ and 3’, yields both 7TM and truncatedforms

NH2• Evidence now shows that these truncated forms may be
important physicologically and pharmacologically• GPCRs interact with G-proteins

CellMembrane– Both α and βγ subunits are released by agonist and eachhas its own transduction pathways• Agonists induce phosphorylation of the receptor byreceptor kinases, leading to β–arrestin binding, which inturn induces a separate transduction cascade involvingMAPK, Src and Akt• Biased signaling results from the differences in theability of an individual ligand to activate G-protein vs β–arrestin pathways

COOH

Intracellular

The future of opioids: Time to abandon pessimism

Once dismissed as an impossibility, approaches are arising to develop mu
opioids lacking many of the adverse effects of current agents, yielding safer
and more efficacious compounds.

The Holy Grail of Opioid research

Heroin: “The non-addictive cure for
the Soldiers Disease”(morphine addiction)

The essence of opioid therapy is selectivity

Selectivity can be achieved by:Route/site of administration
Topical, epidural/intrathecal
Peripherally restricted (agonist or antagonists)

Functional bias at the receptor(Biased Signaling)

Allosteric modulation of transduction(PAM)

Alternative receptor targets(Receptor subtypes)

The essence of opioid therapy is selectivity

Selectivity can be achieved by:Route/site of administration
Topical, epidural/intrathecal
Peripherally restricted (agonist or antagonists)

Functional bias at the receptor(Biased Signaling)

Allosteric modulation of transduction(PAM)

Alternative receptor targets(Receptor subtypes)

Biased Signaling
NH2

ExtracellularCellMembrane
Intracellular
COOH

G-proteinβ-arrestin

The ratio of G-protein/β-arrestin provides a measure of ‘bias’β-Arrestin2 knockout mice
Enhance morphine analgesia

Diminish morphine respiratory depression

Science 286:2495, 1999
JPET 314:1195, 2005

Diminish morphine tolerance

Nature 408:720, 2000

Buprenorphine and β–arrestin-2 recruitment• Buprenorphine does not recruit β–arrestin2 and antagonizes the DAMGO recruitment• At equianalgesic doses in mice, buprenorphine has less respiratory depression than morphineTRV130

JPET 344:708, 2013
R e s p ir a t o r y R a t e

R e s p ir a t o r y R a t e( P e r c e n t o f B a s e li n e )1201008060

400102030405060

T im e ( m in )S a lin e , s .c .

T R V 1 3 0 ( 1 .5 m g /k g )M o r p h i n e ( 2 0 m g /k g )T R V 1 3 0 ( 0 .6 m g /k g )The essence of opioid therapy is selectivity

Selectivity can be achieved by:Route/site of administrationTopical, epidural/intrathecal
Peripherally restricted (agonist or antagonists)

Functional bias at the receptor(Biased Signaling)

Allosteric modulation of transduction(PAM)

Alternative receptor targets(Receptor subtypes)

Positive Allosteric Modulators• No activity alone• Potentiate the activity of orthosteric agonists• Enhance the actions of physiologically released endogenous ligand• Requires appropriate release of endogenous ligand• Advantage of use with exogenous agonists not clear

Burford et al. Br J Pharmacol 172:277, 2015

Crystal Structure of the mouse mu opioid receptor

Cross section

Top20
Manglik et al, Nature, 201220
PMID: 22437502

The essence of opioid therapy is selectivity

Selectivity can be achieved by:Route/site of administration
Topical, epidural/intrathecal
Peripherally restricted (agonist or antagonists)

Functional bias at the receptor(Biased Signaling)

Allosteric modulation of transduction(PAM)

Alternative receptor targets(Receptor subtypes)

The scientific method

Splicing of the Oprm1 gene
E11 Promoter
11 12Exon

E1 Promoter
1 13 145
2 3 15 e d c b a4106789

Full Length 7TM variants• Exon 1 promoter*exon 11• 3’ splicing - unique sequences...

• Mice have 24*…...

• Lengths vary from 1 – 88 aa• Humans have 12• Rats have 13

6TM Exon 11 associated variants• Exon 11 promoter• 5’ splicing eliminates exon 1• Unique exon 11 sequence (~25 aa)
before exon 2 in most variants

1TM variants• 3’ splicing - unique sequences• Mice have 5 (1-127 aa after exon 1)• Humans have 4 (1-90 aa after exon 1)• Rat have 2 (1 - 127 aa after exon 1)...

• Exon 1 promoter• 3’ splicing - unique sequences• Mice have 5• Humans have 3• Rats have 2

Functional Consequences of 3’ Splicing
Splice siteexon exon

D if f e r e n c e S c o r e ( T e s t - P r e )C o n d itio n e d P la c e P r e fe r e n c e
300(E x o n 7 tr u n c a tio n )

200

*1000

WT

T r u n c a t io n

Exon 7 variants facilitate reward and tolerance
Exon 4 variants diminish tolerance

Influence of 7TM variant 3’ splicing on biased signaling
G-protein Biased

Arrestin Biased

Compares a specific drug
against multiple variant

Compares multiple drugs
against a specific variant

Bias Factor+50-50DAMGO
Morphine
β-Endorphin
Methadone
Fentanyl
Levorphanol

MOR-1
1.0
-1.5
-2.1
2.1
-4.4-2.6

MOR-1A MOR-1B1
1.0
1.0
1.2
-1.2
1.0
1.4
2.5
-1.9
-2.5
-6.0
1.6
-1.3

MOR-1E
1.0-11.9
1.1
1.8
-3.0
-1.3

MOR-1O
1.0
2.1
1.1
1.7
-4.3
95.9

DAMGO
Morphine
β-Endorphin
Methadone
FentanylLevorphanol

MOR-1
1.0
1.0
1.0
1.0
1.0
1.0

MOR-1A MOR-1B1-1.9-3.5-1.1-2.8
1.1-1.1-1.6-13.4-1.1-4.7
2.1-1.7

MOR-1E
1.6-5.0
3.7
1.4
2.3
3.1

MOR-1O-10.2
-3.3
-4.3
-12.4
-10.0
24.4

35S-GTPγS binding and β-arrestin-2 bias was calculated for each drug and for each variant and normalized toDAMGO for each variant (top) or normalized to the specific drug and compared across the variants (bottom).

MOR-1DBass

MOR-1CClarinet

MOR-1B1
VIolinMOR-1A
Trumpet

MOR-1
Sax

PotencyVolumeMu opioid analgesia

Drug 2

Drug 1Classifying mu opioid actions

Exons7TM126TM
3112

311TM

SpicedSpicedSpicedLost

Retained

Lost

Exon 11Retained

Lost

Lost

Exon 1/11

Lost

Retained

Lost

Exon 1 KO(Pintar mice)

Knockout models of the mu opioid receptor can be used to geneticallydefine the roles of different set of variants in a drugs activity

27

Sensitivity of mu opioids to loss of 6TM variants

ED50 (mg/kg, s.c.)

7TM

7TM + 6TM6TMShiftWTExon 11 KO

Morphine

1.6

2.6

1.6

Methadone

1.5

1.8

1.2

Fentanyl

0.6

3.2

5

Levorphanol

530

6

Butorphanol

12.4

200

16

Buprenorphine

0.2>10>50IBNtxA

0.53> 20>35-fold

Knockout models indicate:• Morphine and methadone analgesia are independent of 6TM• IBNtxA analgesia is independent of 7TM• Other drugs involve both 6TM and 7TM for analgesia

R a d ia n t h e a t
T a il f li c k80604020W ild T y p eT r ip le K O

00 .1

180
604020AssayED50mg/kg

Tail-Withdrawal (49oC)

0.78

Hot-Plate (54°C)

0.53

Paw-Withdrawal (15%)

0.27

Formalin (late phase)

0.32

Complete Feund’s Adjuvant(mechanical allodynia)

0.35

Spared Nerve Injury(mechanical allodynia)

0.35

0W

OT

IB N tx A ( m g /k g )100

Radiant Heat
Tailflick

E1

1K

100

A n a lg e s ia ( % o f m ic e )A n a lg e s ia

( % o f m ic e )IBNtxA Analgesia

Majumdar al., PNAS 108: 19776, 2011

Weiskopf et al., Pain 155:2063, 2014IBNtxA analgesia• Independent of traditional 7TM mu, delta and kappa receptors• Totally dependent upon 6TM exon 11-associated variants• Is more effective in neuropathic and inflammatory than thermal pain models

Rescue of IBNtxA analgesia
E 1 /E 1 1 K O

W ild t y p e

100* p < 0 .0 0 66040****F e n ta n y l

B u p r e n o r p h in eL evo rp h an o l

0M o r p h in e

20

IB N tx ALentivirus/mMOR-1G vector restores expression

80

IB N tx AWildtype

A n a lg e s ia ( % M P E )Dorsal Horn of Spinal Core
Exon 1/11 KOVectormMOR-1Gw it h L e n t i- m M O R - 1 G

Lentivirus/mMOR-1G vector restores
only IBNtxA analgesia
J Clin Invest 125:2626, 2015

IBNtxA (3-Iodobenzoyl-6β-naltrexamide)

0* p < 0 .0 500153045

T im e (m in )•No respiratory depression

N a lo x o n e

2001000-100Morphine25**p<0.05

IBNtxA*300

Saline** p < 0 .0 0 1

50

Difference Score (Test - Pre)*75

1 0 m g /k g**

1 m g /k g*

IB N tx A

C o n tr o l*

C h r o n ic

1 m g /k g

*C h r o n ic
M o r p h in e

C o n tr o l*

J u m p s /1 5 m inR e s p ir a t o r y R a t e ( % B a s e lin e )

100

100

50

Conditioned Place
Preference

Precipitated
Withdrawal

S a lin e
IB N t x A
M o r p h in e

N a lo x o n e•No physical dependence•No reward behavior

Pharmacological Profiles of 7TM and 6TM Compounds

Morphine(7TM)

IBNtxA(6TM)

Analgesia

++++

++++

Thermal++++++++Inflammatory++++++

Neuropathic+++++

Respiratory depression

++++

-

Constipation+++++Sedation

++++

+/-

Reward++++-Physical Dependence

++++

-

Straub tail++-32

Tolerance

A fixed dose of opioid is administered chronicallyProlonged Mophine Dosing

M o r p h in e d o s e (q 1 2 h )C o n tro l

20

5 m g /k g

( E D 5 0 m g /k g , s .c .)M o r p h in e

1 0 m g /k g
2 0 m g /k g

154 0 m g /k g

1050010203040

M o rp h in e T r e a tm e n t (D a y s )50Opioid Tolerance

PreFrontal
cortexStriatum

ThalamusHypothHippo

PAG

BSSp Cord

Fold change in mRNA levels relative to saline

40

540

40

40

40

40

40

E1-2

7182352

13

55

4

MOR-1A

1683

1240

24

10

6

6

MOR-1B1

374

425

47

714

5

MOR-1B2

2

33

620

48

4108

MOR-1B3
MOR-1B4
MOR-1C
MOR-1D

236942
35
45
150

11625
43

18185831211101117438157
107632311MOR-1H

152

1233

22531

4

MOR-1i

146

72723

9102

MOR-1J

556

634

20

10145MOR-1O

226

226

513

8

7

MOR-1P

426

72049

4118

Morphine(mg/kg)

Summary
Cloning the opioid receptors has permitted the transition to a molecularclassification of receptors and their subtypes
The mu opioid receptor gene Oprm1 undergoes extensive splicing to generate
three major classes of variants:•Full length 7TM variants•Truncated 6TM variants•Truncated 1TM variants
Mu opioids can be classified into three categories:•Dependent upon E1, but not E11 variants
Morphine, Methadone•Dependent upon E11, but not E1 variants
IBNtxA•Dependent upon both E1 and E11 variants
Buprenorphine, fentanyl, M6G , levorphanol

37

Laboratory of Molecular Neuropharmacology
Gavril Pasternak, MD PhD

Ying-Xian Pan, PhD

Susruta Majumdar, PhD

Molecular Pharmacology
Steve Grinnel, PhD
Gina Marrone, PhD
Ankita Narayan, PhD
Valerie Le Rouzic
Amanda Hunkele

Molecular Biology
Zhi-Gang Lu, PhD
Jin XuMing-Ming Xu

Chemistry
Andras Varsadi, PhD
Rajendra Uprety, PhD

Peter F. McManus Charitable Trust
Mayday Foundation

Collaborators
Cornell Medical College
Charles Inturrisi, PhD
Rutgers UniversityJohn Pintar, PhD
Michael Ansonoff, PhD
Long Island University
Grace Rossi, PhD
McGill University
Jeffrey Mogil, PhD
Jeffrey Weiskopf, PhD