Ru-Rong Ji, PhD

Pain Research Division

Department of Anesthesiology

Department of Neurobiology

Duke University Medical Center

July 9, 2015, Ion Channel Retreat, Vancouver

Ion Channel and Pain Targets

Modulation of TRP channels by

resolvins in mouse and human

Omega-3 polyunsaturated fatty acids

Ji et al., Trends Neurosci, 2011

Powerful metabolic products

(Resolvins)

Chronic pain is a major heath problem

• Over 100 million Americans have chronic pain

• The total financial cost of is near 600 billion

a year in the US.

• Annual US expenditure on pain (medical costs

and lost wages) is higher than that for cancer,

heart disease, and diabetes combined.

• Co-morbidity: Chronic pain is associated with depression, anxiety, insomnia, etc.

• Prevention and treatment of this epidemic are equally important.

Pizzo & Clark, N Engl J Med., 2012

American Pain Society

Peripheral mechanisms of pain

Ji et al., Nat Rev Drug Disco., 2014

Nociceptor

Glial

mediators

p38-P

ERK-P

Injury

Microglia

Activation

Astroglia

activation JNK-P

Glutamate

AMPA NMDA TrkB

Primary afferents

Spinal cord dorsal horn neurons

BDNF Glu

GABA-A-R Gly-R

DRG

Inhibition

Excitation

Persistent pain

TNF-a

IL-1b

BDNF

CCL2

IL-1b

Tissue injury

&

Nerve injury

Central mechanisms of pain

Serhan et al., 2008; Nature Reviews Immunology

(RvE1)

(RvD1)

Charles Serhan

DHA

17R-Resolvin D Series

Acetylated COX-2

Aspirin:COX-2

LOX

17S-Resolvin D Series

Lipoxygenase mechanism

LOX

EPA

Aspirin:COX-2

P450

microbial

18R-H(p)EPE

LOX

RvD2

RvE1

RvD1

Biosynthesis of resolvins from PUFA Precursors

Resolvins are potent inhibitors of inflammation

in various animal models

Ji et al., Trends Neurosci, 2011

Vehicle

RvD1, 20 ng (60 pmol)

RvE1, 20 ng (60 pmol)

0

2

4

6

8

10

12

BL 2 h 4 h 24 h

Time after CRG

PW

L (

s)

* * *

Resolvins prevent CRG-induced heat hyperalgesia

Xu et al., Nat Med, 2010

0

20

40

60

Naive CRG CRG+

(RvE1, 20ng)

M

PO

activity

(U m

g1 p

rote

in)

* *

0

20

40

60

80

Pa

w v

olu

me

in

cre

ase

(% o

f B

L)

CRG CRG+

(RvE1, 20ng)

*

Paw edema

RvE1 inhibits CRG-induced inflammation

Neutrophil infiltration

0

5

10

15

20

25

30

Fo

ld o

f n

aïv

e c

on

tro

l

TNF-a

IL-1b

IL-6

MCP-1

CRG CRG+

(RvE1, 20ng)

MIP-1a * * * * *

# # # # #

Naive

CRG, 2 h

RvE1 + CRG, 2 h

3

1

2

4 5

P

P

P

3

1

2

4 5

P

P

P

3

1

2

4 5

P

P

P

a

b

c

Cytokine array

RvE1 inhibits cytokine/chemokine expression

after inflammation

No effect of RvE1 on basal pain

Broad actions of resolvins

Ji et al., Trends Neurosci, 2011

ChemR23 expression in macrophages and microglia

ChemR23 expression in neurons in the PNS and CNS

Ji et al., Trends Neurosci, 2011

ng/ml0.1 1 10Inhib

itio

n r

atio

of

ca

psa

icin

0.0

0.2

0.4

0.6

0.8

1.0

1.2

sec0 400 800

340/3

80

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8 IC50 = 3.2 ng/ml

RvE1

300 nM Capsaicin

10 ng/ml RvE1

50 mM KCl

Control Capsaicin RvE1 + Capsaicin

RvE1 blocks capsaicin-induced signaling in mouse DRG neurons

0

10

20

30

40

50

1 2 3 4 5 6 7 8 9 10

Time (min)

Capsaicin

(500 ng, i.t)

Capsaicin +

RvE1 (10 ng, i.t.)

Du

ratio

n o

f n

ocife

nsiv

e

b

eh

avio

r (s

) * *

*

* *

Patch clamp

recording

6 s

Control

Capsaicin (100 nM)

RvE1 (1 ng ml) + Capsaicin

20

pA

RvE1 blocks TRPV1-mediated

spinal synaptic transmission & pain

0

0.5

1

1.5

2

2.5

3

Frequency

Amplitude

+ Capsaicin (100 nM)

Ra

tio

of

sE

PS

C

*

#

Resolvins are potent endogenous inhibitors of TRPV1/A1

Capsaicin and mustard oil-induced pain

Resolvin E1 (RvE1) blocks TRPV1 current in mouse DRG neurons

GAPDH

TRPV1

1 2 3

ChemR23

4 5 6 7 8 9 10 N

TRPV1-expressing HEK cells

TRPV1 and ChemR23-coexpressing HEK cells

Capsaicin (100 nM)

RvE1 (10 ng/ml)

ChemR23 is sufficient to mediate RvE1’s effect in HEK 293 cells

100 pA

1 min

RvE1 fails to inhibit TRPV1 signaling in b-arrestin 2 KO mice

WT b-arrestin 2 KO

100 pA

1 min

CAP (100 mM) RvE1 (10 ng/ml)

Control RvE1 (10 ng/ml), 15 min

barr2 barr2

20 mm

Minutes after capsaicin injection

0

4

8

12

16

20

0 1 2 3 4 5 6 7 8 9 10

CKO

WT

Nu

mb

er

of

flic

kin

g/l

ick

ing

/bit

ing

1 2 3 4 5 N M

WT barr2 CKO

GAPDH

Nav1.8

barr2

barr1

1 2 3 4 5 N M

TRPV1 signaling is potentiated in b-arrestin 2 CKO mice

TLR7

1 2 3 4 5 6 7 8 9 10 neg

TRPV1

GAPDH

TRPA1

Single-cell PCR

let-7b (30 µM) let-7b (30 µM)

HC030031(100 µM)

10 s

10

0 p

A

30 mm

Ion channel research on Primary sensory neurons of human DRGs

TRPV1 / ChemR23

100 mm

Co-expression of TRPV1 and ChemR23 in human DRG neurons

25 µm 1 n

A

20 s

CAP

RvE1 (100 ng/ml)

Capsaicin. Cap (100 nM)

RVE1

10 n

g/ml

100

ng/ml

100

ng/ml

0

20

40

60

80

100

120

n = 12 / 17

n = 5 / 17n = 3 / 3

% o

f 1

st C

AP

resp

on

se

CAP RvE1

RvE1 inhibits TRPV1 currents in human DRG neurons

• Anti-inflammatory actions

• Pro-resolution actions

• Block TNF-a synthesis and signaling

• Modulate synaptic plasticity

• Powerful endogenous inhibitors of TRPV1/A1

• No effects on basal pain

• Unknown side effects

Limitations

• Unstable

• Difficult to measure

• Expensive to produce

Multiple beneficial effects of resolvins

DHA

17R-Resolvin D Series

Acetylated COX-2

Aspirin:COX-2

LOX

17S-Resolvin D Series

Lipoxygenase mechanism

Epoxidation

NPD1 Protectins

Neuroprotectins

LOX

EPA

Aspirin:COX-2

P450

microbial

18R-H(p)EPE

LOX

RvD2

RvE1

RvD1

Biosynthesis of Protectins from PUFA Precursors

Acknowledgements Temugin Berta

Supported by

NIH grants

R01: NS67686

R01: DE17794

R01: NS54932

R01: DE22743

R01: NS89479

Pfizer/Neusentis/Icagen

Zhen-Zhong Xu Ling Zhang Chul-Kyu Park Ning Lu

Charles Serhan Seok-Yong Lee