Kalpna Gupta, PhD

Vascular Biology Centre

Division of Hematology, Oncology and Transplantation

Department of Medicine

Co-Chair, Tumor Microenvironment, Masonic Cancer Center

Co-Chair, Cellular and Molecular Engineering

University of Minnesota Medical School

Minneapolis, MN

Principal Investigator

Excellence in Hemoglobinopathies Research Award for Sickle Cell Disease,

NHLBI

Promising strategies to treat pain in sickle

cell disease

SCD ASCAT London, Oct 5, 2016

Targeting mechanisms of nociception in sickle cell

disease

Brain

De

sce

nd

ing

path

way?

Asce

nd

ing

pa

thw

ay

Amplification and central sensitization

• “Crises”

• Hypoxia-ischemia reperfusion injury

• Inflammation

• Hemolysis

• Organ damage

Nociceptor sensitization

SK

IN

Spinal

Cord

DRG

PAIN

TLR4

Glial

activation

Mast cell

activation

TNF𝛼

IL-6

PGE1

SP SP

CGRP

Peripheral

nerve

fibers

ROS

SP

Phospho-p38MAPK

Aich A, Beitz A & Gupta K, 2016.

In, Sickle Cell Disease, Inusa B (Ed)

Peripheral Central

Mast cell activation leads to nociceptor activation

and pain

6

Vincent et al. “Plenary Paper” Blood 2013;122:1853-62. Editorial Commentary by Kutlar A “GLEE-ful for sickle pain?”

Higher TLR4 on cutaneous

mast cells of sickle mice

TLR4 activation is involved

in acute lung injury (Ghosh

et al., J Clin Invest 2013)

and endothelial activation

(Belcher et al., Blood 2014).

Imatinib

Chronic myeloid leukemia and sickle cell disease: could Imatinib prevent vaso-occlusive crisis?

Stojanovic et al., Br J Hematol 2011

Homozygous SCD with CML; 5 VOCs in one year Imatinib Tx led to rapid decrease in WBC counts and NO VOC “Each time that the patient forgot to take imatinib for several days, VOC recurred after 2 or 3 days, and stopped immediately when the patient took imatinib again.” “The recurrence of VOC after discontinuation of imatinib was not explained by the recurrence of increased WBC counts, nor by changes in haemoglobin concentration or HbF level, or haemolysis tests, which suggests that another mechanism was involved in the prevention of acute VOC due to sickle cell disease. Thus, tyrosine kinase inhibitors should be added to the list of potential treatments for SCD..”

†† ††

† † †

††

†† ††

*

*

*

* *

*

* P< 0.05 ** p< 0.001 vs. vehicle † P< 0.05, †† p< 0.001 vs. baseline

PW

F –

VF

(1

.0g

fib

er)

Treatment Post-treatment

Mechanical

*

*

*

*

*

*

*

*

*

*

*

*

*

more

pain

less pain 0

2

4

6

8

10

0 BL 1 2 3 4 5 1 2 3 4 5 6 7 8

Vehi…

1 2 3 4 5

Pharmacological Inhibition of TLR4 with TAK242 Ameliorates Hyperalgesia

in a Time Dependent Manner in Sickle Mice

Model for acute pain: Hypoxia/Reoxygenation

H0

Pain testing

baseline (BL)

3h Hypoxia

8% O2 and 92% N2

H

1

H

2

H

3

H4

Pain testing (H/R)

1h

Reoxygenation

At room air

(Cain et al., BJH 2011)

Day 7

Pain testing,

Neurogenic

Inflammation,

Mast Cell Activation

*P< 0.05, ** P< 0.01 vs. vehicle ; † P< 0.05, †† P< 0.01 vs. baseline # P< 0.05, ## P< 0.01 vs. Tx ; $ P< 0.05, $$ P< 0.01 vs. H/R

TAK242 Pretreatment Decreased Hypoxia/Reoxygenation-

evoked Pain and Accelerated Recovery

day 5 days TAK

Genetic deletion of TLR4 reduces cold sensitivity

in sickle mice

Shorter PWL = Increased Pain

* p<0.05, ** p<0.001

Higher PWF = Increased Pain

Heat Cold Cold

TLR4 deletion reduces hypoxia/reoxygenation evoked Injury

hyperalgesia in sickle mice SCD

* p< 0.05, ** p<0.005; vs. BL

† p<0.005 , vs. HbSS-BERK

¶ p<0.05, vs. H/R

Lower Grip force= More Pain

Pharmacological inhibition and genetic deletion of TLR4 reduce

chronic and acute pain. Therefore, therapies targeting TLR4

inhibition may be potentially beneficial in ameliorating sickle

pathobiology and pain.

Nociceptin opioid receptor (NOP/R) as a

potential target to treat pain in SCD

Small-molecule NOP/R Agonist Partial Mu opioid receptor agonist agonist.

However, selectivity for NOP/R is 20 fold higher than mu.

NOP/R agonists show analgesic effect in acute and chronic pain models (Zaveri et al., JPET 2009 and Zaveri et al., JPET 2011).

NOP /MOP agonist drugs in Phase III clinical trials for chronic pain.

AT-200

Zaveri et al. J. Med. Chem. 2004

AT-200

Veh

AT-200 reduced chronic and hypoxia/reoxygenation-evoked hyperalgesia

Lower Threshold = More Pain

Sustained analgesia up to 24h

Vs. Baseline: * p<0.05, ** p<0.005;

10 mg/kg AT-200 s.c.

†

¶¶

*

† ¶

AT-200

Veh

MS

Chronic Acute

Vs. Baseline: * p<0.05; Vs. H/R: ¶ p<0.05, ¶¶

p<0.005; Vs. Veh: † p<0.005

Vang et al., Haematologica 2014

Effect of diet and pleasure on pain

Treatment groups:

• 4 groups of HbSS-Berk mice (n=10/treatment)

• 2 groups of HbAA-Berk mice

− Rodent Diet without mating (n=9) (RD/M-). − Sickle Diet with mating (n=6) [SD/M+].

Rodent Diet (RD) Sickle Diet (SD)

Rodent Diet without Mating (RD/M-) Sickle Diet without Mating SD/M-

Rodent Diet with Mating (RD/M+) Sickle Diet with Mating [SD/M+]

Diet components

Rodent

Diet

Sickle

Diet

Minerals

Magnesium 0.2% 0.48

%

Zinc 70

ppm

225

ppm

Copper 15

ppm

30

ppm

Amino

Acids

Arginine 1% 2.52

%

Cystine 0.4% 0.7%

Aspartic

Acid

1.4% 2.53

%

Glutamic

Acid

3.4% 5.16

%

Rodent

Diet

Sickle

Diet

Vitamins

Vitamin A 15 IU/g 33 IU/g

Vitamin E 110

IU/g

200

IU/g

Folate 4ppm 8ppm

Vitamin B-

12

0.08

mcg/kg

60

mcg/kg

Fatty

Acids

Omega-3

fatty acids

0% 0.26%

Energy density

Protein:

24%

Fat:

18%

Protein:

27.5%

Fat:

26%

†, vs HbSS RD/M-

*, vs BL

High calorie diet and mating attenuate hyperalgesia in sickle mice.

Incre

ase o

f m

echanic

al hypera

lge

sia

M e c h a n ic a l

B L 1 2 30

2

4

6

8

1 0

PW

F (

1.0

g f

ibe

r)

H b S S R D /M -

H b A A R D /M -

H b S S S D /M +

H b A A S D /M +

H b S S S D /M -

H b S S R D /M +

† ††† †††

* * * *

* * * * * * * *

††

† †††

†

*

*

*

T re a tm e n t (w e e k s )

††

Withdrawal of high calorie diet and mating

increases hyperalgesia in sickle mice.

†, vs HbSS RD/M-

*, vs BL

Incre

ase o

f m

echanic

al hypera

lge

sia

M e c h a n ic a l

B L 1 2 30

2

4

6

8

1 0

PW

F (

1.0

g f

ibe

r)

H b S S R D /M -

H b A A R D /M -

H b S S S D /M +

H b S S [S D /M + ]w

H b A A S D /M +

H b A A [S D /M + ]w

† †††

†††

††

* * *

* * * *

T re a tm e n t (w e e k s )

5-HT

Reuptake transporter

Duloxetine

5-HT receptor

Descending inhibitory pathway

NE

NE receptor

Serotonergic and

norepinephrinergic

neurons

First and second

order nerve

terminals

Duloxetine ameliorates hyperalgesia in sickle mice In

cre

ase

of

hyp

era

lge

sia

Male Female

Heat hyperalgesia

† p ≤ 0.05, †† p ≤ 0.01, ††† p ≤ 0.001, †††† p ≤ 0.0001, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ¶ p ≤ 0.05, ¶¶ p ≤ 0.01

† Baseline vs. 3 mg/kg

* Baseline vs. 10 mg/kg

¶ 3 mg/kg vs. 10 mg/kg

Incre

ase

of

hyp

era

lge

sia

Cold hyperalgesia

Curcumin: preclinical studies

• Crosses blood-brain barrier

• Decreased spinal neuroinflammation reduces pain hypersensitivity

• Reduces oxidative stress and inflammatory cytokines reduced thermal hyperalgesia

• Inhibits mast cell activation and anaphylactoid response

• Attenuates oxidative stress and iron accumulation

Curcumin and CoQ10 reduce chronic

hyperalgesia in sickle mice

Valverde et al., Haematologica 2015

7 8 9 10 11 120

100100

200200

400

600

% C

hange o

f P

ain

Thre

shold

high-responders (n=8)moderate-responders (n=8)

non-responders (n=4)

n=8

n=8

n=4

†§†§

†§

†§†§

†§

Sickle Mice Exhibit Variable Response to

electroacupuncture

7 8 9 10 11 120

50100100

150

200300

600

900

Days post treatment

% C

hange o

f P

ain

Thre

shold

High-responders (threshold increase ³ 200%)Moderate-responders (threshold increase 100~200%)Non-responders (threshold increase £100%)

n=5

n=3

n=2

†§ §

Moderate responders

(pain threshold increase100~200 %)

Non-responders

(pain threshold increase ≤ 100 %)

Male (n=10)

§ p<0.05 Vs moderate-responders; † p<0.05 Vs non-responders

Female (n=20)

50% (n=5) High-responders

(pain threshold increase > 200 %)

Days

40% (n=8)

30% (n=3) 40% (n=8)

20% (n=2) 20% (n=4)

Wang et al., Sci Reports 2016

Cannabis to Treat Pain in Sickle Cell Disease:

Compelling evidence from mice and human

• Cannabinoid CP55940, a CB1 and CB2 receptor

agonist attenuates hyperalgesia at a dose of 0.3 mg/kg comparable to 20 mg/kg morphine. Vincent et al., 2015.

• Retrospective survey of 86 SCD pts revealed 52% reported cannabis use decreases pain, anxiety and depression. Howard et al Br J Haematology 2005

• Ongoing proof of principle trial of vaporized cannabis to treat pain in SCD.

Novel analgesics influencing peripheral &

central mechanisms

Brain

De

sce

nd

ing

path

way?

Asce

nd

ing

pa

thw

ay

Amplification and central

sensitization

• “Crises”

• Hypoxia-ischemia reperfusion

injury

• Inflammation

• Hemolysis

• Organ damage

Nociceptor sensitization

SK

IN

Spinal

Cord

DRG

PAIN

TLR4

Glial

activation

Mast cell

activation

TNF𝛼

IL-6

PGE1

SP SP

CGRP

Peripheral

nerve

fibers

ROS

SP

Phospho-p38MAPK

Sickle Cell Disease

Modified from, Aich A, Beitz A & Gupta K, 2016

TAK242

NOP

Duloxetine

Curcumin

Diet

Pleasure

Acupuncture

Cannabis

Imatinib

TAK242

NOP

Curcumin

Diet

Acupuncture

Cannabis

STOP STOP

Lab Members

Ying Wang, PhD

Huy Tran

Jianxun Lei, PhD

Lucile Vincent, PhD

Jinny A Paul, PhD

Yessenia V Guevara,

PhD

Yann Lamarre, PhD

Maureen Reidl, PhD

Joe Cataldo, PhD

Megan Uhelski, PhD

Aditya M Mittal

Lab Members

Julia Nguyen

Barbara Benson

Ritu Jha

Kathryn Luk

Susan Thompson

Lindsey Skubitz

Thu Duong

Jonathan Luk Paul

Deniz Arcan-Fang

Sarita Jarret

Sarah Lam

Ellis Reducinaz

ACKNOWLEDGEMENTS

NHLBI, UO1HL117664, RO1s HL6880, HL103733, HL68802-06S1; HL68802-7S1

Institute for Engineering in Medicine

University of Minnesota Foundation

Dean’s and Department of Medicine Funds

Collaborators

Stephen Nelson, MD

Yvonne Datta, MD

Bin He, PhD

Donald A Simone, PhD

Donald Abrams, MD

Robert P Hebbel, MD

Thank You

Bhubaneswar, Odisha

India

Presents the

3rd Global Congress on Sickle Cell Disease

February 21-24, 2017

Global Sickle Cell Disease Network

www.SickleGlobalCongress2017.com

Pain Sickle cell

disease

Treatable targets to ameliorate pain as well as

the pathobiology evoking pain in SCD.