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.