Hamamatsu Pharma Research (HPR) offers a preclinical efficacy evaluation. HPR has established a non-subjective method to measure "pain" using fMRI.

1. Hamamatsu Pharma Research USA, Inc.
Functional brain imaging of pain
in non-human primates

2.  Japan-based preclinical CRO founded in 2005
 Provides preclinical efficacy study using
Non-human primate (NHP) disease models
 Mission:
To accelerate the drug development process by
providing translational pharmacological studies.
Who is HPR?

3. NHP models:
Clinical relevance
& high translatability
Anatomical similarityPhylogenetic similarity
- Brain structure
- Nervous system
- Reproduction
- Retinal structure etc.
Compatibility w/
Clinical methods
CT
MRI
National Human Genome Research Institute;
Gibbs et al., Science (2007)
mouse
macaque
Modified from Izpisua Belmonte et al., Neuron (2015)
marmoset
human
Cynomolgus macaque
(Macaca fascicularis)

4. Therapeutic areas
Musculoskeletal:
Osteoarthritis
Reproductive
diseases:
Endometriosis
Cerebral ischemia/
Stroke
Ophthalmology:
Glaucoma,
Age-related macular
degeneration (AMD, wet
& dry)
Cardiovascular:
Thrombosis
Gastroenterology:
Inflammatory bowel
disease (IBD),
Irritable bowel
syndrome (IBS)
Image from kidspressmagazine.com
Neurological
disorders:
Parkinson’s disease
Pain:
Acute pain,
Polyneuropathic pain,
Mononeuropathic pain
Pruritus

5. Why pain in NHPs?
Significant unmet medical need
High attrition rate of novel pain
therapeutics development due to:
• Gap between rodent models and human pain
• Lack of objective outcome measures of pain
(i.e. biomarkers)

6. Efficacy of analgesics in preclinical
models of OA & OA patients
Drug/Mechanism Rodents NHP* OA patients
Morphine/-opioid receptor agonist Yes Yes Yes
Diclofenac/COX inhibitor Yes Yes Yes
Aprepitant/NK1 receptor antagonist Yes No No
Duloxetine/serotonin-norepinephrine
reuptake inhibitor
Yes Yes Yes
Pregabalin/α2δ ligand Yes No No
Fatty acid amide hydrolase inhibitor Yes No
TRPV1 antagonist Yes No
Dopamine receptor (D3) agonist Yes No
δ-opioid receptor agonist Yes No
Matrix metalloproteinase inhibitor
(nonselective)
Yes No
*Ogawa et al., European Journal of Pharmacology (2016); Ogawa et al., Osteoarthritis and Cartilage (2016)

7. Outcome measures of pain
Preclinical (rodents):
• Reflexive withdrawal (tail-flick, von Frey etc.)
• Spinal-bulbo-spinal reflexes (licking, vocalization etc.)
Clinical:
• Self report (VAS, NRS etc.)
More objective (& also translatable)
outcome measure(s) needed!
 Functional brain imaging

8. Brain imaging methods
Morton et al., J. Pain Res. (2016)

9. fMRI-BOLD
BOLD = Blood oxygenation level-dependent
Arthurs & Boniface., Trends in Neuroscience. (2002)

10. fMRI-BOLD
OFF ON OFF ON OFF ON
https://www.nature.com/scitable/blog/brain-metrics/what_does_fmri_measure
>10 sets of OFF/ON
Mapping the contrast
onto the brain structure
(“ON” > “OFF”)

11. The Pain matrix
DH: Dorsal horn
RVM: rostral ventral medulla
PAG: periaqueductal gray
ACC: anterior cingulate cortex
IC: insular cortex
PFC: prefrontal cortex
SI: primary somatosensory cortex
SII: secondary somatosensory cortex
Affective
Sensory
Morton et al., J. Pain Res. (2016)

12. Brain activity in response to pain
Lanz et al., J Neural Transm. (2011)
Human subjects (both normal & patients)

13. Brain activity in response to pain
McDermott et al., Neuron. (2019)
Human subjects (healthy control vs. CIP* patients)
CBF measured with fMRI-ASL
*CIP: congenital insensitivity to pain (Nav1.7 loss-of-function)

14. NHP models of pain at HPR
 Oxaliplatin-induced peripheral neuropathy model
(CIPN, polyneuropathic pain)
 Sciatic nerve ligation model
(Mononeuropathic pain)
 Postoperative pain model
 Visceral pain/hypersensitivity model
 Naturally occurring endometriosis model
 Lower back pain model
 Osteoarthritis model
× ｆMRI

15. Chemotherapy-induced
peripheral neuropathy
- oxaliplatin-induced polyneuropathy -

16.  Oxaliplatin, i.v. 5 mg/kg/2 hr., once every 2 weeks (↓).
 Pain (cold allodynia) was assessed with tail withdrawal test.
 Tail withdrawal test:
Distal tail is dipped in 10℃ water and the latency to respond is recorded.
Cut-off time: 20 sec. N=3/vehicle, N=5-6/oxaliplatin.
Oxaliplatin-induced polyneuropathy
0 7 14 21 28 35 42 49 56
0
5
10
15
20
Oxaliplatin 5 mg/kg
Vehicle
** **
* *
*
10 o
C
WithdrawalLatency
(sec)
*P <0.01 vs. vehicle
Neuropathy Induction
*p < 0.05, **p < 0.01 vs. 0 (baseline) Shidahara et al. Pharmacology Research & Perspectives (2016)
Brain activation: fMRI

17. Oxaliplatin-induced polyneuropathy
Brain activity changes in a macaque model of oxaliplatin-induced
neuropathic cold hypersensitivity
Brain activation: fMRI
A 3T-MRI system assessed brain activity before (“intact”, n=4) and 3
days after oxaliplatin infusion (n=4). fMRI scans were also performed 1
hour after treatment with duloxetine 3 days after either the 1st or 2nd
oxaliplatin infusion (n=2).
Nagasaka et al., Scientific Reports (2017)

18. Oxaliplatin-induced polyneuropathy
Non-noxious cold-evoked brain activation: cold hypersensitivity.
Brain activation: fMRI
Nagasaka et al., Scientific Reports (2017)

19. Oxaliplatin-induced polyneuropathy
Brain activation: fMRI
Analgesia following inactivation of the secondary somatosensory
cortex (SII) and the insular cortex (Ins).
Nagasaka et al., Scientific Reports (2017)

20. Oxaliplatin-induced polyneuropathy
Duloxetine analgesia is associated with reduced SII/Ins activity in
oxaliplatin-treated macaques.
***p < 0.001
Nagasaka et al., Scientific Reports (2017)
Brain activation: fMRI

21. Postoperative Pain

22. Proximal
Distal
Postoperative Pain
Pain Assessment
Midline skin and muscle incision, test the following day.
Test sites: proximal and distal to the incision
Incision

23. Increased sensitivity to nonpainful pressure following surgery.
Recovery from pain over time.
N=5, mean ±S.D.; *p < 0.05, **p < 0.01 vs. Pre
Pre 1 2 3 7
0
1
2
3
Proximal
** ****
*
Time Post-Incision (Days)
ResponsePressure(kg)
Pre 1 2 3 7
0
1
2
3
Distal
****
Time Post-Incision (Days)
ResponsePressure(kg)
Hama et al., CNS Neurol Disord Drug Targets (2018)
Postoperative Pain
Pain Assessment

24.  Pain-associated brain activation visualized with 3T-MRI.
 Effect of morphine and pregabalin on activation.
OFF ON
OFF/ON 1 set:
30 sec, 10 frames
30
sec
OFF ON OFF ON
10 sets (~15 min)
ON: 1.5 kg pressure applied to the proximal area
OFF: resting (0.1 kg pressure)
fMRI
(Pre)
15 min
interval
fMRI
(Post)
Morphine (6 mg/kg) OR Pregabalin (20 mg/kg)
fMRI Protocol
Postoperative Pain
Brain activation: fMRI

25.  Before surgery, activation of the Ins with acute noxious pressure (2.5 kg).
 After surgery, activation of the CC and Ins with non-painful pressure (1.5 kg).
Post-surgery
Ins/SII
CC
Pre-surgery
2.5 kg 1.5 kg
Postoperative Pain
Hama et al., CNS Neurol Disord Drug Targets (2018)
Brain activation: fMRI

26.  Pressure-evoked activation of the insular cortex (Ins) and cingulate cortex (CC).
 Activation of both Ins and CC was attenuated with morphine.
 Pregabalin attenuated activation of the CC but not Ins.
No treatment Morphine Pregabalin
Hama et al., CNS Neurol Disord Drug Targets (2018)
Postoperative Pain
Brain activation: fMRI

27. Peripheral mononeuropathy
- Sciatic nerve loose ligation model -

28. Image: Cambridge University Press & Harvard Health Publishing
Neuropathy Endpoint
Ref. Rat models
 Stimuli with von Frey filaments applied
to the plantar surface of the left foot.
 Non-painful stimulation evoked brain
activation observed with fMRI.
PVC tube applied around the
left sciatic nerve.
Filament (1-26 g)
(non-painful to uninjured animals)
Peripheral mononeuropathy

29.  Two to five weeks after nerve injury.
 Brain activation to non-painful stimuli with 3T-MRI.
 Pharmacology: pregabalin or aprepitant vs vehicle.
ON
1 set =
9 sec, 3 frames & 30 sec interval
30 sec ON ON
10 sets ✕ 5 filaments (OFF: 1 g; ON: 4, 8, 15, and 26 g)
ON: Filament pressed on the
plantar surface of the left foot.
Imaging Protocol
30 sec30 sec ON ON
Dose (p.o.)
Nerve injury (cuffing)
fMRI scan
2-5 weeks
1 hr. interval
Brain activation: fMRI
Peripheral mononeuropathy

30. 2W 3W 4W 5W (after surgery)
Brain activation: fMRI
Peripheral mononeuropathy
Ins/SII
CC
Thalamus

31. d
Z value
3.0
1.0
2.0
Vehicle
Aprepitant
Pregabalin
4 g 8 g 15 g 26 g
Brain activation: 4-5 weeks post-injury averaged from 6 macaques
LR

32. 1.96 (p < 0.05)
2.3 (p < 0.01)
Mean ±SEM, n = 6
Ins/SII
 Activation of the Ins/SII to non-painful stimulation in the neuropathic state.
 Pregabalin significantly reduced non-painful activation of the Ins/SII.
LR
Brain activation: fMRI
Peripheral mononeuropathy

33. Visceral pain/hypersensitivity
- post-inflammatory visceral pain induced by DSS -

34. Visceral Pain Model
Induction of colonic inflammation
 Male cynomolgus macaque (N=8)
 Oral administration of 0.25% Dextran Sulfate Sodium (DSS)
in drinking water
 1 cycle = 2 weeks on DSS followed by 2 weeks days off DSS
4W0W 2W 6W 8W 10W
DSS DSS DSS
Mayo & Colonoscopy (every 2 weeks)
Stool consistency & Rectal bleeding (daily)
Water Water Water
12W 13W and after
Remission

35. Active State Remission State
Visceral Pain Model
Induction of colonic inflammation

36. fMRI x Balloon distension
 Inserted into the rectum 5 cm above the anus
under light anesthesia with propofol
 fMRI imaging with increasing volume of air
OFF
ON
10
mL
30
sec
ON
20
mL
30
sec
ON
30
mL
OFF: resting (0 mL)
ON: 10, 20, 30 mL
ON
10
mL
30
sec
ON
20
mL
30
sec
ON
30
mL
Visceral Pain Model

37. Normal
Post-
inflammation
Visceral Pain Model
Ins/SIICC
fMRI x Balloon distension

38. fMRI x Balloon distension (30 mL)
Visceral Pain Model
Pre-drug Morphine (6 mg/kg)
Pre-drug Ramosetron (3 µg/kg)

39. Naturally occurring
endometriosis

40. Macaques with EM demonstrate significant abdominopelvic pressure
hypersensitivity.
Normal Endometriosis
0
1
2
3
mean +/- SD
*** p< 0.003
***
(No endometriosis)
ResponsePressure(kg)
Region where
pressure applied
Region of cyst
(palpation)
Pain
Normal
Yano et al., Hum Reprod (2019)
Pain assessment
Endometriosis model

41. Normal Pre- 10 min 30 min 1 hr
0
1
2
3
mean +/- SD
*p < 0.05, **p < 0.01 vs. Pre-
*
**
Morphine (6 mg/kg, i.m.)
ResponsePressure(kg)
3
(kg)
 Morphine relieves EM-associated hypersensitivity.
 Meloxicam and acetaminophen do not show analgesic effects.
Normal Endometriosis
0
1
2
3
mean +/- SD
*** p< 0.003
***
(No endometriosis)
ResponsePressure(kg)
Normal Pre- 10 min 3
0
1
2
3
**
Morphine (6 mg/kg
ResponsePressure(kg)
Normal Pre- 1 hr 2 hrs 3 hrs
0
1
2
3
mean +/- SD
Meloxicam (0.5 mg/kg, i.m.)
ResponsePressure(kg)
Normal Pre- 1 hr
0
1
2
3
Acetaminophen (10 m
ResponsePressure(kg)
Normal Endometriosis
0
1
2
3
mean +/- SD
*** p< 0.003
***
(No endometriosis)
ResponsePressure(kg)
Normal Pre- 10 min 30 min 1 hr
0
1
2
3
mean +/- SD
*p < 0.05, **p < 0.01 v
*
**
Morphine (6 mg/kg, i.m.)
ResponsePressure(kg)
Normal Pre- 1 hr 2 hrs 3 hrs
0
1
2
3
mean +/- SD
Meloxicam (0.5 mg/kg, i.m.)
ResponsePressure(kg)
Normal Pre- 1 hr 2 hrs 3 hrs
0
1
2
3
mean +/- SD
Acetaminophen (10 mg/kg, p.o.)
ResponsePressure(kg)
Morphine
(6 mg/kg, i.m.)
Meloxicam
(0.5 mg/kg, i.m.)
Acetaminophen
(10 mg/kg, p.o.)
Pain assessment
Endometriosis model
Yano et al., Hum Reprod (2019)

42.  Visualize pressure-induced brain activation with 3T-MRI.
 Effect of morphine on brain activation.
OFF ON
OFF/ON 1 set:
30 sec, 10 frames
30
sec
OFF ON OFF ON
10 sets (~15 min)
ON: 1.5 kg pressure applied to the proximal area
OFF: resting (0.1 kg pressure)
fMRI
(Pre)
15 min
interval
fMRI
(Post)
Morphine (3 mg/kg)
fMRI Protocol
Brain activation: fMRI
Endometriosis model

43.  Significant activation of the insular cortex (Ins) and the thalamus (TH).
 Morphine attenuates pressure-induced activation of Ins and TH.
Pre Morphine
Ins
TH
Endometriosis model
Yano et al., Hum Reprod (2019)
Brain activation: fMRI

44. Effect of dienogest, a progestin,
on cyst size & pressure hypersensitivity
Week 1 2 3 4 5 6 7 8
Dienogest, 0.05 mg/kg, p.o., BID
Endpoints:
 Cyst size, measured with MRI ( )
 Brain activation, measured with fMRI ( )
 Serum estradiol; pressure test ( )
Hormonal therapy
Endometriosis model

45. N = 5, *p < 0.05,**p < 0.01 vs. Pre-.
Pre- 4 W 8 W
0
10
20
30
40
Cystsize(ml)
Pre- 4 w 8 w
0
50
100
150
200
**
**
[Estradiol](pg/ml)
Treatment Time (Weeks)
Pre- 4 W 8 W
0.0
0.5
1.0
1.5
2.0
* *
ResponsePressure(kg)
Cyst size Pressure test Serum estradiol
 Dienogest treatment:
- Reduces cyst size
- Alleviates pain
- Reduces estradiol levels
Endometriosis model
Yano et al., Hum Reprod (2019)
Hormonal therapy

46. Pre 8W
Eight-week treatment with dienogest (0.05 mg/kg, p.o., BID) reduces
Ins and TH activation
Endometriosis model
Ins
TH
Yano et al., Hum Reprod (2019)
Hormonal therapy x fMRI

47. Summary
Pain model IC/SII ACC Thalamus
Oxaliplatin-induced NP
(polyneuropathic)
Yes No No
Postoperative pain Yes Yes No
Visceral pain Yes Yes No
Sciatic nerve
ligation
(mononeuropathic)
Acute Yes Yes No
Chronic
(4W & after)
Yes Yes Yes
Naturally occurring endometriosis Yes No Yes
Yes: activated with stimulation
No: no activation

48. We have established a method to
visualize and quantify brain activation
in response to pain stimuli in NHP
models of various pain.
Different activation patterns observed
in different pain models:
Acute phase: Insular cortex/SII and ACC
Chronic phase: +Thalamus
Summary

49. Limitations of the current method:
Use of propofol for sedation
Comparison b/w resting & evoked pain
(fMRI-BOLD not suitable for chronic pain)
Measurement of chronic/spontaneous pain
Blood flow, fMRI-ASL etc.
Correlation with clinical data
Paths Forward

50. ＜Headquarters/Primate Center＞
e-mail: info@hpharma.jp
URL: www.hpharma.jp (JP)
＜US/San Diego Office (HPR USA)＞
e-mail: info_us@hpharma.jp
URL: www.hpharmausa.com (EN)
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