Poster presented at the IASP (International Association for the Study of Pain) 17th World Congress on Pain; September 12-16, 2018; Boston, Massachusetts

OBJECTIVE• To investigate the potential analgesic effect of olorinab in animal models of colitis

METHODS Study design

• Olorinab or vehicle was administered in mouse and rat models of colitis and in healthy control animals • Olorinab was administered twice daily by oral gavage for 5 days, starting 1 day before induction of colitis• Measurements of in vivo colonic pain and ex vivo colonic afferent mechanosensitivity were conducted on day 5

following instillation of trinitrobenzenesulphonic acid (TNBS)

Animal models of colitis• Colitis was induced C57B/L6J mice and in Sprague Dawley rats by rectal administration of TNBS, as described

in Hughes et al8 (Figure 1) ◦ In each mouse 3.81 mg TNBS in 30% ethanol was administered via enema at a volume of 100 µL ◦ In each rat 12 mg TNBS in 35% ethanol was administered via enema at a volume of 300 µL

Figure 1. Animal models of colitis: mouse and rat.

A

B

TNBS administration C Healthy mouse Colitis mouse (3 days post-TNBS)

Colitis rat (3 days post-TNBS)D Healthy rat

MPO

U/m

g pr

otei

n

Healthy

1000

800

600

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0Colitis (3 days post-TNBS)

Significant histologic damage

0

(130 μg/mL 30% EtOH)

3 7Day:

*

*P < 0.0001, Mann-Whitney test. EtOH, ethanol; MPO, myeloperoxidase; TNBS, trinitrobenzenesulphonic acid.

Olorinab (APD371), a Peripherally Restricted, Highly Selective, Full Agonist of the Cannabinoid Receptor 2 (CB2), Reduces Colitis-Induced Visceral Hypersensitivity in Rats

Joel Castro,1 Jessica Maddern,1 Sonia Garcia-Caraballo,1 Beatriz Lindstrom,2 John Adams,2 Stuart M. Brierley1

1Visceral Pain Research Group, Flinders University, SAHMRI, Adelaide, SA, 5000, Australia; 2Arena Pharmaceuticals, Inc, San Diego, CA, USA

PFR455

Pain assessment in vivo• Noxious distension of the colorectum triggers the visceromotor response (VMR), a nociceptive brainstem reflex

consisting of the contraction of the abdominal muscles,9 used as an indicator of pain• After TNBS treatment in rats, colorectal distension (CRD) was induced using a barostat, and VMR was measured

using an amplifier connected to an analog-to-digital converter (Figure 2)

Figure 2. Pain assessment in vivo by VMR response to CRD in rats.

ADC Healthy control

Colitis

20 mm Hg 40 mm Hg 50 mm Hg 60 mm Hg 80 mm Hg

Ra

w E

MG

(μ

V)

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w E

MG

(μ

V)

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−1000

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AmplifierFilter

Barostat

ADC, analog-to-digital converter; CRD, colorectal distension; EMG, electromyogram; TNBS, trinitrobenzenesulphonic acid; VMR, visceromotor response.

Assessment of colonic mechanosensitivity in vitro• Single-unit extracellular recordings from splanchnic colonic afferent nerves were performed as previously

described8,10,11 (Figure 3)

Figure 3. Single-unit extracellular recordings from mouse splanchnic colonic nerves in vitro.

Focal compression of receptive field(von Frey Hairs; 70-2000 mg)

Mucosal stroking(von Frey Hairs; 10-1000 mg)

Circular Stretch

Krebs superfusion nifedipine(1 μM) plus Indomethacin (3 μM)

Amplifier and Spike Discrimination

Paraffin-filledrecordingchamber

Splanchnic nerve

0510

Spik

es/5

s

152025

30 s

3 g 5 g 7 g 9 g 11 g

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V

1-11 g

Colonic mechanosensory nociceptor recordings

Figure 7. Nociception of mechanical stimuli in the colon is heightened in colitis animals and reduced after treatment with olorinab.

Healthy nociceptors

Olorinab (nM)0 10 100 1000 10000

n = 7

Colitis nociceptors

Olorinab (nM)0 10 100 1000 10000

*

n =10

18

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9

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hano

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onse

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kes/

sec)

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9

6

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Mec

hano

sens

ory

resp

onse

(spi

kes/

sec)

†

‡

*P < 0.05, †P < 0.01, ‡P < 0.001. 1-way analysis of variance with Bonferroni post hoc tests.

SUMMARY

Visceromotor response to colorectal distension• Vehicle-treated colitis animals displayed visceral hypersensitivity, as indicated by significantly elevated VMR to CRD• Healthy control rats treated with olorinab (30 mg/kg) did not show altered visceral sensitivity to CRD• Colitis animals displayed significant colonic inflammation (increase in myeloperoxidase activity) compared with

healthy control animals • Colitis rats administered olorinab (3 mg/kg and 30 mg/kg) had significantly reduced VMR to CRD compared with

vehicle-treated colitis rats (with VMR normalized to healthy control levels)

Colonic nociception• Colonic nociceptors from colitis animals are hypersensitive to mechanical stimuli compared with healthy colonic

nociceptors• Olorinab reverses colonic nociceptor hypersensitivity in colitis animals with no effect on healthy colonic nociceptors

INTRODUCTION• Crohn’s disease and ulcerative colitis, collectively known as inflammatory bowel disease (IBD), are chronic

relapsing gastrointestinal disorders with increasing prevalence worldwide1

• Abdominal pain is reported by up to 60% of patients with IBD, is associated with lower quality of life, and is severe enough to require pain treatment in the majority of cases2

• The cannabinoid 2 (CB2) receptor is expressed throughout the gastrointestinal tract and is an attractive target for IBD3,4

• Olorinab (APD371) has been shown to activate endogenous CB2 receptors in primary rat splenocytes, human HL-60 cells, and primary human B cells5 ◦ Olorinab exhibited >1000-fold selectivity for the CB2 receptor over the CB1 receptor and sustained

efficacy in several animal models of chronic pain5,6 ◦ No adverse psychotropic effects were seen in healthy volunteers who received olorinab7

• Olorinab is in a phase 2 trial for visceral pain associated with Crohn’s disease5,7

REFERENCES1. Wright KL et al. Br J Pharmacol. 2008;153:263-270.2. Wright KL et al. Gastroenterology. 2005;129:437-453.3. Adams JW et al. Poster presented at the American Pain Society Scientific

Summit; March 4-6, 2018; Anaheim, California.4. Han S et al. ACS Med Chem Lett. 2017;8:1309-1313.5. Jones RCW et al. Poster presented at the American Pain Society Scientific

Summit; March 4-6, 2018; Anaheim, California.

6. Hughes PA et al. Gut. 2009;58:1333-1341. 7. Ness TJ, Gebhart GF. Brain Res. 1988;450:153-169. 8. Brierley SM et al. Gastroenterology. 2004;127:166-168.9. Brierley SM et al. Gastroenterology. 2009;137:2084-2095.

ACKNOWLEDGMENTS Medical writing assistance was provided by ApotheCom, San Francisco, CA, and was funded by Arena Pharmaceuticals, Inc. Stuart Brierley is an NHMRC R.D Wright Biomedical Research Fellow.

RESULTS

Visceral hypersensitivity and colonic compliance

Figure 4. Visceral hypersensitivity (A) without change in colonic compliance (B) in rats with induced colitis.

VMR to CRDHealthy animals + vehicle (n = 11)Colitis animals + vehicle (n = 18)

1250A

rea

Und

er C

urve

(Mea

n ±

SEM

)

Pressure (mm Hg)

1000

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20 40 50 60 70 80

*

*

*†

†

Colonic complianceHealthy animals + vehicle (n = 11)Colitis animals + vehicle (n = 18)

Pres

sure

(mm

Hg)

Volume (mL)

120

90

60

30

0

0.25 0.5 0.75 1 1.25 1.5

A B

CRD, colorectal distension; GEE, generalized estimating equation; LSD, least significant difference; SEM, standard error of the mean; VMR, visceromotor response. Healthy + vehicle versus colitis + vehicle: *P < 0.001 and †P < 0.0001. GEE, LSD post hoc test.

Figure 5. No effect of olorinab (30 mg/kg) on VMR (A) or colonic compliance (B) in healthy rats.

1250

Are

a U

nder

Cur

ve(M

ean

± SE

M)

Pressure (mm Hg)

VMR to CRD

1000

750

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250

0

20 40 50 60 70 80

Healthy animals + vehicle (n = 11)Healthy animals + olorinab 30 mg/kg (n = 8)

Pres

sure

(mm

Hg)

Volume (mL)

Colonic compliance

0.25 0.5 0.75 1 1.25 1.5

Healthy animals + vehicle (n = 11)Healthy animals + olorinab 30 mg/kg (n = 8)

120

90

60

30

0

BA

CRD, colorectal distension; GEE, generalized estimating equation; LSD, least significant difference; SEM, standard error of the mean; VMR, visceromotor response.Healthy + vehicle versus healthy + olorinab: not significant. GEE, LSD post hoc test.

Figure 6. Treatment with 3 mg/kg olorinab (A) and 30 mg/kg olorinab (B) in rats with colitis prevents visceral hypersensitivity without an effect on colonic compliance.

Are

a U

nder

Cur

ve(M

ean

± SE

M)

1250

Pressure (mm Hg)

VMR to CRD

1000

750

500

250

0

20 40 50 60 70 80

Healthy animals + vehicle (n = 11)Colitis animals + vehicle (n = 18)Colitis animals + olorinab 3 mg/kg (n = 8)

†

†

†‡

‡

A

B

Pres

sure

(mm

Hg)

Volume (mL)

Colonic compliance

0

0.25 0.5 0.75 1 1.25 1.5

Healthy animals + vehicle (n = 11)Colitis animals + vehicle (n = 18)Colitis animals + olorinab 3 mg/kg (n = 8)

120

90

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30

0

Are

a U

nder

Cur

ve(M

ean

± SE

M)

1250

Pressure (mm Hg)

VMR to CRD

1000

750

500

250

0

20 40 50 60 70 80

Healthy animals + vehicle (n = 11)Colitis animals + vehicle (n = 18)Colitis animals + olorinab 30 mg/kg (n = 9)

Pres

sure

(mm

Hg)

120

Volume (mL)

Colonic compliance

90

60

30

0

0.25 0.5 0.75 1 1.25 1.5

Healthy animals + vehicle (n = 11)Colitis animals + vehicle (n = 18)Colitis animals + olorinab 30 mg/kg (n = 9)

**

* * *

† ‡

‡† ‡

†

†

†‡

‡

CRD, colorectal distension; SEM, standard error of the mean; VMR, visceromotor response.*P < 0.01; †P < 0.001; ‡P < 0.0001. Healthy + vehicle versus colitis + vehicle: gray symbols; colitis + vehicle versus colitis + olorinab: maroon symbols. All post hoc generalized estimating equation using least significant difference.

METHODS Cont. RESULTS Cont.

CONCLUSIONS• Olorinab prevents colitis-induced visceral hypersensitivity, suggesting an antinociceptive role for CB2

receptors in visceral sensory pathways • Olorinab, through its selectivity, may provide a novel therapeutic treatment for IBD-associated abdominal

pain without psychotropic effects and therefore without the potential for dependence or abuse