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CVH: Neurons inhibited by cGMP

Extracellular cyclic GMP (cGMP), the downstream mediator released in response to linaclotide-induced activation of guanylate cyclase-C (GC-C), reduces excitability of murine and human dorsal root ganglion neurons

BACKGROUND

ACKNOWLEDGEMENTS Supported by Ironwood Pharmaceuticals Inc, Forest

Laboratories and NHMRC Australia.

SUMMARY

REFERENCES

RESULTS

• Linaclotide, an investigational GC-C agonist,

improves abdominal pain and bowel symptoms in

humans with Irritable Bowel Syndrome with

Constipation (IBS-C)1.

Colonic DRG neurons from CVH mice displayed a significantly reduced rheobase, firing significantly more action potentials compared with healthy mice at baseline.

In a subpopulation of colonic DRG neurons, cGMP inhibited the neuronal excitability of putative nociceptors, significantly increasing their rheobase and reducing action potential discharge.

This effect was evident in both healthy and CVH DRG neurons, was most apparent in CVH DRG neurons, and occurred at concentrations as low as 100nM cGMP.

In human DRG neurons, cGMP induced an overall reduction in the number of cells responding to hypo-osmotic stimulation.

In addition in human DRG neurons cGMP caused, in a concentration-dependent manner, up to 60% inhibition of the Ca2+ influx induced by hypo-osmotic stimulation.

Mouse DRG studies

AIMS

CONCLUSIONS Exogenous cGMP directly decreases the

excitability of sensory DRG neurons isolated from both mice and humans.

These results complement our previous findings in mice, which demonstrated that cGMP inhibited the peripheral endings of nociceptors within the wall of the colon.

These current findings also provide further mechanistic insight into how linaclotide, through GC-C agonism and the release of cGMP from mucosal epithelial cells, reduces nociceptive signaling from the colon.

Colonic sensory neurons reside in the thoracolumbar (TL: T10-L1) and lumbosacral (LS: L6-S1) dorsal root ganglia (DRG), with afferents projecting via the splanchnic and pelvic nerves respectively. Five different subclasses of mechanosensitive afferent innervate the colorectum via these pathways. Modified from 3.

1) Castro et al., and Brierley 2013, Gastroenterology. 2) Brierley et al., Gastroenterology, 2004, Jul;127(1):166-78. 3) Brierley. Autonomic Neuroscience. 2010. Special Edition, 153, 1-2, 58-68. 4) Brierley. Current Opinion in Pharmacology. 2012. Special edition. 2012

Dec;12(6):632-40. 5) Brierley et al., Journal of Physiology 2011 Jul 15;589(Pt 14):3575-93. 6) de Araujo et al., Brierley & Alewood. Nature Communications.

2014;5:3165.

1,2 Joel Castro, 3 Grigori Y. Rychkov, 4 Andrea Ghetti, 1,2 Andrea M. Harrington, 5 Caroline Kurtz, 5 Ada Silos-Santiago and 1,2,3 Stuart M. Brierley 1 Visceral Pain Group, Discipline of Medicine, SAHMRI, 2Dept of Gastroenterology & Hepatology, Royal Adelaide Hospital. 3 Discipline of Physiology, University of Adelaide, AUSTRALIA. 4 Anabios Inc, USA. 5 Ironwood Pharmaceuticals Inc, USA.

• Linaclotide inhibits colonic nociceptors in vitro 1.

Exogenous cGMP causes greater inhibition of CVH colonic nociceptors

cGMP (µM)

Healthy nociceptors

n=19

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CVH nociceptors

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Exogenous cGMP mimics the effect of linaclotide by inhibiting colonic nociceptors

Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346

Linaclotide causes greater inhibition of colonic nociceptors in mice with CVH

Linaclotide (nM)

CVH nociceptors

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Change from baseline: healthy vs. CVH

Linaclotide (nM)1 30 100 300 1000

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***P<0.001

*P<0.05, ***P<0.001

Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346

• Cyclic GMP (cGMP) is a second messenger

produced in intestinal epithelial cells when

activated by guanylate-cyclase C (GC-C)

agonists 1.

In chronic visceral hypersensitivity (CVH) mice linaclotidehas greater efficacy in reducing nociceptive signaling in

response to noxious colorectal distension (CRD)CVH CRD

Linaclotide + CVH CRD

CVH, N=5.Linaclotide + CVH, N=4.

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T10-T11 T11-T12 T13-L1

Spinal segment

*P<0.05. ***P<0.001

Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346

Phase III clinical trial of 805 IBS-C patients: Linaclotide significantly improves abdominal pain

Castro et al., and Brierley. Gastroenterology (2013; 145:1334–1346

• In vivo intra-colonic administration of linaclotide

also reduces nociceptive signalling from the colon 1.

• cGMP also inhibits colonic nociceptors in vitro

• We performed whole cell patch clamp recordings 5 in current clamp mode from retrogradely traced thoracolumbar (T10-L1) colonic DRG neurons.

• We compared the effect of exogenous extracellular cGMP (100nM-50μM) on the rheobase, or threshold for action potential firing, of DRG neurons from healthy C57BL/6 mice and mice with CVH, 28 days post-TNBS administration 1,6.

• We performed calcium-imaging studies and compared the effects of cGMP (10μM-300μM) on calcium (Ca2+) influx in response to hypo-osmotic stimuli of DRG neurons from healthy donors.

• Pain from the colon and rectum is signalled via

two distinct anatomical spinal afferent pathways 2.

Sacral

Spinal Cord

Lumbar

Thoracic

Key components of the GC-C/cGMP pathway

Abbreviations:GTP: Guanosine triphosphatePKGII: Protein kinase II.NHE3: Sodium/hydrogen exchanger 3.CFTR: Cystic fibrosis transmembrane conductance regulator.

Guanylin

cGMP transporters

Therefore, both linaclotide and exogenous extracellular cGMP inhibit the peripheral endings of colonic nociceptors, with greater efficacy during CVH 1.

However, the effects of exogenous cGMP on sensory neuron function remain to be determined in isolation.

We investigated the effects of exogenous extracellular cGMP on dorsal root ganglion (DRG)

neurons isolated from both mice and humans.

METHODS

Human DRG studies

Mouse DRG studies

Human DRG studies

Human DRG studies

Human DRG neurons were tested with a hypo-osmotic stimuli (70mM NaCl; 5mM KCl, 2mM CaCl2, 1mM

MgCl2, 10mM HEPES, 10mM glucose, pH 7.40, 165 mOsm) followed by capsaicin (200nM).

Neurons that did not respond to either stimuli were classified as “un-responsive’. Neurons responding to

the hypo-osmotic stimuli, but not capsaicin, were termed “mechano-responsive”. Neurons responding to

capsaicin only were termed “capsaicin-responsive”, whilst neurons responding to both stimuli were termed

“Mechano- and Capsaicin responsive”.

Hypo-osmotic stimuli and capsaicin reveal distinct

sub-populations of human DRG neurons

Responsiveness of human DRG neurons to hypo-

osmotic stimuli is dose-dependently decreased by

exogenous extracellular cGMP application

Exogenous extracellular cGMP application causes

greater inhibition in a select sub-population of

human DRG neurons

Human DRG neurons were tested with a hypo-osmotic stimuli (70mM NaCl; 5mM KCl, 2mM CaCl2, 1mM

MgCl2, 10mM HEPES, 10mM glucose, pH 7.40, 165 mOsm) followed by increasing doses (10µM-

300µM) of cGMP (MM-431343). Each dose of cGMP was applied for 2 minutes. After each dose of cGMP

neuronal responsiveness to the hypo-osmotic stimuli was re-tested.

Human DRG neurons were split into specific subpopulations based on their responsiveness to hypo-

osmotic stimuli and capsaicin. This revealed that mechano-responsive neurons were inhibited at lower

concentrations of exogenous extracellular cGMP.

Mouse colonic DRG studies TL colonic DRG neurons from CVH mice display

neuronal hyper-excitability

Healthy mice: Exogenous extracellular cGMP

application inhibits colonic DRG neurons

CVH mice: Exogenous extracellular cGMP application

causes potent inhibition of colonic DRG neurons

Healthy mice: A) A TL colonic DRG

neuron responding to 10pA

depolarizing stimuli in control bath

solution. The rheobase of this neuron

is160pA. B) In the same neuron a 2

min application of 10µM cGMP

increased the rheobase to 280pA. C)

Rheobase is maintained at 280pA

following 2 min 50µM cGMP.

D) Rheobase is partially normalized

after washout of cGMP. E) Group

data showing that cGMP increases

the rheobase of healthy colonic DRG

neurons. Data presented as the

change in rheobase from healthy

control solution baseline.

240 pA

0 pA 0pA

240pA

Healthy mouse colonic DRG neuron CVH mouse colonic DRG neuron

E)

A) CVH: Control solution

Rheobase: 90pA

0 pA

240 pA

B) CVH: 0.1μM cGMP

Rheobase: 140pA

C) CVH: 1μM cGMP

Rheobase: 140pA

D)

A) TL colonic DRG neurons from CVH mice have a significantly reduced rheobase (***P<0.001, Healthy: n=23

vs. CVH: n=13). B) CVH colonic DRG neurons fire more action potentials at 2x rheobase (**P<0.01, Healthy:

n=23 vs. CVH: n=13. C&D) Current clamp recordings of TL colonic DRG neurons in response to depolarizing

stimuli (10pA current steps) from C) Healthy and D) CVH mice.

D) C)

B) A)

CVH mice: A) TL colonic DRG

neuron responding to depolarizing

stimuli (10pA) in control bath solution.

The rheobase of this neuron is 90pA.

B) In the same neuron a 2 min

application of 0.1µM cGMP increases

the rheobase to 140pA. C) Rheobase

is maintained at 140pA following 1µM

cGMP. D) Group data showing that

cGMP dose-dependently increases

the rheobase of CVH colonic DRG

neurons. Data presented as change in

rheobase from CVH control solution

baseline.

Modified from 4.