Analgesic effect of ADX71441, a positive allosteric modulator (PAM) of GABAB receptor in a rat model of bladder pain

Accepted Manuscript Analgesic effect of ADX71441, a positive allosteric modulator (PAM) of GABAB receptor in a rat model of bladder pain Pradeep Kannampalli, Sonia-Maria Poli, Christelle Boléa, Jyoti N. Sengupta PII:

S0028-3908(17)30390-8

DOI:

10.1016/j.neuropharm.2017.08.023

Reference:

NP 6828

To appear in:

Neuropharmacology

Received Date: 29 March 2017 Revised Date:

26 July 2017

Accepted Date: 16 August 2017

Please cite this article as: Kannampalli, P., Poli, S.-M., Boléa, C., Sengupta, J.N., Analgesic effect of ADX71441, a positive allosteric modulator (PAM) of GABAB receptor in a rat model of bladder pain, Neuropharmacology (2017), doi: 10.1016/j.neuropharm.2017.08.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Analgesic effect of ADX71441, a Positive Allosteric Modulator (PAM) of GABAB Receptor in a rat model of Bladder Pain

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Pradeep Kannampalli1, Sonia-Maria Poli2, Christelle Boléa2 and Jyoti N. Sengupta1,3

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Division of Gastroenterology and Hepatology, and 3Department of Pediatric

Gastroenterology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA, 2Addex

Total no of pages: 41 Total no of figures: 8 Total no. of suppl figures: 1

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Total no of tables: 0

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Therapeutics, 14 Chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland.

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*Corresponding author’s address for communication:

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Dr. Jyoti N. Sengupta, PhD., Division of Gastroenterology and Hepatology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. USA. Tel: 414-955-4011 Fax: 414-955-6361

Email: [email protected]

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Abstract Therapeutic use of GABAB receptor agonists for conditions like chronic abdominal pain, overactive bladder (OAB) and gastroesophageal reflux disease (GERD) is severely

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affected by poor blood-brain barrier permeability and potential side effects. ADX71441 is a novel positive allosteric modulator (PAM) of the GABAB receptor that has shown encouraging results in pre-clinical models of anxiety, pain, OAB and alcohol addiction.

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The present study investigates the analgesic effect of ADX71441 to noxious stimulation of the urinary bladder and colon in rats. In female Sprague-Dawley rats, systemic (i.p),

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but not intrathecal (i.t), administration of ADX71441 produced a dose-dependent decrease in viscero-motor response (VMR) to graded urinary bladder distension (UBD) and

colorectal

distension

(CRD).

Additionally,

intra-cerebroventricular

(i.c.v.)

administration of ADX71441 significantly decreased the VMRs to noxious UBD.

In

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electrophysiology experiments, the drug did not attenuate the responses of UBDsensitive pelvic nerve afferent (PNA) fibers to UBD. In contrast, ADX71441 significantly decreased the responses of UBD-responsive lumbosacral (LS) spinal neurons in spinal

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intact rats. However, ADX71441 did not attenuate these LS neurons in cervical (C1-C2) spinal transected rats.

During cystometrogram (CMG) recordings, ADX71441 (i.p.)

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significantly decreased the VMR to slow infusion without affecting the number of voiding contraction. These results indicate that ADX71441 modulate bladder nociception via its effect at the supra-spinal sites without affecting the normal bladder motility and micturition reflex in naïve adult rats.

Key Words: Painful bladder syndrome, interstitial cystitis, GABAB receptor, PAM modulators, visceral nociception.

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1. Introduction Interstitial cystitis or painful bladder syndrome (IC/PBS) is characterized by frequency,

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urgency and pain during bladder filling (Gillenwater and Wein, 1988; Cervigni, 2015). It is estimated that about 12 million people in the US are diagnosed with IC/PBS severely affecting their quality of life (Clemens et al., 2007). In spite of years of research, there is

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no clear understanding about the etiology or pathophysiology of this syndrome and the diagnostic criteria still remain poorly defined (Giannantoni et al., 2012).

Current

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therapeutic options are primarily focused on symptom control and consist of a combination of supportive, behavioral and pharmacologic measures (Fiander 2013). Unfortunately, many of these existing treatments are ineffective in most IC/PBS patients leading to a huge demand for novel drugs with improved therapeutic efficacy and

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minimal side effects.

Various studies have implicated GABA receptors in the transmission and perception of nociceptive information and activation of these receptors has been shown

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to result in effective pain management (Castro-Lopes et al., 1995; Dirig and Yaksh, 1995; Lin et al., 1996; Green and Dickenson, 1997; McCarson and Enna, 2014).

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However, despite considerable progress in developing new compounds, the use of systemic GABAergic drugs is limited by their unwanted side-effects including sedation, respiratory insufficiency, muscle weakness and hypothermia (Jasmin et al., 2004). Furthermore, the physiological functions of GABAergic system appear to be more complex in certain areas of the brain and spinal cord. For example, inflammation was reported to cause a shift in GABAA receptor mediated nociception in rodent model of inflammatory pain (Anseloni and Gold, 2008). Similarly, Orii et al (2003) reported that

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GABAergic neurons modulate the antinociceptive effect of N2O in opposite directions at supraspinal and spinal levels. Baclofen is the only marketed drug targeting GABAB receptors and is primarily used for the treatment of spasticity (Bowery, 2006). The

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centrally mediated analgesic effect of baclofen in several models of experimentallyinduced nociception has been extensively reported (Balerio and Rubio, 2002; Whitehead et al., 2010). Baclofen dose-dependently inhibited bladder contractions and

(Kontani et al., 1988; Miyazato et al., 2008).

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decreased micturition pressure in female rats via its action at pontine micturition center Unfortunately, baclofen has poor

high concentration. difficulty

breathing,

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permeability across the blood-brain barrier in humans thus necessitating the need for This is associated with numerous side effects like drowsiness, seizures,

muscle

weakness,

loss

of

consciousness

and

hypothermia, thereby limiting its widespread applications (Brennan and Whittle, 2008;

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Sullivan et al., 2012).

Positive allosteric modulation (PAM) of the GABAB receptor is a promising alternative to direct activation of the receptor where the allosteric modulator binds to a

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distinct site from that of the orthosteric ligand-binding site to offer the same therapeutic effect (Ong and Kerr, 2005; Conn et al., 2009; Digby et al., 2010; Engers and Lindsley,

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2013). Such allosteric modulation of GABAB has long been recognized as a powerful pharmacological approach to achieve activity-dependent effect of the drug while at the same time avoid potential side effects associated with conventional GABAB receptor agonists (Pin and Prézeau, 2007, Urwyler, 2011). Recently, several PAMs of GABAB receptor were developed and tested in animal models with the main objective of reducing the adverse side effects associated with baclofen (Froestl, 2010).

Unlike

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baclofen that constantly activate the receptor, PAMs for GABAB receptors interact with the binding sites that are topographically distinct from the binding site for the endogenous ligand and exert their effects without significantly interfering with the

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normal downstream signaling mechanisms (Christopoulos, 2002). ADX71441, a new chemical entity which act as PAM of the GABAB receptor, was discovered by Addex Therapeutics following high-throughput screening and medicinal chemistry optimization

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(Kalinichev et al., 2014). In the past few years, several in vitro and in vivo studies were performed that strongly suggested ADX71441 as a potent, selective, reversible and

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orally bioavailable GABAB PAM. Pre-clinical studies have shown that ADX71441 is effective against Charcot-Marie-Tooth Type 1A disease (CMT1A), overactive bladder (OAB), somatic pain, obsessive-compulsive disorder, alcohol binge drinking and anxiety (Kalinichev et al., 2014, 2017a, 2017b; Augier et al., 2017). Kalinichev et al. (2014)

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reported that ADX1441 reduces peritoneal nociception in acetic acid-induced abdominal writhing test indicating that the drug might be effective against visceral nociception. However, the analgesic effect of ADX71441 in a specific visceral nociceptive model has

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not been systematically investigated. In the present study, the main objective was to test ADX71441 in a rat model of bladder nociception and more importantly to examine

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the site of action of the drug along the pain signaling neuraxis.

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2. Materials and methods 2.1 Animals Female Sprague-Dawley rats weighing 275-300g were obtained from Taconic

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Biosciences (Indiana, IN, USA). The surgical procedures and experimental protocols performed in this study were approved by Medical College of Wisconsin’s Institutional Animal Care and Use Committee (AUA 000355) and performed according to the

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guidelines recommended by the International Association for the Study of Pain (IASP). Rats (n=2-3/cage) were kept under controlled conditions with a 12 hours light/dark

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schedule and had free access to unlimited food and water. Rats subjected for CRDinduced VMR were deprived from food, but not water, for 12 hours to empty the descending colon.

ADX71441

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2.2 Drugs and chemicals

(N-(5-(4-(4-chloro-3-fluorobenzyl)-6-methoxy-3,5-dioxo-4,5-dihydro-

1,2,4-triazin-2(3H)-yl)-2-fluorophenyl)acetamide was provided by Addex Therapeutics.

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For the systemic (i.p and i.v.) administration, ADX71441 was dissolved in PEG400:Saline (1:1) solvent and injected to the rats in a single bolus dose. For i.t injection,

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the ADX71441 stock solution was prepared in PEG-400:Saline (1:1) and serially diluted in sterile saline to obtain the target final concentration. Baclofen (Sigma-Aldrich, St.Louis, MO) was dissolved in sterile saline. All drugs were prepared fresh on the day of experiment and appropriate vehicle controls were used as required.

2.3 Surgical procedure for recording EMG and intrathecal (i.t.) drug delivery

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The implantation of EMG recording electrodes and catheter were performed in rats under deep anesthesia by injecting sodium pentobarbital (50mg/kg. i.p.) as previously reported (Kannampalli et al., 2017). Briefly, teflon-coated electrodes (Cooner Wire, Part

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No. AS631, Chatsworth, CA, USA) were implanted into the external oblique muscle of the abdomen to record EMG activity of the muscles. The electrodes were tunneled subcutaneously and externalized at the back of the neck.

For intrathecal i.t. drug

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injection at the lumbo-sacral (LS) segment of the spinal cord, a polyethylene catheter was chronically implanted into the epidural space as reported by Chen et al (2012).

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Briefly, following the EMG electrode implantation, a 2-3 cm midline incision was made on the dorsal surface and the muscles were retracted to expose the L1-L2 vertebral segment. A small laminectomy was performed allowing visualization of the spinal cord. A polyethylene catheter (PE-10) filled with sterile saline was carefully inserted 1-1.5 cm

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caudally so that the tip of the catheter lies exactly at the lumbo-sacral region of the spinal cord. The catheter was then secured to the muscles with 4-0 silk suture and sealed with a stainless steel pin to prevent leakage. The remaining catheter was

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tunneled subcutaneously, externalized at the back of the neck and secured in place with siliastic tubing along with the EMG electrodes. The lumbar midline skin incision was

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then closed with 4-0 silk suture. The placement of the i.t. catheter in the lumbo-sacral segment of the spinal cord was confirmed by injecting 20µl of sterile lidocaine (2%) and observed for a transient hind limb paralysis. Following the placement of i.t catheter, rats were closely monitored for any signs of dehydration, poor weight gain or motor deficit during the post-operative recovery period.

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For intra-cerebroventricular (i.c.v.) drug administration, the rats were placed in a standard rodent stereotactic frame (David Kopff, Tujunga, CA, USA) and the skull was exposed by a midline incision; a small burr hole was drilled unilaterally using the

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Paxinos stereotaxic co-ordinates (A-P: –2.0 mm bregma, 0.8 mm lateral to sagittal suture, 5.0 mm down from the skull surface). A guide cannula (part # 8IC311 GS, PlasticOne, Roanoke, VA, USA) was implanted and secured in place by applying tissue

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glue (3M Vetbond, MN, USA) and capped with a dummy cannula (part # 8IC311DC, PlasticOne, Roanoke, VA, USA).

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For UBD, a midline laparotomy was performed to expose the urinary bladder. Following aspiration of the residual urine, a small incision was made on the dome of the bladder and a PE-100 catheter was inserted into the bladder through the hole and tightly secured around the bladder tissue with the 3-0 silk suture. The bladder was

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distended (0.4ml) to check for any leakage. The other end of the catheter was tunneled subcutaneously and secured at the back of the neck along with the EMG electrodes. The abdominal incision was sutured in layers with 4-0 silk sutures. All rats that

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underwent surgery received analgesic (Carprofen, 5 mg/kg/day, i.m. for 3 days) and antibiotic (Enrofloxacin, 2.5 mg/kg/day, i.m for 3 days) post-operatively. Following the

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surgery, rats were housed separately and allowed to recover for at least 3 days prior to further interventions.

2.4 EMG recordings from the abdominal muscles during colon or bladder distension

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Three days after the electrode and bladder catheter implantation, rats were placed inside the restraining cylinder for 2 hours/day for three days in order to acclimatize them to experimental conditions.

The animals that did not feel comfortable inside the

occurs in our experiments.

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restraining cage even after 3 days of training were excluded from the study, which rarely On the day of VMR recordings, rats were placed in

restraining tubes and a highly compliant latex balloon (~5 cm long and 3.5 cm OD when

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fully inflated) coated with non-reactive bacteriostatic lubricant (Surgilube, Savage Laboratories, Melville, USA) was inserted into the colon through the anal opening for

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colorectal distension (CRD). A stimulus-response function (SRF) to graded intensities of CRD (10, 20, 30, 40, 60 mmHg) was recorded. The duration of distension was 30s with a 180s inter-stimulus interval between two intensities of distensions. The EMG response to CRD was amplified using the amplifier (A-M System, model 1700,

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Carlsborg, WA, USA). For the measurement of VMR to UBD, graded volume (0.5, 1.0 and 2.0 ml) of sterile saline was injected into the bladder through the UBD catheter connected to a pressure transducer and infusion pump. Similar to CRD, the UBD was

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maintained for 60s and the responses to graded volumes of distension were measured with a 180s inter-distension interval. The animals that exhibited intensity-dependent

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increasing EMG responses to graded UBD or CRD during baseline (pre-drug) VMR recording were only included for further investigation. Following a baseline SRF to either CRD or UBD, rats were administered ADX71441 either i.p. or i.t. or i.c.v. and the VMR to CRD/UBD were repeated 5 minutes after the drug administration. Data were recorded real-time using the Spike 4/CED 1401 data acquisition program (CED 1401; Cambridge Electronic Design, Cambridge, UK).

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2.5 Electrophysiology studies 2.5.1 Recording from UBD-sensitive pelvic nerve afferent (PNA) fibers

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The surgical procedure and recordings from the L6-S1 dorsal root was performed as previously described (Sengupta and Gebhart, 1994). Rats were anesthetized with urethane (1.5 g/kg, i.p) and maintained with supplemental doses as required.

The

stroke volume).

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trachea was intubated to mechanically ventilate the rat (55-60 strokes/min and 3-4 ml The femoral artery and vein were cannulated for recording blood The rat was paralyzed with gallamine

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pressure and injecting drug, respectively.

triethiodide (1 mg/kg, i.v.) and supplemental doses were given as required to maintain paralysis during the recording process. Following a midline laparotomy to expose the urinary bladder, the urine was aspirated with a syringe and a small incision was made

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on the dome of the bladder. A PE-100 catheter for urinary bladder distension (UBD) and a pressure transducer (Millar Mikro-Tip®, Model # MPC-500) probe were inserted into the bladder through the incision and tightly secured to the bladder tissue without any

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leakage. The external urethral orifice was sealed with a tissue adhesive (3MTM VetbondTM) and the abdominal incision was sutured in layers with 4-0 silk sutures. The

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LS spinal cord was exposed by a laminectomy between T13 and S2 segment of the spinal cord. The spinal vertebrae was slightly suspended and stabilized by clamping the thoracic and ischial vertebrae. The dorsal skin was reflected laterally and tied to the spinal posts to make a pool for mineral oil. The dura was carefully removed and the spinal cord was covered with warm (37°C) mineral oil. Recordings were made from the distal cut-end of the central processes of the L6 dorsal root as described in our previous

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study (Sengupta et al., 2002).

The UBD-sensitive PNA fibers were identified by

distending the bladder (0.4 ml). Once the recording fiber exhibits increasing response to bladder distension a stimulus-response function (SRF) was constructed by applying

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graded UBD (0.2, 0.4, 0.6 and 0.8 ml). After recording the baseline SRF, ADX71441 (10 mg/kg, i.v.) was administered and a second SRF was constructed 5 minutes after the drug administration to test the effect of the drug on the mechanosensitivity of these

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afferent fibers. The action potentials were amplified through a low-noise AC differential amplifier (model 3000; A-M Systems) and displayed on an oscilloscope. For multi-unit

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recordings, the responses of UBD-sensitive PNA fiber was isolated real time by selectively discriminating the waveform through the window discriminator (modelDDIS1, BAK Instrument) or off-line by using signal waveform analysis software (spike2

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1401 data acquisition software, Cambridge Electronic Design, UK).

2.5.2 Recording from UBD-sensitive lumbo-sacral (LS) spinal neurons The surgical procedure for spinal recordings was the same as described in the previous Recordings were done from both spinal transected and spinal intact rats.

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section.

Cervical (C1-C2) spinal transection was performed by making a small midline incision at

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the neck area and exposing the cervical spinal cord using a retractor.

The dura

membrane was gently removed and 10-15µl of 2% lidocaine was applied to the dorsal surface of the exposed spinal cord.

After 10 min, the spinal cord was completely

transected and the area was covered with a small piece of gelfoam soaked in warm saline and the incision was closed with sutures. Following the spinal transection, rats exhibited a vasodepressor effect, which recovered after 15-30 minutes.

During the

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recovery period the LS spinal cord was exposed and a pool was made as described in the previous section. Recordings from the LS spinal neurons were performed at least one hour after all surgical procedures. The dura membrane was carefully removed and

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the pool was covered with 1.75% agar (<40ºC) in saline. After solidification of the agar, a small window was cut to expose the LS spinal cord. Single barrel carbon fiber filled glass microelectrode (0.4-0.8 mΩ, CARBOSTAR-1, Kation Scientific, MN) was used for

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extracellular recording. The placement of the electrode was 0.1-0.5 mm lateral from the spinal midline and recordings were done in depth between 900-1200 µm from the dorsal

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surface of the spinal cord. To test the effect of ADX71441 on responses of UBDresponsive spinal neurons the same experimental protocol was followed as described in the previous section. Briefly, a SRF to graded UBD was constructed before injecting the drug. A second SRF was constructed 5 minutes after injecting ADX71441 (10

2.6 Statistical Analysis

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mg/kg, i.v.).

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2.6.1 VMR studies: Statistical analysis was performed using SigmaStat (V2.03, SPSS, Inc., Chicago, IL). An SRF to graded CRD/UBD was constructed to test the intensity

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dependent increase in EMG activity. The EMG response to each distension pressure or volume was divided by the EMG response to the highest distension volume (2.0 ml) or pressure (60mmHg) to obtain normalized percentage values. Comparison was performed between pre- and post-ADX71441 responses to respective intensity of distending volume or pressure. Statistical analysis was performed by two way repeated measure of ANOVA followed by Holm-Sidak test for multiple comparisons. The data

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was also subjected to the normality and equal variance test with Shapiro-Wilk test. Values are expressed as mean ± S.E.M. and p<0.05 were considered to be significant.

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2.6.2 Electrophysiology studies: For analysis of PNA fibers and LS spinal neuron’s responses to UBD, the total number of action potentials over a 30s resting period prior to bladder distension and during the distension period (30s) was counted and

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represented as impulses/s. To measure the actual changes in response of the neurons to UBD, the mean firing frequency during the resting period (30s pre-distension) was

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subtracted from the mean firing frequency during bladder distension (30s distension). The difference was calculated for each distension volume tested (0.2-0.8 ml) and the response was divided by the response of the neuron to the highest distension volume (0.8 ml) in order to obtain normalized percentage values. The baseline spontaneous

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firing response was calculated from the mean firing frequency during a 60s resting period devoid of any distension and represented as impulses/s. Statistical analysis was determined by two way repeated measure of ANOVA followed by Holm-Sidak test for

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multiple comparisons. The data was also subjected to the normality and equal variance test with Shapiro-Wilk test. Values are expressed as mean ± S.E.M. and p<0.05 were

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considered to be significant.

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3. Results 3.1 Systemic effect of ADX71441 on the VMR to CRD in awake rats ADX71441, a selective GABAB PAM has been previously reported to exhibit analgesic

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effect in somatic pain model (Kalinichev et al., 2017a, b). However, the analgesic effect of ADX71441 on a specific model of visceral nociception has not yet been studied. In this study, we first systematically examined a dose-dependent analgesic effect of the

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drug against noxious distension of the bladder and colon. In fully awake rats, when VMRs were recorded to CRD, ADX71441 (5, 10, 50 mg/kg, i.p.) dose-dependently

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attenuated the VMR (fig 1). Figures 1A and B illustrates representative examples of CRD-induced EMG activities before and after administration of systemic ADX71441 (10mg/kg, i.p.), respectively. In adult rats, ADX71441 (5 mg/kg) produced a significant (n=6, F (5, 25) = 5.845, p<0.001 vs pre-ADX71441, fig 1C) decrease in the VMR to

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graded CRD from > 20mmHg onwards. A similar effect was also observed at 10 mg/kg (n=6, F (5, 25) = 9.723, p<0.001 vs pre-ADX71441) and 50 mg/kg of ADX71441 (n=6, F (5, 25) = 14.926, p<0.001 vs pre-ADX71441). The reference drug baclofen (GABAB

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agonist) also produced a significant (n=6, F (5, 15) = 5.469, p<0.001 vs pre-BAC, fig 1D) decrease in the VMR to CRD at all distension pressure tested.

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To determine the time course of the visceral analgesic effect of ADX71441, we

tested the EMG response to graded CRD at 5 min, 1, 2 and 3hr post administration of the drug (Supp fig 1A). ADX71441 (10 mg/kg, i.p) produced a significant (n=6, F (5, 25) = 10.507, p<0.001 vs pre-ADX71441, supp fig 1B) decrease in the VMR to graded CRD from 10mmHg onwards. A similar effect was also observed at post 1hr (n=6, F (5, 25) = 8.382, p<0.001 vs pre-ADX71441), post 2 hr (n=6, F (5, 25) = 7.324, p<0.001 vs

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pre-ADX71441) and post 3hr (n=6, F (5, 25) = 6.520, p<0.001 vs pre-ADX71441) of ADX71441 administration. However, the effect of the drug was found to be reduced at 3hr indicating that ADX71441 has a peak analgesic effect for up to 3 hours of

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administration (supp fig 1C).

3.2 Lack of inhibitory effect of ADX71441 on the VMR to CRD at the spinal level Intrathecal (i.t) injection of ADX71441 (10, 50 and 100µg) did not produce inhibition of Figures 2A and 2B show representative EMG

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EMG activities to graded CRD.

responses to colon distension (60mmHg) from the external oblique muscles of the

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abdomen before and after the injection of ADX71441 (50µg, i.t.), respectively. The mean SRFs to graded colon distension are superimposable at all three doses of ADX71441 used and did not exhibit inhibition or facilitation of responses compared to pre-ADX71441 baseline (10 µg, n=6, F (5, 25) =0.445, p=0.584 vs pre-drug; 50µg, n=6,

pre-drug, fig 2C).

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F (5, 25) = 0.157, p=0.861 vs pre-drug, and 100µg, n=6, F (5, 25) = 1.764, p=0.342 vs In contrast, baclofen (1µg, i.t.), a positive control, produced a

significant decrease in EMG at all intensities of distending pressure (n=6, F (5, 25) = This dose of baclofen did not produce any

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9.650, p<0.001 vs pre-BAC, fig 2D).

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sedation and paralysis.

3.3 Central effect of ADX71441 on VMRs to UBD in awake rats Since the main focus of the study is to examine the analgesic effect of ADX71441 against noxious bladder stimulation, in subsequent experiments the effect of the drug was tested to check the influence of the drug on VMR to noxious bladder distension in fully awake rats. In order to examine the site of action of the drug, experiments were

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undertaken systematically by injecting the drug systemically (i.p.), spinally (at LS spinal cord) and supra-spinally (i.c.v.) during the recording of EMG to bladder distension. The doses of ADX71441 in these experiments were selected on the basis of its effect on

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VMR to CRD (see previous sections).

When ADX71441 was injected i.p (5 mg/kg) it produced marked inhibition of EMG activities to isovolumic UBD. Figures 3A and 3B illustrate examples of EMG

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responses to UBD (1.0 ml) before and after i.p. injection of the drug, respectively. The mean EMG response to graded volume (0.5 – 2.0ml) of bladder distension shows a

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significant inhibition of EMG following the drug injection (n=6, F (3, 15) = 8.832, p<0.001 vs pre-drug, fig 3G).

In the next set of experiments, ADX71441 was injected i.t. (10µg bolus) into the LS segment of the spinal cord while recording EMG to graded bladder distension in

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fully-awake rats. Similar to VMR recording to colon distension, EMG responses to UBD following i.t. injection of ADX71441 was unaffected.

Examples of EMG response

patterns before and after the drug injection have been shown in figures 3C and 3D,

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respectively. The analytical data of the mean EMG response to graded volume (0.5 – 2.0ml) of bladder distension does not show significant inhibition of EMG following the

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injection of ADX71441 (n=6, F (3, 15) = 3.034, p=0.062 vs pre-drug, fig 3H). Since ADX71441 did not produce any inhibition of EMG at the spinal level, but

systemically, in subsequent set of experiments the drug was injected i.c.v. while recording EMG responses to bladder distension. Examples of EMG responses patterns to 1.0ml of bladder distension before and after i.c.v. injection of ADX71441 (1µg bolus) have been shown in figures 3E and 3F, respectively. These two figures show that the

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EMG was markedly inhibited after the i.c.v. injection of the drug. The analytical data of the mean EMG response to graded volume (0.5 – 2.0ml) of bladder distension shows a significant inhibition of EMG responses to bladder distension following the injection of

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ADX71441 (n=6, F (3, 15) = 5.326, p<0.05 vs pre-ADX71441, fig 3I).

3.4 Effect of ADX71441 on peripheral and central neurons involved in bladder

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nociceptive signaling

To test the site of action of ADX71441 in pain signaling pathway, we systematically

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examined the effect of the drug on responses of (a) mechanosensitive pelvic nerve afferent (PNA) fibers innervating the bladder, (b) spinal dorsal horn neurons receiving synaptic input from the bladder-sensitive PNA fibers and (c) the descending modulation from the supra-spinal site.

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To examine whether ADX71441 attenuates mechanosensitivity of UBD-sensitive PNAs, we have recorded responses of these afferent nerve fibers to UBD before and after ADX71441 injection (10mg/kg, i.v.).

Figures 4A and 4B show examples of

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volume-dependent excitations of one UBD-sensitive PNA fiber before and after injection of ADX71441, respectively. A total of 6 mechanosensitive PNA fibers from 6 rats were

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tested in these experiments. ADX71441 did not inhibit responses of these fibers phasic isovolumic UBD (n = 6, F (3, 15) = 1.301, p = 0.311 vs pre-ADX71441, fig 4C), neither it attenuated the spontaneous firing of these afferent fibers (n = 6, p = 0.0716 vs pre-drug, fig 4D).

In the second set of experiments, we examined the effect of the drug on L6 spinal dorsal horn neurons responsive to UBD.

The objective was to examine if

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responses of L6 spinal neurons undergo inhibition after the drug injection, which will suggest the modulation of nociceptive signaling from the spinal cord to supraspinal brain structures. In addition, we also wanted to test whether the effect of the drug is

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driven supraspinally by transmitting a descending inhibitory influence to modulate responses of spinal neurons. Therefore, experiments were designed to test the effect of ADX71441 on responses of UBD-responsive L6 spinal neurons either from spinal intact

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rats or from cervical (C1-C2) spinal cord transected rats to remove the supraspinal descending influence.

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We tested 6 UBD-responsive L6 neurons from 6 rats with the spinal cord intact. Figures 5A and 5B illustrate typical response patterns of one such neuron to two volumes (0.4 and 0.6ml) of bladder distension before and after ADX71441 (10mg/kg, i.v.), respectively. The response of the neuron to distension was markedly inhibited The mean SRFs show that ADX71441 significantly

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after the injection of the drug.

inhibited responses of these neurons at every volume of distension (0.2-0.8ml) (n=6, F (3, 15) = 7.610, p<0.01 vs pre-ADX71441, fig 5C). The drug also produced a significant

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decrease in the mean spontaneous firing of these spinal neurons (n=6, p<0.05, fig 5D). In the next step, similar experiments were undertaken, but in spinal cord

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transected rats. Following cervical spinal transection, the rats exhibited vasodepressor effect that was stabilized in about 30 minutes. These animals also exhibited a complete absence of normal voiding contraction due to the lack of supra-spinal control from the micturition centers of the brain. However, their responses to graded UBD remain unaltered following spinal transection. Figures 6A and 6B illustrate responses of an UBD-responsive L6 neuron to two volumes of bladder distension before and after the

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injection of ADX71441 (10 mg/kg, i.v.), respectively. In contrast to that observed in spinal cord intact rats, the analytical data of the mean SRFs before and after the injection of ADX71441 did not exhibit any inhibitory effect of the drug (n=5, F (3, 12) =

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0.349, p = 0.790 vs pre-ADX71441, fig 6C). Similarly, ADX71441 did not decrease the mean spontaneous firing of these neurons from spinal transected rats (n = 5, p = 0.109,

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fig 6D).

3.5 Effect of ADX71441 on the bladder cystometry and VMR during slow infusion

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To address whether the visceral analgesic effect of ADX71441 is independent of its effect on normal bladder function, we tested the effect of the drug on abdominal muscle EMG activities during the slow filling of the bladder. Figures 7A and 7B illustrate representative tracings of the EMG responses and cystometrographic recording during

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slow infusion of the bladder before and after the injection of ADX71441 (10mg/kg, i.p.), respectively. The post-drug mean EMG response indicates that ADX71441 significantly decreases the VMR to slow infusion (n=6, p<0.05, fig 7C). However, the total number

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of voiding contractions following the slow infusion remained unaltered and did not show any significant decrease after ADX71441 injection (n=6, p = 0.173, fig 7D). Baclofen on

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the other hand had a significant inhibitory effect on the voiding contraction during slow filling of the bladder. Figures 8A and 7B illustrate representative tracings of the EMG responses and cystometrographic recording during slow infusion of the bladder before and after the injection of ADX71441 (1 mg/kg, i.p.), respectively.

While the EMG

response was significantly decreased (n=6, p<0.05, fig 8C), baclofen also produced a

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significant decrease (n=6, p<0.05, fig 8C), in the total number of voiding contraction

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during slow infusion of the bladder.

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4. Discussion The present study documents that positive allosteric modulator of GABAB receptor can produce analgesia in a rodent model of bladder nociception. Increasing evidence

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indicate that GABA receptors are involved in regulation of both acute and chronic pain (Enna and McCarson, 2006; McCarson and Enna, 1999; 2014; Hyland and Golubeva, 2015). Several preclinical and clinical reports strongly suggest that the GABAB receptor

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can also function as a potential target for treating bladder dysfunction (Sanger et al., 2002). Currently, baclofen is the only available GABAB receptor agonist used to treat

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muscle spasms in conditions like multiple sclerosis and spinal cord injury (Elbasiouny et al., 2010; Clearfield et al., 2016). Baclofen has also been reported to alleviate symptoms and improve bladder function in patients with functional bladder outlet obstruction (Chen et al., 2016) and in the relief of bladder spasm (Wallace et al., 2013).

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However, its clinical use is limited by its short half-life and entral effects (Leggio et al., 2010; Muzyk et al., 2012; Brennan et al., 2013). In the absence of a reliable orthosteric ligand, positive allosteric modulator (PAM) of GABAB receptor offers an alternative

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option by providing the same therapeutic benefit. ADX71441 is a novel, potent, and selective PAM of GABAB receptor with good permeability across the blood-brain barrier

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(Kalinichev et al., 2017a). Preclinical studies have shown that ADX71441 has anxiolytic and antinociceptive effects and also reduces the signs of OAB in mice and guinea pigs (Kalinichev et al., 2014). Recently, ADX71441 was reported to reduce nociception in a rodent model of osteoarthritis induced pain (Kalinichev et al., 2017b) and was also found to reduce alcohol intake in mice and rats (Hwa et al., 2013; Augier et al, 2017). The objective of the present study is to evaluate the function of this GABAB PAM in the

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modulation bladder nociception and its site of action in the pain signaling neuraxis in rodents. In addition, this study was intended to examine whether the analgesic effect of the drug is associated with altered bladder function, especially by affecting normal

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bladder motility.

In behavioral visceral nociceptive assessment experiments in awake rats, ADX71441 produced a dose-dependent (5 to 50 mg/kg, i.p.) inhibition of EMG

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responses to colon distension, indicating that the drug exerts an analgesic effect to noxious visceral stimulus. Therefore, it appears that a PAM like ADX71441 might

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potentially serve as a promising therapeutic agent in the management of chronic pelvic pain. To test whether ADX71441 alleviates bladder nociception by modulating the activities and/or synaptic transmission of spinal dorsal horn neurons, the drug was injected i.t. at the LS segment of the spinal cord. However, the drug did not produce

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inhibition of VMR when 10µg of ADX71441 was injected; neither did it produce inhibition when the dose was increased to 50 and 100µg. This result suggests that ADX71441 does not act at the spinal cord to reduce nociceptive transmission via the local

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modulation of spinal neurons. In contrast, baclofen (1µg, i.t.) significantly inhibited the EMG responses in these rats. The behavioral result of ADX71441 was supported by

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our electrophysiology experiments where systemic injection of ADX71441 failed to attenuate responses of UBD-responsive spinal neurons from spinal cord transected rats. Therefore, our VMR results in awake rats and electrophysiology recordings of UBD-responsive LS spinal neurons indicate that ADX71441 possibly produces analgesic effect by acting at the supraspinal level. However, recently the drug has been reported to decrease the locomotor activity of the rats on the rotarod test when

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administered orally (Kalinichev et al., 2017a, b). In contrast, Augier et al (2017) reported that ADX71441 attenuates alcohol self-administration and relapse to alcohol seeking in rats without any significant sedative side-effects. While the centrally mediated analgesic

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effect of ADX71441 is convincing, the above observations necessitate the need for an extensive study to investigate the side-effect profile of ADX71441.

In order to confirm the exact site of action of ADX71441, the study was designed

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to systematically eliminate the possible peripheral action of the drug in modulating the functions of pelvic nerve afferent (PNA) fibers innervating the bladder.

The results

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indicate that the drug did not inhibit the spontaneous firing of PNA fibers neither did it attenuate the UBD-induced excitation of these fibers, suggesting that ADX71441 does not attenuate mechanotransduction properties of these primary sensory afferents to produce analgesia. On the other hand, previous studies have shown that orthosteric

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GABAB agonist baclofen can attenuate mechanical responses of afferent fibers including vagus and PNA fibers innervating the esophagus and descending colon, respectively (Page and Blackshaw, 1999; Sengupta et al., 2002). In the present study,

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the effect of baclofen was not tested, because it was not the main objective of the study. To reinforce our behavioral study that has shown that ADX71441 does not exhibit

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analgesia when injected directly into the LS spinal cord, we designed experiments to test the effect of ADX71441 on the responses of UBD-responsive spinal neurons. In these experiments, electrical activities of the LS spinal cord to UBD were recorded either from rats with spinal cord intact or from rats that underwent cervical (C1-C2) spinal transection to remove descending influence on the spinal neurons. In the spinal cord intact rats, systemic injection (10mg/kg, i.v.) of the drug significantly inhibited the

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responses of UBD-responsive LS neurons to bladder distension.

However, this

inhibitory effect of the drug was not observed when the similar experiments were repeated in cervical transected rats. These results support our behavioral experiments

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where the drug was ineffective when injected spinally suggesting a centrally-mediated effect of the drug. Systemic (i.v) administration of ADX71441 in naïve spinal intact rats also produced a significant inhibition of the spontaneous firing of these neurons

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indicating a strong inhibitory effect at the LS spinal cord. However, when tested on the spinal transected rats, ADX71441 did not inhibit the response of these bladder sensitive

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LS spinal neurons to UBD. The spontaneous firing of these neurons also remained unaltered following drug administration indicating a lack of spinal effect. It must be noted that transection of the spinal cord at the cervical (C1-C2) was done to inhibit the descending inhibitory influence on the LS spinal neurons from supra-spinal sites. The

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inhibition of the response of LS spinal neurons by ADX71441 from spinal intact rats can be attributed to its action at supra-spinal sites exerting a descending inhibitory influence on the bladder-sensitive LS spinal neurons.

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ADX71441 was previously reported to improve the symptoms of OAB in mice, by increasing urinary inter-contraction interval, reducing the number of micturition and

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voiding volumes (Kalinichev et al., 2014). In acetic acid induced OAB model in guinea pigs, ADX71441 was found to increase the inter-contraction interval and bladder capacity and reduced micturition frequency (Kalinichev et al., 2014). Based on these reports, we wanted to test whether the visceral analgesic effect of ADX71441 is independent of its effect on normal bladder function by performing cystometrogram studies in fully awake rats by measuring the abdominal EMG activity during slow filling

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of the bladder. The slow filling of the bladder resulted in initiation of voiding contraction and a simultaneous increase in the EMG activity of the external oblique muscles. Following ADX71441 administration, the number of voiding contraction was not

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significantly altered, however, there was a significant attenuation of EMG activity during increasing bladder pressure caused by slow filling of the bladder. Baclofen on the other hand, significantly affected the normal bladder function characterized by a decrease in

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the number of voiding contraction along with decreased EMG activity during slow filling of the bladder. This result indicates that ADX71441 exhibits strong visceral analgesic

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effect without affecting the normal bladder function.

The electrophysiology experiments document that ADX71441 does not modulate responses of PNA fibers, indicating a lack of peripheral analgesic effect in our study. Similarly, the drug does not modulate responses of the UBD-responsive spinal,

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suggesting a lack of analgesic effect at the spinal cord. Instead, it modulates responses of spinal neurons when injected i.c.v., suggesting that the drug exerts descending inhibitory influence to modulate responses of UBD-responsive spinal neurons.

The

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results from electrophysiology experiments correlate well with the findings from the behavioral experiments, since the i.t injection of the drug did not attenuate the VMRs to

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CRD or UBD. On the other hand, i.c.v injection of the drug resulted in a significant inhibition of the VMR to noxious bladder distension. Findings from previously published reports indicate that the analgesic effect of PAMs of GABAB receptors can differ between different PAM and also in different models of nociception in rodent.

For

example, rac-BHFF, another GABAB PAM did not alter the paw-withdrawal latency in mice model of neuropathic pain, although the drug increased the latency significantly in

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naïve sham operated mice (Zemoura et al., 2016).

However, rac-BHFF was found to

potentiate the effect of baclofen in producing the analgesic effect, which suggests that PAM of GABAB receptors might provide an additive effect to reduce the dose of

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baclofen to produce analgesia. On the other hand, the findings from our study indicate that ADX71441 has strong centrally mediated analgesic effect to noxious visceral (colon and bladder) stimulation in naïve rats.

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Interestingly, in our study while the GABAB agonist baclofen exhibited strong analgesic effect at the spinal level, the PAM ADX71441 did not produce any effect at

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the spinal cord. The absence of analgesic effect following intrathecal administration of ADX71441 might be due to the lack of involvement of PAM in modulating bladder nociception at the spinal level although, further studies are warranted to confirm this. In conclusion, our results indicate that ADX71441 exhibits supra-spinally mediated

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analgesic effect in a rodent model of bladder nociception. However, further studies are warranted to determine the side-effect profile and efficacy of ADX71441 in other pre-

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clinical models of experimentally induced bladder nociception.

ACKNOWLEDGMENT

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This work was supported by a grant from Addex Therapeutics awarded to Dr. J. N. Sengupta.

The authors thank Dr. Anjishnu Banerjee for his assistance with the

statistical analysis. This publication was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001436. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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CONFLICT OF INTEREST Authors SP and CB are employed by Addex Therapeutics.

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ETHICS APPROVAL

All experiments were performed according to the approved guidelines of the Institutional Animal care and Use committee at the Medical college of Wisconsin (approval #

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AUA355) and International Association for the Study of Pain (IASP) for humane use of

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laboratory animals.

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Legends Fig.1: Effect of systemic ADX71441 on the VMR to CRD of naïve adult rats. Representative EMG tracing pre- and post administration of ADX71441 to 60mmHg

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CRD (A and B). In both the panels, the top tracing represents the EMG activity of the abdominal external oblique muscle and the bottom tracing indicated the colon distension pressure. VMR is represented as percentage normalized EMG response to

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graded (10–60 mmHg) CRD. Systemic administration of 5, 10 and 50 mg/kg of ADX71441 significantly (n=6, p<0.001 vs pre-ADX71441) decreased the VMR to CRD >

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20 mmHg onwards (C). Similarly, systemic administration (1 mg/kg) of baclofen (BAC) also significantly (n=6, p<0.001 vs pre-BAC) decreased the VMR to CRD from 10mmHg pressure onwards. Values are expressed as mean ± S.E.M of ‘6’ animals in each group.

p<0.05 was considered significant. *, # and $ represents respective post-

BAC baseline.

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ADX71441 or post-BAC response compared with corresponding pre-ADX71441 or pre-

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Fig.2: Effect of intrathecal ADX71441 on the VMR to CRD of naïve adult rats. Representative EMG tracing pre- and post administration of ADX71441 to 60mmHg

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CRD (A and B). In both the panels, the top tracing represents the EMG activity and the bottom tracing indicate the colon distension pressure. VMR is represented as percentage normalized EMG response to graded (10–60 mmHg) CRD. Intrathecal administration of 10, 50 and 100 µg of ADX71441 did not significantly (n=6, p>0.05 vs pre-ADX71441) decrease the VMR to CRD (C) Intrathecal administration (1 µg/kg) of baclofen (BAC) significantly (n=6, p<0.001 vs pre-BAC) decreased the VMR to CRD

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from 10mmHg pressure onwards.

Values are expressed as mean ± S.E.M of ‘6’

animals in each group. p<0.05 was considered significant. * compared with pre-BAC

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baseline

Fig.3: Effect of ADX71441 on the VMR to UBD of naïve adult rats. Representative EMG tracing pre- and post administration of systemic (A and B), intrathecal (C and D) and

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i.c.v (E and F) ADX71441 to 1.0 ml of UBD. In all three panels, the top tracing represents the EMG activity and the bottom tracing indicated the bladder distension

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pressure. VMR is represented as percentage normalized EMG response to isovolumic (0.5-1.5 ml) UBD. Systemic administration of ADX71441 significantly (n=6, p<0.001 vs pre-ADX71441) decrease the VMR to UBD (G). However, intrathecal administration of ADX71441 did not significantly (n=6, p=0.062 vs pre-ADX71441) decrease the VMR to

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UBD (H). ADX71441 (i.c.v) significantly (n=6, p<0.05) decreased the VMR to UBD (I). Values are expressed as mean ± S.E.M of ‘6’ animals in each group. p<0.05 was

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considered significant. * compared with pre-ADX71441 baseline

Fig.4: Example of typical response of a UBD-sensitive PNA fiber from an adult naive rat

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pre- (A) and post (B) administration of ADX71441 to phasic bladder distension. In all panels, the top trace shows the response to UBD represented as a frequency histogram (1s binwidth), the middle trace is the neuron action potential and the bottom trace is the distension volume. (C) Administration of ADX71441 (10 mg/kg, b.w. i.v) did not significantly (n=6, p=0.311 vs pre-ADX71441) alter the response of these fibers to phasic UBD. (D) The spontaneous firing of the PNAs was not significantly (n=6,

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p=0.0716 vs pre-ADX71441) altered following ADX71441 administration. Values expressed as normalized mean ± S.E.M of 6 neurons tested.

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Fig.5: Effect of ADX71441 on the UBD-responsive L6 spinal neurons from adult naïve spinal intact rats. Example of a typical response of an UBD-sensitive L6 spinal neuron pre- (A) and post- (B) ADX71441 administration. In all panels, the top trace shows the

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response to UBD represented as a frequency histogram (1s binwidth), the middle trace is the neuron action potential and the bottom trace is the distension volume. (C)

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Administration of ADX71441 (10 mg/kg, b.w. i.v) significantly (n=6, p<0.01 vs preADX71441) decreased the response of these neurons to phasic UBD. (D) ADX71441 also significantly (n=6, p<0.05 vs pre-ADX71441) attenuated the spontaneous firing of these spinal neurons. Values expressed as normalized mean impulse ± S.E.M of 6

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neurons. p<0.05 was considered significant. * compared with pre-ADX71441 baseline.

Fig.6: Effect of ADX71441 on the UBD-responsive L6 spinal neurons from adult naïve

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spinal transected rats. Example of a typical response of an UBD-sensitive L6 spinal neuron pre- (A) and post- (B) ADX71441 administration. In all panels, the top trace

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shows the response to UBD represented as a frequency histogram (1s binwidth), the middle trace is the neuron action potential and the bottom trace is the distension volume. (C) Administration of ADX71441 (10 mg/kg, b.w. i.v) did not significantly (n=5, p=0.790 vs pre-ADX71441) inhibit the response of these neurons to phasic UBD. (D) ADX71441 did not attenuate (n=5, p=0.109 vs pre-ADX71441) the spontaneous firing of

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these spinal neurons. Values expressed as normalized mean impulse ± S.E.M of 10 neurons. p<0.05 was considered significant. * compared with pre-ADX71441 baseline.

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Fig.7: Effect of ADX71441 on the EMG response to slow infusion of saline into the bladder of adult naïve rats. Example of a typical EMG response to slow infusion pre- (A) and post- (B) ADX71441 administration. In all panels, the top trace shows the EMG

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response to slow infusion and the bottom trace is the cystometrogram indicating the infusion dependent bladder contraction. (C) Administration of ADX71441 (10 mg/kg,

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b.w. i.p) significantly (n=6, p<0.05 vs pre-ADX71441) decreased the EMG response to phasic slow infusion. (D) ADX71441 did not significantly (n=6, p=0.173 vs preADX71441) affected the number of infusion induced voiding contraction (VC). Values expressed as normalized mean EMG response ± S.E.M of 6 animals. p<0.05 was

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considered significant. * compared with pre-ADX71441 baseline.

Fig.8: Effect of baclofen (BAC) on the EMG response to slow infusion (SI) of saline into

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the bladder of adult naïve rats. Example of a typical EMG response to SI pre- (A) and post- (B) baclofen administration. In all panels, the top trace shows the EMG response

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to slow infusion and the bottom trace is the cystometrogram indicating the infusion dependent bladder contraction. (C) Administration of BAC (1 mg/kg, b.w. i.p) significantly (n=6, p<0.05 vs pre-BAC) decreased the EMG response to phasic slow infusion. (D) BAC also significantly (n=6, p<0.05 vs pre-BAC) affected the number of infusion induced voiding contraction. Values expressed as normalized mean EMG

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response ± S.E.M of 6 animals. p<0.05 was considered significant. * compared with preBAC baseline. Supplementary Fig.1 Time course analgesic effect following systemic administration

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(10 mg/kg, i.p.) of ADX71441 on the VMR to CRD of naïve adult rats. Representative EMG tracing pre- and post-5 min, 1hr, 2hr and 3hr of ADX71441 to 40mmHg CRD (A). In the panel, the top 5 tracing represents the EMG activity of the abdominal external

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oblique muscle pre- and post-5 min, 1hr, 2hr and 3hr of ADX71441 administration and the bottom tracing indicates the colon distension pressure. VMR is represented as

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percentage normalized EMG response to graded (10–60 mmHg) CRD. Systemic administration of 10 mg/kg of ADX71441 significantly (n=6, p<0.001 vs pre-ADX71441) decreased the VMR to CRD from 10 mmHg onwards (C). ADX71441 significantly decreased the mean EMG response to 60mmHg CRD post 5 min of administration

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which remained significantly decreased till 2 hr. After 3 hr of ADX71441 administration, the EMG response showed slight recovery but still remained significantly decreased. *

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compared with pre-ADX71441 baseline.

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ADX71441, novel positive allosteric modulators of the GABA(B) receptor with distinct central/peripheral profiles, show efficacy in the monosodium iodoacetate model of

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neuropathic mice. Neuropharmacology. 108, 172-178.

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HIGHLIGHTS IC/PBS significantly affects the quality of life of the patient. ADX71441 is a novel positive allosteric modulator of GABAB receptor.

behavioral and electrophysiological techniques.

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Analgesic effect of ADX71441 was investigated in a rodent bladder pain model using

ADX71441 exhibits supraspinally mediated analgesic effect against bladder pain.

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ADX71441 can be a potential therapeutic option for treating IC/PBS in humans.

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Abstract Therapeutic use of GABAB receptor agonists for conditions like chronic abdominal pain, overactive bladder (OAB) and gastroesophageal reflux disease (GERD) is severely

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affected by poor blood-brain barrier permeability and potential side effects. ADX71441 is a novel positive allosteric modulator (PAM) of the GABAB receptor that has shown encouraging results in pre-clinical models of anxiety, pain, OAB and alcohol addiction.

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The present study investigates the analgesic effect of ADX71441 to noxious stimulation of the urinary bladder and colon in rats. In female Sprague-Dawley rats, systemic (i.p),

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but not intrathecal (i.t), administration of ADX71441 produced a dose-dependent decrease in viscero-motor response (VMR) to graded urinary bladder distension (UBD) and

colorectal

distension

(CRD).

Additionally,

intra-cerebroventricular

(i.c.v.)

administration of ADX71441 significantly decreased the VMRs to noxious UBD.

In

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electrophysiology experiments, the drug did not attenuate the responses of UBDsensitive pelvic nerve afferent (PNA) fibers to UBD. In contrast, ADX71441 significantly decreased the responses of UBD-responsive lumbosacral (LS) spinal neurons in spinal

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intact rats. However, ADX71441 did not attenuate these LS neurons in cervical (C1-C2) spinal transected rats.

During cystometrogram (CMG) recordings, ADX71441 (i.p.)

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significantly decreased the VMR to slow infusion without affecting the number of voiding contraction. These results indicate that ADX71441 modulate bladder nociception via its effect at the supra-spinal sites without affecting the normal bladder motility and micturition reflex in naïve adult rats.