Single-cell RNAseq reveals seven classes of colonic sensory neuron.

OBJECTIVE: Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons. DESIGN: Unbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca2+-imaging. RESULTS: Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca2+-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation. CONCLUSIONS: We identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development.

Visceral and somatic pain modalities reveal NaV 1.7-independent visceral nociceptive pathways.

KEY POINTS: Voltage-gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage-gated sodium channel subtype NaV 1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV 1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality-specific manner and help to direct drug discovery efforts towards novel visceral analgesics. ABSTRACT: Voltage-gated sodium channel NaV 1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV 1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor-specific NaV 1.7 knockout mouse (NaV 1.7Nav1.8 ) and selective small-molecule NaV 1.7 antagonist PF-5198007. NaV 1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV 1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV 1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve-gut preparations in mouse, or following antagonism of NaV 1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage-gated sodium channel α subunits revealed NaV 1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV 1.7 (in NaV 1.8-expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV 1.7 antagonist PF-5198007. Our data demonstrate that NaV 1.7 (in NaV 1.8-expressing neurons) contributes to defined pain pathways in a modality-dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV 1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.

Targeting fatty acid amide hydrolase as a therapeutic strategy for antitussive therapy.

Cough is the most common reason to visit a primary care physician, yet it remains an unmet medical need. Fatty acid amide hydrolase (FAAH) is an enzyme that breaks down endocannabinoids, and inhibition of FAAH produces analgesic and anti-inflammatory effects. Cannabinoids inhibit vagal sensory nerve activation and the cough reflex, so it was hypothesised that FAAH inhibition would produce antitussive activity via elevation of endocannabinoids.Primary vagal ganglia neurons, tissue bioassay, in vivo electrophysiology and a conscious guinea pig cough model were utilised to investigate a role for fatty acid amides in modulating sensory nerve activation in vagal afferents.FAAH inhibition produced antitussive activity in guinea pigs with concomitant plasma elevation of the fatty acid amides N-arachidonoylethanolamide (anandamide), palmitoylethanolamide, N-oleoylethanolamide and linoleoylethanolamide. Palmitoylethanolamide inhibited tussive stimulus-induced activation of guinea pig airway innervating vagal ganglia neurons, depolarisation of guinea pig and human vagus, and firing of C-fibre afferents. These effects were mediated via a cannabinoid CB2/Gi/o-coupled pathway and activation of protein phosphatase 2A, resulting in increased calcium sensitivity of calcium-activated potassium channels.These findings identify FAAH inhibition as a target for the development of novel, antitussive agents without the undesirable side-effects of direct cannabinoid receptor agonists.

Selective 5-HT2C receptor agonists: Design and synthesis of pyridazine-fused azepines.

Heterocycle-fused azepines are discussed as potent 5-HT2C receptor agonists with excellent selectivity over 5-HT2B agonism. Synthesis and structure activity relationships are outlined for a series of bicyclic pyridazino[3,4-d]azepines. By comparison with earlier published work, in vitro assays predict a high probability for achieving CNS penetration for a potent and selective compound 15a, a pre-requisite to achieve in vivo efficacy.

The passive and active contractile properties of the neurogenic, underactive bladder.

OBJECTIVE: To characterize passive and active changes in detrusor activity in a highly compliant bladder. MATERIALS AND METHODS: Bladders from adult female Sprague-Dawley rats were used 5 weeks after lower thoracic (T8) spinal cord transection or a sham-operation. Passive wall properties were assessed by pressure-volume relationships from whole bladders and the tensile response of bladder strips after a rapid (<0.5 s) stretch. Active properties were assessed from the frequency and amplitude of spontaneous contractions of bladder strips, and their response to the inotropic TRPV4 agonist GSK1016790A. RESULTS: Passive bladder wall stiffness of SCT bladders was significantly reduced compared to that of the sham-operated control group (N = 6 and 8, respectively) and SCT bladder strips relaxed more quickly than those from sham-operated rats. The frequency of spontaneous contractions was reduced in SCT rats, and their amplitude, expressed as a ratio of bladder wall stiffness, was greater than in sham-operated rats. GSK1016790A (0.1 µM) significantly increased amplitude in strips from both sham-operated and SCT groups. CONCLUSIONS: There is no evidence of contractile failure in a highly-compliant bladder. The observations of reduced passive bladder wall stiffness and an enhanced rate of stress relaxation lead to the conclusion that increased compliance is marked by altered matrix properties that dissipate muscle force, thereby generating low pressures. Contractile agonists may be effective for improving bladder function in detrusor underactivity.

Serendipity in drug-discovery: a new series of 2-(benzyloxy)benzamides as TRPM8 antagonists.

A new series of 2-(benzyloxy)benzamides are presented that are potent functional antagonists of TRPM8 and possess improved LipE and LE compared to the original lead. They were discovered through a series of compound libraries and we present a powerful visualization method for the chemical space explored with each library. Remarkably this new series originated from the highest risk design strategy where compounds were synthesised with the least degree of similarity to the lead structure.

Altered distribution of interstitial cells and innervation in the rat urinary bladder following spinal cord injury.

Changes in the distribution of interstitial cells (IC) are reportedly associated with dysfunctional bladder. This study investigated whether spinal cord injury (SCI) resulted in changes to IC subpopulations (vimentin-positive with the ultrastructural profile of IC), smooth muscle and nerves within the bladder wall and correlated cellular remodelling with functional properties. Bladders from SCI (T8/9 transection) and sham-operated rats 5 weeks post-injury were used for ex vivo pressure-volume experiments or processed for morphological analysis with transmission electron microscopy (TEM) and light/confocal microscopy. Pressure-volume relationships revealed low-pressure, hypercompliance in SCI bladders indicative of decompensation. Extensive networks of vimentin-positive IC were typical in sham lamina propria and detrusor but were markedly reduced post-SCI; semi-quantitative analysis showed significant reduction. Nerves labelled with anti-neurofilament and anti-vAChT were notably decreased post-SCI. TEM revealed lamina propria IC and detrusor IC which formed close synaptic-like contacts with vesicle-containing nerve varicosities in shams. Lamina propria and detrusor IC were ultrastructurally damaged post-SCI with retracted/lost cell processes and were adjacent to areas of cellular debris and neuronal degradation. Smooth muscle hypertrophy was common to SCI tissues. In conclusion, IC populations in bladder wall were decreased 5 weeks post-SCI, accompanied with reduced innervation, smooth muscle hypertrophy and increased compliance. These novel findings indicate that bladder wall remodelling post-SCI affects the integrity of interactions between smooth muscle, nerves and IC, with compromised IC populations. Correlation between IC reduction and a hypercompliant phenotype suggests that disruption to bladder IC contribute to pathophysiological processes underpinning the dysfunctional SCI bladder.

Identification of 5-HT(2C) mediated mechanisms involved in urethral sphincter reflexes in a guinea-pig model of urethral function.

UNLABELLED: Preclinically, studies investigating urethral function have shown that the clinical benefit of agents such as duloxetine may partly reflect increases in urethral striated muscle activity via a central mode of action. Duloxetine has been shown to inhibit the presynaptic reuptake of 5-HT and norepinephrine in Onuf's nucleus, leading to an increased activity of pudendal motor neurones and a subsequent increase in the strength of urethral sphincter contractions. Preclinical studies have postulated a role for both 5-HT(2C) receptors and 5-HT(2A) receptors in external urethral sphincter (EUS) function, with differences between species that may reflect the differing physiological roles of the EUS in different preclinical species. The present study therefore aimed to investigate the 5-HT receptor subtype involved in EUS function in the guinea-pig. The in vivo data reported in the present study suggest that the effects of clinical agents used to treat stress urinary incontinence, which enhance serotonergic drive, may be mediated, at least in part, via 5-HT(2C) receptors. OBJECTIVE: To elucidate the subtype of 5-HT receptor involved in urethral function using a preclinical model of urethral function. MATERIALS AND METHODS: The effects of the 5-HT(2C) agonist Ro 600-175 were investigated by measuring the urethral pressure profile in anaesthetized guinea-pigs together with antagonists at 5-HT(2A) , 5-HT(2B) and 5-HT(2C) receptor subtypes. RESULTS: Ro 600-175 increased peak urethral pressure in a dose-dependent manner. This effect was reversed by the selective 5-HT(2C) antagonist SB 242084. Neither the 5-HT(2A) antagonist MDL 100907, nor the 5-HT(2B) antagonist SB 240741 had any significant effect on the response. CONCLUSIONS: The clinical benefit of drugs used to treat stress urinary incontinence through enhanced serotonergic and adrenergic drive may be mediated, at least in part, via 5-HT(2C) receptors. Selective 5-HT(2C) agonism increases urethral tone, and hence provides an opportunity for developing new pharmacotherapies for stress urinary incontinence with reduced side-effects.

Kappa agonist CovX-Bodies.

Small peptidic kappa agonists were covalently linked to the reactive lysine of the CovX antibody to create compounds having potent activity at the kappa receptor with greatly extended half-life when compared to the parent peptide as exemplified by compound 20.

Pyrimido[4,5-d]azepines as potent and selective 5-HT2C receptor agonists: design, synthesis, and evaluation of PF-3246799 as a treatment for urinary incontinence.

New pyrimido[4,5-d]azepines 7 are disclosed as potent 5-HT(2C) receptor agonists. A preferred example, 7b had minimal activation at either the 5-HT(2A) or 5-HT(2B) receptors combined with robust efficacy in a preclinical canine model of stress urinary incontinence (SUI) and attractive pharmacokinetic and safety properties. Based on this profile, 7b (PF-3246799) was identified as a candidate for clinical development for the treatment of SUI. In addition, it proved to be critical to build an understanding of the translation between recombinant cell-based systems, native tissue preparations and in vivo preclinical models. This was a significant undertaking and proved to be crucial in compound selection.

In vitro affinity optimization of an anti-BDNF monoclonal antibody translates to improved potency in targeting chronic pain states in vivo.

The role of brain-derived neurotrophic factor (BDNF) signaling in chronic pain has been well documented. Given the important central role of BDNF in long term plasticity and memory, we sought to engineer a high affinity, peripherally-restricted monoclonal antibody against BDNF to modulate pain. BDNF shares 100% sequence homology across human and rodents; thus, we selected chickens as an alternative immune host for initial antibody generation. Here, we describe the affinity optimization of complementarity-determining region-grafted, chicken-derived R3bH01, an anti-BDNF antibody specifically blocking the TrkB receptor interaction. Antibody optimization led to the identification of B30, which has a > 300-fold improvement in affinity based on BIAcore, an 800-fold improvement in potency in a cell-based pERK assay and demonstrates exquisite selectivity over related neurotrophins. Affinity improvements measured in vitro translated to in vivo pharmacological activity, with B30 demonstrating a 30-fold improvement in potency over parental R3bH01 in a peripheral nerve injury model. We further demonstrate that peripheral BDNF plays a role in maintaining the plasticity of sensory neurons following nerve damage, with B30 reversing neuron hyperexcitability associated with heat and mechanical stimuli in a dose-dependent fashion. In summary, our data demonstrate that effective sequestration of BDNF via a high affinity neutralizing antibody has potential utility in modulating the pathophysiological mechanisms that drive chronic pain states.

Pharmacological properties of 2-((R-5-chloro-4-methoxymethylindan-1-yl)-1H-imidazole (PF-3774076), a novel and selective alpha1A-adrenergic partial agonist, in in vitro and in vivo models of urethral function.

2-((R-5-chloro-4-methoxymethyl-indan-1-yl)-1H-imidazole (PF-3774076) is a central nervous system (CNS) penetrant, potent, selective, partial agonist at the human alpha1(A)-adrenoceptor, demonstrating efficacy and selectivity in a range of binding and functional assays. In vivo, PF-3774076 increases peak urethral pressure in anesthetized female dogs in a dose-dependent manner, inducing changes in both the proximal and distal portions of the urethra via a central mechanism of action. The profile of this compound suggests that a CNS penetrant partial agonist at the alpha1(A)-adrenoceptor may offer significant benefit in stress urinary incontinence (SUI). However, despite partial agonism at the alpha1(A)-adrenoceptor and selectivity over alpha1(B)- and alpha1(D)-adrenoceptors, PF-3774076 did not offer the necessary degree of separation over cardiovascular events when assessed in in vivo models of cardiovascular function. This may be due to activation of both peripheral and central alpha1(A)-adrenoceptors. These data indicate that although central, partial alpha1(A)-agonists may offer significant benefit in the treatment of SUI, it may not be possible to achieve the desired level of urethral selectivity over cardiovascular events with this class of agent.

Discovery of a novel azepine series of potent and selective 5-HT2C agonists as potential treatments for urinary incontinence.

A range of heterocycle fused azepines were synthesized in order to find a CNS penetrant, selective 5-HT(2C) agonist for the treatment of incontinence. The pyridazo-azepines such as compound 11 were shown to be potent 5-HT(2C) agonists and have potential for CNS penetration and good in vitro ADME properties but lacked selectivity against 5-HT(2B). Fusing a further heterocycle gave the selective triazolopyrimido-azepines. An example of this series, compound 36, was shown to be potent, selective, metabolically stable in vitro and efficacious in an in vivo model of stress urinary incontinence.

Design and synthesis of pyridazinone-based 5-HT(2C) agonists.

The SAR of a series of pyridazinone derived 5-HT(2C) agonists has been explored and resulted in identification of a compound with excellent levels of 5-HT(2C) functional agonism and selectivity over 5-HT(2A) and 5-HT(2B). This compound displayed good in vivo efficacy in pre-clinical models of stress urinary incontinence, despite having physiochemical properties commensurate with impaired CNS penetration.

Design and synthesis of piperazinylpyrimidinones as novel selective 5-HT(2C) agonists.

This Letter reports the design and synthesis of several novel series of piperazinyl pyrimidinones as 5-HT(2C) agonists. Several of the compounds presented exhibit good in vitro potency and selectivity over the closely related 5-HT(2A) and 5-HT(2B) receptors. Compound 11 was active in in vivo models of stress urinary incontinence.

Novel 2-imidazoles as potent and selective alpha1A adrenoceptor partial agonists.

Novel 2-imidazoles have been identified as potent partial agonists of the alpha(1A) adrenergic receptor, with good selectivity over the alpha(1B), alpha(1D) and alpha(2A) receptor sub-types. Sulfonamide 23 possessed attractive drug-like properties with respect to physicochemical and ADME properties and wide ligand selectivity.

Novel 2-imidazoles as potent, selective and CNS penetrant alpha1A adrenoceptor partial agonists.

A novel series of central nervous system (CNS) penetrant indane 2-imidazoles have been identified as potent, partial agonists of the alpha(1A) adrenergic receptor, having good selectivity over the alpha(1B), alpha(1D) and alpha(2) sub-types. A key structural motif to impart selectivity is a methylene spacer between the indane and a pendant substituent, which includes heterocycles, sulphones and ethers. Introduction of an ortho-halogen to this group led to a lowering of intrinsic efficacy (E(max)).

Barrington's nucleus in the guinea pig (Cavia porcellus): location in relation to noradrenergic cell groups and connections to the lumbosacral spinal cord.

Micturition is largely controlled by Barrington's nucleus in the dorsolateral tegmentum of the pons. This nucleus coordinates simultaneous bladder contraction and external urethral sphincter relaxation, by means of a specific pattern of projections to the lumbosacral spinal cord. The most widely used small animal model in neurourological research is the rat. However, urodynamic studies suggest that, in sharp comparison to rat, guinea pig micturition is very similar to human micturition. Therefore, the present study, using retrograde and anterograde tracing and double immunofluorescence, was designed to investigate the location of Barrington's nucleus in the guinea pig, to identify Barrington's nucleus projections to the spinal cord and to clarify the relationship of Barrington's nucleus to pontine noradrenergic cell groups. Results show that Barrington's nucleus is located in the dorsolateral pons, projects to the intermediolateral and intermediomedial cell groups of the lumbosacral spinal cord and is clearly distinct from the pontine noradrenergic cell groups. These results show that the neuroanatomical circuitry in the spinal cord and brainstem that controls micturition in the guinea pig is similar to that in rat. This means that the differences between rat and guinea pig micturition on a behavioral level are not the result of different neuroanatomical connections in these parts of the central nervous system. These results provide a neuroanatomical basis for further neurourological studies in guinea pig.

A bladder-cooling reflex in the anaesthetised guinea-pig: a model of the positive clinical ice-water test.

INTRODUCTION: In patients with detrusor hyperreflexia, intravesical instillation of ice-cold water results in the development of involuntary bladder contractions at volumes less than normal cystometric capacity. This is referred to as a positive ice-water test (+IWT) and can be reversed by vanilloid receptor agonists and potentiated by menthol. The present study was designed to investigate the existence of an analogous cooling reflex in the guinea-pig bladder that could be used as a small animal model in order to test the effects of drugs on the reflex. METHODS: Bladder pressure and external urethral sphincter electromyogram (EUS EMG) were recorded in alpha-chloralose/urethane anaesthetised guinea-pigs during rapid infusion of cold or warm saline into the bladder with or without prior intravesical exposure to menthol or resiniferatoxin (RTX). RESULTS: The mean control micturition threshold volume (TV) of 2.58 ml at 38 degrees C was reduced to 1.52 ml in response to saline infusion at 3 degrees C (P=0.001). The cold-induced reduction in TV was reproducible during several subsequent repeat infusions at 38 degrees C and 3 degrees C and was accompanied by decreases in bladder voiding pressure. The duration of the micturition reflex was markedly increased following cold compared with warm saline infusion (mean 24.5 s at 3 degrees C, 10.2 s at 38 degrees C, P=0.001) and was associated with oscillations in bladder pressure and concomitant bursting activity in the EUS EMG. During step-wise decreases in infusate temperature from 38 degrees C to 23 degrees C, 15 degrees C, 7 degrees C and 3 degrees C, the threshold infusate temperature to elicit a significant reduction in TV was 15 degrees C. The reduction in TV at 3 degrees C was potentiated by intravesical infusion of 0.6 mM menthol whilst intravesical infusion of 500 nM RTX reversed the reduction in TV at 3 degrees C. DISCUSSION: These data suggest that a bladder-cooling reflex is present in the anaesthetised guinea-pig and represents a useful small animal model of the clinical +IWT.

Residues W320 and Y328 within the binding site of the µ-opioid receptor influence opiate ligand bias.

The development of G protein-biased agonists for the µ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a ß-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/KA) values. [3H]diprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative ß-arrestin2 activity at the W320A mutant, whilst its ß-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more ß-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation.