Mohan Shenoy

Computational Repositioning of the Anticonvulsant Topiramate for Inflammatory Bowel Disease

Computationally predicted repositioning of an anticonvulsant for inflammatory bowel disease is confirmed experimentally. Greening Drug Discovery Recycling is good for the environment—and for drug development too. Repurposing existing, approved drugs can speed their adoption in the clinic because they can often take advantage of the existing rigorous safety testing required by the Food and Drug Administration and other regulatory agencies. In a pair of papers, Sirota et al. and Dudley et al. examined publicly available gene expression data and determined the genes affected in 100 diseases and 164 drugs. By pairing drugs that correct abnormal gene expression in diseases, they confirm known effective drug-disease pairs and predict new indications for already approved agents. Experimental validation that an antiulcer drug and an antiepileptic can be reused for lung cancer and inflammatory bowel disease reinforces the promise of this approach. The authors scrutinized the data in Gene Expression Omnibus and identified a disease signature for 100 diseases, which they defined as the set of mRNAs that reliably increase or decrease in patients with that disease compared to normal individuals. They compared each of these disease signatures to each of the gene expression signatures for 164 drugs from the Connectivity Map, a collection of mRNA expression data from cultured human cells treated with bioactive small molecules that is maintained at the Broad Institute at Massachusetts Institute of Technology. A similarity score calculated by the authors for every possible pair of drug and disease ranged from +1 (a perfect correlation of signatures) to −1 (exactly opposite signatures). The investigators suggested that a similarity score of −1 would predict that the drug would ameliorate the abnormalities in the disease and thus be an effective therapy. This proved to be true for a number of drugs already on the market. The corticosteroid prednisolone, a common treatment for Crohn’s disease and ulcerative colitis, showed a strong similarity score for these two diseases. The histone deacetylase inhibitors trichostatin A, valproic acid, and vorinostat were predicted to work against brain tumors and other cancers (esophagus, lung, and colon), and there is experimental evidence that this is indeed the case. But in the ultimate test of method, the authors confirmed two new predictions in animal experiments: Cimetidine, an antiulcer drug, predicted by the authors to be effective against lung cancer, inhibited tumor cells in vitro and in vivo in mice. In addition, the antiepileptic topiramate, predicted to improve inflammatory bowel disease by similarity score, improved damage in colon tissue of rats treated with trinitrobenzenesulfonic acid, a model of the disease. These two drugs are therefore good candidates for recycling to treat two diseases in need of better therapies—lung cancer and inflammatory bowel disease—and we now have a way to mine available data for fast routes to new disease therapies. Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract for which there are few safe and effective therapeutic options for long-term treatment and disease maintenance. Here, we applied a computational approach to discover new drug therapies for IBD in silico, using publicly available molecular data reporting gene expression in IBD samples and 164 small-molecule drug compounds. Among the top compounds predicted to be therapeutic for IBD by our approach were prednisolone, a corticosteroid used to treat IBD, and topiramate, an anticonvulsant drug not previously described to have efficacy for IBD or any related disorders of inflammation or the gastrointestinal tract. Using a trinitrobenzenesulfonic acid (TNBS)–induced rodent model of IBD, we experimentally validated our topiramate prediction in vivo. Oral administration of topiramate significantly reduced gross pathological signs and microscopic damage in primary affected colon tissue in the TNBS-induced rodent model of IBD. These findings suggest that topiramate might serve as a therapeutic option for IBD in humans and support the use of public molecular data and computational approaches to discover new therapeutic options for disease.

Nerve growth factor regulates VR-1 mRNA levels in cultures of adult dorsal root ganglion neurons.

&NA; Nerve growth factor (NGF) regulates the nociceptive properties including sensitivity to capsaicin of a subset of dorsal root ganglion neurons, which express the high‐affinity NGF receptor, trkA. Capsaicin sensitivity co‐localizes with the expression of a cloned capsaicin receptor, vanilloid receptor type 1 (VR‐1), which displays properties similar to the native capsaicin response. To determine whether VR‐1 mRNA levels are regulated by NGF, VR‐1 mRNA levels and the ability to respond to capsaicin by release of the neuropeptide calcitonin gene related peptide (CGRP) were measured as a function of NGF concentration in cultures of adult dorsal root ganglion neurons. NGF treatment increased both VR‐1 mRNA expression and capsaicin evoked release of CGRP. These effects were inhibited by treatment with the trkA inhibitor k252a.

P2X receptor-mediated visceral hyperalgesia in a rat model of chronic visceral hypersensitivity

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterised by abdominal pain and bloating in association with altered bowel movements. Its pathogenesis and the underlying molecular mechanisms of visceral hyperalgesia remain elusive. Recent studies of somatic and other visceral pain models suggest a role for purinergic signalling mediated by the P2X receptor (P2XR) family. Aims: To examine the role of P2XR signalling in the pathogenesis in a rat model of IBS-like visceral hyperalgesia. Methods: Visceral hypersensitivity was induced by colonic injection of 0.5% acetic acid (AA) in 10-day-old rats and experiments were conducted at 8 weeks of age. Dorsal root ganglion (DRG) neurons innervating the colon were labelled by injection of DiI (1,1′-dioleyl-3,3,3′,3-tetramethylindocarbocyanine methanesulfonate) fluorescence into the colon wall. Results: Visceral hypersensitivity was reversed by TNP-ATP (2′-(or-3′)-O-(trinitrophenyl) ATP), a potent P2X1, P2X3 and P2X2/3 receptor antagonist. Rapid application of ATP (20 μM) induced a fast inactivating current in colon-specific DRG neurons from both control and AA-treated rats. There was a twofold increase in the peak ATP responses in neurons from AA-treated rats. These currents were sensitive to TNP-ATP (100 nM). Under current-clamped conditions, ATP evoked a larger membrane depolarisation in neurons from neonatal AA-treated rats than in controls. P2X3R protein expression was significantly enhanced in colon-specific DRGs 8 weeks after neonatal AA treatment. Conclusions: These data suggest that the large enhancement of P2XR expression and function may contribute to the maintenance of visceral hypersensitivity, thus identifying a specific neurobiological target for the treatment of chronic visceral hyperalgesia.

Molecular and behavioral changes in nociception in a novel rat model of chronic pancreatitis for the study of pain

&NA; The approach to the management of painful chronic pancreatitis has been empirical, primarily due to the lack of information about biological mechanisms producing pain. To facilitate research into pain mechanisms, our aim was to assess a rat model of chronic pancreatitis induced by pancreatic infusion of trinitrobenzene sulfonic acid as a model of painful pancreatitis. Nociception was assessed by measuring mechanical sensitivity of the abdomen and by recording the number of nocifensive behaviors in response to electrical stimulation of the pancreas. Expression of neuropeptides calcitonin gene‐related peptide (CGRP) and substance P (SP) in the thoracic dorsal root ganglia receiving input from the pancreas and nerve growth factor (NGF) in the pancreas were measured. Rats with pancreatitis exhibited marked increase in sensitivity to mechanical probing of the abdomen and increased sensitivity to noxious electrical stimulation of the pancreas. There were significant increases in NGF protein in the pancreas and in expression of neuropeptides CGRP and SP in the sensory neurons from dorsal root ganglia receiving input from the pancreas. We have established quantitative measures of referred nociception and pancreatic hyperalgesia in a rat model of chronic pancreatitis that bears histological similarities to the human disease. This model has considerable construct, face and predictive validity for the human condition. It is of importance for the study of the pathogenesis of pain in this condition and can facilitate the development of new therapeutic options.

Acute pancreatitis results in referred mechanical hypersensitivity and neuropeptide up-regulation that can be suppressed by the protein kinase inhibitor k252a ☆

Although pain is a cardinal feature of pancreatitis, its pathogenesis is poorly understood and treatment remains difficult. Nociceptive sensitization in several somatic pain models has been associated with activation of protein kinases including trkA, protein kinase C, and protein kinase A. We therefore tested the hypothesis that systemic treatment with a kinase inhibitor, k252a, known to inhibit all of these kinases would alleviate pain in an animal model of pancreatitis. Von Frey filament testing of somatic referral regions was evaluated as a method to measure referred pain in a rat model of acute necrotizing pancreatitis induced by L-arginine. Rats with pancreatitis showed increased sensitivity to abdominal stimulation with Von Frey filament. This referred mechanical sensitivity was associated with an 8-fold increase in levels of phosphorylated trkA in the pancreas and with significant up-regulation of both calcitonin gene-related peptide and preprotachykinin mRNA expression in thoracic dorsal root ganglia and with increased calcitonin gene-related peptide and substance P immunoreactivity in spinal cord segment T10. Treatment with the kinase inhibitor k252a suppressed the phosphorylation of trkA in the pancreas as well as reversed both the behavioral changes and the increase in neuropeptide expression associated with pancreatitis.

Nerve growth factor modulates TRPV1 expression and function and mediates pain in chronic pancreatitis.

BACKGROUND & AIMS The pathogenesis of pain in chronic pancreatitis (CP) is poorly understood and treatment remains difficult. We hypothesized that nerve growth factor (NGF) plays a key role in this process via its effects on the transient receptor potential vanilloid 1, TRPV1. METHODS CP was induced by intraductal injection of trinitrobenzene sulfonic acid in rats. After 3 weeks, anti-NGF antibody or control serum was administered daily for 1 week. Pancreatic hyperalgesia was assessed by nocifensive behavioral response to electrical stimulation of the pancreas as well as by referred somatic pain assessed by von Frey filament testing. TRPV1 currents in pancreatic sensory neurons were examined by patch-clamp. The expression and function of TRPV1 in pancreas-specific nociceptors was examined by immunostaining and quantification of messenger RNA levels. RESULTS Blockade of NGF significantly attenuated pancreatic hyperalgesia and referred somatic pain compared with controls. It also decreased TRPV1 current density and open probability and reduced the proportion of pancreatic sensory neurons that expressed TRPV1 as well as levels of TRPV1 in these neurons. CONCLUSIONS These findings emphasize a key role for NGF in pancreatic pain and highlight the role it plays in the modulation of TRPV1 expression and activity in CP.

A Rat Model of Chronic Gastric Sensorimotor Dysfunction Resulting From Transient Neonatal Gastric Irritation

BACKGROUND & AIMS Although several pathophysiologic abnormalities have been noted in functional dyspepsia (FD), their pathogenesis is poorly understood. We hypothesized that chronic gastric hypersensitivity and gastric motor dysfunction seen in FD patients can be modeled in rats by transient gastric irritation during the neonatal period, a time of known neuronal vulnerability to long-term plasticity. METHODS Ten-day-old male rats received 0.2 mL 0.1% iodoacetamide (IA) in 2% sucrose daily by oral gavages for 6 days; controls received 2% sucrose. Rats in both groups were then followed to adulthood (8-10 weeks) at which point behavioral, visceromotor, and great splanchnic nerve responses to graded gastric balloon distention (GD; 20-80 mm Hg) and gastric motor function were tested. RESULTS IA-treated rats exhibited hypersensitivity to GD in a dose-dependent manner, as compared with the control group. The threshold of afferent nerve activation was lower and nerve responses to GD were significantly increased in IA-treated rats. Although IA-treated rats ingested food at a lower rate, gastric emptying was not significantly different between IA and control groups. However, gastric accommodation was significantly reduced in the IA group. No significant gastric pathology was seen in hypersensitive adult rats compared with controls. CONCLUSIONS These studies demonstrate that gastric irritation in the neonatal period can result in chronic gastric hypersensitivity and gastric motor dysfunction in adults even in the absence of significant detectable gastric pathology. Our results offer insight into the pathogenesis of chronic functional dyspepsia and provide a potential model for further study to this important clinical problem.

Human Intestinal Epithelial Cells Express a Novel Receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcαR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcαR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or β-1,4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcαR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.

PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity

Background  Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease.

Substance P and Calcitonin Gene Related Peptide Mediate Pain in Chronic Pancreatitis and Their Expression is Driven by Nerve Growth Factor

CONTEXT Calcitonin gene-related peptide (CGRP), substance P and nerve growth factor play an important role in inflammatory pain in various somatic pain models but their role in chronic pancreatitis has not been well studied. OBJECTIVES The aim of this study was to investigate the effects of intrathecal administration of calcitonin gene-related peptide antagonist and substance P receptor antagonist on pain behavior in a rat model of chronic pancreatitis and to determine whether nerve growth factor drives the up-regulation of expression of these neuropeptides in sensory neurons. METHODS Pancreatitis was induced by retrograde infusion of trinitobenzene sulfonic acid into the pancreatic duct of adult rats. Three weeks post infusion continuous intrathecal infusion of the calcitonin gene-related peptide antagonist alpha CGRP8-37 or neurokinin-1 receptor antagonist CP-96345 or its inactive enantiomer CP-96344 was administered for seven days. The effects of treatment on pancreatic hyperalgesia were assessed by sensitivity of the abdominal wall to von Frey filament probing as well as by the nocifensive response to electrical stimulation of the pancreas. In a separate experiment chronic pancreatitis was induced and pancreas specific dorsal root ganglion neurons labeled with DiI were assessed for calcitonin gene-related peptide and substance P immunoreactivity. RESULTS Intrathecal infusion of calcitonin gene-related peptide and neurokinin-1 receptor antagonists significantly attenuated behavioral pain responses in rats with chronic pancreatitis. Further, treatment of chronic pancreatitis rats with nerve growth factor antibody significantly reduced pancreas specific neurons expressing calcitonin gene-related peptide and substance P in thoracic dorsal root ganglion. CONCLUSIONS Calcitonin gene-related peptide and substance P mediate pancreatic hyperalgesia in chronic pancreatitis and nerve growth factor in turn sustains the up-regulation of these neuropeptides in pancreatic sensory neurons.