High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

A dual role of lysophosphatidic acid type 2 receptor (LPAR2) in nonsteroidal anti-inflammatory drug-induced mouse enteropathy.

Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator that has been found to ameliorate nonsteroidal anti-inflammatory drug (NSAID)-induced gastric injury by acting on lysophosphatidic acid type 2 receptor (LPAR2). In this study, we investigated whether LPAR2 signaling was implicated in the development of NSAID-induced small intestinal injury (enteropathy), another major complication of NSAID use. Wild-type (WT) and Lpar2 deficient (Lpar2-/-) mice were treated with a single, large dose (20 or 30 mg/kg, i.g.) of indomethacin (IND). The mice were euthanized at 6 or 24 h after IND treatment. We showed that IND-induced mucosal enteropathy and neutrophil recruitment occurred much earlier (at 6 h after IND treatment) in Lpar2-/- mice compared to WT mice, but the tissue levels of inflammatory mediators (IL-1ß, TNF-α, inducible COX-2, CAMP) remained at much lower levels. Administration of a selective LPAR2 agonist DBIBB (1, 10 mg/kg, i.g., twice at 24 h and 30 min before IND treatment) dose-dependently reduced mucosal injury and neutrophil activation in enteropathy, but it also enhanced IND-induced elevation of several proinflammatory chemokines and cytokines. By assessing caspase-3 activation, we found significantly increased intestinal apoptosis in IND-treated Lpar2-/- mice, but it was attenuated after DBIBB administration, especially in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Finally, we showed that IND treatment reduced the plasma activity and expression of autotaxin (ATX), the main LPA-producing enzyme, and also reduced the intestinal expression of Lpar2 mRNA, which preceded the development of mucosal damage. We conclude that LPAR2 has a dual role in NSAID enteropathy, as it contributes to the maintenance of mucosal integrity after NSAID exposure, but also orchestrates the inflammatory responses associated with ulceration. Our study suggests that IND-induced inhibition of the ATX-LPAR2 axis is an early event in the pathogenesis of enteropathy.

Is an intraoperative liver function assessment possible? Application of the 13C-methacetin-breath-test during major liver resections - a pilot study.

BACKGROUND: As prevention of posthepatectomy-liver-failure is crucial, there is need of dynamic assessment of liver function, even intraoperatively. 13C-methacetin-breath-test estimates the organ's microsomal functional capacity. This is its first intraoperative evaluation in major liver surgery. METHODS: 30 patients planed for resection of ≥3 liver segments, between March-November 2019, were prospectively enrolled in this "single-center", pilot study. Using the 13C-methacetin-breath-test, liver function was assessed four times: preoperatively, intraoperatively before and after resection and postoperatively. The resulted maximum-liver-function-capacity (LiMAx)-values and delta-over-baseline (DOB)-curves were compared, further analyzed and correlated to respective liver volumes. RESULTS: The intraoperative LiMAx-values before resection were mostly lower than the preoperative ones (-11.3% ± 28%). The intraoperative measurements after resection resulted to mostly higher values than the postoperative ones (42.35% ± 46.19%). Pharmacokinetically, an interference between the two intraoperative tests was observed. There was no strong correlation between residual liver volume and function with a percentual residual-LiMAx mostly lower than the percentual residual volume (-17.7% ± 4.1%). CONCLUSIONS: Intraoperative application of the 13C-methacetin-breath-test during major liver resections seems to deliver lower values than the standard preoperative test. As multiple intraoperative tests interfere significantly to each other, a single intraoperative measurement is suggested. Multicentric standardized measurements could define the "normal" range for intraoperative measurements and control their predictive value.

The Acute Antiallodynic Effect of Tolperisone in Rat Neuropathic Pain and Evaluation of Its Mechanism of Action.

Current treatment approaches to manage neuropathic pain have a slow onset and their use is largely hampered by side-effects, thus there is a significant need for finding new medications. Tolperisone, a centrally acting muscle relaxant with a favorable side effect profile, has been reported to affect ion channels, which are targets for current first-line medications in neuropathic pain. Our aim was to explore its antinociceptive potency in rats developing neuropathic pain evoked by partial sciatic nerve ligation and the mechanisms involved. Acute oral tolperisone restores both the decreased paw pressure threshold and the elevated glutamate level in cerebrospinal fluid in neuropathic rats. These effects were comparable to those of pregabalin, a first-line medication in neuropathy. Tolperisone also inhibits release of glutamate from rat brain synaptosomes primarily by blockade of voltage-dependent sodium channels, although inhibition of calcium channels may also be involved at higher concentrations. However, pregabalin fails to affect glutamate release under our present conditions, indicating a different mechanism of action. These results lay the foundation of the avenue for repurposing tolperisone as an analgesic drug to relieve neuropathic pain.

Pharmacological Evidence on Augmented Antiallodynia Following Systemic Co-Treatment with GlyT-1 and GlyT-2 Inhibitors in Rat Neuropathic Pain Model.

The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30-60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP.

Shedding Light on the Pharmacological Interactions between µ-Opioid Analgesics and Angiotensin Receptor Modulators: A New Option for Treating Chronic Pain.

The current protocols for neuropathic pain management include µ-opioid receptor (MOR) analgesics alongside other drugs; however, there is debate on the effectiveness of opioids. Nevertheless, dose escalation is required to maintain their analgesia, which, in turn, contributes to a further increase in opioid side effects. Finding novel approaches to effectively control chronic pain, particularly neuropathic pain, is a great challenge clinically. Literature data related to pain transmission reveal that angiotensin and its receptors (the AT1R, AT2R, and MAS receptors) could affect the nociception both in the periphery and CNS. The MOR and angiotensin receptors or drugs interacting with these receptors have been independently investigated in relation to analgesia. However, the interaction between the MOR and angiotensin receptors has not been excessively studied in chronic pain, particularly neuropathy. This review aims to shed light on existing literature information in relation to the analgesic action of AT1R and AT2R or MASR ligands in neuropathic pain conditions. Finally, based on literature data, we can hypothesize that combining MOR agonists with AT1R or AT2R antagonists might improve analgesia.

On the Role of Peripheral Sensory and Gut Mu Opioid Receptors: Peripheral Analgesia and Tolerance.

There is growing evidence on the role of peripheral µ-opioid receptors (MORs) in analgesia and analgesic tolerance. Opioid analgesics are the mainstay in the management of moderate to severe pain, and their efficacy in the alleviation of pain is well recognized. Unfortunately, chronic treatment with opioid analgesics induces central analgesic tolerance, thus limiting their clinical usefulness. Numerous molecular mechanisms, including receptor desensitization, G-protein decoupling, ß-arrestin recruitment, and alterations in the expression of peripheral MORs and microbiota have been postulated to contribute to the development of opioid analgesic tolerance. However, these studies are largely focused on central opioid analgesia and tolerance. Accumulated literature supports that peripheral MORs mediate analgesia, but controversial results on the development of peripheral opioid receptors-mediated analgesic tolerance are reported. In this review, we offer evidence on the consequence of the activation of peripheral MORs in analgesia and analgesic tolerance, as well as approaches that enhance analgesic efficacy and decrease the development of tolerance to opioids at the peripheral sites. We have also addressed the advantages and drawbacks of the activation of peripheral MORs on the sensory neurons and gut (leading to dysbiosis) on the development of central and peripheral analgesic tolerance.

Comparisons of In Vivo and In Vitro Opioid Effects of Newly Synthesized 14-Methoxycodeine-6-O-sulfate and Codeine-6-O-sulfate.

The present work represents the in vitro (potency, affinity, efficacy) and in vivo (antinociception, constipation) opioid pharmacology of the novel compound 14-methoxycodeine-6-O-sulfate (14-OMeC6SU), compared to the reference compounds codeine-6-O-sulfate (C6SU), codeine and morphine. Based on in vitro tests (mouse and rat vas deferens, receptor binding and [35S]GTPγS activation assays), 14-OMeC6SU has µ-opioid receptor-mediated activity, displaying higher affinity, potency and efficacy than the parent compounds. In rats, 14-OMeC6SU showed stronger antinociceptive effect in the tail-flick assay than codeine and was equipotent to morphine, whereas C6SU was less efficacious after subcutaneous (s.c.) administration. Following intracerebroventricular injection, 14-OMeC6SU was more potent than morphine. In the Complete Freund's Adjuvant-induced inflammatory hyperalgesia, 14-OMeC6SU and C6SU in s.c. doses up to 6.1 and 13.2 µmol/kg, respectively, showed peripheral antihyperalgesic effect, because co-administered naloxone methiodide, a peripherally acting opioid receptor antagonist antagonized the measured antihyperalgesia. In addition, s.c. C6SU showed less pronounced inhibitory effect on the gastrointestinal transit than 14-OMeC6SU, codeine and morphine. This study provides first evidence that 14-OMeC6SU is more effective than codeine or C6SU in vitro and in vivo. Furthermore, despite C6SU peripheral antihyperalgesic effects with less gastrointestinal side effects the superiority of 14-OMeC6SU was obvious throughout the present study.

The Peripheral Versus Central Antinociception of a Novel Opioid Agonist: Acute Inflammatory Pain in Rats.

Opioid analgesics devoid of central side effects are unmet medical need in the treatment of acute pain (e.g. post-operative pain). Recently, we have reported on 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), a novel opioid agonist of high efficacy producing peripheral antinociception in subchronic inflammatory pain in certain doses. The present study focused on the antinociceptive effect of 14-O-MeM6SU compared to morphine in formalin test of an early/acute (Phase I) and late/tonic (Phase II) pain phases. Subcutaneous 14-O-MeM6SU (253-1012 nmol/kg) and morphine (3884-31075 nmol/kg) dose dependently reduced the pain behaviors of both phases. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid antagonist, abolished the antinociceptive effect of 506 nmol/kg 14-O-MeM6SU. On the other hand, the effects of 14-O-MeM6SU (1012 nmol/kg) and morphine (15538 nmol/kg) were only partially affected by NAL-M, indicating the contribution of CNS to antinociception. Locally injected test compounds into formalin treated paws caused antinociception in both phases. Locally effective doses of test compounds were also injected into contralateral paws. Morphine showed effects in both phases, 14-O-MeM6SU in certain doses failed to produce antinociception in either phase. A NAL-M reversible systemic dose of 14-O-MeM6SU and the lowest systemic effective dose of morphine were evaluated for their sedative effects following isoflurane-induced sleeping (righting reflex). In contrast to morphine, 14-O-MeM6SU in certain antinociceptive doses showed no impact on sleeping time. These data highlight that high efficacy opioids of limited CNS penetration in certain doses mitigate somatic and inflammatory pain by targeting MOR at the periphery.

Analysing the effect of I1 imidazoline receptor ligands on DSS-induced acute colitis in mice.

Imidazoline receptors (IRs) have been recognized as promising targets in the treatment of numerous diseases; and moxonidine and rilmenidine, agonists of I1-IRs, are widely used as antihypertensive agents. Some evidence suggests that IR ligands may induce anti-inflammatory effects acting on I1-IRs or other molecular targets, which could be beneficial in patients with inflammatory bowel disease (IBD). On the other hand, several IR ligands may stimulate also alpha2-adrenoceptors, which were earlier shown to inhibit, but in more recent studies to rather aggravate colitis. Hence, this study aimed to analyse for the first time the effect of various I1-IR ligands on intestinal inflammation. Colitis was induced in C57BL/6 mice by adding dextran sulphate sodium (DSS) to the drinking water for 7 days. Mice were treated daily with different IR ligands: moxonidine and rilmenidine (I1-IR agonists), AGN 192403 (highly selective I1-IR ligand, putative antagonist), efaroxan (I1-IR antagonist), as well as with the endogenous IR agonists agmatine and harmane. It was found that moxonidine and rilmenidine at clinically relevant doses, similarly to the other IR ligands, do not have a significant impact on the macroscopic and histological signs of DSS-evoked inflammation. Likewise, colonic myeloperoxidase and serum interleukin-6 levels remained unchanged in response to these agents. Thus, our study demonstrates that imidazoline ligands do not influence significantly the severity of DSS-colitis in mice and suggest that they probably neither affect the course of IBD in humans. However, the translational value of these findings needs to be verified with other experimental colitis models and human studies.

Inhibition of α2A-Adrenoceptors Ameliorates Dextran Sulfate Sodium-Induced Acute Intestinal Inflammation in Mice.

It has been hypothesized that α2-adrenoceptors (α2-ARs) may be involved in the pathomechanism of colitis; however, the results are conflicting because both aggravation and amelioration of colonic inflammation have been described in response to α2-AR agonists. Therefore, we aimed to analyze the role of α2-ARs in acute murine colitis. The experiments were carried out in wild-type, α2A-, α2B-, and α2C-AR knockout (KO) C57BL/6 mice. Colitis was induced by dextran sulfate sodium (DSS, 2%); alpha2-AR ligands were injected i.p. The severity of colitis was determined both macroscopically and histologically. Colonic myeloperoxidase (MPO) and cytokine levels were measured by enzyme-linked immunosorbent assay and proteome profiler array, respectively. The nonselective α2-AR agonist clonidine induced a modest aggravation of DSS-induced colitis. It accelerated the disease development and markedly enhanced the weight loss of animals, but did not influence the colon shortening, tissue MPO levels, or histologic score. Clonidine induced similar changes in α2B- and α2C-AR KO mice, whereas it failed to affect the disease activity index scores and caused only minor weight loss in α2A-AR KO animals. In contrast, selective inhibition of α2A-ARs by BRL 44408 significantly delayed the development of colitis; reduced the colonic levels of MPO and chemokine (C-C motif) ligand 3, chemokine (C-X-C motif) ligand 2 (CXCL2), CXCL13, and granulocyte-colony stimulating factor; and elevated that of tissue inhibitor of metalloproteinases-1. In this work, we report that activation of α2-ARs aggravates murine colitis, an effect mediated by the α2A-AR subtype, and selective inhibition of these receptors reduces the severity of gut inflammation.

Modulation of excitatory neurotransmission by neuronal/glial signalling molecules: interplay between purinergic and glutamatergic systems.

Glutamate is the main excitatory neurotransmitter of the central nervous system (CNS), released both from neurons and glial cells. Acting via ionotropic (NMDA, AMPA, kainate) and metabotropic glutamate receptors, it is critically involved in essential regulatory functions. Disturbances of glutamatergic neurotransmission can be detected in cognitive and neurodegenerative disorders. This paper summarizes the present knowledge on the modulation of glutamate-mediated responses in the CNS. Emphasis will be put on NMDA receptor channels, which are essential executive and integrative elements of the glutamatergic system. This receptor is crucial for proper functioning of neuronal circuits; its hypofunction or overactivation can result in neuronal disturbances and neurotoxicity. Somewhat surprisingly, NMDA receptors are not widely targeted by pharmacotherapy in clinics; their robust activation or inhibition seems to be desirable only in exceptional cases. However, their fine-tuning might provide a promising manipulation to optimize the activity of the glutamatergic system and to restore proper CNS function. This orchestration utilizes several neuromodulators. Besides the classical ones such as dopamine, novel candidates emerged in the last two decades. The purinergic system is a promising possibility to optimize the activity of the glutamatergic system. It exerts not only direct and indirect influences on NMDA receptors but, by modulating glutamatergic transmission, also plays an important role in glia-neuron communication. These purinergic functions will be illustrated mostly by depicting the modulatory role of the purinergic system on glutamatergic transmission in the prefrontal cortex, a CNS area important for attention, memory and learning.

Dual Alpha2C/5HT1A Receptor Agonist Allyphenyline Induces Gastroprotection and Inhibits Fundic and Colonic Contractility.

BACKGROUND: Allyphenyline, a novel α2-adrenoceptor (AR) ligand, has been shown to selectively activate α2C-adrenoceptors (AR) and 5HT1A receptors, but also to behave as a neutral antagonist of α2A-ARs. We exploited this unique pharmacological profile to analyze the role of α2C-ARs and 5HT1A receptors in the regulation of gastric mucosal integrity and gastrointestinal motility. METHODS: Gastric injury was induced by acidified ethanol in Wistar rats. Mucosal catalase and superoxide dismutase levels were measured by assay kits. The effect of allyphenyline on electrical field stimulation (EFS)-induced fundic and colonic contractions was determined in C57BL/6 mice. RESULTS: Intracerebroventricularly injected allyphenyline (3 and 15 nmol/rat) dose dependently inhibited the development of mucosal damage, which was antagonized by ARC 239 (α2B/C-AR and 5HT1A receptor antagonist), (S)-WAY 100135 (selective 5HT1A receptor antagonist), and JP-1302 (selective α2C-AR antagonist). This protection was accompanied by significant elevation of mucosal catalase and superoxide dismutase levels. Allyphenyline (10(-9)-10(-5) M) also inhibited EFS-induced fundic contractions, which was antagonized by ARC 239 and (S)-WAY 100135, but not by JP-1302. Similar inhibition was observed in the colon; however, in this case only ARC 239 reduced this effect, while neither selective inhibition of α2C-ARs and 5HT1A receptors nor genetic deletion of α2A- and α2B-ARs influenced it. CONCLUSIONS: Activation of both central α2C-ARs and 5HT1A receptors contributes to the gastroprotective action of allyphenyline in rats. Its inhibitory effect on fundic contractions is mediated by 5HT1A receptors, but neither α2-ARs nor 5HT1A receptors take part in its inhibitory effect on colonic contractility in mice.

Interactions between NSAIDs, opioids and the gut microbiota - Future perspectives in the management of inflammation and pain.

The composition of intestinal microbiota is influenced by a number of factors, including medications, which may have a substantial impact on host physiology. Nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics are among those widely used medications that have been shown to alter microbiota composition in both animals and humans. Although much effort has been devoted to identify microbiota signatures associated with these medications, much less is known about the underlying mechanisms. Mucosal inflammation, changes in intestinal motility, luminal pH and bile acid metabolism, or direct drug-induced inhibitory effect on bacterial growth are all potential contributors to NSAID- and opioid-induced dysbiosis, however, only a few studies have addressed directly these issues. In addition, there is a notable overlap between the microbiota signatures of these drugs and certain diseases in which they are used, such as spondyloarthritis (SpA), rheumatoid arthritis (RA) and neuropathic pain associated with type 2 diabetes (T2D). The aims of the present review are threefold. First, we aim to provide a comprehensive up-to-date summary on the bacterial alterations caused by NSAIDs and opioids. Second, we critically review the available data on the possible underlying mechanisms of dysbiosis. Third, we review the current knowledge on gut dysbiosis associated with SpA, RA and neuropathic pain in T2D, and highlight the similarities between them and those caused by NSAIDs and opioids. We posit that drug-induced dysbiosis may contribute to the persistence of these diseases, and may potentially limit the therapeutic effect of these medications by long-term use. In this context, we will review the available literature data on the effect of probiotic supplementation and fecal microbiota transplantation on the therapeutic efficacy of NSAIDs and opioids in these diseases.

Pregabalin-Tolperisone Combination to Treat Neuropathic Pain: Improved Analgesia and Reduced Side Effects in Rats.

The current treatment of neuropathic pain (NP) is unsatisfactory; therefore, effective novel agents or combination-based analgesic therapies are needed. Herein, oral tolperisone, pregabalin, and duloxetine were tested for their antinociceptive effect against rat partial sciatic nerve ligation (pSNL)-induced tactile allodynia described by a decrease in the paw withdrawal threshold (PWT) measured by a dynamic plantar aesthesiometer. On day 7 after the operation, PWTs were assessed at 60, 120, and 180 min post-treatment. Chronic treatment was continued for 2 weeks, and again, PWTs were measured on day 14 and 21. None of the test compounds produced an acute antiallodynic effect. In contrast, after chronic treatment, tolperisone and pregabalin alleviated allodynia. In other experiments, on day 14, the acute antiallodynic effect of the tolperisone/pregabalin or duloxetine combination was measured. As a novel finding, a single dose of the tolperisone/pregabalin combination could remarkably alleviate allodynia acutely. It also restored the neuropathy-induced elevated CSF glutamate content. Furthermore, the combination is devoid of adverse effects related to motor and gastrointestinal transit functions. Tolperisone and pregabalin target voltage-gated sodium and calcium channels, respectively. The dual blockade effect of the combination might explain its advantageous acute analgesic effect in the present work.

The Nonsteroidal Anti-Inflammatory Drug Ketorolac Alters the Small Intestinal Microbiota and Bile Acids Without Inducing Intestinal Damage or Delaying Peristalsis in the Rat.

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) induce significant damage to the small intestine, which is accompanied by changes in intestinal bacteria (dysbiosis) and bile acids. However, it is still a question of debate whether besides mucosal inflammation also other factors, such as direct antibacterial effects or delayed peristalsis, contribute to NSAID-induced dysbiosis. Here we aimed to assess whether ketorolac, an NSAID lacking direct effects on gut bacteria, has any significant impact on intestinal microbiota and bile acids in the absence of mucosal inflammation. We also addressed the possibility that ketorolac-induced bacterial and bile acid alterations are due to a delay in gastrointestinal (GI) transit. Methods: Vehicle or ketorolac (1, 3 and 10 mg/kg) were given to rats by oral gavage once daily for four weeks, and the severity of mucosal inflammation was evaluated macroscopically, histologically, and by measuring the levels of inflammatory proteins and claudin-1 in the distal jejunal tissue. The luminal amount of bile acids was measured by liquid chromatography-tandem mass spectrometry, whereas the composition of microbiota by sequencing of bacterial 16S rRNA. GI transit was assessed by the charcoal meal method. Results: Ketorolac up to 3 mg/kg did not cause any signs of mucosal damage to the small intestine. However, 3 mg/kg of ketorolac induced dysbiosis, which was characterized by a loss of families belonging to Firmicutes (Paenibacillaceae, Clostridiales Family XIII, Christensenellaceae) and bloom of Enterobacteriaceae. Ketorolac also changed the composition of small intestinal bile by decreasing the concentration of conjugated bile acids and by increasing the amount of hyodeoxycholic acid (HDCA). The level of conjugated bile acids correlated negatively with the abundance of Erysipelotrichaceae, Ruminococcaceae, Clostridiaceae 1, Muribaculaceae, Bacteroidaceae, Burkholderiaceae and Bifidobacteriaceae. Ketorolac, under the present experimental conditions, did not change the GI transit. Conclusion: This is the first demonstration that low-dose ketorolac disturbed the delicate balance between small intestinal bacteria and bile acids, despite having no significant effect on intestinal mucosal integrity and peristalsis. Other, yet unidentified, factors may contribute to ketorolac-induced dysbiosis and bile dysmetabolism.

A comprehensive time course and correlation analysis of indomethacin-induced inflammation, bile acid alterations and dysbiosis in the rat small intestine.

It has been proposed that changes in microbiota due to nonsteroidal anti-inflammatory drugs (NSAIDs) alter the composition of bile, and elevation of hydrophobic secondary bile acids contributes to small intestinal damage. However, little is known about the effect of NSAIDs on small intestinal bile acids, and whether bile alterations correlate with mucosal injury and dysbiosis. Here we determined the ileal bile acid metabolome and microbiota 24, 48 and 72 h after indomethacin treatment, and their correlation with each other and with tissue damage in rats. In parallel with the development of inflammation, indomethacin increased the ileal proportion of glycine and taurine conjugated bile acids, but not bile hydrophobicity. Firmicutes decreased with time, whereas Gammaproteobacteria increased first, but declined later and were partially replaced by Bilophila, Bacteroides and Fusobacterium. Mucosal injury correlated negatively with unconjugated bile acids and Gram-positive bacteria, and positively with taurine conjugates and some Gram-negative taxa. Strong positive correlation was found between Lactobacillaceae, Ruminococcaceae, Clostridiaceae and unconjugated bile acids. Indomethacin-induced dysbiosis was not likely due to direct antibacterial effects or alterations in luminal pH. Here we provide the first detailed characterization of indomethacin-induced time-dependent alterations in small intestinal bile acid composition, and their associations with mucosal injury and dysbiosis. Our results suggest that increased bile hydrophobicity is not likely to contribute to indomethacin-induced small intestinal damage.

Hidden Cardiotoxicity of Rofecoxib Can be Revealed in Experimental Models of Ischemia/Reperfusion.

Cardiac adverse effects are among the leading causes of the discontinuation of clinical trials and the withdrawal of drugs from the market. The novel concept of 'hidden cardiotoxicity' is defined as cardiotoxicity of a drug that manifests in the diseased (e.g. ischemic/reperfused), but not in the healthy heart or as a drug-induced deterioration of cardiac stress adaptation (e.g. ischemic conditioning). Here, we aimed to test if the cardiotoxicity of a selective COX-2 inhibitor rofecoxib that was revealed during its clinical use, i.e., increased occurrence of proarrhythmic and thrombotic events, could have been revealed in early phases of drug development by using preclinical models of ischemia/reperfusion (I/R) injury. Rats that were treated with rofecoxib or vehicle for four weeks were subjected to 30 min. coronary artery occlusion and 120 min. reperfusion with or without cardioprotection that is induced by ischemic preconditioning (IPC). Rofecoxib increased overall the arrhythmias including ventricular fibrillation (VF) during I/R. The proarrhythmic effect of rofecoxib during I/R was not observed in the IPC group. Rofecoxib prolonged the action potential duration (APD) in isolated papillary muscles, which was not seen in the simulated IPC group. Interestingly, while showing hidden cardiotoxicity manifested as a proarrhythmic effect during I/R, rofecoxib decreased the infarct size and increased the survival of adult rat cardiac myocytes that were subjected to simulated I/R injury. This is the first demonstration that rofecoxib increased acute mortality due to its proarrhythmic effect via increased APD during I/R. Rofecoxib did not interfere with the cardiprotective effect of IPC; moreover, IPC was able to protect against rofecoxib-induced hidden cardiotoxicity. These results show that cardiac safety testing with simple preclinical models of I/R injury uncovers hidden cardiotoxicity of rofecoxib and might reveal the hidden cardiotoxicity of other drugs.

Chronic treatment with rofecoxib but not ischemic preconditioning of the myocardium ameliorates early intestinal damage following cardiac ischemia/reperfusion injury in rats.

There is some recent evidence that cardiac ischemia/reperfusion (I/R) injury induces intestinal damage within days, which contributes to adverse cardiovascular outcomes after myocardial infarction. However, it is not clear whether remote gut injury has any detectable early signs, and whether different interventions aiming to reduce cardiac damage are also effective at protecting the intestine. Previously, we found that chronic treatment with rofecoxib, a selective inhibitor of cyclooxygenase-2 (COX-2), limited myocardial infarct size to a comparable extent as cardiac ischemic preconditioning (IPC) in rats subjected to 30-min coronary artery occlusion and 120-min reperfusion. In the present study, we aimed to analyse the early intestinal alterations caused by cardiac I/R injury, with or without the above-mentioned infart size-limiting interventions. We found that cardiac I/R injury induced histological changes in the small intestine within 2 h, which were accompanied by elevated tissue level of COX-2 and showed positive correlation with the activity of matrix metalloproteinase-2 (MMP-2), but not of MMP-9 in the plasma. All these changes were prevented by rofecoxib treatment. By contrast, cardiac IPC failed to reduce intestinal injury and plasma MMP-2 activity, although it prevented the transient reduction in jejunal blood flow in response to cardiac I/R. Our results demonstrate for the first time that rapid development of intestinal damage follows cardiac I/R, and that two similarly effective infarct size-limiting interventions, rofecoxib treatment and cardiac IPC, have different impacts on cardiac I/R-induced gut injury. Furthermore, intestinal damage correlates with plasma MMP-2 activity, which may be a biomarker for its early diagnosis.

Glycine transporter inhibitors: A new avenue for managing neuropathic pain.

Interneurons operating with glycine neurotransmitter are involved in the regulation of pain transmission in the dorsal horn of the spinal cord. In addition to interneurons, glycine release also occurs from glial cells neighboring glutamatergic synapses in the spinal cord. Neuronal and glial release of glycine is controlled by glycine transporters (GlyTs). Inhibitors of the two isoforms of GlyTs, the astrocytic type-1 (GlyT-1) and the neuronal type-2 (GlyT-2), decrease pain sensation evoked by injuries of peripheral sensory neurons or inflammation. The function of dorsal horn glycinergic interneurons has been suggested to be reduced in neuropathic pain, which can be reversed by GlyT-2 inhibitors (Org-25543, ALX1393). Several lines of evidence also support that peripheral nerve damage or inflammation may shift glutamatergic neurochemical transmission from N-methyl-D aspartate (NMDA) NR1/NR2A receptor- to NR1/NR2B receptor-mediated events (subunit switch). This pathological overactivation of NR1/NR2B receptors can be reduced by GlyT-1 inhibitors (NFPS, Org-25935), which decrease excessive glycine release from astroglial cells or by selective antagonists of NR2B subunits (ifenprodil, Ro 25-6981). Although several experiments suggest that GlyT inhibitors may represent a novel strategy in the control of neuropathic pain, proving this concept in human beings is hampered by lack of clinically applicable GlyT inhibitors. We also suggest that drugs inhibiting both GlyT-1 and GlyT-2 non-selectively and reversibly, may favorably target neuropathic pain. In this paper we overview inhibitors of the two isoforms of GlyTs as well as the effects of these drugs in experimental models of neuropathic pain. In addition, the possible mechanisms of action of the GlyT inhibitors, i.e. how they affect the neurochemical and pain transmission in the spinal cord, are also discussed. The growing evidence for the possible therapeutic intervention of neuropathic pain by GlyT inhibitors further urges development of drugable compounds, which may beneficially restore impaired pain transmission in various neuropathic conditions.