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.

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.

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.

Efficacy-Based Perspective to Overcome Reduced Opioid Analgesia of Advanced Painful Diabetic Neuropathy in Rats.

Reduction of the opioid analgesia in diabetic neuropathic pain (DNP) results from µ-opioid receptor (MOR) reserve reduction. Herein, we examined the antinociceptive and antiallodynic actions of a novel opioid agonist 14-O-methymorphine-6-O-sulfate (14-O-MeM6SU), fentanyl and morphine in rats with streptozocin-evoked DNP of 9-12 weeks following their systemic administration. The antinociceptive dose-response curve of morphine but not of 14-O-MeM6SU or fentanyl showed a significant right-shift in diabetic compared to non-diabetic rats. Only 14-O-MeM6SU produced antiallodynic effects in doses matching antinociceptive doses obtained in non-diabetic rats. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid receptor antagonist failed to alter the antiallodynic effect of test compounds, indicating the contribution of central opioid receptors. Reduction in spinal MOR binding sites and loss in MOR immunoreactivity of nerve terminals in the spinal cord and dorsal root ganglia in diabetic rats were observed. G-protein coupling assay revealed low efficacy character for morphine and high efficacy character for 14-O-MeM6SU or fentanyl at spinal or supraspinal levels (E max values). Furthermore, at the spinal level only 14-O-MeM6SU showed equal efficacy in G-protein activation in tissues of diabetic- and non-diabetic animals. Altogether, the reduction of spinal opioid receptors concomitant with reduced analgesic effect of morphine may be circumvented by using high efficacy opioids, which provide superior analgesia over morphine. In conclusion, the reduction in the analgesic action of opioids in DNP might be a consequence of MOR reduction, particularly in the spinal cord. Therefore, developing opioids of high efficacy might provide analgesia exceeding that of currently available opioids.

Lack of Small Intestinal Dysbiosis Following Long-Term Selective Inhibition of Cyclooxygenase-2 by Rofecoxib in the Rat.

Intestinal dysbiosis is linked to numerous gastrointestinal disorders, including inflammatory bowel diseases. It is a question of debate if coxibs, selective inhibitors of cyclooxygenase (COX)-2, cause dysbiosis. Therefore, in the present study, we aimed to determine the effect of long-term (four weeks) selective inhibition of COX-2 on the small intestinal microbiota in the rat. In order to avoid mucosal damage due to topical effects and inflammation-driven microbial alterations, rofecoxib, a nonacidic compound, was used. The direct inhibitory effect of rofecoxib on the growth of bacteria was ruled out in vitro. The mucosa-sparing effect of rofecoxib was confirmed by macroscopic and histological analysis, as well as by measuring the intestinal levels of cytokines and tight junction proteins. Deep sequencing of bacterial 16S rRNA revealed that chronic rofecoxib treatment had no significant influence on the composition and diversity of jejunal microbiota. In conclusion, this is the first demonstration that long-term selective inhibition of COX-2 by rofecoxib does not cause small intestinal dysbiosis in rats. Moreover, inhibition of COX-2 activity is not likely to be responsible per se for microbial alterations caused by some coxibs, but other drug-specific properties may contribute to it.

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.

14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [35S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (Emax) and potency (EC50) than morphine in MVD, RVD or [35S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for µ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value.

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.