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The Nonsteroidal Anti-inflammatory Drug Diclofenac Reduces Acid-Induced Heartburn Symptoms in Healthy Volunteers
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The Nonsteroidal Anti-inflammatory Drug Diclofenac Reduces Acid-Induced Heartburn Symptoms in Healthy Volunteers Q28
Takashi Kondo,* Tadayuki Oshima,* Toshihiko Tomita,* Hirokazu Fukui,* Hiroki Okada,‡ Jiro Watari,* and Hiroto Miwa* *Division of Gastroenterology, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan; ‡Minase Research Institute, Pharmacological Research Laboratories, Ono Pharmaceutical Co, Ltd, Osaka, Japan
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BACKGROUND & AIMS:
We investigated the effects of diclofenac, a nonsteroidal anti-inflammatory drug that inhibits prostaglandin production, on induction of esophageal sensation by acid perfusion in healthy men.
METHODS:
We performed a prospective, double-blind, placebo-controlled, 2-period, cross-over study over 3 visits in 12 healthy men. Diclofenac was given 6 hours and 2 hours before an acid perfusion test. During the test, hydrochloric acid (0.15 mol/L) was perfused into the lower esophagus for 30 minutes; we evaluated upper gastrointestinal symptoms using a validated categoric rating scale. Then, we calculated and assessed the acid perfusion sensitivity score (APSS). Biopsy specimens were collected by endoscopy of the distal esophagus before and after acid perfusion; levels of prostaglandin E2 (PGE2) (pg/mg) were measured in the samples using an enzymelinked immunosorbent assay.
RESULTS:
Compared with placebo, diclofenac significantly reduced the APSS for heartburn (82.2 – 12.2 for placebo and 47.5 – 8.9 for diclofenac; P < .01). Of the upper gastrointestinal symptoms, only the APSS for heartburn was reduced significantly by diclofenac. Compared with placebo, diclofenac reduced the overproduction of PGE2 by esophageal tissues after acid perfusion (23.3 – 5.2 for placebo and 11.4 – 3.5 for diclofenac; P < .05). APSS correlated with the development of heartburn and esophageal levels of PGE2 (r [ 0.53; P < .05 for diclofenac vs placebo).
CONCLUSIONS:
Diclofenac attenuated acid-induced heartburn by inhibiting PGE2 overproduction in the esophagus. Esophageal PGE2 might be involved in producing heartburn symptoms. Clinical Trials Registry no: UMIN000014595.
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Keywords: NSAID; Reflux; Treatment; GERD; NERD. Q7 Q8 Q9
he prevalence of gastroesophageal reflux disease (GERD) continues to increase in Western and Japanese populations1–3 with heartburn likely to remain the most common symptom. Although proton pump inhibitors (PPIs) have improved outcomes significantly, a meta-analysis showed that up to 40% of patients with GERD are dissatisfied with standard PPI therapy because their symptoms are not fully resolved.4–7 Furthermore, it is widely recognized that the persistent GERD symptoms resulting from PPI resistance worsen quality of life in patients with nonerosive reflux disease (NERD). Therefore, appropriate management of GERD symptom is very important. One reason why these unmet clinical needs persist is that the pathogenesis of the core symptom, heartburn, is not completely understood.8,9 Therefore, it is important to investigate novel mechanisms of symptom generation for heartburn to provide additional therapeutic options for the treatment and control of heartburn in patients with GERD and NERD.
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Prostanoids are produced during arachidonic acid metabolism in the cyclooxygenase (COX) pathway, and they are established as inflammatory mediators that contribute to inflammation and nociception.10,11 Among the prostanoids, prostaglandin E2 (PGE2) is considered the principal proinflammatory and pro-nociceptive prostanoid,12 and is known to induce sensitization and pain hypersensitivity through activation of prostanoid receptors on peripheral nerve terminals.13–15 Recently,
Abbreviations used in this paper: APSS, acid perfusion sensitivity score; COX, cyclooxygenase; ED, elemental diet; ER, ___________ _________________; GERD, gastroesophageal reflux disease; GI, gastrointestinal; NERD, nonerosive reflux disease; NSAID, nonsteroidal antiinflammatory drug; PGE2, prostaglandin E2; PPI, proton pump inhibitor; RM-ANOVA, _______________________ analysis of variance. Q4 © 2015 by the AGA Institute 1542-3565/$36.00 http://dx.doi.org/10.1016/j.cgh.2015.01.014
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we reported that esophageal PGE2 may be involved in generating heartburn in human beings. Specifically, we showed that acid perfusion in the esophagus induced PGE2 generation in the esophageal mucosa and that the increased PGE2 level was correlated significantly with changes in the esophageal symptom score for heartburn in healthy participants.16 On the basis of this theoretical background, we assumed that inhibiting PGE2 production in the esophageal mucosa might prevent upper gastrointestinal (GI) symptoms, especially heartburn. To confirm this hypothesis, we used the nonsteroidal anti-inflammatory drug (NSAID) diclofenac as an inhibitor of prostaglandin production to premedicate healthy participants before an acid perfusion test, and we then investigated its effect on heartburn symptoms. Therefore, we aimed to investigate whether diclofenac suppressed acid-induced heartburn in healthy participants by inhibiting PGE2 overproduction in the esophagus, and whether esophageal PGE2 levels correlated with the heartburn symptoms. In addition, we explored the effect of diclofenac on several upper GI symptoms.
Methods Study Design
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A prospective, double-blinded, placebo-controlled, 2period, cross-over study was conducted over 3 visits. Eligibility for the study was determined at visit 1, and volunteers were randomized according to a cross-over sequence with diclofenac or placebo. A person who was not involved in the study generated the 2 comparison groups using simple randomization, with an equal allocation ratio, by referring to a table of random numbers. The randomization code was concealed until the end of the trial. At visits 2 and 3, we assessed upper GI symptoms during acid perfusion tests and obtained esophageal biopsy samples. To allow adequate time for complete washout of diclofenac, the interval between visits 2 and 3 was at least 2 weeks. We performed all experiments in accordance with human ethics regulations (Hyogo College of Medicine: no 1162) and obtained written informed consent. This trial is registered with the University Hospital Medical Information Network Clinical Trials Registry (Number UMIN000014595). The trial was conducted according to the principles governing human research in the Declaration of Helsinki. All authors had access to the study data and reviewed and approved the final manuscript.
Participants We enrolled 12 healthy men. The status of daily upper abdominal symptoms was checked with a previously validated questionnaire, the frequency scale for
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the symptoms of GERD.17 At visit 1, we excluded participants based on the following criteria: a frequency scale for the symptoms of GERD total score of greater than 8; any medical history of asthma or allergy to NSAIDs; any history of peptic ulcer or GERD or of upper GI tract surgery; any significant cardiovascular, kidney, liver, neurotic, or psychological disorders; and any subjects taking medications that may affect prostaglandin generation, including NSAIDs, COX-2 inhibitors, or prostaglandin-containing medications. At visit 2, we excluded significant upper GI pathology using endoscopy (eg, esophagitis, Barrett’s esophagus, hiatus hernia, active peptic ulcer disease, and esophageal/gastric cancer).
Trial Protocol We used sustained-release diclofenac sodium (37.5-mg Voltaren sustained release [SR] capsules; Novartis Pharma KK, Tokyo, Japan) in this trial. After a minimum 6-hour fast, Voltaren SR capsule or placebo was given at 6 hours and 2 hours before the acid perfusion test. For the test, participants were placed in the left lateral decubitus position and we performed a transnasal endoscopic examination (GIF-XP260N; Olympus Optical Company, Tokyo, Japan). At this point, we obtained baseline esophageal endoscopic biopsy samples from 3 cm above the esophagogastric junction using biopsy forceps (Radial Jaw 3; Boston Scientific Corporation, MA). After endoscopy, a 5F elemental diet (ED) tube (Nippon Sherwood Medical Industries Ltd, Tokyo, Japan) was inserted via the nasal passage with the distal tip placed 10 cm above the esophagogastric junction. Acid perfusions then were performed, followed by immediate removal of the ED tube and repeated endoscopic biopsies. The endoscopic esophageal mucosa samples were frozen immediately in a 1.5-mL sampling tube with liquid nitrogen and stored in a freezer at 80 C until used to measure PGE2 levels.
Interventions Acid perfusion test. The acid perfusion test was performed by modification of a previously reported method18,19 with participants in an upright sitting position. After confirmation that no participant had heartburn during the initial saline perfusion (8 mL/min for 2 min), hydrochloric acid (0.15 mol/L) was infused for 30 minutes into the lower esophagus via the ED tube. Previous studies have indicated that this acid concentration induces esophageal hypersensitivity in the majority of healthy participants20 and causes hyperalgesia in response to an electrical stimulus in human models.21,22 Infusions were delivered at 8 mL/min for 30 minutes with an automatic infusion pump. Assessment of reflux and upper gastrointestinal symptom severity. During acid perfusion, participants were
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instructed to report when heartburn first was perceived (allowing calculation of the time to first sensation and the duration of heartburn symptom perception). Thereafter, the intensity of several upper GI symptoms was recorded during acid perfusion (for 30 min) at regular 5minute intervals. The upper GI symptoms of interest were heartburn, sour or bitter taste, pricking pain in the stomach, dull pain in the stomach, heavy feeling in the stomach, tight feeling in the stomach, nausea, and fullness. We used a previously validated categoric rating scale,18,23,24 which ranged from 1 (no sensation) to 13 (extremely intense), to assess the intensity of upper GI symptoms associated with acid perfusion. Then, we used the acid perfusion sensitivity score (APSS) to analyze upper GI symptom severity according to the method of Fass et al.19,25 The APSS was calculated as follows: using the duration of the upper GI symptom perception (T) expressed in seconds (s) and the upper GI symptom intensity rating at the end of the acid perfusion (I), APSS ¼ I T / 100 (cm s/100). The symptom scores were recorded by the subjects without prompting for responses. Measurement of prostaglandin E2 levels. After measuring the weight of the lower esophageal mucosal tissue, homogenization (300 Hz/s for 3 min) was conducted with a mixer mill (MM300; Qiagen, Hiden, Germany). The protein level in the supernatant was measured with the BCA protein assay kit (Thermo Fisher Scientific, Inc, MA). The PGE2 level was measured by an enzyme-linked immunosorbent assay using the Prostaglandin E2 EIA Kit-Monoclonal (Cayman Chemical Company, MI) according to the manufacturer’s instructions. The level was determined after absorbance measurement with a microplate reader (Spectra Max 250; Molecular Devices Japan Co, Tokyo, Japan).
Outcomes: Study End Points The primary efficacy outcome for the study was the change in APSS for heartburn, and statistical significance was assessed by the mean difference in the change of scores between the diclofenac and placebo groups. To support the primary outcome, we also assessed the following secondary outcomes: (1) changes in other upper GI symptoms; (2) change in esophageal PGE2 levels; and (3) correlation between esophageal PGE2 levels and APSS.
Sample Size The primary end point of this study was a change in the APSS between diclofenac and placebo. Prior data16 from 14 subjects confirmed that healthy subjects had an average change in APSS of 194 and a SD of 106 between acid and saline infusion. On the basis of these results, we assumed that each expected difference in APSS and SD
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Table 1. Demographic Data of the Study Subjects Placebo/diclofenac Diclofenac/placebo P (N ¼ 6) value (N ¼ 6) Age, ya Sex, male/ female Weight, kga Height, cma BMI, kg/m2a FSSGa
31.2 3.6 6/0 67.8 171.3 23.0 3.2
6.1 4.3 1.8 1.9
35.8 3.7 6/0
.06b —
.85b .45b .46b .76b
71.2 169.0 24.8 3.5
13.8 6.0 3.9 2.6
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BMI, body mass index; FSSG, frequency scale for symptoms of GERD. a Means SD. b Mann–Whitney U test.
was 97 and 53, respectively. The number of subjects with this assumption was 10 in each arm (a ¼ 0.05, 1 b ¼ 0.9). Therefore, a sample size of 12 subjects in this study adequately allowed a power of 90% to detect a difference at a significance level of 0.05 (2-tailed).
Statistical Analysis We compared the time to first sensation and all APSSs for the upper GI symptoms between the diclofenac and placebo groups with the nonparametric Wilcoxon matched-pairs signed-rank test. PGE2 levels before and after the perfusion test were compared between the diclofenac and placebo groups by 2-way RM-analysis of variance (ANOVA), followed by an individual post hoc comparison with the Bonferroni multiple comparison test. Esophageal PGE2 levels after acid perfusion were compared with APSS by the Pearson correlation coefficient. Data are presented as mean standard error. Analyses were performed using SAS 9.2 (TS2M3; SAS Institute Japan Ltd, Tokyo, Japan) and its cooperative system EXSUS version 7.7.1 (CAC Corporation). A P value less than .05 indicated statistical significance.
Results Participants and Recruitment Twelve healthy men originally were enrolled in this study, of whom all candidates were eligible for inclusion. The demographic data of the study subjects are presented in Table 1. One participant was unable to tolerate prolonged endoscopy and was excluded. Therefore, 11 participants completed the study (Supplementary Figure 1).
Effect of Diclofenac on Other Upper Gastrointestinal Symptoms During the acid perfusion test, we also explored several other upper GI symptoms. Of the upper GI symptoms, diclofenac only significantly suppressed APSS
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Figure 1. The effect of diclofenac on upper gastrointestinal symptoms. Compared with placebo, diclofenac significantly suppressed APSS of heartburn during acid perfusion (*P < .05). Compared with placebo, the other upper gastrointestinal symptoms were not affected significantly by diclofenac.
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of heartburn (82.2 12.2 and 47.5 8.9, respectively; P < .01), and had no significant effects on the other upper GI symptoms (Figure 1).
Effect of Diclofenac on Prostaglandin E2 Generation in the Esophageal Mucosa PGE2 levels in the esophageal biopsy samples were significantly higher after acid perfusion (23.3 5.2 pg/mg protein) than before perfusion (7.7 1.2 pg/mg protein) (P < .01, 2-way RM-ANOVA) in the placebo group, which is consistent with our previous report.16 Diclofenac significantly inhibited PGE2 overproduction induced by acid perfusion in the human esophagus (11.4 3.5 pg/mg protein) compared with placebo (23.3 5.2 pg/mg protein) (P < .05, 2-way RM-ANOVA) (Figure 2A). Furthermore, there was a clear correlation between increased esophageal PGE2 levels and APSS of heartburn (r ¼ 0.53, P < .05, Pearson correlation) (Figure 2B).
Drug Administration and Adverse Events All participants were completely symptom free and did not have any adverse events.
Discussion Heartburn is a typical symptom of GERD. Several mechanisms are postulated to explain the pathogenesis of heartburn, including peripheral production of inflammatory mediators, dilated intercellular spaces,26,27 failure of the barrier function within the esophageal mucosa causing increased mucosal permeability,28 and altered nociceptive processing closely associated with visceral
Figure 2. The effect of diclofenac on PGE2 generation in the esophageal mucosa and the correlation between APSS and PGE2 level. (A) In the placebo group, PGE2 levels in the esophageal biopsy samples were significantly higher after acid perfusion than before perfusion (**P < .01). In the diclofenac group, PGE2 levels significantly decreased after acid perfusion compared with the placebo group (*P < .05), and did not change from before acid perfusion (not significant). Data are presented as mean standard error. (B) A positive correlation was found between APSS of heartburn and increased esophageal PGE2 levels (r ¼ 0.53, P < .05).
hypersensitivity.8,29,30 However, many of the mechanisms of heartburn generation remain unknown, which results in persistent unmet clinical need. In this study, we show that premedication of healthy volunteers with the NSAID diclofenac reduced the intensity of heartburn symptoms induced by acid perfusion (Figure 1). Next, we asked the subjects about the presence of several upper GI symptoms during the acid perfusion test because patients with GERD complain of atypical GERD symptoms, such as an epigastric burning
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sensation, epigastralgia, epigastric discomfort, and abdominal fullness.31 We also investigated the effect of diclofenac on these symptoms. Consequently, we found that suppressing PGE2 in the esophagus using diclofenac resulted only in improving heartburn sensation (Figures 1 and 2A). Finally, we confirmed that compared with placebo, diclofenac administration reduced PGE2 levels in the esophageal tissue (Figure 2A), and we detected a significant correlation between the PGE2 and APSS of heartburn (Figure 2B). These results indicated that PGE2 has an important role in esophageal sensation, especially for heartburn, and that the administration of NSAIDs reduced heartburn through a decreased PGE2 level. Thus, manipulating PGE2 may control the symptoms of esophageal reflux. Indeed, we focused on controlling heartburn symptoms with NSAIDs. We propose that NSAIDs could be potential candidates for heartburn control in PPI-refractory GERD and NERD. In addition, we perfused saline to the esophagus for 30 minutes after esophageal biopsy using mini-forceps in a healthy volunteer in a previous report.16 We then confirmed that the small biopsy wound did not affect both the symptom generation of heartburn and the PGE2 level after saline infusion. Therefore, we believe that this study does not necessarily mimic only erosive esophagitis. The anti-inflammatory and analgesic effects of NSAIDs are believed to occur through the inhibition of COX-1 and COX-2, which inhibit peripheral prostaglandin synthesis.32 Diclofenac is an established NSAID that is usedwidely for the treatment of pain and fever caused by inflammation. In this study, we used Voltaren SR capsules, which have a recommended initial daily dose of 37.5 mg twice daily in Japan (approved by the Ministry of Health, Labour and Welfare in Japan). The T max (h) and T1/2 (h) after a single-dose administration of Voltaren SR in human beings are reported to be 7.0 1.0 hours and 1.5 0.4 hours, respectively. Based on these data, we dosed at 6 hours and 2 hours before esophageal acid perfusion to achieve a good analgesic effect on controlling visceral sensation and managing somatic pain (Supplementary Figure 1, visit 2). Continuous administration of NSAIDs is known to cause GI toxicity, including upper GI mucosal injury and bleeding.33,34 In addition, NSAIDs reportedly stimulate acid secretion,35,36 which is mediated predominantly by PGE2 through EP3 receptors.37 Because of the concerns over GI toxicity and increased acid secretion, the analgesic effect of NSAIDs on GI symptoms such as heartburn has not attracted much attention. Basically, these unfavorable effects of NSAIDs are mediated by COX-1 inhibition, so focusing on selective downstream blocking of nociceptive receptors, including PGE receptors, might result in fewer side effects. In fact, there is evidence that pain hypersensitivity is attenuated in EP1-deficient mice38 and that a novel EP1-receptor antagonist, ONO8539, suppresses acid-induced primary and esophageal hypersensitivity in monkeys.39 There is also preclinical evidence that prior systemic treatment with the EP1
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antagonist ZD6416 attenuates acid-induced secondary esophageal hyperalgesia in human beings.22 Thus, acting via the EP1 receptor, PGE2 appears to affect visceral nociceptive processing significantly, and inhibition of this pathway may offer an effective treatment of heartburn without inhibiting the other prostanoids (PGD2, PGF2 , PGI2, and TXA2), which potentially have several systemic biological actions. We hope selective EP1 antagonists may be an effective therapy in the treatment of refractory GERD in the near future. We mainly discussed the peripheral actions of PGE2 and the EP1 receptor.16 However, the EP1 receptor is present not only in the peripheral esophageal tissue,16 but also in the central nervous system.40 In fact, a previous study showed that PGE2 levels were significantly higher after distal esophageal acidification in the brainstem and spinal cord and that the EP1 receptor was expressed both in the central and peripheral nervous tissue in monkeys.39 Sarkar et al20 reported that the EP1 antagonist ZD6416 attenuated secondary esophageal hyperalgesia in human beings. On the basis of these findings, the additional GI symptoms during acid infusion may be partially induced by secondary hypersensitivity at the central level (Figure 1). Furthermore, it is possible that the infused acid may have induced an increase in PGE2 in the gastric and duodenal mucosa, thereby causing additional GI symptoms.41 Therefore, diclofenac may affect these additional GI symptoms by inhibiting PGE2 centrally and peripherally, or by suppressing PGE2 levels in the gastric and/or duodenal mucosa. Limitations of this study included the small sample size (n ¼ 12) and the absence of confirmation in a comparison group of patients with GERD and NERD. Further studies are necessary to investigate the expression of PGE2 and EP1 in the esophagus of patients with GERD and NERD and in patients with noncardiac chest pain or functional heartburn. In addition, further research investigating whether heartburn sensation in patients with GERD and NERD with hypersensitivity could be relieved with diclofenac is needed. These findings have the potential to influence clinical practice and provide a new therapeutic option for controlling heartburn, especially for the subgroup of patients with NERD who typically respond poorly to PPI therapy. In conclusion, this study showed that diclofenac attenuated acid-induced esophageal pain through the inhibition of esophageal PGE2 overproduction. Esophageal PGE2 may play a significant role in heartburn symptom generation, and control of PGE2 may offer a new therapeutic target for heartburn management.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Clinical Gastroenterology and Hepatology at www.cghjournal.org, and at http://dx.doi.org/10.1016/j.cgh.2015.01.014.
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12. Miki T, Matsunami M, Nakamura S, et al. ONO-8130, a selective prostanoid EP1 receptor antagonist, relieves bladder pain in mice with cyclophosphamide-induced cystitis. Pain 2011; 152:1373–1381. 13. Minami T, Okuda-Ashitaka E, Hori Y, et al. Involvement of primary afferent C-fibres in touch-evoked pain (allodynia) induced by prostaglandin E2. Eur J Neurosci 1999;11:1849–1856. 14. Omote K, Kawamata T, Nakayama Y, et al. The effects of peripheral administration of a novel selective antagonist for prostaglandin E receptor subtype EP(1), ONO-8711, in a rat model of postoperative pain. Anesth Analg 2001;92:233–238. 15. Kawahara H, Sakamoto A, Takeda S, et al. A prostaglandin E2 receptor subtype EP1 receptor antagonist (ONO-8711) reduces hyperalgesia, allodynia, and c-fos gene expression in rats with chronic nerve constriction. Anesth Analg 2001; 93:1012–1017. 16. Kondo T, Oshima T, Tomita T, et al. Prostaglandin E(2) mediates acid-induced heartburn in healthy volunteers. Am J Physiol Gastrointest Liver Physiol 2013;304:G568–G573. 17. Kusano M, Shimoyama Y, Sugimoto S, et al. Development and evaluation of FSSG: frequency scale for the symptoms of GERD. J Gastroenterol 2004;39:888–891. 18. Fass R, Naliboff B, Higa L, et al. Differential effect of long-term esophageal acid exposure on mechanosensitivity and chemosensitivity in humans. Gastroenterology 1998;115:1363–1373. 19. Fass R, Yalam JM, Camargo L, et al. Increased esophageal chemoreceptor sensitivity to acid in patients after successful reversal of Barrett’s esophagus. Dig Dis Sci 1997;42: 1853–1858.
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32. FitzGerald GA, Patrono C. The coxibs, selective inhibitors of cyclooxygenase-2. N Engl J Med 2001;345:433–442. 33. Singh G, Rosen Ramey D. NSAID induced gastrointestinal complications: the ARAMIS perspective–1997. Arthritis, Rheumatism, and Aging Medical Information System. J Rheumatol Suppl 1998;51:8–16. 34. Wolfe MM, Lichtenstein DR, Singh G. Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs. N Engl J Med 1999; 340:1888–1899. 35. Savarino V, Mela GS, Zentilin P, et al. Effect of one-month treatment with nonsteroidal antiinflammatory drugs (NSAIDs) on gastric pH of rheumatoid arthritis patients. Dig Dis Sci 1998;43:459–463. 36. Kataoka H, Horie Y, Koyama R, et al. Interaction between NSAIDs and steroid in rat stomach: safety of nimesulide as a preferential COX-2 inhibitor in the stomach. Dig Dis Sci 2000;45:1366–1375. 37. Nishio H, Terashima S, Nakashima M, et al. Involvement of prostaglandin E receptor EP3 subtype and prostacyclin IP receptor in decreased acid response in damaged stomach. J Physiol Pharmacol 2007;58:407–421. 38. Stock JL, Shinjo K, Burkhardt J, et al. The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure. J Clin Invest 2001;107:325–331. 39. Konemura T, Date I, Fujii Y, et al. 746 ONO-8539, a novel prostanoid EP1 receptor antagonist, suppresses acid-induced primary and secondary esophageal hypersensitivity in monkeys. Gastroenterology 2013;144:S134–S135. 40. Kawabata A. Prostaglandin E2 and pain–an update. Biol Pharm Bull 2011;34:1170–1173.
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41. Miwa H, Nakajima K, Yamaguchi K, et al. Generation of dyspeptic symptoms by direct acid infusion into the stomach of healthy Japanese subjects. Aliment Pharmacol Ther 2007;26:257–264.
Mukogawa-cho, Nishinomiya, Hyogo, Japan 663-8501. e-mail: miwahgi@ Q1 hyo-med.ac.jp; fax: (81) 798-45-6661.
Reprint requests Address requests for reprints to: Hiroto Miwa, MD, PhD, Division of Gastroenterology, Department of Internal Medicine, Hyogo College of Medicine, 1-1,
Conflicts of interest This author discloses the following: Hiroki Okada is an employee of Ono Pharmaceutical Co, Ltd. The remaining authors disclose no conflicts. Q3
Q2 Acknowledgments The authors appreciate the excellent technical assistance provided by Mr Takashi Konemura, Mr Ichiro Date, and Ms Noriko Kamiya.
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Supplementary Figure 1. Study flow diagram.
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The Nonsteroidal Anti-inflammatory Drug Diclofenac Reduces Acid-Induced Heartburn Symptoms in Healthy Volunteers Q28
Takashi Kondo,* Tadayuki Oshima,* Toshihiko Tomita,* Hirokazu Fukui,* Hiroki Okada,‡ Jiro Watari,* and Hiroto Miwa* *Division of Gastroenterology, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan; ‡Minase Research Institute, Pharmacological Research Laboratories, Ono Pharmaceutical Co, Ltd, Osaka, Japan
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BACKGROUND & AIMS:
We investigated the effects of diclofenac, a nonsteroidal anti-inflammatory drug that inhibits prostaglandin production, on induction of esophageal sensation by acid perfusion in healthy men.
METHODS:
We performed a prospective, double-blind, placebo-controlled, 2-period, cross-over study over 3 visits in 12 healthy men. Diclofenac was given 6 hours and 2 hours before an acid perfusion test. During the test, hydrochloric acid (0.15 mol/L) was perfused into the lower esophagus for 30 minutes; we evaluated upper gastrointestinal symptoms using a validated categoric rating scale. Then, we calculated and assessed the acid perfusion sensitivity score (APSS). Biopsy specimens were collected by endoscopy of the distal esophagus before and after acid perfusion; levels of prostaglandin E2 (PGE2) (pg/mg) were measured in the samples using an enzymelinked immunosorbent assay.
RESULTS:
Compared with placebo, diclofenac significantly reduced the APSS for heartburn (82.2 – 12.2 for placebo and 47.5 – 8.9 for diclofenac; P < .01). Of the upper gastrointestinal symptoms, only the APSS for heartburn was reduced significantly by diclofenac. Compared with placebo, diclofenac reduced the overproduction of PGE2 by esophageal tissues after acid perfusion (23.3 – 5.2 for placebo and 11.4 – 3.5 for diclofenac; P < .05). APSS correlated with the development of heartburn and esophageal levels of PGE2 (r [ 0.53; P < .05 for diclofenac vs placebo).
CONCLUSIONS:
Diclofenac attenuated acid-induced heartburn by inhibiting PGE2 overproduction in the esophagus. Esophageal PGE2 might be involved in producing heartburn symptoms. Clinical Trials Registry no: UMIN000014595.
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Keywords: NSAID; Reflux; Treatment; GERD; NERD. Q7 Q8 Q9
he prevalence of gastroesophageal reflux disease (GERD) continues to increase in Western and Japanese populations1–3 with heartburn likely to remain the most common symptom. Although proton pump inhibitors (PPIs) have improved outcomes significantly, a meta-analysis showed that up to 40% of patients with GERD are dissatisfied with standard PPI therapy because their symptoms are not fully resolved.4–7 Furthermore, it is widely recognized that the persistent GERD symptoms resulting from PPI resistance worsen quality of life in patients with nonerosive reflux disease (NERD). Therefore, appropriate management of GERD symptom is very important. One reason why these unmet clinical needs persist is that the pathogenesis of the core symptom, heartburn, is not completely understood.8,9 Therefore, it is important to investigate novel mechanisms of symptom generation for heartburn to provide additional therapeutic options for the treatment and control of heartburn in patients with GERD and NERD.
T
Prostanoids are produced during arachidonic acid metabolism in the cyclooxygenase (COX) pathway, and they are established as inflammatory mediators that contribute to inflammation and nociception.10,11 Among the prostanoids, prostaglandin E2 (PGE2) is considered the principal proinflammatory and pro-nociceptive prostanoid,12 and is known to induce sensitization and pain hypersensitivity through activation of prostanoid receptors on peripheral nerve terminals.13–15 Recently,
Abbreviations used in this paper: APSS, acid perfusion sensitivity score; COX, cyclooxygenase; ED, elemental diet; ER, ___________ _________________; GERD, gastroesophageal reflux disease; GI, gastrointestinal; NERD, nonerosive reflux disease; NSAID, nonsteroidal antiinflammatory drug; PGE2, prostaglandin E2; PPI, proton pump inhibitor; RM-ANOVA, _______________________ analysis of variance. Q4 © 2015 by the AGA Institute 1542-3565/$36.00 http://dx.doi.org/10.1016/j.cgh.2015.01.014
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we reported that esophageal PGE2 may be involved in generating heartburn in human beings. Specifically, we showed that acid perfusion in the esophagus induced PGE2 generation in the esophageal mucosa and that the increased PGE2 level was correlated significantly with changes in the esophageal symptom score for heartburn in healthy participants.16 On the basis of this theoretical background, we assumed that inhibiting PGE2 production in the esophageal mucosa might prevent upper gastrointestinal (GI) symptoms, especially heartburn. To confirm this hypothesis, we used the nonsteroidal anti-inflammatory drug (NSAID) diclofenac as an inhibitor of prostaglandin production to premedicate healthy participants before an acid perfusion test, and we then investigated its effect on heartburn symptoms. Therefore, we aimed to investigate whether diclofenac suppressed acid-induced heartburn in healthy participants by inhibiting PGE2 overproduction in the esophagus, and whether esophageal PGE2 levels correlated with the heartburn symptoms. In addition, we explored the effect of diclofenac on several upper GI symptoms.
Methods Study Design
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A prospective, double-blinded, placebo-controlled, 2period, cross-over study was conducted over 3 visits. Eligibility for the study was determined at visit 1, and volunteers were randomized according to a cross-over sequence with diclofenac or placebo. A person who was not involved in the study generated the 2 comparison groups using simple randomization, with an equal allocation ratio, by referring to a table of random numbers. The randomization code was concealed until the end of the trial. At visits 2 and 3, we assessed upper GI symptoms during acid perfusion tests and obtained esophageal biopsy samples. To allow adequate time for complete washout of diclofenac, the interval between visits 2 and 3 was at least 2 weeks. We performed all experiments in accordance with human ethics regulations (Hyogo College of Medicine: no 1162) and obtained written informed consent. This trial is registered with the University Hospital Medical Information Network Clinical Trials Registry (Number UMIN000014595). The trial was conducted according to the principles governing human research in the Declaration of Helsinki. All authors had access to the study data and reviewed and approved the final manuscript.
Participants We enrolled 12 healthy men. The status of daily upper abdominal symptoms was checked with a previously validated questionnaire, the frequency scale for
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the symptoms of GERD.17 At visit 1, we excluded participants based on the following criteria: a frequency scale for the symptoms of GERD total score of greater than 8; any medical history of asthma or allergy to NSAIDs; any history of peptic ulcer or GERD or of upper GI tract surgery; any significant cardiovascular, kidney, liver, neurotic, or psychological disorders; and any subjects taking medications that may affect prostaglandin generation, including NSAIDs, COX-2 inhibitors, or prostaglandin-containing medications. At visit 2, we excluded significant upper GI pathology using endoscopy (eg, esophagitis, Barrett’s esophagus, hiatus hernia, active peptic ulcer disease, and esophageal/gastric cancer).
Trial Protocol We used sustained-release diclofenac sodium (37.5-mg Voltaren sustained release [SR] capsules; Novartis Pharma KK, Tokyo, Japan) in this trial. After a minimum 6-hour fast, Voltaren SR capsule or placebo was given at 6 hours and 2 hours before the acid perfusion test. For the test, participants were placed in the left lateral decubitus position and we performed a transnasal endoscopic examination (GIF-XP260N; Olympus Optical Company, Tokyo, Japan). At this point, we obtained baseline esophageal endoscopic biopsy samples from 3 cm above the esophagogastric junction using biopsy forceps (Radial Jaw 3; Boston Scientific Corporation, MA). After endoscopy, a 5F elemental diet (ED) tube (Nippon Sherwood Medical Industries Ltd, Tokyo, Japan) was inserted via the nasal passage with the distal tip placed 10 cm above the esophagogastric junction. Acid perfusions then were performed, followed by immediate removal of the ED tube and repeated endoscopic biopsies. The endoscopic esophageal mucosa samples were frozen immediately in a 1.5-mL sampling tube with liquid nitrogen and stored in a freezer at 80 C until used to measure PGE2 levels.
Interventions Acid perfusion test. The acid perfusion test was performed by modification of a previously reported method18,19 with participants in an upright sitting position. After confirmation that no participant had heartburn during the initial saline perfusion (8 mL/min for 2 min), hydrochloric acid (0.15 mol/L) was infused for 30 minutes into the lower esophagus via the ED tube. Previous studies have indicated that this acid concentration induces esophageal hypersensitivity in the majority of healthy participants20 and causes hyperalgesia in response to an electrical stimulus in human models.21,22 Infusions were delivered at 8 mL/min for 30 minutes with an automatic infusion pump. Assessment of reflux and upper gastrointestinal symptom severity. During acid perfusion, participants were
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instructed to report when heartburn first was perceived (allowing calculation of the time to first sensation and the duration of heartburn symptom perception). Thereafter, the intensity of several upper GI symptoms was recorded during acid perfusion (for 30 min) at regular 5minute intervals. The upper GI symptoms of interest were heartburn, sour or bitter taste, pricking pain in the stomach, dull pain in the stomach, heavy feeling in the stomach, tight feeling in the stomach, nausea, and fullness. We used a previously validated categoric rating scale,18,23,24 which ranged from 1 (no sensation) to 13 (extremely intense), to assess the intensity of upper GI symptoms associated with acid perfusion. Then, we used the acid perfusion sensitivity score (APSS) to analyze upper GI symptom severity according to the method of Fass et al.19,25 The APSS was calculated as follows: using the duration of the upper GI symptom perception (T) expressed in seconds (s) and the upper GI symptom intensity rating at the end of the acid perfusion (I), APSS ¼ I T / 100 (cm s/100). The symptom scores were recorded by the subjects without prompting for responses. Measurement of prostaglandin E2 levels. After measuring the weight of the lower esophageal mucosal tissue, homogenization (300 Hz/s for 3 min) was conducted with a mixer mill (MM300; Qiagen, Hiden, Germany). The protein level in the supernatant was measured with the BCA protein assay kit (Thermo Fisher Scientific, Inc, MA). The PGE2 level was measured by an enzyme-linked immunosorbent assay using the Prostaglandin E2 EIA Kit-Monoclonal (Cayman Chemical Company, MI) according to the manufacturer’s instructions. The level was determined after absorbance measurement with a microplate reader (Spectra Max 250; Molecular Devices Japan Co, Tokyo, Japan).
Outcomes: Study End Points The primary efficacy outcome for the study was the change in APSS for heartburn, and statistical significance was assessed by the mean difference in the change of scores between the diclofenac and placebo groups. To support the primary outcome, we also assessed the following secondary outcomes: (1) changes in other upper GI symptoms; (2) change in esophageal PGE2 levels; and (3) correlation between esophageal PGE2 levels and APSS.
Sample Size The primary end point of this study was a change in the APSS between diclofenac and placebo. Prior data16 from 14 subjects confirmed that healthy subjects had an average change in APSS of 194 and a SD of 106 between acid and saline infusion. On the basis of these results, we assumed that each expected difference in APSS and SD
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Table 1. Demographic Data of the Study Subjects Placebo/diclofenac Diclofenac/placebo P (N ¼ 6) value (N ¼ 6) Age, ya Sex, male/ female Weight, kga Height, cma BMI, kg/m2a FSSGa
31.2 3.6 6/0 67.8 171.3 23.0 3.2
6.1 4.3 1.8 1.9
35.8 3.7 6/0
.06b —
.85b .45b .46b .76b
71.2 169.0 24.8 3.5
13.8 6.0 3.9 2.6
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BMI, body mass index; FSSG, frequency scale for symptoms of GERD. a Means SD. b Mann–Whitney U test.
was 97 and 53, respectively. The number of subjects with this assumption was 10 in each arm (a ¼ 0.05, 1 b ¼ 0.9). Therefore, a sample size of 12 subjects in this study adequately allowed a power of 90% to detect a difference at a significance level of 0.05 (2-tailed).
Statistical Analysis We compared the time to first sensation and all APSSs for the upper GI symptoms between the diclofenac and placebo groups with the nonparametric Wilcoxon matched-pairs signed-rank test. PGE2 levels before and after the perfusion test were compared between the diclofenac and placebo groups by 2-way RM-analysis of variance (ANOVA), followed by an individual post hoc comparison with the Bonferroni multiple comparison test. Esophageal PGE2 levels after acid perfusion were compared with APSS by the Pearson correlation coefficient. Data are presented as mean standard error. Analyses were performed using SAS 9.2 (TS2M3; SAS Institute Japan Ltd, Tokyo, Japan) and its cooperative system EXSUS version 7.7.1 (CAC Corporation). A P value less than .05 indicated statistical significance.
Results Participants and Recruitment Twelve healthy men originally were enrolled in this study, of whom all candidates were eligible for inclusion. The demographic data of the study subjects are presented in Table 1. One participant was unable to tolerate prolonged endoscopy and was excluded. Therefore, 11 participants completed the study (Supplementary Figure 1).
Effect of Diclofenac on Other Upper Gastrointestinal Symptoms During the acid perfusion test, we also explored several other upper GI symptoms. Of the upper GI symptoms, diclofenac only significantly suppressed APSS
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Figure 1. The effect of diclofenac on upper gastrointestinal symptoms. Compared with placebo, diclofenac significantly suppressed APSS of heartburn during acid perfusion (*P < .05). Compared with placebo, the other upper gastrointestinal symptoms were not affected significantly by diclofenac.
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of heartburn (82.2 12.2 and 47.5 8.9, respectively; P < .01), and had no significant effects on the other upper GI symptoms (Figure 1).
Effect of Diclofenac on Prostaglandin E2 Generation in the Esophageal Mucosa PGE2 levels in the esophageal biopsy samples were significantly higher after acid perfusion (23.3 5.2 pg/mg protein) than before perfusion (7.7 1.2 pg/mg protein) (P < .01, 2-way RM-ANOVA) in the placebo group, which is consistent with our previous report.16 Diclofenac significantly inhibited PGE2 overproduction induced by acid perfusion in the human esophagus (11.4 3.5 pg/mg protein) compared with placebo (23.3 5.2 pg/mg protein) (P < .05, 2-way RM-ANOVA) (Figure 2A). Furthermore, there was a clear correlation between increased esophageal PGE2 levels and APSS of heartburn (r ¼ 0.53, P < .05, Pearson correlation) (Figure 2B).
Drug Administration and Adverse Events All participants were completely symptom free and did not have any adverse events.
Discussion Heartburn is a typical symptom of GERD. Several mechanisms are postulated to explain the pathogenesis of heartburn, including peripheral production of inflammatory mediators, dilated intercellular spaces,26,27 failure of the barrier function within the esophageal mucosa causing increased mucosal permeability,28 and altered nociceptive processing closely associated with visceral
Figure 2. The effect of diclofenac on PGE2 generation in the esophageal mucosa and the correlation between APSS and PGE2 level. (A) In the placebo group, PGE2 levels in the esophageal biopsy samples were significantly higher after acid perfusion than before perfusion (**P < .01). In the diclofenac group, PGE2 levels significantly decreased after acid perfusion compared with the placebo group (*P < .05), and did not change from before acid perfusion (not significant). Data are presented as mean standard error. (B) A positive correlation was found between APSS of heartburn and increased esophageal PGE2 levels (r ¼ 0.53, P < .05).
hypersensitivity.8,29,30 However, many of the mechanisms of heartburn generation remain unknown, which results in persistent unmet clinical need. In this study, we show that premedication of healthy volunteers with the NSAID diclofenac reduced the intensity of heartburn symptoms induced by acid perfusion (Figure 1). Next, we asked the subjects about the presence of several upper GI symptoms during the acid perfusion test because patients with GERD complain of atypical GERD symptoms, such as an epigastric burning
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sensation, epigastralgia, epigastric discomfort, and abdominal fullness.31 We also investigated the effect of diclofenac on these symptoms. Consequently, we found that suppressing PGE2 in the esophagus using diclofenac resulted only in improving heartburn sensation (Figures 1 and 2A). Finally, we confirmed that compared with placebo, diclofenac administration reduced PGE2 levels in the esophageal tissue (Figure 2A), and we detected a significant correlation between the PGE2 and APSS of heartburn (Figure 2B). These results indicated that PGE2 has an important role in esophageal sensation, especially for heartburn, and that the administration of NSAIDs reduced heartburn through a decreased PGE2 level. Thus, manipulating PGE2 may control the symptoms of esophageal reflux. Indeed, we focused on controlling heartburn symptoms with NSAIDs. We propose that NSAIDs could be potential candidates for heartburn control in PPI-refractory GERD and NERD. In addition, we perfused saline to the esophagus for 30 minutes after esophageal biopsy using mini-forceps in a healthy volunteer in a previous report.16 We then confirmed that the small biopsy wound did not affect both the symptom generation of heartburn and the PGE2 level after saline infusion. Therefore, we believe that this study does not necessarily mimic only erosive esophagitis. The anti-inflammatory and analgesic effects of NSAIDs are believed to occur through the inhibition of COX-1 and COX-2, which inhibit peripheral prostaglandin synthesis.32 Diclofenac is an established NSAID that is usedwidely for the treatment of pain and fever caused by inflammation. In this study, we used Voltaren SR capsules, which have a recommended initial daily dose of 37.5 mg twice daily in Japan (approved by the Ministry of Health, Labour and Welfare in Japan). The T max (h) and T1/2 (h) after a single-dose administration of Voltaren SR in human beings are reported to be 7.0 1.0 hours and 1.5 0.4 hours, respectively. Based on these data, we dosed at 6 hours and 2 hours before esophageal acid perfusion to achieve a good analgesic effect on controlling visceral sensation and managing somatic pain (Supplementary Figure 1, visit 2). Continuous administration of NSAIDs is known to cause GI toxicity, including upper GI mucosal injury and bleeding.33,34 In addition, NSAIDs reportedly stimulate acid secretion,35,36 which is mediated predominantly by PGE2 through EP3 receptors.37 Because of the concerns over GI toxicity and increased acid secretion, the analgesic effect of NSAIDs on GI symptoms such as heartburn has not attracted much attention. Basically, these unfavorable effects of NSAIDs are mediated by COX-1 inhibition, so focusing on selective downstream blocking of nociceptive receptors, including PGE receptors, might result in fewer side effects. In fact, there is evidence that pain hypersensitivity is attenuated in EP1-deficient mice38 and that a novel EP1-receptor antagonist, ONO8539, suppresses acid-induced primary and esophageal hypersensitivity in monkeys.39 There is also preclinical evidence that prior systemic treatment with the EP1
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antagonist ZD6416 attenuates acid-induced secondary esophageal hyperalgesia in human beings.22 Thus, acting via the EP1 receptor, PGE2 appears to affect visceral nociceptive processing significantly, and inhibition of this pathway may offer an effective treatment of heartburn without inhibiting the other prostanoids (PGD2, PGF2 , PGI2, and TXA2), which potentially have several systemic biological actions. We hope selective EP1 antagonists may be an effective therapy in the treatment of refractory GERD in the near future. We mainly discussed the peripheral actions of PGE2 and the EP1 receptor.16 However, the EP1 receptor is present not only in the peripheral esophageal tissue,16 but also in the central nervous system.40 In fact, a previous study showed that PGE2 levels were significantly higher after distal esophageal acidification in the brainstem and spinal cord and that the EP1 receptor was expressed both in the central and peripheral nervous tissue in monkeys.39 Sarkar et al20 reported that the EP1 antagonist ZD6416 attenuated secondary esophageal hyperalgesia in human beings. On the basis of these findings, the additional GI symptoms during acid infusion may be partially induced by secondary hypersensitivity at the central level (Figure 1). Furthermore, it is possible that the infused acid may have induced an increase in PGE2 in the gastric and duodenal mucosa, thereby causing additional GI symptoms.41 Therefore, diclofenac may affect these additional GI symptoms by inhibiting PGE2 centrally and peripherally, or by suppressing PGE2 levels in the gastric and/or duodenal mucosa. Limitations of this study included the small sample size (n ¼ 12) and the absence of confirmation in a comparison group of patients with GERD and NERD. Further studies are necessary to investigate the expression of PGE2 and EP1 in the esophagus of patients with GERD and NERD and in patients with noncardiac chest pain or functional heartburn. In addition, further research investigating whether heartburn sensation in patients with GERD and NERD with hypersensitivity could be relieved with diclofenac is needed. These findings have the potential to influence clinical practice and provide a new therapeutic option for controlling heartburn, especially for the subgroup of patients with NERD who typically respond poorly to PPI therapy. In conclusion, this study showed that diclofenac attenuated acid-induced esophageal pain through the inhibition of esophageal PGE2 overproduction. Esophageal PGE2 may play a significant role in heartburn symptom generation, and control of PGE2 may offer a new therapeutic target for heartburn management.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Clinical Gastroenterology and Hepatology at www.cghjournal.org, and at http://dx.doi.org/10.1016/j.cgh.2015.01.014.
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Mukogawa-cho, Nishinomiya, Hyogo, Japan 663-8501. e-mail: miwahgi@ Q1 hyo-med.ac.jp; fax: (81) 798-45-6661.
Reprint requests Address requests for reprints to: Hiroto Miwa, MD, PhD, Division of Gastroenterology, Department of Internal Medicine, Hyogo College of Medicine, 1-1,
Conflicts of interest This author discloses the following: Hiroki Okada is an employee of Ono Pharmaceutical Co, Ltd. The remaining authors disclose no conflicts. Q3
Q2 Acknowledgments The authors appreciate the excellent technical assistance provided by Mr Takashi Konemura, Mr Ichiro Date, and Ms Noriko Kamiya.
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Supplementary Figure 1. Study flow diagram.
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