Experimental study of diclofenac and its biliary metabolites on anastomotic healing

Background Diclofenac increases the risk of anastomotic leakage, but the underlying mechanism is unknown. As diclofenac is excreted largely as biliary metabolites, the aim of this study was to determine the effect of these metabolites on intestinal anastomoses. Methods This was a randomized controlled blinded experiment using 210 male Wistar rats to assess the effect of ‘diclofenac bile’ on the anastomotic complication score, leak rate and anastomotic strength following oral and parenteral administration of diclofenac. Bile duct and duodenal catheterization techniques were used for diversion and replacement of bile, and biliary diclofenac metabolites were determined. Results Replacement of control bile with diclofenac bile resulted in higher anastomotic complication scores (P = 0·006) and leakage in five of 18 animals, compared with one of 18 controls (P = 0·089). In turn, following oral diclofenac administration, replacement of diclofenac bile with control bile reduced anastomotic complications (P = 0·016). The leak rate was seven of 15 versus 13 of 17 without replacement (P = 0·127). After intramuscular administration of diclofenac, the reduction in anastomotic complications was not significant when bile was replaced with control bile (P = 0·283), but it was significant when bile was drained without replacement (P = 0·025). Diclofenac metabolites in bile peaked within 2 h after administration. Administration of diclofenac bile resulted in nearly undetectable plasma levels of diclofenac (mean(s.d.) 0·01(0·01) μg/ml) after 120 min. Following oral diclofenac, bile replacement with control bile did not affect the plasma concentration of diclofenac (0·12(0·08) μg/ml versus 0·10(0·05) μg/ml with diclofenac bile; P = 0·869). Conclusion Altered bile composition as a result of diclofenac administration increases the ileal anastomotic complication rate in rats.


Introduction
Anastomotic leakage is the leading cause of morbidity and mortality after intestinal surgery, with leak rates varying from 3 to 14 per cent 1 . Non-steroidal anti-inflammatory drugs (NSAIDs) have been related to increased leak rates in both animal and retrospective human studies 2 -4 . This is worrisome because NSAIDs are recommended as postoperative analgesics in current guidelines 5 . Clinical evidence is not considered sufficient to ban postoperative use of NSAIDs, as some studies do not report adverse effects 5,6 . Further research is required to delineate the role of NSAIDs in clinical anastomotic leakage 4,7 .
The main mechanism by which NSAIDs are believed to jeopardize anastomotic healing is the cyclo-oxygenase (COX) 2 inhibitory action, which may cause suppression of inflammatory, proliferative or proangiogenic pathways relevant for normal wound healing 2,8 . An alternative explanation could be direct toxicity of intraluminal factors resulting from NSAID treatment (such as drug metabolites and bile products). This is based on a similar relationship found between NSAIDs and small intestinal mucosal damage 9 -11 . It is hypothesized that the NSAID diclofenac may display its detrimental effect on bowel anastomoses through altered and toxic bile composition. At least three observations support this hypothesis. First, NSAID-induced intestinal mucosal damage has been attributed to increased bile toxicity 12 -15 . Biliary excretion of toxic drug metabolites and reduced availability of protective phosphatidylcholine in bile due to competitive binding by NSAID metabolites appear to be responsible for the bile toxicity 9,12,13,16 . Second, diclofenac causes leakage of anastomoses in the ileum and proximal colon, but not in the distal colon of the rat, which can be explained by the fact that most bile components are reabsorbed or converted to non-toxic substances before reaching the distal colon 17 . Third, NSAIDs that are predominantly excreted through bile (such as diclofenac, celecoxib and carprofen) cause 80-100 per cent leakage of experimental ileal anastomoses, whereas naproxen, which is excreted almost entirely through urine in the rat and human, results in less than 15 per cent leakage 18 -20 . In clinical practice, bile toxicity could pose a threat to a large number of patients, as it could be harmful for both small and large bowel anastomoses, considering the altered physiology following partial colectomy.
The aim of this study was to investigate the effect of altered bile composition induced by diclofenac on small bowel anastomotic healing in rats using bile drainage and bile replacement techniques.

Methods
The study was carried out according to the ARRIVE guidelines 21 . It was approved by the Animal Ethics Committee (AEC number 2013-171) and performed in the Central Animal Laboratory of Radboud University.

Animals
In total, 210 adult male Wistar rats (mean(s.d.) weight 313(25) g; Harlan, Horst, The Netherlands) were used (138 to assess anastomotic healing and 72 to donate bile). The animals were acclimatized to laboratory conditions and initially housed, two per cage, at 22-23 ∘ C with a 12-h day cycle, and free access to standard rodent chow (Ssniff ® R/M-H; Bio Services, Uden, The Netherlands) and acidified tap water throughout the experiment. The cages were enriched with a rat retreat and nesting material, and were placed randomly on the shelves. All animals were checked at least twice daily. Humane endpoints were defined; animals were killed if they showed signs of severe discomfort (such as weight loss greater than 20 per cent, distended abdomen or severely reduced activity).

Study design
The study consisted of three experiments ( Table 1). A distal ileum anastomosis model was used in all experiments and rats had surgery on day 0; anastomotic healing was assessed after killing on day 3 17 . This interval was used because most diclofenac-induced leakage occurs before day 3, when the anastomosis is at its weakest, and postponing killing would increase suffering 20 . Rats received 1⋅5 mg/kg diclofenac twice daily either orally (O+) or intramuscularly (IM+), or they received no oral or intramuscular diclofenac (O− or IM−). Cannulation of the common bile duct (CBD) and the duodenum was performed to drain and infuse bile respectively (Fig. 1). Allocation to groups was done randomly by using the online tool at www.randomization .com. In experiment 1, 72 rats were allocated to four groups of 18 each, and 72 rats were used as matched bile donors. In group O−B−, no diclofenac was administered and bile was drained and replaced by control bile from matched donor rats. In group O−B+ rats, the bile was replaced by 'diclofenac bile' from donor rats that had also received 1⋅5 mg/kg diclofenac orally twice daily for 3 days. Rats in group O+B− were administered diclofenac orally, and their bile was replaced with control bile 17 . Rats in group O+B+ were given diclofenac orally and received diclofenac bile from donors.
In experiments 2 and 3, a parenteral route of diclofenac administration was used to explore the effect of avoiding exposure of the gut to drug molecules as a direct result of oral administration 13,14 . In experiment 2, 42 rats were allocated to three groups. Rats in group IM+B= (n = 18) received diclofenac intramuscularly, and no cannulations of the bile duct or duodenum were done. In groups IM+B0 and IM−B0 (n = 12 animals each), bile was drained completely without replacing it to prevent diclofenac metabolites and bile salts from entering the gut. In experiment 3, rats were given diclofenac intramuscularly and either received their own diclofenac bile (group IM+B+; n = 12) or bile was replaced with control bile from donor rats (group IM+B−; n = 12).

Operative procedures
All operations were done by two experienced researchers. For analgesia, 0⋅02 mg/kg buprenorphine (Temgesic ® ; Schering-Plough, Houten, The Netherlands) was given subcutaneously every 12 h, starting 1 h before the operation until 48 h afterwards. No antibiotics were given and animals were not fasted. Anaesthesia was with isoflurane (Abbott, Hoofddrop, The Netherlands), 5 per cent for induction and 2⋅5-3⋅5 per cent for maintenance, in a 1 : 1 mixture of oxygen and pressurized air. Rats were prepared by shaving, skin disinfection with iodine and sterile covers, and then operated on through a 4-5-cm midline laparotomy using strict aseptic technique under a microscope (Wild M650; Wild Heerbrugg, Gais, Switzerland, at 10-16× magnification). Body temperature was kept at 38 ∘ C with a heating pad and lamp.
The procedures started with cannulation of the CBD to drain bile and placement of a catheter in the duodenum to readminister bile, if applicable. This was followed by construction of the bowel anastomosis. The laparotomy wound was closed with a running suture, 3/0 Vicryl ® (Ethicon, Amersfoort, The Netherlands) for the fascia and staples for the skin.

Bile drainage and infusion techniques
The CBD was ligated with 7/0 silk (Pearsalls, Taunton, UK) just before it enters the tail of the pancreas, and a V-shaped hole was made in the duct with iridectomy scissors proximal to the ligation. A custom-made 3-Fr polyurethane cannula (BTPU-040; Instech Laboratories, Plymouth Meeting, Pennsylvania, USA), 20 cm in length, and a silicone ring (BTSIL-047; Instech Laboratories) at 7 and 50 mm from the sharpened tip, was inserted into the bile duct and fixed with 7/0 silk proximal and distal to the silicon ring at 7 mm. A similar, but blunt, cannula was inserted through a purse-string suture (6/0 Vicryl ® ; Ethicon, Norderstedt, Germany) in the duodenum. Cannulas were fixed to the abdominal wall (6/0 Vicryl ® ), tunnelled through the rectus muscle and subcutaneously towards the back between the scapulas, and then connected to a harness (VAHD95AB or VAHD115AB; Instech Laboratories). In turn, the harness was connected to a tether and swivel system (VAH95T+375/22PS or VAHD115T+375/D/22 with BTCOEX-22 tubing; Instech Laboratories), through which the bile was transferred to 50-ml tubes in a cooled (0-10 ∘ C) polystyrene box. When attached to the swivel, rats were still able to move within the entire cage and use nesting material.
In experiment 1, the tube with donor bile was changed twice daily and kept at 4 ∘ C until reinfusion the next day. Because the donor rats in experiment 1 also provided the control bile for experiment 3, and this experiment was conducted later, the bile was kept at −80 ∘ C until reinfusion. Because it involved control bile, there was no concern for loss of toxicity. Bile was infused into the duodenal cannula at room temperature (22-23 ∘ C) using a 20-ml syringe and an infusion pump (IVAC Medical Systems, San Diego, California, USA) at a rate of 0⋅7-1⋅0 ml/h.

Intestinal anastomosis
Anastomoses were created as described previously 17 . Briefly, 1 cm of ileum was resected at 15 cm proximal to the caecum. An inverted anastomosis was made with a single layer of eight interrupted (Lembert) sutures (8/0 Ethilon ® ; Ethicon, Norderstedt, Germany). After operation, rats were housed individually because they were attached to the swivel system.

Assessment of outcome
Animals were killed by asphyxiation with a mixture of 95 per cent carbon dioxide and 5 per cent oxygen, and thereafter 100 per cent carbon dioxide (donor rats), or by cardiac or inferior vena cava puncture combined with cervical dislocation under general anaesthesia with isoflurane (experimental rats). A relaparotomy was done and the anastomosis was inspected macroscopically for anastomotic complications by a researcher blinded to the group randomization.
Bursting pressure and breaking strength were assessed as described previously 17 . Briefly, bursting pressure (mmHg) was determined by infusing anastomotic segments with blue fluid and measuring the maximum pressure sustained before leakage occurred. Subsequently, segments were pulled apart with a tensiometer and the highest force measured before rupture was recorded as breaking strength (newtons).

Analysis of diclofenac excretion profile in bile
To determine whether and when diclofenac products are found in bile, bile samples from four donor rats were obtained fractionally every hour after a single dose of diclofenac 1⋅5 mg/kg orally and analysed by HPLC; for methods used see Appendix S1 (supporting information).

Quantitation of diclofenac metabolites in bile and plasma
Bile samples were obtained as described above and analysed by LC-MS/MS to determine which diclofenac metabolites were most abundant, and when. Plasma samples were collected in experiment 1 to assess whether bile replacement with either diclofenac bile or control bile influenced plasma concentrations through the enterohepatic circulation. Samples of 1 ml were drawn from the tail vein on postoperative day 2, either at 60, 90 or 120 min after diclofenac administration. Any change in plasma level was expected to occur after 60 min, based on biliary excretion time. For a description of methods used, see Appendix S1 (supporting information).

Statistical analysis
Sample size was determined with expected leak rates of 70-80 per cent in groups receiving diclofenac bile and 10-20 per cent in groups receiving control bile or no bile 17    experiment, 18 animals per group were used in experiment 1 and in group IM+B= of experiment 2, and 12 animals in other groups. Absolute leak rates, and not anastomotic complication scores, were used because this facilitates better sample size calculations. The Mann-Whitney U test was used to compare complication scores, and an independent t test to compare strength. Spread of data is presented as the standard deviation. P < 0⋅050 was considered statistically significant. Statistical analysis was performed with IBM SPSS ® Statistics version 22 (IBM, Armonk, New York, USA).

Anastomotic strength
Bursting pressures corresponded with leak rates; values tended to be lower in groups with higher leak rates in experiments 1 and 3 (Fig. 2c). In experiments 1 and 2, breaking strength was the highest in groups that received neither oral or intramuscular diclofenac nor diclofenac bile, with no significant differences (Fig. 2d). A significant difference was observed between group IM+B= and group IM+B0 (mean(s.d.) 0⋅17(0⋅09) versus 0⋅31(0⋅15) N respectively; P = 0⋅006).

Diclofenac excretion profile in bile
HPLC analysis revealed that diclofenac was detected in bile 1 h after oral administration, that levels peaked within 2 h, and that diclofenac remained detectable in bile at least 6 h after administration (Fig. 3a).

Bile production and analysis of diclofenac metabolites in bile
In the donor groups, bile production was comparable at a mean(s.d.) rate of 14⋅9(2⋅4) ml/day in control rats and 14⋅6(2⋅0) ml/day in diclofenac rats (P = 0⋅317). DAG was the most abundant metabolite in bile pooled over 2-8 h after oral administration (Fig. 3b). Diclofenac metabolites were detected almost immediately after intramuscular administration, and levels of DAG, DCF and 4OH-DCF peaked around 2 h after administration (Fig. 3c).

Discussion
This study has demonstrated that increased bile toxicity due to diclofenac administration results in more anastomotic complications in rats. Replacing normal bile with 'diclofenac bile' disturbed anastomotic healing and, in turn, replacement of diclofenac bile with control bile reduced signs of anastomotic leakage following administration of diclofenac. It is suggested that the increase in bile toxicity is caused by biliary excretion of diclofenac metabolites and the subsequent increase of enteral drug and metabolite concentrations.
The composition of bile was shown to be altered rapidly by the presence of diclofenac metabolites after both oral and intramuscular administration. The metabolites, DAG and unconjugated DCF in particular, rapidly enter the gut via biliary excretion, regardless of the route of administration. It is hypothesized that these intraluminal molecules disturb anastomotic healing. In the present study, this was supported by the slightly higher leak rates in oral diclofenac groups compared with those in groups with intramuscular administration.
That altered bile composition and intraluminal drug molecules are detrimental in small bowel anastomosis corresponds with earlier findings 17 , where diclofenac caused leakage of anastomosis in the rat ileum and proximal colon, but not in the distal colon. Bile and drug concentrations are considerably lower in the distal colon and are known to cause most mucosal damage in the terminal ileum 13 . Furthermore, sustained release forms of diclofenac with absorption in more distal intestinal segments appear to divert the mucosal damage from the small to the large intestine 22 . Other studies 10,12,13 of NSAID-induced enteropathy have also attributed the damage to topical effects of drug molecules. Possible underlying mechanisms involve reuptake of active diclofenac molecules, initially causing mitochondrial injury and cell death, and, subsequently, initiation of an excessive inflammatory response by activation of Toll-like receptor 4 on macrophages 11,14,23 . Another possibility is that the diclofenac molecules suppress secretion of mucus, making the anastomosis more vulnerable to leakage, as was recently suggested by Bosmans and colleagues 24 . Although diclofenac is excreted largely as DAG, this metabolite is reactivated by bacterial glucuronidase in the terminal ileum, where it can cause mucosal damage 12,25 . In the present study, the acyl-glucuronide metabolite (DAG) was also abundant in bile. Reactivation of this metabolite in the gut together with biliary excretion of unconjugated forms of diclofenac may lead to high enteral accumulation of potentially harmful drug molecules, which may jeopardize healing from the luminal side of the anastomosis 17,25 .
The finding that healing was still disturbed when diclofenac was administered via the parenteral route and bile was either diverted or replaced by control bile may suggest a systemic role for diclofenac in causing anastomotic leakage. COX-2 inhibition is considered the main feature of systemic diclofenac activity, and could affect essential inflammatory and proliferative pathways 8,26 . Administration of 'diclofenac bile' had little impact on plasma levels of diclofenac through the enterohepatic circulation; thus the effect of diclofenac bile could not be explained by more potent systemic activity.
A valid model was used to study comprehensively the effect of diclofenac and bile composition on the most clinically relevant outcome: anastomotic complications. Comparison of leak severity showed significant differences; comparison of absolute leak rates did not. However, leak rate trends pointed in the same direction. Scoring leakage according to severity and clinical consequences is currently advised in the literature 27 . The lack of significance may have been the result of a slightly smaller effect size than expected from previous studies; the greatest difference was 42 per cent, compared with 50 per cent expected.
The daily amount of bile produced in rats of about 12-16 ml is comparable to the amount in humans of 250-1000 ml per day when adjusted for body surface area. Biliary excretion of diclofenac is poorly studied in humans and depends on genetic polymorphisms in drug metabolism, but is reported to be somewhat lower in humans than in rats 28 . After oral administration (the preferred prescription form of diclofenac) and excretion in bile, total diclofenac concentrations could reach comparable levels in the human gut for anastomotic injury.
Bile toxicity would have most effect on anastomoses that comprise the terminal ileum, where bile is reabsorbed and concentrations are higher, compared with large bowel anastomoses. However, considering the physiological changes that occur following partial or subtotal colectomy, bile toxicity could be comparably relevant for colorectal anastomoses. Clinical cohort studies 2 -4,8 reporting an increased risk of leakage following NSAID administration involved both ileocolic and colorectal anastomoses.
The relevance of the properties of bile and possibly intraluminal drug concentrations in relation to anastomotic healing is a novel perspective on leakage pathophysiology, and underlying mechanisms should be further elucidated. Current evidence provided by retrospective clinical studies and animal models makes it ethically controversial to study NSAIDs in a prospective randomized trial 29 , and experimental studies are suggested to clarify further the role of intraluminal diclofenac metabolite concentrations, the influence of bile salts, and the role of other drug metabolites, such as those of ketorolac and ibuprofen, in leakage pathophysiology.