Effect of a selective nonsteroidal anti-inflammatory inhibitor of cyclooxygenase-2 on the small bowel of rats

The pathogenesis of nonsteroidal anti-inflammatory drug (NSAID) enteropathy is a complex process involving the uncoupling of mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase (COX). Rofecoxib, a selective inhibitor of COX-2, has shown less gastric damage, but the same beneficial effect is not clear in the case of the small bowel. Fifty-seven male Wistar rats (250-350 g) were divided into three groups (N = 19 each) to evaluate the effect of this NSAID on the rat intestine. The groups received 2.5 mg/kg rofecoxib, 7.5 mg/kg indomethacin or water with 5% DMSO (control) given as a single dose by gavage 24 h before the beginning of the experiment. A macroscopic score was used to quantify intestinal lesions and intestinal permeability was measured using [51Cr]-ethylenediaminetetraacetic acid ([51Cr]-EDTA). The extent of intestinal lesion, indicated by a macroscopic score, was significantly lower when rofecoxib was administered compared to indomethacin (rofecoxib = 0.0 vs indomethacin = 63.6 ± 25.9; P < 0.05) and did not differ from control. The intestinal permeability to [51Cr]-EDTA was significantly increased after indomethacin (control = 1.82 ± 0.4 vs indomethacin = 9.12 ± 0.8%; P < 0.0001), but not after rofecoxib, whose effect did not differ significantly from control (control = 1.82 ± 0.4 vs rofecoxib = 2.17 ± 0.4%; ns), but was significantly different from indomethacin (indomethacin = 9.12 ± 0.8 vs rofecoxib = 2.17 ± 0.4%; P < 0.001). In conclusion, the present data show that rofecoxib is safer than indomethacin in rats because it does not induce macroscopic intestinal damage or increased intestinal permeability. Correspondence

• Intestinal permeability Nonsteroidal anti-inflammatory drugs (NSAID) are widely used due to their analgesic, anti-inflammatory and antipyretic properties.The main concern about these drugs is the frequency and severity of their digestive side effects involving the entire gastrointestinal tract.Fecal calprotectin is altered in 44% (1) of patients taking NSAID, and scintigrams suggest small intestine inflammation in 42%, but when fecal excretion of [ 111 In]-labelled leukocytes was used as a measure of inflammation up to 67% (2) of patients were found to have enteropathy.Similarly, intestinal lesions were detected by enteroscopy (3) in 66% of patients and in a post-mortem study, the prevalence of nonspecific intestinal ulceration was 13.5% (4).The intestinal lesions caused by NSAID can mimic inflammatory bowel disease (5) and may lead to chronic bleeding, protein loss, strictures, diverticulitis, appendicitis, internal fistulas, and relapse of inflammatory bowel disease.
The pathogenesis of NSAID enteropathy is complex and not completely understood.There are two main pathogenic mechanisms.The first involves specific biochemical damage to mitochondria (6)(7)(8), with uncoupling of the oxidative phosphorylation reaction, which increases intestinal permeability and calcium release into cytosol, which in turn cause secondary biochemical damage (9,10).The second, and more controversial, mechanism involves the inhibition of cyclooxygenase-1 and/or -2 (COX-1, COX-2) and its products (prostaglandins).However, some studies have shown that COX inhibition is not involved (11,12), while another study claimed that this inhibition is the pivotal event (13).
Early evidence obtained with selective COX-2 inhibitors such as nimesulide (14), celecoxib (15) and rofecoxib (16) suggests that they are indeed associated with significantly improved gastrointestinal tolerability.However, it is not clear whether these agents are less toxic to the small bowel.
Thus, the aim of the present study was to assess the effect of rofecoxib, a selective COX-2 inhibitor, on the small intestine by assessing intestinal permeability and macroscopic damage.
Fifty-seven male Wistar rats (250-350 g) were individually housed in metabolic cages with free access to water and standard laboratory rat chow.The animals were divided into three groups (N = 19 in each group).The groups received water with 5% dimethylsulfoxide (DMSO, control), 7.5 mg/kg indo-methacin, or 2.5 mg/kg rofecoxib, given as a single dose by gavage 24 h before the beginning of the experiment.The drug was initially dissolved in DMSO and then diluted in water so that the final concentration of DMSO was less than 5% and adjusted to pH 7.4.After drug administration, the rats had free access to standard rat food and water.
Several methods can be used to assess intestinal permeability.The dual sugar method uses a disaccharide and a monosaccharide, which permit the assessment of the paracellular and transcellular pathway without a radioactive probe.The disadvantage of this method is the necessity of more sophisticated equipment, such as a mass spectrophotometer or high performance liquid chromatograph.
In the present study, the [ 51 Cr]-ethylenediaminetetraacetic acid ([ 51 Cr]-EDTA) technique was used to measure the changes of intestinal permeability induced by NSAID (17,18) that basically alter the paracellular pathway.Intestinal permeability was assessed on the basis of urinary [ 51 Cr]-EDTA excretion (10,19) after its oral administration.The rats were given 5 µCi [ 51 Cr]-EDTA in 0.5 ml distilled water by gavage, followed by 5 ml water.The animals were then placed in individual metabolic cages with free access to tap water and food for 5 h for collection of urine.The rats were sacrificed by lethal injection (50 mg) of ketamine (Ketalar ® ), laparotomy was performed and the bladder was emptied by puncture.The total 5-h radioactivity excreted into the urine was determined.Data are reported as fractional excretion of the radioactive marker.
The small intestine was gently removed, the intestinal mucosa was exposed by cutting through the contra-mesenteric side, laid out on a piece of cork, and dried with Evan's blue to improve assessment of the macroscopic score 29 h after drug administration, using a stereotactic microscope at 25-50 magnification.The use of a score system is crucial since with the progression of intestinal damage the small ulcers coalesce and form large longitudinal ulcerations, so that the simple count of the number of ulcers does not reflect the actual intestinal damage.In order to correct this bias, a macroscopic score was used to quantify intestinal lesions as previously described (19).The score was based on the number of mucosal ulcers and the largest diameter of the ulcers as follows: a) <1 mm; b) ≥1 and <3 mm; c) ≥3 and <5 mm; d) ≥5 and <10 mm; e) ≥10 mm, multiplied by a factor of 1, 3, 5, 10 and 20, respectively.The total score was calculated as the sum of the values obtained for each item.Results are reported as means ± SEM.Statistical differences in macroscopic score and intestinal permeability among groups were assessed by one-way analysis of variance and Bonferroni's multiple comparison test.
As shown in Figure 1, the macroscopic scores were significantly different among the three groups, i.e., controls, indomethacin and rofecoxib (P < 0.0001).Rofecoxib did not induce intestinal damage.Using a discriminatory test it was possible to show that the macroscopic score was significantly lower when rofecoxib was administered compared to indomethacin (rofecoxib = 0.0 vs indomethacin = 63.6 ± 25.9, P < 0.05) and did not differ from the control group (rofecoxib = 0 vs control = 0, ns).
When an NSAID is prescribed the intestinal damage induced by these drugs is rarely considered, although the intestine is involved in a large number of complications related to this class of drugs.The present study points out the important side effects of NSAID, and demonstrates that it is possible to reduce the risk of intestinal damage by choosing a less toxic NSAID, although this involves a higher cost of treatment.
In agreement with the literature, we showed that indomethacin, a non-selective COX inhibitor, can induce significant damage in the small bowel, as demonstrated by an increase in both the intestinal permeability to [ 51 Cr]-EDTA and the macroscopic score.Nevertheless, we also demonstrated that an equivalent dose of a selective COX-2 inhibitor, rofecoxib, did not disrupt the intestinal barrier as determined on the basis of the same parameters, revealing important differences in the intestinal tolerability of these drugs, which is probably related to two fundamental properties of NSAID: the pK and the inhibition of COX.Rofecoxib is a non-acid NSAID, which does not uncouple the oxidative phosphorylation either in vitro or in vivo (8,15) and does not reduce prostaglandin levels in the gut, since COX-2 is not constitutively expressed in the normal gut (20), a fact that makes it less toxic to the gastrointestinal tract in general.
We showed here that rofecoxib is safer than indomethacin for the small bowel because it does not induce macroscopic intestinal damage or disrupt intestinal permeability, and therefore should be preferred to indomethacin and other common NSAID.

Figure 2 .Figure 1 .
Figure 2. Scatter diagram showing individual mean percent [ 51 Cr]-EDTA excreted into urine for control rats (N = 9) and rats treated by gavage with a single dose of 7.5 mg/kg indomethacin (N = 9) and 2.5 mg/kg rofecoxib (N = 9).The horizontal lines indicate the mean.P < 0.05 for control vs indomethacin and rofecoxib vs indomethacin.Statistical differences in intestinal permeability among groups were assessed by one-way analysis of variance and Bonferroni's multiple comparison test.