Effect of Ketoprofen and ATB-352 on the Immature Human Intestine: Identification of Responders and Non-responders

Abstract

Background and objective: The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a broad spectrum of life-threatening adverse effects on the immature gastrointestinal tract. NSAID derivatives exploiting the beneficial effects of biologically active gases, such as hydrogen sulfide (H2S), have been developed. Herein, we determined the effects of ketoprofen and ATB-352, a H2S-releasing ketoprofen derivative, on selected metabolic pathways previously identified to be significantly altered by indomethacin in the human immature intestine.

Methods: Ketoprofen and ATB-352 were tested on human mid-gestation small intestinal explants maintained in a serum-free organ culture system for 48 hours. The expression levels of the representative genes involved in selected metabolic pathways were measured by real-time PCR after a treatment of 48 hours.

Results: Tested at a concentration that allows more than 80% inhibition of PGE2 production, ketoprofen was found to be less damaging than indomethacin at an equivalent dosage. However, based on the inducibility of cyclooxygenase-2 transcript expression, we were able to discriminate between responder individuals in which the deleterious effects observed with indomethacin were attenuated, and non-responder specimens in which the effects were similar to those observed with indomethacin. ATB-352 did not induce significant changes compared to ketoprofen on these metabolic pathways.

Conclusions: These results show less damaging effects of ketoprofen compared to indomethacin on the immature intestine and indicate that the intestinal response to this NSAID significantly varies between individuals. However, the results did not allow us to demonstrate a specific beneficial effect of H2S release in organ culture.

FIGURE 1

Effects of ketoprofen on the immature intestinal mucosa. A, Inhibition of PGE2 production was evaluated in the presence of 2 concentrations of ketoprofen (mean ± SEM, ^∗∗∗P < 0.005). B, qPCR analysis of transcript levels for representative markers of mucosal homeostasis categories after 48 hours of culture in the presence of 10 μM ketoprofen. C, For comparison, markers expressed relative to the same housekeeping genes in response to 1 μM indomethacin under similar conditions. qPCR data are expressed as ratios of treated over untreated segments (Log2 scale). Values from 6 independent biological samples (mean ± SEM). ^∗P < 0.05 (^#P < 0.06) versus corresponding untreated control segments.

FIGURE 2

Characterization of intestinal ketoprofen responders and non-responders. A, qPCR analysis of PTGS2 transcript levels in the 6 independent explant cultures. B, Based on data from (A) qPCR data for the markers of mucosal homeostasis are shown for the 3 responders and the 3 non-responders to ketoprofen. qPCR data are expressed as ratios of treated over untreated segments for each sample for (A) and for the mean of 3 independent biological samples for each responder group (Log2 scale, ^∗P < 0.05, ^#P < 0.06) versus corresponding untreated control segments (mean ± SEM).

FIGURE 3

Analysis of expression of representative H₂S-targeted genes in ATB-352 or ketoprofen treated intestine. qPCR analysis of TNFα and ICAM1 transcript levels in control (Ctrl), ATB-352 (ATB) or ketoprofen (Keto) treated responder and non-responder explants. Data are expressed as ratios of treated over control explants as the mean of 3 independent biological samples for each group (mean ± SEM). ^∗Denotes a difference (P < 0.05) versus corresponding untreated control explants. NS = non-significant.