The Effect of a Hydrogen Sulphide (H2S)-Releasing Non-Steroidal Anti-Inflammatory Drug (NSAID) on Gastric and Renal Damage and Platelet Aggregation

Melissa Chan, Rory Blackler, Webb McKnight, John Wallace. McMaster University, Hamilton, Ontario, Canada

Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the cyclooxygenase-1 (COX-1) and COX-2 enzymes, which catalyse the formation of prostaglandins and thromboxane A2 (TXA2) from arachidonic acid (AA). NSAIDs act to prevent platelet aggregation by inhibiting TxA2 generation and, therefore, clot formation in vessels. NSAIDs, however, have also been shown to induce significant gastrointestinal (GI) ulceration, bleeding and adverse renal effects. Hydrogen sulphide (H2S) has been shown to have protective and ulcer-healing effects on the GI tract. As such, we investigated the GI, platelet and renal effects of naproxen and an H2S-releasing NSAID, 2-(6-methoxy-napthalen-2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester (ATB-346).

Methods

Male Wistar rats (180-220 g, n = 4-5) were treated twice daily by oral gavage for 4.5 days with vehicle (dimethylsulfoxide/1% carboxymethylcellulose; 5:95), naproxen acid (20 mg/kg) or ATB-346 (31.7 mg/kg). Rats were euthanised by overdose of sodium pentobarbital (65 mg/kg, i.p.) 3 hours after the final dose. Stomach and small intestine were removed for blind scoring of haemorrhagic damage by summing the lengths and areas of lesions (mm2). Blood from all rats was taken into 3.2% tri-sodium citrate (1:9 dilution) and centrifuged to obtain platelet rich plasma (PRP). Using a modified optical multichannel (optimul) platelet aggregometry method (Chan et al., 2012), we assessed the effect of these treatments on platelet aggregation induced by adenosine diphosphate (ADP, 0.1-30 μM) and AA (0.03-1 mM). In other experiments, blood was taken after NSAID dosing, allowed to clot and serum obtained for blood urea nitrogen (BUN) analysis and urine was obtained by cystocentesis for protein and creatinine analysis; all markers of renal dysfunction. Comparisons were made by one-way or two-way ANOVA and data is expressed as mean±S.E.M.

Results

No damage was seen in gastric tissue following ATB-346 or naproxen dosing (p>0.05). In the small intestine, however, naproxen (72.2±10.7, p<0.05) caused significantly more damage than vehicle treatment, whilst ATB-346 (0±0, p>0.05) caused no significant damage. Neither naproxen nor ATB-346 had any effect (p>0.05) on ADP-induced platelet aggregation. In contrast, both naproxen (p<0.01) and ATB-346 (p<0.01) significantly attenuated AA-induced aggregation when compared to vehicle. No difference was seen in BUN, as measured in serum, after naproxen (16.4±0.7 mg/dl, p>0.05) or ATB-346 (18.5±0.3 mg/dl, p>0.05) when compared to vehicle (18.25 mg/dl). Similarly, there was no difference in urine protein/creatinine ratio after dosing with either naproxen (1.5±0.3, p>0.05) or ATB-346 (1.6±0.6, p>0.05) when compared to vehicle (1.4±0.3).

Conclusions

Haemorrhagic damage caused by naproxen dosing in the small intestine was not present when rats were treated with ATB-346, indicating a protective effect of the hydrogen sulphide-releasing NSAID. ATB-346 retained the ability to inhibit platelet COX, as shown from the platelet studies. Our studies were conducted in normally functioning rats, which showed no naproxen-induced renal dysfunction. We therefore propose performing these experiments in animals with co-morbidities, such as hypertension, in order to reveal the effect of ATB-346 in the kidney.

Reference:

Chan MV, Warner TD (2012). Standardised optical multichannel (optimul) platelet aggregometry using high-speed shaking and fixed time point readings. Platelets 23(5): 404-408.