Gut 1998;43:775–782 775 Enantiomers of flurbiprofen can distinguish key pathophysiological steps of NSAID enteropathy in Gut: first published as 10.1136/gut.43.6.775 on 1 December 1998. Downloaded from the rat T Mahmud, S Somasundaram, G Sigthorsson, R J Simpson, S Rafi, R Foster, I A Tavares, A Roseth, A J Hutt, M Jacob, J Pacy, D L Scott, J M Wrigglesworth, I Bjarnason Abstract Non-steroidal anti-inflammatory drugs Background—Non-steroidal anti-inflam- (NSAIDs) are widely used for symptomatic matory drugs (NSAIDs) cause gastro- relief of pain and inflammation, but there is intestinal damage by a non-prostaglandin concern over their gastrointestinal side eVects (PG) dependent “topical” action and by which aVect both the gastroduodenal and the inhibiting cyclooxygenase. small intestinal mucosa.12It is widely believed Aims—To discriminate between these two that this toxicity is a consequence of inhibition eVects by studying some key pathophysi- of cyclooxygenase 1 (COX-1) while the ological steps in NSAID enteropathy fol- analgesic and anti-inflammatory therapeutic 3 lowing administration of (R)- and (S)- eVects are due to COX-2 inhibition. Selective Department of flurbiprofen, the racemic mixture, and an COX-2 inhibition is therefore thought to be Medicine, King’s uncoupler, dinitrophenol. desirable therapeutically. However most con- College School of Methods—The eVects of dinitrophenol, ventional NSAIDs have equal or greater, Medicine and racemic, (R)-, and (S)-flurbiprofen on depending on the assay system, in vitro inhibi- Dentistry, London, UK tor activity for COX-1 than COX-24–6 and T Mahmud mitochondria were assessed in vitro and S Somasundaram on key pathophysiological features of when given at full therapeutic doses, inhibit G Sigthorsson small intestinal damage in vivo (ul- both enzymes. R J Simpson trastructure by electron microscopy, mu- There is, however, circumstantial evidence S Rafi cosal prostanoid concentrations, that COX-1 inhibition by itself may not be the R Foster intestinal permeability, inflammation, only factor in the development of the gastro- M Jacob intestinal damage.7–9 J Pacy and ulcer count) in rats. Firstly, much larger doses I Bjarnason Results—All the drugs uncoupled mito- of aspirin (aspirin doses of 1024 mg/kg chondrial oxidative phosphorylation in produce small bowel lesions in about 30% of 10 Department vitro, caused mitochondrial damage in fasted animals ) are required to cause consist- Rheumatology, King’s vivo, and increased intestinal permeabil- ent small bowel damage than the dose required http://gut.bmj.com/ College School of for eVective COX inhibition in the rat.11 12 Also, Medicine and ity. Dinitrophenol and (R)-flurbiprofen Dentistry, London, UK caused no significant decreases in mu- despite comparable and eVective inhibition of D L Scott cosal prostanoid concentrations (apart gastric COX activities induced by rectal from a decrease in thromboxane (TX) B administration of a variety of NSAIDs in rats Department of 2 13 concentrations following (R)- some fail to cause gastric damage. Secondly, Surgery, King’s there is no clear correlation between inhibition College School of flurbiprofen) while racemic and (S)- flur- of COX and intestinal damage, unlike the anti- on September 30, 2021 by guest. Protected copyright. Medicine and biprofen reduced mucosal prostanoids 14 15 Dentistry, London, UK inflammatory actions of NSAIDs. Thirdly, significantly (PGE, TXB2, and 6-keto- I A Tavares COX-1 knockout mice have less than 1% of PGF1á concentrations by 73–95%). Intesti- nal inflammation was significantly greater normal mucosal prostaglandin concentrations Department of yet do not spontaneously develop gastro- following administration of (S)- Pharmacy, King’s intestinal lesions.16 College, London, UK flurbiprofen and racemate than with dini- Another aspect of the gastrointestinal dam- J M Wrigglesworth trophenol and (R)-flurbiprofen. No small intestinal ulcers were found following age by NSAIDs relates to the “topical” effect(s) Departments of Life of the drugs. This term is used for the dinitrophenol or (R)-flurbiprofen while Sciences and Electron non-prostaglandin mediated eVect of NSAIDs both racemic and (S)-flurbiprofen caused Microscopy, King’s which occurs when high concentrations of the College, Kensington, numerous ulcers. drugs are in contact with the mucosa, following London, UK Conclusions—Dinitrophenol and (R)- A J Hutt flurbiprofen show similarities in their ingestion or via biliary excretion, or during actions to uncouple mitochondrial oxida- drug absorption. The topical eVect can be Department of documented in man by endoscopy where short Medicine, Aker tive phosphorylation in vitro, alter mito- chondrial morphology in vivo, increase term tolerability to NSAIDs can be enhanced University Hospital, by changing the drug formulation or route of Oslo, Norway intestinal permeability, and cause mild administration (enteric coating, NSAID- A Roseth inflammation without ulcers. Concurrent phospholipid formulation, rectal administra- severe decreases in mucosal prostanoids Correspondence to: tion, etc.),91718 abolishing gastric acid seem to be the driving force for the devel- Dr I Bjarnason, Department secretion,19 and by rendering some NSAIDs of Medicine, King’s College opment of severe inflammation and ul- non-acidic by forming an ester linkage to a School of Medicine and cers. Dentistry, Bessemer Road, moiety that contains20–22 or does not contain23 (Gut 1998;43:775–782) London SE5 9PJ, UK. nitric oxide. Accepted for publication Keywords: non-steroidal anti-inflammatory drug; The “ion trapping hypothesis” provides a 4 June 1998 enteropathy; flurbiprofen basis for the “topical” action of NSAIDs but 776 Mahmud, Somasundaram, Sigthorsson, et al not the mechanism.24 25 One suggestion is that and in vivo (n=4); (b) mucosal prostaglandin uncoupling of oxidative phosphorylation may (PG) and thromboxane (TX) concentrations be the biochemical mechanism underlying the (n=8); (c) intestinal permeability (n=8); (d) Gut: first published as 10.1136/gut.43.6.775 on 1 December 1998. Downloaded from “topical” toxicity of NSAIDs.26–28 Most conven- inflammation (n=8); and (e) 25 hour and seven tional NSAIDs and acidic pro-NSAIDs day ulcer count (n=8). (fenbufen and sulindac) uncouple mitochon- For the in vivo experiments animals received drial oxidative phosphorylation.29 30 Further- single doses of 0.5 ml of 3.0 mM DNP (higher more, there are some indications that doses were associated with appreciable mor- non-acidic NSAIDs (such as nitric oxide tality), racemic, (R)-, or (S)-flurbiprofen by containing NSAIDs and nabumetone), which gavage (10 mg/kg each: 1 ml of 12 mM do not uncouple mitochondrial oxidative solution, with a final concentration of dimethyl- phosphorylation,29 are associated with en- sulphoxide (DMSO) less than 5%, to a 250 mg hanced gastrointestinal tolerability.12 20–22 31 rat). The enantiomers were obtained from In this context it has been suggested that Boots Pharmaceutical Company (Nottingham, NSAID damage is a sequential multistage UK) and DNP and racemic flurbiprofen from pathogenic process32 which can be summarised Sigma Chemical Company (Dorset, UK). The as: same dose of racemic, (R)-, and (S)- Biochemical eVects—These include uncoupling flurbiprofen was given (mg/kg) as we sought to of oxidative phosphorylation26–29 (topical ac- achieve a similar topical eVect with all drug tion) and COX inhibition (local and systemic forms. action leading to a decrease in mucosal prosta- The (R)- and (S)-flurbiprofen were 99.7% glandin production). and 99.6% enantiomerically pure, respectively, Transitional stage—Characterised by increased and the racemate was a 1:1 mixture of each. intestinal permeability which could be the con- Animals given the vehicle (1 ml of a 5% sequence of either uncoupling of oxidative solution of DMSO) acted as controls. phosphorylation or decreased mucosal pros- taglandins. MITOCHONDRIAL EXPERIMENTS Tissue reaction—Inflammation (including neu- In vitro trophil chemotaxis, chemokinesis, intercellular Preparation of mitochondria—Coupled mito- adhesion molecule upregulation, neutrophil chondria were obtained as previously adhesiveness and margination, etc.) and ulcers described.39 The validity of using rat liver mito- that are proposed to be the consequences of chondria as surrogate for monitoring chemios- increased intestinal permeability32 33 and the motic uncoupling in the intestine is consistent eVects of decreased prostaglandins on the with the accepted universality of the chemios- microvasculature.34 35 motic coupling mechanism between electron The relative contribution and eVect of transfer and oxidative phosphorylation.40 uncoupling and COX inhibition in the patho- The animals were killed by cervical http://gut.bmj.com/ genesis of stages (2) and (3) is however dislocation and the liver rapidly dissected and unknown. placed in ice cold homogenising solution Commercially available flurbiprofen is mar- (75 mM sucrose, 225 mM mannitol, keted as a racemate, an equal parts mixture of 10 mM 4-morpholine-propanesulphonic acid the (R) and (S) enantiomers. The (S) enanti- (MOPS), 1 mM EDTA, and 5 mg/ml bovine omer accounts for most of the COX inhibition serum albumin (BSA) at pH 7.4), cut finely while the (R) enantiomer is relatively inactive into approximately 1 cm3 pieces with scissors, in this respect in vitro.36 The (R) enantiomer and washed twice with homogenising solution on September 30, 2021 by guest. Protected copyright. might therefore be used to study selectively the to remove excess