A Molecular Biophysical Approach to Diclofenac Topical Gastrointestinal Damage

Abstract

Diclofenac (DCF), the most widely consumed non-steroidal anti-inflammatory drug (NSAID) worldwide, is associated with adverse typical effects, including gastrointestinal (GI) complications. The present study aims to better understand the topical toxicity induced by DCF using membrane models that mimic the physiological, biophysical, and chemical environments of GI mucosa segments. For this purpose, phospholipidic model systems that mimic the GI protective lining and lipid models of the inner mitochondrial membrane were used together with a wide set of techniques: derivative spectrophotometry to evaluate drug distribution at the membrane; steady-state and time-resolved fluorescence to predict drug location at the membrane; fluorescence anisotropy, differential scanning calorimetry (DSC), dynamic light scattering (DLS), and calcein leakage studies to evaluate the drug-induced disturbance on membrane microviscosity and permeability; and small- and wide-angle X-ray scattering studies (SAXS and WAXS, respectively), to evaluate the effects of DCF at the membrane structure. Results demonstrated that DCF interacts chemically with the phospholipids of the GI protective barrier in a pH-dependent manner and confirmed the DCF location at the lipid headgroup region, as well as DCF’s higher distribution at mitochondrial membrane contact points where the impairment of biophysical properties is consistent with the uncoupling effects reported for this drug.

Neutral Diclofenac Causes Remarkable Changes in Phosphatidylcholine Bilayers: Relevance for Gastric Toxicity Mechanisms

“…Experimental data from this study suggested that neutral diclofenac has a deeper preferential location in the DMPC bilayer, putatively establishing H-bonds with the DMPC's phosphate group and disrupting the lipid-lipid interactions in the inner region of the acyl chains. On the other hand, anionic diclofenac seems to have a shallower location, probably because of electrostatic adsorption in the DMPC's choline group, as previously reported (Ferreira et al, 2005;Manrique-Moreno et al, 2009;Fernandes et al, 2018). This hypothesis is in agreement with molecular dynamics simulations that indicate that neutral NSAIDs are located closer to the bilayer center than anionic NSAIDs (Boggara and Krishnamoorti, 2010;Markiewicz and Pasenkiewicz-Gierula, 2011).…”

“…It is noteworthy that the neutral diclofenac-induced effects on the phosphatidylcholine bilayers were often more pronounced and/or distinct than that caused by anionic diclofenac. In fact, the diclofenac-induced decrease of the temperature and cooperativity of the main phase transition of DMPC bilayers at pH 7.4 and 5.0 was previously reported elsewhere (Manrique-Moreno et al, 2009;Fernandes et al, 2018). However, more pronounced alterations were observed with neutral diclofenac in this study, considering equivalent membrane concentrations.…”

“…Beyond the systemic actions of NSAIDs concerning the inhibition of protective PG biosynthesis, NSAID-induced topical actions also seem to be crucial to the occurrence of gastric adverse events. Anionic diclofenac may be harmful by ion trapping in the epithelial cells, as well as by modifying the structural properties of cell and organelle membranes, as previously suggested (Ferreira et al, 2005;Fernandes et al, 2018). By modifying organelle membranes, anionic diclofenac may indirectly inhibit COX isoenzymes (monotopic membrane proteins), causing an additional reduction of protective PGs, which may amplify the gastric toxicity.…”

“…This study is of utmost importance for three main reasons: 1) the gastric mucosal barrier presents a pH gradient from pH ∼2 in the gastric lumen to pH ∼7 in the mucosal epithelium (Bahari et al, 1982); 2) diclofenac is an acidic drug [pK a 5 3.97 (Ferreira et al, 2005)]; thus, both forms will circulate in the gastric mucosa; and 3) although NSAIDs are recommended to be administrated after meals, when the luminal gastric pH rises to 2.5-5 depending on the type of meal (Simonian et al, 2005), both ionized and neutral states of diclofenac may interact with the phospholipid layers, as the pK a of NSAIDs has been reported to increase by ∼1 value in phosphatidylcholine bilayers (de Castro et al, 2001;Pereira-Leite et al, 2018c). In this sense, it is crucial to study the diclofenac interactions with phospholipids at a wider range of pH values than that reported in the literature (Ferreira et al, 2005;Manrique-Moreno et al, 2009;Suwalsky et al, 2009;Fernandes et al, 2018) to completely decipher its topical mechanism of toxicity in the gastric mucosa.…”

Rational Development of Liposomal Hydrogels: A Strategy for Topical Vaginal Antiretroviral Drug Delivery in the Context of HIV Prevention

“…Static exposures were taken below and above the main transition temperature as controlled by a thermostated water bath (stability ± 0.1 °C; Unistat CC, Huber, Offenburg, Germany) in order to obtain the diffraction patterns typical of DPPC lipid phases (L β′ and L α ) and evaluate the effect of drugs in such phases. Data analysis was made as described elsewhere [30].…”