Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.

The use of NSAIDs or COX-2 inhibitors for chemoprevention of colorectal cancer has been suggested for patients at high risk for this disease. However, the gastrointestinal side effects of traditional NSAIDs which consist of bleeding and ulceration, and the cardiovascular effects of COX-2 inhibitors may limit their usefulness. In preclinical studies, our laboratory has shown that the addition of phosphatidylcholine (PC) to the NSAIDs aspirin (ASA) or ibuprofen (IBU) results in a NSAID-PC with fewer GI side effects and also maintained or enhanced analgesic, anti-pyretic and anti-inflammatory efficacy over the unmodified NSAID. Because NSAID-PCs have not been tested for anti-cancer activity, in the present study, ASA-PC and IBU-PC were tested on the SW-480 human colon cancer cell line. SW-480 cells were incubated in media containing 1-5 mM NSAID or NSAID-PC for 2 days. Measurements were made of cell number, cell proliferation (DNA synthesis), and manner of cell death (necrosis and apoptosis). ASA and IBU reduced cell number in a dose-dependent manner with IBU showing a greater potency than ASA. The association of PC to the NSAID resulted in greater reductions of cell number for both NSAIDs. Furthermore, the NSAID-PC formulation had significantly greater efficacy and potency to inhibit cellular DNA synthesis than the unmodified NSAID. PC alone at the doses and times used had no effect on cell number in this cell line, but did have a small effect to reduce DNA synthesis. None of the drugs had a clear effect on cell death by necrosis. Only IBU and IBU-PC caused cell death by apoptosis in SW-480 cells. We conclude that NSAID-PCs have activity to impede the growth of colon cancer cells in vitro, which is due, in major part, to a marked reduction in DNA synthetic activity of these cells. This growth inhibitory effect appears to be independent of COX-2 activity, since it is known that SW-480 cells do not have this inducible COX isoform. Due to its greater efficacy in this model system, IBU-PC should be further evaluated as a chemopreventive agent that is safer for the GI tract than unmodified NSAID.

Cyclooxygenase (COX) catalysis by prostaglandin H synthase (PGHS) is a key control step for regulation of prostanoid biosynthesis. Both PGHS isoforms are integral membrane proteins and their substrate fatty acids readily partition into membranes, but the impact of phospholipids and lipid membranes on COX catalysis and the actions of COX inhibitors are not well understood. We have characterized the COX kinetics and ibuprofen inhibition of the purified PGHS isoforms in the presence of phosphatidylcholine (PC) with varying acyl chain structure and physical state. PC was found to directly inhibit COX activity, with non-competitive inhibition by PC monomers binding away from the COX active site and competitive inhibition by micellar/bilayer forms of PC due to sequestration of the arachidonate substrate. Competitive inhibition by native membranes was observed in a comparison of COX kinetics in sheep seminal vesicle microsomes before and after solubilization of PGHS-1. PC liposomes significantly increase the inhibitory potency of ibuprofen against both PGHS isoforms without changing the reversible character of ibuprofen action or requiring binding of PGHS to the liposomes. These results suggest a useful conceptual framework for analyzing the complex interactions among the PGHS proteins, substrates, inhibitors and phospholipid.