ABSTRACT
(E)-4-(ethoxymethylene)-2-phenyloxazol-5(4H)-one, commonly referred to as oxazolone, is the most potent skin sensitizer in published databases as determined with the murine local lymph node assay. It has been used very widely in immunological research to induce and elicit skin sensitization reactions in experimental animals. Nevertheless, no detailed study on the reactivity of oxazolone with proteins or peptides has been published, which would rationalize its unique sensitization potential from a chemical point of view. Peptide reactivity assays have been proposed as alternatives to animal tests to study the skin sensitization potential of test chemicals. Besides their application to reduce animal experimentation, peptide reactivity assays also allow one to gain mechanistic insights into the reactivity of test chemicals with proteins. In this case study, we applied different peptide reactivity assays to investigate and mechanistically rationalize the reactivity of oxazolone. Its sensitization potential could be linked to the following findings: (i) oxazolone reacts rapidly with the amine group in lysine with an addition-elimination reaction at the ethoxymethylene group to form stable products within minutes at physiological pH; (ii) sequentially different products with cysteine-peptides are formed, the most stable being an S-hippuryl-modification; and (iii) the S-hippuryl-modification can be shuttled to other nucleophilic sites; thus, also Lys residues can subsequently be modified with a hippuryl-moiety. This very rapid and diverse reactivity especially with lysine residues may explain why oxazolone forms sufficient stable novel epitopes on proteins to induce skin sensitization even at very low concentration.
Subject(s)
Oxazolone/analogs & derivatives , Oxazolone/chemistry , Oxazolone/pharmacology , Peptides/chemistry , Skin/drug effects , Animals , Cysteine/chemistry , Gas Chromatography-Mass Spectrometry , Hydrogen-Ion Concentration , Irritants/chemical synthesis , Irritants/chemistry , Irritants/pharmacology , Kinetics , Lysine/analogs & derivatives , Lysine/chemistry , Mice , Oxazolone/chemical synthesis , Sulfhydryl Compounds/chemistryABSTRACT
Packaging small drug molecules, such as non-steroidal anti-inflammatory drugs (NSAIDs) into nanoparticulate systems has been reported as a promising approach to improve the drug's bioavailability, biocompatibility and safety profiles. In the last 20 years, lipid nanoparticles (lipid dispersions) entered the nanoparticulate library as novel carrier systems due to their great potential as an alternative to other systems such as polymeric nanoparticles and liposomes for several administration routes. For ocular instillation nanoparticulate carriers are required to have a low mean particle size, with the lowest polydispersity as possible. The purpose of this work was to study the combined influence of 2-level, 4-factor variables on the formulation of flurbiprofen (FB), a lipophilic NSAID, in lipid carriers currently named as nanostructured lipid carriers (NLC). NLC were produced with stearic acid (SA) and castor oil (CO) stabilized by Tween® 80 (non-ionic surfactant) in aqueous dispersion. A 2(4) full factorial design based on 4 independent variables was used to plan the experiments, namely, the percentage of SA with regard to the total lipid, the FB concentration, the stabilizer concentration, and the storage conditions (i.e., storage temperature). The effects of these parameters on the mean particle size, polydispersity index (PI) and zeta potential (ZP) were investigated as dependent variables. The optimization process was achieved and the best formulation corresponded to the NLC formulation composed of 0.05 (wt%) FB, 1.6 (wt%) Tween® 80 and a 50:50 ratio of SA to CO, with an average diameter of 288 nm, PI 0.245 of and ZP of -29 mV. This factorial design study has proven to be a useful tool in optimizing FB-loaded NLC formulations. Stability of the optimized NLC was predicted using a TurbiScanLab® and the ocular tolerance was assessed in vitro and in vivo by the Eytex® and Draize test, respectively. The developed systems were shown physico-chemically stable with high tolerance for eye instillation.
Subject(s)
Drug Carriers/chemical synthesis , Drug Carriers/toxicity , Eye/drug effects , Flurbiprofen/administration & dosage , Flurbiprofen/toxicity , Nanoparticles/chemistry , Ultrasonics , Animal Testing Alternatives/methods , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Castor Oil/chemistry , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Design , Drug Stability , Drug Tolerance , Flurbiprofen/chemistry , Irritants/administration & dosage , Irritants/chemical synthesis , Irritants/chemistry , Irritants/toxicity , Nanoparticles/administration & dosage , Nanoparticles/toxicity , Particle Size , Polysorbates/chemistry , Predictive Value of Tests , Rabbits , Skin Irritancy Tests/methods , Stearic Acids/chemistry , Surface Properties , Tissue EngineeringABSTRACT
[reaction: see text] The complete carbocyclic skeleton of ingenol is assembled via a route that employs ring-closing metathesis (RCM) to close the strained "inside-outside" BC ring system (i.e., 24 --> 25).
