Energy-Dependent Endocytosis is Involved in the Absorption of Indomethacin Nanoparticles in the Small Intestine

We previously reported that oral formulations containing indomethacin nanoparticles (IND-NPs) showed high bioavailability, and, consequently, improved therapeutic effects and reduced injury to the small intestine. However, the pathway for the transintestinal penetration of nanoparticles remained unclear. Thus, in this study, we investigated whether endocytosis was related to the penetration of IND-NPs (72.1 nm) using a transcell set with Caco-2 cells or rat intestine. Four inhibitors of various endocytosis pathways were used [nystatin, caveolae-dependent endocytosis (CavME); dynasore, clathrin-dependent endocytosis (CME); rottlerin, macropinocytosis; and cytochalasin D, phagocytosis inhibitor], and all energy-dependent endocytosis was inhibited at temperatures under 4 °C in this study. Although IND-NPs showed high transintestinal penetration, no particles were detected in the basolateral side. IND-NPs penetration was strongly prevented at temperatures under 4 °C. In experiments using pharmacological inhibitors, only CME inhibited penetration in the jejunum, while in the ileum, both CavME and CME significantly attenuated penetration. In conclusion, we found a novel pathway for the transintestinal penetration of drug nanoparticles. Our hypothesis was that nanoparticles would be taken up into the intestinal epithelium by endocytosis (CME in jejunum, CavME and CME in ileum), and dissolved and diffused in the intestine. Our findings are likely to be of significant use for the development of nanomedicines

12-OH-Nevirapine Sulfate, Formed in the Skin, Is Responsible for Nevirapine-Induced Skin Rash

Nevirapine (NVP) treatment is associated with a significant incidence of skin rash in humans, and it also causes a similar immune-mediated skin rash in Brown Norway (BN) rats. We have shown that the sulfate of a major oxidative metabolite, 12-OH-NVP, covalently binds in the skin. The fact that the sulfate metabolite is responsible for covalent binding in the skin does not prove that it is responsible for the rash. We used various inhibitors of sulfation to test whether this reactive sulfate is responsible for the skin rash. Salicylamide (SA), which depletes 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in the liver, significantly decreased 12-OH-NVP sulfate in the blood, but it did not prevent covalent binding in the skin or the rash. Topical application of 1-phenyl-1-hexanol, a sulfotransferase inhibitor, prevented covalent binding in the skin as well as the rash, but only where it was applied. <i>In vitro</i> incubations of 12-OH-NVP with PAPS and cytosolic fractions from the skin of rats or from human skin also led to covalent binding that was inhibited by 1-phenyl-1-hexanol. Incubation of 12-OH-NVP with PAPS and sulfotransferase 1A1*1, a human isoform that is present in the skin, also led to covalent binding, and this binding was also inhibited by 1-phenyl-1-hexanol. We conclude that salicylamide did not deplete PAPS in the skin and was unable to prevent covalent binding or the rash, while topical 1-phenyl-1-hexanol inhibited sulfation of 12-OH-NVP in the skin and did prevent covalent binding and the rash. These results provide definitive evidence that 12-OH-NVP sulfate formed in skin is responsible for NVP-induced skin rashes. Sulfotransferase is one of the few metabolic enzymes with significant activity in the skin, and it may be responsible for the bioactivation of other drugs that cause skin rashes