Behavior of tetrahydrolipstatin in biological model membranes and emulsions.

Tetrahydrolipstatin (orlistat) (S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]methyl]dodecyl N-formyl-L-leucinate, a potent inhibitor of pancreatic lipase, is hydrophobic, amphipathic, and water-insoluble. It binds irreversibly to pancreatic lipases and inhibits fat absorption. The focus of this investigation is on the distribution of orlistat in emulsified fat and vesicular membranes such as might be present in the intestine during fat absorption. The models used were unilamellar vesicles and microemulsion particles. [13C]orlistat was synthesized containing 99% 13C in the leucine carbonyl. Spectrawere collected on a Bruker DMX 500 Spectrometer. The chemical shift of the [13C]leucinate carbon was recorded in solvents with increasing hydrogen bonding capacity. The chemical shift moved downfield as H-bonding increased. [13C]orlistat was incorporated into triolein in the presence or absence of water, into sonocated unilamellar egg yolk phosphotidylcholine (EYPC) vesicles, and into microemulsions approximately 300 A in diameter containing triolein and phospholipid in roughly equal molar proportions. [13C] orlistat was soluble in triolein and had a chemical shift at 20 degrees C of 171.46 ppm. When a small amount of water was added, the chemical shift moved down field to 171.69 ppm. When [13C]orlistat was incorporated into EYPC unilamellar vesicles, the chemical shift increased to approximately 172.0 ppm at 25 degrees C, indicating an orientation of [13C]leucinate in orlistat closer to the aqueous interface of vesicles, i.e., more surface oriented. In all systems there was a modest downfield increase in chemical shift as the temperature was raised from 5 degrees to 46 degrees C. When small amounts of [13C]orlistat (1% relative to the emulsion mass) were incorporated into microemulsions, the chemical shift was identical to that in the unilamellar vesicles indicating a surface-like orientation of [13C]orlistat. However, when 3% was incorporated, two peaks appeared, one related to the surface at about 172 ppm, and one related to the core at about 171.65 ppm. Thus, orlistat first partitions into the surface and then when the surface is saturated, it moves into the more hydrophobic core. The fact that the two pools can be resolved using 13C NMR spectroscopy indicates a modestly slow exchange between the core and surface pools. Thus, the potent lipase inhibitor orlistat is ideally situated in the surface layer of emulsion particles and membranes for interaction with enzymes that superficially bind to such surfaces.