Abstract
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.
Highlights
Tetrahydrolipstatin (S)-I-{(ZS,3S)-3-hexyl4-oxooxetan-Z-yl]methyl]dodecylN-formyl-L-leucinate, a potent inhibitor of pancreatic lipase, is hydrophobic, amphipathic, and water-insoluble
Orlistat, a potent inhibitor of pancreatic lipase, is a derivative of the naturally occurring lipase inhibitor lipstatin produced from Strep tomyces toxytricini [1, 2]
Further results indicate that orlistat remains in the GI tract without being absorbed [9].Whereas in animals a nearly complete inhibition of fat absorption can be achieved (6, lo), a more modest inhibition would be a reasonable goal in humans [11, 12]
Summary
[l3ClTetrahydrolipstatin ["C]orlistat was synthesized by Dr Philip F. Four samples of unilamellar vesicles with varying amounts of ['3Clorlistat (1%, 2%, 5%, and 10%)were prepared by weighing out 1.00mg, 2.15 mg, 5.08 mg, and 10.07 mg of ['3C]orlistat, adding them to aliquots of egg PC in chloroform (dry weight of 97 mg), and evaporating the solvent under NP.Each sample was hydrated with 1.6 ml of a 0.56% w/w KCI solution in H 2 0and 0.2 ml D 2 0 ,incubated at 47°C for 15 min, and ultrasonically irradiated using a Branson W-350 sonifier in a pulsed mode at 25-30°C. Three samples of microemulsion particles with varying amounts of [I3C]orlistat (l%,3%, and 6%) were prepared by weighing out 1.52 mg, 5.08 mg, and 10.00 mg of [13C]orlistatand adding them to aliquots of 80 mg of TO and 70 mg of egg PC in chloroform and evaporating the solvent under Ne. The samples were lyophilized in a Labconco Lyph-Lock 6 lyophilizer (Kansas City, MO) for 20 min and hydrated with 10 ml of Tris buffer (0.1 M KCl, 0.01 M Tris-HC1 at pH 8.0, and 0.025% NaN3). A small layer of H 2 0was placed on top of each
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