Behavior of tetrahydrolipstatin in biological model membranes and emulsions.

Ko J, Small DM.

Department of Biophysics, Center for Advanced Biomedical Research, Boston University School of Medicine, MA 02118, USA.

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.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9300776&dopt=Abstract orlistat Xenical online refs
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Quantitative liquid chromatographic-tandem mass spectrometric determination of orlistat in plasma with a quadrupole ion trap.

Wieboldt R, Campbell DA, Henion J.

Analytical Toxicology, Cornell University, New York State College of Veterinary Medicine, Ithaca 14850, USA.

This report evaluates the use of a quadrupolar ion trap for quantitation in a bioanalytical laboratory. The evaluation was accomplished with the cross-validation of an LC-MS-MS quantitative method previously validated on a triple quadrupole mass spectrometer. The method was a multi-level determination of the anti-obesity drug, orlistat, in human plasma. The method has been refined previously on a triple quadrupole instrument to provide rapid sample throughput with robust reproducibility at sub-nanogram detection limits. Optimization of the method on the ion trap required improved chromatographic separation of orlistat from interfering plasma matrix components coextracted during the initial liquid-liquid extraction of plasma samples. The ion trap produces full-scan collision-induced dissociation mass spectra containing characteristic orlistat fragment ions that are useful for quantitation. Data collection on the ion trap required a precursor ion isolation width of 3.0 Da and optimal quantitative results were obtained when three fragment ions were monitored with a 1.8 Da window for each ion. Although a direct cross-validation between the ion trap and the tandem triple quadrupole mass spectrometer was not possible, quantitative results for orlistat comparable to those obtained from the triple quadrupole instrument were achieved by the ion trap with the modified method. The limit of quantitation for orlistat in plasma on the ion trap was 0.3 ng ml(-1) with a linear dynamic range of 0.3 to 10 ng ml(-1). Precision and accuracy varied from 4 to 15% over the quantitation range. The overall results provide an example of the utility of an ion trap in bioanalytical work.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9653954&dopt=Abstract orlistat Xenical online refs
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A 2-methyleneoxetane analog of orlistat demonstrating inhibition of porcine pancreatic lipase.

Dollinger LM, Howell AR.

Department of Chemistry, University of Connecticut, Storrs 06269-4060, USA.

The 2-methyleneoxetane analog 2 of orlistat (OLS, 1) has been synthesized and tested against porcine pancreatic lipase (PPL). Despite the loss of the carbonyl group, a potential site for hydrogen bonding interaction with the enzyme and the key element in the acylation by OLS, 2 has activity comparable to 1.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9871523&dopt=Abstract orlistat Xenical online refs
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Effect of the lipase inhibitor orlistat and of dietary lipid on the absorption of radiolabelled triolein, tri-gamma-linolenin and tripalmitin in mice.

Isler D, Moeglen C, Gains N, Meier MK.

Pharma Division, F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Orlistat, a selective inhibitor of gastrointestinal lipases, was used to investigate triacylglycerol absorption. Using mice and a variety of emulsified dietary lipids we found that the absorption of radiolabelled tripalmitin (containing the fatty acid 16:0), but not of triolein (18:1n-9) or tri-gamma-linolenin (18:3n-6), was incomplete from meals rich in esterified palmitate. Further, the absorption of radiolabelled tri-gamma-linolenin, from both saturated and unsaturated dietary triacylglycerols, was 1.3- to 2-fold more potently inhibited by orlistat than that of triolein and tripalmitin. These radiolabelled triacylglycerols, which have the same fatty acid in all three positions, may not always be accurate markers of the absorption of dietary triacylglycerols. Orlistat was more effective at inhibiting the absorption of radiolabelled triacylglycerols with which it was codissolved than those added separately, which indicates that equilibration between lipid phases in the stomach may not always be complete. The saturation of the dietary lipid had little or no effect on the potency of orlistat. Orlistat provides a novel approach for studying the role of triacylglycerol hydrolysis in the overall process of triacylglycerol absorption.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7632666&dopt=Abstract orlistat Xenical online refs
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Interactions of lipoprotein lipase with the active-site inhibitor tetrahydrolipstatin (Orlistat).

Lookene A, Skottova N, Olivecrona G.

Department of Medical Biochemistry and Biophysics, University of Umea, Sweden.

Lipoprotein lipase (LPL) was rapidly inactivated by low concentrations of the active-site inhibitor tetrahydrolipstatin (THL). The presence of amphiphils (e.g. long-chain fatty acids) or of lipid/water interfaces (lipid emulsions) was required for inhibition to occur. Apolipoprotein CII increased the maximal inactivation rate constant by 1.8-fold in the presence of an emulsion of long-chain triacylglycerols, but had no effect in the presence of an emulsion of tributyrylglycerol. The fully inhibited enzyme had a ratio of THL/LPL of nearly 2, indicating that both subunits of the LPL homo-dimer bound THL. The THL-LPL complex was stable below pH 7.5. At higher pH reactivation occurred indicating that THL was slowly turned over by the enzyme. The apparent reactivation rate constant was increased about threefold by the presence of lipid/water interfaces. Sucrose density gradient centrifugation revealed that THL induces tetramerisation of LPL. This aggregation was reversible on reactivation of the inhibited enzyme. Binding to heparin was not affected by THL. In contrast, binding to lipid droplets and to lipoproteins was increased, indicating exposure of hydrophobic regions in the inhibited LPL. It is suggested that THL induces local conformational changes in LPL, which may involve opening of the putative surface lid structure which covers the active-site.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8020477&dopt=Abstract orlistat Xenical online refs
xenical (orlistat)