[HTML][HTML] Direct measurement of lipase inhibition by orlistat using a dissolution linked in vitro assay
DR Lewis, DJ Liu - Clinical pharmacology & biopharmaceutics, 2012 - ncbi.nlm.nih.gov
DR Lewis, DJ Liu
Clinical pharmacology & biopharmaceutics, 2012•ncbi.nlm.nih.govPurpose To develop a bio-assay that would be able to directly test gastrointestinal and/or
dissolution samples to determine lipase activity and inhibition by Orlistat. Methods Enzyme
assays were performed with porcine pancreatic lipase and para-Nitrophenyl Palmitate
(pNPP) in pH 8.0 reaction buffer at 37 C. Substrate hydrolysis was monitored by absorbance
changes at 410 nm. The dissolution of two Orlistat formulations was tested with a USP II
apparatus. Samples were HPLC analyzed to determine release profile in addition to being …
dissolution samples to determine lipase activity and inhibition by Orlistat. Methods Enzyme
assays were performed with porcine pancreatic lipase and para-Nitrophenyl Palmitate
(pNPP) in pH 8.0 reaction buffer at 37 C. Substrate hydrolysis was monitored by absorbance
changes at 410 nm. The dissolution of two Orlistat formulations was tested with a USP II
apparatus. Samples were HPLC analyzed to determine release profile in addition to being …
Abstract
Purpose
To develop a bio-assay that would be able to directly test gastrointestinal and/or dissolution samples to determine lipase activity and inhibition by Orlistat.
Methods
Enzyme assays were performed with porcine pancreatic lipase and para-Nitrophenyl Palmitate (pNPP) in pH 8.0 reaction buffer at 37 C. Substrate hydrolysis was monitored by absorbance changes at 410 nm. The dissolution of two Orlistat formulations was tested with a USP II apparatus. Samples were HPLC analyzed to determine release profile in addition to being diluted and directly assayed for inhibitory effect.
Results
The lipase-pNPP system demonstrates linearity and Michalis-Menten kinetics with a K m= 2.7±0.2 μM and K cat= 0.019 s− 1. Orlistat showed highly potent and time dependent inhibition with 5 ng/ml effecting 50% activity after 5 minutes in the Lipase-pNPP system. Dissolution studies showed a correlation of the drug release profile to the inhibitory effect of dissolution samples in the assay.
Conclusions
The lipase-pNPP method can be used as an in vitro assay to monitor orlistat inhibition from drug release or dissolution samples.
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