The binding site of sn-1(3)-regioselective Rhizopus oryzae lipase (ROL) has been engineered to change the stereoselectivity of hydrolysis of triacylglycerol substrates and analogs. Two types of prochiral triradylglycerols were considered: 'flexible' substrates with ether, benzylether or ester groups, and 'rigid' substrates with amide or phenyl groups, respectively, in the sn-2 position. The molecular basis of sn-1(3) stereoselectivity of ROL was investigated by modeling the interactions between substrates and ROL, and the model was confirmed by experimental determination of the stereoselectivity of wild-type and mutated ROL. For the substrates, the following rules were derived: (i) stereopreference of ROL toward triradylglycerols depends on the substrate structure. Substrates with 'flexible' sn-2 substituents are preferably hydrolyzed at sn-1, 'rigid' substrates at sn-3. (ii) Stereopreference of ROL toward triradylglycerols can be predicted by analyzing the geometry of the substrate docked to ROL: if the torsion angle phiO3-C3 of glycerol is more than 150 degrees, the substrate will preferably be hydrolyzed in sn-1, otherwise in sn-3. For ROL, the following rules were derived: (i) residue 258 affects stereoselectivity by steric interactions with the sn-2 substituent rather than polar interactions. To a lower extent, stereoselectivity is influenced by mutations further apart (L254) from residue 258. (ii) With 'rigid' substrates, increasing the size of the binding site (mutations L258A and L258S) shifts stereoselectivity of hydrolysis toward sn-1, decreasing its size (L258F and L258F/L254F) toward sn-3.
OBJECTIVE Pancreatic enzyme products were available before the 1938 passage of the Federal Food, Drug, and Cosmetic Act and have to date been marketed without required safety and efficacy testing. Despite a lack of demonstrated bioequivalence, they are often substituted for each other without physician or patient consent or monitoring. We investigated the in vitro variability of key performance parameters among a representative group of currently available pancreatic enzyme formulations.
MATERIALS AND METHODS Three “branded” preparations (Creon 20 Minimicrospheres, Pancrease MT 20, Ultrase MT 20) and 3 “generic” formulations (Pangestyme CN-20, Pancrelipase 20,000 URL, and Lipram CR 20) were evaluated in vitro for physical parameters of the capsules, actual vs. labeled enzyme activity, resistance of the enteric coating to simulated gastric acid, and kinetics of simulated duodenal lipase release. All products were labeled as providing 20,000 units of lipase activity per capsule.
RESULTS All products varied considerably in the percentage relationship between actual and labeled lipase activity. Actual lipase activity exceeded 165% of the label claim in 4 batches of the Pangestyme product and 1 batch of the Lipram product. All batches of the Creon, Lipram, Ultrase, and Pancrease products were found to have residual lipase activity above 80% of their baseline measurements after testing in simulated gastric acid; residual lipase activity varied significantly among batches of the Pangestyme product and was only 1% for the Pancrelipase product. The Creon and Lipram products demonstrated effective protection by the enteric coating at pH <6.0 and rapid release of enzymatic activity at pH ≥6.0. The Pangestyme and Pancrelipase products showed substantial activity of released enzymes already at pH 5.0. Release kinetics were inconsistent between batches for the Ultrase and Pancrease products.
CONCLUSION This study confirms the existence of “branded”-to-“generic,” product-to-product, and batch-to-batch variability among representative pancreatic enzyme formulations with pharmaceutically equivalent labels. The results confirm current cautions regarding pharmacy substitution of pancreatic enzyme products and support the announcement by the US Food and Drug Administration, made subsequent to this study, that as of April 2008 approved new drug applications will be required in order to ensure the quality, potency, and stability of these products.
In a model elaborated earlier to understand and predict the stereopreference ofRhizopus oryzae lipase (ROL) catalyzed hydrolysis of triradylglycerols, we identified the degree of flexibility of the C1′‐X′ bond (X = O for ether, N for amide, C for alkyl, methylene, and a phenylring, respectively) adjacent to C2 of glycerol being responsible for the discrimination of the enantiomers (Kovac et al., Eur. J. Lipid Sci. Technol.2000, 61—72). During catalysis of forward and back reaction — hydrolysis and esterification — in either case the carbonyl carbon of the sn‐1 or sn‐3 fatty acid binds to the active site serine of ROL leading to a covalently bound intermediate, which was simulated in the model. Thus, we assumed that stereoselectivity of ROL in esterification of corresponding 2‐monoradylglycerols with oleic acid in cyclohexane should follow the same model. As predicted by this model 2‐monoradylglycerols with “rigid” phenyl and amide substituents were esterified at thesn‐3 position, and those with “ flexible” ether substituents at thesn‐1 position. However, enantiomeric excess of wild type ROL in esterifying 2‐monaradylglycerols with flexible benzylether and methylene substituents differed by around 50% as compared to hydrolysis experiments with corresponding triradylglyc‐erols. In addition esterification of 2‐monoradylglycerol with flexible ether substituent by ROL L258F/L254F double mutant was essentially non‐selective compared to corresponding triradylglycerol where enantiomeric excess was 58%sn‐1. Whether water activity was a factor determining these discrepancies was investigated for ROL‐ and double mutant enzyme‐catalyzed esterification of the ether and methylene substrates under controlled water activities from 0.02—0.85. In all cases stereoselectivities ob‐served were independent from water activities. In conclusion, the model describing the stereoselective course of aqueous hydrolysis of triradylglycerols catalyzed by ROL in most cases applies to the esterification reaction in organic solvent. Differences in stereoselectivity observed are attributed to reduced possibilities for interaction of 2‐monoradylglycerol substrates with the binding sites of ROL as compared to those of triradylglycerol substrates.
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