G F Walker1, R Ledger, I G Tucker. 1. University of Otago, School of Pharmacy, Dunedin, New Zealand. greg.walker@stonebow.otago.ac.nz
Abstract
PURPOSE: To determine the mechanism by which Carbomer inhibits the enzymatic activity of trypsin in hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and luteinizing hormone-releasing hormone (LHRH). METHODS: Inhibition of enzymatic activity was studied by measuring the formation of metabolites from LHRH and BAEE. Binding of trypsin and substrates to 0.35% (w/v) Carbomer at pH 7.0 was studied by centrifugal filtration. Gel filtration and reverse phase HPLC was used to determine the stability of trypsin. RESULTS: Carbomer reduced the rate of hydrolysis of BAEE and LHRH by trypsin to 34% and 28% of the control activity, respectively. The rate of metabolite formation for both substrates followed pseudo-zero order kinetics in the presence and absence of carbomer. Binding studies showed that 68% of the trypsin protein and 10% of BAEE was bound to carbomer, but no LHRH was bound. No low molecular weight autolysis products of trypsin could be identified by gel filtration. Reverse phase HPLC analysis of the unbound carbomer-treated-trypsin suggests a number of conformational forms of trypsin. The equilibrium binding capacity was 30 microg of trypsin to 1000 microg of carbomer. CONCLUSIONS: Decreased hydrolysis of LHRH and BAEE by trypsin in the presence of carbomer is due to enzyme-polymer interaction.
PURPOSE: To determine the mechanism by which Carbomer inhibits the enzymatic activity of trypsin in hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and luteinizing hormone-releasing hormone (LHRH). METHODS: Inhibition of enzymatic activity was studied by measuring the formation of metabolites from LHRH and BAEE. Binding of trypsin and substrates to 0.35% (w/v) Carbomer at pH 7.0 was studied by centrifugal filtration. Gel filtration and reverse phase HPLC was used to determine the stability of trypsin. RESULTS: Carbomer reduced the rate of hydrolysis of BAEE and LHRH by trypsin to 34% and 28% of the control activity, respectively. The rate of metabolite formation for both substrates followed pseudo-zero order kinetics in the presence and absence of carbomer. Binding studies showed that 68% of the trypsin protein and 10% of BAEE was bound to carbomer, but no LHRH was bound. No low molecular weight autolysis products of trypsin could be identified by gel filtration. Reverse phase HPLC analysis of the unbound carbomer-treated-trypsin suggests a number of conformational forms of trypsin. The equilibrium binding capacity was 30 microg of trypsin to 1000 microg of carbomer. CONCLUSIONS: Decreased hydrolysis of LHRH and BAEE by trypsin in the presence of carbomer is due to enzyme-polymer interaction.
Authors: C M Lehr; J A Bouwstra; W Kok; A G De Boer; J J Tukker; J C Verhoef; D D Breimer; H E Junginger Journal: J Pharm Pharmacol Date: 1992-05 Impact factor: 3.765
Authors: F Haviv; T D Fitzpatrick; R E Swenson; C J Nichols; N A Mort; E N Bush; G Diaz; G Bammert; A Nguyen; N S Rhutasel Journal: J Med Chem Date: 1993-02-05 Impact factor: 7.446