Carbomer inhibits tryptic proteolysis of luteinizing hormone-releasing hormone and N-alpha-benzoyl-L-arginine ethyl ester by binding the enzyme.
OBJECTIVE
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.
Walker
G F
GF
University of Otago, School of Pharmacy, Dunedin, New Zealand. greg.walker@stonebow.otago.ac.nz
Ledger
R
R
Tucker
I G
IG
D000180:Acrylic Resins / Q000378:metabolism* / Q000494:pharmacology*; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010455:Peptides / Q000008:administration & dosage; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism* / Q000494:pharmacology*
D000180:Acrylic Resins; D010455:Peptides; D014361:Trypsin Inhibitors; D007987:Gonadotropin-Releasing Hormone; C006903:carbopol 940; D001120:Arginine; C009727:benzoylarginine ethyl ester; D014357:Trypsin