Abstract
Purpose. To determine the mechanism by which Carbomer inhibits the enzymatic activity of trypsin in hydrolysis of N-α-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 μg of trypsin to 1000 μg of carbomer.
Conclusions. Decreased hydrolysis of LHRH and BAEE by trypsin in the presence of carbomer is due to enzyme-polymer interaction.
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REFERENCES
V. H. L. Lee, R. D. Traver, and M. E. Taub. Enzymatic barriers to peptide and protein drug delivery. In V. H. L. Lee (ed.), Peptide and Protein Drug Delivery. Marcel Dekker, Inc., New York, 1991, pp. 303–358.
D. I. Friedman and G. L. Amidon. Oral absorption of peptides: Influence of pH and inhibitors on the intestinal hydrolysis of Leuenkephalin and analogues. Pharm. Res. 8:93–96 (1991).
T. Fujita, T. Fujita, K. Morikawa, H. Tanaka, O. Iemura, A. Yamamoto, and S. Muranishi. Improvement of intestinal absorption of human calcitonin by chemical modification with fatty acids: synergistic effects of acylation and absorption enhancers. Int. J. Pharm. 134:47–57 (1996).
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, H. N. Nellans, D. J. Hoffman, E. S. Johnson, and J. Greer. Effect of N-methyl substitution of the peptide bonds in luteinizing hormone-releasing hormone agonists. J. Med. Chem. 36:363–369 (1993).
M. Saffran, G. S. Kumar, J. C. Savariar, F. Burnham, F. Williams, and D. C. Neckers. A new approach to the oral administration of insulin and other peptide drugs. Science 233:1081–1084 (1986).
K. Morimoto, E. Kamiya, T. Takeeda, Y. Nakamoto, and K. Morisaka. Enhancement of rectal absorption of insulin in polyacrylic acid aqueous gel bases containing long chain fatty acid in rats. Int. J. Pharm. 14:149–157 (1983).
K. Morimoto, H. Akatsuchi, R. Aikawa, M. Morishita, and K. Morisaka. Enhanced rectal absorption of [Asu1,7]-eel calcitonin in rats using polyacrylic acid aqueous gel base. J. Pharm. Sci. 73:1366–1368 (1984).
C.-M. Lehr, J. A. Bouwstra, W. Kok, A. G. de Boer, J. J. Tukker, J. C. Verhoef, D. D. Breimer, and H. E. Junginger. Effects of the mucoadhesive polymer polycarbophil on the intestinal absorption of a peptide drug in the rat. J. Pharm. Pharmacol. 44:402–407 (1992).
H. L. Lueßen, B. J. de Leeuw, M. W. E. Langemeÿer, A. G. de Boer, J. C. Verhoef, and H. E. Junginger. Mucoadhesive polymers in peroral peptide drug delivery. VI. Carbomer and chitosan improve the intestinal absorption of the peptide drug buserelin in vivo. Pharm. Res. 13:1668–1672 (1996).
H. S. Ch'ng, H. Park, P. Kelly, and J. R. Robinson. Bioadhesive polymers as platforms for oral controlled drug delivery II: Synthesis and evaluation of some swelling, water-insoluble bioadhesive polymers. J. Pharm. Sci. 74:399–405 (1985).
G. Borchard, H. L. Lueßen, A. G. de Boer, J. C. Verhoef, C.-M. Lehr, and H. E. Junginger. The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro. J. Contr. Rel. 39:131–138 (1996).
A. B. J. Noach, Y. Kurosaki, M. C. M. Blom-Rosmalen, A. G. de Boer, and D. D. Breimer. Cell-polarity dependent effect of chelation on the paracellular permeability of confluent Caco-2 cell monolayers. Int. J. Pharm. 90:229–237 (1993).
H. L. Lueßen, B. J. de Leeuw, D. Perard, C.-M. Lehr, A. G. de Boer, J. C. Verhoef, and H. E. Junginger. Mucoadhesive polymers in peroral peptide drug delivery. I. Influence of mucoahesive excipients on the proteolytic activity of intestinal enzymes. Eur. J. Pharm. Sci. 4:117–128 (1996).
J. P. F. Bai, L. L. Chang, and J. H. Guo. Effects of polyacrylic polymers on the lumenal proteolysis of peptide drugs in the colon. J. Pharm. Sci. 84:1291–1294 (1995).
N. L. Burns, K. Holmberg, and C. Brink. Influence of surface charge on protein adsorption at an amphoteric surface: effects of varying acid to base ratio. J. Colloid Interface Sci. 178:116–122 (1996).
W. Norde and J. Lyklem. Thermodynamics of protein adsorption. J. Colloid Interface Sci. 71:350–356 (1979).
J. L. Brash and A.M. Samak. Dynamics of interaction between human albumin and polyethylene surface. J. Colloid Interface Sci. 65:495–504 (1978).
S. T. Tzannis, W. J. M. Hrushesky, P. A. Wood, and T. M. Przybycien. Adsorption of a formulated protein on a drug delivery device surface. J. Colloid Interface Sci. 189:216–228 (1997).
T. Tsai, R. C. Mehta, and P. P. DeLuca. Adsorption of peptides to poly(D,L-lactide-co-glycolide): 1. Effect of physical factors on the adsorption. Int. J. Pharm. 127:31–42 (1996).
T. Tsai, R. C. Mehta, and P. P. DeLuca. Adsorption of peptides to poly(D,L-lactide-co-glycolide): 2. Effect of solution properties on the adsorption. Int. J. Pharm. 127:43–52 (1996).
G. L. Peterson. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 87:386–396 (1977).
G. W. Schwert and Y. Takenaka. A spectrophotometric determination of trypsin and chymotrypsin. Biochim. Biophys. Acta 16:570–574 (1955).
I. Langmuir. The adsorption of gases on plane surfaces of glass, mica and platinum. J. Am. Chem. Soc. 40:1361–1368 (1918).
H. L. Lueßen, J. C. Verhoef, G. Borchard, C.-M. Lehr, A. G. de Boer, and H. E. Junginger. Mucoadhesive polymers in peroral peptide drug delivery. II. Carbomer and polycarbophil are potent inhibitors of the intestinal proteolytic enzyme trypsin. Pharm. Res. 12:1293–1298 (1995).
S. Maroux and P. Desnuelle. On some autolyzed derivatives of bovine trypsin. Biochim. Biophy. Acta 181:59–72 (1969).
E. Varallyay, G. Pal, A. Patthy, L. Sziagyi, and L. Graf. Two mutations in rat trypsin confer resistance against autolysis. Biochem. Biophys. Res. Commun. 243:56–69 (1998).
M. E. Soderquist and A. G. Walton. Structural changes in proteins adsorbed on polymer surfaces. J. Colloid Interface Sci. 75:386–397 (1980).
W. Norde. Adsorption of proteins from solution at the solid-liquid interface. Adv. Colloid Interface Sci. 24:267–340 (1986).
H. Elwing, A. Askendal, and I. Lundstrom. Competition between adsorbed fibrinogen and high-molecular-weight kininogen on solid surfaces incubated in human plasma (the Vroman effect): influence of solid surface wetability. J. Biomed. Mater. Res. 21:1023–1028 (1987).
J.-K. Luey, J. McGuire, and R. D. Sproull. The effect of pH and NaCl concentration on adsorption of β-lactoglobulin at hydrophilic and hydrophobic silicone surfaces. J. Colloid Interface Sci. 143:489–499 (1991).
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Walker, G.F., Ledger, R. & Tucker, I.G. Carbomer Inhibits Tryptic Proteolysis of Luteinizing Hormone-Releasing Hormone and N-α-Benzoyl-L-Arginine Ethyl Ester by Binding the Enzyme. Pharm Res 16, 1074–1080 (1999). https://doi.org/10.1023/A:1018944001869
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DOI: https://doi.org/10.1023/A:1018944001869