Oral delivery of peptides to salmon

Abstract

Purpose. Oral delivery of peptide and protein drugs to fish has potential advantages to the aquaculture industry. However, the bioavailability of proteins and peptides from the intestinal tract is very low. This can be attributed inpart to the extensive lumenal and mucosl proteolytic activities of the intestine. In this study the metabolic activities of the Quinnat salmon (Oncorhynchus tshawytscha) intestinal tract towards bovine serum albumin (BSA), human (hLHRH) and salmon (sLHRH) luteinizing-hormone releasing hormones were investigated. To overcome this metabolic barrier a number of proteolytic inhibitors were investigated to stabilise the protein and peptides drugs in the intestine, improving their opportunity for absorption across the salmon intestine.

Methods. The extent of BSA degradation was determined by the formation of acid soluble peptides from BSA, measured by the method of Lowry. The metabolic stability hLHRH and sLHRH were determined using a capillary electrophoresis (CE) assay. Binding of proteins and proteolytic substrates to the polyacrylic acid, specifically Carbopol ® 934P (CP934P), was studied by centrifugal filtration. Gel filtration and reverse phase HPLC was used to determine the stability of trypsin in the presence of CP934P.

Results. The lower intestinal tract of the salmon is divided into three anatomically distinct sections: anterior, middle and posterior. The lumenal proteolytic activities of the posterior intestinal section towards BSA were approximately half that of the anterior and middle sections. The half-lives of the LHRH analogues in the posterior intestinal sections were 2-fold longer than for the anterior and middle sections. Intestinal luminal proteolytic activities towards BSA and the LHRH analogues correlated with higher activities towards the synthetic substrates of chymotrypsin, trypsin and elastase. LHRH analogues were shown to be rapidly hydrolysed by bovine α-chymotrypsin, whereas bovine trypsin and porcine elastase hydrolysed the LHRH analogues slowly by comparison.

Proteolytic activity of the posterior mucosal homogenates towards BSA was 4-fold higher than for the middle mucosal homogenates. LHRH analogues were hydrolysed by the posterior mucosal homogenate, whereas in the middle mucosal homogenate they were stable. The higher metabolic instability of LHRH analogues in the posterior mucosal homogenate correlated with higher chymotryptic-like activity.

Soybean trypsin inhibitor (SBTI) was the most effective inhibitor, stabilising the LHRH analogues against the lumenal proteolytic activity of the intestine and increasing their half-lives in the anterior, middle and posterior section between 7.5- and 15-fold. CP934P (0.35% w/v, pH 8.0 at 15°C) increased the half-lives of the LHRH analogues in the anterior, middle and posterior sections between 1. 7- and 2. 7-fold. The rates of degradation in the presence of CP934P followed pseudo-zero order kinetics, indicating that the mechanism of inhibition was not autolysis.

CP934P (0.35% w/v, pH 7 at 37°C) inhibited the rate of hydrolysis of N-α-benzyol-L-arginine ethyl ester (BAEE) and hLHRH by trypsin to 34% and 28% of the control activity, respectively. CP934P completely stabilised hLHRH in the presence of bovine α-chymotrypsin. Binding studies showed 68% of the trypsin and 22% of chymotrypsin; 10% of BAEE was bound to CP934P, but no LHRH was bound. Lower specific activities of the unbound CP934P treated proteases indicate that inactivation of the proteases is also occurring. No low molecular weight autolysis products of trypsin could be identified by gel filtration. Reverse phase HPLC analysis of the unbound CP934P treated trypsin suggests a number of conformational forms of trypsin. The equilibrium binding capacity was 30 mg of trypsin to 1g of CP934P.

Conclusions. Chymotryptic-like activity of the salmon intestinal tract is a major metabolic barrier to the oral absorption of the LHRH analogues. The chymotryptic-like activities of the intestinal lumen and the intestinal mucosa are most likely due to the pancreatic secreted chymotrypsin and the intestinal brush-border membrane protease endopeptidase 24.18, respectively. The co-administration of SBTI with LHRH analogues in oral formulations could improve the oral absorption of LHRH analogues in salmon. The inhibition of lumenal proteolytic activity by CP934P is the result of enzyme-polymer interaction, thereby reducing the free concentration of protease and in part denaturing the protease. The posterior intestine of salmon is the most metabolically favourable site for the oral delivery of peptides and proteins; however the LHRH analogues will have to overcome the higher endopeptidase 24.18 activity present in the posterior mucosa.