Trypsin insulin degradation

Drags that structurally resemble nutrients such as polypeptides, nucleotides, or fatty acids may be especially susceptible to enzymatic degradation. For example, the proteolytic enzymes chymotrypsin and trypsin can degrade insulin and other peptide drags. In the case of insulin, proteolysis was shown to be reduced by the coadmmistration of carbopol polymers at 1% and 4% (w/v%), which presumably shifted the intestinal pH away from the optimal pH for proteolytic degradation. 

For example, enzyme degradation of insulin is known to be mediated by the serine proteases trypsin, a-chymotrypsin, and thiol metalloproteinase insulin-degrading... 

Minkowski tried unsuccessfully to prepare an extract of dog pancreas that would reverse the effect of removing the pancreas—that is, would lower the urinary or blood glucose levels. We now know that insulin is a protein, and that the pancreas is very rich in proteases (trypsin and chymotrypsin), normally released directly into the small intestine to aid in digestion. These proteases doubtless degraded the insulin in the pancreatic extracts in Minkowski s experiments. 

Polyacrylic add polymers can inhibit luminal degradation of insulin and other large peptides by trypsin and chymobypsin. They have strong bioadhesive properties, iriuco utg the in situ absorption of insulin [42]. 

Luessen HL, Verhoef JC, Borchard G, Lehr CM, de Boer AG, Junginger HE (1995) 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 Marschutz MK, Bernkop-Schnurch A (2000) Oral peptide drug delivery polymer-inhibitor conjugates protecting insulin from enzymatic degradation in vitro. Biomaterials 21 1499-1507... 

Arg residue, but none, as yet, has been demonstrated. Trypsin cleavage of proinsulin generates small amounts (< 5%) of Arg-(A)-insulin, the form which is present in native insulin at very low levels (< 0.5%). In the absence of carboxypeptid-ase-B, both plasmin and trypsin tend to cleave Lys-Ala or Lys-Thr bonds in proinsulin, resulting in a partially degraded form of insulin that does not occur naturally [40], These serine proteases therefore seem to carry out the rare cleavage at single basic residue sites. 

Oral dosage forms may contain various other additives to increase the solubility and hence oral bioavailability of the drag, such as co-solvents, buffers and surfactants. Newer technologies may also incorporate additives such as enzyme inhibitors, to prevent premature degradation of enzymatically labile drags. For example, the inclusion of trypsin inhibitors, such as soyabean trypsin inhibitor and aprotinin, have been shown to be effective in enhancing the effect of insulin in rats. Penetration enhancers may also be included to facilitate the uptake of poorly absorbed moieies. These are discussed below in Section 6.7.4. 

Bai JPF (1995) The involvement of cytosolic chymotrypsin-like, trypsin-like, and cucumsin-like activities in degradation of insulin-like growth factor I by epithelial tissue. J Pharm Pharmacol 47 674—677... 

The opposite approach, namely, the creation of additional lysine-like sites for cleavage by trypsin has been used in the conversion of cysteine bonds to /S-( -aminoethyl)-cysteine residues by reaction of the thiol groups with jS-bromoethylamine (Lindley, 1959). Enzymatic degradation of such modified proteins, e.g., reduced and /3-aminoethylated insulin, by the ac-... 

Degradation in the gut is caused by endogenous proteases such as trypsin and a-chymotrypsin. Entrapment of macromolecules in liposomes results in better protection against proteolysis [30], Since lipid-soluble molecules are absorbed quickly in the gut, it could be expected that liposome delivery will give enhanced absorption by diffusion. However, this problem is not simple, as illustrated by the lack of uptake of liposomes by enterocytes [30,31]. Insulin entrapped in liposomes is absorbed in this way [32],... 

The spectrophotometric evidence reviewed above for the binding of a proportion of the phenolic hydroxyl groups of the tyrosine residues of native proteins is supported by work on the action of tyrosinase on proteins. Sizer (1946) found that this enzyme oxidizes the tyrosine residues in native trypsin, pepsin, chymotrypsin, casein, peptone, insulin, and hemoglobin. Native ovalbumin, human and bovine serum albumin, tobacco mosaic virus (nucleoprotein), human y- and bovine /3-globulins, and bovine fibrinogen are not susceptible to tyrosinase, but become so after tryptic digestion. It was shown (Sizer, 1947) that for the proteins which are oxidized by tyrosinase in the native state, the observed reaction does indeed occur with the intact proteins and does not require preliminary degradation to tyrosine peptides or free tyrosine. The kinetics of the oxidation of tyrosine by tyrosinase have been studied spectropho-tometrically (Mason, 1948 etc.). 

Specific peptidase and protease systems which involve Mn(II) include thrombin limited-proteolysis of prothrombin [122], insulin protease [123], enkephalin-degrading amino-peptidase [124], camosinase [125,129], ki-ninase [127], and trypsin activation [128]. A metalIo(Mn)-protease is involved in the processing of mitochondrial precursor proteins [130]. Several aminopeptidases are also specifically manganese-dependent, namely Leu-aminopeptidase [131] and prolidase or C-terminal proline dipeptidase [132-135]. Other systems that hydrolyze linear and cyclic G-N bonds include various amino-acylases, deacetylases, amidases and methylene-... 

In a PEC used to deliver a protein, the latter is often used as one of the polyelectrolyte components of the complex. Examples of PECs in which one of the two polyelectrolyte components is an active substance itself, are complexes of chitosan and insulin. The PEC composed of trimethyl chitosan (TMC) and pegylated TMC (PEG-g-TMC) can be obtained simply by mixing the solutions of TMC and insulin at various mass and charge ratios. These PECs were stable in simulated intestinal fluid at pH 6.8. However, they disintegrated in simulated gastrointestinal fluid at pH 1.2. The PECs also protected insulin from temperature-induced denaturation up to 50 °C and from degradation by trypsin. Based on these results, the authors suggested that polyelectrolyte complexation can be a useful technique for fabrication of insulin delivery systems for oral administration. 

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