Proteolytic Degradation Of Insulin Research Articles

Inhibitory effect of emulsifiers in sedds on protease activity: Just an illusion?

AimEvaluation of inhibitory effect of emulsifiers on pancreatic trypsin and α-chymotrypsin. MethodsThe inhibitory effect of Cremophor EL, Cremophor RH 40, Brij O10, Tween 20, polyethylene glycol 8000, polyethylene glycol 400, Carbitol, Pemulen TR-1, Pemulen TR-2, Carbopol Ultrez 20 and Carbopol Ultrez 21 on pancreatic trypsin and α-chymotrypsin was tested. BAEE (Nα-Benzoyl-l-arginine ethyl ester), BTEE (N-Benzoyl-l-tyrosine ethyl ester), casein and insulin were used as substrates for trypsin and α-chymotrypsin. SEDDS containing Pemulen TR-2 were developed, loaded with insulin-DMPG (dimyristoylphosphatidylglycerol) complex, characterized and tested regarding the protective effect towards the proteolytic degradation of insulin. ResultsCremophor EL, Cremophor RH 40, Brij O10, Tween 20, polyethylene glycol 8000, polyethylene glycol 400, Carbitol did not show any inhibitory effect towards trypsin and α-chymotrypsin, whereas Pemulen TR-1, Pemulen TR-2, Carbopol Ultrez 20 and Carbopol Ultrez 21 inhibited the enzymes in a concentration dependent manner. Moreover, Pemulen and Carbopol Ultrez emulsifiers exhibited comparable inhibitory properties (p>0.05). The incorporation of 0.34% w/w of Pemulen TR-2 in SEDDS decreased the degradation rate of the loaded insulin-DMPG complex compared to the blank formulation (p<0.05). ConclusionThe present study revealed that commonly used emulsifiers in SEDDS do not have inhibitory properties on the proteolytic activity of trypsin and α-chymotrypsin. Moreover, it was demonstrated that the incorporation of emulsifiers with inhibitory properties towards trypsin and α-chymotrypsin in SEDDS play a minor role in the protection of embedded drugs from enzymatic degradation.

The role of citric acid in oral peptide and protein formulations: Relationship between calcium chelation and proteolysis inhibition

The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CA’s calcium chelation activity was developed and verified experimentally using an ion-selective electrode. The effects of CA, its salt (citrate, Cit) and the established permeation enhancer, lauroyl carnitine chloride (LCC) were compared by measuring transepithelial electrical resistance (TEER) and permeability of insulin and FD4 across Caco-2 monolayers and rat small intestinal mucosae mounted in Ussing chambers. Proteolytic degradation of insulin was determined in rat luminal extracts across a range of pH values in the presence of CA. CA’s capacity to chelate calcium decreased ∼10-fold for each pH unit moving from pH 6 to pH 3. CA was an inferior weak permeation enhancer compared to LCC in both in vitro models using physiological buffers. At pH 4.5 however, degradation of insulin in rat luminal extracts was significantly inhibited in the presence of 10mM CA. The capacity of CA to chelate luminal calcium does not occur significantly at the acidic pH values where it effectively inhibits proteolysis, which is its dominant action in oral peptide formulations. On account of insulin’s low basal permeability, inclusion of alternative permeation enhancers is likely to be necessary to achieve sufficient oral bioavailability since this is a weak property of CA.

Chemical and alpha-chymotrypsin-mediated proteolytic degradation of insulin in bile salt-unsaturated fatty acid mixed micellar systems.

The proteolytic degradation of porcine zinc insulin by alpha-chymotrypsin was previously found to depend markedly on the state of insulin aggregation (Pharm. Res. 9:864-869, 1992). In this study, the effect of bile salt-unsaturated fatty acid mixed micelles on alpha-chymotryptic degradation of insulin was further characterized. The incorporation of linoleic acid has greatly accelerated insulin degradation with the apparent first order rate constant being linearly related to the concentration of linoleic acid. At a 10 mM linoleic acid concentration solubilized in 10 mM sodium glycocholate, the proteolytic degradation rate constant increased by 16 times, which could not be explained solely by the mechanism of insulin oligomer dissociation. Further, this effect is significantly reduced when the free carboxylic group of linoleic acid is methylated. The catalytic role of mixed micelles on chemical degradation of insulin was found to depend on the concentration of linoleic acid incorporated. When solubilized in the form of mixed micelles, linoleic acid chemically catalyzes peptide bond cleavage in a concentration-dependent manner.

Characterization of in Vitro Transdermal Iontophoretic Delivery of Insulin

In-vitro studies were conducted to characterize the transdermal iontophoretic delivery of insulin, thus avoiding potential complications from various biological variations, which may be encountered during in-vivo studies. The proteolytic degradation of insulin in skin homogenates and degradation under the experimental conditions used was investigated. Appropriate adjuvants were incorporated to minimize the potential degradation problems of insulin. 125I-Insulin was observed to penetrate into and accumulate in the skin during the iontophoresis period. It was then released gradually from this depot, as a mixture of intact and 125-I labelled fragments, into the receptor medium. Drug desorption studies supported the theory of skin depot or reservoir formation. It was found that an electric field could be used to facilitate the desorption of drug from the depot. The post-application flux of insulin (or its fragments) from the skin depot formed during iontophoresis was monitored to study the factors affecting the iontophoretic delivery of insulin. Stripping and delipidization of the skin were noted to increase the skin permeation rate of insulin. The cumulative radioactivity permeated and accumulated in the skin was higher at pH 3.6 than at pH 7.4. The iontophoresis-facilitated transdermal delivery was observed to increase with increasing duration of current application and increasing donor concentration of insulin. Modulation of drug delivery by multiple applications was also found to be feasible.

Proteolytic degradation of insulin and glucagon in rat brain during ontogenesis

The degradation of insulin and glucagon was investigated in rat brain, kidney, and liver during postnatal ontogenesis. It was found that a maximal activity around 13 d is unique for CNS and differs remarkably from time-course in liver and kidney. It is concluded that this result supports the hypothesis of a growth promoting role of insulin in developing rat brain.

Initial site of insulin cleavage by insulin protease.

Exposure of insulin to insulin protease (insulinase, EC 3.4.22.11), a degradative enzyme with considerable specificity toward insulin, results in alterations in the properties of the insulin molecule. Limited degradation by the enzyme results in a decrease in the ability of insulin to bind to membrane receptors with less change in the immunoprecipitability or trichloracetic acid precipitability of the hormone. Limited degradation by insulin protease also alters insulin so that the molecule becomes susceptible to attack by nonspecific endopeptidases which have no effect on unaltered insulin. These data demonstrate the production of an intermediate in the proteolytic degradation of insulin. By labeling with [14C]dansyl chloride, an insulin intermediate with three amino-terminal residues, glycine, phenylalanine, and leucine, was identified. Analysis of this intermediate demonstrated that it was composed of an intact A chain and a B chain cleaved between residues B16 and B17, with the three peptide chains held together by disulfide bonds. Based on these findings, we hypothesize that a stepwise degradation of insulin occurs in vivo and that an early step in the process is the cleavage between B16 and B17 that renders the molecule sucseptible to further degradation by nonspecific proteases.

CARBOHYDRATE METABOLISM IN PREGNANCY: THE DEGRADATION OF INSULIN BY EXTRACTS OF MATERNAL AND FETAL STRUCTURES IN THE PREGNANT RAT1

Cell-free extracts have been prepared from livers, placentas, and fetuses of pregnant rats on day 12, 15, 18 and 21 of gestation and from livers of nonpregnant control animals. With the use of I13l-insulin and nitrogen reference standards, the insulin-degrading capacities of these structures have been compared on the basis of activity per unit mass (specific activity) and activity per total tissue (total potential activity). It has been demonstrated that mechanisms for the proteolytic degradation of insulin are present within the conceptus throughout gestation. Placental specific activity remained constant and at all times equal to that of maternal and control liver. Thus, for pooled placentas, total potential activity paralleled the increase in placental mass to reach onethird the value of maternal liver at term. Fetal specific activity increased late in pregnancy to approximately twice the level found in the other tissues. In isolated fetal parts, activity was greatest in feta skin. At term, the total p...