Degradation Of Calcitonin Research Articles

Effects of polyacrylic polymers on the degradation of insulin and peptide drugs by chymotrypsin and trypsin.

The purpose of this study was to determine whether carbopol polymers, polyacrylic acid polymers, can inhibit lumenal degradation of insulin, calcitonin and insulin-like growth factor I (IGF-I) by trypsin and chymotrypsin and to understand whether reducing the pH of the incubation medium by these polymers results in inhibition. Further, the effects of carbopol polymers on the in-situ absorption of insulin were studied in rats. In saline, carbopol polymers at 1% and 4% (w/v%) inhibited close to 100% of trypsin and chymotrypsin activities against insulin. In 50 mM Tris buffer, carbopol polymers, including 934P, 974P and 971P, at 0.1% only weakly inhibited degradation of calcitonin and insulin by both enzymes; however, as the polymer concentration increased to 0.4%, degradation of insulin, calcitonin, and IGF-I by both enzymes was complete or almost complete. When the Tris buffer was increased to 100 mM, no inhibition was observed at 0.1%. Determination of the final pH of the incubation medium in the presence of polymers revealed that the inhibitory effects of carbopol polymers correlated with the final pH. When the incubation medium has no or low buffer capacity to buffer the protons released by carbopol polymers, these polymers are able to reduce the pH much lower than the optimum pH for the enzyme activities, and thus inhibit proteolytic degradation. When the buffer capacity of the incubation medium increases, the inhibitory effects of carbopol polymers weaken. In-situ absorption of insulin revealed that carbopol polymers improved insulin absorption and induced a significantly greater decline in blood glucose levels. It is concluded that carbopol polymers with strong bioadhesive properties also can inhibit lumenal degradation of peptide hormones, offering multiple advantages for their uses in oral drug delivery.

Use of protease inhibitors to improve calcitonin absorption from the small and large intestine in rats.

The objective of this study was to examine the effects of protease inhibitors on the absorption of calcitonin from different regions of the intestine in rats. The absorption experiments were investigated by in-situ use of closed intestinal loops in rats and stability of calcitonin was examined in mucosal homogenates and intestinal fluids. The intestinal absorption of calcitonin was evaluated by measurement of its hypocalcaemic effect. No substantial hypocalcaemic response was observed when calcitonin was administered into the jejunum or colon. A slight hypocalcaemic effect was observed after administration of calcitonin into the ileum. Of the co-administered protease inhibitors, bacitracin (20mM) strongly promoted calcitonin absorption from the jejunum, ileum and colon. A significant hypocalcaemic effect was also obtained after intestinal administration of calcitonin with soybean trypsin inhibitor (10mgmL(-1)), camostat mesylate (20mM) or aprotinin (2mgmL(-1)). In the stability experiment, bacitracin reduced the degradation of calcitonin in the different intestinal homogenates. Soybean trypsin inhibitor significantly reduced the degradation of calcitonin in the fluids of the small intestine. We also examined the different endopeptidases in gut luminal fluids and the different exopeptidases in gut mucosal homogenates of rats. The ranking order for the total endopeptidase activity of the intestinal fluids was jejunum > ileum > colon. That for total exopeptidase activity of the intestinal mucosa was jejunum > ileum > colon. These results suggest that endo- and exopeptidases might be responsible for the hydrolysis of calcitonin and that protease inhibitors might usefully improve absorption of calcitonin to the systemic circulation from the large intestine.

Chitosan−Pentaglycine−Phenylboronic Acid Conjugate: A Potential Colon-Specific Platform for Calcitonin

Novel drug delivery vehicles based on the biodegradable, mucoadhesive polysaccharide chitosan covalently linked to a boronic acid protease inhibitor have been prepared and characterized. It was anticipated that these conjugates could protect a proteinaceous drug, such as salmon calcitonin, against proteolysis by serine proteases, an obstacle that prevents its oral administration. Specifically, 4-formylphenylboronic acid was linked to chitosan. Three types of conjugates were prepared. In the first, 4-formylphenylboronic acid was directly linked to chitosan. The other two conjugates employed glycylglycine and pentaglycine spacers. Enzyme-inhibition assays toward trypsin and elastase, in the presence of the enzyme chitosanase, demonstrated a strong inhibitory effect for the chitosan-pentaglycine-phenylboronic acid conjugates, while no inhibitory effect could be detected without chitosanase. The chitosan-pentaglycine-phenylboronic acid conjugate with the highest degree of substitution of 4-formylphenylboronic acid was able to decrease the salmon calcitonin degradation rate by trypsin. It is concluded that chitosan-pentaglycine-phenylboronic acid conjugates are a potential multifunctional, colon-specific vehicle for orally administered salmon calcitonin.

Solution stability of salmon calcitonin at high concentration for delivery in an implantable system.

Salmon calcitonin solutions (50 mg/mL and 100 mg/mL) were placed on stability at 37 degrees C for 1 year in a variety of solvent systems including water, ethanol, glycerol, propylene glycol (PG) and dimethyl sulfoxide (DMSO). Calcitonin degradation was monitored by RP-HPLC and size-exclusion chromatography. DMSO and pH 3.3 solutions provided optimum stability. Conformational stability was also monitored by FTIR over the 1 year time course and compared with chemical and physical stability. After 12 months at 37 degrees C, four major conformations were observed: a beta-sheet conformation (pH 3.3, pH 5.0, 70% DMSO and 70% glycerol), an aggregate conformation (pH 7.0 water), a strong alpha-helical conformation (70% EtOH, 70% PG) and a weak alpha-helical conformation (100% DMSO). No correlation between structure and chemical stability was observed in which both the beta-sheet structure (pH 3.3, water) and a loose alpha-helical structure (100% DMSO) demonstrated good stability. However, some correlation was observed between structure and physical stability, where co-solvents inducing an alpha-helical structure resulted in a decrease in gelation. These two structural states associated with improved stability and minimal gelation, indicated that gelation can be reduced or eliminated by the use of pharmaceutically acceptable co-solvents. Finally, salmon calcitonin (50 mg/mL) was formulated in 100% DMSO and delivered from a DUROS implant over 4 months. Delivery at a target dose of 18 microg/day calcitonin at 37 degrees C was confirmed.

High-performance liquid chromatography/tandem mass spectrometry identification of salmon calcitonin degradation products in aqueous solution preparations.

Degradation impurities have been investigated, by means of high-performance liquid chromatography (HPLC)/ mass spectrometry, in commercial ampoules of salmon calcitonin, aqueous solution, stored for up to 2 years. Oxidation derivatives of the cysteine residues in positions 1 and 7, with cleavage of the disulfide bridge, were observed in all samples. Using our reversed-phase separation, these oxidation derivatives coeluted and presented an HPLC behavior identical to the derivative of salmon calcitonin with the disulfide bridge reduced. In a previous study this last compound was tentatively identified, without confirmation by mass spectrometry, as a major degradation product of salmon calcitonin in aqueous solution.

Localization and degradation of calcitonin in renal proximal tubule of rat visualized by immunocytochemistry.

Immunocytochemical and cryoultramicrotomy techniques were applied to label endogenous as well as intravenously injected calcitonin (CT) in rat kidney. Endogenous CT was located to the membranes of the brush border, endocytotic invaginations and vacuoles and dense apical tubules suggesting degradation at the brush border and/or within endocytotic vacuoles after glomerular filtration of endogenous CT. There was no labeling at the basolateral membranes indicating that possible basolateral binding sites are of minor quantitative importance in degradation. After injection of human CT there was an additional labeling of lysosomes whereas injection of insulin did not change the labeling pattern for endogenous CT indicating that the uptake of CT involves specific binding sites. Quantitative immunocytochemistry after injection of h-CT applying monoclonal antibodies against two different fragments of CT showed that the relative amounts of these fragments differed in vacuoles and lysosomes from that observed in the brush border, but no difference was seen in dense apical tubules. This suggests that injected CT may be degraded both at the brush border, within endocytotic vacuoles and lysosomes and that some fragments are more sensitive to hydrolysis in lysosomes than others. Furthermore, it indicates an early formation of dense apical tubules before detectable degradation of CT occurs in endocytotic vacuoles.

Renal uptake and degradation of trapped-label calcitonin

In order to quantitate the role of the kidneys in the clearance and degradation of calcitonin, a trapped-label procedure was used to label human calcitonin. In contrast to conventional [ 125I]calcitonin, the trapped-label preparation allows quantitative measurements of the extent of uptake as well as of degradation in vivo because the final degradation products do not leave the cells. Trapped-label calcitonin activated adenylate cyclase of bone cells and kidney, as did the native hormone. Ten minutes after intravenous injection into rats, 16% of a trace dose was found in the kidneys. Renal recovery increased to 20% after one hour; in addition, 14% of the injected dose was found in the urine. Eighty per cent of the radioactivity in the urine was in high-molecular weight material. After 90 min, the sum of the accumulated radioactivities in the kidneys and the urine reached 40% of the dose. More than 80% of the radioactivity was sedimentable by centrifuging in a density gradient, indicating that intact calcitonin, as well as the degradation products in the cells, were enclosed within membrane-bound vesicles. Two minutes after injection of trapped-label calcitonin, the peak of radioactivity was found in light gradient fractions associated with cell membrane marker enzymes. Between 5 and 15 min, the peak migrated from light fractions to heavy fractions containing lysosomal marker enzymes. After just 2.5 min, 61% of the renal radioactivity was in low-molecular weight degradation products, as determined by gel filtration. The kinetics of renal degradation of calcitonin indicate that substantial amounts of endocytosed calcitonin is degraded before the hormone reaches the lysosomes.

Plasma calcitonin in rats with renal failure.

To study the effect of the kidney on blood calcitonin (CT), rats were made uremic by either total or partial nephrectomy or bilateral ligation of the ureters. Plasma CT concentrations were measured by radioimmunoassay before and after calcium infusion. Uremia induced by total or partial nephrectomy produced an increase in plasma CT. Rats had higher plasma CT levels (49.3 +/- 1.7 pg/ml, mean +/- SE) after total nephrectomy than after ureteral ligation (30.9 +/- 1.5 pg/ml) even though no significant difference was observed between the blood urea nitrogen (BUN) levels of these two groups. These results indicate that the kidney contributes to the degradation of CT.

Calcitonin Fragmentation by Porcine Tissue in vitro Characterized by Gel Filtration Chromatography

The cytosolic fraction of porcine thyroid, liver, kidney and brain tissue contains proteolytic activity capable of calcitonin degradation into peptide fragments. The degree and pattern of calcitonin fragmentation produced by the tissue examined in this study were determined by subjecting aliquots of incubation media to gel filtration chromatography immediately after incubation. Thyroid cytosol produced calcitonin fragmentation in a time-dependent manner. Fragmentation produced by liver, kidney and brain appeared to be quantitatively and qualitatively different from that produced by the thyroid. Hormone degradation produced by thyroid cytosol was substantially inhibited by p-hydroxymercuribenzoate, thimerosal and iodoacetamide but not by pepstatin. The thyroid cytosolic peptidase may be involved in calcitonin turnover and presecretory regulation within the "C' cell. Further, fragmentation within the thyroid may be responsible for the multiple immunoreactive forms of calcitonin which have been detected in the circulation.

Serum calcitonin in patients with chronic renal disease.

The relationship between serum calcitonin, calcium-phosphorus metabolism and renal function was studied in 66 patients with chronic renal disease. Serum calcitonin was significantly elevated both in non-dialysed patients and in patients on chronic hemodialysis. In the non-dialysed patient group a highly significant inverse correlation was found between serum calcitonin and creatinine clearance. Between serum concentrations of calcitonin and phosphorus a significant positive correlation was found. In the dialysed patients a significant inverse correlation was found between serum calcium and serum calcitonin. It is concluded that the elevated serum calcitonin in patients with chronic renal disease might be explained by a reduced renal degradation of calcitonin and/or an increased production due to stimulation by serum phosphorus.

Analysis of sodium fluoride enhancement of calcitonin stimulation of renal cortical adenylate cyclase.

NaF (5 mu) was previously shown to protect calcitonin-responsive adenylate cyclase activity in partially purified membranes from pork renal cortex during solubilization of the enzyme with Lubrol PX (Queener, S. F., Fleming, J. W., and Bell, N. H. (1975) J. Biol. Chem. 250, 7586-7592). NaF has now been found to directly influence the activity of the membrane-bound calcitonin-responsive adenylate cyclase. At low concentrations of NaF causing no direct stimulation of adenylate cyclase activity, NaF produced a synergistic increase with calcitonin but not with parathyroid hormone or glucagon. At maximally stimulating concentrations of NaF, calcitonin produced a further increase in enzyme activity. NaF alone increased the maximal velocity (I’,,) and did not affect the apparent K, of the enzyme for ATP, whereas calcitonin alone did not alter the V,, but lowered the K, for ATP. Concentrations of NaF too low to directly alter either K,,, or V,, significantly increased V,, and decreased K,,, when added with calcitonin. De Ha&n plots (De Ha&, C. (1974) J. Biol. Chem. 249, 2756-2762) of adenylate cyclase activation by calcitonin with and without NaF indicated that NaF increased the maximal response of the enzyme to calcitonin without affecting the affinity of the enzyme for calcitonin. These effects of NaF could not be attributed to alterations in the activity of phosphodiesterase in these membranes. In addition, NaF had no effect on the slope of the Hill plot for activation by calcitonin and, likewise, calcitonin had no effect on the slope of the Hill plot for activation by NaF. NaF did not alter binding of ‘251-calcitonin to renal cortical membranes or the degradation of calcitonin as determined by chromatoelectrophoresis and radioimmunoassay. NaF was shown to increase the heat lability of the enzyme. It is concluded (a) that NaF at low concentrations acts to enhance responsiveness of renal cortical adenylate cyclase to calcitonin without altering binding or degradation of the hormone and (b) that NaF at high concentrations directly stimulates the catalytic component of the enzyme independent of any action of the hormone. These results suggest that NaF acts at different sites to influence the calcitonin-responsive adenylate cyclase system of pork renal cortex.

Characteristics of calcitonin degradation in vitro produced by incubation with porcine thyroid tissue

The possibility of calcitonin (CT) degradation by thyroid tissue in vitro was investigated. Tissue slices or a thyroid supernatant solution was incubated with 131I-labeled porcine CT for 30–60 min at 37°C. Calcitonin degradation and deiodination were determined by chromatoelectrophoresis and/or quso adsorption. Degradation of [ 131I]CT occurred rapidly in the presence of 50–200 mg porcine thyroid tissue; however, little or no hormone deiodination was observed. Porcine liver, kidney and lung slices also degraded [ 131I]CT. The data indicate that a nonspecific proteolytic enzyme, capable of rapid CT degradation, is liberated from thyroid tissue slices in vitro. This substance, which is found in the cytosol fraction of thyroid tissue, is heat labile, has a molecular weight between 25,000 and 100,000, displays an acidic pH maximum for CT degradation and is inhibited by sulfhydryl enzyme inhibitors. The liberation of this substance from thyroid slices may explain the relative unresponsiveness of CT secretion observed in previous in vitro studies.

Degradation of calcitonin during incubation with thyroid slices in vitro.

Division of Pharmacodynamics and Toxicology, College of Pharmacy, 644 Northeast 14th Street, Health Sciences Center, Oklahoma City, Oklahoma 73104, U.S.A. (Received 30 September 1977) The secretion of calcitonin in vitro has recently been examined in various mammalian and submammalian species. It appears that calcium and other calcitonin secretagogues produce a much greater secretory response in vivo than in vitro (Bell, 1970; Feinblatt, Raisz & Kenny, 1973; Gautvik & Tashjian, 1974; Selawry, Becker, Bivins, Snider & Silva, 1975). We have previously suggested that the relative unresponsiveness observed in vitro may be due to calcitonin-induced self-inhibition resulting from the accumulation of calcitonin in a closed incubation system in vitro (Orme & Pento, 1976), However, another possible explanation for the secretory unresponsiveness in vitro may be the occurrence of tissue-induced degradation of the secreted calcitonin. Therefore, the present study examined the influence of thyroid tissue from various species on the degradation of calcitonin

On the nature of degradation of calcitonin by mammalian cells.

SummaryBy the use of immunological reactivity as an indicator, the degradation of synthetic human calcitonin was measured with cells isolated from rat kidney and bone after partial digestion with 0.3% crude collagenase. The degradation of the hormone by bone cells was undetectable (with up to 9 × 105 cells per 0.5 ml). In contrast, renal cells degrade HCT readily. The degradation activity in renal cells appear to be cell bound. Measurement by specific radioimmunoassay of the degradation of HCT in the presence of SCT or PCT suggests competition between three different species of calcitonins for a possible common site of degradation not shared by other peptide hormones tested.

Degradation of radioiodinated porcine and human synthetic calcitonins by sera of different species

The in vitro degradation of porcine and human synthetic calcitonin was measured in the presence of sera of different species (rat, pig, human) using a chromatoelectrophoresis method. Degradation is greater when heterologous sera are used. The degradation was not prevented by enzyme inhibition. Damage was greatest at acid pH and was not removed by heating of the serum. Ammonium sulfate precipitations and euglobulin separation of serum proteins did not result in the isolation of the factor responsible for the degradation of calcitonin. Mercaptoethanol prevented the damage from appearing which suggested that this damage was in part due to breakage of the disulfide bond.

Degradation of calcitonin by homologous thyroid acid proteinase.

An acid proteinase purified from pig thyroid gland rapidly degraded porcine calcitonin labeled with radioiodine and destroyed the biological activity of unlabeled calcitonin. The proteinase was most active at pH below 5.2 and inactive at pH 7.7. Acid extraction of thyroid glands may result in an underestimate of the calcitonin content of the tissues. (Endocrinology 86: 447, 1970)