Can aggregation of insulin govern its fate in the intestine? Implications for oral delivery of the drug (original) (raw)
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pH-responsive modulation of insulin aggregation and structural transformation of the aggregates
Biochimie, 2015
Over the past two decades, much information has appeared on electrostatically driven molecular mechanisms of protein self-assembly and formation of aggregates of different morphology, varying from soluble amorphous structures to highly-ordered amyloid-like fibrils. Protein aggregation represents a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. This has awakened interest in identification of pH-triggered regulators of transformation of aggregation-prone proteins into structures of higher order. The objective of the present study is to elucidate the effects of low-molecular-weight biogenic agents on aggregation and formation of supramolecular structures of human recombinant insulin, as a model therapeutic protein. Using dynamic light scattering, turbidimetry, circular dichroism, fluorescence spectroscopy, atomic force microscopy, transmission electron microscopy, and nuclear magnetic resonance, we have demonstrated that the amino acid L-arginine (Arg) has the striking potential to influence insulin aggregation propensity. It was shown that modification of the net charge of insulin induced by changes in the pH level of the incubation medium results in dramatic changes in the interaction of the protein with Arg. We have revealed the dual effects of Arg, highly dependent on the pH level of the solution e suppression or acceleration of the aggregation of insulin at pH 7.0 or 8.0, respectively. These effects can be regulated by manipulating the pH of the environment. The results of this study may be of interest for development of appropriate drug formulations and for the more general insight into the functioning of insulin in living systems, as the protein is known to release by exocytosis from pancreatic beta cells in a pH-dependent manner.
Molecular Aspects of Insulin Aggregation and Various Therapeutic Interventions
ACS Bio & Med Chem Au, 2022
Protein aggregation leading to the formation of amyloid fibrils has various adverse effects on human health ranging from fatigue and numbness to organ failure and death in extreme cases. Insulin, a peptide hormone commonly used to treat diabetes, undergoes aggregation at the site of repeated injections in diabetic patients as well as during its industrial production and transport. The reduced bioavailability of insulin due to aggregation hinders the proper control of glucose levels in diabetic patients. Thus, it is necessary to develop rational approaches for inhibiting insulin aggregation, which in turn requires a detailed understanding of the mechanism of fibrillation. Given the relative simplicity of insulin and ease of access, insulin has also served as a model system for studying amyloids. Approaches to inhibit insulin aggregation have included the use of natural molecules, synthetic peptides or small molecules, and bacterial chaperone machinery. This review focuses on insulin aggregation with an emphasis on its mechanism, the structural features of insulin fibrils, and the reported inhibitors that act at different stages in the aggregation pathway. We discuss molecules that can serve as leads for improved inhibitors for use in commercial insulin formulations. We also discuss the aggregation propensity of fast-and slow-acting insulin biosimilars, commonly administered to diabetic patients. The development of better insulin aggregation inhibitors and insights into their mechanism of action will not only aid diabetic therapies, but also enhance our knowledge of protein amyloidosis.
Search for New Aggregable Fragments of Human Insulin
Molecules
In this study, three independent methods were used to identify short fragment of both chains of human insulin which are prone for aggregation. In addition, circular dichroism (CD) research was conducted to understand the progress of aggregation over time. The insulin fragments (deca- and pepta-peptides) were obtained by solid-phase synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) as a coupling reagent. Systematic studies allowed identification of the new fragments, expected to be engaged in triggering aggregation of the entire structure of human insulin under physiological conditions. It was found that the aggregation process occurs through various structural conformers and may favor the formation of a fibrous structure of aggregate.
A novel approach using functional peptides for efficient intestinal absorption of insulin
Journal of Controlled Release, 2007
The aim of this study was to evaluate whether oligoarginine, a cell-penetrating peptide (CPP), can improve intestinal absorption of insulin in rats. Peptides composed of six (R 6), eight (R 8) and 10 (R 10) residues of arginine were used as the CPP. No insulin absorption was observed following administration of insulin solution alone; however, insulin absorption increased dramatically after coadministration of the D-form of R 6 (D-R 6) and the L-form of R 6 (L-R 6) in a dose-dependent manner. The effects on insulin absorption were more pronounced for D-R 6 than for L-R 6. Among oligoarginines composed of six, eight, or 10 arginine residues, D-R 8 showed the strongest enhancing effects on insulin intestinal absorption. In contrast, intestinal absorption of other model hydrophilic macromolecules, interferon-β and fluorescein isothiocyanate-labeled dextran 4400, was not affected by coadministration with oligoarginine. Pretreatment by the effective dose of L-R 6 did not induce lactate dehydrogenase leakage or histological damage, suggesting that oligoarginine has no untoward effect on the intestinal mucosa. Our data demonstrate that coadministration of oligoarginine increases intestinal insulin absorption markedly without causing detectable damage in cellular integrity and that the covalent binding between insulin and oligoarginine is not necessary for this effect. We conclude that oligoarginines are likely to become powerful tools for overcoming the low permeability of insulin through the epithelial cell membrane, the major barrier to oral insulin delivery.
Structural Analysis and Aggregation Propensity of Reduced and Nonreduced Glycated Insulin Adducts
Applied Biochemistry and Biotechnology, 2013
The milieu within pancreatic β cells represents a favorable environment for glycation of insulin. Therefore, in this study, insulin samples were individually subjected to glycation under reducing and nonreducing conditions. As monitored by orthophthalaldehyde and fluorescamine assays, the reduced glycated insulin adduct demonstrates extensively higher level of glycation than the nonreduced glycated counterpart. Also, gel electrophoresis experiments suggest a significant impact of glycation under a reducing system on the level of insulin oligomerization. Furthermore, reduced and nonreduced glycated insulin adducts respectively exhibit full and partial resistance against dithiothreitol-induced aggregation. The results of thioflavin T and Congo red assays suggest the existence of a significant quantity of amyloid-like entities in the sample of reduced glycated insulin adduct. Both fluorescence and far-ultraviolet circular dichroism studies respectively suggest that the extents of unfolding and secondary structural alteration were closely correlated to the level of insulin glycation. Moreover, the surface tension of two glycated insulin adducts was inversely correlated to their glycation extents and to the quantity of exposed hydrophobic patches. Overall, the glucose-modified insulin molecules under reducing and nonreducing systems display different structural features having significant consequences on aggregation behaviors and surface tension properties. The particular structural constraints of glycated insulin may reduce the binding interaction of this hormone to its receptor which is important for both insulin function and clearance.
Journal of Pharmaceutical Sciences, 2016
A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.
Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin
Science in China Series C, 2003
Blood glucose lowering assay proved that [B16Ala]insulin and [B26Ala]insulin exhibit potency of acute blood glucose lowering in normal pigs, which demonstrates that they are fastacting insulin. Single-chain precursor of [B16Ala]insulin and [B26Ala]insulin is [B16Ala]PIP and [B26Ala]PIP, respectively, which are suitable for gene expression. Secretory expression level of the precursors in methylotrophic yeast Pichia pastoris was quite high, 650 mg/L and 130 mg/L, respectively. In vivo biological assay showed that the two fast-acting insulins have full or nearly full biological activity. So both [B16Ala]insulin and [B26Ala]insulin can be well developed as fast-acting insulin for clinic use.
Preparation of high-potency, non-aggregating insulins using a novel sulfation procedure
Diabetes, 1983
The marked propensity of insulin to self-associate into large aggregates causes significant mechanical problems in insulin delivery devices and may also stimulate production of a tissue-amyloid A precursor in some patients. Although conventionally prepared sulfated insulin (SI) resists aggregation, clinical application has been limited by major insulin bioactivity losses that occur during synthesis. To eliminate this problem, insulin sulfation was carried out in the organic solvent dimethylformamide in the presence of condensing agents such as N,N'-dicyclohexyl carbodiimide (DCC) and a sulfate donor. With this new procedure, the degree of sulfation could be controlled over an eightfold range by varying the amount of condensing agent. The bioactivity of these new SI derivatives varied between 78% and 87% of unmodified insulin. Insulin aggregation, induced by passage through a syringe and needle, did not occur with derivatives having two or more sulfate moieties per insulin molecule. Diffusion velocity studies using "nonaggregated" insulin solutions demonstrated that aggregates were present in crystalline zinc and sodium porcine insulin. In contrast, SI having more than 0.5 mole sulfate per mole of insulin dialyzed as if it were predominantly in the monomeric form. Results from the studies described in this report now provide the means for selectively designing and preparing specific high-potency, non-aggregating insulins, which may be necessary for optimal use of current and future insulin delivery devices, DIABETES 32:1087-1091, December 1983.
PLOS ONE, 2017
The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.