Insulin chemical degradation

Allen, L.V., Jr., A history of pharmaceutical compounding. Secundum Artem, 11, 3, 2003. Brange, J., Langkjaer, L., Havelund, S., and Vcjlund, A., Chemical stability of insulin. Hydrolytic degradation during storage of pharmaceutical preparations, Pharm. Res., 9, 715, 1991. 

In general, transition metal ions are undesired in protein formulations because they can catalyze physical and chemical degradation reactions in proteins. However, specific metal ions are included in formulations when they are cofactors to proteins and in suspension formulations of proteins where they form coordination complexes (e.g., zinc suspension of insulin). Recently, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (63). 

Other chemical degradation pathways include -elimination, which can lead to racemization and disulphide exchange reactions. The amino acids that may undergo P-elimination include Cys, Ser, Thr, Phe, and Lys (40) and occur especially at alkaline pH (64). The reduction and oxidation of the disulphide bonds are often accompanied by a considerable change in the protein conformation (23,33). An example is the secondary structure of insulin that is disrupted or completely lost when the disulphide bonds are broken (23). The disulphide bond disruption or interchange can also result in an altered three-dimensional structure and therefore a possible loss of activity (40) or aggregation (29). For further details, the reader is directed to the following reviews and book chapters (30,40,62,65). 

In addition, asparagine at the C-terminus of the A chain is replaced by glydne, reducing the likelihood of chemical degradation which might cause cross-linking with the modified C-terminus of the B chain (Fig. 27.10). Insulin Glargine has a duration of action of 24 h. 

J. Brange, L. Langkjaer, S. Havelund, A. Vplund, Chemical Stability of Insulin. 1. Hydrolytic Degradation During Storage of Pharmaceutical Preparations , Pharm. Res. 1992, 9, 715-726. 

Many chemicals important to genetic research have been produced at high levels, e.g. DNA ligase production in E. coli can be enhanced several hundred fold. Other enzymes can also be produced for industrial use and enhanced degradative ability has been generated for instance, enhanced petroleum degraders can be added to oil spills to accelerate clean-up operations. However, it is in the production of medically related compounds that this technology has been most successfully applied as, for example, in the production of insulin. 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

Chemical instability Some drugs, such as penicillin G (see p. 302), are unstable in the pH of the gastric contents. Others, such as insulin (see p. 258), may be destroyed in the Gl tract by degradative enzymes. 

The structure and properties of peptides and proteins depend critically upon the sequence of amino acids in the peptide chain. The first complete amino acid sequence of a protein, that of insulin (51 amino acid residues), was determined by F. Sanger in 1953. The process is now performed using automated protein sequencers, and involves step-by-step identification of amino acids at the N terminus of the protein using a chemical process known as Edman degradation. 

In this chapter, we summarize the general approaches that have been used to successfully achieve the formulation goals for oral delivery minimize enzymatic degradation enhance intestinal absorption maximize blood level reproducibility deliver drug through the gut wall and produce a palatable and acceptable dosage form. Then insulin will be used as an example to show how oral bioavailability has been achieved through chemical modification. 

Intemasal delivery of peptide and protein drugs is severely restricted by pre-systemic elimination due to enz5miatic degradation or mucociliary clearance and by the limited extent of mucosal membrane permeability. a-CyD has been shown to remove some fatty acids from nasal mucosa and to enhance the nasal absorption of leuprolide acetate in rats and dogs. The utility of chemically modified CyDs as absorption enhancers for peptide drugs in rats has been demonstrated. For example, DM-P-CyD was shown to be a potent enhancer of insulin absorption in rats, and a minimal effective concentration of DM-(3-CyD for absorption enhancement exerted only a mild effect on the in vitro ciliary movement.The scope of interaction of insulin with CyDs is limited, because CyDs can only partially include the hydrophobic amino acid residues in peptides with small stability constants. Under in vivo conditions, these complexes will readily dissociate into separate components, and hence the displacement by membrane lipids may further destabilize the complexes. The direct interaction of peptides with CyDs is therefore of minor importance in the enhancement of nasal absorption. Of the hydrophilic CyDs tested, DM- 3-CyD had the most prominent inhibitory effect on the enzymatic degradation of both BLA and insulin in rat nasal tissue homogenates. Because of the limited interaction between peptides and CyDs,... 

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