The stability of proteins and peptides

In Chapter 4 we examined how the stability of dmg formulations may be improved from a knowledge of the principal routes of degradation and the kinetics of breakdown. It has been implicitly assumed that the dmgs under discussion were typical low molecular weight compounds and it is clear that for this type of 

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Pancreatic enzymes strongly effect the stability of proteins and peptides, but... 

Pancreatic enzymes strongly effect the stability of proteins and peptides, but absence of pancreatic juice also changes the absorptive properties of the gastrointestinal tract [33]. 

Biodegradable polymeric nanoparticles have been prepared and used to enhance the stability of proteins and peptides, control their release and pharmacokinetic parameters, furthermore, to improve their bioavailability. For delivery purposes, the polymeric material needs to meet physicochemical and biological requirements, of which, biocompatibility, safety, and biodegradability into non-toxic metabolites are of cmcial importance. The polymers can be easily functionalised towards off opsoni-sation. They are also known to show reduced toxicity in the peripheral healthy tissues. The selection of polymers depends on the method of administration, the bioactive molecules to be loaded, the desired release profile, the intention to target specific tissues, the desired rate of particle degradation, and the biocompatibility. Table 11.3 outlines some of the natural and synthetic polymers currently used in the fabrication of nanoparticles. 

The presence of a solvent, especially water, and/or other additives or impurities, often in nonstoichiometric proportions, may modify the physical properties of a solid, often through impurity defects, through changes in crystal habit (shape) or by lowering the glass transition temperature of an amorphous solid. The effects of water on the solid-state stability of proteins and peptides and the removal of water by lyophilization to produce materials of certain crystallinity are of great practical importance although still imperfectly understood. 

Sequence inversion and racemization have been associated with uncatalyzed formation of the cyclic dipeptides and has been shown to greatly complicate the kinetics of formation. Cyclic dipeptide formation, by uncatalyzed processes, is rapid enough to pose an apparent threat to the stability of proteins and a possible rationale for the posttranslational N-acetylation of proteins that have been observed in higher organisms. The rate of DKP formation will also depend on the carbonyl ester protecting groups or the structures of the peptide-resin linkage in the solid-phase mode. Furthermore, cyclization is a concentration-independent reaction and demands the use of dilute solutions. ... 

Packaging conditions have effects on the stability of protein products. Peptide and protein drugs are high-molecular-weight compounds with unique physicochemical properties. They are extremely sensitive to their microenvironment heat, fight, pH, chemical contaminants, and so on. Trace amounts of metals, plasticizers, and... 

Specific formulation strategies need to be employed for macromolecule compounds. An excellent review of protein stability in aqueous solutions has been published by Chi et al. (92). In addition to solution stability of proteins and peptides, aerosolization may result in significant surface interfacial destabilization of these compounds if no additional stabilization excipients are added. This is due to the fact that protein molecules are also surface active and adsorb at interfaces. The surface tension forces at interfaces perturb protein structure and often result in aggregation (92). Surfactants inhibit interface-induced aggregation by limiting the extent of protein adsorption (92). 

The LTQ-FT mass spectrometer was introduced in late 2003 and, as expected, the main application discussed in the literature is for the analysis of proteins and peptides (Johnson et al., 2004 Syka et al., 2004). A recent book chapter (van der Greef et al., 2004) and a review article (Brown et al., 2005) discussed the application of the LTQ-FT to metabolomics. FTMS applications to dmg metabolism are still very new and dmg discovery research laboratories which have recently purchased the instmment are still in the process of developing and validating methods and approaches. A recent publication describes the depth and flexibility of the experimental setup utilizing accurate mass data-dependent exclusion MS" measurements with a LTQ-FT (Tozuka et al., 2005). We have reported several integrated approaches for determination of metabolic stability, characterization of metabolites and metabolic... 

This chapter reviews a) the characterization of proteins and peptides in a variety of non-aqueous or co-solvent conditions, both acceptable and unacceptable for pharmaceutical applications, b) the applicability of non-aqueous conditions for increasing solubility, stability and activity, and c) novel drug delivery and formulation process technology applications. This review focuses on non-aqueous solutions, suspensions and co-solvent systems that result in miscible conditions. 

The chemical and physical stability of protein and peptide drugs is considered in a separate chapter of this book. Although some newer texts have comprehensively addressed the difficult subject of protein stability, it was felt that no drug stability text would be complete without this subject. 

In order to obtain independent evidence for the involvement of the cyclodextrin cavity, fluorescence measurements were carried out for copper(II) ternary complexes with L- or D-tryptophan. In fact, the fluorescence spectrum of tryptophan has already been shown to be sensitive to the polarity of the microenvironment in which it is located and has been used in many studies as a probe for the conformation of proteins and peptides [53]. As for many fluorophores, the indole fluorescence of Trp is quenched by the copper(II) ion this effect has been used as a measure of the stability constants of copper(II) complexes [54, 55]. In a recent work, it has been shown that the fluorescence of dansyl derivatives of amino acids undergo enantioselective fluorescence quenching by chiral copper(n) complexes and that fluorescence measurements can be used for the study of enatioselectivity in the formation of ternary complexes in solution [56]. Bearing this in mind, we performed the same type of experiments by adding increasing amounts of the [Cu(CDhm)] + complex to a solution of D- or L-tryptophan [36]. The fluorescence titration curve shows that the artificial receptor inhibits the indole... 

Within the framework of a cooperative research project founded by the EC, a procedure has been developed for the formulation of injectable bio-erodible nanoparticles with fairly uniform size distribution and high environmental stability that appear well suited for targeted administration of protein and peptide dmgs. 

This fluorous effect can also be effective for the stabilization of proteins. For example, the selective pairing of peptides that contain perfluorinated chains has been demonstrated, and perfluorinated amino acid side chains have been shown to stabilize the hydrophobic-driven folding of proteins. ... 

Physical and Chemical Integrity of Proteins. The primary sequence of proteins and peptides is comprised of L-amino acids linked together by covalent amide bonds. Substituent group polarity and/or charge is a critical determinant of secondary and tertiary structure and stability. Secondary structures (a-helices and P-sheets) arise from hydrophobic, ionic, and Van der Waals interactions that fold the primary amino acid chain upon itself. Most therapeutic proteins exhibit tertiary structure vital to functionality and are held together by covalent and noncovalent bonding of secondary structures (Figure 5.2). 

Proteins, peptides, and other polymeric macromolecules display varying degrees of chemical and physical stability. The degree of stability of these macromolecules influence the way they are manufactured, distributed, and administered. Chemical stability refers to how readily the molecule can undergo chemical reactions that modify specific amino-acid residues, the building blocks of the proteins and peptides. Chemical instability mechanisms of proteins and peptides include hydrolysis, deamidation, racemization, beta-elimination, disulfide exchange, and oxidation. Physical stability refers to how readily the molecule loses its tertiary and/or sec-... 

The stability of proteins toward covalent degradation pathways can often depend on the protein s folded state. In each pathway, solvent accessibility and varying degrees of structural freedom of the peptide backbone and/or side chains around the labile residue are required for reactions to take place. Accordingly, stabilization of the protein s folded state (i.e., its compact structure) that minimizes solvent accessibility can lower the reaction rate of some covalent protein modifications, extending the shelf life of the protein product. Therefore, the selection of formulation excipients depends on their direct and indirect influence on the rates of covalent protein degradation. 

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