The use of the synthetic form of GnRH aimed to achieve higher stability against degradation of the assembly 19 (Fig. However, in the studies with hGH and LHRH, the concept of amyloids was applied to prepare complexes capable of slowly releasing the active peptide.īased on the “amyloid concept” from these studies, we investigated whether a controlled pH shift in the presence of different concentrations of Zn 2+ would lead to self-assembly and formation of nanostructures and fibrils of a synthetic D-Phenylalanine (Phe) form of GnRH peptide in vitro. The amyloid structures are highly organized stable aggregates, which in most cases are associated with neurodegenerative diseases 18. In both cases, peptides with the ability to form amyloids were investigated. Comparable to hGH, LHRH exhibited a similar tendency to form β-sheet rich aggregates with amyloid-like character 15. The same technique for complexation and precipitation was applied in another study with GnRH, tyroliberin (TRH), dalarelin and ACTH for the formation of Zn 2+-peptide aqueous suspension 17. The lyophilizate was then incorporated into in situ forming implants 16. In this connection, LHRH was complexed and precipitated with Zn 2+, using sodium hydroxide (NaOH) solution for the pH adjustment. The amyloid structures have also been considered as a possible source for sustained release of small peptides, e.g. Various studies have investigated short amyloidogenic peptides and their assemblies that mimic the amyloidosis process 10, 11, 12, 13, 14. The formed structures of Zn 2+-hGH were later found to be fibrils of amyloidogenic nature, able to release monomers upon dilution 9. The Zn 2+-hGH dimer was more stable than the monomeric hGH form 7, 8. The addition of zinc induced the dimerization of human growth hormone (hGH). Similarly, corticotrophin (ACTH) was precipitated with zinc salt 6. The prepared suspension was then further optimized with regards to pH and zinc salt 5.
A precipitation of recombinant Hirudin (rHir) by zinc salt at neutral pH was as well shown to result in Zn 2+-rHir suspension with a prolonged biological activity in rats 4. It further reduced the initial burst release of GLP-1 from the formulation vehicle, indicating its potential for long term release 3. A non-covalently bound adduct of glucagon-like peptide-1 (GLP-1) agonist with zinc acetate could prolong its terminal half-life t 1/2 after subcutaneous application from 2 h to 8.5 h. Recently, a similar approach was adopted with other peptide compounds in order to achieve their controlled and sustained release. The studies found the efficiency of insulin precipitation to be a function of pH and zinc salt concentration.
The development of insulin sustained release formulations for injection originated from studies of the insulin solubility in acetate and phosphate buffer 2. The extended availability of insulin from an insoluble zinc complex has been long known and utilized. Insulin, for instance, is a prominent example for a peptide stored in the pancreas in the form of insoluble zinc complex 1. The in vitro release was slow and continuous over 14 days and not influenced by the oil matrix.Ī non-covalent complexation of peptides with metal ions controlling their release is a method, which resembles their storage in vesicles at physiological conditions. The lyophilized Zn 2+: GnRH assembly was tested as a platform for the sustained delivery of GnRH and incorporated into two different oil vehicle matrices. In contrast to already existing short peptide fibrils, GnRH nanostructures and fibrils form in a Tris-buffered pH environment in a controlled manner through a temperature reduction and a pH shift. Molecular Dynamic (MD) simulation of the 10:1 Zn 2+: GnRH explored the interaction and dimerization processes. Atomic Force Microscopy (AFM) showed the existence of GnRH oligomers and fibrils. Nuclear Magnetic Resonance spectroscopy (NMR) indicated a weak interaction between Zn 2+ and GnRH. Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR–FTIR) revealed the existence of higher order assembly of Zn 2+: GnRH. A synthetic derivative, GnRH, stable against enzymatic degradation, self-assembles and forms nanostructures and fibrils upon a pH shift in the presence of different concentrations of Zn 2+ in vitro.
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