Gonadorelin[6-D-Phe]

A synthetic derivative, GnRH [6-D-Phe], stable against enzymatic degradation, self-assembles and forms nanostructures and fibrils upon a pH shift in the presence of different concentrations of Zn2+ in vitro. Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed the existence of higher order assembly of Zn2+: GnRH [6-D-Phe]. Nuclear Magnetic Resonance spectroscopy (NMR) indicated a weak interaction between Zn2+ and GnRH [6-D-Phe]. Atomic Force Microscopy (AFM) showed the existence of GnRH [6-D-Phe] oligomers and fibrils. Molecular Dynamic (MD) simulation of the 10:1 Zn2+: GnRH [6-D-Phe] explored the interaction and dimerization processes. In contrast to already existing short peptide fibrils, GnRH [6-D-Phe] nanostructures and fibrils form in a Tris-buffered pH environment in a controlled manner through a temperature reduction and a pH shift. The lyophilized Zn2+: GnRH [6-D-Phe] assembly was tested as a platform for the sustained delivery of GnRH [6-D-Phe] and incorporated into two different oil vehicle matrices. The in vitro release was slow and continuous over 14 days and not influenced by the oil matrix.

With respect to the assessment of residue situation and as a part of preclinical trials to determine the biological activities of potential gonadotropin releasing hormone (GnRH) residues in porcine organisms the GnRH agonist Gonadorelin[6-D-Phe] (D-Phe(6)-LHRH) was administered either enterally or intramuscularly (i.m.) to female Goettinger miniature pigs in order to evaluate the GnRH-induced luteinizing hormone (LH) surge. Gilts received an (i) enteral application of 10 mg D-Phe(6)-LHRH via a probang (enteral group, n=7), (ii) i.m. injection of 0.1 mg D-Phe(6)-LHRH (parenteral group, n=5), or (iii) saline injection (control group, n=4). The GnRH and saline applications were repeated every second day with up to seven repetitions. Blood samples were collected via previously fitted jugular catheters immediately before injections, over an 8 h period in 1 h intervals beginning 2 h after injections, and at 24, 26, 28 and 30 h after applications. Enteral application of D-Phe(6)-LHRH induced an LH surge in 23 of 30 treatments. All gilts in the parenteral group exhibited LH release after each D-Phe(6)-LHRH application (P<0.05), whereas no LH surges were observed after saline injection in the control group. A significant (P<0.05) LH rise to mean maximum LH concentrations of 3.25 +/- 0.43 and 3.05 +/- 0.26 ng/ml occurred in both the enteral and parenteral groups, but there was no difference in the time interval after GnRH (2.6 +/- 0.3 vs. 2.3 +/- 0.3 h) and the mean duration of LH peak (6.5 +/- 0.4 and 6.8 +/- 0.3 h) between the treatment groups. In conclusion, (i) enteral application of 10 mg D-Phe(6)-LHRH induced LH release in a physiological range from the pituitary of female minipigs, and (ii) neither an accumulative effect nor a cumulative LH response were found after repeated GnRH application. Furthermore, (iii) in regard to consumer protection and gonadotropin secretion, D-Phe(6)-LHRH residues can be excluded from having long-term effects.

Sustained release lipid microparticles for a potential veterinary application were produced by the means of spray congealing using saturated triglycerides with respective surfactants. The spray congealing process was optimized using unloaded and loaded microparticles, revealing the highest impact of the spray flow on material loss. Yield could be optimized by increasing the spray flow as well as a reduction of the melt temperature from 90 to 75 °C. For the delivery system developed in this study, a release of around 15 days was targeted. The release profile was in first hand determined with the use of model substances (aspartame and tryptophan), before incorporating the decapeptide Gonadorelin [6-D-Phe]. Release could be controlled between 2 and 28 d, which was dependent on stability of microparticles upon incubation, type and concentration of emulsifier, as well as the used triglyceride. Differential scanning calorimetry and X-ray powder diffraction confirmed the crystallization behavior of C14 and C16-triglycerides in combination with various emulsifiers in different modification without impact on release.