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Octreotide is the first synthetic peptide hormone, consisting of eight amino acids, that mimics the activity of somatostatin, a natural hormone in the body. During the past decades, advanced instrumentation and crystallographic software have established X-Ray Powder Diffraction (XRPD) as a valuable tool for extracting structural information from biological macromolecules. The latter was demonstrated by the successful structural determination of octreotide at a remarkably high d-spacing resolution (1.87 Å) (PDB code: 6vc1). This study focuses on the response of octreotide to different humidity levels and temperatures, with a particular focus on the stability of the polycrystalline sample. XRPD measurements were accomplished employing an Anton Paar MHC-trans humidity-temperature chamber installed within a laboratory X’Pert Pro diffractometer (Malvern Panalytical). The chamber is employed to control and maintain precise humidity and temperature levels of samples during XRPD data collection. Pawley analysis of the collected data sets revealed that the octreotide polycrystalline sample is remarkably stable, and no structural transitions were observed. The compound retains its orthorhombic symmetry (space group: P212121, a = 18.57744(4) Å, b = 30.17338(6) Å, c = 39.70590(9) Å, d ~ 2.35 Å). However, a characteristic structural evolution in terms of lattice parameters and volume of the unit cell is reported mainly upon controlled relative humidity variation. In addition, an improvement in the signal-to-noise ratio in the XRPD data under a cycle of dehydration/rehydration is reported. These results underline the importance of considering the impact of environmental factors, such as humidity and temperature, in the context of structure-based drug design, thereby contributing to the development of more effective and stable pharmaceutical products.

2-benzothiazoles and 2-(aminophenyl)benzothiazoles represent biologically interesting heterocycles with high pharmacological activity. The combination of these heterocycles with amino acids and peptides is of special interest, as such structures combine the advantages of amino acids and peptides with the advantages of the 2-benzothiazolyl and 2-(aminophenyl)benzothiazolyl pharmacophore group. In this work, we developed an easy and efficient method for the solid-phase synthesis of 2-benzothiazolyl (BTH) and 2-(aminophenyl)benzothiazolyl (AP-BTH) C-terminal modified amino acids and peptides with high chiral purity.

Relaxin (RLX) is a protein that is structurally similar to insulin and has interesting biological activities. As with all proteins, preservation of RLX’s structural integrity/biological functionality is problematic. Herein, we investigated two methods for increasing the duration of relaxin-2’s (RLX2) biological activity: synthesis of a palmitoyl RLX2 conjugate (P-RLX2) with the use of a Palmitoyl-l-Glu-OtBu peptide modifier, and encapsulation into liposomes of P-RLX2, RLX2, and its oxidized form (O-RLX2). For liposomal encapsulation thin-film hydration and DRV methods were applied, and different lipid compositions were tested for optimized protein loading. RLX2 and O-RLX2 were quantified by HPLC. The capability of the peptides/conjugate to stimulate transfected cells to produce cyclic adenosine monophosphate (cAMP) was used as a measure of their biological activity. The stability and bioactivity of free and liposomal RLX2 types were monitored for a 30 d period, in buffer (in some cases) and bovine serum (80%) at 37 °C. The results showed that liposome encapsulation substantially increased the RLX2 integrity in buffer; PEGylated liposomes demonstrated a higher protection. Liposome encapsulation also increased the stability of RLX2 and O-RLX2 in serum. Considering the peptide’s biological activity, cAMP production of RLX2 was higher than that of the oxidized form and the P-RLX2 conjugate (which demonstrated a similar activity to O-RLX2 when measured in buffer, but lower when measured in the presence of serum proteins), while liposome encapsulation resulted in a slight decrease of bioactivity initially, but prolonged the peptide bioactivity during incubation in serum. It was concluded that liposome encapsulation of RLX2 and synthetic modification to P-RLX2 can both prolong RLX2 peptide in vitro stability; however, the applied chemical conjugation results in a significant loss of bioactivity (cAMP production), whereas the effect of liposome entrapment on RLX2 activity was significantly lower.