Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation

Mariano Venanzi,Marco Savioli,Maribel Guerra‐Vallespi,Emanuela Gatto,Julio Raul Fernandez Masso,Hilda Garay Perez,Lorenzo Antonelli,Ernesto Placidi,Gianfranco Bocchinfuso,Hector Santana,Rita Cimino,Francesca Cavalieri

doi:10.1002/psc.3356

Abstract

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH2 ), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.

Highlights

Despite the relevant progress in the prevention and therapy of tumours, cancer diseases remain one of the most important causes of death
For both the excitation wavelengths, the fluorescence emission spectra of CIGB552 in water and in the therapeutic formulation, show a relatively broad emission band centred at λem = 359 nm, typical of the indole emission in a polar environment (Figure S1)
The aggregation properties of CIGB552 in water and in its therapeutic formulation were investigated by optical spectroscopy and microscopy technique with nanometric resolution

Summary

| INTRODUCTION

Despite the relevant progress in the prevention and therapy of tumours, cancer diseases remain one of the most important causes of death. A new therapeutic peptide, derived from Limulus sp, and based on the amphipathic cyclic pentapeptide LALF32-51, denoted as L2, was investigated as a novel antitumor peptide.[7] With the aim to improve the antitumor activity of L2, some of us from the Center of Genetic Engineering and Biotechnology (CIGB) of Havana (Cuba) synthesized a new linear peptide analogue of L2, denoted in the following as CIGB552.8 L2 was modified by including a proline (P(6)) and a leucine (L(11)) residues in the peptide sequence as D-amino acids, and an acetyl (Ac) group at the N-terminus These modifications were shown to improve the antitumor activity of L2, increasing its apoptosis efficiency and its capacity to inhibit cell proliferation.[8] CIGB552 has proven to be effective in reducing the size of tumour and increasing lifespan in tumour-bearing mice,[9] and in dogs with naturally-occurring cancer.[10] It has been found that CIGB552 decreased the microvessel density in the HT29 xenograft human, suggesting potent anti-angiogenesis properties.[9]. Atomic force microscopy (AFM) imaging experiments and Molecular Dynamics (MD) simulations were carried out to investigate on the morphology of the aggregates and on the structure of both the isolated peptide and the small peptide clusters nucleating the growth of large peptide aggregates, respectively

| MATERIALS AND METHODS

| RESULTS AND DISCUSSION

| CONCLUSIONS