Minimizing acylation of peptides in PLGA microspheres

Abstract

The main objective of this study was to characterize and find mechanisms to prevent acylation of therapeutic peptides encapsulated in glucose-star poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. The effect of addition of divalent cation salts CaCl2, MnCl2 as well as carboxymethyl chitosan (CMCS) on inhibition of acylation of octreotide (Oct), salmon calcitonin (sCT), and human parathyroid hormone (hPTH) was evaluated. Peptide content and integrity inside the degrading microspheres was monitored by reversed-phase high performance liquid chromatography (HPLC) and mass spectrometry during release incubation under physiological conditions. The extent of peptide acylation was strongly inhibited in the formulations containing divalent cations and/or CMCS as excipients, although specific effects were dependent on the specific peptide and excipient combinations. Both inorganic cations improved stability of Oct and hPTH but not sCT. Addition of CMCS alone was ineffective. Combining inorganic cations with CMCS improved stability of Oct and sCT but it had no effect on hPTH stability. The operative stabilization mechanisms are consistent with blocking peptide-PLGA interactions by a) directly competing for PLGA interactions with dications and/or b) increasing peptide affinity in the stabilizer phase within PLGA pores. Hence, inorganic multivalent cations are general stabilizers against peptide acylation, the effect of which may be augmented in certain instances with addition of CMCS.

Reversible hydrophobic ion-paring complex strategy to minimize acylation of octreotide during long-term delivery from PLGA microparticles

“…Nearly 60% of the octreotide was acylated and <20% of native octreotide was released during the In vitro release over a period of 3 months. Similar results has been reported for other peptides and proteins such as bovine serum albumin, human atrial natriuretic peptide, human parathyroid hormone, leuprolide, insulin and salmon calcitonin (Ghalanbor et al, 2012; Ibrahim et al, 2005; Lucke et al, 2002; Na et al, 2003b; Zhang and Schwendeman, 2012). …”

Novel Pentablock Copolymer-Based Nanoparticulate Systems for Sustained Protein Delivery

“…Moreover, the entire spectra of released IgG ranging from 200 to 250 nm were also identical to native IgG, suggesting retention of secondary conformation of IgG during NP preparation and release from NPs. Previous reports suggest that PLA-and/or PGA-based copolymer produces a large molar mass of lactic acid and/or glycolic acid (18,19). These degradation products can stimulate hydrolytic degradation of protein therapeutics.…”

“…However, it is widely reported that protein and peptide can undergo loss of biological activity during formulation, storage, and/or release (12)(13)(14)(15). The presence of hydrophobic/hydrophilic interfaces (16), reduction in pH during polymer degradation (17), and degradation product (lactic acid and glycolic acid)-induced acylation processes (18,19) are potential sources of irreversible aggregation or inactivation of therapeutic proteins inside. The three-dimensional structure of protein is very important for its pharmacological activity.…”

Injectable controlled release depots for large molecules

“…Similarly, we sought to minimize the peptide–polymer interaction by outcompeting the peptide for the polymer carboxylates [44,116, 117]. By adding various water-soluble salts of divalent cations, such as Mn 2+ and Ca 2+ , to suspensions of acid-end group PLGA and the peptide, the octreotide–polymer interaction was strongly inhibited, resulting in a modest decrease in peptide acylation [44].…”

“…This strategy also translated into sharp decreases in octreotide acylation when encapsulated in microspheres when formulation conditions were optimized [116]. The approach was found to be augmented in certain cases when carboxymethyl chitosan was added to the formulation to help retain the divalent cationic salts [117]. …”