S-nitrosoglutathione (GSNO) is a physiological nitric oxide donor and a promising drug for several acute or chronic diseases (e.g. stroke, atherosclerosis, …). However, this nitrosated tripeptide is highly hydrophilic and easily degraded while exposed to oxygen, light, metallic cations or enzymes. The formulation of GSNO, especially for oral delivery, is therefore very challenging.
In this work, three formulations based on the same raw materials (GSNO and Eudragit RLPO) were prepared with emulsion/solvent evaporation methods: nanoparticles (W/O/W, NP-W), microparticles (W/O/W, MP-W) and microparticles (S/O/W, MP-S). These particles were characterized as regards their size (dynamic light scattering/laser diffraction and scanning electron microscopy), zeta potential, GSNO encapsulation efficiency (EE) (fluorimetry), in vitro release (USP IV apparatus), cytocompatibility and intestinal permeability (Caco-2 cells monolayer). Moreover, the three formulations were obtained as dried powders, after optimization of their freeze-drying. Characterization was also performed on these stabilized formulations, including their residual water content (evaluated with a coulometric Karl-Fisher determination).
The three particles were able to encapsulate GSNO with similar EE (NP-W = 28.7 % ±3.3 % (n=11), MP-W = 24.4 % ± 3.7 % (n=7), MP-S = 25.1 % ± 3.6 % (n=5), direct determination). Freeze-drying using sucrose as protectant led to elegant cakes, easy to re-suspend, with similar EE as fresh formulations. On the other hand, particles sizes were not significantly modified by drying (NP-W: 290.7 nm ± 14.5 nm vs 299.7 nm ± 12.7 nm, n=6; MP-W: 70.8 µm ± 7.5 µm vs 68.5 µm ± 5.5 µm, n=3; MP-S: 147.5 µm ± 38.5 µm vs 105.6 µm ± 22.6 µm, n=3). Zeta potential of NP-W remained unchanged and highly positive (around + 55 mV), as expected according to the polymer used. Freeze-dried particles had a residual water content around 5 % and all their characteristics were maintained for at least 6 months after storage at 4°C under inert atmosphere. GSNO release from NP-W was very fast, almost similar to the dissolution profile of the free drug, while MP formulations delayed the release during the 2 first hours of the experiment, with a more progressive profile for MP-S compared to MP-W. All formulations were compatible with Caco-2 cells for up to 8.5 mg/mL of freeze-dried powder (corresponding to 2.8 mg/mL of polymer and 0.017 mg/mL of GSNO, i.e. 50 µM of GSNO). Intestinal permeability of GSNO released from these formulations is currently under evaluation in an in vitro intestinal barrier model.
At the end, three different particles loaded with GSNO were obtained as dried and stable formulations, thus facilitating their preclinical use. Among them, microparticles obtained with a S/O/W process could represent the most interesting lead to explore for GSNO oral delivery. Better results in terms of EE and sustained release might be obtained by reducing the size of GSNO powder (currently 40 µm). Due to GSNO fragility, this step is challenging: experiments are ongoing using supercritical fluid technology.
The PhD thesis of Mr Yi ZHOU is financially supported by the Chinese Scolarship Council.
The CITHEFOR EA3452 lab is supported by the “Impact Biomolecules” project of the “Lorraine Université d'Excellence” (Investissements d’avenir – ANR). The USP IV apparatus was purchased thanks to a “Contrat Plan Etat region” funding.
Audience take away:
• An example of formulation strategy for a very hydrophilic and labile peptidic drug, S-nitrosoglutathione
• A detailed development, characterization and comparison of 3 different polymeric formulations, from the nano- to the micro-meter range, made from the same materials but with different processes
• How this strategy did not work in certain aspects (increasing the encapsulation efficiency) but succeeded in others (more sustained in vitro release, getting stable freeze-dried formulations)