European Journal of Pharmaceutics and Biopharmaceutics
Research paperFreeze-drying of HI-6-loaded recombinant human serum albumin nanoparticles for improved storage stability
Graphical abstract
Introduction
Organophosphate (OP) compounds are considered to be among the most dangerous and toxic pesticides and are still being used in developing countries. Their possible use as warfare agents and in potential terrorist attacks causes a lingering threat calling for effective countermeasures in the form of an effective antidote therapy [1], [2]. Current treatments include the employment of the muscarinic antagonist atropine in combination with acetyl cholinesterase (AChE) reactivators such as HI-6 or obidoxime. HI-6, which is one of the most promising antidotes against OP-poisoning, has already been tested in humans and is available for OP-poisoning therapy in some countries [3]. Unfortunately and due to its hydrophilic structure [4], HI-6 is not able to cross the blood–brain barrier (BBB) whereas in contrast the lipophilic OP compounds easily reach the central nervous system (CNS) where they cause severe symptoms such as a rapid loss of consciousness, seizures, respiratory depressions, and in the worst case, respiratory arrest [1], [5]. To enable the brain transport of this antidote, nanoparticle-bound HI-6 already showed its potential in passing an in vitro BBB model of porcine brain capillary endothelial cells (pBCECs) [6], a process that is considerably enhanced by surface modification of the nanoparticles with apolipoproteins [1], [7].
A major drawback in the application of HI-6 formulations is due to the microbiological and chemical instability of HI-6 in solution as well as of albumin nanoparticles in aqueous dispersions. Previous studies have shown HI-6 to be susceptible to hydrolytic degradation, particularly at physiological pH where it decomposes into formaldehyde, isonicotinic acid, and 2-pyridone [8], [9]. Therefore, prolonged storage in aqueous solutions or nanoparticle dispersions is accompanied by a loss of active ingredient. A proper solution to this is the application of freeze-drying, a mild but effective approach to prolong the stability of these formulations by removing water from a frozen sample by sublimation and desorption under vacuum [10]. So far, lyophilisation has been successfully applied on a variety of nanoparticle formulations, part of them already being loaded with active ingredients [11], [12], [13]. Nevertheless, the production of an elegant cake by several freezing and drying steps may cause physical stress on the nanoparticles consequently resulting in changes in the nanoparticles’ properties such as particle size, size distribution, and drug loading. The need for lyo- and cryoprotectants to ensure the preservation of these particles’ characteristics is consequently often met using sugars (e.g. sucrose and trehalose) or sugar alcohols (e.g. mannitol). Specifically, the use of sugars was shown to positively affect the glass transition temperatures (Tg′ and Tg) and to be beneficial in obtaining stable and amorphous frozen cakes with short reconstitution times as well as low residual moisture contents [11], [12], [13], [14].
The objective of the present study was the stabilisation and preservation of the former investigated HI-6 nanoparticle formulation by freeze-drying. Recombinant albumin as a biodegradable and non-toxic material was chosen as the core material for the nanoparticles and loaded with HI-6 dimethanesulfonate at pH 8.0 by adsorption as previously published [6]. The influence of different excipients on the physicochemical characteristics was investigated using photon correlation spectroscopy. The excipients mannitol, sucrose, and trehalose were chosen due to their cryo- and lyoprotectant capabilities in order to preserve the formulations’ characteristics during the freeze-drying procedure. As levels of residual moisture should be as low as possible (preferably below 1%), the residual moisture of the final product was determined by Karl Fischer titration. The storage stability of the most promising formulation was analysed at different temperatures in order to predict shelf-life. The subsequent functionality testing was performed by the transport over an in vitro BBB model and subsequent reactivation of OP-inhibited AChE.
Section snippets
Chemicals and reagents
Recombinant human serum albumin (rHSA, expressed in rice, lyophilised powder, ⩾96%, 64 kDa) and glutaraldehyde 25% solution were purchased from Sigma–Aldrich Chemie GmbH (Steinheim, Germany). The glutaraldehyde solution was diluted to 8% with purified water. HI-6 dimethanesulfonate (478.5 Da) was supplied by the Bundeswehr Institute of Pharmacology and Toxicology (Munich, Germany). PIC B-7 (sodium heptanesulfonate) and PIC A (tetrabutylammonium phosphate) were the HPLC ion pairing reagents
Results and discussion
HI-6-loaded nanoparticles represent a promising tool to improve the transport of the oxime across the BBB for acute antidotal OP-poisoning therapy. Their preparation has previously been described and found to be optimal at a basic pH [6]. These conditions were maintained in the present study. Unfortunately, aqueous formulations of HI-6 are susceptible to degradation [9] leading to the formulations’ loss of effectiveness. Therefore, the objective of the present study was to prolong the storage
Conclusion
This study shows the ability to freeze-dry HI-6-loaded recombinant human serum albumin nanoparticles in order to prevent the drug’s degradation in aqueous solutions and to prolong the shelf-life. Trehalose 3% and sucrose 3% (w/v) were superior to mannitol as cryo-/lyoprotectants in terms of particle characteristics. The residual moisture content was found to be less than 4.1% for trehalose-containing formulations, which results in a further need of optimisation of the freeze-drying procedure.
Acknowledgements
This work was supported by Grant no. U2.3 – E/UR3G/5G031/5A802 by the German Bundesamt für Wehrtechnik und Beschaffung (BWB), Koblenz, Germany.
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