Mil. Med. Sci. Lett. (Voj. Zdrav. Listy) 2014, vol. 83(1), p. 18-27 ISSN 0372-7025 DOI: 10.31482/mmsl.2014.004 ORIGINAL ARTICLE NON-NEWTONIAN SUSPENSION FORMULATIONS FOR IMPROVED STABILITY AND DELIVERY OF AUTOINJECTABLE CBRN COUNTERMEASURES Andrew P-Z. Clark , Larry A. Cabell, Joe A. McDonough Microencapsulation and Nanomaterials Department, Chemistry and Chemical Engineering Division, Southwest Research Institute, San Antonio, TX Received 28 th October 2013. Revised 23 rd March 2014. Published 7 th April 2014. Summary Autoinjectors are commonly employed by the United States and other countries to deliver emergency therapeutics to counteract the effects of various chemical agent threats, including nerve agents. Autoinjector combination products (autoinjector and drug product) used by the military can have drawbacks, including insufficient thermal stability of drug products, limited aqueous solution concentration of active pharmaceutical ingredients (APIs), and complicated and expensive designs for delivering multiple drugs simultaneously. We have developed a novel Enhanced Formulation (EF) technology that solves these problems, using nanoparticle suspensions in biocompatible cottonseed oil (CSO) instead of aqueous solutions of API. The hydrophobic CSO prevents hydrolytic degradation by limiting exposure to water, and the noninteraction of co-suspended particles simplifies multi-drug cocktail therapies. The high API loading (10% or more) gives the formulations non-Newtonian rheological behavior, with high zero-shear viscosity to resist sedimentation, and shear thinning to allow injection with standard autoinjectors and needles. Key words: Organophosphate; MMB4; nanoparticle; milling; intramuscular injection; formulation; particle size; suspension; non-Newtonian; shear thinning; enhanced formulation; nerve agent antidote; nerve agent intoxication; medical countermeasure; cottonseed oil INTRODUCTION administered by intramuscular (IM) injection: a mus - carinic receptor antagonist (currently atropine), The United States Department of Defense cur - an acetylcholinesterase (AChE) reactivator (currently rently uses autoinjectors for drug delivery in emer - 2-pralidoxime chloride, 2-PAM), and an anticonvul - gency treatment of nerve agent intoxication [1]. sant (currently diazepam) [2]. One dose of this treat - The treatment regimen generally consists of 3 drugs ment can require up to 3 separate autoinjectors, although the more recently developed Antidote Tre - atment Nerve Agent Autoinjector (ATNAA) combi - Southwest Research Institute, nes the atropine and 2-PAM into a single, 6220 Culebra Rd., San Antonio, TX 78238 wet-dual-chamber autoinjector. All currently fielded [email protected] products suffer from inherent limitations, including 210 522 5333 limited aqueous solution concentration, drug-drug 210 522 4632 solution interactions when two or more drugs Clark et al.: Stable Autoinjectable Nanoparticle Suspension CBRN Countermeasures are dissolved in a single chamber as with cocktail drug MATERIALS AND METHODS administration, and implementation of complicated design requirements for wet-dry and wet-wet multi- Materials chamber autoinjectors. Possibly the most critical issue with current key autoinjector products is HPLC-grade acetonitrile, methanol and phosphate inadequate stability of active pharmaceutical buffered saline (PBS) were purchased from Fisher ingredients (APIs) in aqueous solution, leading Scientific and used as received. Reagent-grade to short shelf lives and increased costs to maintain methanesufonic acid and 4-pyridine aldoxime (4-PA) a stockpile. This is particularly critical for bis- were purchased from Sigma-Aldrich and used pyridinium oximes, which constitute an important as received. Cottonseed oil (CSO, USP/NF grade) class of AChE reactivators that are currently being was purchased from Welch, Holme, & Clark Co., Inc. developed for next-generation broad spectrum nerve (Newark, NJ). MMB4 DMS was manufactured agent treatment systems [3,4]. In addition, the pH by SwRI and Cambrex (Charles City, Iowa). of aqueous solutions of these salts must be adjusted Syringes (1, 3, and 5 mL; Monoject) and needles to maximize the stability of dissolved APIs, leading (16 ga 1½ inch and 21 ga 1 in; Becton-Dickinson) to local tolerability issues and patient discomfort were purchased from Fisher Scientific. for products with significantly acidic solutions and high tonicity. Some of the problems caused by HPLC aqueous solutions can be mitigated by using multichamber wet-dry autoinjectors, but these are The quantification of MMB4 and its degradation also complicated and have their own set of products were analyzed by high performance liquid problems, such as complex filling processes, high chromatography (HPLC) (Waters HPLC system). risk / performance ratios, controlling solution Separations were performed on reversed phase kinetics to allow for complete drug injection and columns with UV or diode array detectors. solubility limitations on concentration and delivery speed [5]. For MMB4, HPLC was performed on a Waters 2695 or Agilent 1100 system on a reversed-phase C18 column (Waters Atlantis HILIC, 4.6 x 150mm, In this article, Southwest Research Institute (SwRI) 5 micron) at a flow rate of 1.3 mL/min equipped with presents a formulation which solves many a UV detector set at 297 nm. The mobile phase of the problems with currently available consisted of 80/20 MeCN / 0.05 M Ammonium autoinjector products. Our Enhanced Formulation Formate Buffer (pH 4). The major, minor peaks (EF) [6] consists of drug microparticles and/or of MMB4 DMS and the impurity have retention nanoparticles suspended in biocompatible times of 18, 21 min and 25 min respectively. cottonseed oil (CSO). EF is a non-Newtonian shear- thinning fluid, making it injectable even at high For quantification of the degradation product from solid concentrations (400 mg/mL or more), yet it is MMB4 DMS, HPLC was performed on a reversed- sufficiently viscous so as to prevent significant phase C18 column (Discovery C-18, 3.5 x 150mm, sedimentation of suspended particles. As a hydrophobic Supelco) at a flow rate of 1.5 mL/min with suspension of solid particles, EF makes many APIs photodiode array detector set at 215nm. The mobile far more stable, because they are not subject phase consisted of a gradient of 90/10 (v/v) 25mM to hydrothermal degradation. Shelf life is vastly K2HPO 4, pH 7.0/MeOH (solvent A2) and methanol increased as a result, lowering acquisition costs (solvent B2) with the following conditions: 100% substantially. Suspension of solid state API particles solvent A2 (0 to 20 min), 50% solvent A2/50% also enables facile implementation of drug cocktails solvent B2 (20 to 25 min) and 100% solvent A2 (25.5 by combination of multiple APIs in a single to 30 min). 4-PA was eluted after 7 min. formulation without chemical interactions between drugs. We have demonstrated EF using 1,1’- Particle Size Characterization methylenebis-4-[(hydroxyimino)methyl]pyridinium- dimethanesulfonate (MMB4 DMS) as the API, and The particle size distributions of MMB4 DMS we have developed a cGMP manufacturing process nanoparticles were determined by dynamic light [7]. A clinical supply batch made by this process is scattering (DLS), also known as photon correlation currently under investigation in a Phase I clinical spectroscopy (PCS), using a Brookhaven ZetaPALS trial [8]. 90Plus BI-MAS. This method is discussed in more 19 Clark et al.: Stable Autoinjectable Nanoparticle Suspension CBRN Countermeasures detail in the Results and Discussion section. Samples Physical Properties were prepared by diluting EF with CSO to appro- ximately 50 µg/mL and sonicating in a 100 W bath Rheological experiments were carried out using sonicator for approximately 60 minutes. DLS a Rheometrics RFS-1 fluid analyzer. Injectability experiments were conducted at a controlled was determined using a MTS Insight Materials temperature of 74 °C, and results are reported Testing System, which is an electrodynamic frame as unimodal intensity-weighted median particle sizes. equipped with a load cell (200 lbf max) to measure the applied force as the frame moved the syringe Milling plunger to eject the samples. Four different types of mills were used to reduce the particle size of MMB4 DMS in dry powder form. A benchtop ball mill (Retsch PM100) was used RESULTS AND DISCUSSION in a batch process; stainless steel and ceramic balls were both used separately to mill the MMB4 DMS. Manufacturing of Enhanced Formulation Particle sizes of approximately 72 to 107 µm were achieved using this method. A comminutor impact MMB4 DMS EF was manufactured by a wet mill or Fitzpatrick mill (Model LA1 Fitzmill, milling process. The API was combined with The Fitzpatrick Company, Elmhurst, IL) was used cottonseed oil (CSO) and zirconia beads in an agitator in a batch process with up to 10 consecutive passes bead mill. In this type of a mill an agitator shaft through the mill to produce volume weighted average in the grinding chamber rotates, imparting kinetic particle sizes from approximately 6 to 60 µm. energy to the mixture. A pump and external reservoir A centrifugal impact mill or pin mill (CIM-18-SS, were used to continuously circulate the mixture Munsen Machinery, Utica, NY) was used in a batch through the mill, allowing production of batches process, and was also modified to use a closed-loop much larger than the grinding chamber. The MMB4 system for continuous recirculation. Volume DMS particles were fractured as they impact