Assessment of ionizable, zwitterionic oximes as reactivating antidotal agents for organophosphate exposure.

Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.

Cyanide Scavenging by a Cobalt Schiff-Base Macrocycle: A Cost-Effective Alternative to Corrinoids.

The complex of cobalt(II) with the ligand 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaene (CoN4[11.3.1]) has been shown to bind two molecules of cyanide in a cooperative fashion with an association constant of 2.7 (±0.2) × 10(5). In vivo, irrespective of whether it is initially administered as the Co(II) or Co(III) cation, EPR spectroscopic measurements on blood samples show that at physiological levels of reductant (principally ascorbate) CoN4[11.3.1] becomes quantitatively reduced to the Co(II) form. However, following addition of sodium cyanide, a dicyano Co(III) species is formed, both in blood and in buffered aqueous solution at neutral pH. In keeping with other cobalt-containing cyanide-scavenging macrocycles like cobinamide and cobalt(III) meso-tetra(4-N-methylpyridyl)porphine, we found that CoN4[11.3.1] exhibits rapid oxygen turnover in the presence of the physiological reductant ascorbate. This behavior could potentially render CoN4[11.3.1] cytotoxic and/or interfere with evaluations of the antidotal capability of the complex toward cyanide through respirometric measurements, particularly since cyanide rapidly inhibits this process, adding further complexity. A sublethal mouse model was used to assess the effectiveness of CoN4[11.3.1] as a potential cyanide antidote. The administration of CoN4[11.3.1] prophylactically to sodium cyanide-intoxicated mice resulted in the time required for the surviving animals to recover from "knockdown" (unconsciousness) being significantly decreased (3 ± 2 min) compared to that of the controls (22 ± 5 min). All observations are consistent with the demonstrated antidotal activity of CoN4[11.3.1] operating through a cyanide-scavenging mechanism, which is associated with a Co(II) → Co(III) oxidation of the cation. To test for postintoxication neuromuscular sequelae, the ability of mice to remain in position on a rotating cylinder (RotaRod test) was assessed during and after recovery. While intoxicated animals given CoN4[11.3.1] did recover ∼30 min more quickly than controls given only toxicant, there were no indications of longer-term problems in either group, as determined by continuing the RotaRod testing up to 24 h after the intoxications and routine behavioral observations for a further week.

Long-term stability study of Prussian blue - a quality assessment of water content and thallium binding.

The purpose of this study is to assess the long-term stability of Prussian blue (PB) drug product (DP) and active pharmaceutical ingredient (API) under laboratory storage conditions by monitoring the loss in water content and the corresponding change of the in vitro thallium binding capacity that represents product performance. The bound water content and the in vitro thallium binding capacity of PB DPs and APIs were measured in 2003 and 2013, respectively. Water content, a critical quality attribute that directly correlates to the thallium (Tl) binding capacity was measured by thermal gravimetric analysis (TGA). The thallium binding study was conducted by testing PB in buffered solutions over the human gastrointestinal pH range with thallium concentrations ranging from 600 to 1,500 ppm. Samples were incubated at physiological temperature of 37°C in a shaking water bath to mimic gastric flux and intestinal transport. The binding equilibrium was reached at 24h. Following incubation, each sample was filtered and the free thallium was analyzed using a validated inductively coupled plasma spectroscopic method (ICP). The Langmuir isotherm was plotted to calculate maximum binding capacity (MBC). Compared with 2003, the water content of DP-1 decreased by about 14.1% (from 15.6 to 13.4 mol), and the MBC of DP-1 decreased by about 12.5% (from 714 to 625 mg/g) at pH 7.5. When low concentration of thallium (600 ppm) was used at pH 7.5, the Tl binding remained comparable for both API-1 (286 vs 276 mg/g) and DP-1 (286 vs 268 mg/g). Similarly, the Tl binding remained unchanged for both API-1 (237 vs 255 mg/g) and DP-1 (234 vs 236 mg/g) at pH 5.0. However, at pH 1.0 the binding was reduced 32.3% and 25.9% for API-1 and DP-1, respectively. Since the majority of binding takes place in the upper GI tract where pH around 5 can be expected, and therefore, the Tl binding capacity of PB should be comparable for new and aged samples. The findings that Tl binding changes with the water loss of PB and pH conditions are consistent with our previously published data. The study also represents the first quantitative assessment of the long-term stability of PB. Over last 10 years, PB DPs and APIs have lost about 20% water under ambient laboratory storage conditions which are consistent with a controlled warehouse environment. While the maximum binding capacity of PB to thallium was decreased after about 10 years of long-term storage, it is still very effective, suggesting that the shelf life of PB should be much longer than the manufacturer ascribed expiration date of 2008 under proper storage conditions.

Assessing the chiral switch: approval and use of single-enantiomer drugs, 2001 to 2011.

OBJECTIVES: A "chiral switch" occurs in the pharmaceutical market when a drug made up of 2 enantiomer forms is replaced with a purified single-enantiomer version, often in the context of a patent expiration. We studied the prevalence of chiral switching in the United States over the past decade, including trends in use of, and expenditures on, these products in Medicaid. STUDY DESIGN: Retrospective analysis. METHODS: We used US Adopted Names prefixes (lev/levo/ar/es/dex/dextro) to identify all single-enantiomer drugs approved from 2001 to 2011. From publicly available US Food and Drug Administration (FDA) approval documents, we extracted the characteristics of the pivotal premarket trials for the single enantiomers. Specifically, we evaluated whether the single enantiomer was directly compared with the precursor racemic drug and whether there was evidence of superior efficacy. We used quarterly drug expenditure data from each state Medicaid program to chart trends in use of, and spending on, the single-enantiomer products and their racemic precursors during the study period. RESULTS: From 2001 to 2011, the FDA approved 9 single-enantiomer products: dexlansoprazole, levoleucovorin, levocetirizine, armodafinil, arformoterol, eszopiclone, escitalopram, dexmethylphenidate, and esomeprazole. Of those 9 drugs, 3 had at least 1 pre-approval randomized trial that included the racemic precursor as a direct comparator, but there was no evidence of superiority of the single enantiomer over the racemic at comparable doses. Between 2001 and 2011, US Medicaid programs spent approximately $6.3 billion on these 9 single-enantiomer drugs. CONCLUSIONS: Recently approved single-enantiomer drugs showed no evidence of superior efficacy over the older racemic precursors in the pivotal trials leading to their approval, and in a majority of cases, they were not directly compared.

Good manufacturing practice: manufacturing of a nerve agent antidote nanoparticle suspension.

We have established a current good manufacturing practice (GMP) manufacturing process to produce a nanoparticle suspension of 1,1'-methylenebis-4-[(hydroxyimino)methyl]pyridinium dimethanesulfonate (MMB4 DMS) in cottonseed oil (CSO) as a nerve agent antidote for a Phase 1 clinical trial. Bis-pyridinium oximes such as MMB4 were previously developed for emergency treatment of organophosphate nerve agent intoxication. Many of these compounds offer efficacy superior to monopyridinium oximes, but they have poor thermal stability due to hydrolytic cleavage in aqueous solution. We previously developed a nonaqueous nanoparticle suspension to improve the hydrothermal stability, termed Enhanced Formulation (EF). An example of this formulation technology is a suspension of MMB4 DMS nanoparticles in CSO. Due to the profound effect of particle size distribution on product quality and performance, particle size must be controlled during the manufacturing process. Therefore, a particle size analysis method for MMB4 DMS in CSO was developed and validated to use in support of good laboratory practice/GMP development and production activities. Manufacturing of EF was accomplished by milling MMB4 DMS with CSO and zirconia beads in an agitator bead mill. The resulting bulk material was filled into 5-mL glass vials at a sterile fill facility and terminally sterilized by gamma irradiation. The clinical lot was tested and released, a Certificate of Analysis was issued, and a 3-year International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) stability study started. The drug product was placed in storage for Phase 1 clinical trial distribution. A dose delivery uniformity study was undertaken to ensure that the correct doses were delivered to the patients in the clinic.

Rapid atropine synthesis for the treatment of massive nerve agent exposure.

STUDY OBJECTIVE: We developed and tested a protocol for compounding a large volume of injectable atropine from powder. The resulting protocol could be used by hospitals to rapidly use large amounts of stockpiled atropine. METHODS: The protocol required 2 g of solid (powdered) atropine and 1 L of normal saline solution. The solution was filtered and mixed. One hundred syringes were filled by using a standard syringe-batching system. Modifications, including hand filling, were studied to reduce the time required to synthesize one hundred 3-mL syringes. RESULTS: A single pharmacist was able to reconstitute one hundred 6-mg atropine syringes in 29 minutes using the batching system. The quickest method for a single pharmacist filling syringes by hand was 34 minutes. The cost to the hospital for 5 g of powdered atropine was 11 dollars versus 5,000 dollars for prefilled syringes. CONCLUSION: Large quantities of atropine syringes can be compounded from a powdered form in a timely manner. Additionally, there is a significant cost advantage to using powdered atropine as a hospital stockpile.

Atropine availability as an antidote for nerve agent casualties: validated rapid reformulation of high-concentration atropine from bulk powder.

STUDY OBJECTIVE: Atropine is the preferred antidote for immediate management of toxicity associated with nerve agents or other cholinergic syndromes. A large-scale exposure to a nerve agent or organophosphate insecticide might result in many victims presenting for care within a short period of time. This situation would require the prompt availability of a large amount of atropine to provide treatment. Antidote stocks at many hospitals are inadequate to meet this demand. Atropine that is commercially available comes supplied at concentrations of either 0.4 mg/mL or 1 mg/mL, thereby requiring intravenous administration because of the volume necessary to administer the commonly recommended initial dose of 2 to 6 mg. Moderately ill victims may not require an intravenous line for other care, and in the setting of overwhelmed resources, intramuscular administration is faster and easier to perform. METHODS: To facilitate the delivery of larger atropine doses, we developed a method of fortifying existing injectable atropine with bulk pharmaceutical-grade atropine powder to a concentration of 2 mg/mL, thereby increasing the amount available and facilitating its intramuscular administration. An independent analysis of the resulting formulation was undertaken to assess its potency, absence of pyrogens, and stability. RESULTS: The amount of atropine initially present varied by less than +/-5%, within the range allowed by the US Pharmacopeia for the original product. The product was pyrogen free and maintained its potency at refrigeration temperature for at least 8 weeks after preparation and at room temperature for 4 weeks. Once all materials were available, the compounding of this preparation required about 1 hour to complete. CONCLUSION: Existing atropine stocks can be readily augmented by fortification with powdered atropine accurately and inexpensively. Common pharmaceutical guidelines recommend refrigeration for compounded products such as this if not completely used within 28 days.