Larry A. Cabell

Circulatory Failure During Noninhaled Forms of Cyanide Intoxication

ABSTRACT Our objective was to determine how circulatory failure develops following systemic administration of potassium cyanide (KCN). We used a noninhaled modality of intoxication, wherein the change in breathing pattern would not influence the diffusion of CN into the blood, akin to the effects of ingesting toxic levels of CN. In a group of 300 to 400 g rats, CN-induced coma (CN i.p., 7 mg/kg) produced a central apnea within 2 to 3 min along with a potent and prolonged gasping pattern leading to autoresuscitation in 38% of the animals. Motor deficits and neuronal necrosis were nevertheless observed in the surviving animals. To clarify the mechanisms leading to potential autoresuscitation versus asystole, 12 urethane-anesthetized rats were then exposed to the lowest possible levels of CN exposure that would lead to breathing depression within 7 to 8 min; this dose averaged 0.375 mg/kg/min i.v. At this level of intoxication, a cardiac depression developed several minutes only after the onset of the apnea, leading to cardiac asystole as PaO2 reached value approximately 15 Torr, unless breathing was maintained by mechanical ventilation or through spontaneous gasping. Higher levels of KCN exposure in 10 animals provoked a primary cardiac depression, which led to a rapid cardiac arrest by pulseless electrical activity (PEA) despite the maintenance of PaO2 by mechanical ventilation. These effects were totally unrelated to the potassium contained in KCN. It is concluded that circulatory failure can develop as a direct consequence of CN-induced apnea but in a narrow range of exposure. In this “low” range, maintaining pulmonary gas exchange after exposure, through mechanical ventilation (or spontaneous gasping), can reverse cardiac depression and restore spontaneous breathing. At higher level of intoxication, cardiac depression is to be treated as a specific and spontaneously irreversible consequence of CN exposure, leading to a PEA.

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.

MMB4 DMS Nanoparticle Suspension Formulation With Enhanced Stability for the Treatment of Nerve Agent Intoxication

Various oximes are currently fielded or under investigation in the United States and other countries as a component of autoinjector emergency treatment systems for organophosphate nerve agent chemical weapons. Bis-pyridinium oximes in general have greater efficacy against a broad spectrum of nerve agents, but they have poor stability due to hydrolytic degradation at elevated temperatures. 1,1′-Methylenebis-4-[(hydroxyimino)methyl]pyridinium dimethanesulfonate (MMB4 DMS) is a leading candidate for next-generation nerve agent treatment systems, because it is more stable than other bis-pyridinium oximes, but it still degrades quickly at temperatures often encountered during storage and field use. The primary goal is to increase the stability and shelf life of MMB4 while maintaining the desirable pharmacokinetic (PK) properties of the aqueous formulation. We have developed a formulation to be used in a phase 1 clinical trial consisting of MMB4 micro/nanoparticles suspended in cottonseed oil, a biocompatible vegetable oil. Through various milling techniques, the average particle size can be controlled from approximately 200 to 6000 nm to produce non-Newtonian formulations that are viscous enough to resist rapid particle sedimentation while remaining injectable at a range of concentrations from 5 to 400 mg/mL. The preliminary accelerated stability test shows that MMB4 in these formulations is stable for at least 2 years at temperatures up to 80°C. Preliminary preclinical in vivo studies have demonstrated that all concentrations and particle sizes have desirable PK properties, including high bioavailability and rapid absorption, which is critical to combat potent and fast-acting nerve agents.

International Journal of Toxicology Special Edition Volume of MMB4 DMS

1,10-Methylenebisf4-[(hydroxyimino) methyl]-pyridiniumg dimethanesulfonate (MMB4 DMS), a member of the bisquaternary pyridinium aldoximes group, is currently being developed by the Department of Defense as adjunct therapy in the case of organophosphorus (OP) nerve agent exposure. This issue of International Journal of Toxicology is dedicated to the MMB4 DMS preclinical advanced drug development activities that comprised the investigational new drug (IND) application so as to initiate a first-in-man phase 1 clinical study. The OP nerve agents are among the most lethal chemical weapons. They are chemically stable, easily dispersed, highly toxic, and have rapid effects both when absorbed through the skin and via respiration. The pathophysiology of nerve agent intoxication is well understood. The OP nerve agents irreversibly bind to acetylcholinesterase (AChE), causing the phosphorylation and deactivation of AChE. The clinical effects are secondary to acetylcholine (ACh) excess at cholinergic junctions (muscarinic effects) in the central nervous system (CNS) and at the skeletal nerve–muscle junctions and autonomic ganglia (nicotinic effects). Medical counter measures (MCMs) against nerve agents include a combination of up to 4 drug classes: (1) a carbamate (pyridostigmine bromide [PB]) which when orally administered prior to exposure to a nerve agent serves to sequester a pool of AChE by reversibly inhibiting the enzyme so that it cannot be irreversibly bound by nerve agent; (2) an antimuscarinic (atropine) that antagonizes the effects of ACh at postsynaptic muscarinic receptors in peripheral tissues and CNS and prevents early life-threatening symptoms such as bronchoconstriction, excessive bronchosecretion, and impaired respiratory drive; (3) an oxime that reactivates nerve agentinhibited AChE in peripheral tissues and restores the ability of the enzyme to hydrolyze ACh; and (4) a benzodiazepine (diazepam) for postexposure treatment to prevent or minimize seizure activity though activation of centrally mediated g-aminobutyric acid pathways. The only oxime licensed in the United States for the treatment of nerve agent exposure is 2-pyridine aldoxime methyl chloride, 2-pralidoxime (2-PAM). The antidote treatment nerve agent autoinjector (ATNAA) that contains 2-PAM and atropine is the current MCM for nerve agent poisoning. Although 2-PAM has acceptable efficacy against certain nerve agents (e.g., GB [Sarin] and VX fO-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioateg), it lacks the desired level of efficacy against other agents (e.g., GA [Tabun], GD [Soman], GF [Cyclosarin], and VR N, N-diethyl-2-(methyl-(2-methylpropoxy)phosphoryl)sulfanylethanamine), even when combined with PB pretreatment and atropine and diazepam postexposure. This lack of efficacy toward a broad scope of nerve agents has been a driving factor in the search for more efficacious and safer alternatives to 2-PAM in order to alleviate a serious unmet medical need and, as a result of these efforts, the advanced drug development of MMB4 DMS was initiated. Once realized, the ATNAA will be replaced by the Improved Nerve Agent Treatment System that contains MMB4 DMS and atropine. The preclinical development program conducted in support of the IND submission to the US Food and Drug Administration (FDA) was composed of a series of pharmacology, pharmacokinetic, and toxicology studies. Pivotal INDenabling studies were all conducted in compliance with the FDA’s Good Laboratory Practice (GLP) Regulations and 21 Code of Federal Regulations Part 58 for the conduct of nonclinical laboratory studies. The intramuscular (IM) route of administration was used in the nonclinical studies, because this is the intended route of administration in man. Of the toxicology species tested in the repeated dose studies used to calculate the maximum recommended starting dose and appropriate safety factors, rabbits were the most sensitive species to the effects of MMB4 DMS at equivalent doses. In support of the toxicology studies, full toxicokinetic evaluations were established, and comparative pharmacokinetic studies were conducted as well. Pharmacology studies explored the efficacy of MMB4 DMS against a host of conventional nerve agents,