Circulatory Failure During Noninhaled Forms of Cyanide Intoxication.

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.

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.

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.

Identification of a high-affinity phosphopeptide inhibitor of Stat3.

Stat3 is a latent transcription factor that exhibits elevated activity in a variety of human cancers. To find a lead peptide for peptidomimetic drug development we synthesized and tested phosphopeptides derived from known receptor docking sites and found Y(p)LPQTV as the optimal sequence. SAR studies showed that each residue from pY to pY+3 provided binding energy.