Edward D. Clarkson

Pro-2-PAM therapy for central and peripheral cholinesterases.

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.

Butyrylcholinesterase as a therapeutic drug for protection against percutaneous VX

The administration of purified human plasma-derived butyrylcholinesterase (HuBuChE) as a pretreatment has been demonstrated to enhance survival and protect against decreased cognitive function after exposure to organophosphorus poisons (OPs). Based on efficacy data obtained with guinea pigs and non-human primates and the lack of behavioral side effects, plasma-derived HuBuChE has been granted investigational new drug status by the US Food and Drug Administration. The recent availability of a recombinant form of HuBuChE (rHuBuChE) from the milk of transgenic goats has now allowed us to determine the pharmacokinetics of that material in guinea pigs and use it as a therapy following exposure to the VX. The rHuBuChE was expressed as a dimer and following intramuscular (i.m.) administration had more a rapid adsorption and clearance profile in guinea pigs than the plasma-derived material. Based on those data, we administered rHuBuChE i.m. 1h after a percutaneous exposure of guinea pigs to either 2xLD(50) or 5xLD(50) of VX. Post-exposure therapy with rHuBuChE provided improved survival at both challenge levels, 90% and 33% respectively versus 20% or 0% respectively for animals that did not receive therapy. These studies showed that BuChE can be efficacious as a therapy against percutaneous exposure to VX.

Rapid Decontamination of Chemical Warfare Agents

Publisher Summary Organophosphates (OP) nerve agents are a serious threat to military and civilian personnel. Another serious problem that may be encountered while caring for personnel contaminated with OP nerve agents is the possibility that there will be cross-contamination to the medical personnel treating affected personnel. During combat or terrorist acts, individuals might be exposed to chemical toxins before they don their protective gear. Medical decontamination executes removal and/or neutralization of chemical warfare agents, which, upon penetration of the skin, produces vesication, or for OPs, penetrates to the systemic circulation and inactivates cholinesterases (ChEs). The most important process for the exposed soldier or civilian is to remove the chemical agent from the skin as quickly as possible. The soldier, under harsh conditions, must use the product quickly to minimize transdermal penetration. A decontaminant that inactivates the chemical agent prevents its penetration through the skin and potentially protects a medical worker or buddy from suffering a second hand exposure. Other criteria for the decontaminating system and reagents are that they are as universal as possible and protect against the various classes of chemical agents. In other words, the soldier has a limited amount of space and weight to carry, and cannot carry multiple decontamination schemes. Furthermore, it is unlikely that a soldier would be able to determine the type of agent with which he or she is contaminated in the absence of symptoms.

Median lethal dose determination for percutaneous exposure to soman and VX in guinea pigs and the effectiveness of decontamination with M291 SDK or SANDIA foam.

Soman (GD) and VX are chemical warfare agents that can be absorbed through the skin. We determined the median lethal dose (MLD) for the cutaneous application of GD and VX in anesthetized haired guinea pigs and then tested the ability of a currently fielded decontamination kit, the M291 Skin Decontamination Kit (SDK), and decontaminating foam made by SANDIA Labs to decontaminate areas that have been exposed to cutaneous applications of GD and VX. The fur of guinea pigs was clipped on the left flank 24h prior to exposure. Animals were anesthetized and 5 min later neat GD or neat VX was applied. The MLD for percutaneous exposure to GD was 11.6 mg/kg, and to VX it was 0.10mg/kg. To test the ability of the M291 SDK, either GD or VX was applied and removed 1 min later with the pads of the M291 SDK clasped in a pair of forceps and wiped across the flank of the animal. The MLDs for GD and VX removed with the M291 SDK pads were 76.9 mg/kg and 0.87 mg/kg, respectively. When neat GD or neat VX was applied and removed 1 min later in the same manner with gauze soaked in SANDIA foam (MDF-100), the MLDs were 412 mg/kg and 10.4 mg/kg respectively. These data demonstrate that GD and VX are significantly less potent when applied cutaneously than previously reported for subcutaneous injections and indicate that improvement is needed on the limited protective ratio provided by the M291 SDK.

Rapid Decontamination of Chemical Warfare Agents from the Skin

Medical decontamination of the skin should be suitable for a variety of biological surface detoxification and decontamination schemes for both chemical weapons and pesticides (known as organophosphates), such as those directed against cholinesterases. With the constant threat of chemical warfare, terrorist acts, or spilling of pesticides, the development of improved means of protecting individuals from exposure to these agents, and decontaminating them when they are exposed, is critical. An understanding of skin, the primary barrier to chemical warfare agents, is required to develop novel treatments post-exposure. Therefore, the skin is defined and explored in this chapter.