In the early hours of 21 August, doctors in Damascus area hospitals scrambled—often in vain—to save the lives of Syrian civilians brought to the hospital with foaming mouths and convulsions. Today, a report released by a United Nations inspection team confirms, as many have suspected, that the chemical weapon used in the attack was the deadly nerve gas sarin.
There are medical countermeasures proven to help counteract the poisoning of sarin and other organophosphate-based nerve agents such as soman and VX—some of which were available last month to Syrian victims. But “they have their limitations,” notes David Jett, director of the Countermeasures Against Chemical Threats (CounterACT) program at the US National Institutes of Health (NIH) in Bethesda, Maryland. Certain drug therapies don’t enter the brain well and none offers protection from the long-term effects of sarin exposure. So scientists have ratcheted up their efforts to improve the arsenal of antidotes against this particular chemical weapon and its lasting impact on the nervous system.
Sarin proves so fatal because it inhibits an enzyme called acetylcholinesterase (AChE). This enzyme normally degrades the neurotransmitter acetylcholine, a key signaling molecule that has numerous functions in the body, including facilitating cognitive function and triggering muscle contraction. Without functioning AChE, muscle fibers twitch uncontrollably and neurons in the brain become hyperactive, leading to seizures. If untreated, people exposed to sarin typically die of asphyxiation, as the muscles involved with breathing proceed to fire nonstop.
More than 1,400 people, including an estimated 426 children, died in the August gas attack, according to US intelligence estimates. Syrian doctors had only limited amounts of antidotes against the nerve gas, according to Sawsan Jabri, a trained physician who teaches biology courses at Oakland Community College in Eastern Michigan and serves as a spokeswoman for the US-based Syrian Expatriates Organization. She says that medical staff around Damascus (with whom she is in contact) had a total of some 50,000 ampoules of atropine, a drug that blocks the receptor responsible for binding acetylcholine, thereby preventing nerve and muscle cells from responding to the neurotransmitter. She adds that they also had “very limited amounts” of both pralidoxime (2-PAM)—a compound that reactivates sarin-inhibited AChE—and the anti-anxiety drug diazepam (better known as Valium), which prevents and treats seizures.
These three medicines—atropine, 2-PAM and diazepam—together constitute the ‘gold standard’ of anti-sarin therapies. As a matter of precaution, US military personnel are equipped with kits that contain spring-loaded syringes full of these antidotes, known as autoinjectors, which allow them to self-administer drugs through the muscle soon after, or better yet, before a chemical attack. But the therapeutic window is small, and prophylactic treatment is typically only feasible for military personnel, not civilians. So government defense agencies have long sought more robust and widely applicable alternatives to limit the death toll and mitigate permanent disability among survivors.
“There is a very vibrant research and development program in this area,” Jett says. He notes that the US government has been funding work in this area since “long before the chemical attacks in Syria, and even before the civilian attacks in Tokyo,” referring to the domestic terrorist attack on the Tokyo subway system in 1995, one of the first-ever uses of sarin as a chemical weapon. “We’re on this.”
New fashioned cocktail
Of the trio of drugs used currently to treat sarin poisoning, the one most likely to be replaced first is diazepam. A study published last year—supported in part by the NIH’s CounterACT program, the US Biomedical Advanced Research and Development Authority (BARDA) and the US Department of Defense (DoD)—provided the first human clinical data supporting the use of an autoinjector containing the potent sedative midazolam, which falls in the same benzodiazepine drug class as diazepam. In a trial of 893 individuals with status epilepticus, a life-threatening condition in which the brain is in a state of prolonged seizure (often because of preexisting seizure disorders, brain lesions or other factors), 73% of those who received midazolam via autoinjector from a paramedic had stopped experiencing seizures by the time they arrived at hospital, compared to 63% of people who received intravenous infusions of another benzodiazepine called lorazepam.
The experimental midazolam autoinjector is manufactured by Meridian Medical Technologies, a Maryland-based subsidiary of Pfizer that already provides the US military with autoinjectors of atropine, 2-PAM and diazepam. With evidence in hand from rodent experiments that midazolam protects against the ill effects of sarin and other nerve agents, Meridian is now expected to petition the US Food and Drug Administration (FDA) to approve the product as a defense against nerve gas exposure under the agency’s co-called ‘animal rule’, which provides a path to regulatory clearance when human efficacy studies are not ethical or feasible. However, according to Lauren Starr, a spokeswoman for Pfizer, “it would be premature to comment on this.”
In the future, 2-PAM might also be replaced by improved options. The compound RS194B, discovered by the Nobel Prize–winning chemist Barry Sharpless and his colleagues at the Scripps Research Institute in La Jolla, California, reactivates AChE in a similar way to 2-PAM. But RS194B can also readily penetrate the blood–brain barrier and thereby reinstate AChE function both in the peripheral and central nervous systems—something 2-PAM cannot achieve.
Palmer Taylor, a pharmacologist at the University of California–San Diego, teamed up with Sharpless and others to test RS194B in mice exposed to sarin and other organophosphate chemicals that also block AChE. Reporting last year, the researchers showed that approximately twice as much sarin was needed to kill RS194B-treated mice compared with 2-PAM–treated animals. As an added bonus, RS194B seems to be less toxic than 2-PAM, which means that it might be tolerable at higher doses. Plus, it works in pill form, whereas 2-PAM must be injected.
The last of the three currently used anti-sarin agents, atropine, can reach the brain to act there. But scientists seek compounds with similar effects that do so more quickly and at greater concentrations. Take scopolamine, for instance. Commonly used to treat nausea and motion sickness, this drug is more fat-soluble than atropine, and thus crosses the blood–brain barrier more readily. A few years back, Irwin Koplovitz and Susan Schulz from the US Army Medical Research Institute of Chemical Defense (USAMRICD) in Aberdeen Proving Ground, Maryland, reported that using scopolamine in addition to atropine increased the survival rates of guinea pigs exposed to sarin and allowed for lower drug concentration to achieve the same therapeutic benefit.
Beyond the existing paradigm
Another nerve agent antidote sometimes taken by US troops is pyridostigmine bromide (PB). Approved by the FDA in 2003 for military use against soman, this drug, like the organophosphate nerve gases themselves, inhibits AChE, but it does so in a reversible fashion. As such, it can compete with nerve agents for the active site of the enzyme, thereby shielding AChE from permanent damage. Yet, PB is not thought to be effective against sarin, and it barely traverses the blood–brain barrier, thus providing little to no protection in the central nervous system. An Alzheimer’s drug called galantamine that does enter the brain could help overcome both of these weaknesses.
A team led by Edson Albuquerque from the University of Maryland School of Medicine in Baltimore showed in 2006 that galantamine pretreatment was more effective than PB pretreatment at saving guinea pigs from lethal doses of either soman or sarin. Now, Countervail Corporation, a Charlotte, North Carolina–based firm for which Albuquerque consults, has a Small Business Innovation Research grant from the NIH to further investigate galantamine’s prophylactic potential. The company also has a contract from BARDA to advance galantamine as a protective measure for nerve gas exposure under the brand name AverTox.
Meanwhile, other researchers are pursuing more experimental approaches to keep acetylcholine levels at bay in a chemical attack. For instance, Dan Tawfik, a chemical biologist at the Weizmann Institute of Science in Rehovot, Israel, hopes to use ‘bioscavenger’ drugs that act like sponges to mop up and degrade toxic nerve agents flowing in the blood before they reach organs and other tissues. “The idea is to really remove the agent before it even creates any damage,” Tawfik says.
Using a protein engineering technique known as directed evolution, Tawfik has enhanced the activity of a bioscavenging enzyme called serum paraoxonase 1 (PON1). He and his team injected this enzyme into mice one hour prior to nerve agent exposure, and showed that 63% of the animals survived after two weeks; all the mice that had received atropine and 2-PAM as prophylaxis prior to exposure died. Together with USAMRICD’s Douglas Cerasoli, Tawfik now has funding from the US government to look at PON1’s effects on post-exposure protection.
Another bioscavenger enzyme, and one that even once entered human testing, is butyrylcholinesterase (BChE). The DoD commissioned two contactors, DynPort Vaccine Company and Baxter Healthcare Corporation, to run a 28-person phase 1 trial in 2007 demonstrating the safety and tolerability of BChE derived from human plasma. And two years later, PharmAthene, a DoD-backed biodefense company based in Annapolis, Maryland, tested a recombinant form of this human enzyme produced in the milk of transgenic goats in a 33-person phase 1 trial. However, the low drug yields resulting from either manufacturing process—not to mention the large overhead of maintaining a herd of goats—proved too expensive to cost-effectively produce sufficient quantities of either product.
According to John Troyer, head of chemical defense product development at PharmAthene, the company is now back in the laboratory working on making a new form of BChE in a human cell line. His team is just starting to test the drug’s pharmacokinetics in rodent models, and they still need to assess efficacy. But Troyer has high hopes for this bioscavenger. “Since we’re making a human-like BChE we think that we’re going to be in a pretty good place in terms of performance,” he says. Plus, he adds, “because we are getting high yields, we really believe that our cost of goods are going to be significantly lower than where we were before.”
Many of the new therapeutic avenues under development have been in consideration for a while. But a wholly novel strategy—one not yet published in the literature—comes from Richard Rotundo. The University of Miami Miller School of Medicine neuroscientist has taken a small fragment of a protein that normally anchors AChE in the cell membrane and added a four amino acid sequence that signals for the peptide to travel to the endoplasmic reticulum (ER). The modified peptide then binds to newly synthesized AChE molecules in the ER, the organelle responsible for proper protein folding, where they stabilize the AChE precursors, rescuing them from intracellular degradation. In this way, Rotundo says, the approach can be thought of as a form of enzyme replacement therapy—yet one that uses the body’s own endogenous, but unused, enzyme precursors.
In mouse models, Rotundo has shown that he can boost the percentage of AChE precursors that get folded, assembled and exported correctly from around 20% naturally to 80% or more in animals that received the modified peptides. And in mice subject to nerve agent poisoning, he’s shown the peptide therapy saves more than 90% of animals when given within five minutes of exposure. Rotundo presented the work earlier this year at the International Symposium on Cholinergic Mechanisms in Hangzhou, China.
“We’re actually changing how the proteins fold in vivo to a therapeutic benefit,” Rotundo says. “Pretty soon we’ll be able to put in new [acetylcholine] esterase molecules like you’d change a tire.”
Unfortunately, many of the experimental antidotes and the existing components of the three-drug regimen fail to solve one crucial problem. “What’s missing is neuroprotection,” says John Butera, chief scientific officer of Hager Biosciences, a drug discovery startup based in Bethlehem, Pennsylvania. “Because long after the [nerve] agent is gone, long after the acetylcholine is back down to normal, these other secondary effects start to creep up weeks and months and years down the line.” These long-term symptoms of exposure to sarin and other nerve gases include learning disabilities, cognitive deficits and various psychiatric disorders.
With an eye to long-term neuroprotection, Butera and his colleagues are now looking for compounds that can both reactivate AChE (like 2-PAM does) at the same time that they block a DNA-binding protein called poly(ADP-ribose) polymerase 1 (PARP1). This enzyme is known to be involved in tissue injury and neurodegeneration, and many experimental PARP-1 inhibitors are now in clinical development for the treatment of cancer and stroke.
Of course, none of these experimental therapeutics will likely be ready in time to help Syrian civilians if or when nerve agents are used again during the country’s ongoing civil war. According to Jabri, the Syrian Expatriates Organization spokeswoman, what’s most needed now in Syria are proven sarin antidotes, basic medical supplies and hazmat suits for first responders.
The Syrian crisis does, however, highlight how hard it is to deliver medical countermeasures into the hands of civilians and troops on the ground in a war zone, not to speak of these newer antidotes, should they ultimately prove more effective following further investigation. “What I see coming out of Syria is a situation where the mobilization of those antidotes would be extremely difficult to provide to civilian victims,” says Matthew Redinbo, a structural biologist at the University of North Carolina at Chapel Hill who has studied sarin bioscavengers. “But I think it can be done,” he notes. “Oftentimes our failures are just failures of will, they’re not failures of technology.”
A version of this story appears in the October 2013 issue of Nature Medicine.