Snakebites get renewed attention C&EN Global Enterp 2019.97:31-36. Downloaded from pubs.acs.org by 84.54.57.240 on 02/05/19. For personal use only.
Scientists look for alternative antivenoms that are easy to make and deploy to remote areas
In brief
DINSA SACHAN, special to C&EN
n a sunny September morning in Nemmara, a small township in Kerala, India, Ally Thomas headed out into the woods. Her plan was to cut grass, feed it to her cattle, and attend a prayer service in the evening—a regular day, by all means. Then a sharp, stinging pain shot through her left foot. A snake had bitten her. The serpent slithered away through the grass before she saw it.
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When Thomas reached home, she collapsed with pain. Her family didn’t know what to do. They brought her to a traditional healer, who told her to apply a paste of basil leaves and turmeric on her foot. It didn’t help. Two days later, she experienced excruciating abdominal pain, her urine turned black, and she spat blood. Her family took her to a small private hospital nearby. “They told us she was bitten by a very deadly snake and that she might not survive,” recounts Rohan Thomas, Ally’s son. Doctors at the hospital gave Ally Thomas antivenom, an antidote for snakebites that can save people’s lives if administered quickly enough. In India, doctors give patients a so-called polyvalent antivenom, which targets four of India’s most venomous snake species—Russell’s viper, Indian cobra, saw-scaled viper, and common krait. The hospital hoped giving Thomas this concoction would help her, even though doctors weren’t sure which snake
had bitten her. But after the antivenom was administered, Thomas was still in intense pain. So the next day, her family brought her to the nonprofit Little Flower Hospital and Research Centre, in Angamaly, Kerala. By this time, her limbs were numb and her vision was blurred. Noushad CK, a doctor at the hospital, says Thomas had gone into minor kidney failure, a complication that often arises from the bite of a viper—likely either a Russell’s or pit viper—in this part of the country. Springing to action quickly, Noushad’s team provided Thomas with intravenous fluids to improve her kidney function, and after two days, her condition improved. “It’s a miracle,” Noushad says. Thomas didn’t receive antivenom until three days after she was bitten. It should be given as soon as possible after a bite. Long delays before receiving antivenom normally result in irreversible organ damage or, worse, death. As Thomas sat propped up on pillows
More than 100,000 people die every year from venomous snakebites. In 2017, the World Health Organization recognized snakebites as a neglected tropical disease. Some researchers want to tackle the problem by addressing the shortcomings of current antivenoms. These conventional therapies require refrigeration for storage and trained personnel for delivery, both of which rural areas in the developing world often lack. Also, producing the antivenoms involves inoculating horses with snake venoms, which is expensive, is time consuming, and yields proteins that could trigger allergic reactions in people. Several research groups are now developing antivenom alternatives to try to get around these issues.
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Snakebite hot spots Most venomous snakebites in the world happen in these three regions.
Laos
South and Southeast Asia
Sub-Saharan Africa
Estimated venomous snakebites per year:
Estimated venomous snakebites per year:
231,866–961,508 89,009–417,343 Estimated snakebite deaths per year:
Estimated snakebite deaths per year:
14,902–52,760
3,243–31,751
Notable venomous snakes: Russell’s viper (Daboia russelii), Indian cobra (Naja naja), and Malayan krait (Bungarus candidus)
Notable venomous snakes: Black mamba (Dendroaspis polylepis), black-necked spitting cobra (Naja nigricollis), and puff adder (Bitis arietans)
Mexico, Central America, and Tropical South America Estimated venomous snakebites per year:
71,723–99,268 Estimated snakebite deaths per year:
293–1,760 Notable venomous snakes: Coral snakes (Micrurus), hog-nosed pit vipers (Porthidium), and South American rattlesnake (Crotalus durissus)
Russell’s viper
Black mamba Eastern coral snake
in the intensive care unit of Little Flower, she expressed how fortunate she felt. “I feel I’ve had a rebirth.” Although Thomas lived to tell her tale, many in India don’t. Snakebites kill nearly 50,000 people there every year. But snakebites aren’t India’s problem alone. Africa’s snakebite-related mortality rate is estimated at 30,000 per year, with sub-Saharan Africa being most affected. In total, more than 100,000 people die from venomous snakebites around the world each year. In June 2017, the World Health Organization took notice, recognizing snakebites as a neglected tropical disease. Since then, the world body has established a working group of researchers, physicians, and public health experts to create a plan for improving access to treatment and improving the quality of antivenoms, especially in the developing world. It also approved
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a resolution at the World Health Assembly in May 2018 to create a framework for helping countries tackle snakebites. Many countries struggle with treating snakebites because they lack the infrastructure or logistics planning needed to get antivenoms to people. For example, many clinics in the developing world don’t have adequate facilities for refrigerating antivenom serum. Also, many rural hospitals are often not staffed during the evenings or are remotely located, leaving victims with no choice but to rush to a traditional healer for immediate treatment, just like Thomas and her family did. Another challenge is that some countries lack the expertise to distinguish good antivenoms from bad ones, says JeanPhilippe Chippaux, a French physician who has worked in sub-Saharan Africa. The trouble is, bad ones are often cheap-
er—and more dangerous. They contain high concentrations of preservatives or protein-degradation products, indicating poor storage conditions, Chippaux says. Government officials generally pick the more inexpensive antivenom when deciding which one to stock hospitals with, Chippaux adds. “If the criteria is just price, it is not a good thing.” Moreover, not all antivenoms have gone through clinical trials and have therefore not been regulated. Regulatory concerns aside, some snakebite researchers think antivenoms themselves need an update. The method for making antivenom was standardized in the early 20th century. It involves milking a venomous snake and then immunizing animals, mostly horses, with diluted venom. The horses’ immune systems generate antibodies against the venom molecules over a few months. Technicians then take blood
CR E D I T: SA LE E M H A M EE D/ WI KI MED I A ( RU S S E LL’S V I P E R ) ; SA FA R I T RAVE LP LU S/ WI KI M ED I A ( BL AC K MA M BA ) ; S H UT TE RSTO CK ( CO RA L S N A KE )
Source: PLOS Med. 2008, DOI: 10.1371/journal.pmed.0050218.
plasma from the horses and extract the antibodies to make the antivenom serum. It’s a long and expensive process, and some companies cut corners, especially in purification. “In Africa, the purification of antibodies is not sufficiently achieved, and we have antivenom with high risks of side effects,” Chippaux says. Venoms also contain many components, including peptides, proteins, and enzymes. So antivenoms need to counter combinations of these rather than a single component to be effective. And the antibodies in antivenoms come from horses rather than humans. So injecting them into a person brings with it a risk of an allergic reaction to foreign substances, especially if the purification process hasn’t been carried out properly. Because of these reasons and more, scientists have started to look for alternatives to current antivenoms, keeping in mind the challenges posed by the poor public health infrastructure in countries such as India.
led to a 100% survival rate for rats and mice injected with venom from European adders or Eastern coral snakes (Toxins 2016, DOI: 10.3390/toxins8090248). Pigs that have been given coral snake venom also respond to varespladib. All the animals treated with the compound, either orally or intravenously, survived for the period studied, 120 h. But untreated pigs did not (Toxins 2018, DOI: 10.3390/toxins10110479). The small molecule offers many advantages over conventional antivenoms, Lewin says. It is inexpensive to make and thus would be affordable in resource-poor parts of the world. It would also be shelf stable in tropical climates. Lewin points out that varespladib is thousands of times as potent as a conventional antivenom at inhibiting phospholipase. “Further, it can penetrate tissues that antivenom cannot, such as muscle and neurons.” But varespladib’s biggest advantage is
with manufacturing firms that have global capabilities so that we can keep costs down and get the drugs to the people that need them most,” Lewin says. While Lewin is exploring a small-molecule alternative to standard antivenoms, a team led by Kenneth Shea, a chemist at the University of California, Irvine, is investigating a hydrogel consisting of polymer nanoparticles. The gel works by exploiting a phenomenon that happens when polymer nanoparticles are mixed with a solution containing proteins. The proteins glom on to the particles, creating what is called a corona. Shea and colleagues previously determined that they could get specific proteins to associate with the particles by tuning the composition of their polymers. So the researchers developed nanoparticles that would form coronas of phospholipase A2, selectively sequestering the
C R E D I T: D I N SA SACH A N
Skipping the fridge Some attempts to make alternative antivenoms focus on providing a therapy that is easier to use and easier to store. Current antivenoms are not stable unless refrigerated. “The major gap in antivenom’s utility is that it can only be administered in the hospital, but the vast majority of snakebites—upward of 75%—occur before victims can get to the hospital,” says Matthew Lewin, an emergency physician and the director of the Center for Exploration and Travel Health at the California Academy of Sciences. Lewin’s company, Ophirex, is investigating a therapy that wouldn’t have such storage needs. It is an off-patent compound named varespladib, first developed by the pharmaceutical firm Eli Lilly and Company, for its venom-fighting properties. The small molecule counters phospholipase A2, an enzyme that is found in the venoms of most snakes around the world. The enzyme degrades the lipids in cell membranes, causing cells to break down and a range of symptoms to appear, including paralysis, bleeding, and muscle destruction. Varespladib binds to the hydrophobic active site of the phospholipase enzyme and destabilizes it so it can’t go to work on lipids. The molecule also appears to fight inflammation in the victim’s body. In test tubes, varespladib neutralizes the membrane-munching activity of venoms from 28 snake species, including the black mamba and death adder. Intravenous and subcutaneous injections of the molecule
Ally Thomas nearly lost her life to the bite of a snake in her hometown of Nemmara in Kerala, India. that anyone can give it to a victim right on the spot after a snakebite, Lewin says. Only trained health-care practitioners can administer traditional antivenom therapy to patients because the agents typically have to be given intravenously, and their side effects need to be monitored. Still, Lewin emphasizes that the molecule probably would be used in combination with standard antivenoms. “Antivenoms have a longer half-life, circulating longer in the body, and a broader effect on nonphospholipase components of venom,” Lewin explains. Lewin hopes to begin a Phase I or Pivotal Phase II clinical trial in early 2020, in time for the taipan snakebite season in Papua New Guinea. “We hope to partner
enzyme from snake venoms (J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b10950). Shea likes to describe the polymer nanoparticles as plastic antibodies because the particles are plastic and they tie up the enzyme, preventing it from acting on cells. To find their antivenom nanoparticles, the researchers experimented with different combinations of monomers, such as acrylic acid, N-phenylacrylamide, N-isopropylacrylamide, and N,N′-methylenebis(acrylamide), to produce particles with different polymer compositions. They tested this library of nanoparticles against different types of venoms, and they chose the particles with the highest affinity for phospholipase A2 for further study. JANUARY 28, 2019 | CEN.ACS.ORG | C&EN
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What’s in a snakebite? A closer look at five toxins found in snake venoms.
Snake venom metalloproteinases Snakes with the toxins: Most members of the Viperidae family, including South America’s Bothrops jararaca pit viper Effects of the toxins: The enzymes produce a wide range of damage. For example, they help cause bleeding by breaking down components of capillaries, and they can disrupt blood clotting.
Phospholipase A2 Snakes with the toxins: Most snakes in the Viperidae and Elapidae families Effects of the toxins: The enzymes break down the phospholipids in cell membranes, leading to muscle damage and paralysis.
Shea and his team collaborated with José María Gutiérrez of the University of Costa Rica to test the best-performing nanoparticle in mice. They injected venom from an African cobra into the skin of the mice at a dose that would cause skin necrosis. Mice given several doses of the nanoparticle-based gel at the site of venom injection showed no signs of necrosis after 72 h, while untreated mice did (PLOS Neglected Trop. Dis. 2018, DOI: 10.1371/journal. pntd.0006736). According to further studies, the nanoparticles end up in the liver, where they are quickly captured and cleared from the body. Because the synthetic polymers are stabler than antibodies, this gel could survive in harsh conditions in tropical regions, Shea says. “You don’t have to worry about refrigeration.” But Shea says this so-called nanodote is not intended to replace traditional antivenom either. “It could be injected directly into the site of the bite by the victim or a companion,” he says. The gel would help keep the victim in stable condition until they get medical help, he adds. Before moving to clinical trials, Shea and his team want to test the nanodote against other relevant venomous snake species.
Snake venom serine proteinases Snakes with the toxins: Some snakes in both Viperidae and Elapidae families, including the sharpnosed viper found in southern China Effects of the toxins: These enzymes disrupt multiple aspects of the blood-clotting process.
Three-finger toxins Snakes with the toxins: Most snakes in the Elapidae family, including the Malayan krait (Bungarus candidus) Effects of the toxins: These molecules are potent neurotoxins that can cause paralysis.
Dendrotoxins Snakes with the toxins: Many snakes in the Elapidae family, including the eastern green mamba Effects of the toxins: These neurotoxins block potassium channels on nerve cells. Source: Nat. Rev. Dis. Primers 2017, DOI: 10.1038/nrdp.2017.63.
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While Lewin’s and Shea’s teams are looking for alternatives to antivenoms, some researchers haven’t given up on antivenoms’ main component: antibodies. These scientists are looking to biotechnology to establish less-cumbersome methods of producing venom-deactivating antibodies. The idea is to avoid milking dangerous snakes or injecting venom into horses. One such approach involves developing human monoclonal antibodies. Monoclonal antibodies are genetically engineered proteins that are synthesized by cloned immune cells and are identical to each other in amino acid sequence. Some cancer therapies use such antibodies to target specific proteins on cancer cells. A human monoclonal antibody is capable of targeting a specific venom toxin. An antidote that is a mixture of monoclonal antibodies could target multiple venom toxins to save the lives of snakebite victims. If researchers could find monoclonal antibodies that target specific venom ingredients, they could produce the antibodies on a large scale in mammalian cells. “Everything can be produced in big fermentation tanks in the laboratory,” says Cecilie Knudsen, a researcher at the Technical University of Denmark. “This is faster than immunizing animals.” The process takes days,
C R E D I T: P ROT EI N DATA BA N K ( A LL)
Biotech lends a hand
A long wait ahead
compared with the months that it takes to work with horses. And because the Some antivenantibodies are human, om researchers are they are less likely excited about these to cause the allergic alternatives but think reactions seen with it’s still early days in antivenoms made ustheir development. In ing horses. “Moreover, particular, Chippaux, Polymer nanoparticles (left) capture phospholipase A2 (center) and render it monoclonal antibodies inactive. the French physician, can persist in the huthinks it will take a man body for a longer time—they don’t get is the venom of the coral snake Micrurus while for monoclonal antibodies to make it corallinus, one of the deadliest serpents in cleared out by the immune system the way to the clinic for testing against snakebites. Brazil. foreign antibodies would,” Knudsen says. The venom of a single species of snake, he “The problem with making antivenom Scientists generally have two approachsays, contains an eclectic cocktail of toxins, against coral snake is obtaining venom to es to find a human monoclonal antibody and making antibodies against every one of immunize the horse. It doesn’t have enough them would be a marathon task. that targets a specific venom protein. venom in the venom gland,” Ho says. Hybridoma technology involves injecting Julien Potet, a policy adviser for the Instead of milking these snakes for their the target protein into mice, isolating the nonprofit group Doctors Without Borders, paltry supply of venom, the team wanted to says in the near term, these alternatives genes for the mouse antibody that forms find a way to make the proteins themselves. could work in conjunction with current in response to the protein, then tweaking They examined the amino acid sequences the gene in specific ways to humanize it. antivenom therapies. For example, he says, of the venom’s five most important toxins monoclonal antibodies “could be added to and then mapped their epitopes—the parts the conventional animal antibody prepaof the toxins to which an antibody would rations in order to fortify them against actually bind. They then constructed genes selected snake venoms that are hard to that encoded just those epitopes. neutralize with existing products.” The researchers injected pieces of DNA In general, these novel antidotes will containing these genes into mice so that have to prove a lot before they’d be adthe animals’ cellular machinery would opted in the field, experts say. First, Potet says, other researchers will —Paulo Lee Ho, biochemist, Instituto synthesize and churn out need to confirm in the lab Butantan proteins containing the epitopes. The rodents’ immune that the antidotes neutralize systems then started to their targeted venoms. After The other route is to use phage display, produce antibodies against that, scientists will need to which won the 2018 Nobel Prize in ChemN these epitopes. Basically, the investigate any possible toxistry, to screen a library of human antiscientists used the pieces of icities of the novel agents. body genes to find the antibody that binds O O DNA to immunize the mice And then large-scale clinical a target protein. O against the venom—like trials will be needed, and Knudsen and her colleagues from HO O NH2 those are expensive. injecting horses with the acDenmark and Costa Rica used the latter tual snake toxins. “You need detailed studmethod to find human monoclonal anVarespladib The team found that ies that involve thousands tibodies against the venom of the black immunizing the mice first with these DNA of people for these novel antidotes bemamba, a highly venomous snake from pieces and then with a booster shot of cause venom works differently on differsub-Saharan Africa. They looked for proteins containing just the epitopes was ent people’s bodies, based on individual antibodies that could bind to so-called fairly successful. The animals’ survival rate height, body weight, and amount of venom dendrotoxins inside the black mamba’s when injected with the snake venom was injected,” says Priyanka Kadam, founder venom. The team made two prototype 60%. All the untreated mice died. of the India-based nonprofit group Snakeantidotes and tested them in mice, findHo wants to improve the survival rate. bite Healing and Education Society and a ing that treated mice survived injections He thinks that the researchers haven’t yet member of the WHO panel on venomous of the mamba venom (Nat. Comm. 2018, isolated epitopes for all the significant snakebites. DOI: 10.1038/s41467-018-06086-4). toxins in the coral snake’s venom. “You’ve Unfortunately, most of the antivenoms Black mamba venom contains another got to keep in mind that venom is very that are supplied to sub-Saharan Africa group of deadly components called α-neucomplex.” and India aren’t clinically tested. “Like for rotoxins. Knudsen’s team next wants to Ho thinks this epitope immunization other neglected tropical diseases, there is develop antibodies against those molemethod could work well for snakes that very limited funding for the development cules. The researchers’ goal is to find anhave limited amounts of venom or highly of antivenoms,” Potet says. “For many tibodies for all the key mamba toxins and venomous serpents that are difficult to producers, the money is just not there to then mix them together into one antidote. handle. To produce antivenoms for people finance the clinical trials. Only a couple of Meanwhile, Paulo Lee Ho, a biochemist rather than mice, the team would have to the dozen existing antivenoms intended at Instituto Butantan, in São Paulo, and for use in sub-Saharan Africa have been his team are also harnessing biotechnology apply its technique to horses and harvest the antibodies produced after immunizing evaluated in robust randomized clinical techniques to derive an antivenom against them. trials.” And that doesn’t stop countries a particularly tricky snake. Their target
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“You’ve got to keep in mind that venom is very complex.”
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with lackadaisical regulatory bodies from registering antivenoms anyway. “It’s a shame,” Potet says. Meanwhile, Chippaux, who has treated snakebites in sub-Saharan Africa for decades, believes there is actually no need for novel antidotes. Just improving the quality and quantity of conventional antivenoms could significantly help Africans. “There is a need for a greater quantity of good-quality antivenom. While fewer than 100,000 doses are sold annually in sub-Saharan Africa, actual needs are estimated to be higher than 1 million doses,” he says. Potet also urges antivenom manufacturers to keep improving the current inventory. “This is not rocket science, and the investments that need to be made are probably not very high, but they need to happen,” Potet says. He points to incremental improvements at all stages of antivenom production, including how the venom immunization mixture is prepared and how the final product gets purified. “Not many producers have focused on these incremental improvements,” he says. In 2016, the WHO commissioned an assessment of the antivenoms sold in Africa and the facilities, many of which are in India, where they are produced. Manufac-
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turers were asked to submit information about their products to the organization. WHO officers also inspected the facilities to check if they followed best practices of production—for example, they determined whether the horses were kept in
ment is finalized, we will be able to select the antivenoms that we’d like to test in our clinical trial,” Potet says. No matter which of these efforts—improving current antivenom production, developing antivenom alternatives, or
“Like for other neglected tropical diseases, there is very limited funding for the development of antivenoms.” —Julien Potet, policy adviser, Doctors Without Borders healthy conditions. The WHO hasn’t yet disclosed its findings but plans to release a list of trusted suppliers after this assessment. “This will clarify which antivenoms are the most capable of neutralizing the venoms of the different African snake species,” Potet says. Moreover, Doctors Without Borders is gearing up to host and implement clinical trials of existing antivenoms in sub-Saharan Africa because it wants to find the most effective antivenoms for African snake species. The organization will start after the WHO completes its assessment of African antivenoms. “Once this assess-
creating human-antibody-based antivenoms—is most successful, it’s clear that changes are needed. “Ninety-five percent of people affected by snakebites come from impoverished backgrounds, and they aren’t aware of their fundamental rights, including the right to basic health care,” Kadam says. “These are voiceless victims, and not a priority for policy makers.” She says the developing world badly needs a novel antidote that could reduce treatment costs. “It would be a game changer.”
Dinsa Sachan is a freelancer writer based in New Delhi.
