For more than fifty years, the only drugs for Chagas disease, a parasitic infection that leads to deadly cardiac and gastrointestinal outcomes, have been difficult to get ahold of, plagued by unwanted side effects, and have not been very effective. Now, by using a naturally infected nonhuman primate model, researchers have identified a new compound that may be the best hope yet for treating Chagas disease.
Host: Stephanie DeMarco
Michael Levy at the University of Pennsylvania
Rick Tarleton at the University of Georgia
Gregory Wilkerson at the University of North Carolina, Chapel Hill
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DDN Dialogues is a new podcast from Drug Discovery News. Join us as we explore the stories behind the latest advances in drug discovery research.
Stephanie DeMarco: Hello everyone! Welcome back to a new episode of DDN Dialogues! I’m your host, Stephanie DeMarco.
In today’s show, we’re taking a virtual trip down to South Texas, specifically to a primate research center just outside Austin.
There, we’ll meet some very special monkeys who — like many animals that live at least part of their lives outdoors — have a taste for bugs. While these primates aren’t picky about their insect snacks, one of the common ones they eat is the kissing bug.
Kissing bugs may conjure up images of romance and passion, but the reality is much darker. They often carry a deadly hitchhiker inside of them: the parasite Trypanosoma cruzi. A T. cruzi infection can lead to a life-threatening case of Chagas disease both in animals and humans. While we have drugs to treat this disease, the drugs themselves can have severe side effects, and if people don’t get the drugs soon after they’re infected, the treatment is not very effective.
But now, scientists may have discovered a new compound that could be a game-changer for Chagas disease treatment, and they did it with the help of a few of these bug-eating monkeys down in Texas.
Our story starts just after dark, in a home in rural Peru or Mexico or the southern United States.
With walls built of adobe and a thatched roof overhead, these homes have many openings for kissing bugs to sneak through while the inhabitants are sleeping. These insects, which also go by the names reduviid or triatomine bugs, feed on blood.
But they don’t infect people with T. cruzi by biting them. No, their most common route of infection is a little more gross.
Michael Levy: It will suck blood for quite a long time, 10 or 20 minutes, and then it defecates while it's sucking blood. And if it has this parasite — the parasite is in the gut of the insect — and it comes out with the insect’s poo, and then it gets on the skin. And then it can get into the body in a number of ways.
DeMarco: That’s Michael Levy, an epidemiologist who studies Chagas disease transmission at the University of Pennsylvania. Oftentimes, people will rub or scratch where the kissing bug bit them. This will inadvertently transfer the parasites from the insect’s poo into their victim’s eyes, mouth, or other mucus membranes. This allows the parasite to enter the blood stream, and that’s where T. cruzi really starts to wreak havoc.
Rick Tarleton is an immunologist and parasitologist who has studied T. cruzi and Chagas disease at the University of Georgia for almost forty years, and he told me just how much damage these parasites can do.
Rick Tarleton: The parasites cycle through an intracellular and extracellular stage while they're in mammals. They have to invade cells in order to replicate. That continuous cycling in and out of host cells and the immune response to that is what causes the disease, Chagas disease. Parasite has a predilection for infection in muscle, so the primary presentation of Chagas disease is heart disease and sometimes gut disease. And this is related to the destruction of muscle tissue by the persistence of the parasite and the immune response to it for years — if not decades — in an infected host.
Levy: Although it sounds like a very unlikely set of occurrences — this bug biting you and pooping on you and this parasite getting into your bloodstream — it's actually an extraordinarily common and prevalent disease throughout the Americas.
DeMarco: The World Health Organization estimates that around six to seven million people living in South America, Central America, Mexico, and the southern United States have Chagas disease .
Tarleton: I suspect it's at least twice that because detection is not routinely done. And it is one of the largest causes of infectious myocarditis in the world as a result of its presence in so many individuals in this area.
DeMarco: Currently, doctors treat Chagas disease with one of two drugs: nifurtimox and benznidazole. But because of the drugs’ severe side effects and variable effectiveness, researchers have been searching for better ones. But in the past 50 years since these drugs were developed, no other drug candidate has come close to even the minimal success of nifurtimox and benznidazole.
Tarleton: It is an understudied parasite, so there's not that many people that work on it. Too, it is a disease of poverty, so those who have the infection or disease are not influential. There's not likely to be a lot of money to be made in this area, so these large pharmaceutical companies in general aren't interested in it.
DeMarco: Tarleton and his team spent many years investigating the interplay between the immune system and the parasite, but a research trip to Argentina inspired him to look beyond T. cruzi’s basic biology to see how his research could translate into new treatments.
Tarleton: We worked with a group of scientists including cardiologists and human subjects, initially doing immunology, but starting to understand that interesting immunological questions didn't always — our answers to those didn't always translate into utility for infected individuals. Rodolpho Vialti, a cardiologist who unfortunately died a few years ago, was a big influence there into convincing me that, really one of the things that needed to be done was better drug development.
And so, we started to work in that area, thinking we could make a difference because we understood the parasite and the infection, and we used different animal models to understand it. And that if we worked with really good medicinal chemists who understood the chemistry of drug design, that we might collectively be able to make an impact.
DeMarco: To make this drug discovery goal a reality, Tarleton and his team joined forces with the scientists at Anacor Pharmaceuticals, which is now owned by Pfizer. At the time, Anacor had a library of boron-containing compounds that had already shown promise for treating other parasitic diseases in many different animal models.
Tarleton: The initial experiments started from some compounds that Anacor Pharmaceuticals had previously screened, and they had some interesting hits from that. But the group they had collaborated with had sort of given up on it, and they asked us, would you be willing to test this, this one compound that looked pretty promising in mice, and so we did. And we found that it worked quite well, better than some of the other compounds that are in use now. And so that was sort of the hint to go down that road, but that road broadens very quickly into a very large number of compounds.
DeMarco: Tarleton’s team screened multiple compounds in vitro and in mice and found that the drugs were extremely good at killing parasites without having any harmful effects on the rodents. With a promising compound from Anacor’s library in hand, they requested a few similar compounds to see if they could find one with even better parasite-killing properties. One of those compounds was a benzoxaborole called AN15368.
Tarleton: The other compounds that we were looking at had great activity; this one had fantastic activity.
It's a pro drug. And it looks like the pro drug is likely preferentially taken up by host cells and then into parasites, where it's cleaved into the active drug compound, and that cleavage doesn't happen in the host cell itself. And that's a really important aspect because the inactive compound basically goes into the parasite and gets activated by a parasite molecule, so that is likely one of the reasons why its toxicity is not worse.
At this point in our study, we were starting to realize that this looked a lot like some other compounds that worked with related organisms like African trypanosomes. And so we were able to look at the targets of those compounds and say, is the target the same here? And at least one of the targets is a splicing factor, CPSF3. If the parasite essentially can't make new proteins, that's not a good situation, and it seems to be fairly selective in that activity. So that's also great.
DeMarco: While this was promising, researchers have cured mice with Chagas disease before. Getting those drugs to translate to humans has been largely unsuccessful.
Tarleton: There have been a number of clinical trials of new drugs for treating Chagas disease, they’ve failed, simply put, because they haven't been as extensively evaluated using the tools that are available before they were tested in humans.
DeMarco: But Tarleton and his team found those tools, which brings us back to those monkeys in South Texas.
To learn more about these very special primates, I spoke with Greg Wilkerson, who is now a veterinary pathologist at the University of North Carolina, Chapel Hill. At the time of this study, he led the nonhuman primate research for this paper at the Michale E. Keeling Center for Comparative Medicine and Research at the MD Anderson Cancer Center.
Greg Wilkerson: Around 2012, 2013, the entire nonhuman primate research industry became interested in Chagas disease as a background artifact to a lot of the studies that were going on. We were doing some testing, and we noticed that we had a fair percentage of animals that were coming up positive. And as we started to follow this over the next couple of years, we realized we were getting about 8 to 12 animals per year newly infected, and they're getting infected through exposures to the insect vector of this parasite.
Kissing bugs are native to southern United States, in Texas in particular, and the monkeys are housed indoor-outdoor. It betters the life of the research animals to have indoor-outdoor sort of contact. And these animals eat bugs as part of their normal life. They enjoy it. They catch them. They eat them, and by eating these bugs that are infected with this parasite, they can become infected.
Over the years, we've come to the conclusion that the vast majority of these infections are coming from the environment, and in short of taking them out of South Texas or completely confining them indoors, we're not gonna be able to get rid of it.
DeMarco: As other nonhuman primate facilities in south Texas became aware of the T. cruzi infections affecting their monkeys, they found new homes for these animals or placed them on projects where their infection wouldn’t affect the study results. But, Wilkerson and his team at the Keeling Center had the resources to watch over their monkeys with Chagas disease.
Wilkerson: We came to the conclusion that it was of no harm to keep these animals within the colony. They were still reproducing just fine. They weren't spreading it to one another. And so I actually took these animals and sort of characterized them. There were animals that have been infected for as short as six months and some that had been infected for as long as 12 years. So, I had a wide variety of animals. I knew the exact time period that they were infected because once I diagnosed them as infected, I actually had blood saved back from these animals. We do yearly physicals on them, and I went backwards in time and found out the exact year that they became infected. So, I had all this documented, and after I had a nice little pool of animals, I went and started knocking on doors, such as it was through email. And, I came across Dr. Tarleton, and he's well well known within the Chagas research field, and he was definitely interested.
Tarleton: Some people look at this, and worry about the ethics of working in nonhuman primates. But the thing is, these are animals that are already infected with T. cruzi and some of them are clinically ill from that.
Wilkerson: I've used the analogy, you know, lemons into lemonade, because for some people within the industry, these animals were not very much use within the biomedical community, but for Chagas they're a perfect model.
DeMarco: With this pretty much perfect model of Chagas disease and the opportunity to potentially treat some very sick monkeys, Tarleton, Wilkerson and their teams joined forces to test their new compound. So, the researchers enrolled 22 infected rhesus macaques in the study. Three monkeys served as controls and 19 monkeys received the oral AN15368 treatment for 60 days in a row.
Wilkerson: They all were brought into the housing facility indoors during the 60 days of test administration, but we gave them, several weeks to get used to being indoors. With that time period, we also started training them so that they were getting treats. Every day at 11 o'clock, they got a marshmallow sandwich, you know, a little marshmallow on two pieces of graham cracker, or the next day, it would be some frozen yogurt. And we found that we were able to put the drug within those treats. So as far as they know, they were just getting treats for 60 days.
DeMarco: I love that they like marshmallows with graham crackers, like a s’more!
Wilkerson: That’s not part of their normal feeding routine, but you know, when you're trying to administer a drug, you're not sure how it's gonna taste? We do go out of the way a little bit!
We didn't see any nausea and any weight loss. Actually, they gained weight, as you might imagine getting treats for 60 days. So they did well during the treatments. And the control animals got treats, but they did not have the AN15368 added to their treats. But best we can tell, the monkeys were unable to detect it, and the reason I say that is we've had other drugs in the past, and you'll see the monkeys, they'll learn that that's a tainted treat. And they will take that treat, and they'll dig around the center part where the drug may be hidden. They'll nibble around it. They'll smell it, and this drug just didn't seem to be detected by any of the monkeys on the study. So that was really nice in that regard.
DeMarco: After the monkeys got their last marshmallow or froyo treat on day 60, the researchers collected their blood to look for parasites. They looked at multiple time points until 145 days after their last dose.
Wilkerson: Because this parasite is complex and likes to hide within cells of the body, we had to sacrifice nine of the 19 animals. They were euthanized, and we did a very very extensive necropsy. And then we collected tissues. We analyzed those tissues using molecular techniques.
DeMarco: That is when the truly hard work began. Finding parasites in tissue is easy, but proving a negative — that the drug killed every single parasite — is a lot more challenging. Tarleton and his team analyzed the tissues from the control monkeys and the treated monkeys with every test they knew.
Tarleton: We just kept going back to those same tissues and say, okay, we need to screen more of this, screen more of this, and we just never detected anything until we satisfied ourselves that okay, we've screened enough here. That took a while, and you know, fingers were crossed every time we did a new set of screens, but they turned out great.
DeMarco: Levy, who was not affiliated with the study, was also really impressed with the thoroughness of these screens.
Levy: Just the barrage of assays that they had at their disposal to get as close as possible to that test of cure, that elusive, 'we got rid of the parasite test' is a real strength.
DeMarco: Now, you might be wondering. What about the other 10 monkeys in the study? What happened to them?
Wilkerson: The other 10, though, they're the interesting story. They actually were returned to the breeding colony. One died within about a year and a half, not related in any way, shape, or form to the drug, but the other nine of the 10 that we put back are still in the colony to date. And for three and a half years, the nine that remained out there have been free of, of the parasite T. cruzi, which is, which is really part of the exciting part of this study.
A lot of these studies don't have the ability to put back a subset of their monkeys. They're able to look at them for 145 days and go, “Yay, it worked.” But you really want to know, especially with this parasite, have you controlled it in the long term, and three and a half years later, we're feeling like we we've pretty much controlled it.
DeMarco: Tarleton and his team also monitored the antibodies in the blood of these monkeys over the course of the same three and a half years. While the control monkey that didn’t receive treatment continued to produce antibodies against T. cruzi, the treated monkeys did not.
Wilkerson: It proved that the drug worked well for three and a half years. But it also cleared some of our monkeys. Again, these monkeys that may have had manifestations of cardiac disease or health issues later, we basically got nine animals fixed, treated using this novel treatment, which is great.
DeMarco: Moving forward, Tarleton and his team are testing AN15368 in additional toxicology studies and determining if they can easily manufacture the drug under GLP conditions.
Tarleton: Those studies are ongoing. During that time, we hope to secure additional funding for human clinical trials, and we're exploring partnerships that would allow for that first, assuming that manufacturing and toxicology studies support it, that we can be prepared for a human clinical trial with this drug.
DeMarco: While Tarleton and his team will continue studying this compound, Chagas disease researchers like Levy are eager to see what comes next.
Levy: Oh yeah. There's been so many years of mediocre drugs and poorly done trials that this was a huge breath of fresh air. I probably got overly excited when I first read this paper when it came out.
DeMarco: Having worked with the monkeys on this study, Wilkerson too is hopeful for the future of AN15368.
Wilkerson: So I've worked with some vaccines. I've worked with some other drugs, and we've initially seen some parameters of success only to be disappointed in the end. And this one has consistently surprised me, especially when we started going back six months later and testing those monkeys that went back out into the colony, and they were still negative, that we still could not find the parasite in these animals. I became a believer. I was hopeful, but I didn't want to get over exuberant about the possibilities of it. But I've really become a believer that this may be a great therapeutic going forward, for everyone, humans-animals alike.
DeMarco: Maybe now, after fifty years of research, we finally have the most promising treatment for Chagas disease yet. Tarleton is ready to see it through.
Tarleton: There hasn't been a lot of high impact work done on this disease, and so the opportunity to make an impact was something that has driven me at least in the last 10 or 15 years and through this project, and may keep me off the retirement list for a few more years.
DeMarco: That’s all we have for this episode of DDN Dialogues. I’d like to thank Rick Tarleton, Greg Wilkerson, and Michael Levy for talking with me, and thank you so much for listening! Until next time, I’m your host Stephanie DeMarco. This episode of DDN Dialogues was reported, written, and edited by me, with additional audio editing by Jessica Smart. To never miss an episode, subscribe to DDN Dialogues wherever you get your podcasts.
And, if you ever find yourself outside in South Texas, say a little thank you to the bug-eating monkeys for their contribution to what hopefully will someday be a treatment for Chagas disease.