Richard Plemper, PhD, specializing in molecular biology and biochemistry, distinguished University Professor of the Institute for Biomedical Sciences at Georgia State University, talks about research and work being done to take steps towards a new antiviral drug that could help ease the symptoms of influenza when it’s a mutated strain that can’t be stopped with a vaccine or other flu medications.
Talk about this antiviral drug you’re working on against the flu.
Plemper: Influenza, as you know, is a major and recurring clinical issue, with roughly 20 to 60 thousand case fatalities yearly in the United States alone and obviously globally, many more that affect predominantly older adults, in normal seasonal influenza years, but as we know, in pandemic years, that can be much larger, the death rates, and can also affect other groups of the population. For example, children can become much more exposed. Recognizing that as a major threat, we have been working for quite some time on developing influenza antivirals. This particular drug that we are favoring at the moment is a compound that inhibits the viral polymerase, so that’s the enzyme that the virus needs to amplify its genetic information, essentially to replicate and produce new infectious particles. This compound prevents activity of the polymerase, it stops the application process so the virus cannot produce new genomes that then can be packaged into new viral particles. The important thing is that the virus struggles to escape from this inhibitor. As you may have heard, we have since October 2018 a new class of influenza drugs that is effective, but that the virus rapidly escapes from inhibition. Meaning the virus genetically changes, it mutates and develops resistance, and the resistance can come up very rapidly. And resistant virus obviously is not inhibited any longer, so the drug loses its efficacy, loses its ability to block the virus. What we pursued as one of our major objectives was to identify a compound that creates or establishes a high barrier against viral resistance. That means it makes it very difficult for the virus to find a way around it. Naturally, that is in addition to potent inhibition of the virus. But this difficulty of the virus to escape is what we think is a major asset of our compound.
And what is the compound?
Plemper: The compound is an analog of the building blocks of the normal substrates that the virus uses to produce its genomic RNA. It’s called N4 hydroxycytidine or NHC. Cytidine is one of these building blocks. And our compound is a chemical analog; meaning it has modifications that don’t change it so much that the vial polymerase wouldn’t recognize it as the natural building block. But once it has incorporated it into the viral genome, then it impairs the replication process because it’s chemically slightly different.
So current medications for the flu, slowly, over time, the virus builds up resistance to them… How can this compound compare to those?
Plemper: What we have done is, in the initial tests, we perform viral adaptation studies. That means you grow virus in the lab in the presence of compound, in the presence of increasing concentrations. What you try to do is to force the virus to learn to replicate in the presence of the compounds, to adapt to it. In the clinic that would be the classical situation that you have a patient taking medication and feeling better, stopping taking the medication before the virus or the infection is entirely cleared. Then the pathogen starts replicating again, but in the presence of low drug concentrations. And that is essentially the breeding ground for resistance to emerge. Because if you completely block replication, then no mutation can come up. But if replication in the presence of a some compound, creating selective pressure, then resistance emerges. That is the same with bacteria diseases and escape, for example from penicillin, what we all know about and are warned against, right? Always take your full drug course. Deliberately using such a setting in the lab, we have compared how does the virus respond to our compound in comparison with licensed therapeutics, for example Xofluza or Tamiflu. Then we ask wether the virus can replicate again? Can we see replication? But we also drill down on a genetic level and use next generation sequencing, deep sequencing, to look at the whole genome composition of the virus population and try to identify candidate resistance mutations. What we saw is that for the licensed therapeutics resistence emerged as predicted, providing kind of the experimental control. After only a small number of passages, on average something between five to 10 passages, the virus escaped and signature resistance mutations, that are mutations that are known clinically and also in the research community, showed up. And those become then very quickly genetically dominant. That means all viruses in the population in the test dish that we are looking at carry this mutation. By comparison, even after extensive adaptation to our compound, we could not identify any resistance mutations, no mutation that became dominant and took over the virus population. That was a clear sign that the barrier is high. In biology, one needs to be very careful to say something cannot happen because there could be a constellation that may create more favorable conditions for a mutation that we did in the lab, despite our best efforts. Really a large amount of clinical data will ultimately address the question. But what we feel comfortable to say is that the barrier is high, based on our data. Which is reassuring, because even if in the field the virus finds a way to escape, and resistance can ultimately be mounted, the frequency with which this appears is obviously much lower if the barrier is higher. That, plus the assumption that if the virus needs to go to great length to escape, it may need more than one mutation. That’s typically how the barrier can be high? Let’s say it’s not one mutation that mediates resistance, but a combination of many that a virus may need to find. The statistical likelihood that that happens is much lower than a single mutation, which means that the frequency of escape is much, much lower. Even if that happens in the field, it obviously would remain to be seen whether such a virus would be less fit than the nonresistant parent virus. That means if a virus is impaired, if its fitness, is reduced, it may be clinically less of a concern because it cannot defend itself quite as well against the antiviral response that we mount.
Just to clarify, this compound compared to other current treatments – it’s more likely to fight the replications before it inhibits the reputation, so the virus doesn’t…
Plemper: Escape. To understand we need to look at the chemistry of the compound. The compound is very, very similar to the natural building block. What the substance is, the normal cytidine that the virus needs to incorporate into its nucleic acid. Influenza is an RNA virus, which means it has an RNA genome and must incorporate nucleotides, including cytidine, when it amplifies its genomic RNA. To escape, it would be imperative for the virus to find a way to differentiate between the inhibitor and the natural building block. It would be very easy for the virus to create a mutation that prevents incorporation of all cytidine. But of course, that’s fatal, right? If the virus cannot incorporate cytidine then it cannot produce RNA. Such an escape route is simply not viable because the virus would inactivate itself, thus such a mutation could never come up. The challenge for the virus, the biochemical challenge, would be to identify a mutation that lets it distinguish the compound from the natural cytidine. And then, of course, prevent selectively only incorperation of the analog compound. And because chemically both substances are so similar, that seems to be a real challenge, a biochemical challenge that we at least could not trigger the virus to solve. Therefore, we think if the virus finds mutations that block the compound, block incorporation of the inhibitor, they probably also either block or at least impair incorporation of the natural building block to such a degree that the overall polymerization efficiency is too low for a virus to survive.
I was reading that you did some studies. Can you talk a bit about that?
Plemper: Yes of course. Drug development starts with identifying the clinical need and appropriate inhibitor candidates. If we have isolated candidates that are antiviral in cultured cells, we need to ask then also the question how long is the drug actually present in the body? Because when we take drugs, concentrations change ouver time due to, in particular, liver function, which is responsible for removing substances from the bloodstream – even if this is an antiviral drug that we don’t want to be removed rapidly. When we see that drug levels in an organism are good, we can ask how active is the compound against the virus? How good is it to block the virus in a relevant animal model of influenza disease, which requires testing it in animals as a necessary stepping-stone before one can consider clinical trials and testing it in human patients. There are different animal models, but what one really requests in an animal model is that it as closely as possible recapitulates hallmarks of human disease. Because that is ultimately what we want to treat. And so we tested our compound in ferrets that were infected with influenza viruses, different influenza virus strains. One was a virus that was isolated during the 2009 pandemic, which was the last major influenza pandemic that we had. Everybody remembers the swine origin influenza, right? We probably have a good chance that some of us here actually were infected at the time. That means, for our tests we used a pandemic influenza virus that replicates quite aggressively and causes strong symptoms in the ferret hosts. The ferrets develop influenza similar to humans; they develop fever, they shed the virus. That means a lot of virus is produced in the respiratory tract and can then also be spread, or be transmitted from animal to animal through droplets. They can develop respiratory symptoms like sneezing and discharge. And of course, they also have high, high viral titers, meaning many viral particles are in the respiratory tract. One can follow this in investigating nasal lavages, but viral titers are also very high in the lung, for example. We then treated these animals with the drug, and importantly we treated them therapeutically. That means we first give them the virus, then let disease develop, and then start the treatment, because that is really what happens in the clinic. Not many people would take a drug prophylactically; before they have any symptoms. The important parameters are then how late can we start treatment after infection and have an effect? And what is the minimal concentration that we have to give, which tells us how much drug is necessary to see benefit, like reduction of clinical signs? And then lastly, it was an important objective for us in the development program to address how can we administer the drug? We think that it is important for the patient population that we want the drug to benefit, mostly older adults suffering from serious but seasonal influenza, that the compound is orally available. That means that people can take it, for example, as a pill by mouth rather than that it requires injection. Clearly, oral bioavailability will improve compliance of the patient to actually take the drug. Accordingly, this is how we administered the compound in our studies, we gave it to the ferret orally, not as pill, but as a liquid directly given by mouth. And then we followed clinical signs. We could demonstrate that the compound was efficacious, meaning it lowered viral burden rapidly. It alleviated clinical signs, for example fever went down faster. Every other clinical sign we examined also disappeared significantly faster than control animals that didn’t receive the compound but were dosed placebo, and recovered faster from the infection. And very importantly, the compound was well tolerated. Naturally the last we want is the therapeutic itself to be toxic make people feel worse or have any side effects that negatively effect the patient well-being, independent of its antiviral effect. In the animal models, we can follow appearance as sign of toxicity, but can also go deeper and follow, for example, the blood chemistry after treatment, such as liver enzymes and ask whether anything abnormal comes up. So far, the ferrets we treated in our studies tolerated the drug very, very well.
So how far away do you see this going to human trials?
Plemper: The pipeline to human trials is that first the drug needs to be tested under controlled conditions, and then needs formal toxicology studies. That means the drug needs to be given to accepted toxicology species. That’s typically a rodent species like, for example, rats, and typically dogs are the second species over a 28-day time course. And that is followed very closely, and then the highest tolerated concentrations of the concentration that does not induce any serious adverse effects is determined. And that knowledge then drives the application to the FDA to test the drug in the first human trial, which would address first safety, how well is the drug tolerated in humans, starting with very low doses, and then then raising those slowly. This influenza inhibitor has completed the 28-day two species tests under formal conditions, and is at the moment at the stage of assembling the safety package, which will then be evaluated by the FDA. Dependent on the FDA’s position taken to these drugs, they may request more tests or they may be satisfied with what has been provided, and clinical trials and safety trials could start as early as this summer to fall.
So summer 2020?
Plemper: Yes. I talked a lot about experimental therapeutic testing, experimental drug development, but this compound is, from our perspective, quite advanced towards actual clinical use. At the moment influenza is obviously a major clinical problem, but what is at the moment in the news and in the forefront of our mind is obviously corona viruses with the current outbreak that may become a corona virus pandemic. What is exciting about this compound is that it does not only block influenza viruses, but it actually has what we call a broader indication spectrum. That means it is active against different viruses. My lab does not work specifically with corona viruses, which belongs to an entirely different viral family, but colleagues tested the same drug against coronaviruses and have actually shown good activity also against coronavirus infection in a mouse model. Combined with the encouraging toxicology data that creates a therapeutic possibility if needed, depending on how the current threat plays out, plays out right now in real time with us watching this. This compound could be a potential candidate for fast tracking and use as an emergency medication against the coronavirus outbreak.
OK So they actually did already test this compound?
Plemper: It has been tested. But again, it has been tested in animal models and in cell culture. There, it was very active. As I said, we’ve known for a while that the compound has a broadened indication spectrum and is active against a number of pathogens. Obviously now coronaviruses are or have moved to the forefront of our minds. Therefore it was expedited for testing in the coronavirus animal model, and it was efficacious based on the data released so far. Will it be active in humans? I mean we need to look deep into the crystal ball and decide. Taking together what we know about the compound, what we have done in terms of testing against influenza and the early animal model against coronavirus is encouraging. I think it’s clearly one of the candidates very worthwhile looking at.
And what implications do you believe this will have for the medical field or any particular individual going through the flu?
Plemper: For influenza the primary objective is to prevent severe respiratory infection. Typically with influenza, you develop bronchitis. But you can get an infection also of the lower respiratory tract. What we are really concerned about is that the virus spreads to the small airways, resulting in viral pneumonia. Then that is in particular to older adults a very quickly life-threatening disease. One of the major objectives is to prevent this progression to real severe infection of the small airways. That doesn’t mean that there won’t be any clinical signs and symptoms, but life-threatening diseases is avoided. That is one of the major fields of use that we see against seasonal influenza. But then with influenza and coming back once more to coronavirus, what we see also is that that viruses change, they evolve, and new or potentially more pathogenic forms can emerge that can cause or at least threaten to cause a global pandemic with major health implications for almost everyone globally. And therefore for influenza, what we think we need to do is add to our arsenal of therapeutics that improve preparedness so that we not only start developing drugs when a pandemic is upon us, because then it’s simply too late, considering the timeline of drug development, but that we actually have more options, more anti-viral options to fight a pandemic with a highly pathogenic avian influenza virus that spills over into the human population and is more pathogenic than normal seasonal viruses. These viruses immediately cause more severe disease, and spreads rapidly, if they are able to very efficiently transmit from human to human. That is really the major fear. And we feel that we need compounds that give us additional therapeutic options to stop such a pandemic should it happen, to stop that in its tracks.
Any side effects so far that you’ve seen? Any people with certain conditions that this wouldn’t necessarily be good for?
Plemper: That is difficult to say before we really have formal toxicology results in humans. Like every drug, of course, the effect and also side effects, depend on concentration. In drug development, what we look at is what’s the therapeutic window or the specificity index. What is the concentration we need for the medical benefit, in this case to block the virus, and then at what concentration does the drug have adverse effects, so the body responds negatively to it? Side effects are unavoidable, but of course what we want is that the ratio between those two concentrations is as large as possible, meaning very low concentrations are anti-viral but toxic concentrations are very high. That being said, every drug if it’s taken in too high a concentration, will cause toxic effects. At some point, we overload the system and if we move towards, say liver failure, then of course, that leads to catastrophic outcomes.
Anything I didn’t ask you that you feel people should know?
Plemper: We work on antiviral therapeutics, but I want to encourage everyone to vaccinate if a vaccine is available. People may say, well, if you have a therapeutic, then we don’t need to vaccinate. Plus, they don’t like the needle prick; the yearly flu shot. But we really are not developing therapeutics to provide an alternative option. We develop therapeutics because vaccines, in particular against influenza, vaccines can fail or be counter-indicated. And so not everyone who takes a vaccine is actually protected, or some people cannot be vaccinated because they have pre-existing conditions or they simply made the decision to not vaccinate. These are vulnerable to infection and come down with disease. That’s where we see the need for antivirals. But I would encourage everyone who can take the vaccine, to vaccinate, because prophylaxis is always better than treatment and trying to curb a disease. But if vaccination is not an option, was ineffective, or a shot has been missed, then we try to provide mitigation that prevents progression to potentially catastrophic disease.
END OF INTERVIEW
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