Posted on 11/14/2023 9:56:47 AM PST by Red Badger
In 2022, the U.S. National Institutes of Health (NIH) placed a large bet on an experimental drug developed to limit brain damage after strokes. The agency committed up to $30 million to administer a compound called 3K3A-APC in a study of 1400 people shortly after they experience an acute ischemic stroke, a perilous condition in which a clot blocks blood flow to part of the brain.
The gamble seemed warranted. Lab studies, most by a longtime grantee, prominent University of Southern California (USC) neuroscientist Berislav Zlokovic, had generated promising data. A small safety study of the drug, sponsored by a company Zlokovic co-founded called ZZ Biotech, was also encouraging. Analyses of data from the phase 2 trial hinted that the treatment reduced the number of tiny, asymptomatic brain hemorrhages after stroke patients received either surgery to remove the clot, the clot-busting drug tissue plasminogen activator (tPA), or both.
For many years, scientists have tried to reduce the brain cell death, bleeding, and inflammation that can follow a stroke, some of which results from disruption of the blood-brain barrier—a system of tiny blood vessels that delivers oxygen and nutrients but shields the brain from toxic substances. tPA, the only approved stroke drug in the United States and Europe, can vastly reduce death and disability by clearing a stroke’s blockage, but the drug, too, can cause dangerous brain bleeding. 3K3A-APC could help mitigate such damage and prevent brain cells from dying, ZZ Biotech said.
Because of its potential to address an unmet medical need, the U.S. Food and Drug Administration (FDA) gave the compound “fast track” status, with the prospect of “accelerated approval and priority review.” ZZ Biotech says the new trial should start within a few months.
But a 113-page dossier obtained by Science from a small group of whistleblowers paints a less encouraging picture. The dossier, which they submitted to NIH, highlights evidence from the phase 2 trial that the experimental remedy might have actually increased deaths in the first week after treatment: Six of the 66 stroke patients who received 3K3A-APC died within this period, compared with one among 44 in the placebo group, although the death rate evened out after a month. Patients who received the drug also trended toward greater disability and dependency at the end of the trial, 90 days after treatment.
Deepening the concern, the dossier also highlights evidence that dozens of papers from Zlokovic’s lab—including many supporting the idea that the compound was ready for human testing—contain seemingly doctored data that suggest scientific misconduct. The whistleblowers say apparent changes to images used for protein identification and other purposes seem to skew results in favor of the scientist’s hypotheses, which include influential ideas about the blood-brain barrier and its role in stroke and Alzheimer’s disease, as well as how 3K3A-APC supposedly affects it.
Zlokovic’s institution, USC, will confidentially review the content of the dossier, a spokesperson said, adding, “USC takes any allegations relating to research integrity seriously.” Zlokovic declined requests for an interview about the whistleblowers’ findings. But an attorney representing him told Science in a statement that Zlokovic “is committed to fully cooperating” with the USC inquiry. Without providing specifics, the statement noted that some elements of the dossier are “based on information and premises Professor Zlokovic knows to be completely incorrect,” or pertain to experiments not completed in his lab.
But speaking to Science anonymously, four former members of Zlokovic’s lab say the anomalies the whistleblowers found are no accident. They describe a culture of intimidation, in which he regularly pushed them and others in the lab to adjust data. Two of them said he sometimes had people change lab notebooks after experiments were completed to ensure they only contained the desired results. “There were clear examples of him instructing people to manipulate data to fit the hypothesis,” one of the lab members says.
Given the dossier findings, its authors want all clinical testing of 3K3A-APC halted for now. Multiple neurologists and neuroscientists who reviewed the dossier for Science agree. “To have a fourfold increase in mortality in the first few days of giving the drug really gives me pause,” says Wade Smith, a neurologist at the University of California, San Francisco. Smith found the whistleblower report so disturbing that he couldn’t sleep the night after he read it.
Because the drug has to be given soon after a stroke, Smith and others point out, hundreds of patients—or their family members by proxy—might have just hours or even minutes to decide whether to join the trial. Given what Smith calls possible “scientific fraud” in the preclinical research supporting 3K3A-APC’s supposed protective effects, he thinks the trial should not go forward until NIH, USC, and other organizations can address the whistleblowers’ allegations. “If we’re wrong about the trial, and we really upset some people, well, then I’m sorry,” Smith says. “But the opposite is unfathomable.”
All told, the whistleblowers raise concerns about images from 35 basic research studies Zlokovic’s team has published, as well as data from two reports on the phase 2 trial of 3K3A-APC. The publications have a single common author: Zlokovic. In 29 of them—including the main report on the phase 2 trial—he occupied the last author slot, denoting his senior role. No other author is on even half of the dossier’s papers.
Some of Zlokovic’s collaborators argue that extensive work outside his lab supports the promise of the potential drug enough to press on with the phase 3 trial. But the lead whistleblower, Vanderbilt University neuroscientist Matthew Schrag, hopes NIH will delay it after seeing the dossier and initiate a sweeping examination of the challenged papers. “Numerous articles appear to warrant retraction and it is likely that this has involved a range of grants as well,” the document’s introduction notes. NIH told Science it takes research integrity concerns very seriously, but otherwise declined to comment.
Much of the data described in the dossier “is clearly, undeniably, the result of misrepresentation,” says a leading neuroscientist who studies some of the same topics as Zlokovic. “That saddens me, because he’s a very respected member of this community.” The researcher insisted on anonymity, concerned about becoming embroiled in a controversy that he predicts will threaten Zlokovic’s career.
A CELEBRATED AND ECLECTIC neuroscientist and biotech entrepreneur, Zlokovic has rarely hit a false note—as an M.D.-Ph.D. researcher, institute leader, entrepreneur, and even a talented amateur opera singer. Throughout his steady climb into the academic stratosphere, the charismatic physician—“Betza” to friends and close colleagues—always found time to maintain his vocal gifts.
“Science requires clear and perfect language, while music is a universal language,” he told the Cure Alzheimer’s Fund, one of his patrons. He proved that maxim a few years ago at a neuroscience conference reception, belting out a credible version of O Sole Mio to the evident delight of his youthful audience.
Zlokovic even credits opera as opening a door to his lifelong research interests. During a fellowship at Queen Elizabeth College in London, his department chair asked him to attend a dinner party. “He invited me because he knew I could sing,” Zlokovic told AAAS, publisher of Science, in 2014 when the organization made him one its prestigious Fellows. He apparently charmed the dinner guests, including eminent physiologist Hugh Davson, an expert in the blood-brain barrier. Davson inspired Zlokovic to study the role of the barrier in neurological problems of aging, including Alzheimer’s and an often-related condition, cerebral amyloid angiopathy (CAA), in which protein deposits replace the smooth muscle fibers of blood vessel walls and weaken them.
Trained as a doctor at the University of Belgrade in 1978, Zlokovic stayed on there to complete a Ph.D. in physiology in 1983. He later joined the faculty at USC, then spent more than a decade at the University of Rochester before returning to USC in 2012 to direct the Zilkha Neurogenetic Institute, created in 2002 with $20 million from the W. M. Keck Foundation and a matching grant from married philanthropists Selim Zilkha and Mary Hayley. (Zilkha inspired the other “Z” in ZZ Biotech.)
Under Zlokovic’s leadership, the USC institute has expanded to more than 30 labs and grown its annual funding more than 10-fold, exceeding $39 million in 2022. NIH grants to Zlokovic have totaled about $93 million. A prodigious fundraiser, in the past decade alone he has added at least $28 million from private sources, according to USC.
Hard-driving and prolific, Zlokovic has pioneered work on pericytes, cells that surround the brain’s capillaries and help maintain the blood-brain barrier. He has also linked the blood-brain barrier to Alzheimer’s disease, partly by showing it helps move beta-amyloid proteins, widely viewed as a cause of the disease, out of the brain. That work won him a share of the $100,000 Potamkin Prize in 2009 from the American Academy of Neurology.
Last year, the Journal of Molecular Neuroscience published an analysis of key “influencers” who have explored the role of the blood-brain barrier in mild cognitive impairment—early symptoms of dementia. Zlokovic, it concluded, dominated that field of research. Science also used Dimensions Analytics, a database of scholarly research from the U.K. company Digital Science, to examine Zlokovic’s influence in related research categories. The data show that for decades, he has led the world in citations to studies of the influence of the blood-brain barrier and pericytes on stroke or Alzheimer’s (see chart, below).
Zlokovic and a collaborator, Scripps Research biochemist John Griffin, also dominate studies of an enzyme called activated protein C (APC). It acts as an anticoagulant in the body, and they argue that the molecule protects against blood clots and inflammation in the brain’s blood vessels, suggesting it could lead to a treatment for stroke. In 2007, Zlokovic helped launch ZZ Biotech to pursue that hope, and according to the company, now owns a roughly 3% stake in it. The firm has worked to turn a safer and more effective version of APC, created in Griffin’s lab, into a drug. 3K3A-APC, a form of APC in which three amino acids have been changed, is the result.
Company CEO Kent Pryor said last year that for people who experience acute ischemic stroke, “3K3A-APC is a potential game changer.”
ON PUBPEER, a website where scientists and data sleuths publish concerns about possible image doctoring and other forms of scientific misconduct—often anonymously—doubts about Zlokovic’s research began to surface in 2017. Schrag, who previously uncovered possible image manipulation in other lines of Alzheimer’s research, agreed to help Science scrutinize some of the claims and other work by Zlokovic.
He concluded that work from the lab warranted a close look. Over a few weeks Schrag examined Zlokovic’s publications, many in leading journals, and the 3K3A-APC clinical trial reports. He also recruited Kevin Patrick, a forensic image analyst who is not a scientist and uses the pseudonym “Cheshire” on social media. (Patrick, who agreed to have his identity revealed publicly for the first time in this article, made additional findings.) Mu Yang, a neurobiologist at Columbia University, also contributed. Schrag and Yang worked independently from their respective universities. The dossier they compiled also folds in comments about Zlokovic’s research posted to PubPeer by Patrick and microbiologist and forensic image analyst Elisabeth Bik, among others.
Science did not pay the dossier authors or anyone else for scrutinizing Zlokovic’s work. According to Schrag, he, Patrick, and Bik might file a federal whistleblower lawsuit to receive a portion of any NIH funds the government claws back from USC if federal authorities deem Zlokovic’s work fraudulent.
Molecular biologist Mike Rossner—president of Image Data Integrity, a former Journal of Cell Biology editor, and a consultant on image manipulation—also evaluated the Zlokovic dossier. And Bik reviewed images in it that she had not personally posted to PubPeer. Both agree the dossier shows strong evidence of errors or misconduct in many of Zlokovic’s papers. Some images, including in studies about 3K3A-APC and APC, appear doctored in ways that could affect the interpretation of the data, the two say.
For example, a 2013 study in The Journal of Neuroscience suggests that 3K3A-APC confers a range of protections for brain cells. According to the whistleblowers, key Western blots—which use antibodies to visualize specific proteins within a tissue sample—seem to have been improperly copied and flipped horizontally. And a 2022 mouse study in Frontiers in Neuroscience showing that 3K3A-APC protects brain cells and the blood-brain barrier from stroke damage includes a crucial image that appears to have been duplicated, in altered form, from a 2019 Nature Neuroscience paper on a different topic.
The dossier authors and others who reviewed it for Science note that some apparently duplicated images could be simple mistakes. Other anomalies might be innocent digital artifacts. For example, Western blots sometimes gain unnatural-looking qualities during the publication process. Doubts about an image in a paper can often be resolved only by comparing the original, uncropped, high-resolution version against the published example, and Zlokovic did not respond to Science’s request for original images. But everyone who has seen the findings says they raise serious and far-reaching questions about his lab practices, research results, and the pending clinical trial.
ONE OF SEVEN neuroscientists who reviewed the dossier is Stanford University’s Thomas Südhof, a Nobel laureate who has seen some of his own papers criticized on PubPeer. (He conceded some errors and rejected other critiques as unfounded.) He cautions against uncritically accepting every apparent image anomaly as evidence of misconduct. “I’m not implying that some of the key papers underlying the clinical trials have fraudulent elements,” Südhof says.
Even if some duplicated images are innocent errors, he says, they suggest a worrisome carelessness by Zlokovic and his co-authors. Others are “very hard to explain” as accidental, Südhof adds. He was particularly struck by a 2004 Nature Medicine paper that appears to show a single blood vessel cross section copied and pasted digitally in two other places within an image (see image, above). Südhof calls it “almost impossible” to explain as unintended.
Chris Schaffer, a Cornell University biomedical engineer, says he was most taken aback by a pair of papers published 5 years apart that seem to use the same image to represent different results (see image, below). In a 2004 paper the image purportedly shows how natural APC prevents brain cells from dying. But a 2009 paper includes what appears to be the same image as evidence that the ZZ Biotech compound also protects the brain but without causing hazardous bleeding, a drawback of natural APC. Data from that paper helped set the dose of 3K3A-APC initially tested in people.
The dossier suggests that cellular features had been removed from a raw image before its use in the 2004 paper, and that the original image was used in the later paper. The whistleblower analysis was so persuasive that it left Schaffer shaken: “I immediately felt nauseous,” he says. “The integrity of the scientific record is so fundamental to what we do that seeing this kind of data anomaly is distressing.”
Schaffer, an expert in optical imaging for neuroscience, took the dossier’s analysis of the two papers even further. When he adjusted the image contrast, details missed by the whistleblowers swam into view: In the 2004 paper, superimposed square boxes cover the nuclei of some brain cells. The boxes may mask signs of nuclear fragmentation indicating that the supposedly protected cells were dying, he suggests. “It’s hard to imagine those boxes emerging as a digital artifact,” Schaffer says. “They’re perfect squares.”
The Cornell scientist says whoever apparently manipulated the image might have wanted to show “cleaner,” more consistent data or, in a less charitable interpretation, tried to obscure signs that APC and 3K3A-APC didn’t actually protect brain cells.
Another neuroscientist who reviewed the dossier but declined to be named for fear of courting a legal dispute was shocked to see apparently manipulated images in two papers he had peer reviewed. “One of them is so clear in retrospect that I should have spotted it,” he says. “But we are not trained as referees to spend much time looking for such things.” The scientist adds: “Overall, I broadly agree with the conclusions [in the dossier]. My main residual question is, why? Why would one bother to go to these lengths to change images, when the guy has the resources to generate loads of great papers without doing this?”
THE FOUR FORMER lab members who allege Zlokovic pushed them and others to manipulate data paint a picture of a pressure cooker environment in which their boss expected new data almost every week, always in line with his hypotheses. All worked with Zlokovic for years and published with him, and they gave similar descriptions of the research environment. Science also spoke briefly to a fifth former lab member, Angeliki Nikolakopoulou, the first author on three papers in the dossier and now principal scientist at Bionaut Labs, a Los Angeles biotech company. “The only thing I can tell you after being a member of his lab for 8 years is that there is no misconduct,” she said, then hung up.
In lab meetings, the accusers say, researchers were discouraged from speaking up and contributing intellectually to the lab’s work, which was tightly controlled by Zlokovic. “It’s science. So normally you would express your opinions,” one notes. Instead, that person says, newcomers soon learned that speaking up meant facing “humiliation”—a term three of the insiders used—and dismissal of their comments. Except to answer questions, one researcher says, “we were all silent.”
All four say Zlokovic routinely castigated junior scientists when his desired experimental results were not obtained. “If you are not in agreement with him, you will lose the lead authorship on a paper or on a project,” one of the scientists says. “Of course, this is important for your career.” Another says, “If the data does not look like the hypothesis, we were afraid to even bring it to the lab meeting.”
One researcher described how a group of lab members approached Zilkha’s human resources department about the “toxic environment.” The complaint was rejected because they insisted on remaining anonymous for fear of retaliation.
Several former lab members provided details of experimental data from Zlokovic’s lab that they say were falsified. These included experiments referenced in the whistleblower dossier. In some cases, they said, data points that would have invalidated the desired results were removed. “It was not real science. He already knew what he wanted to say” before the experiment was completed, one says. “I started hating science. … It made me sick.”
Two of the insiders also say Zlokovic sometimes had his team improperly alter existing notebooks. Normally these notebooks—in which scientists record details of their work as it proceeds—provide a ground truth for an experiment’s methods and results. As a result, they’re also often central to misconduct investigations.
But two of the former lab members say that after an experiment was completed and its results published, Zlokovic sometimes admonished his scientists to make sure the notebooks were “clean.” That was understood to mean pasting into them printouts of the published results and methodology or omitting contrary details that challenged the paper’s conclusions. Zlokovic explained that those changes were needed in case of an “audit,” according to the two scientists.
Two of the former lab members say they have wrestled with whether to speak out for years, knowing it might damage their own careers. “This is a moment in life when I must choose between what is right and what is easy,” one says. “The easy option would be not speaking with you. I decided that when I lay on my deathbed one day, I might regret not doing what is right.”
AFTER A LONG HISTORY of failed stroke drugs, a panel of researchers and physicians in 1999 established rigorous criteria for clinical trials of stroke treatments—known by the acronym STAIR, for the Stroke Treatment Academic Industry Roundtable that established them. The panel wanted to help ensure that only clearly promising treatments would be tested in stroke victims.
In a 2013 paper in the journal Stroke, Zlokovic and colleagues assessed 3K3A-APC research to see whether it met the 10 STAIR criteria. For example, a potential stroke drug had to show promise in both sexes of two animal species—in that case, rats and mice. And both animal behavior and tissue samples had to validate the drug’s efficacy after a stroke. 3K3A-APC easily met all the STAIR criteria, they concluded. Among other evidence cited, it reduced the volume of brain tissue damaged by the stroke and stopped some bleeding caused by tPA.
Much of the evidence that 2013 paper relied on is now in question, however.
Science provided the dossier to USC neurologist Patrick Lyden, principal investigator for the 3K3A-APC phase 2 trial and head of the planned phase 3 study. Lyden, who works at the Zilkha institute and was an author of the Stroke paper certifying 3K3A-APC as ready for clinical trials, said in a statement that the alleged problems in Zlokovic’s papers were outweighed by the support for the drug from other sources.
But even if 3K3A-APC truly meets the STAIR criteria, the drug’s phase 2 trial, known as RHAPSODY, was problematic, according to the whistleblowers and others. They say the trial might have unintentionally favored 3K3A-APC. Stroke patients were given the ZZ Biotech drug or a placebo after standard care—tPA, the surgical removal of the clot, or both. But according to the final report on the RHAPSODY trial, among patients who got both standard treatments, the placebo group received tPA, on average, more than 2 hours later than those given ZZ Biotech’s experimental drug. A table in the published final trial report seems to indicate that some of the placebo patients even got tPA outside of the American Stroke Association–approved window of no more than 4.5 hours after acute ischemic stroke.
“Even minutes [of delay before getting tPA] are considered a significant difference,” says neuroscientist Andreas Charidimou of Boston University, who reviewed the dossier. That disparity “pushed the data to show benefits in the experimental drug.”
In response to that concern, Lyden gave Science a different version of the phase 2 trial data, showing no delays in the placebo patients receiving tPA. But in the revised data table, patients who had clots removed surgically followed by a placebo waited, on average, more than 2 hours longer than the surgical patients later treated with 3K3A-APC. Some of the placebo patients had surgery beyond the study’s prescribed 6-hour limit. Although there was no indication that care was purposely delayed, the trial’s prespecified criteria indicate those patients should not have been eligible for the study.
Charidimou, a veteran stroke investigator, says the longer the delay before the surgery, the less successfully it prevents brain damage. So Lyden’s new data table, like the published version, showed that the experiment favored patients treated with 3K3A-APC, Charidimou contends.
Despite either possible advantage, 3K3A-APC didn’t show a statistically significant advantage over the placebo in brain-hemorrhage volume. Its superiority in the rate of hemorrhages was barely significant only for the tiniest, asymptomatic bleeds, detected using brain scans. And in addition to the six deaths soon after the compound’s use, some neurologists are concerned that more patients in the treatment group than placebo group experienced potentially damaging brain swelling in the days after the intervention—the opposite of the drug’s intended effect.
Lyden challenged those negative interpretations, saying that in part because of the trial’s small size there were no “statistically significant differences in safety outcomes,” and the phase 3 trial would better evaluate any drug side effects.
Science also shared the dossier with Pryor, ZZ Biotech’s CEO, and Griffin, 3K3A-APC’s co-developer. Both defended the drug.
“While Prof. Zlokovic’s lab has run the majority of the stroke animal models using 3K3A-APC, results from other laboratories have shown similar findings and everything we have seen is consistent with the externally established mechanism of the drug,” Pryor said in his statement. He added that the evidence shows it’s safe enough for further testing. “If anything is uncovered between now and then that would change our feelings, we have time to halt the study commencement.”
In Griffin’s statement to Science, he said ample evidence from several labs supports the promise of 3K3A-APC. “There is no basis for implying there is any need to delay continuation of the RHAPSODY trials because of any important deficiency in current fundamental knowledge about 3K3A-APC,” he said, calling the dossier’s claims “unjustified assertions.”
Griffin is a co-author on 11 of the questioned Zlokovic papers, published over 20 years. Telling Science he stands by the “fundamental principal conclusions” of those involving APC, Griffin says he can’t vouch for specific images flagged as potentially altered because he lacks access to the originals for comparison. Griffin calls Zlokovic “a brilliant scientist … of unimpeachable integrity.”
SEVERAL SUBFIELDS of neurological research could face a reckoning if Zlokovic’s work comes unraveled for sloppiness or misconduct. All of the papers challenged in the dossier pertain to some degree to Zlokovic’s 4-decade interest in the blood-brain barrier, which has become increasingly important in research on CAA, Alzheimer’s, stroke, and other neurological conditions. For example, NIH funding for studies concerning both “blood-brain barrier” and “Alzheimer’s” shot up from $13 million in 2006 to $241 million last year. The agency’s funding also rose dramatically for work examining the blood-brain barrier and stroke or pericytes.
The basic-science papers described in the whistleblower dossier, excluding a recently posted preprint, have been cited more than 8400 times. On average, those papers have been 27 times as influential as comparable work in the same fields published during the same years, according to Dimensions. They have been cited in 49 patents by 30 companies, universities, and foundations—indicating broad interest in commercializing discoveries that are now in question.
To Schaffer, the dossier findings “unquestionably trigger the need for a robust investigation [of all the questioned papers] that goes all the way back to raw data and includes interviews of the scientists who conducted this work.” The goal would be to see which of Zlokovic’s contributions rest on solid data from his own lab or others, Schaffer adds. “Until that investigation has been conducted, the scientific community should use caution in building on these results.”
Prior to the dossier’s creation, Patrick and Bik had conveyed concerns to several journals that published some of the Zlokovic papers, requesting an examination. In six cases, corrections were made. After Science provided the dossier to Zlokovic, he or colleagues also responded on PubPeer to comments on several other papers, saying corrections were in process.
It remains to be seen just how damaging an invalidation of Zlokovic’s work would be to other research on Alzheimer’s and CAA, and to drug development for stroke. That’s “one of the million-dollar questions,” Charidimou says. “We need to clarify which of these findings are replicable and correct, and which are completely off.”
Any impacts on research might take years to play out. But in the coming weeks NIH and FDA will face a pressing issue: whether to postpone or halt human testing of 3K3A-APC. Like NIH, FDA declined to comment on the matter.
If the trial goes forward, NIH should force Zlokovic and his close collaborators “to recuse themselves from the conduct of the trial,” says University of Calgary neurologist Eric Smith. “If any unconflicted investigators are left,” he adds, they should “justify to an independent oversight committee that there is sufficient support for the scientific rationale, independent of the Zlokovic work, to continue.”
The evidence would need to be compelling, he says. “As a site investigator for stroke trials, I would not agree to participate in the phase 3 trial based on what I know now.”
With reporting by Madeleine Sherer of the Investigative Reporting Workshop at American University and Science Contributing Correspondent Cathleen O’Grady. Aaron Sorensen of Digital Science provided technical assistance.
This story was supported by the Science Fund for Investigative Reporting.
How can we help but ask ourselves WHAT is on the up and up anymore?
Money has long corrupted medical research. Many “definitive studies” are financed by pharmaceutical companies with a vested interest in the outcomes. Those “studies” are then published in “prestigious journals” who’s editors have become rich by the lavish pharmaceutical advertisements placed in those journals. The FDA and the CDC, Lysenko pawns of the Democrats, are simply no longer trusted by the majority of independent physicians.
Uh-oh!
Look at profit motive.
Research that shows something commonly available is much more likely to be devoid of greed taint.
Thanks, badger.
Ping to the rest of you.
Should we ping Dingbat?
This sure looks like misinformation— after all, it’s attacking SCIENCE™!
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