The compound, part of a class of molecules called salicylanilides, was designed in the laboratory of Professor Kim Janda, PhD, the Ely R. Callaway, Jr. Professor of Chemistry and director of the Worm Institute for Research and Medicine at Scripps Research, in La Jolla, CA.

“It has been known for 10 or 15 years that salicylanilides work against certain viruses,” Janda says. “However, they tend to be gut-restricted and can have toxicity issues.”

Janda’s compound overcomes both issues, in mouse and cell-based tests, acting as both an antiviral and an anti-inflammatory drug-like compound, with properties that auger well for its use in pill form.

Salicylanilides were first discovered in Germany in the 1950s and used to address worm infections in cattle. Versions including the drug niclosamide are used in animals and humans today to treat tapeworm. They have also been studied for anti-cancer and antimicrobial properties.

The modified salicylanilide compound that Janda created was one of about 60 that he built years ago for another project. When the SARS-CoV-2 virus became a global pandemic in early 2020, knowing that they may have antiviral properties, he started screening his old collection, first in cells with collaborators from Sorrento Therapeutics and The University of Texas Medical Branch, and later, after seeing promising results, working with Scripps Research immunologist John Teijaro, PhD, who conducted rodent studies.

One compound stood out. Dubbed simply “No. 11,” it differs from the commercial tapeworm medicines in key ways, including its ability to pass beyond the gut and be absorbed into the bloodstream — and without the worrisome toxicity.

“Niclosamide is basically digestive-track restricted, and that makes sense, because that’s where parasites reside,” Janda says. “For that reason, simple drug repurposing for a COVID treatment would be counterintuitive, as you want something that is readily bioavailable, yet does not possess the systemic toxicity that niclosamide has.”

About 80 percent of salicylanilide 11 passed into the bloodstream, compared to about 10 percent of the antiparasitic drug niclosamide, which has recently entered clinical trials as a COVID-19 treatment, Janda says.

The experiments showed that of the many modified salicylanilides he had built in his laboratory, No. 11 affected pandemic coronavirus infections in two ways. First, it interfered with how the virus deposited its genetic material into infected cells, a process called endocytosis. Endocytosis requires the virus to form a lipid-based packet around viral genes. The packet enters the infected cell and dissolves, so the infected cell’s protein-building machinery can read it and churn out new viral copies. No. 11 appears to prevent the packet’s dissolution.

“The compound’s antiviral mechanism is the key,” Janda says. “It blocks the viral material from getting out of the endosome, and it just gets degraded. This process does not allow new viral particles to be made as readily.”

Importantly, because it acts inside cells rather than on viral spikes, questions about whether it would work in new variants like Delta and Lambda aren’t a concern, he adds.

“This mechanism is not dependent on the virus spike protein, so these new variants coming up aren’t going to relegate us to finding new molecules as is the case with vaccines or antibodies,” Janda says.

In addition, No. 11 helped quiet potentially toxic inflammation in the research animals, Janda says, which could be important for treating acute respiratory distress associated with life-threatening COVID infections. It reduced levels of interleukin 6, a signaling protein which is a key contributor of inflammation typically found in advanced stages of COVID-19.

Better medications against COVID-19 are urgently needed, as highly infectious new variants drive renewed surges of illness and death globally. But Janda says salicylanilide No. 11 was created long before the pandemic.

After fighting an unpleasant bacterial infection called Clostridioides difficile about 10 years ago,he saw a clear need for better treatment options. Multi-drug-resistant strains of C. difficile have become a major cause of drug-resistant diarrheal disease outbreaks in health care institutions globally, and among people using antibiotics. As director of the Worm Institute, which focused on parasitic infections, Janda was very familiar with salicylanilides, and knew of their antimicrobial properties. His laboratory created a “library” of modified salicylanilides several of which showed strong efficacy against C. difficile, and the collection was subsequently licensed by pharmaceutical firm Sorrento Therapeutics. Among them was salicylanilide 11.

“Salicylanilide 11 actually was placed on the back burner in my laboratory against C. difficile because it’s not as gut-restricted as we would like it to be,” Janda says. “But salicylanilide 11 has got a lot of really positive things going for it as a potential therapeutic for COVID.”

Now, in the journal Scientific Reports, researchers describe the putative molecular regulators of this pathological chronic reductive stress — a microRNA network.

Redox balance is vital for health. Oxidative stress has long been linked to heart failure, the progressive weakening of the heart muscle that can lead to death, though attempts at antioxidant therapy have been ineffectual. The finding that reductive stress can also lead to heart pathology may help personalize treatment of heart failure patients, leading to better outcomes.

Human microRNAs, or miRNAs, are short, non-coding RNAs with about 22 bases. They act to regulate gene expression by a complementary pairing with specific messenger RNAs of the cell. That pairing silences the messenger RNA, preventing them from being translated into a protein. Thus, miRNAs are a fine-tuned controller of cell metabolism or the cell’s response to stress and adverse challenges, like oxidative stress in the heart.

The current research, led by Rajasekaran Namakkal-Soorappan, Ph.D., associate professor in the UAB Department of Pathology, used mice that overexpress Nrf2, pronounced “nerf-two,” in cardiomyocytes to identify the miRNA network.

Nrf2 is a master transcriptional regulator that confers short-term protection — by helping express genes for antioxidant activity — for heart muscle cells when reactive oxygen and nitrogen species are created as blood flow returns after a heart attack. However, persistent Nrf2 activation can paradoxically result in reductive stress.

The researchers had recently shown that Nrf2 deficiency inhibited the expression of several miRNAs in the heart, suggesting a relationship between Nrf2 expression and miRNAs. So, they now decided to look for changes in miRNA levels in three mouse models — one with normal Nrf2 and two that constitutively overexpress Nrf2, at either low or high levels. Overexpression of both leads to pathological heart remodeling.

Comparison of miRNA levels from the three models identified a subset of miRNAs that appeared to be a direct and dose-dependent target of Nrf2, and thus putative regulators of reductive stress. Namakkal-Soorappan calls these miRNAs reductomiRs, pronounced “reducto-meers.”

The researchers also identified dose-dependent genes that were differentially expressed in the hearts of mice that overexpress Nrf2. Because miRNAs silence gene expression at the post-transcriptional level, the researchers believed that this distinct subset of genes could represent reductomiR targets for negative regulation.

Next, they looked for a link between the reductomiRs and the genes. In normal function, Nrf2 promotes the expression of genes that contain a DNA sequence called the “antioxidant response element” located near their promoters. With genomic software tools, the researchers probed the DNA of the mouse genome to find sequences for miRNAs that also had an antioxidant response element near their promoters.

They then used bioinformatics tools to identify 19 miRNAs that exhibited complementary sequences to the seed sequences in 61 down-regulated differentially expressed genes. These 19 miRNAs thus appear to be reductomiRs that mediate Nrf2-responsive myocardial reductive stress. Other computational tools also were used to create an integrative, Nrf2-responsive miRNA-mRNA functional network that shows putative nodes of differentially expressed genes.

Namakkal-Soorappan calls the reductomiRs only putative mediators because the analyses in the current study resulted from bioinformatics. Needed next, he says, are mechanistic studies to confirm functions of the mediators.

Co-first authors of the study, “Identification of Nrf2-responsive microRNA networks as putative mediators of myocardial reductive stress,” are Justin M. Quiles and Mark E. Pepin, UAB Department of Pathology, Division of Molecular and Cellular Pathology.

Co-authors, along with corresponding author Namakkal-Soorappan, are Sini Sunny, Sandeep B. Shelar, Anil K. Challa and Adam R. Wende, UAB Department of Pathology; Brian Dalley and John R. Hoidal, University of Utah; and Steven M. Pogwizd, UAB Department of Medicine Comprehensive Cardiovascular Center.

Support came from National Institutes of Health grants 2HL118067, HL118067, AG042860, HL133011, HL007918 and HL137240; American Heart Association grant BGIA 0865015F; a University of Utah Center for Aging Pilot Grant; the University of Utah; UAB; and an Alexander von Humboldt Foundation postdoctoral fellowship.

Many individuals with kidney dysfunction develop anemia, or a shortage of healthy red blood cells that carry oxygen to the body’s tissues. Some treatments for anemia are linked with serious cardiovascular side effects, but a new class of oral drugs called hypoxia inducible factor-prolyl hydroxylase inhibitors may be comparable to placebo in these trials. These drugs act on the pathway involved in the production of erythropoietin that stimulates red blood cell formation.

To evaluate the efficacy and cardiovascular safety of one such inhibitor — called roxadustat — Robert Provenzano, MD (Wayne State University School of Medicine) and his colleagues analyzed data pooled from three phase 3 studies of roxadustat in patients with chronic kidney disease and anemia.

In total, 2,391 patients received roxadustat and 1,886 received a placebo. Roxadustat treatment boosted levels of hemoglobin (the protein in red blood cells responsible for transporting oxygen): roxadustat- vs. placebo-treated patients showed an average change in hemoglobin averaged over weeks 28 to 52 of 1.9 vs. 0.1 g/dL. Roxadustat also reduced the need for red blood cell transfusions in the first 52 weeks, and there were no increased risks of mortality, heart attacks, strokes linked to the drug.

“Roxadustat was shown to be effective, with an acceptable safety profile,” said Dr. Provenzano. “As an oral agent, roxadustat addresses the significant unmet need in treating anemia in patients with kidney disease.”

Study co-authors include Lynda Szczech, MD, Robert Leong, MD, Khalil Saikali, MD, Ming Zhong, PhD, Tyson T. Lee, PhD, Dustin J. Little, MD, Mark T, Houser, MD, Lars Frison, PhD, John Houghton, and Thomas B. Neff, MD.

Disclosures: RP serves as a consultant for AstraZeneca, DaVita, and FibroGen. LS, RL, KGS, MZ, TTL are employees of FibroGen and hold stock and/or stock options in FibroGen. MTH, LF, DJL, and JH are employees of AstraZeneca and hold stock and/or stock options in AstraZeneca.

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In the general population, low blood vitamin D levels have been associated with higher risks of various diseases, including type 2 diabetes and kidney disease. Sun H. Kim, MD, MS (Stanford University School of Medicine) and her colleagues conducted a secondary analysis of the Vitamin D and type 2 diabetes (D2d) study to evaluate the effects of vitamin D supplementation on kidney health in individuals with pre-diabetes, a condition that increases risk for type 2 diabetes, which in turn is the leading cause of kidney disease.

The study randomized 2,423 adults with overweight/obesity and pre-diabetes to vitamin D₃ 4000 IU per day or placebo, for a median treatment duration of 2.9 years. “The D2d study is unique because we recruited individuals with high-risk pre-diabetes, having 2-out-of-3 abnormal glucose values, and we recruited more than 2,000 participants, representing the largest vitamin D diabetes prevention trial to date,” said Dr. Kim.

During the trial, there were 28 cases of kidney function worsening in the vitamin D group and 30 in the placebo group, and the average change in kidney function during follow-up was similar in both groups. “Our results did not show a benefit of vitamin D supplements on kidney function. About 43% of the study population was taking outside-of-study vitamin D, up to 1000 IU daily, at study entry, though. Among those who were not taking any vitamin D on their own, there was a suggestion for vitamin D lowering the amount of urine protein over time, which means that it could have a beneficial effect on kidney health. Additional studies are needed to look into this further.”

Dr. Kim added that vitamin D supplementation is popular, and it’s difficult for clinical trials of vitamin D supplementation to show a benefit if the population studied is not vitamin D deficient. “The majority of the study population had sufficient blood vitamin D levels and normal kidney function,” she said. “Benefits of vitamin D might be greater in people with low blood vitamin D levels and/or reduced kidney function.”

Study co-authors include Irwin G. Brodsky, MD, Ranee Chatterjee, MD, MPH, Sangeeta R. Kashyap, MD, William C. Knowler, MD, DrPH, Emilia Liao, MD, Jason Nelson, MPH, Richard Pratley, MD, Neda Rasouli, MD, Ellen M. Vickery, MS, Mark Sarnak, MD, MS, and Anastassios G. Pittas, MD, MS.

Disclosures: The authors reported no relevant financial disclosures associated with this publication.

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