Hebrew University researchers say Tricor (fenofibrate) could potentially transform a devastating disease into a much more manageable form of mild respiratory disease in a multiple patient test where 14 out of 15 severe patients were disconnected from ventilators.
Tricor lowers alipids and reduces inflammation in coronavirus patients in 48 hours and eliminates the need for oxygen support within five to seven days, a Hebrew University study published Monday said.
Recent vaccination efforts around the world have been hampered by multiple variants that challenge existing vaccines. While the infection generally produces a mild disease, in some patients it can turn into a severe inflammatory disease, which requires medical intervention.
Recently, Professor Yaakov Nahmias’ team at the Hebrew University of Jerusalem reported that the novel coronavirus strain causes an abnormal buildup of lipids, which are known to initiate severe inflammation in a process called lipotoxicity.
Last year, the team identified the lipid-lowering drug TriCor (fenofibrate) as an effective antiviral, showing that it reduces damage to lung cells and blocks virus replication.
These results have since been confirmed by several international research groups.
Now, the Hebrew University team is reporting promising results from an interventional clinical trial led by Nahmias and coordinated by Prof. Shlomo Maayan, head of the Infectious Diseases Unit at Barzilai.
In addition, 14 of the 15 severe patients did not require oxygen support one week after treatment, while historical data shows that the vast majority of severe patients treated with the standard of care require long-term respiratory support, ”he added.
These results are promising as TriCor was approved by the FDA in 1975 for long-term use and has a strong safety record.
“There are no silver bullets,” said Nahmias, “but fenofibrate is much safer than other drugs proposed to date, and its mechanism of action makes it less likely to be variant-specific.”
“All patients treated with Tricor were discharged within less than a week of starting treatment and were discharged to complete the 10-day treatment at home, with no drug-related adverse events reported,” as noted Maayan .
These preliminary results offer promise of alleviating the substantial health burdens experienced by patients surviving the acute phase of COVID19, ″ he said. The researchers pointed out that although the results have been extremely promising, only randomized placebo-controlled trials can serve as a basis for clinical decisions.
“We have entered the second phase of the study and are actively recruiting patients,” said Nahmias, noting that two Phase 3 studies have already been conducted in South America, the United States and Israel.
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for a pandemic, which has cost over 1.9 million lives worldwide so far (Dhama et al., 2020; World Health Organization, 2020; Wu et al., 2020). The emergence of new virus variants with higher transmissibility rates is seeing rapid increases in infection rates and deaths across the world.
Several vaccines have undergone accelerated approval and are being rolled out worldwide (Baden et al., 2021; Voysey et al., 2021). While the clinical data are very promising, the vaccines are not recommended or suitable in all patient groups, e.g., children, those with hyperimmune disorders, and those using immunosuppressants (Meo et al., 2021), and with the global spread of viral variants of concern, e.g., Alpha-B.1.1.7, Beta-B.1.351, Gamma-P.1, and Delta-B.1.617.2, it is presently unclear whether the current vaccines will offer sufficient protection to emerging strains (Meo et al., 2021).
While in a few countries vaccination programs are progressing at speed, vaccine uptake rates are variable and for most low middle-income countries, significant proportions of the population are unlikely to be vaccinated until 2022. Furthermore, while vaccination has been shown to reduce infection rates and severity of disease, we are as yet unsure of the strength and duration of the response. Therapies are still urgently needed to manage COVID-19 patients who develop symptoms and/or require hospitalization.
The virus gains entry to human cells by the receptor-binding domain (RBD) of the viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) on human cells (Clausen et al., 2020; Hoffmann et al., 2020). Although other receptors of the virus have been identified (Cantuti-Castelvetri et al., 2020; Daly et al., 2020), drugs that block virus binding to ACE2 may substantially reduce virus uptake, thereby reducing/relieving symptoms in patients with an active infection or reduce transmission of the virus to uninfected individuals.
While the rapid escalation of the SARS-CoV-2 epidemic leaves insufficient time to develop new drugs via traditional pipelines, drug repurposing offers an expedited and attractive alternative. Drugs which are repurposed are available for immediate clinical use and their pharmacokinetic and safety profiles are usually well described.
This has already proven true, with the identification that dexamethasone reduces mortality of SARS-CoV-2 patients (RECOVERY Collaborative Group et al., 2021) and remdesivir decreases the time needed for patients to recover from infection (Beigel et al., 2020). In these cases, although the drugs are technically being repurposed, their use still depends on the drug’s recognized mechanism of action. It is less obvious which drugs might have a novel mechanism of action and interfere with SARS-CoV-2 binding and cellular entry mediated by ACE2. To this end, we recently developed an assay to measure the viral spike protein’s RBD binding to ACE2 (Lima et al., 2021).
Structural studies have shown that ACE2 is a dimer and that there may be multiple spike RBDs interacting with each ACE2 dimer (Yan et al., 2020). Molecular dynamic simulations have suggested considerable flexibility in ACE2 and this might allow multiple ACE2 dimers to bind to each spike trimer (Barros et al., 2021). If this were to be the case, the dimerization of ACE2 would lead to multiple contacts with each spike trimer, increasing the avidity of the binding.
Alternatively, dimerization of ACE2 might sterically hinder the protomers from binding to the spike protein. It therefore seems reasonable that the extent of ACE2 dimerization might affect the avidity of RBD binding. Furthermore, dimerization has been shown to affect the internalization of other receptors.
For example, dimerization of EGF or FGF receptors promotes their endocytosis (Wang et al., 2005; Opalinski et al., 2017) and different mechanisms of internalization may exist for monomeric and dimeric GH receptors (Gent et al., 2002). This led to the hypothesis that drugs that altered dimerization of ACE2 might affect viral infection by endocytosis. In order to test this hypothesis, we developed an assay to measure dimerization of ACE2, making use of the NanoBIT protein interaction system (Dixon et al., 2016).
This is based on a modified luciferase (NanoLuc) which has been split into two catalytically incomplete components, LgBIT and SmBIT, that must bind together to form an active luciferase. LgBIT and SmBIT associate with low affinity but when fused to other proteins that interact with each other, colocalization of the fusion proteins allows an active luciferase to be formed (Dixon et al., 2016). Here, we have used this system to measure dimerization of ACE2 and screened a library of approved drugs (FMC Library (Khanim et al., 2011)) using an unsupervised approach to identify drug candidates for repurposing.
Our experiments demonstrated that fenofibric acid (Supplementary Figure S1), the active metabolite of the oral hyperlipidaemic drug fenofibrate, apparently induced ACE2 dimerization and destabilized the spike RBD inhibiting binding of spike RBD to ACE2. Importantly and as hypothesized, fenofibrate-induced changes in RBD-ACE2 interactions correlated with significantly lower infection levels (< 60%) and viral release in cell culture models using live SARS-CoV-2. Our data combined with unpublished data from other groups and the existing clinical knowledge of fenofibrate identify it as a strong candidate for treating SARS-CoV-2 infections.
Discussion
The development of new more infectious SARS-CoV-2 variants has resulted in a rapid expansion in infection rates and deaths in several countries around the world, especially the United Kingdom, US, and Europe. While vaccine programs will hopefully reduce infection rates and virus spread in the longer term, there is still an urgent need to expand our arsenal of drugs to treat SARS-CoV-2-positive patients. Using an unsupervised approach, we have identified that the off-patent licensed drug fenofibrate has the potential to treat SARS-CoV-2 infections. The drug was identified through a screen of approved drugs to identify those which alter the dimerization of ACE2. Clofibrate was identified as a hit in this screen and testing of other fibrates led to the identification of fenofibrate as being the most likely to be effective as an antiviral agent. Fenofibric acid also appears to affect the stability of spike protein RBD and inhibit binding to ACE2. Importantly, these effects on RBD by fenofibric acid/fenofibrate correlated with decreases in SARS-CoV-2 infection rates in vitro using two different virus assays (staining for spike protein and plaque formation) in two independent laboratories.
The ACE2 dimerization assays depend on the colocalization of LgBIT and SmBIT brought about by the formation of ACE2 dimers. No signal was observed using protein kinase A subunits that do not interact with ACE2 and overexpression of unlabeled ACE2 suppressed the signal from the NanoBIT reporters, giving confidence that the assay measures the interaction of ACE2 protomers. Although described here as a dimerization assay, the assay may not discriminate between dimer formation and higher-order oligomers, and drugs showing activity in the dimerization assay could alternatively elicit conformational changes in ACE2 complexes which improve the interaction of the NanoBIT reporters. We also acknowledge that although ACE2 is well-established as a membrane protein and this is supported by our own binding assays, we have not formally shown that the NanoBIT-tagged proteins are located on the cell membrane. All the fibrates tested showed some activity in the dimerization assays, but the most pronounced effects were observed with fenofibric acid. Following oral administration of fenofibrate, the ester prodrug is completely converted to the free acid (Figure 6) in a reaction thought to be catalyzed by carboxylesterases. The prodrug fenofibrate (the isopropyl ester of fenofibric acid) was inactive in the dimerization assay, suggesting that the free carboxylic acid is necessary.
Potential mechanisms by which fenofibrate may improve the treatment of SARS-COV-2 infections. Fenofibric acid, the metabolite of fenofibrate, stimulates ACE2 dimerization, destabilizes the RBD, and exerts metabolic effects which are likely to reduce infection. Fenofibric acid possesses anti-inflammatory properties which are likely to blunt the immune response and correspondingly alleviate symptoms. Lastly, fenofibric acid inhibits platelet activation and aggregation, which is anticipated to reduce the hemodynamic problems seen in SARS-COV2 patients.
In addition to effects on ACE2, DSF showed that all the fibrates destabilized the viral spike protein RBD and lowered its “melting” temperature. The most potent effects were again seen with fenofibric acid. These results were corroborated with a modified “CETSA” assay which measured RBD aggregation after thermal denaturation. The effects of fenofibric acid on RBD may contribute to its inhibition of the binding of RBD to ACE2 in ELISA and cell-binding studies performed at 37°C.
When measured in cells at 0°C, the fibrates did not inhibit binding to ACE2; this temperature is likely to prevent melting, providing a potential explanation for the lack of activity of fibrates in binding assays at lower temperatures. To provide evidence for a dual mechanism of action of fenofibrate on both RBD and ACE2, we compared preincubation of cells or RBD with fenofibric acid to experiments when binding of RBD was measured when all three reagents were coincubated.
Preincubation of fenofibrate with either ACE2-expressing cells or RBD increased the inhibition of RBD binding by fenofibric acid, consistent with the drug having effects on both RBD and ACE2. Taken together, these data prompted us to evaluate whether fenofibric acid or fenofibrate would reduce infection by SARS-CoV-2.
To provide robust data evaluating the potential of fenofibric acid/fenofibrate to inhibit infection by SARS-CoV-2, the drugs were evaluated independently in two separate laboratories using different viral infection assays performed on Vero cells and two separate SARS-CoV-2 isolates, both of which are identical to the original Wuhan strain (hCOV-19/England/2/2020 and Italy/UniSR1/2020).
In both cases, fenofibrate/fenofibric acid was found to significantly reduce infection rates. Fenofibrate/fenofibric acid decreased the number of Vero cells staining positive for viral spike protein at 24 h indicating inhibition of primary infection. The number of cells infected 48 h after infection was also significantly reduced, demonstrating the potential for sustained inhibition of infection.
This was further confirmed by PCR which showed a reduction in viral mRNA released by the cells into the culture supernatant. Likewise, we saw significant reductions with fenofibric acid/fenofibrate in plaque formation assays which are considered the gold-standard assay for measuring infectivity by SARS-CoV-2. Several assays demonstrate that the reduced viral infection was not due to a cytotoxic effect of the fibrates in the host cells.
Considering that fenofibrate is used in the treatment of hypercholesterolaemia and hyperlipidaemia, the effect of several statins on SARS-CoV-2 infection was also assessed. These included both hydrophilic (pravastatin; rosuvastatin) and lipophilic statins (pitavastatin; simvastatin). None of the statins inhibited viral infection, suggesting that the antiviral effect was not mediated by inhibition of cholesterol synthesis.
The differences we observed in potency between fenofibrate and fenofibric acid in the two antiviral assays may reflect different strains of the virus or different methodologies. Although we cannot presently fully explain these, it is clear that fenofibrate or its metabolite fenofibric acid demonstrated anti-SARS-CoV-2 activity.
Fenofibric acid was identified as a potential antiviral agent through its effects on ACE2 dimerization, but it remains to be clarified to what extent the effects of fenofibrate/fenofibric acid on dimerization contribute to its antiviral activity. The mechanism by which increased dimerization could inhibit viral infection was not investigated and several explanations are plausible. It was not possible to measure the effect of fibrates on dimerization of ACE2 in streptavidin precipitation assays.
This may reflect the insensitivity of this latter method or that fenofibrate alters the conformation of ACE2 rather than inducing dimerization. Structural studies have shown that ACE2 adopts “open” and “closed” conformations (Yan et al., 2020) which may be detected by the NanoBIT reporters. The open and closed conformations may also affect RBD binding to each ACE2 protomer or the number of spike proteins that can bind to an ACE2 dimer, thereby affecting the avidity of the virus for cells. Conformational changes in ACE2 may also affect its susceptibility to proteolysis by TMPRSS2.
The suggestion that the antiviral activity of fenofibrate depends at least in part on effects on ACE2 also offers advantages over drugs that inhibit viral proteins. Mutations in the viral genome are less likely to affect the antiviral activity of drugs which target human rather than viral proteins. Excitingly, fenofibrate also destabilized the RBD and reduced its binding to ACE2. It is highly likely that this contributes to the reduced infection in cells treated with fenofibrate.
This also suggests that fenofibrate has multiple mechanisms of action, making it less likely that resistance to it will quickly emerge and fenofibrate may retain activity against newly emerging strains of SARS-CoV-2. However, our data suggest that the antiviral activity of fenofibrate measured in the infection assays presented here is not mediated by the transcription factor PPARα. The efficacy of fibrates in the treatment of hyperlipidaemia depends on their ability to activate PPARα However, GW6471, a PPARα antagonist (Xu et al., 2002), did not prevent fenofibrate from inhibiting viral infection.
To our knowledge, this is the first experimental evidence that fenofibrate can modulate RBD and ACE2 proteins and inhibit SARS-CoV-2 infection. Importantly, others have also proposed its therapeutic use in SARS-CoV-2. These proposals are based on pharmacological effects of fenofibrate that are additional to the ones we have identified here (summarized in Figure 6). Fenofibrate increases the levels of the glycosphingolipid sulfatide and this has been proposed to reduce SARS-CoV-2 infection (Buschard, 2020).
SARS-CoV-2 infection is associated with overproduction of cytokines, such as TNF-α, IFN-γ, IL-1, IL-2, and IL-6, and subsequently a cytokine storm that induces several extrapulmonary complications including myocardial injury, myocarditis, acute kidney injury, impaired ion transport, acute liver injury, and gastrointestinal manifestations such as diarrhea and vomiting (Gupta et al., 2020; Lee and Choi, 2021).
Similar to dexamethasone, fenofibrate has been shown to suppress airway inflammation and cytokine release including TNF-α, IL-1, and IFN-γ in both mouse and human studies (Madej et al., 1998; Delayre-Orthez et al., 2008; Stolarz et al., 2015). Fenofibrate has also been shown to have antithrombotic and antiplatelet activities (Jeanpierre et al., 2009; Lee et al., 2009) reduce fibrinogen levels and increase clot permeability, thereby enhancing fibrinolysis (Undas et al., 2006).
These properties may reduce or prevent hypercoagulability seen in the late stage of disease in many SARS-CoV-2 patients (Rogosnitzky et al., 2020). A meta-analysis has also suggested that fenofibrate may be useful in the treatment of hepatitis C infection (Grammatikos et al., 2014). Lastly, we note a preprint from the group of Nahmias that has also suggested that fenofibrate may have clinical effects against SARS-CoV-2 infection which depends on the PPARα mediated alterations in host cell metabolism (Ehrlich et al., 2020).
Based on the data in this preprint, two clinical trials have been registered using fenofibrate in SARS-CoV-2 patients requiring hospitalization (Hospital of the University of Pennsylvania (NCT04517396) and Hebrew University of Jerusalem (NCT04661930)). The metabolic effects of fenofibrate may be mediated not only by its cognate target, PPARα, but also by activation of AMPK (Murakami et al., 2006) which regulates protein synthesis and autophagy pathways through mTORC1.
Given the current acceleration in infection and death rates observed in several countries, we strongly advocate clinical trials of fenofibrate in patients with SARS-CoV-2 requiring hospitalization. Fenofibrate has a relatively safe history of use, the most common adverse effects being abdominal pain, diarrhea, flatulence, nausea, and vomiting. The half-life of fenofibric acid is 20 h (Desager et al., 1996), allowing convenient once daily dosing.
The recommended doses in the United Kingdom (up to 267 mg) provide plasma concentrations (Cmax 70 μM; Css 50 µM) comparable to those at which we and others have seen antiviral activity, Finally, if proven effective, fenofibrate is available as a “generic” drug and consequently is relatively cheap, making it accessible for use in all clinical settings, especially those in low and middle-income countries. Preliminary data indicate that fenofibrate is equally effective against the B.1.1.7 variant (data not shown) implying that mutations in S protein are unlikely to affect the efficacy of fenofibrate. There are a number of medical conditions which contraindicate the use of fenofibrate, such as significantly impaired kidney function, and these could potentially limit its use in the treatment of COVID patients.
There are also a number of drug interactions with fenofibrate which are potentially severe, although some of these may be avoided by temporarily withholding the interacting drug. Appropriate risk-benefit analysis will be necessary once the clinical antiviral activity of fenofibrate is defined to identify which SRS-COV2 patients can safely be treated with fenofibrate. While further studies to clarify the precise mechanism of the antiviral activity of fenofibrate are ongoing, our data support the clinical evaluation of fenofibrate in the community infection setting and also in patients requiring hospitalization.
One possibility is that fenofibrate is tested in newly diagnosed symptomatic patients, who do not require hospitalization, in whom reduction in viral infection levels by fenofibrate would reduce disease severity and the spread of infection to other individuals.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8377159/
