Researchers have identified older drugs with the potential to help accelerate patient recovery from SARS-CoV-2 infection

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University of New Mexico researchers who combed through a “library” of previously approved drugs believe they have identified a medication with the potential to help speed a patient’s recovery from SARS-CoV-2 infection.

“The gist of it is we think we found a drug that is on par with remdesivir and is much cheaper,” said Tudor Oprea, MD, Ph.D., professor of Medicine and Pharmaceutical Sciences and chief of the UNM Division of Translational Informatics. Remdesivir is a relatively new antiviral medication that has been shown to shorten hospital stays for those recovering from the novel coronavirus.

In a paper published this week in ACS Pharmacology & Translational Science, Oprea and his colleagues, in partnership with a team at the University of Tennessee Health Science Center led by professor Colleen Jonsson, Ph.D., reported that an older antimalarial drug called amodiaquine was effective in eradicating the virus in test tube experiments.

Tudor Oprea, MD, Ph.D.It was one of three promising candidates identified in a process that entailed studying the molecular characteristics of about 4,000 drugs approved for human use by the Food and Drug Administration and other agencies. The researchers hoped to find drugs that would target known vulnerabilities in the virus.

The other two drugs—an anti-psychotic called zuclophentixol and a blood pressure medication called nebivolol also cleared the virus in the experiments, said Oprea, who served as the corresponding author on the new paper.

The researchers think any of these three drugs could be combined with remdesivir or a related antiviral drug called favipiravir to mount a more potent attack on the virus.

Combining two drugs could mean that lower doses of each could be administered, lessening the likelihood of adverse reactions, he said.s Administering two drugs also makes it less likely that the virus would develop a mutation rendering it immune from the treatment.

“Think of it as a whack-a-mole game,” Oprea said. “Instead of having one hammer, you have two hammers, which is more effective. We’re trying to give the scientific community two hammers instead of one.”

Many compounds that show antiviral activity in a laboratory setting don’t have the same effect in living organisms, Oprea notes, so the next step is to mount clinical trials to see whether the medications work in COVID-positive patients.

The UNM drug screening process started with Oprea and his colleague Larry Sklar, Ph.D., Distinguished Professor in the Department of Pathology.

They used computational methods to identify candidate drugs by gauging their similarity to hydroxychloroquine, a since-discredited antimalarial medication that had been widely touted as a COVID-19 treatment.

Because of molecular variations in some of the drugs, more than 6,000 combinations were assessed.

Likely candidates were forwarded to Steven Bradfute, Ph.D., assistant professor in the Center for Global Health, who tested the compounds against samples of the virus in his Biosafety Level-3 laboratory.

Later, the experiments were repeated by the University of Tennessee scientists to provide independent confirmation of the findings – and they used an additional test that reveals the drugs’ potency against the virus, Oprea said.

Amodiaquine, first made in 1948, is on the World Health Organization’s List of Essential Medicines. It has a good safety profile and is widely used in Africa to treat malaria. Zuclophentixol has been used to treat schizophrenia since the 1970s, while nebivolol has been used for hypertension since the late 1990s.


In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China [1]. Despite containment measures, SARS-CoV-2 spread in Asia, Southern Europe, then in America and in Africa.

The global number of cumulative cases in world was 13,646,660 and 809,747 deaths in August 24, 2020 (https://www.coronavirus-statistiques.com/stats-globale/covid-19-cases-europe/). Currently, 54 countries are affected in Africa with 1,003,435 cumulative cases and 20,398 reported deaths (August 24, 2020) (https://who.maps.arcgis.com/apps/opsdashboard/index.html#/0c9b3a8b68d0437a8cf28581e9c063a9).

African countries see slower dynamic of COVID-19 cases and deaths. Several hypotheses could explain the later emergence and spread of COVID-19 pandemic in Africa, like delay in systematic SARS-CoV-2 detection and appropriate epidemiological surveillance, limited international air traffic, climate conditions, demographic conditions with less people above 65 years old, genetic polymorphisms of the cell entry receptor for the SARS-CoV-2 (angiotensin converting enzyme 2, ACE-2) [2].

Another hypothesis that may explain this later emergence in Africa, and more particularly in malaria endemic areas, would be the use of antimalarial drugs. Antimalarial drugs could be effective against SARS-CoV-2. In 2002, the World Health Organization (WHO) recommended the use of artemisinin-based combination therapy (ACT) in the treatment of uncomplicated falciparum malaria in Asia, South America and Africa.

The combinations artesunate-amodiaquine (Burundi, Cameroon, Democratic Republic of Congo, Gabon, Ivory Coast), artesunate-mefloquine (Cambodia, Brazil), artemether-lumefantrine (Benin, Central African Republic, Malawi, South Africa) or dihydroartemisinin-piperaquine (Thailand, Vietnam) are currently used. Although chloroquine is no longer used to treat falciparum malaria due to high level of resistance, it remains the first-line treatment in combination with primaquine for vivax malaria in some African countries, such as Ethiopia, South Africa and Sudan, in American countries, such as Brazil, Colombia, Guyana, Nicaragua, Peru, Venezuela), in Eastern Mediterranean countries, such as Afghanistan, Pakistan, Sudan, in south east Asian countries, such as India, Myanmar.

Antimalarial drugs are potential candidates to be repurposed in both COVID-19 prophylaxis and therapy [3]. Are antimalarial drugs effective against SARS-CoV-2?

Chloroquine, a quinoline, has been shown to be effective in vitro against SARS-CoV-2 in Vero E6 cells (African green monkey kidney cells) with median effective concentration (EC50) at micromolar range [[4], [5], [6]].

Hydroxychloroquine, an analogue of chloroquine used in autoimmune diseases such as rheumatoid arthritis and lupus, has also demonstrated in vitro antiviral activity against SARS-CoV-2 with EC50 at micromolar range [4,6,7].

Twenty-three clinical trials have been conducted in China to investigate the efficacy and safety of chloroquine and hydroxychloroquine in the treatment of COVID-19 [[8], [9], [10]].

Although the clinical assay was not performed according to the randomized double blind method, preliminary data indicated that chloroquine phosphate has demonstrated efficacy in treatment of COVID-19 with few severe adverse reactions in more than 100 patients by shortening hospital stay and improving the clinical evolution [9].

Hydroxychloroquine could shorten time to clinical recovery [11,12].

Effects of hydroxychloroquine were potentiated in vitro and in vivo by azithromycin [7,13]. Ferroquine, a ferrocenic analogue of chloroquine with anti-malarial activity [14], was shown to be an effective inhibitor of SARS-CoV-1 replication with EC50 of 1.4 μM [15].

In 2002, the World Health Organization (WHO) recommended the use of artemisinin-based combination therapy (ACT) in the treatment of uncomplicated P. falciparum malaria (artemether-lumefantrine, artesunate-amodiaquine, dihydroartemisinin-piperaquine or artesunate-mefloquine).

Marketed artemisinin derivatives exhibited in vitro anti-viral activity at micromolar concentrations against human cytomegalovirus [[16], [17], [18]].

Amodiaquine, a quinoleine antimalarial and one of the partner of artemisinin derivative in ACT, was found to be active in vitro at micromolar concentration against SARS-CoV-1 (2.5 μM) but was inactive in vivo on SARS-CoV-1 in BALB/c mice [19].

Another quinoline, mefloquine, which has been used in combination with artesunate for the treatment of uncomplicated falciparum malaria, exerts in vitro cytopathic effects on Vero cells infected by SARS-CoV-2 at 10 μM [20]. Pyronaridine, a quinoline component of the EU-approved antimalarial Pyramax (pyronaridine-artesunate), was effective in vitro against Ebola virus with EC50 of 1.14 μM and protected mice when administered 1 h after infection [21].

Taken together, these reports suggest that antimalarial drugs may have antiviral effects and be effective against SARS-CoV-2. Chloroquine, hydroxychloroquine, ferroquine, quinine, mefloquine, desethylamodiaquine (the metabolite of amodiaquine), lumefantrine, pyronaridine, piperaquine and dihydroartemisinin (the metabolite of artemisinin derivatives) were assessed in vitro against a clinically isolated SARS-CoV-2 strain.

Discussion

Chloroquine and its analogues ferroquine and hydroxychloroquine showed in vitro activities at low-micromolar range with EC50 of 2.1 ± 0.7 μM (SI > 47.6), 1.5 ± 0.3 μM (SI > 66.7) and 1.5 ± 0.3 μM (SI = 13.6), respectively. The EC50 values for chloroquine and hydroxychloroquine are lower than those obtained in a previous work on Vero E6 cells at MOI of 0.2 (7.1 and 17.3 μM, respectively) [4].

EC50 values depend on several methodological conditions like MOI, duration of incubation [6]. Ferroquine has already shown in vitro anti-coronavirus activity against feline coronavirus with EC50 of 2.9 μM and SARS-CoV-1 virus with EC50 of 1.4 μM [15].

These concentrations were consistent with concentrations observed in human plasma and lungs. Chloroquine given at 100 mg day in the prophylaxis of malaria leads to a plasma concentration of 0.01–0.4 mg/l, ie 0.03–1.25 μM [24].

Chloroquine has an excellent diffusion and tissue concentration which would lead to chloroquine levels 200 to 700 times higher in the lung than in the blood (a concentration which can go up to 280 mg/kg in the lung) [25]. An oral uptake of 400 mg of hydroxychloroquine led to a Cmax of 1.22 μM [26].

Hydroxychloroquine accumulated 30 times more in lungs than in blood (around 0.3 μM vs 7.8 μM at 6 h) [27]. An oral uptake of 800 mg of ferroquine led to a mean Cmax (maximum blood concentration) value of 155 ng/ml (around 0.6 μM) and t1/2 (elimination half-life) of 10.9 days [28].

No data is available on ferroquine accumulation in lungs. However, as ferroquine is an analogue of chloroquine, we can assume that it may accumulate at least 10 times than in blood.

The antiviral activity of chloroquine and its analogues against SARS-CoV-2 are compatible with oral uptake at doses administered in malaria treatment. Chloroquine and hydroxychloroquine inhibited SARS-CoV-2 entry [4,5]. Chloroquine impaired the terminal glycosylation of ACE-2 receptor required for virus entry that resulted in reduced binding affinities between SARS-CoV-1 and its ACE-2 receptor and blocked SARS-CoV-1 entry in human cells [29].

The spike viral protein of SARS-CoV-2 used the ACE-2 receptor for entry, but also sialic acids and gangliosides. In silico analyses showed that the viral spike protein was not able to bind gangliosides in the presence of chloroquine or hydroxychloroquine [30].

Additionally, the ORF8 viral protein could bind to the porphyrin. At the same time, viral orflab, ORF10 and ORF3a proteins could attack the heme to dissociate the iron to form the porphyrin and inhibit the human heme metabolism leading to a decrease of hemoglobin amount which carry oxygen and carbon dioxide.

Chloroquine could inhibit the binding of ORF8 to porphyrin and prevent the attack of the 1-beta chain of hemoglobin by orflab, ORF10 and ORF3a proteins [31]. Besides its antiviral activity, chloroquine and hydroxychloroquine have anti-inflammatory effects by decreasing the expression of various pro-inflammatory cytokines including interleukin 6 (IL6), tumor necrosis factor-alpha (TNF) and interferon gamma (INFγ) by mononuclear cells [32].

These cytokines were considerably increased in the cytokine storm due to COVID-19 [33]. Many clinical trials on hydroxychloroquine alone or in combination with azithromycin to treat COVID-19 are in progress. The efficacy of hydroxychloroquine alone or in combination with azithromycin has been controversial.

Hydroxychloroquine showed antiviral activity Vero E6 cells (African green monkey kidney cells) [[4], [5], [6],34] but not in a model of reconstituted airway epithelium [34]. Moreover, neither hydroxychloroquine alone or in combination with azithromycin showed significant effect on the viral load levels in comparision with placebo [34].

Some studies showed that early treatment with hydroxychloroquine alone or in combination with azithromycin was associated with a reduced risk of hospitalization, reduced risk of death and shorter duration of viral presence [35], [36], [37], [38], [39].

Early treatment with hydroxychloroquine decreased the level of secreted inflammatory cytokines (IL6, TNF and INFγ) [40]. Conversely, some studies showed that treatment of mild-to-moderate or mild-to-severe COVID-19 with hydroxychloroquine alone or in combination with azithromycin did not improve clinical status or duration of viral shedding in comparison with standard care [[41], [42], [43], [44], [45]]. Moreover, therapeutic interventions using high dosage chloroquine and/or in combination with macrolides may have severe side-effects including cardiac toxicity.

A prophylactic approach with chloroquine at lower dosage could be administrated in vulnerable persons with comorbidities at-risk of severe COVID-19 or in health workers [46]. Chloroquine at 100 mg daily was used for decades for the antimalarial chemoprophylaxis.

Several trials on prophylaxis with chloroquine are currently in progress (NCT04349371, NCT04303507). Chloroquine could be evaluated alone or in combination with antibiotics like doxycycline in prophylactic trials. Indeed doxycycline showed in vitro antiviral activity against SARS-CoV-2 (EC50 = 5.6 μM) and low toxicity [47].

Doxycycline at 100 mg daily was used for many years for the antimalarial chemoprophylaxis and combining chloroquine to doxycycline in daily prophylaxis did not increase the risk of adverse effects [48].

Desethylamodiaquine, the metabolite of amodiaquine, showed the best in vitro efficacy with EC50 of 0.52 ± 0.2 μM (SI = 166). Amodiaquine was used in combination with artesunate in the treatment of uncomplicated malaria in Africa (306 mg amodiaquine base and 100 mg artesunate).

Amodiaquine given at 612 mg day led to a plasma concentration of 753 ng/ml of desethylamodiaquine (around 1.9 μM) and a t1/2 of 8.9 days [49].

About 0.07% of the administered dose was found in rat lung [50]. This suggests that for an uptake of 612 mg in human, 428 μg would be found in lungs. Amodiaquine, was found to be active also in vitro against SARS-CoV-1 with EC50 of 2.5 μM but was inactive in vivo on SARS-CoV-1 in BALB/c mice [19].

Chloroquine, effective in vitro with EC50 of 2.5 μM, was also inactive in vivo on SARS-CoV-1 in BALB/c mice.

Amodiaquine also inhibited dengue virus type 2 replication with EC50 of 1.08 μM and EC90 of 2.69 μM [51]. The antiviral activity of desethylamodiaquine against SARS-CoV-2 is compatible with oral uptake of amodiaquine at doses commonly administered in malaria treatment.

Amodiaquine can only be recommended as treatment and not as prophylaxis due to risk of hepatitis and agranulocytosis during long-term administration [52].

Mefloquine showed anti-SARS-CoV-2 activity with EC50 of 1.8 μM and EC90 of 8.1 μM. These results are consistent with previous study which showed that mefloquine at 10 μM inhibited completely cytopathic effect onVero E6 cells infected by SARS-CoV-2 [20].

Mefloquine administered at malaria therapeutic dose (1250 mg) lead to a blood concentration of 1648 ng/ml (around 4 μM) in healthy males [53]. A study on postmortem cases showed that mefloquine levels are 10 times higher in the lung than in the blood (a concentration which can go up to 180 mg/kg in the lung) [54].

The antiviral activity of mefloquine against SARS-CoV-2 is compatible with malaria oral therapeutic doses. But mefloquine can cause neuropsychiatric adverse effects [55].

Pyronaridine showed effective antiviral activity with EC50 of 0.72 μM and EC90 of 0.75 μM. Pyronaridine tetraphosphate given at 720 mg day led to a plasma concentration of 271 ng/ml (around 0.3 μM) in human and a t1/2 of 33.5 days [56]. A single oral dose of 2 mg (10 mg/kg) in rats led to a blood Cmax of 223 ng/ml and a lung Cmax of 36.4 μg/g (165 more concentrated) [57].

The antiviral activity of pyronaridine against SARS-CoV-2 is compatible with malaria oral therapeutic doses. Acute and sub-acute toxicity was less than that of chloroquine. Cardiovascular toxicity was also less than that of chloroquine [58]. Pyronaridine was well tolerated: around 38% of adverse events versus 56% for chloroquine [59].

Quinine showed medium antiviral in vitro activity with EC50 of 10.7 ± 3.0 μM and EC90 of 38.8 ± 34 μM. A 600 mg single oral dose of quinine sulphate led to blood Cmax around 3.5 mg/l (around 8.5 μM) [60]. In rat, after intravenous dose of 10 mg/kg of quinine, the observed concentration lung/blood ratio was 246 [61].

The in vitro effective concentration in lungs to cure SARS-CoV-2 is achievable in human. If its clinical efficacy in human would be confirmed, quinine could be administered in intravenous in patients before cytokine storm.

Quinine can cause haemolytic anemia in patients with G6PD deficiency and severe side-effects including cardiac toxicity [52]. Additionally, quinine could be associated with doxycycline against COVID-19, as is done in malaria treatment [62].

Lumefantrine, piperaquine and dihydroartemisinin showed low antiviral activity with EC50 of 24.7, 33.4 and 20.1 μM, respectively. A single oral dose of lumefantrine (480 mg) led to Cmax of 1.1 μM [63]. A single oral dose of 1280 mg of piperaquine and 160 mg of dihydroartemisinin in fed participants led to plasma Cmax of 596 ng/ml and 324 ng/ml, respectively [64].

No data is available on drug accumulation in lungs for these antimalarial. The ratio Cmax/EC50 or Cmax/EC90 were too low to reach effective concentrations to inhibit SARS-CoV-2 in human. However, ACT (artemether-lumefantrine, artesunate-amodiaquine, dihydroartemisinin-piperaquine, artesunate-mefloquine or artesunate-pyronaridine), evaluated at plasma concentrations expected after oral uptake at recommended doses used in uncomplicated malaria treatment, showed an in vitro inhibition of SARS-CoV-2 replication that ranged from 30 to 70% [65].

The combination mefloquine-artesunate was found to be the most effective in vitro against SARS-CoV-2.

Conclusion

Chloroquine, hydroxychloroquine, ferroquine, desethylamodiaquine, mefloquine, pyronaridine and quinine showed in vitro antiviral effective activity against SARS-CoV-2 with IC50 and IC90 compatible with drug oral uptake at doses commonly administered in malaria treatment.

These in vitro activities are higher than those obtained with drugs which are evaluated in clinical trials worldwide like remdisivir (23 μM), lopinavir (26.6 μM) or ritonavir (>100 μM) [66]. However, these results must be taken with caution regarding the potential use of antimalarial drugs in SARS-CoV-2 infected patients: it is difficult to translate in vitro study results to actual clinical treatment in patients.

Experts agree on the in vitro activity of chloroquine or hydroxychloroquine against SARS-CoV-2 but disagree on hydroxychloroquine efficacy in COVID-19 treatment, which remains controversial [67,68]. In vivo evaluation in animal experimental models is now required to confirm the antiviral effects of these antimalarial drugs on SARS-CoV-2.

The antiviral effects of some antimalarial drugs could partially explain the later emergence and spread of COVID-19 pandemic in Africa. It could be necessary now to compare the antimalarial use and the dynamics of COVID-19 country by country to confirm the potential effects of antimalarial drugs.

Based on our results, we would expect that countries which commonly use artesunate-amodiaquine or artesunate-mefloquine report fewer cases and deaths than those using artemether-lumefantrine or dihydroartemisinin-piperaquine.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477610/


More information: Giovanni Bocci et al, Virtual and In Vitro Antiviral Screening Revive Therapeutic Drugs for COVID-19, ACS Pharmacology & Translational Science (2020). DOI: 10.1021/acsptsci.0c00131

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