FDA 승인 기생충약물 ivermectin은 시험관내 SARS-CoV-2 복제를 억제

항바이러스 연구

Antiviral Research

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Volume 178, June 2020, 104787

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The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro

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Highlights

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Ivermectin is an inhibitor of the COVID-19 causative virus (SARS-CoV-2) in vitro.

Ivermectin은 시험관내에서 COVID-19 원인 바이러스 (SARS-CoV-2)의 억제제입니다.

 

단일 처리로 세포배양에서 48시간에 바이러스를 ~ 5000배 감소시킬 수 있습니다.

A single treatment able to effect ~5000-fold reduction in virus at 48 h in cell culture.

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Ivermectin is FDA-approved for parasitic infections, and therefore has a potential for repurposing.

Ivermectin은 기생충 감염에 대해 FDA 승인을 받았으므로 용도변경 가능성이 있습니다.

 

Ivermectin은 필수 의약품의 WHO 모델 목록에 포함되어 널리 사용 가능합니다.

Ivermectin is widely available, due to its inclusion on the WHO model list of essential medicines.

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요약 Abstract

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가능한 치료법을 테스트하기 위해 여러 임상시험이 현재 진행중이지만 COVID-19 발병에 대한 전 세계적 대응은 모니터링/격리로 크게 제한되었습니다.

FDA에서 승인한 이전에 시험관내에서 광범위한 항바이러스 활성을 보인 것으로 밝혀진 항기생충 Ivermectin이 Vero-hSLAM에 한 번 추가된 원인 바이러스 (SARS-CoV-2)의 억제제라고 여기에 보고합니다.

SARS-CoV-2 감염 2시간 후 세포는 48시간에 바이러스 RNA를 ~ 5000배 감소시킬 수 있습니다.

따라서 Ivermectin은 인간에게 가능한 이점에 대한 추가 조사를 보장합니다.

Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post infection with SARS-CoV-2 able to effect ~5000-fold reduction in viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans.

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1. Introduction 소개

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Ivermectin은 FDA가 승인한 광범위한 항기생충제 (Gonzalez Canga 등, 2008)로 최근 몇 년 동안 다른 그룹과 함께 광범위한 바이러스에 대해 항바이러스 활성을 갖는 것으로 나타났습니다 (Gotz 등 ., 2016; Lundberg 등, 2013; Tay et al., 2013; Wagstaff 등, 2012) in vitro.

원래 인간 면역결핍 바이러스 -1 (HIV-1) 인테그라제 단백질 (IN)과 IN 핵 수입을 담당하는 임포틴 (IMP) α / β1 이종 이합체 사이의 상호작용 억제제로 확인된 Ivermectin은 IN 핵 수입 및 HIV-1 복제를 억제하는 것으로 확인된 이후 (Wagstaff 등, 2012).

이버멕틴의 다른 작용이 보고되었지만 (Mastrangelo 등, 2012), ivermectin은 숙주 (예, Kosyna 등, 2015; van der Watt et al., 2016)) 및 바이러스의 핵 유입을 억제하는 것으로 나타났습니다.

유인원 바이러스 SV40 대형 종양 항원 (T-ag) 및 뎅기 바이러스 (DENV) 비 구조 단백질 5를 포함한 단백질 (Wagstaff 등, 2012, Wagstaff 등, 2011). 중요한 것은 DENV 1-4 (Tay 등, 2013), West Nile Virus (Yang 등, 2020), Venezuelan equine encephalitis virus (VEEV) (Lundberg 등)와 같은 RNA 바이러스에 의한 감염을 제한하는 것으로 입증되었습니다.

., 2013) 및 인플루엔자 (Gotz 등, 2016), 이 광범위한 스펙트럼 활동은 감염 동안 IMPα / β1에 대한 다양한 RNA 바이러스의 의존성 때문이라고 믿어집니다 (Caly et al., 2012; Jans et al. , 2019).

Ivermectin은 in vitro 및 in vivo에서 DNA 바이러스 가성 광견병 바이러스 (PRV)에 대해 유사하게 효과적인 것으로 나타 났으며, 이버멕틴 치료는 PRV에 감염된 쥐의 생존을 증가시키는 것으로 나타났습니다 (Lv 등, 2018).

쥐에서 Zika 바이러스 (ZIKV)에 대한 ivermectin의 효능은 관찰되지 않았지만, 저자는 연구 한계가 ivermectin의 anti-ZIKV 활성의 재평가를 정당화한다고 인정했습니다 (Ketkar et al., 2019).

마지막으로, ivermectin은 2014-2017년 태국에서 DENV 감염에 대한 3상 임상 시험의 초점이었으며, 1일 경구 투여량이 안전하고 바이러스 NS1 단백질의 혈청 수준을 현저히 감소시키는 것으로 관찰되었지만 바이러스 혈증이나 임상적 이점의 변화는 관찰되지 않았습니다 (아래 참조) (Yamasmith et al., 2018).

현재 COVID-19 유행병인 SARS-CoV-2의 원인 물질은 중증 급성 호흡기 증후군 코로나 바이러스 (SARS-CoV)와 밀접한 관련있는 단일 가닥 양성 센스 RNA 바이러스입니다.

SARS-CoV 단백질에 대한 연구는 SARS-CoV Nucleocapsid 단백질의 신호의존적 핵 세포질 폐쇄에서 감염 동안 IMPα/β1에 대한 잠재적 역할을 밝혀냈습니다 (Rowland et al., 2005; Timani et al., 2005; Wulan 등, 2015), 이는 숙주 세포 분열에 영향을 미칠 수 있습니다 (Hiscox et al., 2001; Wurm et al., 2001).

또한 SARS-CoV 보조 단백질 ORF6은 거친 ER / Golgi 막에서 IMPα / β1을 격리하여 STAT1 전사 인자의 항바이러스 활성을 길항하는 것으로 나타났습니다 (Frieman et al., 2007).

종합하면 이 보고서는 이버멕틴의 핵 운반 억제 활성이 SARS-CoV-2에 효과적일 수 있음을 시사했습니다.

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SARS-CoV-2에 대한 이버멕틴의 항 바이러스 활성을 테스트하기 위해 우리는 2시간 동안 0.1의 MOI에서 SARS-CoV-2 분리 호주/VIC01/2020으로 Vero/hSLAM 세포를 감염시킨 다음 5μM 이버멕틴을 추가했습니다.

상청액과 세포 펠렛을 0 ~ 3일에 수확하고 RT-PCR로 SARS-CoV-2 RNA의 복제를 분석했습니다 (그림 1A / B).

24시간에 비히클 DMSO에 비해 ivermectin으로 처리한 샘플의 상층 액 (방출된 비리 온을 나타냄)에 존재하는 바이러스 RNA가 93% 감소했습니다.

유사하게 세포 관련 바이러스 RNA의 99.8% 감소 (비 방출 및 비포장 비리 온을 나타냄)가 이버멕틴 처리로 관찰되었습니다.

48시간까지 이 효과는 대조군 샘플에 비해 이버멕틴 처리된 바이러스 RNA의 ~ 5000배 감소로 증가했으며, 이는 이버멕틴 처리가 본질적으로 모든 바이러스 물질의 효과적인 손실을 48시간까지 초래했음을 나타냅니다.

이 아이디어와 일치하여 72시간에 바이러스 RNA의 추가 감소는 관찰되지 않았습니다.

이전에 관찰한 바와 같이 (Lundberg 등, 2013; Tay 등, 2013; Wagstaff 등, 2012), 샘플 웰 또는 병렬 테스트에서 테스트한 모든 시점에서 이버멕틴의 독성이 관찰되지 않았습니다. 약물 단독 샘플.

 

Ivermectin is an FDA-approved broad spectrum anti-parasitic agent (Gonzalez Canga et al., 2008) that in recent years we, along with other groups, have shown to have anti-viral activity against a broad range of viruses (Gotz et al., 2016; Lundberg et al., 2013; Tay et al., 2013; Wagstaff et al., 2012) in vitro. Originally identified as an inhibitor of interaction between the human immunodeficiency virus-1 (HIV-1) integrase protein (IN) and the importin (IMP) α/β1 heterodimer responsible for IN nuclear import (Wagstaff et al., 2011), Ivermectin has since been confirmed to inhibit IN nuclear import and HIV-1 replication (Wagstaff et al., 2012). Other actions of ivermectin have been reported (Mastrangelo et al., 2012), but ivermectin has been shown to inhibit nuclear import of host (eg. (Kosyna et al., 2015; van der Watt et al., 2016)) and viral proteins, including simian virus SV40 large tumour antigen (T-ag) and dengue virus (DENV) non-structural protein 5 (Wagstaff et al., 2012, Wagstaff et al., 2011). Importantly, it has been demonstrated to limit infection by RNA viruses such as DENV 1-4 (Tay et al., 2013), West Nile Virus (Yang et al., 2020), Venezuelan equine encephalitis virus (VEEV) (Lundberg et al., 2013) and influenza (Gotz et al., 2016), with this broad spectrum activity believed to be due to the reliance by many different RNA viruses on IMPα/β1 during infection (Caly et al., 2012; Jans et al., 2019). Ivermectin has similarly been shown to be effective against the DNA virus pseudorabies virus (PRV) both in vitro and in vivo, with ivermectin treatment shown to increase survival in PRV-infected mice (Lv et al., 2018). Efficacy was not observed for ivermectin against Zika virus (ZIKV) in mice, but the authors acknowledged that study limitations justified re-evaluation of ivermectin's anti-ZIKV activity (Ketkar et al., 2019). Finally, ivermectin was the focus of a phase III clinical trial in Thailand in 2014–2017, against DENV infection, in which a single daily oral dose was observed to be safe and resulted in a significant reduction in serum levels of viral NS1 protein, but no change in viremia or clinical benefit was observed (see below) (Yamasmith et al., 2018).

The causative agent of the current COVID-19 pandemic, SARS-CoV-2, is a single stranded positive sense RNA virus that is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV). Studies on SARS-CoV proteins have revealed a potential role for IMPα/β1 during infection in signal-dependent nucleocytoplasmic shutting of the SARS-CoV Nucleocapsid protein (Rowland et al., 2005; Timani et al., 2005; Wulan et al., 2015), that may impact host cell division (Hiscox et al., 2001; Wurm et al., 2001). In addition, the SARS-CoV accessory protein ORF6 has been shown to antagonize the antiviral activity of the STAT1 transcription factor by sequestering IMPα/β1 on the rough ER/Golgi membrane (Frieman et al., 2007). Taken together, these reports suggested that ivermectin's nuclear transport inhibitory activity may be effective against SARS-CoV-2.

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To test the antiviral activity of ivermectin towards SARS-CoV-2, we infected Vero/hSLAM cells with SARS-CoV-2 isolate Australia/VIC01/2020 at an MOI of 0.1 for 2 h, followed by the addition of 5 μM ivermectin. Supernatant and cell pellets were harvested at days 0–3 and analysed by RT-PCR for the replication of SARS-CoV-2 RNA (Fig. 1A/B). At 24 h, there was a 93% reduction in viral RNA present in the supernatant (indicative of released virions) of samples treated with ivermectin compared to the vehicle DMSO. Similarly a 99.8% reduction in cell-associated viral RNA (indicative of unreleased and unpackaged virions) was observed with ivermectin treatment. By 48 h this effect increased to an ~5000-fold reduction of viral RNA in ivermectin-treated compared to control samples, indicating that ivermectin treatment resulted in the effective loss of essentially all viral material by 48 h. Consistent with this idea, no further reduction in viral RNA was observed at 72 h. As we have observed previously (Lundberg et al., 2013; Tay et al., 2013; Wagstaff et al., 2012), no toxicity of ivermectin was observed at any of the timepoints tested, in either the sample wells or in parallel tested drug alone samples.



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Fig. 1. Ivermectin is a potent inhibitor of the SARS-CoV-2 clinical isolate Australia/VIC01/2020. Vero/hSLAM cells were in infected with SARS-CoV-2 clinical isolate Australia/VIC01/2020 (MOI = 0.1) for 2 h prior to addition of vehicle (DMSO) or Ivermectin at the indicated concentrations. Samples were taken at 0–3 days post infection for quantitation of viral load using real-time PCR of cell associated virus (A) or supernatant (B). IC50 values were determined in subsequent experiments at 48 h post infection using the indicated concentrations of Ivermectin (treated at 2 h post infection as per A/B). Triplicate real-time PCR analysis was performed on cell associated virus (C/E) or supernatant (D/F) using probes against either the SARS-CoV-2 E (C/D) or RdRp (E/F) genes. Results represent mean ± SD (n = 3). 3 parameter dose response curves were fitted using GraphPad prism to determine IC50 values (indicated). G. Schematic of ivermectin's proposed antiviral action on coronavirus. IMPα/β1 binds to the coronavirus cargo protein in the cytoplasm (top) and translocates it through the nuclear pore complex (NPC) into the nucleus where the complex falls apart and the viral cargo can reduce the host cell's antiviral response, leading to enhanced infection. Ivermectin binds to and destabilises the Impα/β1 heterodimer thereby preventing Impα/β1 from binding to the viral protein (bottom) and preventing it from entering the nucleus. This likely results in reduced inhibition of the antiviral responses, leading to a normal, more efficient antiviral response.

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To further determine the effectiveness of ivemectin, cells infected with SARS-CoV-2 were treated with serial dilutions of ivermectin 2 h post infection and supernatant and cell pellets collected for real-time RT-PCR at 48 h (Fig. 1C/D). As above, a >5000 reduction in viral RNA was observed in both supernatant and cell pellets from samples treated with 5 μM ivermectin at 48 h, equating to a 99.98% reduction in viral RNA in these samples. Again, no toxicity was observed with ivermectin at any of the concentrations tested. The IC50 of ivermectin treatment was determined to be ~2 μM under these conditions. Underlining the fact that the assay indeed specifically detected SARS-CoV-2, RT-PCR experiments were repeated using primers specific for the viral RdRp gene (Fig. 1E/F) rather than the E gene (above), with nearly identical results observed for both released (supernatant) and cell-associated virus.

Taken together these results demonstrate that ivermectin has antiviral action against the SARS-CoV-2 clinical isolate in vitro, with a single dose able to control viral replication within 24–48 h in our system. We hypothesise that this is likely through inhibiting IMPα/β1-mediated nuclear import of viral proteins (Fig. 1G), as shown for other RNA viruses (Tay et al., 2013; Wagstaff et al., 2012; Yang et al., 2020); confirmation of this mechanism in the case of SARS-CoV-2, and identification of the specific SARS-CoV-2 and/or host component(s) impacted (see (Yang et al., 2020)) is an important focus future work in this laboratory. Ultimately, development of an effective anti-viral for SARS-CoV-2, if given to patients early in infection, could help to limit the viral load, prevent severe disease progression and limit person-person transmission. Benchmarking testing of ivermectin against other potential antivirals for SARS-CoV-2 with alternative mechanisms of action (Dong et al., 2020; Elfiky, 2020; Gordon et al., 2020; Li and De Clercq, 2020; Wang et al., 2020) would thus be important as soon as practicable. This Brief Report raises the possibility that ivermectin could be a useful antiviral to limit SARS-CoV-2, in similar fashion to those already reported (Dong et al., 2020; Elfiky, 2020; Gordon et al., 2020; Li and De Clercq, 2020; Wang et al., 2020); until one of these is proven to be beneficial in a clinical setting, all should be pursued as rapidly as possible.

Ivermectin has an established safety profile for human use (Gonzalez Canga et al., 2008; Jans et al., 2019; Buonfrate et al., 2019), and is FDA-approved for a number of parasitic infections (Gonzalez Canga et al., 2008; Buonfrate et al., 2019). Importantly, recent reviews and meta-analysis indicate that high dose ivermectin has comparable safety as the standard low-dose treatment, although there is not enough evidence to make conclusions about the safety profile in pregnancy (Navarro et al., 2020; Nicolas et al., 2020). The critical next step in further evaluation for possible benefit in COVID-19 patients will be to examine a multiple addition dosing regimen that mimics the current approved usage of ivermectin in humans. As noted, ivermectin was the focus of a recent phase III clinical trial in dengue patients in Thailand, in which a single daily dose was found to be safe but did not produce any clinical benefit. However, the investigators noted that an improved dosing regimen might be developed, based on pharmacokinetic data (Yamasmith et al., 2018). Although DENV is clearly very different to SARS-CoV-2, this trial design should inform future work going forward. Altogether the current report, combined with a known-safety profile, demonstrates that ivermectin is worthy of further consideration as a possible SARS-CoV-2 antiviral.

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2. Methods

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2.1. Cell culture, viral infection and drug treatment

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Vero/hSLAM cells (Ono et al., 2001) were maintained in Earle's Minimum Essential Medium (EMEM) containing 7% Fetal Bovine Serum (FBS) (Bovogen Biologicals, Keilor East, AUS) 2 mM L-Glutamine, 1 mM Sodium pyruvate, 1500 mg/L sodium bicarbonate, 15 mM HEPES and 0.4 mg/ml geneticin at 37 °C, 5% CO2. Cells were seeded into 12-well tissue culture plates 24 h prior to infection with SARS-CoV-2 (Australia/VIC01/2020 isolate) at an MOI of 0.1 in infection media (as per maintenance media but containing only 2% FBS) for 2 h. Media containing inoculum was removed and replaced with 1 mL fresh media (2% FBS) containing Ivermectin at the indicated concentrations or DMSO alone and incubated as indicated for 0–3 days. At the appropriate timepoint, cell supernatant was collected and spun for 10 min at 6,000 g to remove debris and the supernatant transferred to fresh collection tubes. The cell monolayers were collected by scraping and resuspension into 1 mL fresh media (2% FBS). Toxicity controls were set up in parallel in every experiment on uninfected cells.

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2.2. Generation of SARS-CoV-2 cDNA

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RNA was extracted from 200 μL aliquots of sample supernatant or cell suspension using the QIAamp 96 Virus QIAcube HT Kit (Qiagen, Hilden, Germany) and eluted in 60 μl. Reverse transcription was performed using the BioLine SensiFAST cDNA kit (Bioline, London, United Kingdom), total reaction mixture (20 μl), containing 10 μL of RNA extract, 4 μl of 5x TransAmp buffer, 1 μl of Reverse Transcriptase and 5 μl of Nuclease free water. The reactions were incubated at 25 °C for 10 min, 42 °C for 15 min and 85 °C for 5 min.

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2.3. Detection of SARS-CoV-2 using a TaqMan Real-time RT-PCR assay

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TaqMan RT-PCR assay were performed using 2.5 μl cDNA, 10 μl Primer Design PrecisonPLUS qPCR Master Mix 1 μM Forward (5′- AAA TTC TAT GGT GGT TGG CAC AAC ATG TT-3′), 1 μM Reverse (5′- TAG GCA TAG CTC TRT CAC AYT T-3′) primers and 0.2 μM probe (5′-FAM- TGG GTT GGG ATT ATC-MGBNFQ-3′) targeting the BetaCoV RdRp (RNA-dependent RNA polymerase) gene or Forward (5′-ACA GGT ACG TTA ATA GTT AAT AGC GT -3′), 1 μM Reverse (5′-ATA TTG CAG CAG TAC GCA CAC A-3′) primers and 0.2 μM probe (5′-FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-286 NFQ-3′) targeting the BetaCoV E-gene (Corman et al., 2020). Real-time RT-PCR assays were performed on an Applied Biosystems ABI 7500 Fast real-time PCR machine (Applied Biosystems, Foster City, CA, USA) using cycling conditions of 95 °C for 2 min, 95 °C for 5 s, 60 °C for 24 s. SARS-CoV-2 cDNA (Ct~28) was used as a positive control. Calculated Ct values were converted to fold-reduction of treated samples compared to control using the ΔCt method (fold changed in viral RNA = 2^ΔCt) and expressed as % of DMSO alone sample. IC50 values were fitted using 3 parameter dose response curves in GraphPad prism.

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Funding

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This work was supported by a National Breast Cancer Foundation Fellowship, Australia (ECF-17-007) for KMW and an National Health and Medical Research Council (NHMRC), Australia Senior Prinicple Research Fellow (SPRF) (APP1103050) for DAJ.

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