key: cord-0807685-t9p50x9d
authors: Foo, Caroline S.; Abdelnabi, Rana; Vangeel, Laura; De Jonghe, Steven; Jochamns, Dirk; Weynand, Birgit; Neyts, Johan
title: Ivermectin does not protect against SARS-CoV-2 infection in the Syrian hamster model
date: 2022-02-23
journal: bioRxiv
DOI: 10.1101/2022.02.22.481472
sha: 4c6f4a004d0c4ffd9eb316b630b2af18c4a70bcc
doc_id: 807685
cord_uid: t9p50x9d

Ivermectin, an FDA-approved antiparasitic drug, has been reported to have in vitro activity against SARS-CoV-2. An increasing off-label use of Ivermectin for COVID-19 has been reported. We here assessed the effect of Ivermectin in Syrian hamsters infected with the SARS-CoV-2 Beta (B.1.351) variant. Infected animals received a clinically relevant dose of Ivermectin (0.4 mg/kg subcutaneously dosed) once daily for four consecutive days after which the effect was quantified. Ivermectin monotherapy did not reduce lung viral load and even significantly worsened the SARS-CoV-2-induced lung pathology. Additionally, it did not potentiate the activity of Molnupiravir (Lagevrio™) when combined with this drug. This study contributes to the growing body of evidence that Ivermectin does not result in a beneficial effect in the treatment of COVID-19. These findings are important given the increasing, dangerous off-label use of Ivermectin for the treatment of COVID-19.

Main text 24 Two years after it was declared a global pandemic by the World Health Organization, the 25 coronavirus disease (COVID-19) continues its devastating impact, having claimed officially 26 over 5.9 million lives as of 22 nd Feb 2022 [1] . The etiological agent, the severe acute respira-27 tory syndrome coronavirus 2 (SARS-CoV-2), first emerged in Wuhan, China in December 28 2019 and has since evolved into variants emerging from different regions of the world, 29 namely the Alpha, Beta, Delta, and more recently Omicron variants of concern (VoC) [2, 3] . 30 These VoC have been implicated in resurgence of infections and mortality, with increased 31 transmissibility and potential escape from both vaccine-and natural infection-induced im-32 munity [3] [4] [5] . 33 Ivermectin is an FDA-approved, broad-spectrum antiparasitic drug with a range of other ac-34 tivities, including antiviral and host immunomodulation effects [6] . This widely-accessible 35 drug has been attracting increasing off-label use for COVID-19 since its in vitro activity against 36 SARS-CoV-2 was reported [7] , which is of growing concern. In an in vivo model of mouse hep-37 atitis virus, a type 2 family RNA coronavirus similar to SARS-CoV-2, Ivermectin reduced the 38 viral load and protected mice from disease [8] . In Syrian hamsters infected with SARS-CoV-2, 39 treatment with Ivermectin did not result in a direct antiviral activity, but rather apparently 40 showed some immunomodulatory effects, improved lung pathology and clinical presenta-41 tions [9] . Ivermectin is being assessed as a treatment for COVID-19 in a large number of clin-42 ical trials, with no clear evidence of its clinical benefit so far [10].

Molnupiravir (Lagevrio TM , EIDD-2801) is the orally bioavailable counterpart of the ribonu-44 cleoside analogue N4-hydroxycytidine (NHC, EIDD-1931), which was initially developed for 45 the treatment of influenza [11] . NHC exerts a broad-spectrum antiviral activity against mul-46 tiple RNA viruses of different families by its incorporation into viral RNA, resulting in the 47 accumulation of deleterious mutations in the nascent viral RNA, and consequently, error ca-48 tastrophe [12] . In pre-clinical infection models in mice, Syrian hamsters, and ferrets, Mol-49 nupiravir demonstrated efficacy against SARS-CoV-2 [13] [14] [15] , including against several VoC 50 [16] . The drug has been approved in many regions for treatment of COVID-19.

Here, we evaluate the antiviral efficacy of Ivermectin against SARS-CoV-2 Beta (B.1.351) VoC 52 in a hamster infection model alone or in combination with Molnupiravir; this combination 53 was inspired by the observation of a pronounced combined activity of Molnupiravir and 54 Favipiravir [17] . 55 To that end, we tested the efficacy of single treatment with Ivermectin (0.4 mg/kg, subcuta-56 neous, once daily (QD)) or Molnupiravir (150 mg/kg, oral BID) or the combination of both. 57 Briefly, 6-8 weeks female SG hamsters were treated with the intended dose of each compound 58 or the vehicle (i.e. the control group) for four consecutive days starting one hour before in- Figure 1B ) and lung infectious virus titers by 1.9 71 (P=0.0002, Figure 1C ) log10/mg lung tissue, which is similar in potency to the single treatment 72 with Molnupiravir (150 mg/kg, BID). No significant weight loss or toxicity signs were ob-73 served in any of the treated groups ( Figure 1D ). However, the average %body weight change 74 on the day of the sacrifice (compared to day zero) in the Ivermectin single treatment group 75 (average of -1.7%) was markedly lower than the vehicle-treated group (average of 2.6%), 76 Figure 1D . 77 The median histopathology score in the single Ivermectin treatment group (5.8) was signifi-78 cantly higher than the vehicle treated control (median score of 4.5, P=0.04) (Figure 2A) . A 79 significant improvement in lung pathology was observed in the single Molnupiravir-treated 80 group (median score of 3.5, P=0.04) while the combined Molnupiravir/Ivermectin treatment 81 resulted in a median histopathology score of 3.8 ( Figure 2A ). Hematoxylin/eosin (H&E)-82 stained images of lungs of the Ivermectin-treated hamsters revealed severe peri-vascular in-83 flammation with vasculitis and significant multifocal bronchopneumonia ( Figure 2B ). On the 84 other hand, the lungs of animals treated with Molnupiravir as a single treatment showed very 85 limited peribronchial and peri-vascular ( Figure 2B ).

In our study, Ivermectin did not result in any antiviral activity against SARS-CoV-2 in Syrian 87 hamsters as monotherapy. This is consistent with a previous study published as well in ham-88 sters, whereby Ivermectin was dosed once subcutaneously at 0.4 mg/kg (an antiparasitic 89 dose used in the clinical setting) and monitored for four days [9] . However, unlike the previ-90 ous study, we did not notice an improvement in SARS-CoV-2-induced lung pathology with 91 Ivermectin. Transcriptomic profiling of the lungs was performed in the previous study to 92 compare expression levels of inflammatory genes with vehicle and Ivermectin treatment, as 93 well as a descriptive histopathological analysis which indicated that SARS-CoV-2-infected, 94 Ivermectin-treated hamsters exhibited reduced degrees of edema and congestion, with 95 greater numbers of mononuclear cells in the alveolar spaces [9] . In our study, a cumulative 96 lung score was performed based on ten different parameters, including edema and conges-97 tion, which had similar scorings in both vehicle-treated and Ivermectin-treated groups. Ad-98 ditionally, no mononuclear cells in the alveolar spaces were observed. These differences in 99 findings may be due to slight hamster age differences or virus strain differences used for in-100 fection, namely the SARS-CoV-2 Beta variant was used in our study and the ancestral SARS-101 CoV-2 strain (BetaCoV/France/IDF00372/2020) was used in the previous study [9] . 102 Additionally, Ivermectin did not potentiate the activity of Molnupiravir in our hands, indicat-103 ing that it is unlikely to have a significant role in directly or indirectly modulating SARS-CoV-104 2 replication. A Cmax of 80.2 ng/ml was measured in Syrian hamsters when treated once sub-105 cutaneously with 0.4 mg/kg of Ivermectin [20] , comparable to that measured in humans 106 when dosed at similar levels orally [9, 21] . Taken together with other available evidence, it 107 suggests that in order to achieve therapeutic activity against SARS-CoV-2 (IC50 of 1750 ng/ml 108 [7]), the dosing levels of Ivermectin in humans would need to be substantially increased from 109 the approved clinical dose to potentially toxic doses [22] [23] [24] . This is in line with the fact that 110 several studies claiming the clinical benefits of Ivermectin in COVID- 19 In conclusion, we report that Ivermectin, at a clinically relevant dose, does not reduce lung 114 viral load nor improve lung pathology in Syrian hamsters infected with SARS-CoV-2 Beta var-115 iant. In fact, virus-induced lung pathology increases in severity in Ivermectin treated animals. 116 Moreover, the drug does not potentiate the antiviral activity of Molnupiravir (Lagevrio TM ). 117 This study contributes to the growing body of evidence that Ivermectin does not results in a 118 clinical beneficial effect in the treatment of COVID-19 at the current approved dose. Hence, 119 we strongly advice against the increasing, dangerous off-label use of Ivermectin for the treat-120 ment of COVID-19. group showed peribronchial inflammation with intra-bronchial cell debris (blue arrows), perivascular 153 inflammation with endothelialitis (red arrows) and bronchopneumonia (green arrows) surrounded by 154 intra-alveolar hemorrhage. Image of the lung from Ivermectin-treated animal showed severe peri-vas-155 cular inflammation with vasculitis (red arrows), limited and focal peri-bronchial inflammation (blue 156 arrows) and significant and multifocal bronchopneumonia (green arrows). On the other hand, lung from 157 Molnupiravir (EIDD-2801) revealed very limited peribronchial (blue arrows) and peri-vascular (red 158 arrows) inflammation. Scale bar 100 µM. 

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We thank Carolien De Keyzer, Lindsey Bervoets, Thibault Francken, Stijn Hen-179 drickx, Niels Cremers for excellent technical assistance. We are grateful to Piet Maes for kindly provid-180 ing the SARS-CoV-2 strain used in this study. We thank Prof. Jef Arnout and Dr. Annelies Sterckx (KU 181 Leuven Faculty of Medicine, Biomedical Sciences Group Management) and Animalia and Biosafety De-182 partments of KU Leuven for facilitating the animal studies. We thank the Histology department of KU 183 Leuven for technical support for histopathological analyses. We also thank Fran Berlioz-Seux, Rob Jor-184 dan and Betsy Russell for helpful discussion.