Antiviral effect of high-dose ivermectin in adults with COVID-19: A proof-of-concept randomized trial
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Abstract
Background
There are limited antiviral options for the treatment of patients with COVID-19. Ivermectin (IVM), a macrocyclic lactone with a wide anti-parasitary spectrum, has shown potent activity against SARS-CoV-2 in vitro. This study aimed at assessing the antiviral effect of IVM on viral load of respiratory secretions and its relationship with drug concentrations in plasma.
Methods
Proof-of-concept, pilot, randomized, controlled, outcome-assessor blinded trial to evaluate antiviral activity of high-dose IVM in 45 COVID-19 hospitalized patients randomized in a 2:1 ratio to standard of care plus oral IVM at 0·6 mg/kg/day for 5 days versus standard of care in 4 hospitals in Argentina. Eligible patients were adults with RT-PCR confirmed SARS-CoV-2 infection within 5 days of symptoms onset. The primary endpoint was the difference in viral load in respiratory secretions between baseline and day-5, by quantitative RT-PCR. Concentrations of IVM in plasma were measured. Study registered at ClinicalTrials.gov: NCT04381884.
Findings
45 participants were recruited (30 to IVM and 15 controls) between May 18 and September 9, 2020. There was no difference in viral load reduction between groups but a significant difference was found in patients with higher median plasma IVM levels (72% IQR 59–77) versus untreated controls (42% IQR 31–73) (p = 0·004). Mean ivermectin plasma concentration levels correlated with viral decay rate (r: 0·47, p = 0·02). Adverse events were similar between groups. No differences in clinical evolution at day-7 and day-30 between groups were observed.
Interpretation
A concentration dependent antiviral activity of oral high-dose IVM was identified at a dosing regimen that was well tolerated. Large trials with clinical endpoints are necessary to determine the clinical utility of IVM in COVID-19.
Funding
This work was supported by grant IP-COVID-19-625, Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, Argentina and Laboratorio ELEA/Phoenix, Argentina.
1. Introduction
COVID-19 treatment guidelines panel. Coronavirus disease 2019 (COVID-19) treatment guidelines.
].
]. More recently, several viral infections like Dengue, Zika, and Influenza were shown to be susceptible in vitro most likely through host-based mechanisms [
]. A potent activity against SARS-CoV-2 was reported in Vero-hSLAM cell cultures using high concentrations of IVM [
- Caly L.
- Druce J.D.
- Catton M.G.
- Jans D.A.
- Wagstaff K.M.
]. In a model of SARS-CoV-2 viral kinetics with acquired immune response to investigate the dynamic impact of timing and dosing regimens, the most significant effects for ivermectin were identified with earlier and longer exposure at high doses; in this regard, repeated daily doses of ivermectin at 600 µg/kg had meaningful impact whereas doses of 300 µg/kg had significantly lower effects [
- Kern C.
- Schöning V.
- Chaccour C.
- Hammann F
]. Doses of 300 µg/kg were recently found not to be superior to placebo in a randomized clinical trial in Colombia [
- López-Medina E.
- López P.
- Hurtado I.C.
- Davalos D.
- Ramirez O.
- Martinez E.
- et al.
]. IVM is prescribed in weight-based regimens, most frequently at 200 µg/kg, with a proposed link between Cmax and toxicity [
- Chaccour C.
- Hammann F.
- Rabinovich N.R.
]. Higher dose regimens are under evaluation due to their potential utility for new indications and dosing strategies [
- Navarro M.
- Camprubà D.
- Requena-Méndez A.
- Buonfrate D.
- Giorli G.
- Kamgno J.
- et al.
,
- Smit M.R.
- Ochomo E.O.
- Aljayyoussi G.
- Kwambai T.K.
- Abong’o B.O.
- Chen T.
- et al.
]. Single dose regimens of up to 2000 µg/kg have been used in a trial in healthy volunteers without clinically significant safety issues [
- Guzzo C.A.
- Furtek C.I.
- Porras A.G
].
To evaluate the antiviral activity and safety profile of high dose IVM in COVID-19 patients we completed a proof-of-concept randomized controlled clinical trial in hospitalized patients. To achieve further insights into the potential clinical utility of IVM in COVID-19, the relationship between pharmacokinetic (PK) (IVM plasma concentrations) and pharmacodynamic (PD) (dynamic of the viral load) aspects was investigated. Here we present the results of the trial with descriptions on the impact of IVM on SARS-CoV-2 viral load in respiratory secretions.
2. Methods
2.1Â Study design
Pilot, multicenter, randomized, open label, outcome assessor blinded, controlled study to assess the antiviral activity and safety of a 5-day regimen of high dose IVM versus no treatment in a 2:1 allocation ratio, in patients with COVID-19. All patients in both groups received standard of care which at that moment in the study area included hospitalization of all symptomatic cases. The trial was done at 4 hospitals in the metropolitan area of Buenos Aires, Argentina.
Ethical approval was obtained from the Institutional Independent Ethics Committees and national regulatory agencies. All participating individuals provided written informed consent. The trial was done in accordance with the principles of the Declaration of Helsinki and is registered with ClinicalTrials.gov, NCT04381884. This study conformed to the CONSORT 2010 guidelines. The funding sources had no role on the design, analysis or decision to publish the results of this study.
2.2Â Participants
Participants were COVID-19 patients aged 18 to 69 years-old with RT-PCR confirmed infection, hospitalized and not requiring intensive care. Eligibility criteria included COVID-19 symptoms onset ≤ 5 days at recruitment, absence of use of drugs with potential activity against SARS-CoV-2 (hydroxychloroquine, lopinavir, remdesivir and azithromycin); and those drugs were not permitted during the first week of the trial. Exclusion criteria included the use of immunomodulators within 30 days of recruitment, pregnancy, breast feeding and poorly controlled comorbidities. Patients of child-bearing age (men and women) were eligible if agreed to take effective contraceptive measures during the study period and for at least 30 days after the last study drug administration.
2.3Â Randomization and masking
A blocked randomization with random block sizes (of 3 or 6 allocations) and stratified by center was used. The randomization list was developed prior to study initiation and by means of a centralized eCRF/IWRS web system (Jazz Clinical, Buenos Aires, Argentina). For reproducibility, a random seed of 1701214029 was used. Once the availability of the informed consent and the verification of all eligibility criteria had been confirmed, the assignment was communicated to the investigators on the computer screen and by email. The patients and center personnel were not blinded to the allocated group. The outcome assessors (personnel in charge of viral load determinations) were blinded to the allocated group upon receiving the samples labeled with the randomization number and the visit number.
2.4Â Procedures
- Navarro M.
- Camprubà D.
- Requena-Méndez A.
- Buonfrate D.
- Giorli G.
- Kamgno J.
- et al.
,
- Smit M.R.
- Ochomo E.O.
- Aljayyoussi G.
- Kwambai T.K.
- Abong’o B.O.
- Chen T.
- et al.
,
- González Canga A.
- Prieto A.M.S.
- Diez Liébana M.J.
- Martinez N.
- Vega M.
- Vieitez G
,
- Matamoros G.
- Sánchez A.
- Gabrie J.A.
- Juárez M.
- Ceballos L.
- Escalada  
- et al.
]. Nasopharyngeal swabs were collected at baseline and 24, 48 and 72Â h and on day 5 for SARS-CoV-2 viral load quantification. Blood samples were obtained by venipuncture for plasma IVM concentrations 4Â h after drug intake on treatment days 1, 2, 3, and 5 (aiming at measuring peak plasma levels) and on day 7 (aiming to evaluate potential drug accumulation) in the IVM group. Blood samples were obtained from participants in both groups for hematologic and chemical parameters.
2.5Â Outcomes
The primary outcome measure was the difference in SARS-CoV-2 viral load between baseline and day-5 in both groups. Secondary outcomes included clinical evolution at days 7 and 30, relationship between IVM plasma concentrations and the primary outcome, and frequency and severity of adverse events.
2.6Â SARS-CoV-2 viral load measurements
- Han M.S.
- Byun J.H.
- Rim J.H.
]. The performance of the assay includes: (i) efficiency = 99%, (ii) reproducibility with a coefficient of variation (CV) between 1·01 and 2·31, (iii) repeatability with a CV between 0·27 and 1·89%, (iv) dynamic range from 10 to 1 × 108 copy per reaction, (v) specificity = 100% tested against SARS-CoV-2 negative samples and a panel of respiratory viruses. All these parameters were determined according to the guidelines for in vitro quantitative diagnostic assays as were reported previously [
,
- Bustin S.A.
- Benes V.
- Garson J.A.
- Hellemans J.
- Huggett J.
- Kubista M.
- et al.
].
2.7Â Measurement of IVM plasma concentration profiles
- Lifschitz A.
- Virkel G.
- Sallovitz J.
- Sutra J.F.
- Galtier P.
- Alvinerie M.
- et al.
]. An aliquot of plasma was combined with moxidectin (used as internal standard). After an acetonitrile-mediated chemical extraction, IVM was converted into a fluorescent molecule using N-methylimidazole and trifluoroacetic anhydride (Sigma Chemical, St Louis, MO, USA). An aliquot (100 μL) of this solution was injected directly into the HPLC system (Shimadzu Corporation, Kyoto, Japan) and analyzed using a reverse phase C18 column (Kromasil, Eka Chemicals, Bohus, Sweden, 5 μm, 4·6 mm × 250 mm) and an acetic acid 0·2% in water/methanol/acetonitrile (1·6/60/38·4) mobile phase at a flow rate of 1·5 mL/min at 30 °C. Fluorescent detector was set at 365 nm (excitation) and 475 nm (emission wavelength). The coefficient of determination (r2) of the calibration curve was 0·995. The mean absolute drug recovery percentage was 94%. The precision of the method showed a coefficient of variation below 8·1%. The limit of drug quantitation was 0·3 ng/mL. Drug concentrations in experimental plasma samples were obtained by peak area integration using the Solution Software (Shimadzu Corporation, Kyoto, Japan).
2.8Â Pharmacokinetic and pharmacodynamic analysis of the data
where λ is the decay rate constant and S is the slope [].
2.9Â Statistical analysis
,
]. Based on these grounds and aiming for a sample size with the ability to detect a low effect size (0·3) of the intervention (IVM) in the difference between baseline and day-5 viral load values compared to untreated controls given the absence of preliminary or historical data; sample size for a full-scale trial for two study groups with a significance level of 5% and 80% power, a 2:1 randomization and inflated for 10% lost-to-follow-up was calculated in 342 participants and a pilot trial would be at least 31 [
]. In view of the presumed effect of IVM on the replication of SARS-CoV-2 and the limited available information of viral dynamics at the time of study design (April 2020), the sample size of the pilot trial according to standardized size effects [
], was calculated for a 2:1 randomization to be 45 patients, including 30 participants in the IVM arm and 15 controls without consideration to the center-based stratification.
Baseline characteristics of the two groups (control and ivermectin) were compared with Student`s T– test and Chi square. Difference in viral load between baseline and day-5 in the two groups as well as the comparison between the viral decay rate of both groups was compared by the non-parametric Mann–Whitney test. The clinical evolution at day-7 was evaluated by Fisher’s Exact Test. Finally, the relationship between IVM plasma concentrations with viral load reduction and viral decay rate were measured by Spearman rank test. When difference across three groups by Kruskal-Wallis was significant, pairwise comparisons with Dunn`s multiple comparisons test were used. Two randomly occurring single missed values of viral load in two different participants were assumed as “missing completely at random†type of values and estimated by regression analysis using the interpolation of all the existing data from that particular curve. In all cases, p-values <0·05 were considered statistically significant. All analysis were performed with GraphPad Prism version 5·00 for Windows (La Jolla California USA).
2.10Â Role of the funding source
The sponsors of the study participated in study design, but had no role in primary data collection, data analysis, data interpretation, writing of the report, or the decision to submit for publication. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.
3. Results
Table 1Baseline characteristic of the study population.
Numeric variables are reported as median (IQR) or mean ± standard deviation• Categoric variables are reported as counts (%). Overweight: Body mass index (BMI) 25–29•9 kg/m2; Obesity I: BMI 30–34•9 kg/m2; Obesity II: BMI 35–39•9 kg/m2; Obesity III: BMI >40 kg/m2.
Fig. 2Viral load by quantitative RT-PCR on upper respiratory tract secretions since baseline in patients receiving IVM 0•6 mg/kg/day for 5 days versus untreated controls.
3.1Â Data are mean (SD). Day-1 indicates baseline measurements
Fig. 3Viral load reduction between baseline and day-5 (median and IQR) in untreated controls and IVM treated patients discriminated by their median IVM plasma concentrations.
All treated patients receiving IVM 0·6 mg/kg/day for 5 days.
Fig. 4Viral load decay rates by quantitative RT-PCR on upper respiratory tract secretions in untreated controls and IVM treated patients according to median plasma concentrations of IVM. Data are expressed as median (IQR).
- Yousaf X.Z.
- Al-shokri S.D.
- Al-soub H.
- Mohamed M.F.H.
- Rj A.
- Jt H.
- et al.
].
Table 2Summary of events in safety population.
AE: adverse event. SAE: serious adverse event. *: hyponatremia; **: includes the SAE (hyponatremia) and ALT and AST elevation, both in the same patient.
4. Discussion
- Caly L.
- Druce J.D.
- Catton M.G.
- Jans D.A.
- Wagstaff K.M.
]. Findings on IVM plasma concentrations are in agreement with human SARS-CoV-2 viral kinetic models identifying the need for high doses, but contradict those concerns stating that those drug concentrations would not be achievable at safe doses [
- Kern C.
- Schöning V.
- Chaccour C.
- Hammann F
,
- Chaccour C.
- Hammann F.
- Ramon-Garcia S.
- Rabinovich N.
]. The extensive pattern of IVM distribution to lung tissue has been well characterized in cattle and pigs, with the later also achieving in nasopharyngeal tissue higher levels than plasma [
- Lifschitz A.
- Virkel G.
- Sallovitz J.
- Sutra J.F.
- Galtier P.
- Alvinerie M.
- et al.
,
- Errecalde J.
- Lifschitz A.
- Vecchioli G.
- et al.
]. Considering that similar volumes of distribution have been reported for IVM in both cattle and humans and the systemic availability observed in this clinical trial, it is reasonable to estimate median IVM levels >395Â ng/g in lung tissue. A similar pattern of IVM distribution to lung tissue has been recently simulated using a minimal physiologically based PK model [
- Jermain B.
- Hana P.O.
- Cao Y.
- Lifschitz A.
- Lanusse C.
- Rao G.G.
].
- Wang Y.
- Zhang D.
- Du G.
- Du R.
- Zhao J.
- Jin Y.
- et al.
], highlighting the relevance of adequate timing of implementation of antiviral treatment as has been shown in a recently published pilot double-blind trial randomized trial of IVM for non-severe COVID-19 that identified statistically significant differences in the duration of anosmia and trends towards lower viral load with treatments started within 72Â h of symptoms onset [
- Chaccour C.
- Casellas A.
- Blanco-Di Matteo A.
- Pineda I.
- Fernandez-Montero A.
- Castillo P.
- et al.
].
- Navarro M.
- Camprubà D.
- Requena-Méndez A.
- Buonfrate D.
- Giorli G.
- Kamgno J.
- et al.
]. Diet is a key variable affecting oral bioavailability of IVM, with increased plasma concentrations achieved with fed state [
- Muñoz J.
- Ballester M.R.
- Antonijoan R.M.
- Gich I.
- RodrÃguez M.
- Colli E.
- et al.
,
- Guzzo C.A.
- Furtek C.I.
- Porras A.G
]. The interaction of IVM with ABC transporters as P-glycoprotein and the modulation of P-glycoprotein activity after oral administration is well known [
- Lespine A.
- Martin S.
- Dupuy J.
- Roulet A.
- Alvinerie M.
,
- Ballent M.
- Lifschitz A.
- Virkel G.
- Sallovitz J.
- Lanusse C.
]. Thus, variable constitutive and/or induced level of expression and activity of intestinal P-glycoprotein in treated patients, may have contributed to the observed large variability in the pattern of IVM absorption and systemic exposure.
- Smit M.R.
- Ochomo E.O.
- Aljayyoussi G.
- Kwambai T.K.
- Abong’o B.O.
- Chen T.
- et al.
]. In that trial, IVM was associated with dose-dependent mild transient visual disturbances in
- Smit M.R.
- Ochomo E.O.
- Aljayyoussi G.
- Kwambai T.K.
- Abong’o B.O.
- Chen T.
- et al.
]. The frequency of adverse events reported by study participants (43% of those in the IVM group and 33% of the untreated controls) (Table 2), likely reflects events related and unrelated to the study drug which as expected were more frequent in the treated groups, although most of them of severity grades 1 and 2.
- Vegvari C.
- Hadjichrysanthou C.
- Cauët E.
- Lawrence E.
- Cori A.
- De Wolf F.
- et al.
]. For that reason, key components for adequate endpoints are sensitive quantifiable measurements of the underlying cause as the quantitative RT-PCR [
- Vegvari C.
- Cauët E.
- Hadjichrysanthou C.
- Lawrence E.
- Weverling G.
- Wolf F.
- et al.
]. As it has been proposed in an influenza model of antiviral candidate drugs evaluation 25, viral decay rates proved to be a critical parameter of antiviral activity. Additionally, as it has been clearly demonstrated for acute viral infections, early treatment initiation plays a critical role [
- Vegvari C.
- Cauët E.
- Hadjichrysanthou C.
- Lawrence E.
- Weverling G.
- Wolf F.
- et al.
,
- Arabi Y.
- Assiri A.Y.
- Assiri A.M.
- Balkhy H.
- Bshabshe A.
- Jeraisy M.
- et al.
]. The clinical relevance of these findings remains to be confirmed in trials with clinical endpoints. Beyond clinical aspects, lowering viral burden might influence infectivity, although there is conflicting data regarding the relationship between burden of viral shedding and infectivity [
- Wölfel R.
- Corman V.M.
- Guggemos W.
- Seilmaier M.
- Zange S.
- Müller M.A.
- et al.
]. The proposed antiviral mechanism of IVM is through its ability to inhibit the nuclear import of viral proteins mediated by IMPα/β1 heterodimer [
- Caly L.
- Druce J.D.
- Catton M.G.
- Jans D.A.
- Wagstaff K.M.
], and it has also been suggested that IVM could promote defense mechanisms such as pyroptosis in infected epithelial cells [
- Draganov D.
- Gopalakrishna-pillai S.
- Chen Y.
- Zuckerman N.
- Moeller S.
- Wang C.
- et al.
]. Drugs such as ivermectin can be used to target viral entry or viral replication mechanisms in host cells, as well as to modulate the innate immune responses, to achieve indirect antiviral activity in vivo. Mechanisms essential for viral infection, such as nuclear transport or intracellular signal transduction, among others, have been indicated as better targets to identify broad-spectrum antiviral agents, with some advantages over direct-acting antivirals targeting viral components [
].
In summary, our findings support the hypothesis that IVM has a concentration dependent antiviral activity against SARS-CoV-2 and provides insights into the type of evaluations to be considered in the assessment of antiviral drugs for the control of COVID-19. Follow-up trials to confirm our findings and to identify the clinical utility of IVM in COVID-19 are warranted.
Acknowledgments
We thank Romina Guelho for technical assistance. Natalia PacÃfico, Belén Bargallo, Julián GarcÃa, Andrés Benchetrit, Guillermo Onis, Luis Vanzetti, Liucó ZubeldÃa Brenner, Natalia DÃaz and Francisco Moioni for patient care. Javier Toibaro for assistance in statistical analysis. Graciela Ciccia, Ignacio Demarco and Alicia Bagnoli for assistance in administrative project management.
Contributors
Study design: AK, AL, ROC, CL, SG, ES
Writing protocol: AK, MT, ES, MAT
Literature search: AK, MT, MM, AL, PF
Data collection: RV, RS, JF, IB, DFO, LA, LC, MG
Database maintenance and supervision: MAT, MT
Laboratory analyses: DFA, GAC, FF, MM, LC, LA, GA
Data analysis: AL, MT, PF, AK
Data interpretation: CL, AM, AK
Writing: AK, AL, ROC, MM, CL
Coordination: ROC, ES, AM, CL, DFA, AK
Supervision of the project: AK, CL, SG
Data sharing
De-identified individual clinical and laboratory data and a data dictionary will be made available to others after 3 months of trial publication upon request to the corresponding authors, only for research, non-commercial purposes to individuals affiliated with academic or public health institutions.
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