key: cord-0726236-m0fhb453
authors: Lionis, C.; Karakasiliotis, I.; Petelos, E.; Linardakis, M.; Diamantakis, A.; Symvoulakis, E.; Panopoulou, M.; Kampa, M.; Pirintsos, S. A.; Sourvinos, G.; Castanas, E.
title: A mixture of essential oils from three Cretan Aromatic Plants (thyme, Greek sage and Cretan dittany, CAPeo) inhibits SASR-CoV-2 proliferation: in vitro evidence and a Proof-of-Concept intervention study in mild ambulatory COVID-19-positive patients
date: 2021-01-15
journal: nan
DOI: 10.1101/2021.01.11.20248947
sha: 785103c30977e21d4045ebdbef77019af6ba48d3
doc_id: 726236
cord_uid: m0fhb453

The need for therapeutic regimens for the non-critically ill patients of the COVID-19 pandemic remains unmet. In this line, repurposing existing drugs, against known or predicted SARS-CoV-2 protein actions, has been advanced, while natural products have also been tested. Previous work has shown that a Cretan Aromatic Plant (Thymbra capitata (L.) Cav., Salvia fruticosa Mill. and Origanum dictamnus L.) essential oil mixture (CAPeo) has a remarkable in vitro antiviral activity against Influenza A & B and Rhinovirus 14 strains, decreasing the symptoms of upper respiratory tract infections, while proven safe in experimental animals and humans. Here, we tested CAPeo in VERO cells infected with SASR-CoV-2. We report that this mixture, at similar concentrations as those previously reported, exhibits a remarkable antiviral activity. Administration of 1 ml of a 1.5% CAPeo in olive oil, in a Proof-of-Concept intervention study in SARS-CoV-2-positive, exhibiting mild COVID-19 symptoms, humans resulted in a significant amelioration of general and local symptoms of the disease. We conclude that CAPeo may be a valuable addition for the prevention and/or treatment of mild COVID-19 ambulatory patients, pending a confirmation through a prospective randomized controlled trial in humans.

Since the outbreak of COVID-19 pandemics, in 2019, a previously unseen international effort has been 49 undertaken for the identification of the underlying cause (the SARS-CoV-2 virus) and the detailed analysis 50 of its genome (O'Leary and Ovsepian, 2020 ). An international effort is actually directed towards an 51 efficient therapy (Baum et al., 2020; Hansen et al., 2020; Weinreich et al., 2020) , or the development of 52 efficient vaccines (Polack et al., 2020; Voysey et al., 2020) . A number of established pharmaceutical 53 molecules and patients' plasma have been tested as drug candidates for the treatment of with 54 variable results (see (Bolarin et al., 2020; Wang et al., 2020a; Wang et al., 2020b) and references herein). 55

Among the multitude of products tested against COVID-19 disease, a number of natural products, 56

including herbal extracts, have also been assayed (critically reviewed in (Benarba and Pandiella, 2020) and 57 references herein), targeting mainly the viral proteases. 58

Recently, we have reported that a combination of three aromatic plants essential oil (CAPeo) (Thymbra 59 capitata (L.) Cav., Origanum dictamnus L., Salvia fruticosa Mill., , and references 60 herein) is efficient against upper respiratory tract viral infections, in humans (Duijker et al., 61 2015; Anastasaki et al., 2017) . In vitro studies revealed the efficacy of CAPeo against Influenza A & B and 62

Human Rhinovirus 14, and reported an action through the inhibition of the nuclear translocation of viral 63

nucleoproteins (Tseliou et al., 2019) , resulting in impaired viral protein transcription. Furthermore, we 64

have reported the safety of CAPeo, both in humans (administered in the form of soft gels, 1 ml/day of a 65

1.5% essential oil combination in extra virgin olive oil, (Duijker et al., 2015) ) and in experimental animals 66 (Kalyvianaki et al., 2020) . In the present study, we have assayed the efficiency of CAPeo mixture on the 67 proliferation of SARS-CoV-2 in VERO cells. We report a remarkable antiviral activity of CAPeo, at 68

concentrations compatible with those obtained after the recommended dose administration in humans. 69 Moreover, we performed a Proof-of-Concept intervention study in mild COVID-19-positive humans and 70 report that CAPeo can significantly ameliorate the general and local symptoms of the disease. We suggest 71

that CAPeo, pending additional confirmation of results through a prospective randomized controlled trial, 72 may represent a valuable addition for the prevention and/or therapeutic management of mild COVID-19 73 ambulatory patients. 74 75

CAPeo production and use 77 Spanish oregano (Coridothymus capitatus (L) Rchb. F. synonym of Thymbra capitata (L) Cav.), dictamnus 78 or Cretan dittany (Origanum dictamnus L) and sage (Salvia fruticosa Mill., Salvia pomifera L., were 79 cultivated under total Good Agricultural Practice and high precision agriculture, based on an Ecological 80

Niche Modelling tool, we have recently developed (Bariotakis et al., 2019) , in order to maximize their 81 essential oil composition and content. A constant genotype of plants, specified by a barcoding of each 82 batch, was used. Essential oils were prepared by steam distillation of the dried plant leaves, under GMP 83

conditions. The final extract, contained 4 parts Corydothymus Capitatus (L) extract, 2 parts Salvia Fruticosa 84

Mill. extract and 1 part Origanum Dictsmnus L extract. It was analyzed by Gas Chromatography-Mass 85

Spectroscopy (GC-MS), in a Shimadzu, QP 5050A apparatus. The mixture of essential oils contains 86 carvacrol (53%) eucalyptol (13%) and β-Caryophyllene (3%). Concentrations of the compounds p-Cymene, 87

γ-Terpinene, Borneol and α-Terpineol were 1.32, 1.17, 1.68 and 1.06% respectively, while the 88 concentrations of the remaining 15 compounds were less than 1%. For the complete analysis of 89 compounds, please refer to previous reports (Duijker et al., 2015; Kalyvianaki et al., 2020) . These 90 concentrations refer to the stock essential oil mixture, while a concentration of 1.5% in DMSO (Sigma-91

Aldrich) was used in the present study. This refers to the dilution 1/1, mimicking the suggested daily dose 92 of the CAPeo extract in humans (1 ml of a 1.5% of CAPeo in olive oil, for the management of upper 93 respiratory tract infections (Duijker et al., 2015; Anastasaki et al., 2017) ). As the pharmacokinetics and 94 bioavailability of CAPeo are under investigation, we have used bibliography data, suggesting a variable 95 absorption of phenolic compounds ranging from 27 to 0.0006% and a blood recovery ≤1% for the majority 96 of compounds (Scalbert et al., 2002; Manach et al., 2005) . Therefore, to mimic available concentrations in 97 humans, different dilutions (1:10, 1:100 and 1:1000 of the clinically administered concentration -15 mL 98 extract/L, 1 mL/day-) in DMSO) were used in the present study. The same concentrations were used in a 99

previous study, to determine the protective and therapeutic effect of CAPeo in cells infected with other 100 upper respiratory viruses (Tseliou et al., 2019) . 101

SARS -CoV-2 (isolate 30-287) was obtained through culture in Vero E6 cells, from an infected patient, in 103

Alexandroupolis, Greece. Virus stock was prepared by infecting fully confluent Vero E6 cells in DMEM, 104 10% fetal bovine serum (FBS), with antibiotics at 37 o C, 5% CO2. Four days after inoculation, the 105 supernatant was frozen at −80°C until use. Titration was carried-out in 96 -well plates using Vero E6 cells 106

and TCID50 was calculated according to the method of Reed and Muench (Reed and Muench, 1938) . Plates 107 were incubated at 37°C for 4 days, and the cytopathic effect (CPE) was scored by observation under an 108 inverted phase contrast microscope. 109

Infections were carried out in 96-well plates, using SARS -CoV-2 (m.o.i. of 0.1) on Vero E6 cells. Cells were 111 treated with different concentrations of CAPeo, as described above, in a volume of 15 μl, per 150 μl of 112 medium, for 48h. Cell morphology was observed with phase contrast, in an inverted microscope, to record 113 CPE. Culture supernatants were also collected and analyzed using real-time RT-PCR. 114

To determine viral load, RNA was extracted from 96-well supernatants (100 μl) using NucleoSpin Dx Virus 116

according to the manufacturer (Macherey Nagel). Multi-target real-time RT-PCR was performed using 117 COVID-19 SARS-Cov-2 Real-TM according to the manufacturer (Sacace Biotechnologies, Como, Italy). 118

Seventeen (17) medical history, smoking habits, symptoms and signs has been recorded in a pre-tested questionnaire for 128 all study participants (Table 1) . 129

Data were collected on day 1, day 4, day 7 and day 14; following the initial face-to-face consultation, data 130 collection and consultations were performed, either remotely (by phone), or via home visits, by trained 131 medical personnel. The severity of symptoms was assessed through the utilisation of a seven-point Likert 132 scale, with data recorded on Day 1 and taken as the baseline. A seven-point Likert scale allowed recording 133 of reported symptoms starting from 1 (minor) to 2 (very mild) and 3 (mild), 4 (somewhat moderate) and 134 5 (moderate) and culminating to 6 (severe) and 7 (very severe). The main outcome defined to assess the 135 clinical effectiveness of the CAPeo was symptom reduction, in terms of severity and frequency, defined 136 as total number of symptoms over the 14-day period, and with measurement on day 4, day 7 and day 14. 137

Five patients reported symptoms prior to their confirmed diagnosis; the corresponding interval varied 138 from 2 to 4 days. For the purposes of reporting and given the small interval prior to the confirmed 139 diagnosis and study inclusion, the date of the confirmed diagnosis, i.e., day 1 of the study was considered 140 as day 1 for symptoms, and severity and frequency assessment. 141 CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The potential antiviral effect of CAPeo against SARS-CoV-2 was assessed both at cellular and molecular 152 level, in vitro. Cells infected with SARS-CoV-2 present a CPE effect. CPE of infected VERO cells, was 153 monitored in the presence of different concentrations of CAPeo ( Figure 1A ). As shown, cells were viable 154

and presented a morphology similar to that of non-infected cells, up to a concentration of 1/100 CAPeo, 155

where minimal CPE was present. At lower concentrations, CPE was obvious and cell morphology was more 156 similar to the vehicle (DMSO)-treated cells. Similar results were found in cells preincubated with the same 157 concentrations of CAPeo, for 2h, before infection with SARS-CoV-2 (0.1 m.o.i.). 158

To avoid drug carry-over effects during TCID50 and to assess more accurately viral RNA production, we 159 used quantitative real time PCR for the determination of viral RNA presence in culture medium. Real-time 160 PCR analysis of three different SARS-CoV-2 genes (N, E, and SARS-CoV/SARS-CoV-2 common E region), 161

from the supernatant of infected cells, treated with different concentrations of CAPeo, is shown in Figure  162 1B. Results (expressed as % of non-treated cells) showed that at a concentration of 1/10, CAPeo 163 significantly reduced the viral release to the medium by >80%. The effect, albeit smaller (~35%), persisted 164 at concentrations of CAPeo 1/100 of the suggested dose in humans, but is absent at concentrations 165 1/1000. The calculated IC50 of CAPeo, with a logistic curve fitting, is 1/60 of the proposed dose for viral 166 growth and ~1/250 for maintenance of the cell phenotype. Interestingly, similar results ( Figure 1C ) were 167

found when VERO cells were preincubated with different concentrations of CAPeo for 2h prior infection, 168

suggesting that CAPeo, in addition to a possible therapeutic action, might be dotted with a prophylactic 169 effect against SARS-CoV-2 virus. 170

Results, presented above, suggest a direct SARS-CoV-2 inhibitory effect of CAPeo in cells. In order to 173 provide a proof of concept on the effect of the preparation in humans, we have performed a small 174 intervention study in 17 COVID-19-positive individuals, with mild symptoms, not necessitating 175 hospitalization, in the context of a primary care center. No adverse effects were noted in any patient, in 176 accordance with our previous observations in humans (Duijker et al., 2015; Anastasaki et al., 2017) and 177 experimental animals (Kalyvianaki et al., 2020) . The average age of enrolled patients was 34.4±11.0 years 178 and underlying morbidities were reported in 29.4% of them; co-morbidities included dyslipidaemia, 179 obesity, COPD and Hashimoto disease. Based on the questionnaire used, infection source was established 180

to be either cohabitation (23.5%) or close contact with a confirmed case (58.8%). 181

On day 1, these individuals have consulted their General Practitioner (GP), with a variety of symptoms, 182 both general and local ( Figure 2A ). Interestingly, general symptoms (fever, which in our population was 183 mild, lower than 37.5°C, in all that two individuals, headache, fatigue and myalgias) were similar to those 184

previously reported in two of the largest studies, focusing on symptom duration for COVID-19 outpatients 185 in the US (Tenforde et al., 2020) and on the clinical presentation of mild-to-moderate forms of COVID-19 186 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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in Europe (Lechien et al., 2020) . However, in our group, the frequency of nasal congestion, cough, anosmia 187

and ageusia was lower. 188

In our study group, we have observed that the number of symptoms (4 at day1, 2 at day 4 and 1 at day 7) 189 and the frequency of symptoms significantly decreased after CAPeo administration (Table 2, Figure 2B,  190 left panel). The severity of symptoms, measured as the sum of symptoms in the seven-point Likert scale 191 (see Material and Methods) was also decreased, following CAPeo administration (Wilcoxon tests; p<0.01, 192 Table 2 ). Interestingly, at day 14, almost all symptoms completely regressed, with the notable exception 193 of anosmia and ageusia, and mild ENT symptoms (nasal congestion and cough, in a small number of 194 patients). EC20, EC50 and EC80, calculated with a logistic fit from data shown in Table 2 (Rohatgi, 199 2020) . Data from (Tenforde et al., 2020) are reported in Figure 2B , right panel. Fourteen days after 19 testing, a number of symptoms persist; they include headache, fatigue, myalgias, anosmia, ageusia and 201 respiratory distress. Concerning the common general symptoms ( Figure 2C ), we observe a notable, 202 significant difference in the frequency of occurrence of headache, fatigue and myalgias, while fever was 203 absent in both groups and a similar proportion of patients with persisting anosmia and ageusia. 204

Symptoms evolution was compared to that reported by (Tenforde et al., 2020) ; the authors report 205 symptoms at days 1 and 14. In another study, we have used also as reference (Allen et al., 2020) , including 206 a much larger number of cases, symptoms were self-reported, daily, but without the implication of a 207 medical examiner. We also used these data for comparison, as the frequency of symptoms was 208 significantly different from the two other studies (Lechien et al., 2020; Tenforde et al., 2020) , at day 1. A 209 complete resolution of headache, a major symptom in COVID-19, was found in our group, with T1/2 of 5.1 210 days, while it persisted in 14% of cases in (Tenforde et al., 2020) and was almost absent in (Allen et al., 211 2020) . Fatigue was also completely resolved in our group with T1/2 of 5.8 days, while it persisted in 35% of 212 patients in the study of (Tenforde et al., 2020) and at about 15% in (Allen et al., 2020) . Fever, another 213 major symptom in day 1, completely resolved in our group, with T1/2 of 2.7 days, as compared to ~9 days 214

in (Allen et al., 2020) . At 14 days, fever was also absent in studies by (Allen et al., 2020; Tenforde et al., 215 2020) . Therefore, CAPeo seems to ameliorate general symptoms very quickly, better than the reference 216

population. In addition, as shown in Figure 2B , the majority of symptoms, both general and local, 217

completely resolve at the end of the first week. A special notion applies to anosmia and ageusia. Both in 218 our group and in the European multi-center study (Allen et al., 2020) , these symptoms progressively 219

increase, peaking at day 4 and at days 4-6 respectively. Thereafter, at day 14, these two symptoms persist 220 in 17%, 23% and 21% in our group, in (Tenforde et al., 2020) and in (Allen et al., 2020 ) studies, respectively. 221

As mentioned, and according to protocol, we included three subjects in the analysis since they were family 222 members (father, mother and sister of one patient), working together and living in the same house. They 223 received the CAPeo for prophylactic use, on the basis of the decision of the family doctor, despite having 224 tested negative for SARS-CoV-2. One of them (mother) presented symptoms two days after the baseline, 225

and was re-tested and found positive, and she was enrolled in the study, while the remaining two (father 226 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 15, 2021. ; and sister) did not report symptoms on any of the days of observation. We consider this an important 227

reporting aspect in terms of establishing guidelines for sequential testing, managing oligo-or 228 asymptomatic patients, in outpatient settings and to inform future clinical study design across settings, as 229 highlighted by a recent report (Wernhart et al., 2020) . The median incubation period of five days creates 230 a false sense of safety, but also presents a challenge in terms of study inclusion and sound trial conduct 231 and reporting (Lauer et al., 2020) . 232 233 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The COVID-19 pandemics imposed a number of, not yet resolved, problems to the International Scientific 235

Community. Thanks to the combined world-wide scientific effort and the analysis of SARS-CoV-2 virus 236 (O'Leary and Ovsepian, 2020), successful and safe vaccines are now begin to emerge (Polack et al., 237 2020; Voysey et al., 2020) . Although multiple molecules are at various stages of preclinical and clinical 238 development, there largely remains an unmet need for prophylactic and therapeutic regimens to combat 239 the disease, with proposed measures being scarce and non-specific (Bolarin et al., 2020; Wang et al., 240 2020a), with the exception of monoclonal antibodies (Baum et al., 2020; Hansen et al., 2020; Weinreich et 241 al., 2020) and dexamethasone (Cain and Cidlowski, 2020; Recovery Collaborative Group et al., 2020) , which 242

primarily target hospitalized patients in intensive care units. In this respect, the need for non-expensive 243 therapeutic regimens, safe and effective in non-critically-ill patients and efficient for their management 244 in ambulatory settings, remain unmet. Accordingly, drug repurposing, for candidates acting against known 245 or predicted SARS-CoV-2 protein actions have been advanced (reviewed and discussed in (Asselah et al., 246 2020; Cadegiani, 2020; Khan et al., 2020) ), while natural products have also been tested (reviewed in 247 (Benarba and Pandiella, 2020) ). Finally, quercetin has been proposed as an alternative for dexamethasone 248 (Pawar and Pal, 2020) . Here, we suggest CAPeo as a potential novel agent, for the safe and effective 249 therapeutic management of ambulatory mild cases of COVID-19. 250

CAPeo, a 1.5% of essential oils of Thymbra capitata (L.) Cav., Salvia fruticosa Mill. and Origanum dictamnus 251 L. in olive oil, has been advanced by our group in 2015, and found to be effective in reducing the severity 252 and duration of symptoms of viral upper respiratory tract infections (Duijker et al., 2015; Anastasaki et al., 253 2017) . It presents remarkable anti-viral properties against Influenza A and B strains and HRV14 (Tseliou et 254 al., 2019) , while it is safe in both experimental animals (Kalyvianaki et al., 2020) and humans (Duijker et 255 al., 2015) . Its properties have been recently reviewed in reference . Here, we extend 256 these previous findings, by providing in vitro evidence about its antiviral activity against SARS-CoV-2 257 infected VERO cells. CAPeo was effective at concentrations compatible with the expected circulating 258 concentrations of CAPeo constituents (Scalbert et al., 2002; Manach et al., 2005) , and similar with the 259 previously reported in vitro antiviral activity in Influenza strains and HRV14 (Tseliou et al., 2019). 260 Interestingly, as shown in Figure 1 , CAPeo mixture was both prophylactic and therapeutic in vitro, at 261 concentrations up to 1/100 the suggested per os dose in humans, preserving the viability, cell phenotype 262 and viral RNA presence in the culture medium. In this respect, calculated EC50 through a logistic fit was 263 estimated as 1/60 of the proposed dose for viral growth, and ~1/250 for cell phenotype, compatible with 264 the expected concentrations of CAPeo constituents in human plasma (Scalbert et al., 2002; Manach et al., 265 2005) . 266

CAPeo contains 25 different micro-constituents (please refer to Supplemental Table 4 of Reference 267 (Duijker et al., 2015) , for an exhaustive presentation of concentrations of specific constituents). The main 268 compounds are carvacrol (53%), eucalyptol (13%), β-Caryophyllene (3%), p-Cymene (1.32%), γ-Terpinene 269

(1.17%), Borneol (1.68%) and α-Terpineol (1.06%). As reviewed recently , none of 270 these compounds have been reported as anti-virals. However, work in progress in our group has identified 271 specific viral targets for some of these constituents, with a direct impact on viral replication. 272 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Based on the encouraging in vitro results, and having ensured the safety of CAPeo in experimental animals 273 (Kalyvianaki et al., 2020) and humans (Duijker et al., 2015) , we have further performed a Proof-of-Concept 274 intervention study in humans. Due to the very low incidence of COVID-19 positive cases in Crete, at the 275 time of the study, only seventeen (17) eligible ambulatory patients, positive for COVID-19 by real-time  276 quantitative PCR, were enrolled, and tested for the severity and duration of general and local symptoms, 277 for 14 days. We have chosen this interval as previous studies report a self-resolution of mild COVID-19 278 cases in 14 (Tenforde et al., 2020) , or 14-21 days (Allen et al., 2020) and the persistence of virus in upper 279 respiratory tract samples for about 10 days (Singanayagam et al., 2020) . A concrete limitation of this study 280 is that we do not have a control group; we have therefore compared the evolution of disease symptoms 281

with the few studies reporting the evolution of symptoms in non-hospitalized patients (Allen et al., 282 2020; Tenforde et al., 2020) . As discussed previously (Allen et al., 2020; Lechien et al., 2020) , symptoms 283 may vary significantly, related to the ethnicity of participants. At the beginning of the study (patient 284 consultation and positive real-time PCR result), the main general symptoms include headache, myalgia, 285 weakness and fever, in accord with previous investigations (Allen et al., 2020; Lechien et al., 2020; Tenforde 286 et al., 2020) . However, in our group, fever was low <37.5°C in all but one patient, and the frequency and 287 severity of other symptoms, such as gastrointestinal (diarrhea, respiratory and ENT symptoms were low, 288

possibly because of the low viral load, but without excluding the possibility of simply witnessing an effect 289 prevalent simply because of the small group size. 290 Symptoms evolution was compared to that reported symptoms at days 1 and 14, by (Tenforde et al., 2020) 291 and (Allen et al., 2020) , including a larger number of cases, in which symptoms were self-reported, daily, 292 but without the participation of a medical examiner. In our group, treated with CAPeo, we report a 293 complete resolution of headache, fatigue and fever, major general symptoms in COVID-19. In contrast, 294

headache, and fatigue persisted in the studies of (Allen et al., 2020; Tenforde et al., 2020) , while fever was 295 equally resolved. We have therefore concluded that CAPeo ameliorates general symptoms very quickly, 296

better than an untreated population. In addition, as shown in Figure 2B , the majority of symptoms, both 297 general and local, almost completely resolve at the end of the first week. Although we have not analyzed 298 in depth the underlying mechanism of action for this beneficial effect of CAPeo in the evolution of 19, in addition to the possible direct anti-viral effect reported in cells, another mechanism of action might 300 be its anti-inflammatory effect, previously reported in experimental animals (Kalyvianaki et al., 2020) and 301

humans (Duijker et al., 2015) . Anosmia and ageusia, however, evolved both in our group and in (Allen et 302 al., 2020) , presenting a maximum at day 4 and at days 4-6 respectively, and persist in 17%, 23% and 21% 303 in our group, in the study of (Tenforde et al., 2020) and in the population reported by (Allen et al., 2020) 304 respectively. Whether this is due to a late recovery of nasal and buccal mucosa, or in the persistence of 305

virus in a small percentage of patients (Singanayagam et al., 2020) is not clear yet. 306

In conclusion, our findings suggest that CAPeo, a mixture of essential oils of three Cretan aromatic plants, 307 possesses a potent antiviral activity, in addition to Influenza and HRV14 (Tseliou et al., 2019) , against SARS-308

CoV-2, in which it also possesses a prophylactic activity. In addition, our reported here proof-of-concept 309

intervention study in humans shows that it significantly reduces general and local symptoms of mild 310 COVID-19 patients. If these results will be confirmed in a planned prospective clinical study, CAPeo might 311 be a novel, inexpensive, therapeutic agent in cases of ambulatory COVID-19 patients. 312

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 15, 2021. ; . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.11.20248947 doi: medRxiv preprint Frequency of symptoms, common to both studies at Day 1, in our group and in (Tenforde et al., 2020) . B. 479

Heatmaps of symptom frequency in our CAPeo-treated group (left panel) and the population reported by 480 (Tenforde et al., 2020 ) (right panel). C. Evolution of selected symptoms in our CAPeo-treated group (red 481 curves). T1/2 for the resolution of symptoms was calculated with a logistic regression fit, with Origin Pro 482

2018. For comparison, the frequency of symptoms in the reference population reported by (Tenforde et 483 al., 2020) is also presented (green curves). In panels B-C, the frequency of symptoms was extracted from 484 Figure 1 of (Tenforde et al., 2020) , with the online resource WebPlotDigitizer (Rohatgi, 2020) . 485 486 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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