Impact of Doxycycline on COVID-19 Patients with Risk Factors: DYNAMIC, a Multi-Centre, Randomised, Placebo-Controlled, Double-Blind Trial

Background: The DYNAMIC study is based on three properties of tetracyclines. (1) Tetracyclines are known to chelate zinc from matrix metalloproteinases. It is possible their chelating activity may help inhibit COVID-19 infection by limiting its ability to replicate in the host. (2) As seen with dengue virus, tetracyclines may also be able to inhibit the replication of positive polarity single-stranded RNA viruses, such as COVID-19. (3) Tetracyclines are also modulators of innate immunity (anti-inammatory activity), a property that has been used to treat inammatory skin diseases for many years. They could therefore participate in limiting the cytokine storm induced by COVID-19. Moreover, the lipophilic nature of tetracyclines and their strong pulmonary penetration could allow them to inhibit viral replication at this level. Among the tetracyclines, doxycycline has three advantages: its long safety history (side effects are uncommon with no notable risks), its short treatment duration and its low cost. Methods: The trial will involve 330 patients who are positive for SARS-CoV-2 infection and have one or more risk factors for worsening the disease. These patients will be included in six hospitals covering the whole of France. For 14 days they will be given either 200mg of doxycycline a day or a placebo. Our hypothesis is a considerable reduction in the number of patients hospitalised due to COVID-19 thanks to the treatment of doxycycline. Discussion: This study could have an impact on general practitioners’ management of COVID-19 patients with risk factors prior to hospital admission. If kept at home under experimental treatment, these patients would help reduce the risk of spreading SARS-CoV-2 among the population. This treatment would therefore contribute to supporting the deconnement strategy by blocking the viral infection early and reducing the infectious period. The latest Chinese meta-analysis performed on 43 studies involving 3,600 patients provides an overview of the clinical characteristics, laboratory results, chest imaging results, disease severity and case fatality rate of COVID-19 patients [5]. The dominant clinical features of COVID-19, which can be present in a variety of combinations, are: fever, cough, asthenia, rhinorrhoea, headache, dysgeusia, dysosmia and diarrhoea [6,7]. The most frequently reported laboratory abnormalities are a decreased lymphocyte count, elevated C-reactive protein (CRP) and D-dimer levels and elevated lactate dehydrogenase [6]. The vast majority of patients with COVID-19 have a good prognosis, but there are critical situations and even deaths [8]. Most of these critically ill and deceased patients did not develop severe clinical manifestations in the early stages of the disease. Some of the patients only had a mild fever, cough or muscle pain, with their condition suddenly deteriorating in the latter stages of the disease. Acute Respiratory Distress Syndrome (ARDS) and multi-organ failure occur rapidly, resulting in death within a short time [9]. pandemic body mass index

plaques by disrupting the conformational changes in the viral envelope that are necessary for virus entry [26]. They have also shown that at normal human body temperature and fever conditions, doxycycline signi cantly inhibited the serine protease of the virus and a concentration-dependent decrease in viral replication was observed [27].
Most recently, on 14 April 2020, Bruno Pradines' team demonstrated the antiviral activity of doxycycline on SARS-CoV-2 in vitro [28]. In addition, tetracyclines are modulators of innate immunity (anti-in ammatory activity), a property that has been used to treat in ammatory skin diseases for many years. These modulatory effects are apparent on several targets of innate immunity. They can decrease the expression of NFB and the release of in ammatory cytokines such as TNF-α, IL-1β and IL-6, and inhibit the formation of in ammatory granulomas and the release of free radicals, independently of their antibiotic mechanism [19]. Furthermore, a recent publication has shown that species-independent coronaviruses induce mast cell proliferation in the respiratory submucosa, which in turn produces in ammatory agents such as histamine and protease, in addition to in ammatory cytokines such as IL-1 and IL-33 [29].
Two other studies have shown that chemically modi ed tetracyclines can induce mast cell apoptosis and activation of protein kinase C, thereby lowering the levels of in ammatory agents [30]. These teams have suggested that tetracyclines can be used to treat in ammatory disorders, including those induced by coronaviruses [29,30]. It should also be noted that, because of their anti-in ammatory capabilities, tetracyclines have also been documented to have in vitro activity on other viral infections such as HIV, West Nile virus and viral encephalitis [31]. Doxycycline may therefore contribute to limiting the cytokine storm induced by SARS-CoV-2. Lastly, the lipophilic nature of tetracyclines and their strong pulmonary penetration could enable them to inhibit viral replication directly at the in ammatory site.
The interest of doxycycline also lies in its long safety history (side effects are uncommon with no notable risks), its short treatment duration, which will be 14 days in our study (corresponding to the period during which SARS-CoV-2 can induce serious signs in the patient) and its low cost.
As already mentioned, it is often elderly patients with comorbidities who worsen during the second week of the disease and have to be hospitalised for respiratory deterioration or even put on mechanical ventilation. The French High Council for Public Health (HCSP) has determined the patients with risk factors [14] who have become the target population of our study in France. We have added obesity as a risk factor since it seems to be present in the literature for COVID-19-positive patients in intensive care units [32]. Our hypothesis is to offer this treatment as soon as a patient with a risk factor is con rmed as being COVID-19-positive by a PCR (polymerase chain reaction) test, with only a few symptoms, with no serious signs and before the onset of oxygen dependence, to reduce or even stop the cytokine storm, and thus the progression to a serious form of the disease, which will in turn avoid the need for hospitalisation.
The importance of nding medicines to keep patients at home and relieve hospital overcrowding is crucial during this pandemic. However, this outpatient trial will be carried out in hospital due to the required access to equipment and team facilities dedicated to clinical research.
Our study will be a multi-centre, randomised, placebo-controlled study to determine the e cacy of doxycycline in this context, by measuring the decrease in the number of patients hospitalised compared to the control arm.

Methods/design
Objectives The primary objective is to evaluate the decrease in the number of patients hospitalised after at least 48 hours of experimental treatment or requiring hospitalisation for COVID-19. The 48-hour delay allows a more objective attribution of the clinical course to advanced treatment. The embedded second primary objective is to evaluate the decrease in the number of patients requiring mechanical ventilator support.
The secondary objectives are to evaluate: -The decrease in the number of patients with a SARS-CoV-2-positive PCR test 7 days after the start of the experimental treatment.
-The decrease in the total length of hospital stay.
-The decrease in the length of hospitalisation in the intensive care unit.
-The decrease in the duration of mechanical ventilator support.
-The decrease in the rate of deaths related to SARS-CoV-2 infection.
-An assessment of the safety of doxycycline.

Outcomes
The main criterion is an e cacy criterion corresponding to the percentage of patients hospitalised after at least 48 hours of experimental treatment. The criterion corresponding to the embedded second primary objective corresponds to the percentage of patients requiring mechanical ventilator support. These three criteria are monitored in the two arms from 48 hours of treatment to 28 days corresponding to the end-of-study visit.
For the secondary outcomes: E cacy: These outcomes correspond to the report of the: -Total length of hospital stay in the intensive care unit.
-Duration of mechanical ventilator support.
-Number of deaths related to SARS-CoV-2 infection.
Safety: Report of the number of adverse events (AE) and serious adverse events (SAE) over 28 days.
Apart from the rst and the last secondary criteria, the other criteria will be recorded at 28 days. If the patient is hospitalised during the study period, this data will be recorded three months after the beginning of the treatment. For all of these secondary outcomes, percentages will be calculated.
For all of the outcomes, the data will be reported as percentages with con dence intervals and a p value.

Study design
The DYNAMIC study is a superiority study of the e cacy of doxycycline. It is a prospective, phase III, randomised, double-blind, placebo-controlled, multi-centre national study to evaluate the use of doxycycline in patients with at least one risk factor for worsening COVID-19.
As the study is double-blind, the study treatment and its placebo will be managed by the pharmacist of the coordinating centre, CHU Nantes. The oral antiin ammatory dose of 200 mg of doxycycline or the matching placebo will be taken once a day in the evening one hour before going to bed, for 14 days. It should be noted that the placebo being used is marketed by COOPER; it is made of lactose and is very similar to the doxycycline tablet. The treatment (doxycycline or placebo) will be dispensed at the inclusion/randomisation visit. No dose adjustment is required. The treatment will end if the patient is hospitalised between day 0 (D0) and day 14 (D14).
Six French university hospitals: CHU Nantes, AP-HP [Assistance Publique -Hôpitaux de Paris; Hôpital Avicenne], CHU Bordeaux, CHU Caen, CHU Dijon and CHU Grenoble, will include 330 patients to provide at least 280 analysable patients. The inclusion period may be three to six months depending on the recruitment rate. Advertising, approved by the ethics committee, will be displayed to improve the recruitment rate. The treatment period is 14 days per patient and the last patient visit will be at 28 days (+/-2 days) from the start of treatment.

Randomisation
Randomisation will be strati ed by centre and by severity factors (1 versus ≥2). It will be performed according to a 1:1 ratio and balanced by blocks; the block size was random. The random numbers will be generated by computer. The software used to collect the data from the electronic report form allocated the patients automatically. Randomisation was performed according to the IWRS (Interactive Web Response System).
Subjects are randomised into blocks as the allocation progresses, with a block being a sub-group of a predetermined size which contains a random allocation of patients. The software used for the randomisation is R version 9.4. An email alert will notify the investigator's team and the pharmacist that a patient has been randomised. For the pharmacist, the allocation will be revealed. The randomisation key is known only to the biostatistician and the data managers, to make it impossible for the investigator to assign a particular treatment. It should be noted that the biostatistician who carries out the randomisation is different to the biostatistician who will perform the statistical analysis.

Study population
The study population is made up of male or female patients over 45 years of age, suspected of having COVID-19 by their treating physician or by a physician from a COVID-19 unit at a participating centre, with one or more risk factors for worsening the disease. They may be included in the study after a positive SARS-CoV-2 PCR test performed at the hospital. We chose 45 years as the minimum age in this study because this is the low end of the range for COVID-19positive patients likely to be admitted to intensive care units [33]. In order to be eligible, these patients must also have one or more risk factors for worsening the disease as described by the French High Council for Public Health [14]: 70 years of age or older, cardiovascular history (stroke, coronary artery disease, complicated hypertension, cardiac surgery, NYHA class III or IV heart failure), poorly controlled insulin-dependent diabetes or complicated secondary diabetes, respiratory disease likely to undergo decompensation due to viral infection, patients with chronic renal failure on dialysis, and cancer patients under treatment. We have also added BMI (BMI>30) as a risk criterion, based on recent data from the literature [10][11][12][13]. Exclusion criteria are vulnerable populations and contraindication of the use of doxycycline. Box 1 presents all of the inclusion/exclusion criteria.
Furthermore, patients cannot be included in any other interventional research, unless they are hospitalised, and provided there is no drug interaction with any other experimental treatment. Patients who are hospitalised will be considered as a failed patient for the primary objective. These patients will then be followed up only for the management of this SAE. If hospitalisation occurs between D2 and D7, the PCR will not be performed at D7 and the corresponding objective will not be applied to this patient.

Study schedule
The plan for the study described in this section is presented in Figure 1 and the owchart in Figure 2. The screening is performed by COVID-19 emergency facilities or the COVID-19 unit of the investigating centre. To increase the recruitment rate, advertisements will be displayed in GP waiting rooms.
A 45-year-old-patient suspected of having COVID-19, with one or more characteristic clinical signs, and having one or more risk factors for worsening the disease is sent to hospital for a SARS-CoV-2 test. In the waiting room of the department specially dedicated to SARS-CoV-2 PCRs, a poster presenting DYNAMIC will be displayed. If the PCR is positive, either the patient who has seen the poster will contact the DYNAMIC team or the investigation team will contact the patient because of the PCR result. The hospital investigator explains the DYNAMIC study to the patient orally, checks the eligibility criteria, provides information on DYNAMIC and obtains the patient's signed informed consent. If the version of the informed consent changes during the patient's participation in the study, they will be informed of this immediately during the telephone calls held during the trial, and the new consent will be signed at the next consultation. The Supplementary Material le contains the French informed consent form that patients sign prior to their inclusion in the trial (the updated protocol is version 1.8 as of 28 October 2020). Furthermore, blood samples for serology and immunological markers will be collected, stored and kept if the patient signed the biocollection informed consent. This informed consent form states that the samples may be used for scienti c research. This biocollection and its consent procedure have been registered with French ethics committee CPP (Comité de protection des Personnes) Ouest IV under number DC-2011-1399. After a clinical examination, patients will be randomised either in the doxycycline arm or in the placebo arm. The patients, the investigators and their teams will be blind and will not know the assigned treatment. As Karanicolas et al. pointed out in their article, the biostatisticians will also be blinded until the analyses are performed to reduce the study bias [34].
On D3 and D14, a telephone call will be made by the investigator or their team. A clinical assessment of the patient will be carried out using the questionnaire shown in Figure 3.
The patient will answer daily this questionnaire and if their answers indicate warning signs they will call the investigators or the emergency department. If necessary, a face-to-face consultation will be conducted immediately after this call to accurately assess the extent to which the disease has worsened.
On D7, a face-to-face follow-up visit will be performed. After a clinical examination, a SARS-CoV-2 qualitative PCR test will be carried out. On D28, the face-toface end-of-study visit will be performed. A blood sample will be kept for serology and other immunological markers. The patient will return any unused therapeutic units and the empty blister packs.
An early end-of-study visit will be carried out corresponding to the end-of-study visit (D28 visit), with the exception of serology if the patient leaves the study before 14 days. For included patients hospitalised before 28 days, the experimental treatment is stopped as soon as they are hospitalised and they are considered a failure. Clinical data related to their hospitalisation will be reported in the CRF. For patients hospitalised after 28 days and up to 90 days posttreatment, they will be followed up until discharged. This follow-up will include the collection of vigilance data and various hospitalisation data (date of end of hospitalisation, length of hospitalisation in an intensive care unit, duration of mechanical respiratory support, possible death).
During the trial, any drug judged necessary for the patient's well-being, which should not interfere with the evaluation of the test drug, may be administered at the investigator's discretion. Absorption of doxycycline is impaired by antacids containing aluminium and calcium, as well as magnesium, bismuth subsalicylate and iron-and zinc-containing preparations. The study drug should therefore be taken at a different time to antacids. Other prohibited treatments are those prohibited by the Summary of Product Characteristics of doxycycline and are listed in the exclusion criteria (see Box 1). However, in the absence of a change to the therapeutic management of a patient arriving at hospital according to their treatment arm, a blinding procedure is not required in this protocol.
Treatment will be stopped if hospitalisation occurs.

Statistical methods
The embedded testing strategy will be used to successively test: -The decrease in the percentage of patients requiring hospitalisation related to SARS-CoV-2 infection after at least 48 hours of treatment.
-A decrease in the percentage of patients requiring mechanical ventilator support if the rst null hypothesis is rejected.
An intermediate futility analysis will be carried out after 100 patients have been evaluated, with the objective of stopping the clinical trial if there is no probability that the null hypothesis will be rejected at the end of the full study. The sole response to this analysis is whether or not to stop the trial because of a lack of e cacy. The Peto method will be used with an alpha risk of 1 per 1,000 at the intermediate analysis and of 5% at the nal analysis [35]. As already quoted, the biostatistician will be part of the blinded team. They will give the results of this intermediate analysis to the Data and Safety Monitoring Committee (DSMC), which may or may not unblind depending on the results. The analysis will be strati ed by centre and number of severity factors (1 versus ≥ 2). The severity factors are ≥ 70 years, BMI > 30, cardiovascular history (stroke, coronary artery disease, complicated hypertension, cardiac surgery, NYHA class III or IV heart failure), poorly controlled insulin-dependent diabetes or complicated secondary diabetes, respiratory disease likely to undergo decompensation due to viral infection, patients with chronic renal failure on dialysis, and cancer patients under treatment at the time of inclusion.
A modi ed intention-to-treat analysis will be applied. It will include all eligible randomised to participate in the trial. It will exclude patients who withdrew their consent prior to the assessment of the rst primary endpoint, randomised patients who were incorrectly included (inclusion or exclusion criteria not met) and patients who did not receive any dose of treatment.
A per-protocol analysis will also be performed, including all patients evaluated in their treatment group who received more than 75% of the total treatment dose.
For the main criteria (percentage of patients), we will use a Mantel-Haenszel test to compare strati ed percentages across centres and the number of risk factors [36,37]. For the quantitative secondary criteria, a mixed linear model will be used to take into account the risk factors and the centre effect (random Sample size It is estimated that 25% of the eligible patients in the study will require hospitalisation. In the rst wave, hospitalisation rates ranged from 18.1% [38] to 31.4% [39], we chose the mean. It is hypothesised that with doxycycline treatment, this rate would decrease to 12%. Indeed, the article concerning the effect of doxycycline on dengue patients: "Dengue Patients Treated With Doxycycline Showed Lower Mortality Associated with a Reduction in IL-6 and TNF Levels" [40], shows that the group on doxycycline had a lower mortality rate than those in the untreated group (11.2% [13/116] vs 20.9% [24/115], respectively, p=0.05). In addition, doxycycline administration resulted in a signi cant drop in IL-6 and TNF. These results therefore con rm our hypothesis of reducing the hospitalisation rate to 12%.
An embedded testing strategy is used for the primary objective, and as the EMA report stated, no adjustment is needed for multiplicity [41].
For the second embedded hypothesis, it is estimated that 23% (source: https://www.data.gouv.fr/fr/datasets/donnees-hospitalieres-relatives-a-lepidemie-decovid-19/) of hospitalised patients are admitted to intensive care units. With the recruitment level we have, we will have a power of 80% to show a decrease from 23% to 10%.
Under these hypotheses, with a 5% alpha risk in a bilateral situation and a power of 80%, we would need to evaluate a total of 280 patients in both arms. If the rst hypothesis is rejected, the power of the test of the second hypothesis would then be 83.5%. Three hundred and thirty (330) patients will be included to compensate for study dropouts and non-assessable patients.
There have been no reports of overdose. Adverse events reported for other tetracyclines following renal impairment (hepatic toxicity, hyperazotemia, hyperphosphataemia, acidosis), are unlikely to occur with doxycycline due to the lack of a change in blood levels in relation to the functional value of the kidney. For the placebo, the main expected effects are digestive disorders (excess gas, feeling of abdominal bloating, abdominal cramps and pain, and diarrhoea). As regards the pathology, patients with an uncomplicated viral infection of the upper respiratory tract may have non-speci c symptoms such as fever, fatigue, cough (with or without discharge), anorexia, malaise, muscle aches, sore throat, dyspnoea, nasal congestion or headache. Rarely, patients may also experience diarrhoea, nausea and vomiting [6,42], and loss of taste or smell [43]. The disease can worsen to pneumonia, acute respiratory distress syndrome [44], sepsis and septic shock [45] .
All SAEs, whether expected or unexpected, require the completion of an SAE report. The investigator must ensure that the information entered in this report is accurate and clear. The SAE should be reported to the sponsor immediately (within 24 hours of being highlighted by the investigator). After receiving an unexpected SAE report, the sponsor will notify the authorities. Furthermore, a DSMC has been set up; it is a consultative committee responsible for reviewing the safety of a study on behalf of the study sponsor and coordinator/principal investigator. Members of the committee who are competent in the eld of clinical trials (pathology methodology and pharmacovigilance) are not involved in the study.
The DSMC is a referral point for pharmacovigilance if a suspected unexpected serious adverse reaction (SUSAR) or SAE poses particular analytical di culty, or if a doubt arises concerning the risk/bene t of the study. It will also make a decision on the outcome of the futility analysis. If the study is ended early following a decision of the DSMC or the study sponsor, the regulatory authorities and the ethics committee will be informed by post within a maximum of 15 days. In any event, written con rmation will be sent to the coordinating investigator of the study (specifying the reasons for early termination) and to the principal investigator of each centre, if applicable. All patients involved in the study will be informed and will be required to attend their early discharge visit.

Ethical, regulatory and dissemination aspects
The clinical study will be conducted in accordance with the relevant versions of the French Public Health Code, national and international good clinical practice guidelines, and the Declaration of Helsinki.
In accordance with French law, the study protocol was submitted to French regulatory authority the ANSM. This clinical study was submitted to and approved by the ethics committee of Boulogne-Billancourt (CPP Ile de France VIII) on 13 May 2020. Requests for substantial modi cations should be addressed by the As required, the sponsor has provided an insurance policy to cover the nancial consequences of its civil liability in accordance with the regulations. This protocol was created thanks to a Scienti c Committee. The Scienti c Committee is coordinated by Professor B Dréno and its members include external and internal experts in COVID-19, a general practitioner, experts in clinical trials and a methodologist. An inspection or audit may take place as part of this study, performed by the sponsor and/or the regulatory authorities. Inspectors will check the documents, logistics, records and any other resources the authorities consider to be associated with the clinical trial and that may be located at the trial site itself.
The trial results will be published in international, medical and scienti c journals and presented at national and international conferences. The investigators will follow the rules and guidelines of the International Committee of Medical Journal Editors [46]. In practice, the Scienti c Committee will be among the authors of the publication, as will the investigators who included the patients in the trial.

Discussion
The COVID-19 pandemic is affecting all continents. Saturation of the hospital care system is feared in many countries, and the rst and the second lockdown of the French population and practically the whole world was decided in an attempt to avoid hospital overcrowding.
Researchers are looking for a life-saving drug. To date, 3,904 clinical studies have been published at https://clinicaltrials.gov/ (viewed on 13 November 2020) since the beginning of the pandemic. Of these studies, 2,217 are intervention research. However, there are only 113 studies that deal with outpatient clinical research. The great strength of our study is that it tests doxycycline on at-risk patients on an outpatient basis before the cytokine wave. Our target is therefore the early onset of the viral infection and it involves very close work with general practitioners. As the French National Academy of Medicine has indicated, the mobilisation of general practitioners during the rst months of 2020 prevented hospital emergency departments from being overwhelmed by requests for rstline treatment, thus con rming the complementarity of private and public sector resources [47]. Now, a lockdown easing strategy is in place and its effectiveness will mainly be supported by general practitioners. They are the key players in the strategy for detecting new cases of COVID-19 and thus play a crucial role in our study.
In its "Clinical management of severe acute respiratory infection (SARI) when COVID-19 disease is suspected" guidance document, WHO proposes that elderly patients and those with comorbidities such as cardiovascular disease and type II diabetes should be hospitalised even if they do not show signs of severity. By avoiding hospitalisation, the impact of our treatment could be to relieve congestion by allowing healthcare personnel to focus on serious cases and freeing up resuscitation places. Moreover, this will prevent the need to cancel at least 20% of their usual activity in surgery and medicine, which is currently the case in French hospitals in autumn 2020.
This experimental treatment could support the decon nement strategy by blocking the viral infection early in cases of infection, by reducing the infectious period, and by making home care possible for infected patients with a treatment whose tolerance has been proven for nearly sixty years, even in elderly patients.
The medico-economic interest is also very important. The treatment is inexpensive at around 3 euros per 15 tablets, and we hope to avoid the transfer of patients to other intensive care units, which are very expensive for the community.
Recently, in Italy, Bonzano et al. reported the bene t of doxycycline in patients with COVID-19 [48]. Their retrospective study, albeit on only six patients, suggests promising results that our randomised clinical trial will be able to strengthen by removing the bias of a retrospective study on a few cases.
Furthermore, Yates et al. have presented a series of four high-risk, symptomatic, COVID-19-positive patients with known pulmonary disease, treated with doxycycline with subsequent rapid clinical improvement.
Doxycycline is an attractive candidate. If the results are as positive as we hope, this treatment could be given to the entire population, potentially for prevention of the disease.

Trial status
The updated protocol is version 1.8 as of 28 October 2020.
The rst patient inclusion is expected by 1 December 2020. With an inclusion period of six months, the last patient may be included on 1 May 2021 and the study will end after their follow-up visits one month later.

ARS
Agence régionale de Santé All patients participating in the study will be given oral and written information about this trial and will sign the informed consent form.
An independent ethics committee, the Comité de Protection des Personnes of Boulogne-Billancourt, CPP Ile de France VIII, issued a favourable opinion for this clinical trial and gave its informed consent on 28 April 2020.

Consent for publication
The Supplementary Material le contains the French informed consent form, and information materials are available from the corresponding author on request.

Availability of data and materials
Any data required to support the protocol can be supplied on request. The data from the completed trial will not be shared and will only be transmitted to the sponsor. Data collected during the test may be processed electronically in accordance with the requirements of the CNIL (compliance with French Reference Methodology MR001).

Declaration of competing interests
The authors declare that they have no competing interests. Flowchart Study