Drug Repositioning of Protease Inhibitors in Combinational Therapy As a Treatment for COVID-19

Drug repositioning of protease inhibitors in combinational therapy as a treatment for COVID-19

Max Malmberg

Bachelor project in pharmacology, 15 hp, Spring 2021

Examinator: John Sedin Division of Pharmacology Department of Pharmaceutical biosciences Faculty of Pharmacy Uppsala University

Abstract There is currently no standardized treatment of COVID-19. Despite several vaccines being deployed, the need for an effective treatment in hospitalized patients is heavily sought after. In this systematic review, six clinical trials were analyzed for their findings on the effect of commercially available anti-viral protease inhibitors in treatment of COVID-19. The aim of the study was to evaluate the effect on the hospitalization period and mortality of patients suffering from COVID-19 and if HIV protease inhibitors could be repositioned to treat COVID-19 in combinational therapy. The findings suggest that protease inhibitors targeting the hepatitis C virus (HCV) proteases could potentially be effective at reducing the hospitalization period and that the effect can be further enhanced by using them in combination with drugs targeting the RNA polymerases. Further studies are needed confirm these findings. Lopinavir/ritonavir which was the most common protease inhibitor included in the study did not reduce the hospitalization period or the mortality significantly. It is unclear whether the

HCV protease inhibitors could reduce mortality. Further studies regarding this outcome are warranted.

Table of contents

BACKGROUND...... 4

SARS-COV-2 ...... 4 THE VIROLOGY OF SARS-COV-2 ...... 4 VACCINES ...... 5 DRUG RE-POSITIONING ...... 5 EARLIER EXAMPLES OF RE-POSITIONED DRUGS USED TO TREAT COVID-19 ...... 6 CURRENT MEDICAL TREATMENT ...... 6 ANTI-VIRAL MEDICATION ...... 7 Cell Entry inhibitors ...... 7 Polymerase inhibitors ...... 7 Interferons ...... 7 Protease inhibitors ...... 7 MEASURES OF CLINICAL EFFICACY ...... 8 AIM...... 9 METHOD ...... 9 RESULTS ...... 13

FIRST CLINICAL STUDY USING HCV PROTEASE INHIBITOR DANOPREVIR TO TREAT COVID-19 PATIENTS (47)...... 13 A TRIAL OF LOPINAVIR-RITONAVIR IN ADULTS HOSPITALIZED WITH SEVERE COVID-19 (48)...... 14 TRIPLE COMBINATION OF INTERFERON BETA-1B, LOPINAVIR-RITONAVIR, AND RIBAVIRIN IN THE TREATMENT OF PATIENTS ADMITTED TO THE HOSPITAL WITH COVID-19: AN OPEN-LABEL, RANDOMIZED, PHASE 2 TRIAL (49)...... 15 INTERFERON Β-1B IN TREATMENT OF SEVERE COVID-19: A RANDOMIZED CLINICAL TRIAL (50)...... 16 THE EFFECTS OF ARBIDOL (UMIFENOVIR) ON COVID-19:A RANDOMIZED CONTROLLED TRIAL (51)...... 17 SOFOSBUVIR AND DACLATASVIR COMPARED WITH STANDARD OF CARE IN THE TREATMENT OF PATIENTS ADMITTED TO HOSPITAL WITH MODERATE OR SEVERE CORONAVIRUS INFECTION (COVID-19):A RANDOMIZED CONTROLLED TRIAL (52)...... 18 DISCUSSION ...... 19

COMBINATIONAL TREATMENTS ...... 19 STANDARD TREATMENT OF CARE ...... 20 STUDY DESIGN ...... 20 THE UNKNOWNS OF COVID-19, FUTURE STUDIES ...... 22 OTHER STUDIES ...... 22 CONCLUSION ...... 23 POPULÄRVETENSKAPLIG SAMMANFATTNING ...... 24 REFERENCES ...... 25

Background On the 30th of December 2019, a report of “viral pneumonia” was issued by the Wuhan municipal health commission in People’s Republic of China (1). Unbeknownst to the world this was the beginning of a global pandemic that as of this writing has claimed over 3.4 million lives (2).

SARS-CoV-2 SARS-CoV-2 is a single-stranded RNA virus that causes the disease COVID-19 (3, 4). SARS-CoV-2 spreads through droplets released via coughing, but contaminated surfaces can also lead to an infection provided that the virions come in contact with a mucous membrane (3). SARS-CoV-2 is very similar to the previous coronavirus that caused an outbreak in 2002 (4). SARS-CoV-2 is part of a zoonotic viral family named corona viruses that normally cause mild symptoms in mammals as well as birds (5). SARS-CoV-2 is not the first virus in this family to be troublesome and other strains such as MERS-CoV have led to deadly outbreaks in the past (6). It is therefore not unimaginable to assume new infectious strains will appear in the future.

The virology of SARS-CoV-2 Because treatment of COVID-19 is currently limited to treating the symptoms of the disease it is important to understand how and why certain symptoms appear. After inhalation of droplets containing SARS-CoV-2, the virions incorporate themselves into type 1 & 2 pneumocytes. The pneumocytes express a high amount of angiotensin converting enzyme type 2 (ACE-2) which act as binding sites for the spike proteins on the virus (7–9). The ACE-2 enzymes are engulfed back into the cell and the virus then hijacks the cells ribosomes to start translation of their single-stranded RNA into at least 27 different proteins (4, 5). These proteins are cut by proteases, two of which are of extra importance, papain-like protease, PLpro and chymotrypsin-like protease, 3CLpro who are responsible for most of the polypeptide cleaving (10, 11). Important parts that are cut by these proteases include the RNA-dependent RNA polymerase, of which there are multiple and the helicase (11). The proteins are cut into 4 structural proteins (Spike S, envelope E, membrane M and nucleocapsid N) and 8 auxiliary proteins. One of them, the S-glycoprotein is responsible for the binding to the ACE-2 enzyme like a ligand. The S-glycoprotein therefore crucial for the replication of the virus (12). The function of the N protein is the encapsidation of the viral ssRNA, while the E and M proteins ensures that the mRNA is incorporated into new virions, ready to be released into the body (11). The function of the auxiliary proteins are not fully understood but some seem to modulates host response to infection (11). When the virions release, the cell bursts, different kinds of inflammatory mediators, mainly Interleukin-1 (IL-1), Interleukin-6 (IL-6) and Tumor Necrosis Factor alfa (TNF- α) are now present in the plasma which causes an inflammatory response and fever. This leads to vasodilation causing plasma to leak into the interstitial space as well as into the alveoli causing a decreased gas exchange and in some cases a collapse of the

alveoli. This leads to shortness of breath, heavier breathing, and an increased breathing frequency in response to the bodies reduced oxygen supply. When the immune system fights a viral infection, it does so by destroying infected cells. If enough cells break down their remnants may form a thick sludge which is then coughed up. Over time, if the body fails to fight off the virus, septic shock, caused by decreased blow flow to the internal organs may set in (5, 13–15). The severity of symptoms differs depending on the age of the infected. Young people tend to develop less severe symptoms than elders who present more atypical symptoms such as confusion and kidney failure (16). A observational study indicated oxygen saturation level below 93% on hospital admission was predictive of death in older patients (17). First symptoms appear after roughly 5 days, for people that develop severe symptoms, death can happen in as short as 6 days with a median time span of 14 days (18). Early studies showed SARS-CoV-2 to have a mortality rate between 11-15%, a later meta- analysis showed numbers in the low 5% range (19–21). These differences could be explained by differences in study populations.

Vaccines There are several vaccines being deployed to counter the spread of SARS-CoV-2, but these vaccines have several flaws that needs to be addressed. Vaccines are prophylactic, therefore cannot be used to treat an ongoing infection. Vaccines are specific which means that mutations in the virus will inevitably lead to the vaccine becoming ineffective. The mutations are bound to happen due to the high virulence of the virus (22–26). Vaccines often needs to be stored differently than other medications, as is the case with the Pfizer-BioNtech vaccine that must be stored in very low temperatures (27). This does pose a logistical problem in countries with less developed infrastructure. Furthermore, not everyone can be vaccinated due to medical reasons such as previous vaccinations causing severe allergic reactions or patients being immunocompromised. Due to the nature of viruses, new mutations will inevitably arise which may make current vaccines ineffective. The implication of this is that no matter how good a vaccine is, there will always be a need for an effective treatment method. Finally, vaccines are not 100% effective which further cements the fact that effective treatment must be made available (28).

Drug re-positioning The cost of time and resources involved in researching and deploying new drugs is high, it is therefore important to consider already established drugs for new indications. The term “drug re-positioning” refers to the method of finding and re-purposing old drugs, and/or drug candidates which may be used to treat illnesses they were not originally designed for (29). One of the most famous examples of drug re-positioning is sildenafil which was designed to treat hypertension but is today known for its ability to treat erectile dysfunction (29). Drug re-positioning is an important factor when discussing ongoing and time sensitive problems such as the ongoing corona pandemic. By identifying antivirals that may be

used to treat COVID-19 we can reduce economical loss as well as loss of lives that is associated with the disease.

Earlier examples of re-positioned drugs used to treat COVID-19 There are a couple of drugs that have already been re-purposed for treatment of COVID-19, among them is the earlier mentioned remdesivir (30) originally designed to treat a number of emerging viral diseases such as Ebola and Marburg disease (31). Remdesivir is now used in the united states for treatment of COVID-19 patients that need extra oxygen but have no need for mechanical respiration (32). Early drugs being proposed for drug re-positioning was hydroxychloroquine and ritonavir/lopinavir but was found by the World Health Organization (WHO) to be ineffective as of July 2020 (33). Because most of the protease inhibitors used today are not used in monotherapy, this study will primarily look towards the use of protease inhibitors in combinational therapy.

Current medical treatment As there are no internationally defined standard of care (SoC) for COVID-19, different countries may treat the disease differently. In the United States, patients do not receive any medication until hospitalization is needed (32). For patients receiving supplemental oxygen, remdesivir and dexamethasone are recommended treatment options (32). The treatment procedure for COVID-19 usually consists of dexamethasone, remdesivir and hydroxychloroquine along with a protease inhibitor with a CYP3A4 inhibitor (lopinavir/ritonavir or darunavir/atazanavir+ritonavir). Although lopinavir/ritonavir and hydroxychloroquine have been declared ineffective for use in monotherapy by WHO as of July 20 2020 (33). Remdesivir is a nucleoside analogue that imitates adenosintriphosphate (ATP), an important molecule for energy metabolism. By breaking the early RNA-chains in the virus, replication cannot continue (30). Remdesivir has shown severe kidney toxicity in apes and is therefore not recommended to patients with eGFR < 30ml/min (30). This poses a problem as older people are more severely affected by COVID-19 than young people who on average have a higher degree of kidney function (34, 35). Dexamethasone is a glucocorticoid steroid that suppresses the body’s inflammatory response (36) and has proven to be highly effective in reducing the mortality in COVID-19 patients (37). Despite this, dexamethasone alone is inadequate in treating COVID-19 and therefore only serves an important part of the treatment. Hydroxychloroquine is a Disease-modifying antirheumatic drug (DMARD) that is used in the treatment of malaria, it acts by disrupting the immune systems signaling and cytokine production (38). This drug has the same area of use as dexamethasone but has since it use began to show to be ineffective and possibly counter-productive for use as it may inhibit the effects of remdesivir (30, 39).

Anti-viral medication All anti-viral medication work by inhibiting the viral replication (40), albeit in different ways. Anti-viral drugs are often used in combinational treatment. The reason for this is often the use of synergistic effects, but it also relates to the virus’s ability to quickly mutate. By using multiple attack vectors, the longevity of anti-viral drugs can be prolonged, at least in theory. To understand how and why protease inhibitors are important for treating viral diseases, a short explanation of what mechanisms of action antiviral medication utilize is warranted along with their downsides.

Cell Entry inhibitors For a virus to replicate it must first enter the cell, this is often facilitated via cell membrane proteins. By inhibiting a virus ability to enter the cell the infection can be slowed down. Neuraminidases are viral transmembrane enzymes that facilitates the entry into a host cell, allowing RNA to pass the cell membrane (40). The currently available neuraminidase inhibitors are designed to treat influenza-viruses which belong to a different family than SARS-CoV-2. There are several other cell entry inhibitors indicated for other viruses like HIV, Hepatitis B and D. However, as cell entry inhibitors are inherently most effective in the early stages of an infection and as the symptoms of COVID-19 can appear already 9 days post-infection, the effectiveness of cell entry inhibitors might be limited (5, 40).

Polymerase inhibitors During the SARS-CoV-2 replication process, the RNA-dependent RNA-polymerase converts viral RNA into parts to form new virions. By introducing “broken” building blocks that incorporates into the forming polypeptide chain the replication is terminated which in turn slows down the rate of replication (13, 40). Each family of viruses has different polymerases, and depending on the type of virus (DNA, RNA, single-stranded or double-stranded) they can very so much that no single polymerase inhibitor works on all of them. Despite this, the mechanism of action is largely the same and therefore make interesting candidates for repositioning.

Interferons Interferons are a family of proteins that has the function of modulating cell growth and immune function (40). The mechanism which by the immunomodulating interferons act is by binding to ganglioside receptors expressed on the cell membrane causing the production of enzymes which stops viral mRNA translation (40). Side effects are common and may mimic the symptoms of influenza, worsening the general health of a patient.

Protease inhibitors At the end of viral replication, polypeptides are assembled into complete virions. This assembly is carried out by the viral protease. The viral proteases are unique to the virus

and drugs targeting the protease are therefore less likely to be toxic. Three common protease inhibitors, all of which are indicated for HIV treatment, darunavir, lopinavir and ritonavir all work by binding to protease and inactivating their active site. Ritonavir differs in that is also inhibits cytochrome P450 3A enzymes and is therefore often used as a pharmacokinetic booster to increase the effect of other antiviral drugs that are broken down by the above-mentioned enzyme system (40, 41). Not all protease inhibitors are indicated for HIV, some newer drugs also target the hepatite C virus protease NS3/4A protease, although these are a relatively new addition to the drug group (42). Protease inhibitors are mainly used in combinational therapy and serves as an important part of the synergistic cocktail that is HIV-treatment (40). Protease inhibitors do have some problematic side-effects, mainly gastro-intestinal problems such as nausea and vomiting. Combined with the use of pharmacokinetic boosters causing many pharmacological interactions (40), protease inhibitors should be used carefully. Despite this, their specificity does lend them to make a viable component in combinational therapy due to their synergistic effects which enables lower dosage minimizing unwanted side effects. Several protease inhibitors has shown ligand affinity for the SARS-CoV-2 protease in vitro (43). This study looks at the effects of protease inhibitors designed to treat HIV. The Federal Drug Agency FDA of the United States currently has 10 substances approved for use, these are Amprenavir, Atazanavir, Darunavir, Fosamprenavir, Indinavir, Lopinavir, Nelfinavir, Ritonavir, Saquinavir and Tipranavir (44). Two of these compounds, amprenavir and nelfinavir are not available on the EU market (45, 46).

Measures of clinical efficacy When trying to validate if a certain drug works or not, a decision must be made on what study outcome to look at. A good outcome must be able to give insight into the aim of a study. When it comes to treating COVID-19, two outcomes are highly relevant. Firstly, the mortality rate after the intervention of a new drug. Secondly, the time spent in hospital. The mortality rate has some benefits as an outcome. Though it might seem dire, COVID-19 is a deadly disease and a crude measurement like mortality rate is therefore a good indicator of the efficacy of a certain drug combination. It is also easy to measure, this is very important as this study tries to analyze a variety of clinical trials which may look very different. The hospitalization period also has benefits as an outcome, like mortality rate, it is also easy to measure. It tells us how quickly an individual on average, may recover from COVID-19, giving us insight into the efficacy of a drug combination.

Aim The aim of this study is to investigate if FDA approved HIV protease inhibitors could be used to treat COVID-19 in combinational therapy. - Can FDA approved HIV protease inhibitors be used in combinational therapy to reduce the mortality compared to the standard treatment of COVID-19? - Can FDA approved HIV protease inhibitors be used in combinational therapy to reduce the hospitalization period compared to the standard treatment of patients with COVID-19?

Method This study was conducted by performing a systemic review. Articles were collected from PubMed. The basic criterions for an article to be included in the systematic review was that they were written in English, were peer-reviewed and was not a duplicate of an earlier collected article. Specific criterions were that an article had to contain at least one of the outcomes, mortality rate and/or the hospitalization period. The article had to be a clinical trial and be available in free full text; this was done by using the search filters in PubMed. The articles selected had to try to treat COVID-19, this was important, because these drugs are not originally designed to treat COVID-19. The protease inhibitors included had to be commercially available, that is, already on the market in some country as a licensed drug. The protease inhibitor had to be used in combinational treatment, but not in studies comparing it to vaccines. Finally, a potential article had to validate SARS-CoV-2 infection via a PCR-test. A comprehensive list of the criterions can be found in Table 1. Articles had to include FDA approved protease inhibitors targeting the HIV protease. The following substances were used both as search phrases and in the selection process: amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir. The full list of search phrases, along with the date on which they were conducted, and their yields were compiled into a table which can be seen in Table 2. To efficiently analyze which articles were to be included, a system to quickly sift through search results had to be used. The selection process was done in three parts, Selection 1 through 3. Selection 1 sought to include articles with titles that had at least one of the inclusion criteria while not being excluded. Duplicates were removed before being included in Selection 1. Selection 2 sifted through the abstracts of articles from Selection 1. Finally, articles in Selection 3 were read in full text, and if they fulfilled all the criterions were included in the study. This process is illustrated in Figure 1. A total of 9 articles was identified that filled some of the listed criteria based on their title. After going through the abstracts and later the full text, only 6 articles were included in the study. This study has two outcomes: 1. Hospitalization period, defined as the time spent in hospital until discharge. The qualification for a patient to be discharged from the hospital may be different on a country per country basis, no weight is therefore put on the qualifications. Rather, the study only looked at discharge as-is.

2. Mortality. Different countries may define deaths by COVID-19 differently, in order to be able to make a comparison between the different studies, this discrepancy must be considered when interpreting the statistics. Results were presented on an article-by-article basis, this was because the composition of drugs used in each article compares one or multiple combinational therapies with either the standard treatment in or another combinational treatment. It was therefore difficult to assess and to establish a conclusion without a clear separation of which drugs were compared to what. Furthermore, the size of the study population differs to such an extent that some articles were not comparable to each other.

(Table 1. A list of criteria used to include or exclude articles) Inclusion criterions Exclusion criterions Article written in English Duplicate of article from earlier search Peer-reviewed Vaccine comparison One of the outcomes must be looking at mortality and/or hospitalization period Clinical trial Available in free full text Treatment of COVID-19 Protease inhibitor must be commercially available Protease inhibitor must FDA approved Protease inhibitor must be in a combinational treatment Protease inhibitor must be included in study Confirmed SARS-CoV-2 infection through PCR. Protease inhibitor must be indicated for HIV

(Figure 1. A flow chart describing the selection process).

(Table 2. Display of how many articles were retrieved from a specific search phrase in PubMed along with the total amount of articles retrieved to be included in the study). Date Search Phrase # of Selection Selection Selection articles 1 2 3 2021-04-13 Protease inhibitor 29 6 5 4 AND COVID-19 2021-04-16 Covid-19 AND 103 1 - - (combinational therapy OR protease inhibitor) 2021-04-26 Combinational therapy 97 1 - - AND COVID-19 2021-04-26 Amprenavir AND 0 - - - COVID-19 2021-04-26 Atazanavir AND 2 - - - COVID-19 2021-04-26 Darunavir AND 0 - - - COVID-19 2021-04-26 Fosamprenavir AND 0 - - - COVID-19 2021-04-26 Indinavir AND 0 - - - COVID-19 2021-04-26 Lopinavir AND 28 - - - COVID-19 2021-04-26 Nelfinavir AND 0 - - - COVID-19 2021-04-26 Ritonavir AND 27 - - - COVID-19 2021-04-26 Saquinavir AND 0 - - - COVID-19 2021-04-26 Tipranavir AND 0 - - - COVID-19 2021-04-26 Combination AND 112 1 1 1 COVID-19 2021-04-26 Treatment AND covid- 99 0 0 0 19 AND combination Total 497 9 6 6

Results

First clinical study using HCV protease inhibitor danoprevir to treat COVID-19 patients (47). The aim of the study was to investigate the potential of danoprevir, a hepatite C (HCV) NS3/4A protease inhibitor as a treatment for COVID-19 compared to SoC which consisted of ritonavir/lopinavir with or without interferon-α. The study is an open-label, single arm study. The study was conducted between February 17th and March 19th of 2020. The study took place in Nanchang, Jiangxi, People’s Republic of China and included 11 patients. Patients were included under the category named moderate COVID-19 patients under Chinese guidelines. Patients had to be between 18 and 75 years old, diagnosed with SARS-CoV-2 via PCR and showed respiratory symptoms as well as CT-scan lung imaging confirming pneumonia. Patients were split into two groups, naïve and experienced patients which referred to previous exposure to SoC treatment. Basic information of the groups can be seen in Table 3. For a patient to be discharged, four criterions had to be fulfilled according to People’s Republic of China’s national standards. This meant that a patient had to have had a normal body temperature for 3 days in a row, significantly recovered respiratory systems, lung imaging that showed obvious absorption and recovery of acute exudative lesion as well as two negative PCR- tests with a minimum of a one-day interval between the tests. The Patients received treatment until they were discharged from the hospital. The experimental treatment consisted of danoprevir 100mg twice daily + ritonavir 100mg twice daily. 6 of the 11 patients also received 5 million international units, IU of interferon-α via nebulization. The “control” arm consisted of patient 7-11 and received lopinavir/ritonavir 100/50 mg twice daily + 5 million IU of interferon-α via nebulization. At day 11, the first patient was put on only danoprevir and ritonavir 100/100 mg twice daily, and by day 14, the whole “control” arm was receiving this treatment. An overview of which patient was on which medication can be seen in Figure 2. The study showed that after receiving danoprevir, patients tested negative for SARS- CoV-2 after a median of 2 days. The naïve patients were hospitalized 7 and 9 days respectively while the experienced group did not meet the criteria for discharge until being given danoprevir due to symptoms not improving, even after a two-week period. After receiving danoprevir, the median time to discharge was 8 days. No patients died during the study and a mortality rate therefore cannot be calculated. Due to small population size, a statistical analysis was not performed.

(Table 3. Group adherence, information regarding age, sex, hospitalization period as well as mortality. † The experienced group not receiving danoprevir had not recovered by day 10 and were put on danoprevir, after that they took an average of 8 days to be released from hospital). Group Age (yr) Sex Participants Hospitalization Mortality median, (Male, (N) period (days) (%) (min- Female) max) Experienced 44, (18– 3, 6 9 Unverifiable, Not 66) 8† calculated Naïve 18 & 44 1, 1 2 7, 9 Not calculated

(Figure 2. Timeline describing which patients took what drug(s) and when they received each respective drug(s). Figure is taken from study (47)).

A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19 (48). The aim of the study was to compare the efficacy of ritonavir/lopinavir with SoC. The study was a randomized, controlled, open-label study. The study was conducted between January 18th and February 3rd of 2020. The main outcome was time to improvement of two points on a 7-point scale or time to discharge.

The study was conducted in Wuhan, Hubei, People’s Republic of China and included 194 patients. The requirement for inclusion was a positive PCR-test, CT-scan showing pneumonia and an oxygen saturation below 94%. Patients had to be at least 18 years of age and not pregnant. Criteria for exclusion was any medical condition stopping the patient from following the protocol safely. Patients were split into two groups, with 95 and 99 patients in the respective group. The median age of both groups was 58. An overview of patient statistics can be seen in table 4. No requirements for discharge were listed, presumably the national standards. The trial group received lopinavir/ritonavir 400/100 mg twice daily + SoC for 14 days. The control group received SoC only, which consisted of antibiotics, vasopressor medication, renal replacement therapy as well as supplemental oxygen. The study showed that patients receiving ritonavir/lopinavir + SoC had a numerically lower hospitalization duration than the control group that only received SoC (12 vs 14 days median). However, no significant difference could be discerned between the two groups (1 day difference, 95% CI 0 to 3). As for mortality, the 28-day mortality between the two groups was 16.7% vs 25% for the trial and control group, respectively (-5.8 percent difference 95% CI = -17.3 to 5.7) which was not significant.

(Table 4. Group adherence, information regarding age, sex, hospitalization period as well as mortality). Group Age yr Sex (male, Participants Hospitalization Mortality median, female) % (N) period (days, (%) (IQR) median) Trial 58, 50–68 61, 39 95 12 16.7 Group Control 58, 48–68 59, 41 99 14 25 Group

Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to the hospital with COVID-19: an open-label, randomized, phase 2 trial (49). The aim of the study was to compare the efficacy and safety of a triple-combination of interferon β-1b, lopinavir/ritonavir and ribavirin compared to lopinavir/ritonavir alone. The study was a multicenter, prospective, open-label, randomized phase 2 trial. The study was conducted between February 10th and March 20th of 2020. The study was conducted in 6 different hospitals in Hong Kong, Hong Kong Special Administrative Region of the People’s Republic of China. The study included 127 patients who were divided into two groups that received either the triple combination or ritonavir/lopinavir in a 2 to 1 split. The criteria for inclusion into the study was that patients had to be at least 18 years of age, a positive PCR-test, at least a NEWS2 score of 1 and had shown symptoms for fewer than 14 days. The primary outcome was a negative PCR-test. Secondary outcomes were a NEWS2 score of 0 for at least 24h, hospitalization duration and 30-day mortality. NEWS2 is a system for ranking a patient’s vital signs as well as conscious level where higher scores means that the

patient is more ill. The mean age of the triple combinational group and the control group were 51 and 52 respectively, with an approximately equal differentiation in sex between the two groups. An overview of patient adherence and characteristics can be seen in table 5. The triple combinational group received lopinavir/ritonavir 400/100 mg twice daily, ribavirin 400 mg twice daily as well as interferon β-1b 8 000 000 IU every other day. The regiment lasted 14 days. The control group received lopinavir/ritonavir 400/100 mg twice daily. Ribavirin is a nucleoside analogue that inhibits viral RNA replication in an infected cell. Within the combinational group, 52 patients were admitted less than 7 days from symptom onset and 34 patients were admitted after more than 7 days from symptom onset. The study showed that the triple combinational group had a significantly shorter median time to show a negative PCR-test 7 vs 12 days (p = 0.0010), which was the primary endpoint. The median hospitalization duration was also shorter in the triple combinational group that received treatment within < 7 days of symptom onset than in the control group, 8 (6-12.5 min, max) vs. 15 (9-16 min, max) days (p = 0.003). The patients that received treatment > 7 days after symptom onset showed a statistically insignificant difference in hospitalization duration 13 (8-15 min, max) days in the combinational group vs 13.5 (12.3-21.8 min, max) days in the control group (p = 0.090). When combining the two combinational therapy subgroups, hospitalization duration was still significantly better in the combinational group 9 (7-13, min max) days vs 14.5 (9.3-16 min,max) days in the control group (p = 0.016). As for mortality, no patients lost their lives during the study. (Table 5. Group adherence, information regarding age, sex, hospitalization period as well as mortality. * P ≤ 0,05 compared to the control group). Group Age yr Sex Participants Hospitalization Mortality (median, (male, (N) period (days, (%) IQR) female) median) % Triple 51, 31–61 52, 48 86 9* Not combination calculated group Control 52, 33–62 56, 44 41 14.5 Not group calculated

Interferon β-1b in treatment of severe COVID-19: A randomized clinical trial (50). The aim of the study was to evaluate the efficacy and safety of interferon β-1b when treating severely ill COVID-19 patients. The study was an open label, randomized clinical trial. The study was conducted between April 20th and May 20th of 2020 with a study period of two weeks. The primary outcome of the study was time to clinical improvement which was defined as a two-point improvement on a 6-point scale. Secondary outcomes were intensive care unit ICU admission, intubation rates, hospitalization duration, ICU stay as well as 28-day mortality.

The study took place in Tehran, Islamic Republic of Iran. The criterions for inclusion were that patients had to be at least 18 years of age, test positive for SARS-CoV-2 via PCR-test and show signs of pneumonia. Patients had to have an oxygen saturation less than 93%. The criteria for exclusion were previous uncontrolled underlying disease, psychiatric disorders, pregnancy as well as lactation. The study included 66 patients who were split in a 1:1 ratio into a control and trial group. The median age was 60 (47- 73) and 61 (50-71) for the trial and control group, respectively with an approximately equal differentiation in sex between the two groups. All patients required respiratory support and over 80% needed supplemental oxygen via facemask. An overview of patient adherence and characteristics can be found in table 6. The control group received SoC which consisted of either lopinavir/ritonavir 400/100 mg twice daily, or atazanavir/ritonavir 300/100 mg daily. As well as hydroxychloroquine 400 mg twice daily on the first day of treatment which was later lowered to 200 mg twice daily for 7-10 days. The trial group received interferon β-1b on top of the SoC. The study showed that the time to clinical improvement was significantly shorter in the trial group 9 (6-10) days vs. 11(9-15) days in the control group (p = 0.002, HR = 2.3 with a 95% CI 1.33 to 3.39). The hospitalization duration was shorter, albeit barely significantly shorter in the trial group than in the control group, 11 (9-13) vs. 13 (10-17) days respectively (p = 0.05). The 28-day mortality rate was 6.06% and 18.18% in the trial and control group respectively (p = 0.12). (Table 6. Group adherence, information regarding age, sex, hospitalization period as well as mortality. * P ≤ 0,05 compared to the control group). Group Age yr Sex (male, Participants Hospitalization Mortality (median, female) % (N) period (days, (%) IQR) average) Trial 60, 47–73 60, 40 33 11* 6.06 group Control 61, 50–71 58, 42 33 13 18.18 group

The effects of Arbidol (Umifenovir) on COVID-19:a randomized controlled trial (51). The aim of the study was to determine the effect of umifenovir on COVID-19. The study was an open label, randomized controlled trial. The study took place between April 20th and June 18th of 2020. The primary outcome of the study was hospitalization duration and clinical improvement 7 days after admission. Secondary outcomes 30-day mortality rate and CT-scan changes after 30 days as well as amount needing invasive mechanical ventilation. The study took place in Tehran, Islamic Republic of Iran and included 100 patients who were split in a 1:1 ratio into a trial and control group. The mean age of the trial and control group was 56.6 (standard deviation, SD = 17.8) and 56.2 (SD = 14.8) years of age, respectively. The proportion of male and female study participants was 66% vs. 34% in the trial group and 54% vs. 46% in the control group. An overview of group adherence and characteristics can be seen in table 7. The criteria for inclusion were

being at least 18 years of age, non-pregnant, confirmed SARS-CoV-2 infection via PCR or CT-scans showing pneumonia and an oxygen saturation level below 94%. The criteria for exclusion were medical reasons such as allergies to umifenovir, abnormal liver, kidney, and blood coagulation function as well as patients with severe heart disease. The control group received Lopinavir/ritonavir 400/100 mg twice daily as well as hydroxychloroquine 400 mg on day one. The trial group received umifenovir 100 mg three times daily for 7-14 days depending on patient illness as well as hydroxychloroquine 400 mg twice daily on day one. Umifenovir is a cell entry inhibitor. The study shows that the hospitalization duration was significantly shorter in the trial group than in the control group which was 7.2 vs 9.6 days respectively (p = 0.02). The need for invasive mechanical ventilation was not significantly different between the two groups. Oxygen saturation levels were significantly different between the trial and control group, 94% vs. 92% respectively after 7 days (p = 0.02). Three patients died during the study, one in the trial group and two in the control group, measuring 2% and 4% respectively. (Table 7. Group adherence, information regarding age, sex, hospitalization period as well as mortality. * P ≤ 0,05 compared to the control group). Group Age yr Sex (male, Participants Hospitalization Mortality (median, female) % (N) period (days, (%) SD) average) Trial 56.6, 17.8 66, 34 50 7.2* 2 group Control 56.2, 14.8 54, 46 50 9.6 4 group

Sofosbuvir and daclatasvir compared with standard of care in the treatment of patients admitted to hospital with moderate or severe coronavirus infection (COVID-19):a randomized controlled trial (52). The aim of the study was to compare the efficacy of a common hepatitis C drug combination to SoC. The study is an open label, multicenter, randomized controlled trial. The study was conducted between March 26th and April 26th of 2020. The primary outcome was clinical recovery within 14 days of enrolment which was defined as fever- free, normal respiratory rate as well as an oxygen saturation above 94% without the need for supplemental oxygen for at least 24h. Secondary outcomes were mortality, need for mechanical ventilation and hospitalization duration. The study took place in Tehran and Gorgan, Islamic Republic of Iran and included 66 patients. Patients were split into a trial and a control group on a 1:1 basis. The median age for the trial and control group was 58 (IQR, 38-65) and 62 (IQR, 49-70) respectively. The trial group contained significantly more men than the control group 61% vs 42% for the two groups, respectively. An overview of group adherence and characteristics can be seen in table 8. The criteria for inclusion were patients being at least 18 years of age, positive PCR-test as well as CT-scan indicating moderate to severe COVID-19. Patients also needed to display symptoms in the form of fever, elevated respiratory rate, oxygen saturation below 94% for no more than 8 days. The

criteria for exclusion were known allergies to drugs used, pregnancy or lactation and any earlier experimental treatment to COVID-19. A heart rate of < 60 beats per minute, evidence of multiorgan failure, requiring invasive mechanical ventilation, eGFR < 50mL/1.73 m2/min as well as not taking amiodarone which is indicated for heart arrhythmias. The control group received hydroxychloroquine 200 mg twice daily with or without lopinavir/ritonavir 200/50 mg twice daily which was the SoC. The trial group received sofosbuvir 400 mg and daclatasvir 60 mg once daily as well as SoC. Sofosbuvir is a RNA-dependent RNA-polymerase inhibitor, targeting the NS5B protein and is indicated for treatment of HCV. Daclatasvir is an inhibitor of HCV NS5A protein which plays part in the RNA replication. The study shows that the primary outcome that is, clinical recovery within 14 days was achieved in 88% vs. 67% in the trial and control arm respectively (p = 0.076). The duration of hospitalization was significantly shorter in the trial vs control arm, 6 (IQR, 4-8) vs. 8 (IQR, 5-13) days (p = 0.029). There was no significant difference in the need for mechanical ventilation, 3 vs 7 (p = 0.303) or mortality between the trial and control group, 3 vs. 5 deaths (p = 0.708). (Table 8. Group adherence, information regarding age, sex, hospitalization period as well as mortality. * P ≤ 0,05 compared to the control group). Group Age yr Sex (male, Participants Hospitalization Mortality (median, female) % (N) period (days, (%) IQR) average) Trial 58, 38–65 61, 39 33 6* 9.09 group Control 62, 49–70 42, 58 33 8 15.16 group

Discussion

Combinational treatments Five of the six studies included protease inhibitors approved by the FDA. The substance not approved by the FDA was a Chinese substance, danoprevir which is sold under the brand name Ganovo. It is used to treat hepatite C and has shown good results in the treatment thereof (53). The lopinavir/ritonavir combination showed a numerical difference in hospitalization duration compared to SoC, however this difference was not statistically significant. This is not unsurprising as this drug is normally used in combination with other anti-viral drugs when treating HIV. WHO also declared it ineffective in treating COVID-19. The danoprevir study showed that protease inhibitors can be effective(47). Comparing the danoprevir study with the sofosbuvir/daclatasvir study shows that HCV protease inhibitors yields similar results on the hospitalization period to NS5A+NS5B inhibitors. The age group of both studies are comparable (46, 51). This suggests that protease inhibitors could be a viable option when treating COVID-19, although HIV protease inhibitors might not be a viable option..

Moving on to the studies that included interferons show that interferons alone are much less effective than the direct-acting anti-viral drugs with the exception of lopinavir/ritonavir alone (50). A decreased hospitalization period can be observed when the interferons are used together with lopinavir/ritonavir as well as ribavirin (49). Because the other studies show modest results from both lopinavir/ritonavir and interferons the assumption that most of the effect on the hospitalization period comes from the RNA-dependent RNA-polymerase inhibitor ribavirin must be made (47, 49). The study comparing umifenovir with lopinavir/ritonavir shows that umifenovir is more effective than lopinavir/ritonavir in reducing the hospitalization period. With comparable results to the HCV protease inhibitors as well as the NS5A/NS5B inhibitors in reducing the hospitalization period. No further comparisons can be made between this and the other studies due to umifenovir belonging to a separate group of anti-viral drugs not present in the other articles. Despite this, because umifenovirs attack vector is different from all the other drugs mentioned, it is reasonable to assume umifenovir could be used in combinational treatment assuming no severe drug interactions. As for mortality, only 4/6 articles included mortality data. Two of these studies (47, 50) showed a very high or very low mortality rate compared to the remaining two studies. The umifenovir study only had three deaths which is insufficient to conclude anything. The patients in (48) had a much higher mortality than was reported in the other articles as well as what the literature indicates. Which suggests that these patients had a worse clinical status compared to the patients in the other studies. The mortality results from (48) should therefore be considered with care. The studies suggest that lopinavir/ritonavir is not effective in reducing the mortality rate alone, this is consistent with our current understanding of treatment regimen (54). No comparison could be made as to its effect in combinational treatments. As for the HCV protease inhibitor, no mortality data could be retrieved as no patients died during the study. The results from the NS5A/NS5B inhibitors study were significant and concluded that the daclatasvir in combination with sofosbuvir decreases the mortality. Further studies on both HCV protease inhibitors and HCV NS5A/NS5B inhibitors effect on mortality is warranted before any conclusions can be made. The effects of protease inhibitors yielding results in the treatment of COVID-19 was not a complete surprise as an earlier study showed that several protease inhibitors had ligand affinity for the SARS-CoV-2 protease (43).

Standard treatment of care The standard of care of COVID-19 in the collected articles were largely the same. All, except one included lopinavir/ritonavir and all but two articles also included hydroxychloroquine. The doses of these drugs were roughly equal in their respective article. The addition of vasopressors, antibiotics and other supportive care differed on a patient-to-patient basis and no obvious pattern could be discerned.

Study design The main problem with this study is that the protease inhibitors in many cases are part of the SoC, this muddles the results and makes it hard to determine which combinations are useful. A perfect study would compare the combinational therapies with placebo,

although ethical reasons most likely prohibit this from ever happening. None of the studies were double blinded which lowers their credibility. This is an effect of the setting in which the studies were performed and is something that future studies can solve. Some of the included articles discuss the issue of being underpowered, this is of course also related to the setting, especially as all the included articles were conducted in the first half of 2020. The results that the control groups report differs between studies even when similar treatment is given. This suggests that there are factors unaccounted for, what these factors are is unknown. When coming to conclusion with study results, the level of evidence must be considered. A common way of classifying the power of evidence a certain study has, an evidence hierarchy pyramid can be utilized. The evidence hierarchy can then be used to compare the results of different studies (55). When it comes to designing clinical trials, the highest level of evidence power that can be achieved is done by doing a double- blinded randomized controlled trial (56). However, when dealing with a pandemic of a deadly virus that has no standardized treatment, hospital beds quickly filling up and no vaccines in sight, some problems arise that impacts the quality of a study. Firstly, ethical issues such as giving placebo to a sick patient can result in their death. It is therefore difficult to find a “clean” control group. This obviously complicates the interpretation of results. Secondly, the design of a study might not be perfectly planned due to the environment in which the study takes place, that is doctors trying to save their patients. With that said, these studies are still valuable as they give insight into determining what further studies might be of interest. While conducting the study, FDA approved protease inhibitors was used, it could be argued that a more complete list of protease inhibitors could be used instead. The time and resource constraints of this study made that approach unfeasible. However, while conducting the study, searching for specific compounds yielded limited results. This could be to various reasons, but most likely studies on the FDA approved HIV protease inhibitors have not yet been conducted and/or published. Using EMA approved drugs would therefore most likely not yield more relevant articles. When motivating the choice of study outcomes to include it is easy to understand why a reduced mortality rate equals a good result, it is, however not obvious why a reduced hospitalization period can be equally good if not better. A scenario can be imagined where a certain drug does not reduce the mortality rate of a disease, but out of those that does survive the disease, a shorter hospitalization period is observed. A result like this would mean that more patients could be treated for a set time interval which in turn results in more lives saved, even if the drug does not directly increase an individual patient’s chance of survival. Hospitalization duration is also an outcome that is often included in clinical trials, possibly increasing the number of articles that could be included. A few outcomes that are common within health sciences are Quality-adjusted life years (QALY), time to discharge, morbidity, mortality, and cost-effectiveness. For a more COVID-19 specific situation, outcomes such as oxygen saturation levels, the concentration of a certain substance in blood plasma, urine or the filtration rate in the kidneys or liver may be of greater value. Some of these outcomes were seen in the included articles. Being this specific however, would severely limit the number of articles that could be included and hamper the real-world application of new insight as these values does not necessarily indicate anything useful. Because COVID-19 is such a new disease with a constant influx of new information, published articles might already be obsolete by the time they come out, or they might

use legacy procedures that has since the study was conducted, been abandoned. The most pressing of these issues is the lopinavir/ritonavir + hydroxychloroquine combination which has been deemed ineffective but due to the time it takes for articles to be reviewed and published is included in all the collected articles. While conducting this systematic review, it became obvious that it is not very well placed in time, meaning that this type of study would yield more conclusive results if it were performed two years from now. However, this can also be seen as a strength as early studies like this one can be valuable tools to guide future research. The study could have benefited from using more than one database as there could potentially be more viable articles that could help answer the aim of the study. There are, as of June 7th, 2021, no systematic reviews on the topic of protease inhibitors other than lopinavir/ritonavir and COVID-19 could be found on PubMed which means that this study adds value to the subject.

The unknowns of COVID-19, future studies Despite the global effort to solve the COVID-19 pandemic, much about the disease is still unknown. For example, the mortality rate seems to differ depending on when and where a study took place. Furthermore, older patients are more vulnerable than younger patients. This does pose a problem as earlier studies included mainly middle-aged people who on average, have less effective organ systems. Older patients also tend to have a higher amount of comorbidity than younger individuals which increases the mortality in this patient group. Studies seem to indicate that earlier intervention of protease inhibitors yields a greater effect. The same observation can be seen in (49), where patients receiving treatment > 7 days after symptom onset showed no significant differences from the control group as to hospitalization period. The symptoms of COVID-19 such as shortness of breath does not show until the disease has progressed for some time, making studies of early intervention difficult as patients are not admitted to hospitals until their symptoms are severe enough. This is of course not optimal, perhaps future studies in other controlled environments such as nursing homes where you could treat individuals who have not yet showed any symptoms could shed more light on this subject. Further studies on the efficacy of protease inhibitors targeting the hepatitis C proteases in combination with an RNA-polymerase inhibitor and a cell entry inhibitor on a large-scale basis could yield greater insight of the efficacy of targeted anti-viral cocktails. Especially since these drugs are readily available and might therefore work as SoC until new drugs, specifically designed for the treatment on COVID-19 are readily available on the market.

Other studies There is a large study currently being conducted that included as of June 8th, 2020, 754 participants. The study is named DisCoVeRy with the aim of evaluating the efficacy and safety of 5 different therapies for the treatment of COVID-19 (57). The DisCoVeRy trial will hopefully yield better answers to the questions proposed in this study. WHO are currently conducting two studies, “Solidarity I and II” which are both trying to answer what treatments strategies are effective in the treatment of COVID-19.

Conclusion In conclusion, this study shows that HCV protease inhibitors could possibly serve an important part of the treatment of COVID-19, however, further studies on their efficacy are needed before widespread clinical use can be motivated. Protease inhibitors in combinational therapy can potentially reduce the hospitalization period, however the data is insufficient as to their role in reducing the mortality of COVID-19.

Populärvetenskaplig sammanfattning I slutet av december 2019 publicerades den första varningen om en ny typ av ”lunginflammation” som börjat drabba lokalbefolkning i Wuhan, Kina. I maj 2021 så hade samma ”lunginflammation” tagit livet på över 3,4 miljoner människor. Sjukdomen som senare blev känt som COVID-19 eller Corona i folkmun orsakas av ett nytt, mycket smittsamt virus i virusfamiljen coronavirus. Viruset är besläktat med SARS och MERS som tidigare orsakat problem i världen. Flera vaccin mot sjukdomen har kommit ut på marknaden och många länder hoppas ha vaccinerat stora delar av sin befolkning till slutet av 2021. Huvudproblemet ligger i att det tar lång tid att få ut fungerade vaccin till befolkningen, även om det gått fort relativt en normal vaccinframställning. Alla människor kan heller inte ta vaccinen, vilket betyder att delar av befolkning inte kommer kunna vara helt skyddade från viruset. Det finns också logistiska problem med vaccinen, exempelvis så måste Pfizer-BioNtech’s vaccin förvaras mycket kallt för att behålla sin effektivitet. Detta är mycket problematiskt i länder med mindre utvecklad infrastruktur. Utöver detta så är vaccin inte användbara för behandling av de som är sjuka med COVID-19. Behandlingsmetoder för att rädda de som redan ligger inne på sjukhusen är därför av ytterst vikt. Idag saknas en standardiserad behandlingsmetod för de som drabbas av sjukdomen. I väntan på nya läkemedel som specifikt kan behandla COVID-19 så bör vi utreda ifall något av de läkemedel som vi redan har tillgängliga är effektiva mot sjukdomen. Detta kallas för ”drug repositioning” och är mycket värdefullt, inte bara för att rädda liv men också eftersom dessa läkemedel redan finns på marknaden, så är de oftast också billiga och lätta att skala upp produktionen på vilket sparar både tid och pengar. Den här systematiska översiktsartikeln tittar på ifall läkemedel ur gruppen antivirala proteashämmare som normalt används för att behandla HIV kan användas för att minska dödligheten och sjukhusvistelsetiden hos patienter med en bekräftad COVID-19 infektion. Resultaten visar att proteashämmare ämnade mot HIV inte verkar har en signifikant effekt. Proteashämmare ämnade mot hepatit C har potentiellt en positiv effekt på sjukhusvistelsetiden och kan med fördel kan kombineras med andra typer antivirala läkemedel. Mer studier som tittar på proteashämmarnas effekt på dödligheten och sjukhusvistelsetiden av sjukdomen behövs för att bekräfta något om detta.

References 1. Listings of WHO’s response to COVID-19 [Internet]. [cited 2021 Mar 30]. Available from: https://www.who.int/news/item/29-06-2020-covidtimeline

2. WHO Coronavirus (COVID-19) Dashboard | WHO Coronavirus Disease (COVID-19) Dashboard [Internet]. [cited 2021 Mar 30]. Available from: https://covid19.who.int/table

3. Säkerhetsdatablad smittämnen - SARS-CoV-2 [Internet]. Folkhälsomyndigheten; [cited 2021 Mar 30]. Available from: https://www.folkhalsomyndigheten.se/contentassets/a7f4650efc06407c918feb41c693fbc6/saker hetsdatablad-smittamnen-sars-cov-2.pdf

4. Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J Virol [Internet]. 2020 Mar 17 [cited 2021 May 19];94(7). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081895/

5. Yuki K, Fujiogi M, Koutsogiannaki S. COVID-19 pathophysiology: A review. Clin Immunol Orlando Fla. 2020 Jun;215:108427.

6. Middle East respiratory syndrome coronavirus (MERS-CoV) [Internet]. [cited 2021 Mar 30]. Available from: https://www.who.int/news-room/fact-sheets/detail/middle-east-respiratory- syndrome-coronavirus-(mers-cov)

7. Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol. 2020 Feb 24;1–8.

8. Li F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol. 2016 Sep 29;3(1):237–61.

9. Tortorici MA, Veesler D. Structural insights into coronavirus entry. Adv Virus Res. 2019;105:93–116.

10. Snijder EJ, Decroly E, Ziebuhr J. The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing. Adv Virus Res. 2016;96:59–126.

11. Perlman S, Netland J. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol. 2009;7(6):439–50.

12. Liu Z, Xiao X, Wei X, Li J, Yang J, Tan H, et al. Composition and divergence of coronavirus spike proteins and host ACE2 receptors predict potential intermediate hosts of SARS‐CoV‐2. J Med Virol [Internet]. 2020 Mar 11 [cited 2021 May 19]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228221/

13. V’kovski P, Kratzel A, Steiner S, Stalder H, Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol. 2021 Mar;19(3):155–70.

14. Ulhaq ZS, Soraya GV. Interleukin-6 as a potential biomarker of COVID-19 progression. Med Mal Infect. 2020 Jun;50(4):382–3.

15. Hojyo S, Uchida M, Tanaka K, Hasebe R, Tanaka Y, Murakami M, et al. How COVID-19 induces cytokine storm with high mortality. Inflamm Regen [Internet]. 2020 Oct 1 [cited 2021 May 22];40. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527296/

16. Atzrodt CL, Maknojia I, McCarthy RDP, Oldfield TM, Po J, Ta KTL, et al. A Guide to COVID- 19: a global pandemic caused by the novel coronavirus SARS-CoV-2. FEBS J. 2020;287(17):3633–50.

17. Gómez-Belda AB, Fernández-Garcés M, Mateo-Sanchis E, Madrazo M, Carmona M, Piles- Roger L, et al. COVID-19 in older adults: What are the differences with younger patients? Geriatr Gerontol Int. 2021 Jan;21(1):60–5.

18. Rothan HA, Byrareddy SN. The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak. J Autoimmun. 2020 May;109:102433.

19. Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet. 2020 Feb 15;395(10223):507–13.

20. Li L, Huang T, Wang Y, Wang Z, Liang Y, Huang T, et al. COVID‐19 patients’ clinical characteristics, discharge rate, and fatality rate of meta‐analysis. J Med Virol [Internet]. 2020 Mar 23 [cited 2021 May 20]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228329/

21. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet. 2020 Feb 15;395(10223):497–506.

22. Chen T-M, Rui J, Wang Q-P, Zhao Z-Y, Cui J-A, Yin L. A mathematical model for simulating the phase-based transmissibility of a novel coronavirus. Infect Dis Poverty. 2020 Feb 28;9(1):24.

23. Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia. N Engl J Med. 2020 Mar 26;382(13):1199– 207.

24. Majumder MS, Mandl KD. Early Transmissibility Assessment of a Novel Coronavirus in Wuhan, China [Internet]. Rochester, NY: Social Science Research Network; 2020 Jan [cited 2021 May 19]. Report No.: ID 3524675. Available from: https://papers.ssrn.com/abstract=3524675

25. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet Lond Engl. 2020;395(10225):689–97.

26. Zhao S, Lin Q, Ran J, Musa SS, Yang G, Wang W, et al. Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data- driven analysis in the early phase of the outbreak. Int J Infect Dis. 2020 Mar;92:214–7.

27. Pfizer-BioNTech COVID-19 Vaccine Storage and Handling Summary. :3.

28. Thompson MG. Interim Estimates of Vaccine Effectiveness of BNT162b2 and mRNA-1273 COVID-19 Vaccines in Preventing SARS-CoV-2 Infection Among Health Care Personnel, First Responders, and Other Essential and Frontline Workers — Eight U.S. Locations, December 2020–March 2021. MMWR Morb Mortal Wkly Rep [Internet]. 2021 [cited 2021 Jun 7];70. Available from: https://www.cdc.gov/mmwr/volumes/70/wr/mm7013e3.htm

29. Sleigh SH, Barton CL. Repurposing Strategies for Therapeutics. Pharm Med. 2010 Jun 1;24(3):151–9.

30. Veklury (remdesivir) [Internet]. Läkemedelsverket / Swedish Medical Products Agency. 2020 [cited 2021 May 27]. Available from: https://www.lakemedelsverket.se/sv/behandling-och- forskrivning/lakemedelsmonografier/sok-monografier/veklury-remdesivir

31. COVID-19 | Gilead [Internet]. [cited 2021 Mar 30]. Available from: https://www.gilead.com/purpose/advancing-global-health/covid-19/about-veklury

32. Therapeutic Management [Internet]. COVID-19 Treatment Guidelines. [cited 2021 Mar 30]. Available from: https://www.covid19treatmentguidelines.nih.gov/therapeutic-management/

33. WHO discontinues hydroxychloroquine and lopinavir/ritonavir treatment arms for COVID-19 [Internet]. [cited 2021 Apr 8]. Available from: https://www.who.int/news/item/04-07-2020- who-discontinues-hydroxychloroquine-and-lopinavir-ritonavir-treatment-arms-for-covid-19

34. Mueller AL, McNamara MS, Sinclair DA. Why does COVID-19 disproportionately affect older people? Aging. 2020 May 29;12(10):9959–81.

35. Cheng Y, Luo R, Wang K, Zhang M, Wang Z, Dong L, et al. Kidney disease is associated with in-hospital death of patients with COVID-19. Kidney Int. 2020 May;97(5):829–38.

36. Dexamethasone Krka - FASS Vårdpersonal [Internet]. [cited 2021 Mar 30]. Available from: https://fass.se/LIF/product?userType=0&nplId=20161029000106

37. Group TRC. Dexamethasone in Hospitalized Patients with Covid-19. N Engl J Med [Internet]. 2020 Jul 17 [cited 2021 Apr 27]; Available from: https://www.nejm.org/doi/10.1056/NEJMoa2021436

38. Schrezenmeier E, Dörner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology. Nat Rev Rheumatol. 2020 Mar;16(3):155–66.

39. Effect of Hydroxychloroquine on Clinical Status at 14 Days in Hospitalized Patients With COVID-19: A Randomized Clinical Trial | Critical Care Medicine | JAMA | JAMA Network [Internet]. [cited 2021 Apr 27]. Available from: https://jamanetwork.com/journals/jama/fullarticle/2772922?utm_campaign=articlePDF&utm_m edium=articlePDFlink&utm_source=articlePDF&utm_content=jama.2020.22240

40. Ritter J, Flower RJ, Henderson G, Loke YK, MacEwan D, Rang HP. Rang and Dale’s pharmacology. 9th Ed. Edinburgh: Elsevier Health Sciences; 2019.

41. Ritonavir Accord - FASS Vårdpersonal [Internet]. [cited 2021 Apr 27]. Available from: https://fass.se/LIF/product?userType=0&nplId=20160702000051#pharmacodynamic

42. de Leuw P, Stephan C. Protease inhibitors for the treatment of hepatitis C virus infection. GMS Infect Dis. 2017 Nov 28;5:Doc08.

43. Hamed M, El-Hasab M, Mansour FR. Direct acting anti-hepatitis C combinations as potential COVID-19 protease inhibitors. VirusDisease. 2021 Apr 29;1–7.

44. Commissioner O of the. HIV and AIDS: Medicines to Help You. FDA [Internet]. 2020 Jan 7 [cited 2021 May 19]; Available from: https://www.fda.gov/consumers/free-publications- women/hiv-and-aids-medicines-help-you

45. Anonymous. Viracept [Internet]. European Medicines Agency. 2018 [cited 2021 Jul 9]. Available from: https://www.ema.europa.eu/en/medicines/human/EPAR/viracept

46. Anonymous. Agenerase [Internet]. European Medicines Agency. 2018 [cited 2021 Jul 9]. Available from: https://www.ema.europa.eu/en/medicines/human/EPAR/agenerase

47. Chen H, Zhang Z, Wang L, Huang Z, Gong F, Li X, et al. First clinical study using HCV protease inhibitor danoprevir to treat COVID-19 patients. Medicine (Baltimore). 2020 Nov 25;99(48):e23357.

48. Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, et al. A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19. N Engl J Med. 2020 May 7;382(19):1787–99.

49. Hung IF-N, Lung K-C, Tso EY-K, Liu R, Chung TW-H, Chu M-Y, et al. Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial. Lancet Lond Engl. 2020 May 30;395(10238):1695–704.

50. Rahmani H, Davoudi-Monfared E, Nourian A, Khalili H, Hajizadeh N, Jalalabadi NZ, et al. Interferon β-1b in treatment of severe COVID-19: A randomized clinical trial. Int Immunopharmacol. 2020 Nov;88:106903.

51. Nojomi M, Yassin Z, Keyvani H, Makiani MJ, Roham M, Laali A, et al. Effect of Arbidol (Umifenovir) on COVID-19: a randomized controlled trial. BMC Infect Dis. 2020 Dec 14;20(1):954.

52. Sadeghi A, Ali Asgari A, Norouzi A, Kheiri Z, Anushirvani A, Montazeri M, et al. Sofosbuvir and daclatasvir compared with standard of care in the treatment of patients admitted to hospital with moderate or severe coronavirus infection (COVID-19): a randomized controlled trial. J Antimicrob Chemother [Internet]. 2020 Aug 19 [cited 2021 Apr 26]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454592/

53. Xu X, Feng B, Guan Y, Zheng S, Sheng J, Yang X, et al. Efficacy and Safety of All-oral, 12- week Ravidasvir Plus Ritonavir-boosted Danoprevir and Ribavirin in Treatment-naïve Noncirrhotic HCV Genotype 1 Patients: Results from a Phase 2/3 Clinical Trial in China. J Clin Transl Hepatol. 2019 Sep 28;7(3):213–20.

54. Repurposed Antiviral Drugs for Covid-19 — Interim WHO Solidarity Trial Results. N Engl J Med. 2021 Feb 11;384(6):497–511.

55. Turner M. UC Library Guides: Evidence-Based Practice in Health: Hierarchy of Evidence [Internet]. [cited 2021 May 5]. Available from: https://canberra.libguides.com/c.php?g=599346&p=4149721

56. Misra S. Randomized double blind placebo control studies, the “Gold Standard” in intervention based studies. Indian J Sex Transm Dis AIDS. 2012;33(2):131–4.

57. Ader F, Discovery French Trial Management Team. Protocol for the DisCoVeRy trial: multicentre, adaptive, randomised trial of the safety and efficacy of treatments for COVID-19 in hospitalised adults. BMJ Open. 2020 Sep 21;10(9):e041437.