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Survival of the hospitalized patients with COVID‐19 receiving atorvastatin: A randomized clinical trial

Abstract As statins decrease the progression of sepsis and its related mortality, this study aimed to evaluate the effect of atorvastatin on survival and symptom improvement in hospitalized patients with COVID‐19. This randomized controlled trial was performed on 156 hospitalized patients with COVID‐19 in Bojnourd city in 2021. Patients were randomly divided into comparison (standard therapy: hydroxychloroquine + Kaletra®) and intervention groups (atorvastatin 20 mg, SD, plus standard therapy). The main outcomes were the rate of symptom improvement, duration of hospitalization, need for intubation, and mortality rate. In this study, seven patients died, two patients (2.6%) in the comparison group and five (6.6%) in the intervention group. The mean hospitalization days (p = 0.001), the pulse rate (p = 0.004), and the frequency of hospitalization in the ICU ward (18.4% vs. 1.3%) were longer and greater in the intervention group. The remission probability in the comparison group was greater (p = 0.0001). The median hospitalization days in the intervention group was longer (p < 0.001) and remission in the comparison group occurred 1.71 times sooner (hazard ratio = 1.70, 95% confidence interval = 1.22–2.38, p = 0.002). Totally, adding atorvastatin to the standard regime in this study increased hospitalization days and imposed negative effects on symptom improvement in hospitalized patients with COVID‐19.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, in December 2019 and has rapidly become a pandemic. 1 It is primarily transmitted by respiratory droplets and close contact, 2 and its incubation period lasts 14-33 days. 3 Asymptomatic patients are able to spread this disease. 4 Infected patients who show mild to moderate symptoms may develop severe pneumonia and acute respiratory distress syndrome, septic shock, multiple organ failure, and even death. The most common sign and symptoms of this disease are fever (98%), dry cough (76%), myalgia or fatigue (44%), and dyspnea (55%), and the less common symptoms are sputum production (28%), headache (8%), hemoptysis (5%), and diarrhea (3%); and chest computerized tomography scans show pneumonia. 5,6 Laboratory markers include leukopenia (25%), lymphopenia (63%), thrombocytopenia (5%), and high lactate dehydrogenase (73%). 6 Cytokine storm is the manifestation of COVID-19 severity which is accompanied by an increase in the plasma level of interleukin (IL)-2, -4, -6, -7, and -10, granulocyte colony-stimulating factor (GCSF), inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP1α), tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ). Furthermore, a significant decrease in lymphocyte counts, especially CD8 + T cells, and an increase in neutrophil counts are seen. Thereafter, dynamic cytokine storms and neutrophil-to-lymphocyte ratio (NLR) are predictors of developing severe COVID-19. 2 The pathogenesis of COVID-19 is categorized into two main courses. The former is a replication cycle that is modulated by viral proteins and includes identification, fusion, entry, and replication of this virus. The latter is the infection progression phase with an inflammatory and immune response to the virus that causes tissue damage. Thus, both virus and host factors are important for the pathogenesis of this disease which is considered a promising target for COVID-19 therapy. In this regard, probable effective drugs are classified into "target virus" and "target host" categories. 7 At the time of writing this article, just remdesivir has been approved by the Food and Drug Administration (FDA) for the treatment of COVID-19 requiring hospitalization 8 and some drugs including tocilizumab, sotrovimab, propofol-liuro 1%, bamlanivimab and etesevimab, casirivimab and imdevimab, baricitinib, COVID-19 convalescent plasma, fresenius propoven 2%, and REGIOCIT have issued emergency use authorization declaration in certain conditions. 9 Furthermore, since the outbreak of COVID-19, some old drugs are suggested to manage this disease in clinical trials based on the in vitro and in vivo studies. It seems drug repositioning is able to help in controlling this urgent outbreak faster and more economically. 7 A group of Iranian specialist physicians in treating COVID-19 recommended antiviral medications to treat hospitalized patients with COVID-19. At the time of designing this clinical trial study, the sixth edition of this recommendation was proposed. The recommended antiviral monotherapy regime was chloroquine (500 mg q12h on the first day followed by 250 mg q12h, orally) or hydroxychloroquine (HQ; 400 mg q12h on the first day followed by 200 mg q12h, orally) for 7-14 days based on the clinical progress. In combination therapy, lopinavir/ritonavir (400/100 mg q12h, orally) was administrated with chloroquine (500 mg SD, orally) or HQ (400 mg SD, orally) for the first day that was followed just with lopinavir/ritonavir (400/100 mg q12h, orally) for 7-14 days. In combination therapy with atazanavir/ritonavir, atazanavir/ritonavir (300/100 mg SD, orally) plus chloroquine (250 mg q12h, orally) or HQ (200 mg q12h, orally) were administrated from the first day for 7-14 days. 10 In addition to cholesterol-lowering ability, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) are able to diminish inflammation and induce immunomodulatory, antioxidative, and anti-atherosclerotic effects. 11,12 They inhibit liver production of C-reactive protein by reducing interleukin-6-induced C-reactive protein production in human hepatocytes. 13 Atorvastatin reduces C-reactive protein-induced chemokine secretion, ICAM-1 upregulation and chemotaxis in adherent human monocytes. 14 Rosuvastatin reduces plasma concentrations of pro-inflammatory cytokines TNFalpha and IFN-gamma and decreases TNF-alpha and IFN-gamma production in stimulated T-lymphocytes. It inhibits the Th-1-immune response. 15 Besides these pleiotropic effects, these drugs showed promising therapeutic effects against various infectious diseases. In a preclinical study, adding simvastatin, fluvastatin, or pravastatin increased antitubercular activity of rifampicin, isoniazid, and pyrazinamide. 16 In preclinical models, they showed favorable antiviral effects. For instance, atorvastatin restricts human immunodeficiency virus (HIV) replication in CD4 + T cells, 17 lovastatin inhibits respiratory syncytial virus (RSV) replication, and virus-induced cell-to-cell fusion, 18 and simvastatin shows anti CMV effects. 19 Not only preclinical studies but also do clinical and retrospective studies prove honorable anti-infective and antiviral effects of statins.
They reduced the mortality rate in elderly patients with communityacquired pneumonia and sepsis, 20 bacteriemia, 21  . Written informed consent was obtained from all patients or their first-degree family members after receiving an explanation of the study. The sample size was determined through clinical significance. Based on the expert opinions, a 1-day decrease in the length of stay in the hospital was considered significant. Therefore, the sample size was determined to consist of 50 patients for each group using the G-power software with a confidence interval (CI) of 95% and a power of 80%. However, given the probability of a 25% drop in samples, at least 75 patients were assigned to each group.

| Participants
COVID-19 patients were diagnosed based on the WHO criteria and clinical symptoms/signs by an infectious diseases specialist. 10 Those who need hospitalization and did not meet the following exclusion criteria were included in this study. Exclusion criteria were including age <16 years old, need for hospitalization in an intensive care unit (ICU) at admission, a history of type 1 diabetes, ketoacidosis, uncompensated heart failure, severe renal failure (GFR < 30 ml/min), metabolic acidosis, severe respiratory failure need to intubation, and sensitivity to atorvastatin. Furthermore, pregnant and lactating women were not included. with HQ (400 mg SD, orally) for the first day that was followed just with lopinavir/ritonavir (400/100 mg q12h, orally) for 7-14 days.

| Randomization, interventions, and follow-up
Patients received drugs till they discharged from the hospital.
Other supportive care such as fluid therapy, treatment of electrolyte disorders, and antibiotic therapy was considered according to the hospital protocols. The duration of the study was from the hospitalization of studied patients until the time of discharge from the hospital or death.
Patient demographic data, baseline diseases, symptoms at the time of disease presentation, vital signs, and laboratory data at the time of hospital admission were recorded. Patients were daily monitored in terms of changes in the vital signs, hemodynamic parameters, oxygenation status, laboratory data, and treatment strategies. The need for supplemental oxygen therapy and also invasive or noninvasive respiratory supports were evaluated regularly.

Based on the European Society of Cardiology (ESC) and
European Atherosclerosis Society (EAS) guidelines for the management of dyslipidemias, 23 emergency termination conditions during the study included an increase in liver enzyme levels (defined as more than three times the upper limit of normal) and new clinically diagnosed myopathy, as identified by treating clinicians.

| Outcome measures
The primary outcome was the length of stay in the hospital from admission to discharge through survival analysis. Therefore, all eligible patients had been followed till they get better and were discharged from the hospital. Those with uncompleted data were considered as the right-censored. Also, the secondary outcomes in this trial were the need for hospitalization in the ICU during the study and paraclinical findings.

| Statistical analysis
After discharge of all patients from the hospital, the time until complete remission of symptoms and recovery was assessed.
Patients who had incomplete information or who died at the time of discharge from the hospital were considered right-censored. Cox proportional-hazards regression models were used to estimate the relationship between the administration of atorvastatin and remission. In cox regression, the proportional hazard (PH) assumption is very important. This assumption means that the relative hazard is constant over time for the study groups. Before modeling, PH assumption for all predictor variables was checked through the Schoenfeld residuals analysis test. Then, each of the predictors was examined separately using cox regression. Variables that were significantly associated with remission in the univariate cox regression model were entered into the multivariate cox regression model.
The results were reported as hazard ratio (HR) with a 95% CI.
Kaplan-Meier analysis was applied to plot and estimate remission probabilities. Comparison of demographic and clinical characteristics of the studied groups was performed using appropriate analytical tests. All analyzes were done by SPSS version 24 software.

| RESULTS
The flow diagram of participants in this clinical trial is shown in  Table 1. Furthermore, the available baseline values of some laboratory tests of some patients are included in Table 1. The results showed that there was no significant difference between the two groups in terms of age, sex, respiratory rate, blood pressure, body temperature, cough, dyspnea, and diarrhea (p > 0.05).
It seems two groups were not statistically different in platelets count, hemoglobin, plasma creatinine, aspartate transaminase, alanine transaminase, alkaline phosphatase, total bilirubin, direct bilirubin, and lactate dehydrogenase level (p > 0.05). Unfortunately, the data about CPK at the baseline was missed.  Table 2). Figure 2 shows the Kaplan-Meier plot for the atorvastatin and atorvastatin-free groups. The patients receiving standard treatment (97.4%) were more likely to recover than those receiving atorvastatin (93.4%), which is a statistically significant difference (p = 0.0001). Table 3 shows the mean and median length of hospital stay of the patients. The median length of hospital stay in the intervention and control groups was 7 and 4 days, respectively. In other words, 50% of the patients in the intervention group after 7 days and in the control group after 4 days from the start of the study have been discharged from the hospital.
Before performing the cox models (PH cox models), the PH assumption was checked and confirmed for all the studied variables.
The crude and adjusted HRs for the relationship between the studied variables and survival time is shown in Table 4. Variables that were significant in the univariate/crude model were entered into the multivariate cox regression model to adjust or control them. In univariate analysis, it was observed that only there was a statistically significant relationship between age and groups with improvement, in such a way that with increasing age of the patients, the improvement decreases by 1% and also the improvement in the control group   Furthermore, remission in the intervention group occurred later than the standard group.
Our findings are consistent with this study that emphasized the deleterious effects of statins on COVID-19 clinical outcomes including prolonged hospital stay (≥7 days) and/or need for invasive mechanical ventilation although this study didn't show any association between statin use and mortality. 24 Furthermore, the results from the CORONADO study verify our data that reported routine use of GHAFOORI ET AL.
| 3165 statins increased COVID-19 related mortality in inpatients with type 2 diabetes. 25 Peymani et al. 26  A cohort study showed statin use in residents diagnosed with COVID-19 was accompanied by less severe symptoms and improvement of clinical outcomes. 29 Based on another retrospective study by Daniels et al., 30  opposes the ACE action. It converts AngI to Ang(1-9) and AngII to Ang(1-7) causing vasodilator and anti-inflammatory properties. 33 Recently, ACE2 which is expressed in the respiratory airways, intestine, kidney, heart, and pancreas has been proposed as SARS-CoV-2 virus entering receptor. 34 On the other side, ACE2 overexpression is associated with reduced severity of acute respiratory distress syndrome. 35  interactions. 39 Both lopinavir and ritonavir are potent CYP3A inhibitors and their concurrent administration with statins raises statin concentrations and the risk of toxicity. 40 It is a predictable drug interaction and based on references atorvastatin 20 mg/day 41 was selected herein, however, it was better we evaluated enrolled patients for rhabdomyolysis, an outcome that was missed in this study, and its measuring was just limited to symptomatic patients during the study, while Davoodi et al. 28  The effects of more potent doses of statins with tight control on the inclusion criteria and side effects, and evaluation in a more targeted population of patients with COVID-19 may clarify, support, or reject this data in the future. It is recommended that critical care scores, such as SOFA or qSOFA scores, be added to the data to more accurately understand and assess the true situation between the two groups of patients. Furthermore, it might be beneficial to test and verify the effect of statins in outpatients who are in the earlier phase of the COVID-19, to prevent the start of the inflammatory process, rather than trying to stop it.