Aprotinin is a potent multi-target drug for the combination therapy of moderate COVID-19 cases

Objectives. COVID-19 is a contagious multisystem inflammatory disease caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have studied the efficacy of Aprotinin (nonspeciﬁc serine proteases inhibitor) and Avifavir or Hydroxychloroquine (HCQ) combinations for the therapy of COVID-19. 
Methods. Three prospective single-center (cohorts 1 – 3) studies included participants with moderate COVID-19-related pneumonia, laboratory-confirmed SARS-CoV-2 and admitted to the hospitals. Patients received combinations of intravenous (IV) Aprotinin (1,000,000 KIU daily, 3 days) and HCQ (cohort 1), inhalation (inh) treatment with Aprotinin (625 KIU 4 times per day, 5 days) and HCQ (cohort 2) or IV Aprotinin (1,000,000 KIU daily for 5 days) and Avifavir (cohort 3). 
Results. In the cohorts 1 – 3, the combination therapy showed 100% efficacy in preventing the transfer of patients (n = 30) to the intensive care unit (ICU). The effect of combination therapy in the cohort 3 was the most prominent and the median time to SARS-CoV-2 elimination was 3.5 days (IQR 3.0 – 4.0), normalization of CRP concentration was 3.5 days (IQR 3 – 5), of D-dimer concentration - 5 days (IQR 4 – 5); body temperature - 1 day (IQR 1 – 3), improvement in clinical status or discharge from the hospital - 5 days (IQR 5 – 5), and improvement in lung lesions of patients on 14 day - 100%. 
Conclusions. The administration of Aprotinin combinations prevented the transfer of moderate COVID-19 patients to the ICU for mechanical ventilation (ALV) or non-invasive ventilation (NIV) and by shortening of their hospital stay.


Introduction
SARS-CoV-2 spread globally and for the start of November 2020 the total number of infections in the world exceeded 46 million in 190 countries, with more than 1.198 million dead [1]. Given that COVID-19 poses a serious threat to public health and the economy around the world, the urgent need exists for a new effective drugs for the treatment and prophylaxis of SARS-CoV-2 infections.
COVID-19 is a multisystem disease as SARS-CoV-2 infects cells using an angiotensin-converting enzyme 2 (ACE2) receptor, which is highly expressed in different tissues -lungs, kidneys, gastrointestinal tract, liver, vascular endothelial and arterial smooth muscle cells [2]. COVID-19 prognosis has largely been influenced by multiorgan involvement, largely due to the elevated concentration of fibrinogen and D-dimer in patients' blood, which activates hypercoagulation. D-dimers are not normally present in human blood plasma, their level became elevated due to the formation and degradation of fibrin clot in vivo, when the coagulation system has been activated, for instance because of the presence of thrombosis or disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT), pulmonary embolism, ect [3]. Therefore elevated D-dimer along with C-reactive protein (CRP) have a high predictive value for venous thromboembolism (VTE) in critically ill COVID-19 patients. In several studies authors have monitored an association between COVID-19 pneumonia and VTE [4]. It was suggested that the prevalence of VTE was 25%, with a sensitivity, specificity and negative predictive value of D-dimer cutoff value of 1.5 μg/mL [5]. CRP is another valuable marker to anticipate the possibility of aggravation of mild COVID-19 -the exacerbated patients have much higher levels of CRP (median 43.8 mg/L, IRQ 12.3 -101.9) as compared to mild (median 12.1 mg/L, IRQ 0.1 -91.4) COVID-19 patients [6]. Higher plasma CRP levels indicate severe COVID-19 pneumonia and a longer stay in hospital [7].
Currently, two broad categories of drugs for COVID-19 are in clinical use: (1) drugs that can directly target SARS-CoV-2 virus replication cycle and (2) drugs inciting innate antiviral immune responses or alleviating damage induced by the dysregulated inflammatory responses [8]. The SARS-CoV-2 replication inhibitors Favipiravir (Avifavir) [6,9] and Remdesivir (Veklury®) [9] were among the first drugs repurposed for COVID-19 treatment. Timely use of these antiviral drugs (up to 3-7 days after infection) have prevented the progression to more severe respiratory disease.
Due to the complex nature of SARS-CoV-2 pathogenesis and multiorgan involvement, combination of direct virus-targeted and host-targeted drugs is clinically beneficial for the therapy of COVID-19. One of the promising drug candidates for the combination therapy of COVID-19 is Aprotinin, a natural protease inhibitor with a long history of clinical use since 1960s and good safety profile [10]. Firstly, Aprotinin is a broad-spectrum antiviral drug as it is a nonspecific inhibitor of the serine proteases, especially trypsin, chymotrypsin, plasmin and kallikrein. The inactivation of kallikrein blocks factor XIIa formation and had a negative impact on the intrinsic pathway of coagulation, fibrinolysis and thrombin generation, leading to the attenuation of the pro-inflammatory response [11][12][13]. Secondly, Aprotinin possesses a specific mechanism of action for SARS-CoV-2 as it inhibits transmembrane serine protease 2 (TMPRSS2), a host cell protease responsible for the cleavage and activation of the spike protein of SARS-CoV-2. This mechanism of Aprotinin's action suggests that it can prevent SARS-CoV-2 penetration into susceptible cells [14]. Recently, it was shown that Aprоtinin also inhibits replication of SARS-CoV-2 by downregulating cellular protease during replication cycles [15].
These data raise the hypothesis that Aprotinin is a typical multi-target drug or a "magic shotgun" [16] and can represent an efficient treatment for moderate and severe forms of COVID-19. Our first pilot clinical trials confirmed this assumption and showed that Aprotinin is effective as a drug candidate for the prevention [17] and treatment [18] of COVID-19. Therefore, we conducted a prospective clinical study and evaluated the efficacy of Aprotinin and HCQ or Avifavir combination therapy for moderate COVID-19 patients.

Study design and patients
An open non-comparative study of the efficacy and safety of Aprotinin in patients hospitalized with COVID-19 was based on the recommendations of the WHO research and development program [ 19] and the Russian Ministry of Health [20]. Patients aged 18 and older, admitted to hospital with moderate COVID-19-related pneumonia ( Table 1)

Efficacy end points
The primary efficacy end point of the study was laboratory-confirmed time to normalization of the following parameters: elimination of SARS-CoV-2 (defined as two negative results from a RT-PCR assay with at least a 24-hour interval); CRP and D-dimer concentrations. Clinical manifestations, including persistent fever ˃ 38°C, respiratory rate, oxygen saturation, oxygen therapy requirement and biological parameters, including CRP, D-dimer, neutrophil, lymphocyte, and platelet counts, INR, prothrombin and fibrinogen, were recorded at baseline and at discharge from the hospital. The median time to improve the clinical state by 2 points was determined according to the WHO-OSCI. Not to be resuscitated meant that patient was not eligible for transfer to the ICU. Chest CT was done with a single inspiratory phase with patients in the supine position. Radiologists classified the CT scan as typical, equivocal, or negative for COVID-19 and described the main CT features: ground glass opacity, crazy-paving pattern and consolidation. A semi-quantitative scoring system was used to estimate the pulmonary involvement of the observed abnormalities based on the area involved: mild (˂ 25%), moderate (25 -50%), severe (51-75%) or diffuse (˃ 75%) involvements [21].

Statistical analysis
The sample size was based on the exact single stage Phase II assessment at one-sided α=0·05 and 80% power [22]. Continuous variables with a normal distribution were expressed as mean (SD) and with a non-normal distribution as median with interquartile range (IQR) and compared using 2-tailed, paired t test for parametric data and Wilcoxon rank-sum test for nonparametric data. The categorical variables were presented as absolute and relative (in percentage) frequency and compared using chi-square test.
The effect of Aprotinin for COVID-19 patients was done on a full analysis set (including all patients who signed informed consent and received at least one administration of study drug). The efficacy time points (time to viral clearance, time to CRP normalization (≤ 10 mg/L), time to D-dimer normalization (<253 ng/mL), time to temperature normalization (< 37°C), time to improvement in clinical status) were done using Kaplan-Meier curves. Groups comparison was used log-rank test (p-value ˂ 0.05 was considered significant). We used the R (version 3.6.2) for analyses.

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The efficacy of Aprotinin combinations for the normalization of thrombosis markers (D-dimer and fibrinogen) in patients' blood are presented in Table 2 and Figure 2. Initially increased D-dimer levels (525 -855 ng/mL, Table 1) quickly returned back to normal within median 4.5 (IQR 3 -6),  and 5 (IQR 4 -5) days for cohorts 1, 2 and 3 patients, respectively (Table 2, Figure 2A). The difference between cohorts 2 and 3 is statistically significant (p=0.002). Before initiation of Aprotinin combination therapy, patients in cohorts 1 -3 had elevated fibrinogen levels (5·0 -9·8 g/L, Table 1), which returned back to normal on Day 4, presumably as a result of the therapy ( Figure 2B). The dynamics of changes in indicators of INR (prothrombin, neutrophils, and leukocytes) defined normal both when patients were admitted and discharged from the hospital (Figure 3).
The dynamics of INR and prothrombin changes used to monitor blood-thinning anticoagulants and to check blood clotting problem defined normal at the admission and discharge of the patients from the hospital (Figures 3A and 3B). Patients presented a normal value for neutrophils and leukocytes when admitted to the hospital and discharged from the hospital after aprotinin combination therapy ( Figures 3C   and 3D).

Discussion
Currently, there is no particular antiviral therapy suggested for patients admitted to the hospital due to COVID-19-associated pneumonia and requiring oxygen therapy to reduce their transfer to ICU for ALV or NIV. Remdesivir and Favipiravir are the first known drugs repurposed for the treatment of COVID-19 and registered by the US FDA [23] and Russian Ministry of Health [24], respectively. But we did not found the conclusive clinical evidence on its impact on the main markers of the COVID-19 severity -CRP and D-dimer concentration in patients' blood. At the same time, we demonstrated that Aprotinin combination therapy was significantly associated with normalization of CRP level in moderate COVID-

Authors' contributors
OSR and ENS were the Principal Investigators responsible for the recruitment of patients, study treatment, and data collection in compliance with the Protocol. AAI, VGL, EVY and AVI conceived this project, suggested a variants of its organization and controlled the implementation. VNA, NPS, SVP and ANE developed the clinical trial protocol, worked on the statistical aspects of the study and the analysis of the results. EAM and NVK organized the clinical trial and collection of the data. RNK developed the preclinical study design, organized its implementation, did literature search and edited the manuscript.
DVK developed the technology and coordinated the production of the substance. AVI carried out scientific management of the project, wrote and edited the manuscript. All the authors read and approved the final manuscript.

Declaration of interests
The Sponsor of the study -Aviron LLC, Skolkovo Innovative Centre, Moscow, Russian Federation. AAI, AVI, NPS, and DVK are reporting patent RU 2731932 (09.09.2020). Other authors declare no competing interests.

Appendix.
Exclusion criteria were the following: refusal of the patient to participate, patients with respiratory rate > 35 per min that does not decrease after the body temperature drops to normal or sub-febrile values; blood oxygen saturation ≤ 93% at rest; partial pressure of oxygen in arterial blood (SpO2) < 60 mm Hg;    patients.
Normal concentration of CRP is ≤ 10 mg/L. The plot A shows time since initiation of treatment (days).
The plot B shows time from inclusion (days).

Figure 2. Effect of Aprotinin and HCQ or Avifavir (cohorts 1 -3) combination therapy on dynamics of normalization of D-dimer (A) and fibrinogen (B) concentrations in COVID-19 patients.
Normal concentration of D-dimer is ≤ 253 ng/mL. Normal range of fibrinogen is 2 -4 g/L. The plot shows time from inclusion (days).