key: cord-269659-a1gf76bu
authors: Hong, W.; Chen, Y.; You, K.; Tan, S.; Wu, F.; Tao, J.; Chen, X.; Zhang, J.; Xiong, Y.; Yuan, F.; Yang, Z.; Chen, T.; Peng, P.; Tai, Q.; Wang, J.; Zhang, F.; Li, Y.
title: Celebrex adjuvant therapy on COVID-19: An experimental study
date: 2020-05-11
journal: nan
DOI: 10.1101/2020.05.05.20077610
sha: 
doc_id: 269659
cord_uid: a1gf76bu

Background: The world is under serious threat with the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19). However, there is no effective drug for the treatment of COVID-19. Based on analyses of available data, we deduced that the excessive prostaglandins E2 (PGE2) accumulation mediated by cyclooxygenase-2 (COX-2) was the key pathological basis of COVID-19. Methods: The urine PGE2 levels were measured by mass spectrometry. An experimental study about Celebrex to treat COVID-19 was conducted based on routine treatment. A total of 44 confirmed COVID-19 patients were enrolled (Experimental group n=37, Control group n=7). Patients in experimental group were given Celebrex once or twice a day (0.2 g/time) for 7-14 days. The dosage or duration was modified for individuals. Clinical outcomes of Celebrex adjuvant therapy were evaluated by vital signs, laboratory tests, and computed tomography upon the discontinuance of Celebrex. Results: We found that the concentrations of PGE2 in urine samples of COVID-19 patients were significantly higher than that of healthy individuals (mean value is 170 ng/ml vs 18.8 ng/ml, p<0.01) and positively correlated with the progression of COVID-19. Among the experimental group (ordinary n=29, severe n=7, critical n=1), 25 cases were treated with full dose and 11 cases with half dose of Celebrex, and 1 case with Ibuprofen. The remission rate were 100%, 82% and 57% in full dose, half dose and control group respectively. Celebrex significantly reduced the PGE2 levels and promoted recovery of ordinary or severe COVID-19. Conclusion: Our study suggests that Celebrex adjuvant treatment may be helpful for the therapy of COVID-19.

The severe acute respiratory distress syndrome (ARDS) caused by SARS-CoV, MERS-CoV and SARS-CoV-2 infections is a major factor of mortality. It has been found that the nucleocapsid protein (N) and spike glycoprotein (S) of SARS-CoV can directly bind to the promoter of cyclooxygenase-2 (COX-2) gene, which drives overexpression of COX-2 in a dose-dependent manner. [1, 2] Homologous analysis showed that there were 90.6% and 75.8% similarity of the N and S protein between SARS-CoV and SARS-CoV-2. Therefore, it is possible that the SARS-CoV-2 infection might also hold the potential to induce COX-2 overexpression in lung epithelial cells, resulting in a significant accumulation of prostaglandins, especially prostaglandin E 2 (PGE 2 ).

Excessive PGE 2 levels may participate in COVID-19 pathology with one of the following mechanisms: 1) binding to the EP2 receptor causing fever, pain, acute inflammation, and enhanced vascular permeability; 2) binding to the EP3 receptor leading to edema, inflammatory mucus secretion, increased viscosity of exudates covered alveoli, bronchioles blockage, thus hindering blood oxygen exchange; [3] 3)

binding to the EP4 receptor, causing bronchial contractions and spams, [4] increasing airway resistance, causing respiratory and hemodynamic disorders, ARDS and multiorgan failures; 4) inhibition of T lymphocyte functionality, by promoting amplification, differentiation and proliferation of Th1 and Th17 subtypes through EP4 receptor causing contact hypersensitivity of bronchioles;[5] 5) PGE 2 and thromboxane A2 (TXA2) activate platelet aggregation and thrombosis, contributing to pulmonary hypertension in ischemia-reperfusion lung injury. [6] Under the circumstances of this international emergency and the situation of having no effective drugs for COVID-19, it makes great sense to explore the pathological mechanisms of COVID-19 and to establish an integrated strategy for diagnosis and treatment with available drugs using the discovered key pathological target(s).

Here, we propose that excessive PGE 2 may be a key in the pathology of COVID-19 and that COX-2 is the critical target for therapy. To test this hypothesis, the urinary PGE 2 levels were determined in COVID-19 patients to verify its correlation with disease status. And Celebrex, a specific inhibitor of COX-2, was to be used for experimental study.

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This was a prospective study done at Guangzhou Eighth People's Hospital. Patients with SARS-CoV-2 infection were confirmed by next-generation sequencing or realtime RT-PCR according to a previously published protocol.

[7] Based on the "Diagnosis and Treatment Guideline for COVID-19" of China, the clinical criteria for classification of OVID-19 stage in Chinese Guideline are listed as following. Mild:

The clinical symptoms are mild and no pneumonia manifestation can be found in CT imaging; Ordinary: Fever and respiratory tract symptoms, etc. and pneumonia manifestation can be seen in CT imaging; Severe: Meeting any of the following: 1) Respiratory distress, RR≥30 breaths/min; 2) Oxygen saturation ≤ 93% at a rest state; 3)

Arterial partial pressure of oxygen (PaO2)/oxygen concentration (FiO2) ≤ 300mmHg;

Critical: Meeting any of the following: 1) Respiratory failure occurs and mechanical ventilation is required; 2) Shock occurs; 3) Complicated with other organ failure that requires Intensive care unit (ICU) care.

A total of 44 confirmed COVID-19 patients, who were admitted to hospital from January 23 to February 15, 2020, were enrolled into this study (Table 1 and Table S1 ).

One of the 44 patients were classified as critical case (2.2%), 7 of the 44 patients were classified as severe type cases (16%) and the others were ordinary type cases (81.8%).

The enrolled patients are fully aware of the purpose, benefits and potential risks, and signed the informed consent prior to this study. This investigational study design was approved by the Medical Ethics Committee of Guangzhou Eighth People's Hospital (Approve number: AF/sc-02/01.6). The study was registered on the Chinese Clinical Trials Registry, ChiCTR2000031630.

The clinical information including the physical, laboratory tests and chest computed tomography (CT) of COVID-19 patients were collected.

The patients in experimental group were treated with Celebrex (Celecoxib, Pfizer, Dalian, China) in combination with routine treatments suggested by the guideline.

The usage and dose of Celebrex was once or twice a day (0.2 g/time) for 7-14 days by . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 11, 2020. . oral. The dosage or duration of medication was subject to change based on each individual case. Routine treatments were according to the National Guideline, including isolation, nursing, bed rest, symptomatic and supportive treatment, antibiotics, antiviral medication, glucocorticoids, oxygen therapy and/or assisted breathing.

Based on the comparison of sequential chest CT images, as well as the changes of symptoms and laboratory test results, the clinical outcomes will be classified with three categories included remission, constant and exacerbation. Remission was defined as dissipating/clarifying of the mass opacities in chest CT images, decreasing the D-dimer, CRP, serum alanine aminotransferase and aspartate aminotransferase levels and improvement of lymphopenia and neutrophilia; the constant and exacerbation outcomes were evaluated accordingly with the changes of those parameters. The length of Celebrex treatment was determinate by the individual conditions. The discharge standards according to the national guideline are listed as following: 1) with normal body temperature for more than 3 days; 2) with significantly recovered respiratory symptoms; 3) lung imaging shows obvious absorption and recovery of acute exudative lesion; 4) with negative results of the nucleic acid tests of respiratory pathogens for consecutive two times (sampling interval at least 1 day). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The differences between two groups of data were compared by Student's-t test, and the statistical results were expressed by mean ± standard error (mean ± SEM). The statistical analysis was conducted by using GraphPad Prism 7 software. p <0.05 was considered statistically significant.

The concentrations of PGE 2 in urine samples were determined by a method of mass spectrometry ( Figure S1 ). Our data showed that the PGE 2 levels in COVID-19 patients, who were hospitalized within two days, were significantly higher than the ones of healthy individuals (170±40 ng/ml vs 18.8±3.8 ng/ml, p<0.01) (Figure 1 ). We determined that the normal threshold of PGE 2 concentration in urine is lower than 20 ng/ml and that 100 ng/ml is considered to be significant as a risk line.

Since the PGE 2 was mainly generated by COX-2, then a COX-2 specific inhibitor (Celebrex) was used to treat COVID-19 patients based on the routine treatment. On

March 19, 2020, 25 cases (6 severe, 19 ordinary) were given full dose (0.2g, twice a day) of Celebrex, and all cases showed an improved outcomes after discontinuation.

There were 11 cases (2 severe/critical, 9 ordinary) that had received half dose (0.2g, once a day) of Celebrex, and all the ordinary cases showed improvement, while the 2 severe/critical cases were exacerbated after discontinuation of treatment. Whereas in control group (n=7), 4 cases were improved and 3 cases were exacerbated at day 10 after admission in hospital (Table 1 and Table S1 ). Our results indicated that Celebrex treatment with a conventional dose (0.2 g, twice a day) might effectively promote the recovery of ordinary and severe cases of COVID-19.

It has been reported that 15.7% of the ordinary COVID-19 cases progressed to a severe stage under routine treatments.

[9] With Celebrex treatment, none of the 29 ordinary cases progressed to a severe classification (Table S1 ). Two cases were chosen to represent the control (case C1) and experimental group (case E20) respectively ( Figure 2) . The PGE 2 level of C1 remained at a high level (>1000 ng/ml) during day 5-9 ( Figure 2A ). While the case E20 treated with Celebrex decreased steadily from over 1000 ng/ml (day 2) to 100 ng/ml (day 3-4) ( Figure 2B ). Except the improvements of symptoms and laboratory tests, the chest CT images (at day 4-10)

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The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.05.20077610 doi: medRxiv preprint showed that the improvement of pulmonary opacification and pneumonia of case E20 was faster than the control group C1 case in the same period ( Figure 2C and D) . It suggested that Celebrex might promote the recovery process of ordinary COVID-19

patients.

In addition, none of the severe COVID-19 cases with full dose of Celebrex treatment progressed to critical illness. The comparison of the control (case C2) and experimental cases (case E6) with similar severe pulmonary opacification diagnosed by CT imaging was illustrated (Figure 3 ). The PGE 2 levels of case C2 fluctuated around 1000 ng/ml at day 5-12 after admitted to hospital with routine treatment ( Figure 3A ). In contrast, the PGE 2 levels of case E6 in Celebrex group were steady decrease, and remained at lower level than 100 ng/ml ( Figure 3B ). Indeed, the chest CT images (at day 3, 6 and 11) of case E6 showed that the ground glass-like opacities were clarified continually, and significantly faster than the control case C2 ( Figure 3C and D). It suggests that Celebrex might reverse the progress of severe COVID-19 and prevented the progression to a critical stage.

Moreover, there were two patients (experimental case E3 and E5), who were hospitalized and received routine treatment for 12 and 15 days respectively, progressed from ordinary to severe illness. After taken Celebrex based on routine treatment, their PGE 2 levels were controlled and the pneumonia were gradually improved ( Figure 4 ). These findings indicated that Celebrex may also be effective on patients who progressed from ordinary into severe type under routine therapy.

Altogether, our findings suggested that the increase of PGE 2 may be critical for progression of COVID-19 and Celebrex intervention may effectively promote the recovery of ordinary and severe types of COVID-19.

ARDS is the leading cause of death of the critical SARS-CoV, MERS-CoV and SARS-CoV-2 infected patients. Autopsy reports indicate that the pathological manifestation of lung injury were diffuse alveolar injuries, including fibrin mucus exudation, ground-glass edema membrane formation, and alveolar epithelial cell detachment.[10-12] However, the pulmonary fibrosis and consolidation caused by . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.05.20077610 doi: medRxiv preprint SARS-CoV-2 was less severe than SARS-CoV, but the alveolar mucous secretion was more severe.

[13] Therefore, it was speculated that the COVID-19 patient's alveolar blood and gas exchange was seriously blocked, which led to ARDS. The patient eventually died from respiratory and multi-organ failures.

Prostaglandin is a class of small lipid molecules transformed from arachidonic acid by COX-1 and COX-2, and PGE 2 is one of the most active molecule. We confirmed that the urine PGE 2 concentration of COVID-19 patients was significantly higher than healthy individuals. Celebrex, a specific inhibitor of COX-2, effectively decreased the level of urinary PGE 2 and promoted the recovery of COVID-19. However, we also found that, in some cases, discontinuation of Celebrex might lead to increased PGE 2 and pneumonia relapse, such as experimental cases E1 and E9, whose PGE 2 levels were rebounded and increased quickly accompanied with slightly worsen of pulmonary opacification ( Figure S2 ). These observations suggested that the duration of Celebrex treatment should be determined according to each patient's condition to reduce the risk of disease relapse, deterioration and pulmonary fibrosis.

The half-life of PGE 2 in vivo is about one minute.

[17] After going through the lung, liver and kidney organs via blood circulation, up to 90% of PGE 2 will be degraded.

Extremely high concentrations of PGE 2 (up to 2500 ng/ml) were detected in the urine of COVID-19 patients, such as control case C1 and C2 (Figure 2 and 3) , so it is conceivable that the PGE 2 levels in lung tissues and blood could be much higher. This kind of "prostaglandin storm" occurs in the very early and progressive stages of COVID-19. While very few immune cytokines could be detected in blood or urine during this early period, which could be occurred at the critical stage.

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The copyright holder for this preprint this version posted May 11, 2020. Taken together, COX-2 overexpression accompanied with PGE 2 accumulation may be a key in the molecular pathology of COVID-19. Celebrex, a specific COX-2 inhibitor, may be an effective drug for the treatment on COVID-19. However, our study was not a rigorous randomized, double-blind and controlled clinical trial. There is needed another well-designed large-scale clinical trial to validate this hypothesis.

Our study may provide useful information for the treatment of COVID-19.

This study design was approved by the Medical Ethics Committee of Guangzhou Eighth People's Hospital.

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The copyright holder for this preprint this version posted May 11, 2020. . * Data are mean (SD) or n (%); Reduced denominators indicate missing data. Percentages may not total 100 because of rounding. BID twice a day; QD once a day.

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The copyright holder for this preprint this version posted May 11, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.05.20077610 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.05.20077610 doi: medRxiv preprint The representation of sequential chest CT images illustrated therapeutic outcomes of these two cases under Celebrex intervention respectively.

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Nucleocapsid protein of SARS-CoV activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa B and CCAAT/enhancer binding protein

Spike protein of SARS-CoV stimulates cyclooxygenase-2 expression via both calciumdependent and calcium-independent protein kinase C pathways

Prostaglandin E2-EP3 signaling induces inflammatory swelling by mast cell activation

All authors declared no conflict of interests.

This study was supported by The Emergency and Special Research Project for 

The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.This project was conducted in the national emergency period; we would like to thank the Guangdong General Police Hospital, Guangdong He Yi Company, Shenzhen Giant Future Company and Guangzhou Zhongke Baier Company for donating protective uniforms, masks, bio-safe transport equipment and other materials, as well as the many volunteers for their selfless dedication and hard work.