key: cord-0847048-w5zxzpj7 authors: SHI, C.; WANG, C.; WANG, H.; YANG, C.; CAI, F.; ZENG, F.; CHENG, F.; LIU, Y.; ZHOU, T.; DENG, B.; LI, J.; ZHANG, Y. title: Clinical observations of low molecular weight heparin in relieving inflammation in COVID-19 patients: A retrospective cohort study date: 2020-04-01 journal: nan DOI: 10.1101/2020.03.28.20046144 sha: d8a3c1fcef5f6368eb1d55864373bb44c19b7db8 doc_id: 847048 cord_uid: w5zxzpj7 Abstract: Background On March 11, 2020, the World Health Organization declared its assessment of COVID-19 as a global pandemic. Effective therapeutic drugs are urgently needed to improve the overall prognosis of patients, but currently no such drugs are available. Methods Patients in the study were divided into a heparin and a control group based on whether low molecular weight heparin (LMWH) was used. D-dimer, C-reactive protein (CRP), peripheral blood lymphocyte percentage, interleukin-6, and other indices in 42 patients with novel coronavirus pneumonia were retrospectively analyzed to compare and evaluate the progress of patients before and after LMWH treatment. Results Compared to the control group, D-dimer levels in the heparin group significantly increased before treatment, and there was no significant difference after treatment. There was no significant difference in the change of CRP levels between the two groups of patients before and after LMWH treatment, and levels for both groups were significantly lower after, compared to before, treatment. Compared to the control group, patients in the heparin group had a higher percentage of lymphocytes after treatment and lower levels of interleukin-6; these differences were statistically significant. Conclusions Under conventional antiviral treatment regimens, LMWH can improve hypercoagulability, inhibit IL-6 release, and counteract IL-6 biological activity in patients. LMWH has potential antiviral effects and can help delay or block inflammatory cytokine storms. It can also increase the lymphocytes (LYM%)of patients and has the potential for treatment of COVID-19. Coronavirus is an enveloped, non-segmented, positive-sense, single-stranded RNA virus that causes common colds and severe respiratory diseases, 1 such as Middle East respiratory syndrome (MERS) 2 and severe acute respiratory syndrome (SARS) 2 . In December 2019, a series of unexplained pneumonia cases appeared in Wuhan, Hubei . 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 April 1, 2020. . https://doi.org/10.1101/2020.03.28.20046144 doi: medRxiv preprint Province, China, and their clinical manifestations suggested viral pneumonia. 4 Deep sequencing of lower respiratory tract samples identified a novel coronavirus named SARS-CoV-2 and the disease it caused was named COVID-19. 5 Its clinical manifestations include fever, cough, sputum, chest distress or asthma, fatigue, myalgia, diarrhea, nausea, and vomiting. 6, 7, 8 Severe cases may progress rapidly to acute respiratory distress syndrome, metabolic acidosis, septic shock, coagulopathy, and organ failure (e.g., liver, kidneys, and heart), which all pose a serious threat to human health. On March 11, 2020 , the World Health Organization (WHO) declared its assessment of COVID-19 as a global pandemic. SARS-CoV-2 is characterized by a long incubation period, high infectivity, and multiple routes of transmission. 9, 10 According to real-time WHO statistics, the total number of confirmed cases of COVID-19 worldwide as of March 28, 2020 has exceeded 600,000, with more than 28,000 deaths. However, no effective medicines are currently available, and it can only be treated symptomatically. As the worldwide patient base continues to expand and the number of severely and critically ill patients increases rapidly, determining the mechanism through which patients progress from mild to severe and from severe to critical is the main avenue for discovering effective treatment strategies. Lymphopenia and inflammatory cytokine storms are typical abnormalities observed in highly pathogenic coronavirus infections (such as SARS and MERS), 12 and are believed to be associated with disease severity. 12, 13, 14 Multiple studies have shown that cytokine storms are important mechanisms of disease exacerbation and death in patients with COVID-19. 12, 13, 14 IL-6 levels are significantly higher in severely ill patients with COVID-19 compared to those with mild cases. 15 A cytokine storm may occur when cytokines reach a certain threshold in the body. 16 Reducing the release or activity of IL-6 can prevent or even reverse the cytokine storm syndrome caused by the virus, thereby improving the condition of patients with COVID-19. In recent years, a large number of studies have revealed that low molecular weight heparin (LMWH) has various non-anticoagulant properties that play an anti-inflammatory role by reducing the release and biological activity of IL- 6. 17,18,19 . 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 April 1, 2020. . https://doi.org/10.1101/2020.03.28.20046144 doi: medRxiv preprint However, the anti-inflammatory effects of LMWH in COVID-19 are not currently known. To the best of our knowledge, this is the first retrospective cohort study, to analyze the relieving effect of LMWH in patients with COVID-19. This study aims to review and analyze the treatment course of patients with COVID-19 to investigate the anti-inflammatory effects of heparin and delay disease progression to provide guidance for subsequent clinical practice. All cases in this study were located at Union Hospital, Tongji Medical College, (2) patients with liver and kidney insufficiency or congenital heart disease; (3) patients who had been treated with LMWH in the previous three months; (4) patients with a previous history of mental illness; (5) pregnant or lactating women; (6) patients clinically classified as critically ill or housed in the intensive care unit (ICU); (7) patients allergic to LMWH or contraindicated for LMWH. . 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 April 1, 2020. . https://doi.org/10.1101/2020.03.28.20046144 doi: medRxiv preprint Basic patient information collected in this study from electronic medical records included complete blood count, coagulation function, cytokines, serum biochemical tests (including liver function, kidney function, lactate dehydrogenase, C-reactive protein (CRP), and electrolytes), and disease progression. Two researchers also independently reviewed the data collected forms to double check the data collected. Data analysis was performed using SPSS 22.0 statistical software. Data were expressed as mean ± standard deviation (SD). GraphPad 6.0 software was used for plotting. Differences between groups were evaluated using the T-test for measurement data, the chi-square test for count data, and the Kruskal-Wallis nonparametric test between groups (independent samples) and within groups (related samples). Differences of p < 0.05 were considered statistically significant. As shown in Table 2 , the heparin group consisted of 13 males and eight females aged between 42 and 91 years (median age was 69.0 years), and the control group consisted of 14 males and seven females aged between 40 and 84 years (median age was 69.0 years); there was no significant difference between the two groups. There were no significant differences in comorbidities, such as hypertension, diabetes, cardiovascular disease, and carcinomas, between the two groups. Similarly, there were no significant differences in novel coronavirus pneumonia onset symptoms, including fever (body temperature ≥ 37.3°C), cough, sputum, chest distress or asthma, myalgia, fatigue, anorexia, diarrhea, and nausea and vomiting. Similarly, there was no significant difference in antiviral treatment between the two groups. These results indicate that the general characteristics of the two groups of patients were consistent and comparable. LMWH has no effect on the days to conversion to negative and the duration of hospitalization of patients with COVID-19 As shown in Table 2 , the number of days to conversion to negative (time from . 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 April 1, 2020. Notably, all patients showed improvement after treatment. As shown in Figure 2A -F, there was no significant difference in lymphocyte percentage between the heparin and control groups before and after LMWH treatment. However, patients in the heparin group had a significantly increased percentage of lymphocytes after LMWH treatment. In addition, the changes in lymphocyte percentages in patients in the heparin group before and after LMWH treatment were significantly different to those in the control group. The results suggest that LMWH increases lymphocyte percentage in patients with COVID-19, indicating that LMWH has some anti-inflammatory effects. Compared with the control group, platelets were significantly increased after LMWH treatment. Similarly, the changes in platelet levels before and after LMWH treatment in the heparin group were significantly different from those in the control group. However, there was no significant difference in RBC, WBC, neutrophil% and monocyte% levels between the two groups. As shown in Figure 2G -N, before patients were administered LMWH, the levels of D-dimer and fibrinogen degradation products (FDP) in patients in the heparin group were significantly higher compared to those in the control group, indicating that these patients may have been in a hypercoagulable state. After the administration of LMWH, D-dimer and FDP levels in the heparin group significantly decreased, and there was no significant difference with the control group. The D-dimer and FDP levels of patients in the heparin group before and after LMWH administration were significantly different. The results indicate that LMWH improves the hypercoagulable state in patients with COVID-19. However, there was no significant difference in TT, . 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 April 1, 2020. . https://doi.org/10.1101/2020.03.28.20046144 doi: medRxiv preprint APTT, PT, FIB, ATⅢ and INR levels between the two groups. As shown in Figure 2O , there were no significant differences in CRP levels between the two groups of patients before and after LMWH treatment, and both were significantly lower compared to before treatment. Similarly, there were no significant differences in CRP levels between the two groups of patients. To further investigate the anti-inflammatory effects of LMWH, we performed statistical analyses on the levels of inflammatory cytokines in the two groups ( Figure 3 ). There were no significant differences between the levels of IL-2, IL-4, IL-10, TNF-α, and IFN-γ in the heparin group and those of the control group before and after LMWH treatment. Similarly, the changes in IL-2, IL-4, IL-10, TNF-α, and IFN-γ levels were not significantly different between the two groups. Notably, there was no significant difference in IL-6 levels between the heparin and control groups before LMWH treatment. After LMWH treatment, IL-6 levels in the heparin group were significantly reduced compared to the control group. Similarly, the changes in IL-6 levels in the heparin group before and after LMWH treatment were significantly different to those in the control group. The results suggest that the anti-inflammatory effects of LMWH may be associated with the reduction of IL-6 levels in patients with COVID-19. On March 11, 2020, the WHO declared its assessment of COVID-19 as a global pandemic. Although its case fatality rate is only 2.27%, 12 the number of severely and critically ill patients is increasing rapidly as the global patient base continues to expand. Studies have shown that cytokine storms are associated with deterioration in several infectious diseases, including SARS and avian influenza. 20 Cytokine storms are an important mechanism of exacerbation in patients. 21 In recent years, numerous studies have revealed that heparin has various non-anticoagulant properties. LMWH can exert anti-inflammatory effects by reducing the release and biological activity of IL-6. However, the anti-inflammatory effects of heparin in COVID-19 are not yet . 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 April 1, 2020. 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 April 1, 2020. . https://doi.org/10.1101/2020.03.28.20046144 doi: medRxiv preprint consider this parameter as an evaluation index for disease progression. The average values of d-dimer and FDP before treatment was greater in the heparin group than in the control group (3.75,1.23,P ＜ 0.01;14.35,4.05,P ＜ 0.01). This result is consistent with the goals of LMWH treatment in the heparin group. The analysis of differences showed that LMWH has a better effect on lowering D-dimer and FDP levels. Several studies have recommended CRP and lymphocytes (LYM%)as indices for evaluating the effectiveness of clinical drugs or treatments. 37,38, 39 In the analysis of differences in the present study, there was no statistically significant difference in CRP between the groups, indicating that LMWH treatment has no effect on this parameter. In the analysis of differences of LYM%, the mean value of the heparin group was higher than that of the control group (11.10% and 3.08%, respectively). LYM% was higher in the heparin group after treatment compared to the control group (p < 0.001), which is consistent with the results of Derhaschnig et al. 40 This suggests that LMWH can increase LYM% in patients with COVID-19 and improve their condition. There are two possible reasons for this; on one hand, LMWH is a glycosaminoglycan 41 that partially inhibits the SARS-related coronavirus strain HSR1. 42 Mycroft-West et al. also showed that LMWH can bind to the SARS-CoV-2 surface protein (Spike) S1 Receptor Binding Domain and block the replication of the virus, thus, showing potential antiviral effects. 43 Additionally, LMWH can reduce lymphocyte death caused by the direct viral infection of lymphocytes. 13 On the other hand, preliminary research has confirmed that proinflammatory cytokines, such as TNFα and IL-6, can induce lymphopenia. 14 Therefore, decreases in the number or activity of relevant inflammatory factors may have some significance for the increase in LYM%. Studies have shown that cytokine storms are important mechanisms of exacerbation in patients. 21 IL-6 levels in severely ill patients with COVID-19 are significantly higher than in patients with mild cases, 15 and a cytokine storm may occur when cytokines reach a certain threshold in the body. 16 The transition from a mild to severe condition in patients with COVID-19 may be caused by cytokine storms. Reducing IL-6 release or activity can prevent or even reverse the cytokine storm syndrome caused by the . 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 April 1, 2020. 45 Multiple studies have shown that LMWH may reduce the release of IL-6 in the body by regulating plasma AT 46 or APC 47 levels and inhibiting the expression of nuclear factor κB (NF-κB). 17, 18, 19 In the present study, we performed statistical analysis of the levels of inflammatory cytokines in two groups of patients, and the results showed that IL-6 significantly decreased in the heparin group compared to the control group, whereas the changes in other inflammatory factors were not statistically significant; these results are consistent with the conclusions above. In addition, a study by Mummery et al. found that LMWH can bind to IL-6, competitively reduce the binding of IL-6 to SIL-6R and sgp130, 48 and block signal transduction, thereby inhibiting the biological activity of IL-6. This indicates that LMWH reduces the release of IL-6 while also reducing its biological activity, which also explains the increase of LYM% in the heparin group. The present study found that under conventional antiviral treatment regimens, LMWH could improve hypercoagulability, inhibit IL-6 release, and inhibit IL-6 biological activity in patients. LMWH has potential antiviral effects, helps delay or block inflammatory cytokine storms, increases LYM%, and may be suitable for COVID-19 treatment. In addition, to further confirm the conclusions of this study, we conducted a prospective clinical study to evaluate the efficacy and safety of enoxaparin sodium in the treatment of hospitalized adult patients with COVID-19 ( Chinese Clinical Trial Registry，number:chiCTR2000030700), with the expectation of providing a more powerful reference for treatment. This study has some limitations. First, due to the retrospective design, we were unable to control the time intervals between examinations of various indices in patients and the LMWH dosing schedule, and we could not estimate the effective dose and timing of LMWH. Second, there were no critical cases in the two groups of patients in the trial; the treatment outcome of all cases was improvement and discharge and there were no deaths. Therefore, the reference value for the treatment outcomes of critically ill patients was limited. Finally, the sample size and single-center design may have . 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 April 1, 2020. . https://doi.org/10.1101/2020.03.28.20046144 doi: medRxiv preprint limited our findings. CS, JP and YZ conceptualized and designed the study, and CS and YZ had full access to all data, and took responsibility for data integrity and accuracy of the analysis. CS, CW and HX wrote the manuscript. CY, FC and FZ reviewed the manuscript. FC, YH, TT and BD performed the statistical analysis. All authors contributed to data acquisition, analysis and interpretation, and approved the final version for submission. All authors declare no competing interests. The data used to support the findings of this study are included within the article. No funding was received. This work was supported by the National Key Research and Development Plan of China (2017YFC0909900). . 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 April 1, 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. The copyright holder for this preprint this version posted April 1, 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. The copyright holder for this preprint this version posted April 1, 2020. There were no significant differences in age, sex, comorbidities, onset symptoms, time from hospitalization to virus shedding, length of hospital stay, antiviral treatment and disease progression between the two groups. Data are median (IQR) or n(%). p values are comparing heparin group and control. NA=not applicable. Data are expressed as mean ± standard deviation (SD) (n = 21). C1 vs. H1 or C2 vs. H2, a p < 0.05, aa p < 0.01, aaa p < 0.001; C1 vs. C2 or H1 vs. H2, b p < 0.05, bb p < 0.01, bbb p < 0.001; C3 vs. H3, c p < 0.05, cc p < 0.01, ccc p < 0.001. (C1: control group, indices at admission; C2: control group, indices at discharge; C3: control group, changes in indices during hospitalization; H1: heparin group, indices before LMWH treatment; H2: heparin group, indices after LMWH treatment; H3: heparin group, changes in indices before and after LMWH treatment.) 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 April 1, 2020. . https://doi.org/10.1101/2020.03.28.20046144 doi: medRxiv preprint two groups of patients with COVID-19. Data are expressed as mean ± standard deviation (SD) (n = 21). a p < 0.05, aa p < 0.01, aaa p < 0.001; C1 vs. C2 or H1 vs. H2, b p < 0.05, bb p < 0.01, bbb p < 0.001; C3 vs. H3, c p < 0.05, cc p < 0.01, ccc p < 0.001. (C1: control group, indices at admission; C2: control group, indices at discharge; C3: control group, changes in indices during hospitalization; H1: heparin group, indices before LMWH treatment; H2: heparin group, indices after LMWH treatment; H3: heparin group, changes in indices before and after LMWH treatment.) . 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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