Zinc sulfate in combination with a zinc ionophore may improve outcomes in hospitalized COVID-19 patients

Introduction COVID-19 has rapidly emerged as a pandemic infection that has caused significant mortality and economic losses. Potential therapies and prophylaxis against COVID-19 are urgently needed to combat this novel infection. As a result of in vitro evidence suggesting zinc sulphate may be efficacious against COVID-19, our hospitals began using zinc sulphate as add-on therapy to hydroxychloroquine and azithromycin. Aim To compare outcomes among hospitalized COVID-19 patients ordered to receive hydroxychloroquine and azithromycin plus zinc sulphate versus hydroxychloroquine and azithromycin alone. Methodology This was a retrospective observational study. Data was collected from medical records for all patients with admission dates ranging from 2 March 2020 through to 11 April 2020. Initial clinical characteristics on presentation, medications given during the hospitalization, and hospital outcomes were recorded. The study included patients admitted to any of four acute care NYU Langone Health Hospitals in New York City. Patients included were admitted to the hospital with at least one positive COVID-19 test and had completed their hospitalization. Patients were excluded from the study if they were never admitted to the hospital or if there was an order for other investigational therapies for COVID-19. Results Patients taking zinc sulphate in addition to hydroxychloroquine and azithromycin (n=411) and patients taking hydroxychloroquine and azithromycin alone (n=521) did not differ in age, race, sex, tobacco use or relevant comorbidities. The addition of zinc sulphate did not impact the length of hospitalization, duration of ventilation or intensive care unit (ICU) duration. In univariate analyses, zinc sulphate increased the frequency of patients being discharged home, and decreased the need for ventilation, admission to the ICU and mortality or transfer to hospice for patients who were never admitted to the ICU. After adjusting for the time at which zinc sulphate was added to our protocol, an increased frequency of being discharged home (OR 1.53, 95 % CI 1.12–2.09) and reduction in mortality or transfer to hospice among patients who did not require ICU level of care remained significant (OR 0.449, 95 % CI 0.271–0.744). Conclusion This study provides the first in vivo evidence that zinc sulphate may play a role in therapeutic management for COVID-19.

Zinc sulfate in combination with a zinc ionophore may improve outcomes in hospitalized COVID-19 patients

INTRODUCTION
The World Health Organization has declared a pandemic due to the spread of the coronavirus disease of 2019  caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) [1,2]. Despite limited and conflicting data, the U.S. Food and Drug Administration authorized the emergency use of hydroxychloroquine for the treatment of COVID-19 with or without azithromycin. Hydroxychloroquine was thought to be efficacious partly based on in vitro activity against SARS-CoV-2 [3]. However, clinical data in humans has yielded mixed and disappointing results [4][5][6][7]. In spite of this, hydroxychloroquine may still have a role at blocking coronavirus replication, when used in combination with zinc, which inhibits coronavirus RNA polymerase activity.
Zinc is an essential trace element that is required for the maintenance of adaptive and innate immune responses [8].

ACCESS
The benefits of zinc have previously been recognized for its therapeutic use against other respiratory viruses including those that cause the common cold [9][10][11]. Zinc has also been observed to improve pneumonia in children and its deficiency is associated with pneumonia in the elderly [9,12,13]. Implicating a role for zinc in COVID-19, zinc inhibits viral RNA-dependent RNA polymerase, and has been shown to do this in vitro against SARS-CoV [14]. When combined with a zinc ionophore, such as hydroxychloroquine, cellular uptake is increased making it more likely to achieve suitably elevated intracellular zinc concentrations for viral inhibition [10,15]. This combination is already being tested as a prophylactic regimen in a prospectively followed cohort (NCT04326725) and in a randomized clinical trial (NCT04377646). Other trials are also investigating this regimen for therapeutic efficacy (NCT04370782, NCT04373733).
As New York became the epicentre of the pandemic, hospitals in the area quickly adopted investigational therapies, including the use of hydroxychloroquine and azithromycin. Given this proposed synergistic effect of zinc with hydroxychloroquine, practices at NYULH changed and the addition of zinc sulphate 220 mg PO BID along with hydroxychloroquine 400 mg once followed by 200 mg PO BID with azithromycin 500 mg once daily became part of the treatment approach for patients admitted to the hospital with COVID-19. This study sought to investigate outcomes among patients who received hydroxychloroquine and azithromycin alone compared to those who received triple therapy of hydroxychloroquine and azithromycin plus zinc sulphate.

METHODS
We performed a retrospective analysis of data from patients hospitalized with confirmed SARS-CoV-2 infection at NYU Langone Health. Data was collected from electronic medical records (Epic Systems, Verona, WI, USA) for all patients being treated with admission dates ranging from 2 March 2020 through 11 April 2020. Patients were admitted to any of four acute care NYU Langone Health hospitals across New York City. COVID-19 positivity was determined by real-time reverse-transcriptase PCR (RT-PCR) of nasopharyngeal or oropharyngeal swabs.
Patients were included in the study if they were admitted to the hospital, had at least one positive test for COVID-19, were ordered to receive hydroxychloroquine and azithromycin, and had either been discharged from the hospital, transitioned to hospice or expired. Patients were excluded from the study if they were never admitted to the hospital or if there was an order for other investigational therapies for COVID-19, including tocilizumab, nitazoxanide, rituximab, anakinra, remdesivir or lopinavir/ritonavir during the course of their hospitalization to avoid potential confounding effects of these medications. We collected demographics as reported by the patient and any past medical history of hypertension, hyperlipidemia, coronary artery disease, heart failure, chronic obstructive pulmonary disease, asthma, malignancy other than non-melanoma skin malignancy and diabetes. We also recorded vital signs on admission, the first set of laboratory results as continuous variables, and relevant medications as categorical variables, including NSAIDs, anticoagulants, antihypertensive medications and corticosteroids ordered at any point during the course of the hospitalization. We compared clinical outcomes of patients who received a combination of zinc sulphate plus hydroxychloroquine, a zinc ionophore, to those that received hydroxychloroquine without zinc supplementation.

Statistics
Patients were categorized based on their exposure to hydroxychloroquine (400 mg load followed by 200 mg twice daily for 5 days) and azithromycin (500 mg once daily) alone or with zinc sulphate (220 mg capsule containing 50 mg elemental zinc twice daily for 5 days) as treatment in addition to standard supportive care. Descriptive statistics are presented as mean and standard deviation or mean and interquartile range for continuous variables and frequencies for categorical variables. Normality of distribution for continuous variables was assessed by measures of skewness and kurtosis, deeming the dataset appropriate for parametric or nonparametric analysis. A two-tailed Student's t-test was used for parametric analysis, and a Mann-Whitney U test was used for nonparametric data analysis. Pearson's chi-squared test was used to compare categorical characteristics between the two groups of patients. Linear regression for continuous variables or logistic regression for categorical variables was performed with the presence of zinc as the predictor variable and outcome measures [duration of hospital stay, duration of mechanical ventilation, maximum oxygen flow rate, average oxygen flow rate, average FiO2, maximum FiO2, admission to the intensive care unit (ICU), duration of ICU stay, death/ hospice, need for intubation, and discharge destination], as dependent variables. Data was log transformed where appropriate to render the distribution normal for linear regression analysis. Multivariate logistic regression was used to adjust for the timing that our protocol changed to include zinc therapy using admission before or after 25 March as a categorical variable. P values less than 0.05 were considered to be significant. All analyses were performed using STATA/SE 16.0 software (STATA Corp.).

Study approval
The study was approved by the NYU Grossman School of Medicine Institutional Review Board. A waiver of informed consent and a waiver of the Health Information Portability Privacy act were granted.

RESULTS
Patients taking zinc sulphate in addition to hydroxychloroquine and azithromycin (n=411) and patients taking hydroxychloroquine and azithromycin alone (n=521) did not differ in age, race, sex, tobacco use or past medical history (Table 1). On hospital admission, vital signs differed by respiratory rate and baseline systolic blood pressure. The  (Table 3). However, after excluding all non-critically ill patients admitted to the intensive care unit, zinc sulphate no longer was found to be associated with a decrease in mortality (Table 3). Thus, this association was driven by patients who did not receive ICU care (OR 0.492, 95 % CI 0.303-0.799). We also found that the addition of zinc sulphate was associated with likelihood of discharge to home in univariate analysis (OR 1.56, 95 % CI 1.16-2.10) ( Table 3). We performed a logistic regression model to account for the time-period when the addition of zinc sulphate to hydroxychloroquine plus azithromycin became utilized at NYULH. After adjusting for this date (25 March), we still found an association for likelihood of discharge to home (OR 1.53, 95 % CI 1.12-2.09) and decreased mortality or transition to hospice however the other associations were no longer significant ( Table 3). The decrease in mortality or transition to hospice was most striking when considering only patients who were not admitted to the ICU (OR 0.449, P value: 0.002) ( Table 3).

DISCUSSION
While practicing at the epicentre of the pandemic in the United States, we were faced with unprecedented challenges of adopting investigational therapies quickly into clinical practice. Initially, antiviral options at our institution consisted of clinician preference for either ritonavir/lopinavir or hydroxychloroquine plus azithromycin. After the findings of ritonavir/lopinavir, we noticed an increase in the use of hydroxychloroquine plus azithromycin [16]. Our providers within the infectious diseases division, clinical pharmacy and hospitalists discussed the use of zinc sulphate as an addition to hydroxychloroquine, based on the potential synergistic mechanism, and low risk of harm associated with this therapy.
There has been significant interest in the use of zinc sulphate to treat and prevent COVID-19 infection and its use is being considered in several trials (NCT04326725, NCT04377646, NCT04370782, NCT04373733, NCT04351490) [9,17,18]. To our knowledge, we provide the first in vivo evidence on the efficacy of zinc sulphate in addition to hydroxychloroquine in COVID-19 patients. The main finding of this study is that the addition of zinc sulphate to hydroxychloroquine and  azithromycin was found to be associated with a decrease in mortality or transition to hospice among patients who did not require ICU level of care, but this association was not seen for patients who were treated in the ICU. This result may reflect one of the proposed mechanisms by which zinc sulphate may provide protection against COVID-19. Zinc has been shown to reduce SARS-CoV RNA-dependent RNA polymerase activity in vitro [14]. As such, zinc may have a role in preventing the virus from progressing to severe disease, but once the aberrant production of systemic immune mediators is initiated, known as the cytokine storm, the addition of zinc may no longer be effective [19]. To further examine the ability of zinc to prevent viral replication and disease progression, future studies should measure viral RNA levels in clinical specimens before and after zinc administration. Our findings suggest a potential protective effect of zinc, potentially enhanced by a therapeutic synergistic mechanism of zinc sulphate with hydroxychloroquine, if used early on in presentation with COVID-19.
After adjusting for the timing of zinc sulphate treatment, the negative associations between zinc and the need for ICU and invasive ventilation were no longer significant but we did still observe a trend. This observation may be because patients with COVID-19 were initially sent to the ICU quicker, but as time went on and resources became more limited, clinicians began treating COVID-19 patients on general medicine floors for longer periods of time before escalating to the ICU. Future studies are needed to confirm or refute the hypothesis that the addition of zinc sulphate to a zinc ionophore such as hydroxychloroquine may reduce the need for ICU care in patients with COVID-19.
This study has several limitations. First, this was an observational retrospective analysis that could be impacted by confounding variables. This is well demonstrated by the analyses adjusting for the difference in timing between the patients who did not receive zinc and those who did. In addition, we do not know whether the observed added benefit of zinc sulphate to hydroxychloroquine and azithromycin on mortality would have been seen in patients who took zinc sulphate alone or in combination with just one of those medications since few patients at our hospitals received zinc sulphate as stand-alone therapy. The optimal dose and formulation of zinc supplementation necessary to inhibit RNA-dependent RNA polymerase activity of coronaviruses also remains unknown. Early reports suggested higher doses may be necessary, ranging from 50 to 150 mg elemental zinc/ day, and such doses have been proved to be safe for short periods of time when used for other viruses or in improving immune reconstitution [20][21][22][23]. In addition, although other formulations of zinc supplementation exist, in the form of acetate or gluconate, our hospital formulary had sulphate, which generally has a higher elemental amount of zinc per tablet. Therefore, further investigation is required to determine whether sulphate is the preferred formulation when used to treat coronaviruses, or if another formulation may be better tolerated. Given the recent concern regarding the potential for side effects associated with hydroxychloroquine, future studies should examine whether zinc sulphate would provide benefit as a stand-alone therapy or in combination with another zinc ionophore. In addition, as zinc is hypothesized to inhibit viral RNA polymerase, future studies are needed to determine if zinc synergistically acts with other antiviral medications. We also do not have data on the time at which the patients included in the study initiated therapy with hydroxychloroquine, azithromycin and zinc. Those drugs would have been started at the same time as a combination therapy, but the point in clinical disease at which patients received those medications could have differed between our two groups. Finally, the cohorts were identified based on medications ordered rather than confirmed administration, which may bias findings towards favouring equipoise between the two groups.

CONCLUSION
Zinc sulphate added to hydroxychloroquine and azithromycin associates with a decrease in mortality or transfer to hospice among patients who do not require ICU level of care and an increased likelihood to be discharged directly home from the hospital. In light of study limitations, this study alone is not sufficient to guide clinical practice. Rather, these findings suggest a potential role for zinc sulphate in COVID-19 patients and support the initiation of future randomized clinical trials investigating zinc sulphate against COVID-19.

Funding information
The authors received no specific grant from any funding agency.