Efficacy and safety of Levamisole treatment in clinical presentations of non-hospitalized patients with COVID-19: a double-blind, randomized, controlled trial

In late December 2019, a novel human coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19), was identified as the cause of a series of pneumonia cases in Wuhan, Hubei Province in China [1‐3]. The outbreak has rapidly spread, resulting in an epidemic throughout China, as well as other countries around the world. On March 12th 2020, the World Health Organization (WHO) announced the outbreak of COVID-19 as a pandemic [4]. Predominant symptoms at the initiation of the disease include the following: fever, cough, myalgia, chills, dyspnea and pneumonia. Less common Symptoms are sputum, headache, hemoptysis and diarrhea [5].

Since the structure of the SARS-CoV and SARS-CoV-2 was similar, the biochemical interactions and the pathogenesis were also expected to be similar [6]. However, the pathogenic mechanism may proceed differently in the novel SARS-CoV-2 and the intermediate host of SARS-CoV2 is yet to be identified. In order to enter into the host cell, the virus needs to bind to the angiotensin-converting enzyme 2 (ACE-2) receptors in the type II pneumocytes in the lungs and form a viral endosome through a clathrin-mediated endocytosis [7‐9]. When internalized, the fusion of virus with lysosomes occurs under low endosomal and lysosomal pH [10]. The binding of the SARS-CoV-2 to the ACE-2 receptors triggers an inflammation cascade in the lower respiratory tract and causes a systemic inflammatory state. Infection of human cells by SARS-CoV-2 results in inflammatory cascade by virus-infected antigen-presenting cells (APCs). APCs present the outsider antigen to CD4 + -T-helper (Th1) cells, and release interleukin-12 which further stimulate B-cells to produce antigen-specific antibodies [11, 12]. Currently, few approved prophylactic or therapeutic medications are available for COVID-19 diseases. Some therapeutic agents are used off-label, alone or in combination [13].

Levamisole is a synthetic low-molecular weight agent which belongs to the anti-helminthic class of medications. Levamisole can enhance cellular immunity depending on the dosage and timing of the administration [14, 15]. In in-vitro models, it has been reported that the combination of Levamisole and ascorbic acid can reverse the depressed helper/inducer subpopulation of lymphocyte in measles virus. The abnormality in lymphocytes number could be reproduced in Levamisole treated lymphocytes in-vitro [15, 16].

Through improving the cellular immunity, Levamisole showed efficacy in various autoimmune and inflammatory diseases [17]. Interestingly, this anti-helminthic medication is able to phagocytose the immune cells to the normal level via its immune regulatory properties. Furthermore, Levamisole improved the function of human interferon and modulates T-cell immunity. The efficacy of this drug has been further documented in the management of some malignancies and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, vitiligo, and viral warts [18]. Levamisole inserts its promising effects on cellular and humoral immunity through regulating the circulating pro-inflammatory cytokines such as interleukins (IL) 6 and 8. Levamisole has also been used in patients with recurrent aphthous ulcers and it is believed to normalize the CD4 + / CD8 + cell ratio and elevates the serum levels of IgA and IgM.

Therefore, considering the pathophysiology of COVID-19 and the immunomodulatory properties of Levamisole, we conducted this double-blind, randomized controlled trial to evaluate the efficacy and safety of Levamisole when compared to the routine standard of care in non-hospitalized patients with mild to moderate COVID-19.

This prospective, double-blind, randomized controlled clinical trial was performed in adult patients aged between 18 to 60 year old with mild to moderate COVID-19 (room-air oxygen saturation > 94%) who referred to the infection clinic of Shahid Sadoughi Hospital, Yazd, Iran. The diagnosis of COVID-19 was based on the results of polymerase chain reaction (PCR) (using the real-time PCR method with Pishtazteb kit, Pishtazteb company, Iran) and radiological manifestations of COVID-19 chest computed tomography (CT) scan during a 4 month period from late April 2020 to mid-August 2020. Patients were randomly assigned into two arms of the study by permutation block method: 25 patients in the control group and 25 patients in the intervention group. This study was approved by the Ethics Committee of Yazd University of Medical Sciences (Ethics ID: IR.SSU.REC.1399.063).

The present study was a randomized controlled clinical trial evaluating the effectiveness of Levamisole in combination with routine care in patients suffering from COVID-19. The results of the current study have demonstrated that patients receiving the combination of Levamisole with routine standard of care had significantly higher chance of better clinical status including cough and dyspnea on day 14 when compared to the placebo. However, the effect-size of this finding has uncertain clinical importance. No significant difference in fever, headache, asthenia, dizziness, myalgia, and nausea was observed on days 1, 3, 7, and 14 between groups.

Currently, the coronavirus COVID-19 pandemic is a global dilemma. Since the main pathogenic mechanism of the disease is unclear, many drugs have been evaluated in an attempt to relieve the symptoms [21]. One of the suggestive agents used for systemic treatment of COVID-19 is Levamisole because of its wide variety of immunological effects including regulation of neutrophils, macrophages, and T-cell activity. Moreover, this drug also modulates the human interferon (IFNs), interleukin-6 (IL-6) and IL-8 [22, 23] and enhances the serum level of immunoglobulin A (IgA) and IgM [24]. The combination of Levamisole with ascorbic acid has shown to reverse the depressed helper/inducer subpopulation of lymphocyte in an in-vitro study. In vitro observations have further shown that the abnormality in lymphocytes could be reproduced in Levamisole-treated lymphocytes. Therefore, Levamisole could be suggested for the treatment of COVID-19 [23, 25].

As with the previous study in China [14], more than half of the infected patients in the current study were men (60%). The most common clinical presentations of 41 infected patients with COVID-19 in Wuhan, China, were the following: fever (98.0%), cough (76.0%), dyspnea (55.0%), myalgia (44.0%), sputum production (28.0%), headache (8.0%), hemoptysis (5%), and diarrhea (3%) [14]. The most common clinical presentations in the current study were fever (88.0%), cough (78.0%), and dyspnea (54.0%). While less common symptoms were asthenia (18.0%), headache (8.0%), dizziness (6.0%), myalgia (6.0%), and nausea (6.0%) at the initiation of the trial. Nevertheless, no significant difference between groups were observed.

Pro-inflammatory state is the second phase of COVID-19 disease and commonly associated with elevation in several inflammatory cytokines including IL-6 and IL-8 that results in acute lung injury, cytokine storm and systemic inflammation [15]. Thus, inflammatory response reduction could be considered as a potential therapeutic target against COVID-19 disease in this phase. Several studies are currently underway to elucidate the potential molecular mechanisms and employ some interventions to suppress this phase of COVID-19 [15, 16]. Heretofore, no specific antiviral treatment is available for COVID-19. While accurate information about the effectiveness of different lmmunomodulators are limited, anti-inflammatory and immunomodulatory therapies has the potential for COVID-19 patients in the pro-inflammatory phase [16].

In severe cases of COVID-19, the plasma level of IL-6 is especially high [26]. Using Tocilizumab (TCZ), a human IL-6 blockade agent, indicated a reduction in lung lesion opacity, oxygen demand, decrease in C-reactive protein, and normalization of lymphocytes count in 90.5, 75, 84.2, and 52.6% in COVID-19 patients, respectively [27]. Levamisole is another anti-IL-6 agent. This drug blocks pro-inflammatory activity of IL-6 and could be effective in the management of COVID-19 patients.

Although the findings of the present study are interesting, care should be taken in implementing these results and the limitations of the study should be considered. First, the main limitation was the small size of the subject groups. It should be pointed out that until now; the efficacy of Levamisole in COVID-19 management has not been evaluated. Thus, this primary evaluation is the first trial of Levamisole to gain an insight for future studies. Second, because of the urgent circumstances in which the study was done, the effect of Levamisole on SARS-CoV-2 viral load were not assessed. Third, while the dose of Levamisole was chosen based on previous data, the effective dose for COVID is yet to be determined. The last limitation is that other laboratory variables which could be used in identifying additional predictors of patients’ outcomes were not collected.

Given the small sample and the lack of power to detect an efficacy outcome difference, we cautiously interpret these data to suggest the possibility of a beneficial effect and the absence of harm. Thus, we conclude that further efficacy evaluation in the form of a larger clinical trial is warranted.

Previous investigation found that lmmunomodulators play an important role in management of patients infected with COVID-19 [21, 28]. Levamisole is a safe and successful immunomodulatory drug, suggesting that it may also attenuate the exaggerated immune response associated with COVID-19 severity. Our results indicated that Levamisole can efficiently improve some of the Clinical Status when compared to placebo.

The results of the current study have demonstrated that adding Levamisole to the routine standard of care significantly enhanced the overall clinical status including cough and dyspnea in COVID-19 patients when compared to the placebo/routine care. However, the effect-size of this finding has uncertain clinical importance.