Lactoferrin as Antiviral Treatment in COVID-19

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Lactoferrin (Lf), also known as lactotransferrin (Ltf), is a multifunctional protein of the transferrin family.

Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, such as milk, saliva, tears, and nasal secretions. Lactoferrin is also present in secondary granules of PMNs and is secreted by some acinar cells.

A group of Italian researchers in 2021 demonstrated via a clinical trial that Lactoferrin could be used as an antiviral to treat COVID-19.
 
The Italian study team conducted an in vivo preliminary study to investigate the antiviral effect of oral and intranasal liposomal bovine Lf (bLf) in asymptomatic and mild-to-moderate COVID-19 patients. From April 2020 to June 2020, a total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided into three groups. Thirty-two patients (14 hospitalized and 18 in home-based isolation) received only oral and intranasal liposomal bLf; 32 hospitalized patients were treated only with standard of care (SOC) treatment; and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, untreated, healthy subjects were added for ancillary analysis.
 
Interestingly, liposomal bLf-treated COVID-19 patients obtained an earlier and significant (p < 0.0001) SARS-CoV-2 RNA negative conversion compared to the SOC-treated and untreated COVID-19 patients (14.25 vs. 27.13 vs. 32.61 days, respectively). Liposomal bLf-treated COVID-19 patients showed fast clinical symptoms recovery compared to the SOC-treated COVID-19 patients.

In bLf-treated patients, a significant decrease in serum ferritin, IL-6, and D-dimers levels was observed. No adverse events were reported.
 
These findings led the COVID-19 Supplements study team to speculate a potential role of bLf in the management of mild-to-moderate and asymptomatic COVID-19 patients.
 
The study findings were published in the peer reviewed International Journal of Environmental Research And Public Health.
https://www.mdpi.com/1660-4601/18/20/10985/htm

The ability of bLf to hinder viral infection is generally attributed to its binding to cell surface anionic components and/or viral particles. BLf is able to competitively bind to HSPGs, components of the host cell surface and identified as initial interaction sites for enveloped viruses such as SARS-CoV-2 [7,54,55], thus hindering viral adhesion and internalization [34]. Moreover, bLf can also bind directly to surface spike glycoproteins of SARS-CoV-2 particles, as demonstrated in silico [34].

In order to explore the helpful antiviral and immunomodulating effects of bLf and its possible role in the management of asymptomatic and mild-to-moderate COVID-19 patients, we designed a preliminary clinical trial to investigate the effect and safety of this natural glycoprotein belonging to innate immunity.

We focused our research on asymptomatic and mild-to-moderate COVID-19 patients, considering them a transmission reservoir with possible evolution to the most severe disease form [56]. Li et al. [57], analyzing the viral shedding dynamics in asymptomatic and mildly symptomatic patients infected with SARS-CoV-2, observed a long-term viral shedding, also in the convalescent phase of the disease, where specific antibody production to SARS-CoV-2 may not ensure viral clearance after hospital discharge.

In their study, the median duration of viral shedding appeared shorter in pre-symptomatic patients (11.5 days) compared to asymptomatic (28 days) and mild symptomatic cases (31 days) [57]. Accordingly, we documented a significantly reduced mean time to rRT-PCR SARS-CoV-2 RNA negative conversion in a liposomal bLf-treated group compared to SOC-treated and untreated patients, suggesting a favoring of gradual viral clearance and clinical symptoms recovery with a potential decrease in the risk of transmission and contagion.

This result is in agreement with the data obtained by Rosa et al. [58] in a survey based on real-life clinical practice conducted on ambulatory asymptomatic, paucisymptomatic, and moderate symptomatic COVID-19 patients treated with bLf, alone or as a supplementary agent. BLf oral administration, unloaded in liposomes, induces a time to SARS-CoV-2 RNA negativization similar to that observed with liposomal bLf (15 versus 14.25 days).

Although there are currently rare satisfactory markers for predicting worsening of the disease, some cytokines, including IL-6, IL-10, and TNF-α, and D-dimers levels have been described as biomarkers related to severe SARS-CoV-2 infection [59,60,61,62]. In our study, we identified suitable dysregulated blood parameters to consider as treatment target markers.

Indeed, we found a statistically significant difference in liposomal bLf-treated COVID-19 patients regarding some blood parameters, including IL-6, D-dimers, and ferritin. Even if this result appears very interesting, it requires a randomized clinical trial on a larger number of patients to be confirmed.

Mainly, high serum IL-6 levels are considered to be associated with higher disease severity; IL-6 inhibitors, such as tocilizumab, have been used to treat severe COVID-19 patients [63,64].

The ability of bLf to down-regulate pro-inflammatory cytokines, such as IL-6, has already been established in both in vitro [65] and in vivo [66] models as well as in clinical trials [67].

To our knowledge, even though in a small sample size, this should be the first evidence showing an IL-6 down-regulation in COVID-19 patients after liposomal bLf treatment.

We also observed a statistically significant decline in D-dimers levels, crucial to define disease prognosis, possibly leading to a reduction in SARS-CoV-2 complications related to coagulation disturbance. Recently, it has been shown that hLf can regulate the activation of plasminogen and control the coagulation cascade with a remarkable antithrombotic activity [44].

This property could be relevant, considering that COVID-19 is a prothrombotic disease and that the severity of the coagulation parameters’ impairment is related to a poor prognosis. In light of this view, SARS-CoV-2 is able to activate a prominent prothrombotic state rarely observed in viral diseases. Patients affected by severe COVID-19 pneumonia are at a higher risk of imbalance of coagulation parameters and are, thus, treated with low molecular weight heparin or unfractionated heparin at doses registered for prevention of venous thromboembolism [45].

Our clinical experience could lead us to speculate a potential protective and safe role of an early treatment of liposomal bLf in COVID-19 patients to control the risk of a thromboembolic evolution of the disease. Lf can exert negative regulatory effects on cell migration through the inhibition of plasminogen activation and via regulation of fibrinolysis [44]. In addition, we observed an increased platelet count after liposomal bLf treatment. Indeed, COVID-19 induces thrombocytopenia, as SARS-CoV-2 seems to entrap megakaryocytes and block the release of platelets [68]. Liposomal bLf, by rebalancing platelet count, induces COVID-19 viral clearance.

Ferritin, besides reflecting levels of iron stores in healthy individuals, also performs an acute-phase protein up-regulated and elevated in both infectious and non-infectious inflammation. This has been reported to be relevant in COVID-19 for assessing disease severity and patient outcome [69,70]. In particular, serum ferritin concentration shows significantly higher values in COVID-19 patients with a worse outcome compared to those with a good outcome [71].

Iron chelators, such as Lf, have been repeatedly proposed as a potential therapeutic target during infections [72], and even in COVID-19 we assessed the reduction of ferritin levels during liposomal bLf administration, demonstrating its anti-inflammatory activity together with its iron chelating ability, which is pivotal for bacterial and viral replication and at the basis of its antibacterial and antiviral activity [23,31,36,37].

Liver function is known to be disturbed in COVID-19, and a meta-analysis showed that 16% and 20% of patients with COVID-19 had ALT and AST levels higher than the normal range [73]. Liver biochemistry abnormality in COVID-19 patients could be ascribed to several factors, such as direct hepatocyte injury by the virus, drug-induced liver injury, hypoxic-ischemic microcirculation disorder, and underlying liver diseases [62]. In our study, we observed that liposomal bLf reduced transaminase levels, even if at a not significant level, thus decreasing the risk of liver injury among COVID-19 patients, which is a frequent complication in severe forms of SARS-CoV-2 [74].

Regarding clinical symptoms recovery, in the liposomal bLf-treated COVID-19 patients we observed a gradual reduction of all symptoms, with the exception of fatigue, which persisted in 18.75% of patients (Table 3). We explained this result considering patient age and concomitant comorbidities, which could create a bias to identify COVID-19 symptoms. On the other hand, in SOC-treated patients, we observed a partial symptoms recovery and, particularly, the persistence of anosmia and ageusia in all affected patients, even at the end of treatment (Table 4).

Concerning liposomal bLf safety, we reported gastrointestinal complaints in two patients as occasional findings that did not lead to treatment discontinuation. Therefore, we concluded that bLf was safe and well tolerated among our study population.
Regarding the SOC regimen group we observed limited adverse events related to the administration of hydroxychloroquine, even though its use is controversial, as several meta-analyses pointed out its intrinsic safety risks, especially in vulnerable populations, and its inefficacy in mitigating COVID-19 symptoms.

Indeed, hydroxychloroquine has been related to gastric symptoms, retinopathy, and cardiomyopathy [75,76]. As a matter of fact, in a paper by Mercuro et al. [48], patients who received hydroxychloroquine for the treatment of pneumonia associated with COVID-19 were at high risk of corrected QT (QTc) prolongation, and the combination with azithromycin was associated with greater changes in QTc [48].

Overall, the use of hydroxychloroquine alone or in combination with azithromycin was not effective in treating COVID-19 hospitalized patients. In addition, it was associated with higher mortality rates in comparison with the control group [76].

In our analysis, we used formulations containing bLf embedded in liposomes. The inclusion of bLf in preserving structures, such as liposomes, reduces gastric and intestinal enzymatic digestion while maintaining its integrity and, therefore, its biological functionality [77,78].

Indeed, the bLf at 5% of iron saturation form is best suited to obtain the maximum chelating effect. Liposomal bLf is intranasally and orally administered, because it exerts two main functions dependent on topical or oral systemic administration. The topical intranasal administration (about 16 mg/nostril/day) is related to bLf binding with HSPGs of host cells and spike glycoproteins.

This binding establishes a protective barrier against viral infection. Conversely, oral systemic administration of bLf (1 g/day) is related to the anti-inflammatory and anti-thrombotic activities. Of note, the anti-inflammatory activity also decreases intracellular iron overload, which, in turn, facilitates viral multiplication.

One of the limitations of our study was the small sample size of COVID-19 patients. Further studies on larger samples are needed to better evaluate the role of bLf in treating SARS-CoV-2 and to define the best treatment: bLf alone or as a supplementary agent.
Considering the risk of COVID-19 relapse [79], we also suggest additional long-term studies to evaluate the maintenance of viral clearance via continuous administration of bLf.

Only after the randomized clinical trials to confirm its efficacy could bLf be considered as an effective treatment in asymptomatic and mild-to-moderate COVID-19 patients. This could not only improve patient outcomes and prevention of hospital recovery, but also hinder chronic consequences of infection and disease transmission, especially by shortening the period of infectiousness.

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