Lactoferrin Versus Placebo in ICU Patients (RCT)

Clinical Research to Study the Effect of Lactoferrin Versus Placebo in Improving Clinical Outcomes in ICU Patients, a Double-blinded RCT

Introduction: Lactoferrin has several uses due to its effects. It has anti-inflammatory, antioxidant, immunomodulatory, antibacterial, antifungal, and antiviral effects. Its safety is proven by food and drug administration. Aims: The objective is to study the effect of lactoferrin on improving clinical outcomes in ICU patients when compared to placebo, and also to evaluate its safety. Patients and populations: A sample of 650 patients (325 patients in both groups A, and B) who will be admitted to ICU departments in Mansoura university hospital will be used to represent the population in ICU. Methods: A sample of 650 participants was randomized 1:1 into two groups (group A (325 patients), and group B (325 patients)). This study is a double-blind, randomized controlled clinical trial. Randomization was performed by independent clinical pharmacists working in hospital ICU departments.

Introduction 1.1. Lactoferrin molecule Lactoferrin (LF) is iron linking milk protein, as its name suggests (lacto refer to milk protein, and ferrin means iron. LF helps to modulate iron levels in the body [1-3]. LF is a part of the milk whey protein. The colostrum (the first milk produced in mothers after delivery) has seven times more LF than mature milk [4]. LF is found in the body everywhere needing immunity against microbes. it is found in many organs like kidneys, lungs, gallbladder, pancreas, intestine, liver, prostate, and also in the body fluids like saliva, tears, sperm, cerebrospinal fluid, urine, bronchial secretions, vaginal discharge, synovial fluid, umbilical cord blood, blood plasma, and the immune cells [1,2,4]. LF has many beneficial effects in the body. It has antioxidant, immunomodulatory, anti-inflammatory, antimicrobial, and antiviral effect [5,6] as shown in figure 1 [7]. Figure 1: different effects of lactoferrin [7] 1.2. Lactoferrin as antioxidant molecule The body is affected by several factors such as pathogens, environmental pollutants, and toxins. This leads to the development and accumulation of reactive oxygen species (ROS) in the body which is known as oxidative stress. ROS can cause many diseases like cancer; so, antioxidant effect produced by several molecule as LF can stop the harm induced by ROS [8,9]. The target is to maintain the balance between antioxidant molecule and ROS. LF is a type of antioxidant molecules which enhance the activity of endogenous antioxidant pathways [10]. LF act as a neuroprotective agent in Parkinson's disease and Alzheimer's disease [11-13]. It also protects against osteoporosis by inhibition of osteoblast activity and its antioxidant effect [14,15]. LF improve glucose metabolism in patients with type 2 diabetes mellitus via enhancement of the insulin-mediating response [16-19] and prevent obesity due to imbalance in body fat metabolism [17,20]. It also reduces blood pressure in hypertension [21]. 1.3. Lactoferrin as antipathogenic and immunomodulating molecule LF has antibacterial activity against Gram-positive and Gram-negative bacteria, as it kills pathogens, prevents the biofilm formation by Staphylococcus aureus or Pseudomonas aeruginosa [22,23]. LF also prevent viral, bacterial, fungal and protozoal gastrointestinal tract infections [22,24]. LF enhance the treatment of Helicobacter pylori gastric infection [25]. LF also protect against endotoxemia, bacteremia, sepsis and necrotic enteritis after partial bowel resection [26] and in neonates [27-29]. LF act as antibacterial agent by killing the bacterial cells, modulation of the immune system. In addition, LF binds to iron lead to its absence in media causing inhibition of bacterial growth, which protects the body from infection [30]. LF possess an antiviral activity and enhances the effect of antiviral drugs [31]. LF act as antiviral agent by blocking the pathogen's surface receptors and prevent it from binding to the target cell, for example, binding to angiotensin converting enzyme II receptors which used by severe acute respiratory syndrome- coronavirus 2 (SARS-CoV-2) to pass cell membrane and thus inhibit virus entry into the cell [32-34]. LF also has antifungal effect against dermatophytes and enhance the effect of antifungal drugs [35,36]. In addition, LF has an antiparasitic effect in treatment of toxoplasmosis and malaria [37]. It also has an immunomodulating effect, stimulating the body to synthesize cytokines and chemokines as well as accelerating the maturation of the immune system cells [3,38,39]. 1.4. Lactoferrin as anti-inflammatory molecule LF possess also anti-inflammatory activity in non-infectious disorders such as allergies, arthritis, cancer [40] and inflammatory colitis [41,42]. LF has anti-inflammatory effect by inhibition of ROS to prevent lipid peroxidation by chelating iron. LF also enhance the production of anti-inflammatory cytokines (IL-10), and suppressing the production of pro-inflammatory cytokines (IL-6, IL-8, IL-1b, and tumor necrosis factor (TNF-α)). In addition, LF enter into mast cells and interact with the inflammatory proteases (chymase, cathepsin G, and tryptase) [43]. Lactoferrin mainly inhibit the production of ROS and further lipid peroxidation by chelating iron which is essential for their production so inhibiting the inflammatory pathway [44]. 1.5. Lactoferrin for anemia of inflammation LF treats inflammatory disorders by increasing ferroprotein production, and by decreasing IL-6 level to redistribute endogenous iron between blood and tissue. So, LF is used as therapy for anemia caused by inflammation [45-47]. 1.6. Safety of lactoferrin use LF is safe for use. Its safety is proved by the Food and Drug Administration [48]. However, bovine LF, as an ingredient in cow's milk, may cause an hypersensitivity, So, lactoferrin use has a risk of allergic reaction, and contraindicated in case of hypersensitivity to cow's milk proteins, and in lactose intolerance [49,50]. Other side effects of LF are mild include: stomach pain, vomiting, and constipation [51]. Aim of the study The objective is to study the effect of lactoferrin on improving clinical outcomes in ICU patients when compared to placebo, and also to evaluate its safety. Methods 3.1. Study design The research will be a double-blind, randomized controlled clinical trial to evaluate the efficacy and safety of a drug. Randomization will be 1:1 and achieved by independent clinical pharmacists who are working in ICU departments of the hospital. Both patients and investigators will be blinded for identification of treatment, and placebo groups. Allocation of patients into their groups will be made after checking for meeting inclusion and not meeting exclusion criteria within 24 hours of the admission to ICU. All patients will receive the standard of care at admission. 3.2. Patients and populations A sample of 650 patients (325 patients in both groups A, and B) who will be admitted to ICU departments in Mansoura university hospital will be used to represent the population in ICU. 3.3. Intervention Doses of LF from 100 to 4500 mg daily for its indications are safe without significant toxicities [52]. Patients will be randomized (1:1) into two groups (group A, and group B). A dose of 200 mg orally twice daily (400 mg per day), shows improvements in platelet count, lymphocyte count, and serum hemoglobin level [53]. So, this dose will be chosen in this study. One group will receive a two sachet of 100 mg lactoferrin enterally (either orally or by Ryle tube) every 12 hours (400 mg daily) for 28 days plus the standard of care. another group will receive the standard of care only. 3.4 Statistical analysis and sample size Statistical analysis: Intention-to-treat (ITT) strategy will be used in this study. Categorical variables will be presented as proportion and percent. Continuous variables will be presented as mean (standard deviation) if normally distributed or as a median (IQR) or (25th-75th percentile) for non-normally distributed data Mann-Whitney test or t-test will be used to compare baseline characteristics and outcomes between the two groups. In comparison between the two groups, Mann-Whitney test will be used to compare proportions for non-parametric data (nominal or categorical), and to compare medians for non- normally distributed continuous parametric data. While t-test will be used only to compare means in case normally distributed continuous parametric. So, distributions of continuous data will be tested in order to know the correct test to be used in comparison of parametric data between the two groups. Investigators will report the 95% confidence interval and the P-value for our statistical tests with level of statistical significance will be p-value < 0.05. Regression analysis will be performed, if there is a statistically significant differences between the baseline characteristics including age, gender, No. of comorbidities (DM, hypertension (HTN), ischemic heart disease (IHD), atrial fibrillation (AF), COPD) in order to exclude the effect of these confounding variables on the study outcomes Investigators will compare the 28-day all-cause mortality rate, incidence of hypersensitivity reactions, and need for invasive mechanical ventilation (IMV) using the Mann-Whitney test. while t-test will be used to compare the day of death, duration of need for oxygen therapy and IMV, and duration of ICU stay, and all parameter measured in the study if they will be normally distributed, but if they will be non-normally distributed, Mann-Whitney test will be used instead. . Statistical analysis will be achieved with SPSS software, version 26. Sample size: The power of trial will depend on the primary outcome (28-day mortality). The proportion of ICU Population to all hospital population is about 25% A total sample sizes of 634 patients would achieve at least 80 % (0.8) power to detect a risk difference of 0.2 (20%) in the 28-day all-cause mortality (primary outcome) between alternative hypothesis and the null hypothesis (proportions of two groups are 0.5) with a significance level (α) of 0.05 and 95% confidence level proportion in Clincalc.com calculator [55]. To compensate for the estimated loss-to-follow-up and increase the study power more than 80%, Investigators will increase the sample size in both groups to be 650 patients (317 in each study group). The mortality data was estimated from the average mortality in August, September, and October 2023 at the Mansoura University Hospital ICU departments among all hospitalized patients. Mortality rate is found to be about 720 cases in these 3 months (240 cases / month) in ICU patients receiving the standard of care. The online system has been used to obtain mortality rate in these three months. The hypothesis is that LF will decrease mortality by 20% so mortality rate will be decreased from 240 to 192 per month. Data Quality and Safety Investigators will collect the data from hospital system directly into an excel sheet, Patient confidentiality will be kept before, during and after the study. Patients who will be discharged before 28 days of hospital stay, will be communicated at day 28 in order to know mortality at day 28. Publishing the study results and funding Due to size limitations in publishing the research as one paper in a journal. Investigators aim to divide this research into three papers and publish these papers in peer-review journals (3 stage publications). Funding there is no funding source for this study Conflict of interest The investigators declare no relevant conflict of interest Ethical Considerations Ethical approval will be taken from the Institutional Review Board (IRB), faculty of medicine, Mansoura university, Research Ethics Committees in faculty of medicine and pharmacy, Tanta university. Benefits of the intervention to the patients outweigh expected risks. Informed consents will be obtained from all participants in this research. Privacy of participants and confidentially of data will be maintained. Any unexpected risk appeared during the research will be cleared to participants, the IRB, and ethical committees on time. All study procedures will obey the standard of the Declaration of Helsinki (1964) principles

Lactoferrin 100 mg sachets with a dose of 200 mg (2 sachets) orally twice daily (400 mg per day) In addition to standard of care

Inclusion Criteria: patients admitted to ICU, of any age and gender. Exclusion Criteria: inability to give informed consent by patients or their relative, history of hypersensitivity to milk products, history of lactoferrin use in the past 6 months, patients with lactose intolerance, patients with no enteral access to administer LF either orally or by Ryle tube, patients who are expected to die within 48 hours, and patients with poor oral absorption as in case of shock and resected bowel.

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