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 compared to control and standard therapy, and also to evaluate its safety.

Patients and populations:

A sample of 660 patients (330 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 660 participants was randomized 1:1 into two groups (group A (330 patients), and group B (330 patients)).

This study is a single blind, randomized controlled clinical trial. Randomization was performed by independent clinical pharmacists working in hospital ICU departments.

1. Introduction

   1.1. Lactoferrin molecule

   Lactoferrin (LF) is iron linking milk protein. 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 by mothers after delivery) has seven times more LF than mature milk [4]. LF 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 immune cells [1,2,4]. LF has many beneficial effects in the body. It has antioxidant, immunomodulatory, anti-inflammatory, antimicrobial, and antiviral effects [5]. Figure 1: different effects of lactoferrin [5]

   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. LF can stop the harm induced by ROS [6, 7], and enhance the activity of endogenous antioxidant pathways [8].

   LF acts as a neuroprotective agent in Parkinson's disease and Alzheimer's disease [9]. It also protects against osteoporosis by inhibition of osteoblast activity and due to its antioxidant effect [10]. LF improves glucose metabolism in patients with type 2 diabetes mellitus via enhancement of the insulin-mediating response [11] and prevents obesity due to imbalance in body fat metabolism [12]. It also reduces blood pressure in hypertension [13]. So, LF can act as anti-hyperglycemic, and anti-hypertensive agent.

   1.3. Lactoferrin as antipathogenic and immunomodulating molecule LF has antibacterial activity against Gram-positive and Gram-negative bacteria, as it kills pathogens, and prevents the biofilm formation by Staphylococcus aureus or Pseudomonas aeruginosa [14]. LF also prevents viral, bacterial, fungal and protozoal gastrointestinal tract infections [15]. It enhances the treatment of Helicobacter pylori gastric infection [16], and also protects against endotoxemia, bacteremia, sepsis and necrotic enteritis after partial bowel resection [17] and in neonates [18]. LF possess an antiviral activity and enhances the effect of antiviral drugs [19].

   It acts as antiviral agent by blocking the pathogen's surface receptors and prevents it from binding to the target cell, for example, its binding to angiotensin converting enzyme II receptors which used by SARS-CoV-2 to pass cell membrane and thus inhibit virus entry into the cell [20].

   LF also has antifungal effect against dermatophytes and enhance the effect of antifungal drugs [21]. In addition, it has an antiparasitic effect in treatment of toxoplasmosis and malaria [22].

   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, 23].

   LF possesses also anti-inflammatory activity in non-infectious disorders such as allergies, arthritis, cancer [24] and inflammatory colitis [25]. Its anti-inflammatory effect is due to the inhibition of ROS to prevent lipid peroxidation by chelating iron. It also enhances the production of anti-inflammatory cytokines (IL-10) and suppresses the production of pro-inflammatory cytokines (IL-6, IL-8, IL-1b, and TNF-α). In addition, It can enter into mast cells and interact with the inflammatory proteases (chymase, cathepsin G, and tryptase) [26].

   1.5. Lactoferrin for anemia of inflammation

   LF treats inflammatory disorders by increasing ferroprotein production and decreasing IL-6 level to redistribute endogenous iron between blood and tissue. So, LF is used as therapy for anemia caused by inflammation [27].

   1.6. Safety of lactoferrin use

   The safety of LF is proved by the Food and Drug Administration. However, bovine LF, as an ingredient in cow's milk, may cause 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 [28]. Other side effects of LF include stomach pain, vomiting, and constipation [29].

2. Aim of the study

   The objective is to study the effect of lactoferrin on improving clinical outcomes in ICU patients, and also to evaluate its safety.

3. Methods

   3.1. Study design

   The research will be a single-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. The patients 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 total of 660 patients who will be admitted to ICU departments in Mansoura University Hospital will represent the population in ICU.

   3.3. Intervention

   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.

   A sample of 660 patients (330 patients in both 2 groups (group A: lactoferrin and group B: standard group) who will be admitted to ICU departments in Mansoura university hospital will be used to represent the population in ICU. The standard group (B) will be divided into three subgroups of 110 patients in each. Subgroup B1 will receive standard antioxidant drug (Acetyl cysteine 600 mg / 12 hr) orally to be compared with lactoferrin A1 subgroup.

   - Subgroup B2 will receive standard antimicrobial drugs to be compared with lactoferrin A3 subgroup.

   - Subgroup B3 will receive standard anti-inflammatory, and immunomodulatory drug (dexamethasone I.V 8 mg / day) to be compared with lactoferrin A2 subgroup.

   3.4. Outcomes and parameters

   Primary outcomes:

   • 28-day mortality (efficacy), and

   • incidence of any allergic or hypersensitivity reactions (safety).

   Secondary outcomes:

   • the day of death,

   • need for invasive mechanical ventilation (IMV),

   • duration of need for oxygen therapy, and IMV,

   • duration of ICU stay, and

   • kidneys, and liver functions.

   • Glasgow coma score (GCS) and

   • sequential organ function assessment (SOFA) score [31].

   • The changes in biochemical markers

   Blood sample collection:

   About 5 ml venous blood samples will be withdrawn by sterile venipuncture and centrifugated at 3000 rpm for 15 min, then sera will be kept frozen at 8 oC until analysis of • CBC

   • Inflammatory marker (TNF-α)

   • Oxidant marker (TAC)

   • Liver functions

   • Kidney functions In addition, arterial blood gases (ABG), vital signs, and Oxygen saturation were measured.Most of these parameters are measured in previous researches [32-39]

   Subgroup analysis will be conducted in order to compare lactoferrin with other drugs with relevant activity as shown below:

1- A comparison between the antioxidant effect of lactoferrin vs standard antioxidant drug (Acetyl cysteine) indicated by Anti-oxidant marker (TAC)

2- A comparison between the adjuvant antimicrobial effect of lactoferrin vs standard antimicrobial drugs as indicated by the WBCs and its differential counts (neutrophils, lymphocytes) in case of infections.

3- A comparison between the anti-inflammatory effect of lactoferrin vs standard anti-inflammatory, and immunomodulatory drug (dexamethasone) as indicated by the inflammatory marker (TNF- α, CRP)

3.5 Statistical analysis and sample size

Statistical analysis:

Per-protocol 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, t-test or Chi-square test will be used to compare baseline characteristics and outcomes between the two groups. In comparison between the two groups, Chi-square test will be used to compare proportions for non-parametric data (nominal or categorical). Mann-Whitney test will be used 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. ANOVA test will be used in case of comparison between more than 2 groups (as in case of comparison of antioxidant therapies).

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 Chi-square 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 [62]. 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 660 patients (330 in each study group).

The mortality data will be estimated from the average mortality in January, February, and March 2025 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 [32-39]. The hypothesis is that LF will decrease mortality by 20% so mortality rate will be decreased from 240 to 192 per month.

4. 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.

5. Publishing the study results and funding

Due to size limitations in publishing this big research as one paper in one journal. Investigators aim to divide this research into five papers and publish these papers in peer-reviewed journals (5 stage publications).

Funding There is no funding source for this study

Conflict of interest The investigators declare no relevant conflict of interest

6. Ethical Considerations

Ethical approval will be taken from

1. Institutional research board (IRB), faculty of medicine, Mansoura University,

2. Research ethics committee, faculty of 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 the confidentiality of data will be maintained. Any unexpected risks that 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

Inclusion Criteria:

• The inclusion criteria for patients to participate in this research include adult (age ≥ 18 years) patients admitted to ICU for any reason or disease.

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.

After the end and publication of the study

After the end and publication of the study

all will be accessible