Trial Information

Bovine Lactoferrin as a Natural Regimen of Selective Decontamination of the Digestive Tract in Patients With Prolonged Mechanical Ventilation

Background Nosocomial infection Nosocomial infection, especially those caused by bacteria
resistant to antibiotics, is a major threat to critical care medicine. A systemic review
revealed that 10-20% of patients receiving mechanical ventilation (MV) more than 48 hours
will develop ventilator associated pneumonia (VAP), patients who develop VAP are twice as
likely to die compared with the similar patients without VAP, and patients who develop VAP
have in additional hospital costs more than $ 10,019 [1].

The same problem occurs in Respiratory Care Centers (RCCs) which are designed to care the
patients with prolonged mechanical ventilation (PMV), MV for more than 21 days, in Taiwan.
One report from Taiwan showed that nosocomial infection rate in RCC is about 40% and the
patients develop nosocomial infection have lower weaning rate and higher mortality compared
with the patients without infection [2]. It has been estimated that 50% to 60% the
nosocomial infection occurring each year in the United Sates are caused by
antibiotic-resistant bacterial strains [3]. This high rate of resistant strains increases
the morbidity, mortality and medical costs of infection. Preventing infection and limiting
the emergence of antibiotic-resistant strains are two important issues in critical care
medicine.

Selective Decontamination of Digestive Tract (SDD) One of the current hypotheses of
nosocomial infection is that colonization of the nasopharynx and oropharynx predisposes
patients to the development of nosocomial pneumonia, and the bacterial overgrowth in the
intestinal tract increases gut wall permeability, leading to bacterial translocation and
sepsis. Selective decontamination of the digestive tract (SDD), which aims to eradicate
colonization of potential pathogens from the oropharynx and gastrointestinal tract, is one
of the strategies to reduce ventilator associated pneumonia (VAP) and sepsis in critically
ill patients. Controversy exists about the effectiveness of SDD in reducing mortality and
preventing antibiotic resistance [4-6]. The present SDD regimens may create selective
pressure and induce the emergence of methicillin-resistant Staphylococcus aureus (MRSA),
Gram-negative bacilli harboring extended-spectrum β-lactamases (ESBL), and even Candida [7,
8]. Therefore, SDD research must aim to find an ideal regimen effective on most pathogens
while leaving the anaerobic flora undisturbed.

Lactoferrin Lactoferrin (LF) is an iron-binding glycoprotein found in milk and various
external secretions such as saliva, tears, airway secretion, and the granules of
neutrophils, implying a important role in innate immunity. This protein has a number of
biological functions, including antimicrobial, anti-tumor, antioxidant, and immunomodulatory
effects. Partial degradation of LF by pepsin in stomach, may give rise to peptides termed
lactoferricin with more potent antimicrobial activity. LF and lactoferricin have been shown
to inhibit the growth of a number of pathogenic bacteria including antibiotic-resistant
strains, fungi and even viruses in both in vitro and in vivo studies [9,10]. In mouse
experiments, oral administration of bovine LF reduced bacterial infections in the
gastrointestinal tract [9] while promoting the growth of bacteria with low iron requirements
such as Lactobacillus and Bifidobacteria, which are generally believed to be beneficial to
the host [11].

Bovine lactoferrin (bLF) and human lactoferrin (hLF) have high (77%) amino acid homology,
with bLF exhibiting even higher in vitro antimicrobial activity than hLF.5 Bovine
lactoferrin has been granted GRAS (generally recognized as safe) status by the US Food and
Drug Administration [12] and on this basis is added to infant formula by many manufacturers
with no reported adverse effects. Despite many promising in vitro and animal experimental
data, clinical information on bLF is scarce, with no studies investigating the effects of
bLF supplementation in patients with prolonged mechanical ventilation.

Our previous studies We previously demonstrated that both recombinant porcine LF (pLF)
produced from yeast [13] and a synthetic 20-residue porcine lactoferricin peptide [11]
exhibit antimicrobial activity in vitro. Its bactericidal activity was four times more
effective than that of human lactoferricin [14].

In our recent report [15], we performed pathogen challenges of the digestive tract of a
transgenic milk-fed animal model to test if porcine LF (pLF) is an effective SDD regimen.
Transgenic mice expressed recombinant LF in their milk at 120 ± 13.6 mg/L during the
lactation stage and fed normal CD-1 mice pups for 4 weeks. The pups were subsequently
challenged with pathogenic Escherichia coli, Staphylococcus aureus and Candida albicans. The
groups that were fed pLF transgenic milk demonstrated statistically significant improvements
in weight gain, lower bacterial numbers in intestinal fluid, blood and liver, and healthier
microvilli in the small intestinal tissue, lower proinflammatory cytokines when compared to
the control groups that were fed normal milk. Results showed that recombinant pLF expressed
in the milk of transgenic mice and fed to mice pups led to broad spectrum antimicrobial
activity in the digestive tract and protected the mucosa of the small intestine from injury,
implying that porcine LF can be used as an effective selective decontaminant of the
digestive tract.

Aim of this study This study is a prospective, randomized, double-blind, placebo-controlled
clinical trial examining whether oral supplementation with bLF reduces nosocomial infection,
sepsis and even mortality in patients with prolonged mechanical ventilation.

Methods Patients Between July 1, 2010, and June 30, 2012, we will enroll about 280 patients
with mechanical ventilation for more than 21 days and no evident signs of infection on
admission to our Respiratory Care Centers (RCCs) for clinical study. The study has been sent
to the Ethics Committees of China Medical University Hospital for approval. Patients or
guardians provide written informed consent after our explanation. Exclusion criteria were
informed consent lacking/refused, ongoing antibiotics treatment for infection, predicted
mortality in 7 days. All patients will be followed up until death or discharge from our
hospital.

Objectives The primary objective is to evaluate the effectiveness of bLF in the prevention
of the first episode of nosocomial infection and sepsis of bacterial or fungal origin after
admission to RCC. Secondary objectives are assessments of the incidence of pneumonia,
urinary tract infection and sepsis, mortality prior to discharge (overall and
sepsis-attributable), weaning rate from MV, total days of MV, alteration of immune system,
cytokines and liver function, and adverse effects or intolerance.

Study design Lactoferrin and placebo will be masked as drug A and B in the factory.
Randomization will be stratified by center and patients will be randomized into A or B
groups by a random-number table sequence after informed consents are obtained. No patients,
research nurses, investigators, or other medical staffs in RCC will be aware of the
assignment during the study period.

Patients will receive either bLF (10 mg/Kg/day) (Westland Co-operative Dairy Company, New
Zealand) or placebo (starch) as control. The dosage of bLF is based on the mean hLF intake
that very low body weight neonates ingest with mother's fresh milk in the first 2 weeks of
life (30-150 mg/d) [16] and bLF 200 mg bid is found to be effective to suppress Helicobacter
pylori [17]. Drug administration will begin within 24 hours after RCC admission and will
last for 6 weeks or until discharge. Medication and nutritional support will be prescribed
as the medical routine.

Systematic surveillance of adverse events (eg, vomiting, feeding intolerance, skin rashes)
will be performed through daily examination. Weekly surveillance of liver function, complete
blood counts, CD4/CD8, cytokines (TNF-alpha, IFN-gamma, IL-1, IL-2, IL-12, IL-18) will be
performed [18,19].

Definition of outcomes The diagnosis of sepsis is based on the detection of clinical signs
and symptoms by the physicians in charge, presence of laboratory findings consistent with
sepsis, and isolation of a causative organism from blood or body fluids. Patients with an
episode of sepsis continue to receive follow-up until death or discharge from RCC for
secondary outcomes [16]. VAP is characterized by the presence of signs of respiratory
infection (fever, leukocytosis and purulent respiratory secretions), and a new and
persistent infiltrate on chest X-ray in patients undergoing MV [20]. Urinary tract
infections is diagnosed by isolation of a pathogen from urine collected by suprapubic
puncture or bladder catheterization, with growth of more than 100 000 bacteria/mL or more
than 10 000 fungi/mL. Success of weaning from MV is defined as no need of MV for more than 5
days.

Statistical Analysis Sample size analysis predicted that a total of 131 MV patients in each
group would be needed to detect a relative difference between treated and non-treated
patients of at least 60% (decrease from 20% to 6%) for mortality rate based on two-sided
test with type I error of 0.05 or less and 80% power of 80%. Quantitative variables will be
expressed as mean and standard deviation. Categorical variables will be represented by
percentages. Univariate analyses exploring associations between individual risk factors and
primary/secondary outcomes were performed using Fisher's exact test for dichotomous
variables and Student's t test for continuous variables. A multivariate logistic regression
model is performed to investigate the effect of relevant risk factors and relative
contributions of the various risk factors. Goodness of fit is evaluated through the
log-likelihood of the fitted model. Odds ratios and their 95% confidence interval are
calculated in the logistic regression model to describe the strength of the relationship
between the categorical risk factors and outcomes. All tests are two-sided, and a p-value of
0.05 is considered to be statistically significant. All analysis is carried out using SAS
software version 6.12 (SAS Institute Inc, Cary, NC).

EXPECTED FINDINGS

Our findings can be expected to answer the following questions as follows:

1. Can oral supplement of bLF prevent nosocomial infection in patients with PMV?

2. Can oral supplement of bLF reduce the incidence of nosocomial infection and reduce the
use of antibiotics in patient with PMV?

3. Can oral supplement of bLF reduce mortality of the patients with PMV?

4. Can oral supplement of bLF increase the weaning rate from MV in patients with PMV?

5. What are the changes of immune system after oral supplement of bLF in patients of PMV?

EXPECTED CONTRIBUTION FROM CURRENT PROJECT Preventing infection and limiting the emergence
of antibiotic-resistant strains are two important issues in critical care medicine. To find
a natural antimicrobial peptide as an adjuvant therapy for antimicrobial agents in treatment
of the antibiotic-resistant strains, or as a regimen to increase immunity and to prevent
infection is one of the directions of researches. LF is one of the promising agents in many
vitro and animal studies. The study is trying to examine if LF is an ideal natural SDD
regimen for the prevention of nosocomial pneumonia or sepsis in patients with PMV. If the
result is positive, LF can be used clinically to increase the survival of the critically ill
patients, increase weaning rate from MV, reduce the prescription of antibiotics and finally
medical cost.

REFERENCES (List without PMID No)

12. CFSAN/Office of Food Additive Safety. Agency response letter: GRAS notice No. GRN
000077. US Food and Drug Administration Web site. http://
www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm154188.htm. August 14, 2001. Accessed May 21, 2009.