Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR PREVENTING AND TREATING THE INSULT-INDUCED
METABOLIC IMBALANCE IN HUMANS AND OTHER ANIMALS
Related Applications
This application is based on provisional application Serial No.
60/045,521 filed May 3, 1997 entitled "Use of Lactoferrin for Prophylaxis and
Therapy of the Systemic Inflammatory Response System in Animals and
Humans" which is incorporated herein by reference. This application also
relates
to U.S. Serial No. 08/724,586, filed 9/30/96, entitled "Cloning, Expression
and
Uses of Human Lactoferrin", which in turn is a continuation of U.S. Serial No.
08/238,445, filed 5/5/94, which in turn is a CIP of U.S. Serial No.
08/132,218, filed
10/6/93, which in turn is a continuation of U.S. Serial No. 07/489,186, filed
3/8/90,
all of which are incorporated herein by reference.
Field of the Invention
The present invention relates generally to the iron binding protein
lactoferrin. fn particular, it relates to the use of lactoferrin to treat or
prevent insult-
induced metabolic imbalance in humans and animals, and its use for the
manufacture of a medicament for the treatment or prevention of insult-induced
metabolic imbalance in humans and animals.
Background of the Invention
Homeostasis is a state of equilibrium in the internal environment. The
integrity of such system is continuously disturbed by stimuli that tend to
create an
internal imbalance. In response to prolonged stimuli, the compensatory
mechanisms often do not restore the balance. This may, consequently lead to
the
activation of self-perpetuating, autodestructive mechanisms including death.
The
. central pathway involved in the insult-induced metabolic imbalance may
depend
in part on the nature of the stimuli, but the hypo- or hyper-thermia appears
to be
. common for many forms of insult. The energy balance of the internal
environment
is controlled by the central nervous system (CNS) and regulated by the
decrease
(chills) or increase (fever) of our body temperature. Whether the insult is
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microbial infection, inflammation or trauma the internal environment responds
to
those insults by activating thermoregulatory mechanisms that coincide with the
production and release of many immunomodulatory substances. Cytokines.
prostaglandins, and different growth factors and hormones are released from .
specific cells to restore the internal metabolic balance, which largely
depends on
the energy equilibrium. .
The significance of lactoferrin in health and disease has been the subject
of several reviews. A most recent publication entitled "Lactoferrin: Molecular
Structure and Biological Function" has been published in 1995 by B. Lonnerdal
and S. lyer in Ann. Rev. Nutr., 15:93-110.
Lactoferrin is a multifunctional protein expressed in a variety of cell types
under different mechanisms of control. The primary function of lactoferrin
seems
to be a protection against pathogenic bacteria. By virtue of sequestering
iron,
~lactoferrin may control development of potential infections. In addition, it
can kill a
wide variety of Gram-negative and Gram-positive bacteria by direct interaction
with the cell surtace, a mode of action that is not dependent on iron.
Lactoferrin
is thought to be an important component of the defense system, active at
mucosal surfaces, including the gastrointestinal tract. Various
immunoregulatory
and anti-infective roles for lactoferrin have been reviewed by J. Brock in an
article
entitled "Lactoferrin: a multifunctional immunoregulatory protein?" and
published
in immunology Today (1995), 16:417-419.
Although, considerable data from in vitro experiments indicate several
physiological rotes for lactoferrin, there is no firm evidence concerning its
actual
physiological function from in vivo studies. For example, in a review by Roy
D.
Byens and Werner R. Bezwoda entitled "Lactoferrin and the inflammatory
response" and published in the book: Lactoferrin: Structure and Function, pp
133-
141, (1994), a relationship between plasma lactoferrin and granulocyte
activity in
sepsis is mentioned. However, the biological function of the significant
amounts
of lactoferrin in plasma of septic patients is as yet incompletely understood.
!n another review entitled "The role of lactoferrin as an anti-inflammatory
molecule" by Bradley E. Britigan, Jonathan S. Serody, and Myron S. Cohen and
published in the book: Lactoferrin: Structure and Function, pp 143-156,
(1994),
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the role of factoferrin in inflammation is suggested to be played at two
different
levels: (i) as an antioxidant, capable of binding free iron, and (ii) as an
endotoxin
scavenger, capable of reducing lipopolysaccharide (LPS)-induced toxicity.
Furthermore, the ability of lactoferrin to bind LPS in vitro has been
confirmed by
E. Elass-Rochard, A. Roseanu, D. Legrand, M. Trif, V. Salmon, C. Motas, J.
' Montreuil and G. Spik in an article entitled "Lactoferrin-lipopolysaccharide
interaction: involvement of the 28-34 loop region of human lactoferrin in the
high
affinity binding to Escheria coli 05585 lipopolysaccharide'; published in
Biochem.
J. (1995) 312:839-845. However, in vivo studies have to confirm lactoferrin's
role
in those internal metabolic responses during inflammatory processes.
In another article entitled: °Lactoferrin can protect mice against a
lethal
dose of Escherichia coli in experimental infection in vivo" by T. Zagulski, P.
Lipinski, A. Zagulska, S. Broniek and Z. Jarzabek, published in 1989 in Br. J.
Exp. Path., 79:697-704, the use of lactoferrin is disclosed to increase the
survival
of mice injected with a lethal dose of bacteria. However there is no
disclosure that
the intravenously administered lactoferrin has any effect on the gut function
and
structure to give such protection.
Relevant patents are also silent as to the role of lactoferrin in insult-
induced metabolic activity.
U.S. Patent 4,977,137 of Nichols et al. discloses milk lactoferrin as a
dietary ingredient which promotes growth of the gastrointestinal tract of
human
infants and newborn nonhuman animals immediately on birth. Nichols discusses
the use of lactoferrin in the management of short gut syndrome, an anatomical
dysfunction rather than an insult-induced metabolic imbalance.
U.S. Patent 5,240,909 of Nitsche relates to the use of lactoferrin as an
agent for the prophylactic and therapeutic treatment of the toxic effects of
endotoxins. Nitche discloses that the lactoferrin used according to his
invention
has the ability to neutralize endotoxin and must have bound to it either iron
or
another metal to be effective.
U.S. Patent 5,066,491 of Stott et al. encompasses a method of disease
treatment utilizing a therapeutically effective product produced from ordinary
milk
whey.
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Summary of the Invention
The method of the present invention provides a' novel use of the iron
binding protein lactoferrin to prevent or treat insult-induced metabolic
imbalance
in humans and other animals. In one embodiment of the present invention there
is provided a method to use lactoferrin to modulate such metabolic imbalance
through the gastrointestinal tract. In a further embodiment, the present
invention
relates to the use of lactoferrin for the manufacture of a medicament for the
prevention or treatment of insult-induced metabolic imbalance in humans and
animals. In yet a further embodiment, the present invention relates to the use
of
lactoferrin for the manufacture of a medicament for the modulation of such
metabolic imbalance through the gastrointestinal tract.
Brief Description of the Drawings
Fig. 1 Illustrates histological sections of mouse jejunum stained with
hematoxylin and eosin to visualize intestinal structures during 21 days
treatment
with lactoferrin (a) or saline (b).
Fig. 2 Illustrates the cumulative weight gain and daily food consumption in
mice that have been treated with either lactoferrin or saline during
experimental
infection with Trichinella spiralis.
Fig. 3 Illustrates histologicai sections of mouse jejunum stained with
hematoxylin and eosin to visualize intestinal structures during experimental
infection with Trichinella spiralis a) infected, lactoferrin-treated, b)
infected, saline-
treated.
Fig. 4 Illustrates histological sections of mouse jejunum stained with
hematoxylin and eosin to visualize intestinal structures during experimental
endotoxemia a) non-infected, saline control, b) non-infected, lactoferrin-
treated,
c) infected, saline-treated, d) infected, lactoferrin-treated.
Table 1. Illustrates jejunal responses to glucose and chloride
secretagogues after long term treatment with lactoferrin.
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Table 2. Illustrates jejunal responses to glucose and chloride
secretagogues following infection with Trichinella spiralis
Description of the Preferred Embodiments
The maintenance of homeostasis is essential for cellular integrity and the
wellness of our body. Inflammation is one of those compensatory mechanisms in
response to external insult. When excessive in magnitude or duration, however,
the otherwise beneficial effects of inflammation may be deleterious, impacting
negatively on the recovery or healing of the host. As illustrated below, acute
inflammation caused by an external insult may be repaired or it may develop
into
a slow progressing chronic condition such as inflammatory bowel disease (IBD),
rheumatoid arthritis (RA) or into fast progressing systemic inflammatory
response
syndrome (SIRS), including sepsis, septic shock, and multiple organ failure
(MOF):
(Sepsis, Septic shock, MOF) (IBD, RA)
SYSTEMIC INFLAMMATORY CHRONIC INFLAMMATION
RESPONSE SYNDROME
2o T T
I NSU LT ------------------~ ACUTE I N F LAMMATION -----------------~ RE PAI R
(Infection (Initial Metabolic Imbalance) (Damage Control)
Trauma)
When tissue damage occurs, particularly if it is induced by infection during
trauma, the vascular effects of the repair mechanisms are immediate. The
tissue
becomes inflamed at the site of injury, with the tissue spaces and the
lymphatics
blocked by fibrin clots. The fluid barely flows through the inflamed tissue,
therefore the spread of bacteria and/or their toxic products is delayed.
Unlike the
immune responses, which may take days to develop, the vascular effects of
inflammation occur in seconds and coincide with the burst of inflammatory
cytokines, such as tumor necrosis factor (TNFa) and interleukin-1 (IL-1 Vii),-
from
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activated monocytes/macrophages. Subsequent to the release of cytokines is an
acute increase in neutrophils in the blood. Large numbers of neutrophils begin
to
invade the tissues that attract these cells. Although generally beneficial to
the
host, inflammatory processes are intrinsically destructive to the surrounding
tissues and potentially can result in major tissue injury. Furthermore, the
inflammatory response can spread from the local environment and induce
generalized systemic response which may become self-nPrnPt~ ~at~.,., r",
overproduction of pro-inflammatory cytokines.
According to the present invention, the feedback control mechanism of
inflammation depends on the presence of factoferrin. By blocking the
deleterious
effects of the pro-inflammatory cytokines, lactoferrin provides a feedback
mechanism for the metabolic imbalance during development of systemic
inflammation, and thus, can be used in accordance with the present invention
in a
method for treating or preventing such insult-induced metabolic imbalance.
The gastrointestinal tract may be viewed as an ecological system that acts
to maintain balance between the host and the bacterial flora. Two major host
components appear to be involved in maintaining this balance. The first is a
non-
specific structural barrier provided by the epithelial layer of the
gastrointestinal
mucosae. The second component involves functional immunological elements
found in the mucosal and submucosal compartments, e.g., gut associated
lymphoid tissue. When the gut integrity is disrupted by invasive pathogens or
by
trauma, a myriad of pro-inflammatory mediators is released from cells in the
gut
wall that exert actions in the tissue or gut lumen. The gut responses to such
insults are immediate to protect the internal environment from enteric
bacteria
crossing the gut barrier and inducing systemic responses. Therefore, it is of
great
importance to preserve the structure and function of the gastrointestinal
tract
during initial stages of such metabolic imbalance to avoid the systemic
translocation of the enteric bacteria.
According to the present invention the gastrointestinal tract is considered
the key organ to provide compensatory mechanisms to any type of insult-induced
metabolic imbalance. The present invention, by in vivo experiments on gut,
shows that lactoferrin attenuates the severity of the insult-induced metabolic
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imbalance, thereby protecting the development of severe hypo- or hyperactivity
that often leads to chronic inflammation or systemic inflammatory response
syndrome. Furthermore, it has been found that lactoferrin can be used in
accordance with the present invention in a method for protecting intestinal
functions during insult-induced metabolic imbalance. The evidence presented
herein confirms that lactoferrin administered either enterally or parenterally
helps
to maintain physiological balance in normal and pathologic gut situation.
Insult is defined herein as any intervention in the internal environment
including microbial, viral or parasitic infections; stress; trauma;
insufficient or
excessive nutrient intake; invasive or non-invasive medical procedures, any of
which cause metabolic imbalance, a state of disturbed integrity of the
internal
system. Usually, such metabolic imbalance is referred to as hypo- or hyper-
activity of the internal system.
The present invention is to use lactoferrin administered to the gut for
treating or preventing metabolic imbalance during insult-induced hypo- or
hyper-
activity often manifested by inflammation as a result of such insult.
Lactoferrin is one of the most abundant proteins found at mucosal
surfaces and within secondary granules of neutrophiles in all vertebrates. The
highest concentration of lactoferrin has been found in mammary glands of
lactating females. The sequence homology between human and other species
lactoferrins is between 50% to 70%. Bovine milk lactoferrin, which is
commercially available, is about 69% identical to its human counterpart. Due
to
this fact, the clinical application of bovine lactoferrin in humans is limited
to oral
administration; any type of systemic administration of bovine lactoferrin in
humans would cause highly antigenic reaction.
Lactoferrin for use in a present invention may be human lactoferrin from
human breast milk or extracted from milk of other animals such as bovine
lactoferrin from cow's whey. Due to severe limitations on availability of
large
quantities of human breast milk and the FDA requirements, it may be difficult
to
develop a commercial production of clinically acceptable natural human
lactoferrin. Consequently, recombinant DNA technology is considered the best
solution to obtaining large quantities of reliable human or bovine
lactoferrins
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which would be consistent in production, uniform in its biological properties,
and
non-pathogenic.
The preferred lactoferrin is lactoferrin expressed in a yeast expression
system such as Pichia pastoris or Hansenula polymorpha, or in an eukaryotic
expression system. The preferred lactoferrin is described in U.S. Serial No.
08/724,586, filed 9/30/96, entitled "Cloning, Expression and Uses of Human
Lactoferrin" and PCT/US95/05653, filed 05/05/95. Other recombinant
lactoferrins
are described in U.S.Patents 5,571,691; 5,571,697; and 5,571,896, all of which
are incorporated herein by reference.
Lactoferrin is administered in accordance with the present invention either
enterally, preferably orally, in the form of a powder, solution or gel, or
parenterally, preferably intravenously, in the form of an injectable solution,
as an
aid to treat or prevent metabolic imbalance. Preferable formulations or
medicaments of the present invention comprise lactoferrin alone or in
combination with carriers such as, saline, silica, talcum, stearic acid, its
magnesium or calcium salt, polyethyleneglycol, and fatty emulsions and
suspensions that will be readily apparent to the skilled artisan. The
lactoferrin is
preferably present in the formulation at a level of 0.01 milligram to 2
milligram ,
more preferably between 0.1 to 1 milligram, based on 1 milliliter or 1 gram of
the
carrier. An effective amount of lactoferrin varies depending on the individual
treated, severity of the insult-induced metabolic imbalance and the form of
administration. Preferable in treating mammals, a single or twice daily dose
of
0.01 milligram to 20 milligrams, more preferable 0.1milligram to 1 milligram
of
lactoferrin per kilogram of body weight or per 1.0 square inch of targeted
area is
administrated.
The effectiveness of lactoferrin in the treatment or prevention of insult-
induced metabolic imbalance according to the present invention is demonstrated
below in different types of insults in a mouse model: (i) parasitic infection
with
Trichinella spiralis, and (ii) LPS endotoxemia. Injection with LPS, a
derivative of
the cell wall of Gram negative bacteria, is commonly used as the insult for
study
of sepsis or MOF. The insults were chosen either at excessive levels to
exemplify
the effect of lactoferrin under acute conditions such as sepsis or multiple
organ
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failure or at lower levels to illustrate the effect of lactoferrin on stress
or trauma in
mice. Also, the route of administration of lactoferrin or saline by gavage,
intravenously or intraperitoneally exemplify the effects of noninvasive or
invasive
medical procedures on the internal environment. The following objectives were
evaluated: (i) safety of a long term oral administration of lactoferrin, (ii)
effects of
lactoferrin on the gut caused by intestinal infection with Trichinella
spiralis, and
(iii) the effects of iactoferrin on the metabolic activity during LPS-induced
endotoxemia, all in a mouse model.
The procedures and methods for determining the physiological function of
the gut under different type of insults are summarized as follows. These
procedures have been developed to measure the state of the gut as normal,
returned to normality, or pathologic using saline-treated animals as a
control.
Human lactoferrin has been used replaceable with its bovine counterpart. Only
male CF-1 mice (Harlan, Houston, TX), are used throughout this investigation.
Mice are housed in groups of three per cage and are given stock diet (F6
Rodent
Diet 8664, Teklad, Madison, WI) and water at libitum. Daily food consumption
and body weight are measured each morning. Both food intake and body weight
is expressed as the average for each group. The electrophysiological
parameters
are measured on jejunal segments obtained from mice under anesthesia. Briefly,
jejunum, beginning 1 cm distal to the ligament of Treitz, is removed, rinsed
in
Krebs-Ringer bicarbonate (KRB) solution, pH 7.4, and slit open along the
mesenteric border. Consecutive one-cm full thickness segments are taken from
the proximal part of the intestine and mounted as a flat sheet between two
Ussing
half chambers with an aperture of 0.512 cm2. Tissues, bathed on their mucosal
and serosal sides with 10 ml KRB solution, are voltage clamped at zero
transepithelial potential using a VCC-600 voltage current clamp (Physiologic
Instruments, San-Diego, CA). A continuous record of short circuit current with
respect to time is obtained and recorded on a BD-41 Kipp & Zonen recorder
(DeIft,Holland). To measure tissue resistance, a current that generates an
extra
1 rnV potential difference across the tissue from a pulse generator in the
voltage
clamp apparatus is passed every two min for 0.1 sec. Resistance is calculated
using Ohm's law (V=IR). Changes in short circuit current (olsc) induced by CI-
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secretagogues [serotonin (5rHT) and carbamylcholine (CCh)] and by glucose are
presented as the maximal elevation and are expressed as oA/cm2.To perform
histological evaluation the jejunal segments are fixed in 10% formalin and
embedded in paraffin using standard techniques. Section 5 um thick are cut and
stained with hematoxylin and eosin and with periodic acid/Schiff (PAS) to
visualize brush border and epithelial mucin. Sections are examined by light
microscopy and photographs are taken with a Nikon Optiphot microscope.
Student t-test and two way ANOVA (Snedecor 1980) are used to compare
means among the different groups. The results are expressed as means t SE. P
value of 0.05 or less was considered significant.
The following examples are presented herein to illustrate the present
invention:
I. Long term administration of lactoferrin using gavage as a non-invasive
route of administration.
EXAMPLE 1
Naive mice were gavaged daily with human lactoferrin (1 mg/100p1 saline)
for 21 days each morning. Their control counterparts were given 1001 of saline
for the same time period. After 21 days of treatment all mice were killed and
jejunai segments were obtained for electrophysiological measurements and
histological examination.
Food consumption and cumulative weight gain in mice fed lactoferrin were
similar in both lactoferrin and saline-treated mice. Basal
electrophysiological
parameters of mouse jejunum [intestinal transepithelial resistance (R),
potential
difference (PD) and short circuit current (SCC)] were not altered by long term
administration of lactoferrin. Likewise, intestinal glucose absorption, and CI-
secretion induced by 5-HT (serotonin), CCh, or histamine were not affected by
three weeks administration of lactoferrin (Table 1 ). Also, the histology of
the
intestine was not altered in mice fed lactoferrin (Fig. 7 ).
The results show that there are no adverse effects of enteral
administration of lactoferrin on gut structure and function.
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II. The effects of lactoferrin on the intestinal metabolic activity induced by
infection with Trichinella spiralis.
EXAMPLE 2
Human lactoferrin (1mg1100N1 saline) was given to mice orally by gavage
for three consecutive days. Their counterparts were given 100N1 of saline. On
day
four, one half of all animals from each group were infected with 600
Trichinella
spiralis larvae. For seven additional days mice receive either lactoferrin or
saline.
At this time all mice were killed and jejunal segments were removed for
electrophysiological and histological studies.
When lactoferrin was given to mice for three days prior to infection with
Trichinella spiralis and seven days thereafter, the reduction in food
consumption,
normally caused by the infection, was not as marked (Fig 2). The same effect
was evident in the cumulative weight gain. Mice treated with iactoferrin
gained
weight following infection at the same rate as noninfected mice, while
infected
animals given saline lagged in their weight gain when compare to all other
groups.
Resistance of the intestinal tissue seven days after infection was
comparable among infected and noninfected groups. The group of mice fed
lactoferrin and infected with the parasite showed an increase in SCC, when
compared with all other groups, however the difference was not statistically
significant. Jejunal PD in the infected mice fed with lactoferrin also was
higher,
although not significant, when compared to that of infected mice given saline.
Intestinal glucose absorption was significantly reduced in infected groups,
regardless of treatment (Table 2). Although infection reduced secretion,
jejunum
from mice that received lactoferrin before and after inoculation with
Trichinella
spiralis showed a greater capacity to secrete CI- in response to CCh (but not
to 5-
HT) than did mice given saline (Table 2).
At day seven post-infection with Trichinella spiralis, the inflammatory
process in the intestinal mucosa was at its peak. Histological sections of
jejunum
stained with hematoxylin and eosin showed that lactofen-in had a protective
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on the intestinal epithelium (Fig. 3). Infection with Trichinella spiralis
caused a
diffused enteritis in control mice with swelling of the villi and enlargement
of the
crypts. The intestinal epithelium exhibited severe vacuolar degeneration,
papiliarization, pseudostratification and shedding of epithelial cells. In the
lactoferrin treated mice vacuolar degeneration was confined to scattered,
single
cells, while papiliarization and pseudostratification were absent.
Inflammatory cell
infiltration of the mucosa appeared similar in the lactoferrin-treated mice
and in
their control counterparts. However, intestinal stromal swelling was reduced
in
the lactoferrin-treated mice. Also, the number of mitotic cells was 2-3 times
higher
in iactoferrin treated animals than that of saline controls. Staining
intestinal
sections for carbohydrates demonstrated that lactoferrin significantly
enhanced
the production of mucin, a common marker of goblet cells. The following
average
counts of goblet cells per villi were obtained from the histological section
by light
microscopy: a) saline-treated, noninfected mice 7.1 (n=2), b) lactoferrin-
treated,
noninfected mice 10.9 {n=3), c) saline-treated, infected mice 10.2 (n=2), d)
lactoferrin-treated, infected mice 16.2 (n=3). The increase in the intestinal
goblet
cell number in lactoferrin-treated, noninfected mice was 53% when compared
with their saline-treated, noninfected counterparts. A similar increase (58%)
was
observed in lactoferrin-treated, infected mice when compared with their saline
treated, infected counterparts.
It is evident that the intestinal damage due to parasitic infection is
significantly reduced in lactoferrin-treated animals. These important
observations
suggest that lactoferrin protects intestinal function during insult-induced
metabolic
imbalance.
EXAMPLE 3
This experiment was designed to test the effect of lactoferrin on the
establishment of Trichinella spiralis larvae in mouse intestine, after
inoculation
with infective worms. Mice were given human lactoferrin (1mg/100 NI saline)
orally by gavaging for three consecutive days. The control group was given
saline. On day four, following three days of treatment, mice were infected
with
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600 Trichinella spiralis larvae. Twenty four hours later worms were collected
from
the gut and counted.
The number of Trichinella spiralis infective larvae that established in the
intestine of mice fed lactoferrin supplemented diet for three days prior to
infection
was lower than in their counterparts (152 ~ 10.8; n=6 versus 211.8 ~ 25.6;
n=6).
it is evident that lactoferrin attenuates the establishment of infective
larvae in
mouse intestine.
II. The effects of lactoferrin on the metabolic activity in endotoxemic mice.
The administration of lactoferrin was performed in a non-invasive
procedure such as gavage or in an invasive procedure such as intravenously or
intraperitoneally.
EXAMPLE 4
The effect of human lactoferrin on the development of LPS-induced
endotoxemia in mice was determined by examining survival of mice upon
challenge with a lethal dose of LPS. Naive mice were injected once
intraperitoneally with 150N1 of saline solution lactoferrin (7.5mg/mouse) one
hour
before or after LPS challenge. This illustrates both a prophylactic and
therapeutic
treatment. Their control counterparts were given 150N1 of saline. Bacterial
LPS
(E.coli, Serotype 0111:B4) was given intraperitoneally at the lethal dose of
1.5x106 endotoxic units per mouse. The survival of mice was monitored over the
period of time of four weeks (n=6 per each group).
Treatment Survival
LPS 16.6
Lactoferrin followed by
LPS
ro h lactic a lication 83.3
LPS followed by Lactoferrin
thera eutic a lication 66.6
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A single intraperitoneal dose of lactoferrin (7.5mg) administered 1 hour
before or after LPS challenge significantly increased the survival of mice.
EXAMPLE 5
The effectiveness of oral administration of lactoferrin on survival of mice .
subjected to lethal injection of LPS was determined in the following
experiment.
Naive mice were gavaged with 150N1 of saline solution of bovine lactoferrin
(7.5mg/dose) for three days prior or after LPS challenge. Their control
counterparts were given 1501 of saline. Bacterial LPS (E.coli, Serotype
0111:B4)
was given intravenously at the lethal dose of 1.5x106 endotoxic units per
mouse.
The survival of mice was monitored over the period of time of four weeks (n=6
per each group).
Treatment Survival /( o)
LPS 37.5
Lactoferrin followed by
LPS
ro h lactic a lication 50
LPS followed by Lactoferrin
thera eutic a lication 83.3
Oral administration of lactoferrin for three days prior or after intravenous
administration of LPS increased significantly the survival of mice.
EXAMPLE C
Also, the protective effect of lactoferrin on survival of mice subjected to
lethal injection of LPS was determined by administering lactoferrin
intravenously.
Naive mice were injected intravenously with 150N1 of saline solution of bovine
lactoferrin (7.5mg/dose) for three days prior or after LPS challenge. Their
control
counterparts were given 150NI of saline. Bacterial LPS (E.coli, Serotype
0111:B4)
was given intravenously at the lethal dose of 1,5x106 endotoxic units per
mouse.
The survival of mice was monitored over the period of time of four weeks (n=6
per each group).
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Treatment ~ Survival (%)
LPS 37.5
Lactoferrin followed by
LPS
ro h lactic a lication 100
LPS followed by Lactoferrin
thera eutic a lication 100
Intravenous administration of lactoferrin for three days prior or after
intravenous administration of LPS provides total protection from a lethal dose
of
LPS.
EXAMPLE 7
The effect of lactoferrin on electrophysiological parameters of gut function
in mice challenged with a lethal dose of LPS was determined in the following
experiment. Naive mice (n=6 per each group) were injected intraperitoneally
with
a lethal dose of LPS (1.5x106 endotoxic units per mouse). One hundred
microliters (100N1) of saline solution of human lactoferrin (10mg/ml) was
given to
mice enterally by gavaging at 1 hour, 3 hours and 6 hours post-LPS challenge.
Their counterparts were given saline. Twenty four hours later the blood
samples
were collected, mice were sacrificed and jejunal segments removed for
electrophysiological and histological measurements.
Treatment of mice with lactoferrin had no effect on the electrophysiological
characteristics of jejunal epithelium. Resistance (R) of the intestinal tissue
following LPS challenge was significantly lower in both lactoferrin treated
mice
and saline control. Mice fed lactoferrin and challenged with LPS showed a
moderate increase in short circuit current when compared with saline control.
PD
was comparable for all groups. Glucose absorption was about 30% higher for
LPS-challenged animals with no significant difference between lactoferrin
treated
and saline control. Cl- secretory response to 5-HT and CCh was also elevated
in
both LPS groups.
The intestinal epithelium of mice injected with LPS exhibited severe
vacuolar degeneration in control animals with shortening and shrinking of the
villi
and expansion of the crypts. There were heavy inflammatory infiltrates in the
CA 02288982 1999-11-02
WO 98/50076 PCT/US98/09053
tunica mesenteris of control animals (Fig 4). In the lactoferrin-treated mice
vacuolar degeneration was less pronounced with the epithelium resembling the
highly polarized, resorbtive epithelium of non-infected mice.
This protective effect of lactoferrin on gut function and structure during
experimental endotoxemia correlates with significant reduction of cytokines in
plasma. It has been shown that lactoferrin attenuates the release of TNFa and
IL
1 a into plasma by more than 50% when measured 2 hours post LPS challenge.
However, only an insignificant decrease in the cancentration of those
cytokines
was observed in the intestinal tissue homogenates.
It is evident , that lactoferrin helps to maintain the physiological function
of
the gut during LPS-induced metabolic imbalance.
EXAMPLE 8
The thermoregulatory effects of lactoferrin were tested on endotoxemic
mice by measuring body temperature in conjunction with production of pro-
inflammatory cytokines. Naive mice were injected intravenously with 1501 of
saline solution of bovine lactoferrin (7.5 mg/mouse). Their control
counterparts
were given 150N1 of saline. One hour later all mice were challenged
intravenously
with LPS at the dose of 3.75x105 endotoxic units per mouse. Two hours later
mice were anesthetized, temperature was measured and blood was collected.
The concentration of pro-inflammatory cytokines in plasma and tissue
homogenates was measured by Enzyme-linked immunoabsorbent assay (ELISA)
using specific antibodies for each antigen.
Treatment TNFa IL-1 ~i IL-6 Temperature
/ml c/ml pg/ml C
Saline 0.000.00 11.521.40 9.313.6 38.160.21
Lactoferrin 1.601.60 1.450.93 12.12.2 36.330.24
LPS 694.4125.9 128.3114.2 23,5662,466 35.160.21
Lactoferr./LPS280.536.8 26.1 4.43 7,1441,403 36.91 0.08
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WO 98/50076 PCT/US98/09053
Statistically significant differences were found between lactoferrin-treated
mice and their control counterparts for all parameters measured (n=6 per each
group). Lactoferrin attenuates the release of TNFa, IL-1 ~3, and IL-6 into
plasma
and modulates the decrease of body temperature that is due to the insult-
induced
' metabolic imbalance. Moreover, the effect of lactoferrin on the
thermogenesis is
confirmed by the fact that lactoferrin interplays with the major
thermoregulatory
factor nitric oxide during insult-induced hypo- or hyper-activity.
It is evident from all the foregoing examples that lactoferrin given either
orally or systemically is capable of modulating the gut function during insult
induced metabolic imbalance by protecting intestinal epithelium. We have
demonstrated that such protection of gut function have inhibitory systemic
effect
on development of autodestructive mechanisms including death. Both
prophylactic and therapeutic applications of lactoferrin have been shown to be
effective in accordance with the present invention.
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