Note: Descriptions are shown in the official language in which they were submitted.
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W098/06425 1 PCT/SE97/013M
TREATMENT AND PREVENTION OF INFECTIONS, INFLAMMATIONS
AND/OR TUMOURS WITH LACTOFERRIN AND/OR LACTOFERRICIN
FIELD OF THE INVENTION
The present invention relates to a pharmaceutical
composition comprising lactoferrin and/or lactoferricin
for treatment and/or prevention of infections, inflamma-
tions and/or tumours, to the use of lactoferrin and lac-
toferricin in the production of a pharmaceutical composi-
tion for treatment and/or prevention of infections, in-
flammations and tumours, and to a method for treatment
and/or prevention of infections, inflammations and/or tu-
mours comprising administration of lactoferrin and/orlactoferricin.
BACKGROUND OF THE INVENTION
It is known that human milk in several ways is anti-
inflammatory. Goldman et al. pointed out that human milkis poor in initiators and mediators of inflammation but
rich in anti-inflammatory agents (see Goldman A. S., et
al., Anti-inflammatory properties of human milk, Acta
Paediatr. Scand. 75:689-695, 1986). Human milk contains
several soluble anti-infective components, such as spe-
cific secretory IgA (SIgA) antibodies and non-specific
components, including lactoferrin (LF) (see e.g. Hanson
L. A., et al., Protective factors in milk and the devel-
opment of the immune system, Pediatrics 75:172-176,
1983).
Lactoferrin is a single chain metalbinding glycopro-
tein with a molecular weight of 77 kd. It occurs in three
isoforms: LF-a, LF-~, and LF-y. These three variants have
the same physical, chemical and antigenic characteris-
tics, but differ in their functional properties.
The iron-binding lactoferrin is also present in spe-
cific granules of polymorphonuclear leucocytes and in
other exocrine secretions than milk such as saliva, tears
and bronchial mucus, as well as cervical secretion, amni-
otic fluid, decidua, and trophoblasts (see e.g. Montreuil
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J., et al., Isolement d'une lactosiderophiline du lait de
femme, CR Acad. Sci. Paris 250 D:1736-37, 1960; Montreuil
J., et al., Preparation et propriétés de la lactosidero-
philine (lactotransferrine) du fait de femme, Biochim.
Biophys. Acta 45:413-421, 1960; and Masson P. L., et al.,
Lactoferrin an ironbinding protein neutrophilic leuco-
cytes, J. Exp. Med. 130:643-656, 1969). Lactoferrin is
associated with host defense at mucosal surfaces through
its antibacterial and iron-binding properties.
Human lactoferrin is found in colostrum and mature
milk at levels of 2-5 g/l.
Bovine lactoferrin shares 68% and 64% amino acid
identity with human lactoferrin and murine lactoferrin,
respectively.
Lactoferricin is a pepsin-cleaved fragment of human
and bovine lactoferrin. It has recently been found to
contain the structural domain responsible for the bacte-
ricidal properties of lactoferrin (see e.g. Bellamy W.,
et al., Identification of the bactericidal domain of lac-
toferrin, Biochim. Biophys. Acta 1121:130-136, 1992, and
Bellamy W., et al., Antibacterial spectrum of lactofer-
ricin B, a potent bactericidal peptide derived from the
N-terminal region of bovine lactoferrin, J. Appl. Bact.
73:472-479, 1992).
Lactoferrin receptors are found on many types of
cells including monocytes and macrophages (Broxmeyer H.
E., et al., Specificity and modulation of the action of
lactoferrin, a negative feedback regulator of myelopoi-
esis, Blood 55:324-333, 1980), lectin-stimulated human
peripheral blood lymphocytes (Mazurier J., et al., Ex-
pression of human lactotransferrin receptors in phytohe-
magglutinin-stimulated human peripheral blood lympho-
cytes. Isolation of the receptors by anti-ligand-affinity
chromatography, Eur. J. Biochem. 179:481-487, 1989),
brush-border cells (Hu W. L., et al., Lactotransferrin
receptor of mouse small-intestinal brush border. Binding
characteristics of membrane-bound and Triton X-100-
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solubilized forms, Biochem. J. 249:435-441, 1988i Cox T.
M., et al., Iron-binding proteins and influx of lron
across the duodenal brush border. Evidence for specific
lactotransferrin receptors in the human intestine. Bio-
chim. Biophys. Acta 588:120-128, 1979; Mazurier J., et
al., Visualization of lactotransferrin brush-border re-
ceptors by ligand-blotting. Biochim. Biophys. Acta
821:453-460, 1985; and Wei-Lu Hu, et al., Isolation and
partial characterization of a lactotransferrin receptor
from mouse intestinal brush-border, Biochemistry 29:535-
540, 1990), tumor cell lines, e.g. HT-29, HL-60, K562,
(see e.g. Roiron D., et al., Lactoferrin-binding sites at
the surface of HT29-D4 cells. Comparison with transfer-
rin. Eur. J. Biochem. 186:367-373, 1989; Miyazawa K., et
al., Effect on lactoferrin binding to monocyte/macro-
phage-differentiated HL-60 cells. J Immunol. 146:723-729,
1991; and Yamada Y., et al., Lactoferrin binding by leu-
kemia cell lines, Blood 70:264-270, 1987).
In addition to the role of lactoferrin as an essen-
tial growth factor for both human B- and T-lymphocytic
cell lines (see e.g. Hashizume S., et al., Identification
of lactoferrin as an essential growth factor for human
lymphocytic cell lines in serum-free medium, Biochem.
Biophys. Acta 763:377-382, 1983) and as an inducer of
growth of HT-29 cells (see e.g. Anuric M., et al., Effect
of lactoferrin on the growth of a human colon adenocarci-
noma cell line - comparison with transferrin. In Vitro
20:543-548, 1984), lactoferrin is a negative regulator of
myelopoiesis (see e.g. Broxmeyer H. E., et al., Specific-
ity and modulation of the action of lactoferrin, a nega-
tive feedback regulator of myelopoiesis, Blood 55:324-
333, 1980, and Gentile P., et al., Suppression of mouse
myelopoesis ~y administration of human lactoferrin in
vivo and the comparative action of human transferrin,
Blood 61:982-993, 1983). This latter function is mediated
through suppression of IL-1 and GM-CSF release from mono-
cytes and macrophages (see e.g. Broxmeyer H. E., et al.,
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Lactoferrin acts on I-A and I-E/C antigen subpopulations
of mouse peritoneal macrophages in the absence of T lym-
phocytes and other cell types to inhibit production of
granulocyte-macrophage co~ony stimulatory factors "in vi-
tro", J. Immunol. 133:306-314, 1984, and Zucali J. R., et
al., Lactoferrin decreases monocyte-induced fibroblast
production of myeloid colony-stimulating activity by sup-
pressing monocyte release of interleukin-1, Blood
74:1531-1536, 1989).
After binding of bacterial lipopolysaccharides (LPS)
to macrophages, T-cells and cultured human monocytes,
these cells synthesize tumor necrosis factor-a (TNF-a),
interleukin-1 (IL-1), interleukin-6 (IL-6), and colony-
stimulating factor (CSF) (see e.g. Arai K., et al., Cy-
tokines: coordinators of immune and inflammatory re-
sponses, Ann. Rev. Biochem. 59:783-836, l9gO; Hirano T.,
et al., Biological and clinical aspects of interleukin 6,
Immunol. Today 11:443-449, 1990; and Shalaby M. R., et
al., Endotoxin, tumor necrosis factor-a and interleukin-1
induce interleukin-6 production "in vivo", Clin. Immunol.
Immunopath. 53:488-498, 1989). Cells participating in the
inflammatory response carry several different LPS-binding
receptors (Lei M-G., et al., Specific endotoxic lipopoly-
saccharide-binding proteins on murine splenocytes. II.
Membrane localization and binding characteristics, J. Im-
munol. 141:1006-1011, 1988 and Couturier C., et al.,
Binding sites for endotoxin (LPS) on human monocytes, J.
Immunol. 147:1899-1904, lg91). Such cells also have re-
ceptors for lactoferrin. An interaction between LPS and
lactoferrin has been observed, the complex being bound to
the cells also via LPS receptors (see Miyazawa K., et
al., Effect on lactoferrin binding to monocyte/macro-
phage-differentiated HL-60 cells. J Immunol. 146:723-729,
1991). Recently, it was showed that lactoferrin exerted
an inhibitory effect on the production of IL-1 and TNF-a
in LPS stimulated monocytes (see Crouch P. M., et al.,
Regulation of cytokine release from mononuclear cells by
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W O 98/06425 5 PCTtSE97/01344
the iron-binding protein lactoferrin, Blood 80:235-2~0,
1992).
It has earlier been shown (see Mattsby-Baltzer I. et
al., Lactoferrin or a fragment thereof inhibits the endo-
toxin-induced interleukin-6 response in human monocytic
cells, Pediactric Research 40:257-262, 1996) that human
and bovine lactoferrin as well as bovine lactoferricin
suppress LPS-induced IL-6 response when added to fresh
monocytes or cultured monocytic cells. Human lactoferrin
has also been reported to suppress TNF-~ induced
IL-6 response when added to fresh monocytes or cultured
monocytic cells.
According to the present invention it has now been
found that lactoferrin and lactoferricin have an in vivo
effect on all kinds of inflammation, i.e. not only when
IL-6 is involved, as well as on infections, such as uri-
nary tract infection, and tumours.
DESCRIPTION OF THE INVENTION
Thus, an object of the present invention is to pro-
vide a pharmaceutical composition for treatment and/or
prevention of infections, inflammations and/or tumours
comprising an effective amount of lactoferrin and/or lac-
toferricin.
Another object of the present invention is use of
lactoferrin and/or lactoferricin in the production of a
pharmaceutical composition for treatment and/or preven-
tlon of infections, inflammations and/or tumours.
A third object of the present invention is to pro-
vide a method for treatment and/or prevention of infec-
tions, inflammations and/or tumours by administration of
an effective amount of lactoferrin and/or lactoferricin.
The characterising features of the invention will be
evident from the following description and the appended
claims.
In order to treat a patient, suffering from an in-
fection, an inflammation or a tumour, with the pharmaceu-
,, . ~ .
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tical composition according to the invention, the pharma-
ceutical composition, comprising an effective amount of
lactoferrin and/or lactoferricin, is preferably adminis-
tered systemically, and most preferably orally.
The infections treatable with the pharmaceutical
composition according to the present inventions include
infections caused by all kinds of pathogens, such as bac-
teria, viruses, fungi, etc.
Inflammation is a phenomenon marked by abnormal
"redness'r and swelling of tissues and organs, pain and
heat in affected areas, capillary dilation, leucocyte in-
filtration, etc. Inflammation is primarily caused by ex-
posure to bacterial and other noxious agents and physical
injury. Inflammation is mediated by a variety of cytoki-
nes and other chemical signals. These mediators of in-
flammation include tumor necrosis factor-a (TNF-a), in-
terleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8
(IL-8), and various colony-stimulating factors (CSFs).
As used herein, "treatment" refers to preventing,
curing, reversing, attenuating, alleviating, minimizing,
suppressing or halting the deleterious effects of a dis-
ease state, disease progression or other abnormal condi-
tion, including urinary tract infections.
"Prevention" refers to minimizing, reducing or sup-
pressing the risk of developing a disease state or pro-
gression or other abnormal or deleterious conditions.
A "patient" is a subject at risk for or suffering
from a disease state, disease progression or other abnor-
mal or deleterious condition.
An "effective amount" is an amount sufficient to
treat or prevent a disease state, disease progression or
other abnormal or deleterious condition.
"Systemic administration" can be undertaken by oral,
nasal, intravenous, intraartery, intracavitary, intramus-
cular, subcutaneous, transdermal, suppositories
(including rectal) or other routes known to those of
skill in the art. Preferably, the pharmaceutical composi-
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tion according to the present invention is formulated for
oral administration.
The lactoferrin and lactoferricin used according to
the present invention can e.g. be obtained through isola-
tion and purification from natural sources, such as humanmilk, through use of genetic engineering techniques, such
as recombinant expression or direct production in geneti-
cally altered animals, or through chemical synthesis. The
lactoferricin can also be obtained by enzymatic degrada-
tion of lactoferrin (hydrolysate).
The lactoferrin used according to the present inven-
tion is preferably human lactoferrin or bovine lactofer-
rin, and it is preferably administered as a hydrolysate.
The lactoferricin used according to the present in-
vention is preferably human lactoferricin or bovine lac-
toferricin.
The pharmaceutical composition comprising lactofer-
rin and/or lactoferricin according to the present inven-
tion is particularly well suited for treatment and/or
prevention of urinary tract infection and colitis, but
several other inflammatory and infectious diseases are
also treatable according to the present invention, such
as inflammatory bowel diseases, rheumatoid arthritis,
conditions caused by the virus ~IV-l, conditions caused
by the virus CMV, and conditions caused by the fungus
Candida albicans.
The pharmaceutical composition according to the pre-
sent invention is also well suited for preventive medical
care by reducing the risk of developing urinary tract in-
fection or other inflammatory or infectious diseases inpatients with an increased risk of attracting such com-
plications.
The pharmaceutical composition according to the pre-
sent invention may also comprise other components, such
as pharmaceutically acceptable carriers, vehicles, pre-
servatives, lubricators etc., which is well known to per-
sons skilled in the art.
.
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According to the present invention it is also possi-
ble to include lactoferrin and/or lactoferricin, in an
effective amount, in any kind of food or beverage in-
tended to reduce infections and/or inflammations in pa-
tients running an increased risk of such conditions dueto an underlying disease or a medical treatment.
According to the present invention it is also possi-
ble to include lactoferrin and/or lactoferricin, in an
effective amount, in an infant formula food intended to
inhibit harmful effects of bacteria, such as weight loss
caused by inflammation induced by bacteria, viruses or
fungi in infants.
EXAMPLES
The invention will now be further explained in the
following examples. These examples are only intended to
illustrate the invention and should in no way be consid-
ered to limit the scope of the invention.
In the examples reference is made to the accompany-
ing drawings on which:
Fig. 1 a - d illustrate bacterial recovery from the kid-
ney (a and b) and bladder (c and d), respectively,
of C3H/Tif and C3H/HeN mice infected with E. coli
in the urinary tract and perorally given human
lactoferrin (LF hum), bovine lactoferrin (LF bov),
or PBS, 30 min after the injection of bacteria.
The samples represented by symbols below the line
were culture negative.
Fig. 2 a and b illustrate the kinetics of the urinary
leucocyte influx in E. coli infected C3H/Tif and
C3H/HeN mice treated with human lactoferrin (LF
hum), bovine lactoferrin (LF bov), or PBS.
Fig. 3 a and b illustrate the kinetics of the urinary IL-
6 response in E. coli infected C3H/Tif and C3H/HeN
mice treated with human lactoferrin (LF hum), bo-
vine lactoferrin (LF bov), or PBS, 30 min after
the injection of bacteria.
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Fig. 4 lllustrates the serum IL-6 response 24 h after ex-
perimentally induced urinary tract infection in
C3H/Tif and C3H/HeN mice treated with human lac-
toferrin (LF hum), bovine lactoferrin (LF bov), or
PBS, 30 min after the injection of bacteria.
Fig. 5 illustrates the cytokine concentration in serum
from mice with experimentally induced colitis af-
ter treatment with bovine lactoferrin (LF bov)
compared to a control group not receiving lac-
toferrin.
Example 1: Treatment of urinary tract infection in mice
by oral administration of human lactoferrin
The antibacterial and anti-inflammatory properties
of lactoferrin were explored by studying the effects of
lactoferrin given to mice (C3H/Tif and C3H/HeN) with ex-
perimentally induced urinary tract infection (UTI).
In order to induce urinary tract infection (UTI) in
the mice, the animals were injected with 100 ~l of a bac-
terial solution containing 2xlO9 E. coli-bacteria/ml di-
luted with phosphate-buffered saline (PBS) directly into
the bladder via a catheter according to Svanborg-Edén et
al (see C. Svanborg-Edén et al Infect. Immun. 55:1224-
1232, 1987).
A solution containing 10 mg/ml of either human lac-
toferrin, bovine lactoferrin, or bovine lactoferricin was
orally administered (50 ~1) to the mice 30 min after the
instillation of bacteria.
Urine samples from the mice were collected 0, 2, 5,
and 24 hours after infection. 50 ~l of each of the undi-
luted urine samples were cultured. The number of leuco-
cytes in uncentrifuged urine was analyzed for each sam-
ple. The remaining urine from each animal at each sam-
pling time was centrifuged and saved for IL-6 analysis.
After 24 h the mice were bled and killed. The blad-
der and kidneys were taken out aseptically. The organs
were homogenized, and serial dilutions thereof (bladder
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1/1, 1/10, kidneys 1/1, 1/10, 1/100, 1/1000) were cul-
tured on Drigalsky plates.
The results are illustrated below in Table 1 and in
Figures 1-4.
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WO 98/06425 PCT/SE97/01344
r_
~ ~ a~ a
~ C 'O ~ 110 0 ~ ~ r- O O O O
3 E , ~ E~ Co o
U~ ~ ,_ U~
~ ~ 1-- o ~ -- o o
v ~ ~ o o ~ o ~ ~ e E
~~1 ~ O ~ ~ O
o o~ ~ ~~~ ~ O O
o o
O ~ ~
~ ~ o ~ ~ ~
v~ c O ~ c c ~ ~ ' v
o o ~~ 3
~ a
.. , ~ ~ ~
~ O ~ O
O ~ ~ ~ o ~n -~J ~ -~ ~
3 ~ O ~ O
o u~ ~ o u~
~V
o a)
) a) ,~ o ~) ~ Ll ~ L
L a) J~ ~) ~ U~ ) O O O Ll ~) Ll
a) a~h ~ ~ O -r~l ~) ~ ~
~- Q ~ ~ 3 ~ -~ Q, Q
a~ Ll ,~ L ,~ ~
a~ -~ ~ ~ H '- H t~
CA 02263416 1999-02-11
W098/0~25 12 PCT/SE97/01344
The data shown in Table 1 clearly shows the effect
of the treatment with lactoferrin and lactoferricin.
From the table and the figures it is evident that
orally administered lactoferrin (both human and bovine)
significantly decreased the number of bacteria in the
urinary tract of the infected mice, compared to the con-
trol group.
In Figures 2 a and b the kinetics of the urinary
leucocyte influx is illustrated (** in Figure 2 a signi-
fies p<0.01, Mann-Whitney test), and in Figure 3 a and b
the IL-6 response in urine is illustrated (* in these
figures signifies p<0.05, Mann-Whitney test). These fig-
ures clearly shows that the local inflammatory response
was reduced after 24 h.
The systemic cytokine response, viz. IL-6 response
in serum, after 24 h is illustrated in Figure 4, and this
response was also reduced in the lactoferrin treated ani-
mals.
In conclusion these results demonstrate that oral
administration of lactoferrin or lactoferricin is sys-
temically effective by preventing infection and inflamma-
tion in the urinary tract by an as yet unidentified
mechanism.
Example 2: Treatment of experimental colitis by oral ad-
ministration of human lactoferrin
Acute colitis was induced in C57BI/6J mice by giving
5% dextransulphate in the drinking water for 6 days. Hu-
man lactoferrin was orally given to ten mice twice a day
in a dose of 1 mg/mouse, starting from day 3 of the ex-
periment. Two control groups (in total 17 mice) were
given the same volume of drinking water or bovine serum
albumin (BSA) (2 mg per mouse and day). 30% of the mice
in the lactoferrin treated group presented gross rectal
bleeding on day 5 and 6 compared to 100% in the control
group (p = 0.0007, Fischer's test). Moreover, the colon
length was significantly reduced in the control groups
. . . . . . .
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compared with the lactoferrin treated group, indicating a
more advanced inflammation of the colon tissue in the
controls (p = 0.041, Mann-Whitney test). High concentra-
tions of lactoferrin were found in serum of the lactofer-
S rin treated group.
In an other experiment using 3% dextransulphate the
systemic TNF-a response was reduced in the lactoferrin
treated mice after 10 days (p < 0.0006). The result is
illustrated in Figure 5.
In summary, the results demonstrate that oral ad-
ministration of LF reduces some of the clinical symptoms
of experimental colitis.
