U.S. patent application number 12/493919 was filed with the patent office on 2009-10-15 for lactoferrin.
This patent application is currently assigned to Fonterra Corporate Research and Development Ltd.. Invention is credited to Jillian Cornish, Neill Ward Haggarty, Kate Patricia Palmano, Ian Reginald Reid.
Application Number | 20090259025 12/493919 |
Document ID | / |
Family ID | 28673156 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090259025 |
Kind Code |
A1 |
Cornish; Jillian ; et
al. |
October 15, 2009 |
Lactoferrin
Abstract
A pure lactoferrin polypeptide containing no more than two metal
ions per molecule, or a mixture of the polypeptide and a fragment
thereof. The polypeptide or the mixture stimulates skeletal growth
and inhibits bone resorption. Also disclosed is a method of
treating a bone-related disorder with the polypeptide or the
mixture.
Inventors: |
Cornish; Jillian; (Auckland,
NZ) ; Reid; Ian Reginald; (Auckland, NZ) ;
Palmano; Kate Patricia; (Palmerston North, NZ) ;
Haggarty; Neill Ward; (Palmerston North, NZ) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Fonterra Corporate Research and
Development Ltd.
NZMP & Health & Nutrician Unit
Auckland UniServices Ltd.
|
Family ID: |
28673156 |
Appl. No.: |
12/493919 |
Filed: |
June 29, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12098253 |
Apr 4, 2008 |
|
|
|
12493919 |
|
|
|
|
10205960 |
Jul 26, 2002 |
|
|
|
12098253 |
|
|
|
|
Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61P 19/00 20180101;
C07K 14/79 20130101; A23L 33/19 20160801; A61P 19/10 20180101; A61P
3/02 20180101; A23L 33/18 20160801; A23V 2002/00 20130101; A23L
33/16 20160801; A61K 38/00 20130101; A23V 2002/00 20130101; A23V
2250/54248 20130101; A23V 2250/1592 20130101; A23V 2200/306
20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/79 20060101
C07K014/79 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 3, 2002 |
NZ |
518121 |
Claims
1. A pure lactoferrin polypeptide containing no more than two metal
ions per molecule, wherein the polypeptide stimulates skeletal
growth and inhibits bone resorption.
2-37. (canceled)
Description
RELATED APPLICATION
[0001] This application claims priority to New Zealand Application
Ser. No. 518121, filed Apr. 3, 2002, the content of which is
incorporated herein by reference.
BACKGROUND
[0002] Lactoferrin is an 80kD iron-binding glycoprotein present in
most exocrine fluids, including tears, bile, bronchial mucus,
gastrointestinal fluids, cervico-vaginal mucus, seminal fluid, and
milk. It is a major constituent of the secondary specific granules
of circulating poly-morphonuclear neutrophils. The richest source
of lactoferrin is mammalian milk and colostrum.
[0003] Lactoferrin circulates at a concentration of 2-7 .mu.g/ml.
It has multiple postulated biological roles, including regulation
of iron metabolism, immune function, and embryonic development.
Lactoferrin has anti-microbial activity against a range of
pathogens including Gram positive and Gram negative bacteria,
yeasts, and fungi. The anti-microbial effect of lactoferrin is
based on its capability of binding iron, which is essential for the
growth of the pathogens. Lactoferrin also inhibits the replication
of several viruses and increases the susceptibility of some
bacteria to antibiotics and lysozyme by binding to lipid A
component of lipopolysaccharides on bacterial membranes.
SUMMARY
[0004] This invention relates to a lactoferrin polypeptide that is
capable of stimulating skeletal growth and inhibiting bone
resorption.
[0005] Specifically, this invention features a pure lactoferrin
polypeptide containing no more than two (i.e., 0, 1, or,
preferably, 2) metal ions per molecule. A "pure" polypeptide is a
polypeptide free from other biological macromolecules and at least
65% (e.g., at least 70, 75, 80, 85, 90, 95, or 99%) pure by dry
weight. The purity of a polypeptide can be measured by any
appropriate standard method, for example, by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis. The
lactoferrin polypeptide can be a naturally occurring polypeptide, a
recombinant polypeptide, or a synthetic polypeptide. Variants of a
wild-type lactoferrin polypeptide (e.g., a fragment of the
wild-type lactoferrin polypeptide containing at least 2 (e.g., 4,
6, 8, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700) amino acids,
or a recombinant protein containing a lactoferrin polypeptide
sequence) that maintain the biological activity of a wild-type
lactoferrin polypeptide are within the scope of the invention. A
lactoferrin polypeptide of the invention can be of a mammalian
origin, e.g., from human or bovine milk. The metal ion bound to the
polypeptide can be an iron ion (as in a naturally occurring
lactoferrin polypeptide), a copper ion, a chromium ion, a cobalt
ion, a manganese ion, a zinc ion, or a magnesium ion.
[0006] A lactoferrin polypeptide of the invention can be used to
stimulate skeletal growth (e.g., by promoting proliferation of
osteoblasts and chondrocytes) and inhibit bone resorption (e.g., by
inhibiting osteoclast development). A preparation of a lactoferrin
polypeptide of the invention (e.g., lactoferrin isolated from
bovine milk) can contain polypeptides of a single species, e.g.,
every molecule binding two iron ions. It can also contain
polypeptides of different species, e.g., some molecules binding no
ion and others each binding one or two ions; some molecules each
binding an iron ion and others each binding a copper ion; some
molecules each being a biological active lactoferrin polypeptide
(full-length or shorter than full-length) that contains 0, 1, or 2
metal ions and others each being a fragment (same or different) of
the polypeptide; or all molecules each being a fragment (same or
different) of a full-length lactoferrin polypeptide that contains
0, 1, or 2 metal ions. For example, a mixture of full-length
lactoferrin polypeptides and various fragments of full-length
lactoferrin polypeptides can be prepared from a hydrolysate, e.g.,
a partial digest such as a proteinase digest, of full-length
lactoferrin polypeptides. Otherwise, it can be obtained by mixing
full-length lactoferrin polypeptides with various fragments of
full-length lactoferrin polypeptides (e.g., synthetic fragments). A
mixture of various fragments of full-length lactoferrin
polypeptides, on the other hand, can be prepared, for example, by
complete digestion (i.e., no full-length polypeptides remain after
digestion) of full-length lactoferrin polypeptides, or by mixing
different fragments of full-length lactoferrin polypeptides.
[0007] The invention further features a nutraceutical composition,
which can be milk, juice, a soft drink, a snack bar, or a dietary
supplement. The nutraceutical composition contains a lactoferrin
polypeptide of the invention or a mixture of the polypeptide and
fragments of the polypeptide in an amount higher than the naturally
occurring amount. Lactoferrin has been found to stimulate
osteoblast and chondrocyte proliferation and inhibit osteoclast
development. Thus, a nutraceutical composition of this invention is
useful for preventing and treating bone disorders such as
osteoporosis and rheumatoid or osteo-arthritis. The nutraceutical
composition can further include an adequate amount of another
bone-enhancing agent, such as calcium, zinc, magnesium, vitamin C,
vitamin D, vitamin E, vitamin K2, or a mixture thereof.
[0008] In addition, this invention features a pharmaceutical
composition that contains a lactoferrin polypeptide of the
invention or a mixture of the polypeptide and fragments of the
polypeptide and a pharmaceutically acceptable carrier. Optionally,
the pharmaceutical composition also includes another bone-enhancing
agent. The invention also encompasses the use of a lactoferrin
polypeptide or a mixture of the polypeptide and fragments of the
polypeptide described above for the manufacture of a medicament for
preventing and treating bone diseases.
[0009] This invention provides a method of preventing and treating
bone-related disorders (e.g., by stimulating skeletal growth and
inhibiting bone resorption). The method includes administering to a
subject in need thereof an effective amount of a lactoferrin
polypeptide of the invention or a mixture of the polypeptide and
fragments of the polypeptide. The method can further include
concurrently administering to the subject an effective amount of
another bone-enhancing agent.
[0010] The details of one or more embodiments of the invention are
set forth in the accompanying description below. Other features,
objects, and advantages of the invention will be apparent from the
detailed description, and from the claims.
DETAILED DESCRIPTION
[0011] This invention is based on the unexpected discovery that
lactoferrin stimulates osteoblast and chondrocyte proliferation and
inhibits osteoclast development. Thus, it is useful for preventing
and treating bone disorders.
[0012] A lactoferrin polypeptide of the invention is a pure
polypeptide containing no more than two metal ions per molecule.
Practically, the measurement of the ion/lactoferrin ratio for a
preparation of lactoferrin can be in the range of 0-2.5. It can be
isolated from a natural source (e.g., mammalian milk), or produced
using genetic engineering or chemical synthesis techniques
well-known in the art. The following is an exemplary procedure for
isolating lactoferrin from bovine milk:
[0013] Fresh skim milk (7 L, pH 6.5) is passed through a 300 ml
column of S Sepharose Fast Flow equilibrated in milli Q water, at a
flow rate of 5 ml/min and at 4.degree. C. Unbound protein is washed
through with 2.5 bed volumes of water and bound protein eluted
stepwise with approximately 2.5 bed volumes each of 0.1 M, 0.35 M,
and 1.0 M sodium chloride. Lactoferrin eluting as a discreet pink
band in 1 M sodium chloride is collected as a single fraction and
dialysed against milli Q water followed by freeze-drying. The
freeze-dried powder is dissolved in 25 mM sodium phosphate buffer,
pH 6.5 and subjected to rechromatography on S Sepharose Fast Flow
with a sodium chloride gradient to 1 M in the above buffer and at a
flow rate of 3 ml/min. Fractions containing lactoferrin of
sufficient purity as determined by gel electrophoresis and reversed
phase HPLC are combined, dialyzed and freeze-dried. Final
purification of lactoferrin is accomplished by gel filtration on
Sephacryl 300 in 80 mM dipotassium phosphate, pH 8.6, containing
0.15 M potassium chloride. Selected fractions are combined,
dialyzed against milli Q water, and freeze-dried. The purity of
this preparation is greater than 95% as indicated by HPLC analysis
and by the spectral ratio values (280 nm/465 nm) of .about.19 or
less for the iron-saturated form of lactoferrin.
[0014] Iron saturation is achieved by addition of a 2:1 molar
excess of 5 mM ferric nitrilotriacetate (Foley and Bates (1987)
Analytical Biochemistry 162, 296-300) to a 1% solution of the
purified lactoferrin in 50 mM Tris, pH 7.8 containing 10 mM sodium
bicarbonate. Excess ferric nitrilotriacetate is removed by dialysis
against 100 volumes of milli Q water (twice renewed) for a total of
20 hours at 4.degree. C. The iron-loaded (holo-) lactoferrin is
then freeze-dried.
[0015] Iron-depleted (apo-) lactoferrin is prepared by dialysis of
a 1% solution of the highly purified lactoferrin sample in water
against 30 volumes of 0.1 M citric acid, pH 2.3, containing 500
mg/L disodium EDTA, for 30 h at 4.degree. C. (Massons and Heremans
(1966) Protides of the Biological fluids 14, 115-124). Citrate and
EDTA are then removed by dialysis against 30 volumes of milli Q
water (once renewed) and the resulting colourless solution
freeze-dried.
[0016] A lactoferrin polypeptide of the invention can contain an
iron ion (as in a naturally occurring lasctoferrin polypeptide) or
a non-iron metal ion (e.g., a copper ion, a chromium ion, a cobalt
ion, a manganese ion, a zinc ion, or a magnesium ion). For
instance, lactoferrin isolated from bovine milk can be depleted of
iron and then loaded with another type of metal ion. For example,
copper loading can be achieved according to the same method for
iron loading described above. For loading lactoferrin with other
metal ions, the method of Ainscough, et al. ((1979) Inorganica
Chimica Acta 33, 149-153) can be used.
[0017] In a preparation of a lactoferrin polypeptide of the
invention, the polypeptides can be of a single species, or of
different species. For instance, the polypeptides can each contain
a different number of metal ions or a different species of metal
ions; or the lengths of the polypeptides can vary, e.g., some are
full-length polypeptides and some are fragments, and the fragments
can each represent a particular portion of a full-length
polypeptide. Such a preparation can be obtained from a natural
source or by mixing different lactoferrin polypeptide species. For
example, a mixture of lactoferrin polypeptides of different lengths
can be prepared by proteinase digestion (complete or partial) of
full-length lactoferrin polypeptides. The degree of digestion can
be controlled according to methods well known in the art, e.g., by
manipulating the amount of proteinase or the time of incubation. A
complete digestion produces a mixture of various fragments of
full-length lactoferrin polypeptides; a partial digestion produces
a mixture of full-length lactoferrin polypeptides and various
fragments.
[0018] A lactoferrin polypeptide or a mixture of the polypeptide
and fragments of the polypeptide described above is used to prepare
a nutraceutical composition of this invention for preventing and
treating bone-related disorders. Examples of such disorders
include, but are not limited to, osteoporosis, rheumatoid or
osteo-arthritis, hepatic osteodystrophy, osteomalacia, rickets,
osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis,
osteopenia, fibrogenesis-imperfecta ossium, secondary
hyperparathyrodism, hypoparathyroidism, hyperparathyroidism,
chronic renal disease, sarcoidosis, glucocorticoid-induced
osteoporosis, idiopathic hypercalcemia, Paget's disease, and
osteogenesis imperfecta. The nutraceutical composition can be a
dietary supplement (e.g., a capsule, a mini-bag, or a tablet) or a
food product (e.g., milk, juice, a soft drink, a herbal tea-bag, or
confectionary). The composition can also include other nutrients,
such as a protein, a carbohydrate, vitamins, minerals, or amino
acids. The composition can be in a form suitable for oral use, such
as a tablet, a hard or soft capsule, an aqueous or oil suspension,
or a syrup; or in a form suitable for parenteral use, such as an
aqueous propylene glycol solution, or a buffered aqueous solution.
The amount of the active ingredient in the nutraceutical
composition depends to a large extent on a subject's specific need.
The amount also varies, as recognized by those skilled in the art,
dependent on administration route, and possible co-usage of other
bone-enhancing agents.
[0019] Also within the scope of this invention is a pharmaceutical
composition that contains an effective amount of a lactoferrin
polypeptide or a mixture of the polypeptide and fragments of the
polypeptide described above, and a pharmaceutically acceptable
carrier. The pharmaceutical composition can be used to prevent and
treat bone-related disorders described above. The pharmaceutical
composition can further include an effective amount of another
bone-enhancing agent. The pharmaceutically acceptable carrier
includes a solvent, a dispersion medium, a coating, an
antibacterial and antifungal agent, and an isotonic and absorption
delaying agent. An "effective amount" is the amount required to
confer therapeutic effect. The interrelationship of dosages for
animals and humans (based on milligrams per meter squared of body
surface) is described by Freireich, et al. (1966) Cancer Chemother.
Rep. 50: 219. Body surface area can be approximately determined
from height and weight of the subject. See, e.g., Scientific
Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. Effective
doses also vary, as recognized by those skilled in the art,
dependent on route of administration, excipient usage, and the
like.
[0020] A lactoferrin polypeptide of the invention or a mixture of
the polypeptide and fragments of the polypeptide can be formulated
into dosage forms for different administration routes utilizing
conventional methods. For example, it can be formulated in a
capsule, a gel seal, or a tablet for oral administration. Capsules
can contain any standard pharmaceutically acceptable materials such
as gelatin or cellulose. Tablets can be formulated in accordance
with conventional procedures by compressing mixtures of the
lactoferrin polypeptide or a mixture of the polypeptide and
fragments of the polypeptide with a solid carrier and a lubricant.
Examples of solid carriers include starch and sugar bentonite. The
lactoferrin polypeptide or a mixture of the polypeptide and
fragments of the polypeptide can also be administered in a form of
a hard shell tablet or a capsule containing a binder, e.g., lactose
or mannitol, a conventional filler, and a tableting agent. The
pharmaceutical composition can be administered via the parenteral
route. Examples of parenteral dosage forms include aqueous
solutions, isotonic saline or 5% glucose of the active agent, or
other well-known pharmaceutically acceptable excipient.
Cyclodextrins, or other solubilizing agents well-known to those
familiar with the art, can be utilized as pharmaceutical excipients
for delivery of the therapeutic agent.
[0021] The efficacy of a composition of this invention can be
evaluated both in vitro and in vivo. See, e.g., the examples below.
Briefly, the composition can be tested for its ability to promote
osteoblast and chondrocyte proliferation in vitro. For in vivo
studies, the composition can be injected into an animal (e.g., a
mouse) and its effects on bone tissues are then accessed. Based on
the results, an appropriate dosage range and administration route
can be determined.
[0022] The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present invention to its fullest extent. All
publications recited herein are hereby incorporated by reference in
their entirety.
Lactoferrin Promotes Proliferation of Primary Rat Osteoblasts
[0023] Osteoblasts were isolated by collagenase digestion from
20-day fetal rat calvariae, as previously described by Lowe and
co-workers (Lowe, et al. (1991) Journal of Bone and Mineral
Research 6, 1277-1283). Calvariae were dissected aseptically, and
the frontal and parietal bones were stripped of their periosteum.
Only the central portions of the bones, free from suture tissue,
were collected. The calvariae were treated twice with phosphate
buffered saline (PBS) containing 3 mM EDTA (pH 7.4) for 15 minutes
at 37.degree. C. in a shaking water bath. After washing once in
PBS, the calvariae were treated twice with 3 ml of 1 mg/ml
collagenase for 7 minutes at 37.degree. C. After discarding the
supernatants from digestions I and II, the calvariae were treated
further two times with 3 ml of 2 mg/ml collagenase (30 mins,
37.degree. C.). The supernatants of digestions III and IV were
pooled, centrifuged, and the cells washed in Dulbecco's modified
Eagle's medium (DME) with 10% fetal calf serum (FCS), suspended in
DME/10% FCS, and placed in 75 cm3 flasks. The cells were incubated
under 5% CO2 and 95% air at 37.degree. C. Confluence was reached by
5-6 days, at which time the cells were subcultured. After
trypsinization using trypsin-EDTA (0.05%/0.53 mM), the cells were
rinsed in minimum essential medium (MEM) with 5% FCS and
resuspended in a fresh medium, then seeded at 5.times.10.sup.4
cells/ml in 24-well plates (0.5 ml cell suspension per well, i.e.,
1.4.times.10.sup.4 cells/cm.sup.2). The osteoblast-like character
of these cells has been established by demonstration of high levels
of alkaline phosphatase activity and osteocalcin production [as
described by Groot, et al. (1985) Cell Biol Int Res 9, 528] and a
sensitive adenylate cyclase response to parathyroid hormone and
prostaglandins [as described by Hermann-Erlee, et al. (1986) Ninth
International Conference on calcium regulating hormones and bone
metabolism, p 409].
[0024] Proliferation studies (cell counts and thymidine
incorporation) were performed both in actively growing and
non-actively growing cell populations. To produce actively growing
cells, sub-confluent populations (24 h after subculturing) were
placed in fresh MEM containing 1% FCS and a lactoferrin sample. To
produce non-actively growing cells, sub-confluent populations were
placed in serum-free medium with 0.1% bovine serum albumin plus a
lactoferrin sample. Cell numbers were analyzed at 6, 24, and 48
hours after the addition of lactoferrin samples (i.e., purified
lactoferrin, holo-lactoferrin, and apo-lactoferrin) prepared as
described above. The cell numbers were determined after detaching
cells from the wells by exposure to trypsin/EDTA (0.05%/0.53 mM)
for approximately 5 minutes at 37.degree. C. Counting was performed
in a haemocytometer chamber. [.sup.3H]-thymidine incorporation into
actively growing and non-actively growing cells was assessed by
pulsing the cells with [.sup.3H]-thymidine (1.mu.Ci/well) two hours
before the end of the incubation. The experiment was terminated at
6, 24, or 48 hours by washing the cells in MEM containing cold
thymidine followed by the addition of 10% trichloroacetic acid. The
precipitate was washed twice with ethanol:ether (3:1), and the
wells desiccated at room temperature. The residue was redissolved
in 2 M KOH at 55.degree. C. for 30 min, neutralized with 1 M HCl,
and an aliquot counted for radioactivity. For both cell counts and
thymidine incorporation, each experiment at each time point was
performed at least 4 different times using experimental groups
consisting of at least 6 wells.
[0025] The mitogenic response of the purified lactoferrin sample
was found to be very potent, as shown by a markedly increased rate
of osteoblast cell proliferation (i.e., increase in thymidine
incorporation into DNA of growing cells). The potent osteogenic
response seen above was compared with that of insulin-like growth
factor 1 (IGF-1), a well-recognized osteoblast mitogen. IGF-1
showed a maximal effect of 1.25 times the control in the same
osteoblast cell culture system, whereas lactoferrin's effect was
2.26 times that of the control for the highest dose tested (10
.mu.g/ml).
Lactoferrin Promotes Proliferation of Chondrocytes
[0026] Chondrocytes were isolated by removing cartilage (full-depth
slices) from the tibial and femoral surfaces of sheep under aseptic
conditions. Slices were placed in Dulbecco's Modified Eagles (DME)
media containing 5% FBS (v/v) and antibiotics (penicillin 50 g/L,
streptomycin 50 g/L and neomycin 100 g/L) and chopped finely with a
scalpel blade. Tissue was removed and incubated at 37.degree. C.
with firstly pronase (0.8% w/v for 90 minutes) followed by
collagenase (0.1% w/v for 18 hours) to complete the digestion.
Cells were isolated from the digest by centrifugation (10 minutes
at 1300 rpm), resuspended in DME/5% FBS, passed through a nylon
mesh screen of 90 Fm pore size to remove any undigested fragments,
and recentrifuged. The cells were then washed and resuspended twice
in the same media, seeded into a 75 cm2 flask containing DME/10%
FBS, and incubated under 5% CO.sup.2/95% air at 37.degree. C.
Confluence was reached by 7 days, at which time the cells were
subcultured. After trypsinization using trypsin-EDTA
(0.05%/0.53mM), the cells were rinsed in DME/5% FBS and resuspended
in a fresh medium, then seeded into 24-well plates
(5.times.10.sup.4 cells/mL, 0.5 mL/well). Measurement of thymidine
incorporation was performed in growth-arrested cell populations as
for the osteoblast-like cell cultures described above. Lactoferrin
was found to stimulate chondrocyte proliferation at concentrations
above 0.1 .mu.g/ml.
Lactoferrin Promotes Proliferation of Osteoblasts in Organ
Culture
[0027] Neonatal mouse organ culture has been previously described
(Cornish, et al. (1998) Am J Physiol 274, E827-E833). Briefly,
two-day old neonatal mice were subcutaneously injected with
radioactively labeled 45Ca. Three days later, the calvariae were
excised and placed on mesh grids in Petri dishes containing 0.1%
bovine serum albumin/Media 199. Lactoferrin was added, and the
calvariae were incubated for 48 hours. Four hours before the end of
the incubation period, [.sup.3H]-thymidine was added. The
experiment was terminated, and 45Ca release and thymidine
incorporation were measured. Lactoferrin was found to stimulate DNA
synthesis, which reflects the proliferation of cells of the
osteoblast lineage.
Lactoferrin Signals Via MAP Kinase in Osteoblasts
[0028] This methodology has been previously described (Grey, et al.
(2001) Endocrinology 142,1098-1106). Specifically, primary rat
osteoblasts prepared as described above were seeded in 6-well
tissue culture plates at an initial density of 5.times.10.sup.4
cells/ml in MEM 5% FCS, and grown to 80-90% confluence. After serum
starvation overnight, cells were treated at room temperature with
lactoferrin in MEM/0.1% BSA. In experiments designed to determine
the effect of inhibitors of signal transduction on
lactoferrin-induced p42/44 MAP kinase phosphorylation, the cells
were pre-treated with the inhibitor for 30 min prior to addition of
lactoferrin. After treatment for the indicated period of time, the
treatment medium was aspirated, the cells were washed in ice-cold
PBS and then scraped in ice-cold HNTG lysis buffer (50 mM HEPES, pH
7.5, 150 mM NaCl, 1% Triton, 10% glycerol, 1.5 mM MgCl.sub.2, 1 mM
EDTA) containing a cocktail of protease and phosphatase inhibitors
(1 mM PMSF, 1 .mu.g/ml peptatin, 10 .mu.g/ml leupeptin, 10 .mu.g/ml
aprotinin, 1 mM sodium vanadate, 500 mM NaF). The lysates were
briefly vortexed, centrifuged at 13,000 rpm at 4.degree. C., then
stored at -70.degree. C. until analyzed. Protein content of the
cell lysates was measured using the DC protein assay (BioRad,
Hercules, Calif.). Equal amounts of the whole cell lysate (30-50
.mu.g) were subjected to 8% SDS-PAGE, transferred to nitrocellulose
membranes, and immunoblotted overnight at 4.degree. C. with an
anti-phospho-p42/44 MAP kinase antibody (1:1000). As a control for
protein loading, the same filters were stripped and re-probed with
an antibody against total p42/44 MAP kinase (1:400). Incubation
with the HRP-conjugated secondary antibody was for 1 h at room
temperature, and the membranes were analyzed by ECL. Immunoblots
were repeated at least 3 times. Lactoferrin was found to induce
phosphorylation of p42/p44 MAP kinases in osteoblasts in a dose-
and time-dependent manner at concentrations of 1-100 .mu.g/ml.
Lactoferrin Stimulates Bone Growth in Vivo
[0029] The mouse model used in these studies have been previously
described (Cornish, et al. (1993) Endocrinology 132, 1359-1366).
Injections (0 mg, 0.04 mg, 0.4 mg and 4 mg) of lactoferrin were
given daily for 5 days, and the animals were sacrificed one week
later. Bone formation was determined by fluorescent labeling of
newly formed bone. Indices of bone resorption and of bone mass were
determined by conventional light microscopy, assisted by image
analysis software. Local injection of lactoferrin in adult mice
resulted in increased calvarial bone growth, with significant
increases in bone area after only 5 injections.
Application 1
[0030] Set yoghurts of between 14 and 17% solids, with or without
fruit added, can be prepared as follows:
[0031] Medium heat skim milk powder (between 109-152 g) and ALACO
stabilizer (100 g) are reconstituted with approximately 880 ml of
50.degree. C. water. Anhydrous Milk Fat (20 g) is then added and
mixed for 30 min. The mixture is then heated to 60.degree. C.,
homogenized at 200 bar, and then pasteurized at 90.degree. C. After
cooling to a temperature between 40-42.degree. C., a starter
mixture and the freeze-dried protein preparation described above
(up to 50 mg of lactoferrin at 95% purity or an equivalent quantity
from a not so highly purified source) is added. If desired, fresh
fruit may also be added at this point. The mixture is then filled
into containers, incubated at 40.degree. C. until pH 4.2-4.4 is
reached, and then chilled in a blast cooler.
[0032] An alternative method for preparing the same set yoghurts is
by dry blending the indicated quantity of lactoferrin or the
indicated quantity as a dose rate, into the dry milk solids, prior
to its use in the yoghurt formulation.
Application 2
[0033] Dry blends of either skim or whole milk powder with calcium
and the freeze dried lactoferrin preparations can give dairy based
formulations or compositions which can be used either as functional
foods or as functional food ingredients. Such compositions can be
used as reconstituted milks, milk powder ingredients, dairy
desserts, functional foods, cheeses or butter or beverages, and
nutraceuticals or dietary supplements. Blending the dry ingredients
in ratios of milk powder:calcium:active lactoferrin agent between
90:9.5:0.5 and 94:5.95:0.0001 provide compositions suitable for
such uses.
Application 3
[0034] Blended compositions of milk powder, calcium, and the
lactoferrin rich ingredient can be used as bone health functional
foods, bone health food ingredients, or as a food ingredient for
delivery of bone health nutrients in a range of health foods.
[0035] For such compositions, the calcium and protein contents of
the compositions need to be adjusted to required, allowable
nutritional limits. Commercially available ingredient milk powders
typically contains between 300 and 900 mg calcium per 100 g powder,
depending upon their sources. A source of calcium may be added to
the powder to extend the calcium content up to 3% by weight of the
ingredient milk powder as a blend. The protein level of
commercially available ingredient milk or dairy-based protein
powders varies depending upon the type of the ingredient, the
method of its manufacture, and its intended use. Ingredient milk
powder typically contains between 12% and 92% protein. Examples are
commercially available skim and whole milk powders, food grade
caseins, caseinates, milk protein concentrate powders, spray dried
ultrafiltered or microfiltered retentate powders, and the milk
protein isolate products. The lactoferrin rich preparation may be
incorporated into a protein and calcium blend to give nutritional
milk powders that can be used as ingredients in healthy foods and
drinks. Such blends provide ingredients suitable for use in
preparing yoghurts and yoghurt drinks, acid beverages, ingredient
milk powder blends, pasteurized liquid milk products, UHT milk
products, cultured milk products, acidified milk drinks,
milk-and-cereal combination products, malted milks, milk-and-soy
combination products. For such uses, the blend can have a
composition where the calcium content is between 0.001% and 3.5%
(w/w), the protein composition is between 2% and 92%, and
lactoferrin as the osteoblast proliferating agent is added at
levels between 0.000001% and 5.5%.
OTHER EMBODIMENTS
[0036] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0037] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the scope of the following claims.
* * * * *