Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2090377
ANTI-CANCER THE~APEUTIC COMPQ~I~ION8 FOR PROPHYLA$IS OR FOR
TREATMENr OF C~EMICALLY INDUCBD CANCEa
Rela~ed Applications
This application is a division and a continuation-in-part
of U.S. Serial No. 417,246 filed October 4, 1989, which i5 a
continuation-in-part of U.S. 289,971 which in turn, is a
continuation-in-part of U.S. 188,276. This application i8 also
a continuation-in-part of U.S. application Serial No.
filed April 10, 1992, which i8 a continuation-in-part of U.S.
application 563,794 filed May 3, 1990, which is a continuation-
in-part of said U.S. Serial No. 289,971 filed December 23, 1988.
The contents of these applications is included by reference in
their entirety.
Background and prior art
In U.S. Application 298,971 filed December 23, 1988 and U.S.
Application 417,2467 filed October 4, 1989, of which this
application i8 a continuation-in-part and division, and also in
Bounous et al ~Dietary Whey Protein Inhibits the Development of
Dimethyl-hydrazine induced Nalignancy" (1) we described
experiments showing that continuous feeding of whey protein
(w.p.c.) in the diet inhibits the development (number and size
of tumours) in the colon of a mouse over a period of 24 weeks of
DMH treatment. This anti-tumour effect could be caused by
increased resistance of target cells to the carcinogen and/or a
direct inhibitory effect of w.p.c. on the cancer cells. A
subsequent series of experiments (2) where animals were fed
Purina diet for the first 20 weeks of DMH and then switched to
w.p.c. diet for the remaining 8 weeks of DMH treatment, suggested
some inhibitory effect of w.p.c. feeding on cancer cells.
Most recently (3) a group of French scientists confirmed n
vitro a direct inhibitory effect of w.p.c. on human cancer cells.
Indeed similar studies in vitro with human breast cancer cells
have shown that bovine serum albumin (BSA) is the factor exerting
inhibition of cancer cell replication (4).
We have also shown that this activity of w.p.c. is
specifically dependent upon the glutamylcysteine groups
(substrate for GSH synthesis) present in the BSA fraction of
w.p.c. (U.S. #563,794). Interestingly, the introduction of the
2090377
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cyateine delivery system OTZ (ozothiazolidine-4-carboxilate),
while enhancing GSH levels in normal cells, was found to result
in feedback inhibition of the GSH cycle in human tumour cells
(5). This differential effect of OTZ was recently confirmed in
YiY_ (6). The previously described direct inhibitory e~fect o~
w.p.c. (3) and more specifically of ~SA (4) could be explained
therefore by the release during incubation of a potent cysteine
delivery system such as glutamylcysteine.
We have therefore reached the following conclusions:
1) BSA i8 the protein fraction of w.p.c. that we found to
be primarily responsible for the GSH promoting activity of w.p.c.
This activity which we believe to be the basis for the i~muno
enhancing and antieareinogen effeet of w.p.e., is specifieally
dependent upon the glutamyleysteine groups (substrate for GSH
synthesis) present in the BSA fraction of w.p.c. (U.S. ~563,794).
2) The molecular weight of BSA is 66,267 hence quite
different from the MW of the anti-eaneer faetor patented by
Villadsen (MM 500-20,000). (7).
(3) Our earlier findings (1,2) eould be explained as
follows: During DMH treatment, w.p.e. feeding by inereasing
eellular GSH proteets the target cells against the effeets of the
eareinogen. In addition, inereased availability of substrate
for GSH synthesis eould inhibit replieation of formed eancer
cells.
4) We have now established the importanee of a high level
of serum albumin (BSA) in the w.p.e. in providing a substrate for
GSH synthesis. We ean eonelude that dietary whey protein
eoneentrate in undenatured form and eontaining 2 10% BSA exerts
an anti-tumour effeet.
Summarv of Invention
Aeeording to the present invention there is provided an
anti-eaneer eomposition of a biologieally aetive whey protein
eomposition compri6ing a suitable coneentration of whey protein
coneentrate wherein the whey protein concentrate contains the
proteins whieh are present in an undenatured state and wherein
the biological activity of the undenatured whey protein
concentrate is based on the overall amino acid and associated
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small peptides patterns resulting from the contribution of all
its protein components.
Further, according to the present invention there ls
provided a method of treatment of patients having lesions
resulting from cancer cells comprising the administration of
undenatured whey protein concentrate in an amount sufficient to
inhibit the replication of such cells.
Brief Description of the Drawings
Figure l illustrates the liver glutathione content in male
mice C57BL/6NIA fed undenatured whey protein (U-Lacp), denatured
whey protein (D-Lacp), casein, egg white protein or purina diet-
fed counterparts at age 10 weeks, 27, 20 and 21 months.
Figure 2 illustrates the heart glutathione content of male
mice C57BL/6NIA fed undenatured whey protein (U-Lacp), denatured
whey protein (D-Lacp), casein, egg white protein or purina diet-
fed counterparts at age 10 weeks, 17, 20 and 21 months.
Figure 3 illustrates the effect of various sources of whey
protein concentrate and casein (20 g/100 g. diet) on spleen PFC
response to 5 x 106 SRBC in mice.
Detailed Description
We will first describe the experiments in U.S. Applications
298,971 filed December 23, 1988 and U.S. 417,246 filed October
4, 1989 of which this application is a continuation-in-part.
Definitions
(a) Whey Protein
Whey proteins are the group of milk proteins that remain
soluble in "milk serumN or whey after precipitation of caseins
at pH 4.6 and 20C. The major whey proteins in cow's milk are
beta-lactoglobulin (,B L), alpha-lactalbumin (~ L), immunoglobulin
and serum albumin (SA).
The product of industrial separation of this protein mixture
from whey is called "whey protein concentrate" (WPC) or isolate.
The WPC used in most of our experiments is from bovine milk
(Lacprodan-80 from "Danmark Protein A.S."). Use in its
undenatured state is indicated as U-Lacp, and in its denatured
state is indicated as D-Lacp. Lactalbumin (L) is the term
traditionally used to define WPC.
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(b) C - casein;
(c) SRBC = Sheep red blood cells;
(d) PFC 2
Plaque forming cells (spleen):
enumeration of PFC in spleen i5 used to assess
the humoral immune response to SRBC in~ection;
(e) GSH = Glutathione (L-gamma-glutamyl-L-
cysteinylglycine);
(f) DNH = 1,2-Dimethylhydrazine;
(g) The defined formula diets tested varied only in the
type of protein;
(h) Whey of bovine milk contains approximately 6 g per
litre protein, most of the lacto6e, mineral and water
soluble vitamins.
Diets used in these 6tudies: referred to below in Table 3
Diets are prepared in the following way: 20 g of
selected pure protein, 56 g of product 80056 protein free diet
powder containing corn syrup, corn oil, tapioca starch, vitamins
and minerals (Mead-Johnson Co. Inc., U.S.A.), 18 g cornstarch,
2 g wheat bran; 0.05 g Nutramigen vit-iron premix (Bristol-Myers,
Ontario, Canada), 2.65 g KCl; 0.84 g NaCl. The carbohydrate and
lipid components of our formula diets were the same. The only
variable in the various purified diets was the type of protein
(20 g protein/100 g diet). At this concentration in diet all the
different proteins tested provided the daily requirements of
essential amino acids for the growing mouse (8). Vitamins and
minerals were the same in each set of experiments and were added
in the amount necessary to provide daily requirements for the
growing mouse (9, 10). Table 1, below, indicates the variation
in suggested vitamin requirements for mouse diets and their
contents in some of our formulations. Therefore all the formula
diets used in our experiments were designed to provide adequate
nutrition as demonstrated by normal body growth, serum protein
(9) and by the absence of hair loss, dermatitis, cataract,
ataxia, fatty liver etc. The latter symptoms were of course
present in very old mice and were related to the aging process.
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TABI~E 1
VITAMIN ANJ~ ~RAL CONl~IETS (amount /lOOe diet~
TEST JACKSON (9)
DIETS (range of amount
recommended in
Jackson labora- AIN 76(2)
tones dietfi~
VITAMINS:
Vitamin A, IU 1295 .........1800 24 - 550 400
Vitamin D, IU 260 .......... 360 14 - 506 100
Vitamin E, IU 11.6 ............ 18 1 - 2.7 5.0
Vitamin K, mg 0.06 ......... 0.09 - 0.005
Thiamine(Vitamin Bl),mg ............... 0.34 0.63 0.22 - .99 0.60
Riboflavin(VitaminB2),mg .............. 0.38 0.69 0.24 - 1.1 0.60
Vitamin B6, mg 0.26 ......... 0.36 0.1 - 0.55 0.70
VitaminB12, mB 0.0012 ......Ø054 .0039-.0055 0.001
Niacin, mg .................5.1 9.2 2.6 - 14.3 3.0
Folic acid, mg 0.063 .......Ø12 0.05 - 0.27 0.2
Pantothenic acid,mg1.93 ......... 3.38 1 - 5.5 1.6
Biotin, mg ...............Ø031 0.058 0.019-0.165 0.02
Vitamin C, mg 53.3 ............65
Choline, mg .................. 44 76 49 - 145 100
Inositol, mg ................19.8 19.8
AIN 76
MINERALS:
Calcium, mg ................. 430 # 520
Phosphorus, mg 260 ......... # 400
Magnesium, mg 63.2 ........ # 50
Iron, mg ................. 7.9 3.5
Zinc, mg ................ 3.57 # 3.0
Copper, mg ................ 0.47 # 0.60
Iodine, mg ................ 0.023 0.02
Sodium, mg ................. 232 100
Pot~ssium, mg 997 360
# after minerals analysis
(9) Hoag W.G., Dickie M.M. "Nutfltion: in Grff~ E.L. (~3d) Biology of the laboratory
mouse McGraw-Hill NY
1966 pp 39-43. Jackson was our supplier.
(10) The mouse in biomedical research, vol m Eds Foster-H.L., SeaU J.D., Fo~ J.B.,
Academic press 1983, NY pp 57-58
- 2090377
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mmunization for laque assaYs
The diet-fed mice were immunized by an intravenous injection
of 5 x lo6 wa~hed sheep red blood cells obtained weekly from
Institut Armand-Frappier, Laval des Rapides, Quebec, Canada.
Plaaue formina cell (PFC~ as6ay
The method used for assaying IgM plaque forming cell~ was
essentially the one described by Cunningham and Szenberg (11),
with certain minor modifications. Spleen cell suspensions were
prepared by gently tamping the spleen through a 50-mesh stainless
steel screen, and collecting the cells in balanced salt solution
(BSS) supplemented with 10~ heat-inactivated calf serum (Grand
Island Biological Company, Montreal, Que~ec, Canada). The spleen
cells were washed and made up to 15 ml with BSS. Sheep red blood
cells were washed twice and made up to a 20% concentration.
Guinea pig serum (Grand Island Biological Company, Montreal,
Quebec, Canada) as a source of complement wa6 diluted 1/15 with
BSS. All stock 60lution6 were kept on ice water until used. The
test consi6ted of mixing 0.05 ml of 6pleen cells, 0.15 ml of
sheep red blood cells and 0.75 ml of the complement 601ution in
a test tube at 37C. The whole mixture was immediately withdrawn
and put into slide chambers, sealed with warm paraffin wax, and
incubated at 37C for 45 to 60 min. The number of plaque forming
cells was counted and their total number per spleen estimated by
multiplying the number of plaque forming cells in each sample
(0.05 ml spleen cells) by 300. The values are expressed per
total organ rather than per lo6 spleen cells, ~ince this appears
to reflect more accurately the functional status of the spleen
per se.
Mice were assayed for the plaque forming cell response to
sheep red blood cells normally on the fifth day after
immunization when the response was shown to peak or, in the
kinetic study, on days 3, 4, 5 and 6 post- immunization.
Statistics
The mean plaque forming cell values were compared among the
dietary groups using either Student's ~est, when two groups were
being compared, or the analysis of variances (ANOVA) for more
2~90377
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than two groups. ~ecause of the heterogeneity of variance~ among
groups, the adjustment given by Brown and Forsythe was used.
S~leen alutathione content
Ninety milligrams of mouse spleen were weighed using a
Mettler PM-300 balance and samples varied from 90 mg by less than
5 mg (5%). The samples were then homogenized in 5-sulfosalicylic
acid (5% w/v). Homogenates were centrifuged for 5 min in a
microfuge at 10,000 x g. The assay was carried out using the
supernatants on the same day according to the methods of Anderson
(12). Values are expressed as ~mol per g/wet tissue.
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Table 2
AMINO ACID COMPOSITION
(g/100 g protein)
Amino ~cid Whey Protein Egg White
Concentrate * Protein **
Aspartic acid11.3 7.9
Threonine 7.2 4.4
Serine 6.1 7.9
Glutamic acid20.1 14.1
Proline 6.6 3.8
Glycine 2.0 3.7
Alanine 5.4 7.6
Valine 6.5 7.8
Isoleucine 6.7 6.5
LRucine 11.2 8.8
Tyrosine 2.9 4.2
Phenylalanine3.1 6.4
Lysine 9.5 6.0
Histidine 1.9 2.2
Arginine 2.7 5~9
Methionine 2.2 3.9
Cysteine 2.4 2.4
Tryptophan 1.7 1.5
* Lacprodan-80 from Danmark Protein A/S, Copenhagen, Denmark,
1986; used in our experiments.
** Values calculated from "Amino Acid Content of Foods",
U.S.D.A., 1957. Values from cysteine analyzed by Sigma on
samples used = 2.38 g/100 g protein and in our laboratory
= 2.4 g/100 g protein.
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Tissue Glutathione Assay:
Ninety milligrams of mouse heart or liver were homogenized
in 5-sulfosalicylic acid (5% w/w). Homogenates are centrifuged
for 5 minutes in a microfuge at lO,ooO x g. The assay i8 carried
out using the supernatants on the same day according to the
method of Anderson (12), Values are expressed as ~mol/g wet
tissue.
After three months on either diet initiated at age 17
months, GSH content was found to be higher in the liver and heart
of U-Lacp (undenatured whey protein Lacprodan-80) fed mice
compared to the D-Lacp (denatured whey protein Lacprodan-80),
casein, egg white protein or Purina diet-fed counterparts
(Figures 1 & 2). The GSH values in heart and liver of mice fed
Purina laboratory chow was similar at age 10 weeks, 17, 20, 21
months. The U-Lacp diet appears to enhance the GSH content of
heart and liver above '~normal" values after 3 and 4 months of
continuous feeding (Figures 1 & 2).
In addition, after three weeks on the U-Lacp diet, spleen
GSH content is increased during the antigen driven clonal
expansion of the lymphocytes in young adult C3H/HeN mice as
compared to a decline in controls fed D-Lacp, casein or egg white
protein diets. In old C57BL/6NlA mice, long term feeding of
U-Lacp diet results in a moderate but sustained increase in liver
and heart GSH levels (Figures l and 2). The GSH enhancing
activity of WPC is restricted to its undenatured form (ULacp).
This property is not solely due to the high cysteine content of
WPC because another protein source with similar cysteine content
(egg white) (see Table 2) does not exhibit this biological
activity. This property of U-Lacp does not depend specifically
on its nutritional efficiency as evaluated by body weight, serum
proteins, and food consumption, but appears to depend on the
primary, secondary and tertiary structure of the protein in its
native form.
Some of the previously discussed methods of increasing
intracellular levels of glutathione concentration are either
toxic (13) or dangerous owing to the risks related to the initial
phase of glutathione depletion (14, 15). The methods involving
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the use of gamma-glutamylcyst~e)ine ~16), athiazolidine (17) or
glutathione esters (18) (U.S. patent ~4,784,685) offer an
interesting possibility for short term intervention.However,
their long term effectiveness in producing sustained elevation
of cellular glutathione has not been shown, nor has the possible
toxicity of their long term use been disproved. Indeed,
glutathione and glutathione disulfide were found to be positive
in the most commonly used short term tests for carcinogenicity
and mutagenicity (13). Relevant to our invention are recent data
indicating specifically that a lack of the GSH precursor,
cysteine, rather than a decrease in biosynthetic enzyme
activities is responsible for the deficiency of GSH noted in
aging animals (19). Similarly, the fall in cytosolic GSH in the
liver of chronic ethanol fed rats does not appear to be caused
by a limitation in the capacity of gamma- glutamylcysteine
synthetase activity (20).
Data in Figures 1 and 2 show that the concentration of liver
and heart glutathione in control Purina fed mice remains very
constant over time. On the other hand a moderate but sustained
elevation of tissue GSH was noted in mice fed the nutritionally
equivalent whey protein (U-Lacp) diet. Only minuscule quantities
of glutathione and no breakdown products that can be readily
attributed to glutathione are excreted in urine (21). The
magnitude of change in cellular glutathione concentration that
can be achieved may be quite limited, perhaps reflecting the
critical importance of this molecule and the attendant tight
regulatory control. Glutathione itself serves as a negative
feedback on the GSH synthetic enzymes, which obviously limits
cellular capacity to increase GSH concentration (22).
Glutathione reductase maintains GSH in its predominant reduced
form (2 90%). This serves both to maintain this functional state
and also to control cellular concentration since reduced
glutathione (GSH) cannot cross the membrane, whereas the oxidized
form (GSSG) can and does afflux, resulting in decreased total
glutathione. Besides these enzymes, gamma glutamyltranspeptidase
(GGT) is important in GSH metabolism. GGT serves as a salvage
pathway for glutamyl moieties at the cell membrane level, passing
2090377
them back into the cytosol to be used in GSH synthesis. Increased
activity of this enzyme has been associated with elevated GSH
concentration in a number of cell lines and malignant tiseues
(23, 24).
It is advantageous to include Vitamin Bl (thiamine) in a
diet that results in elevated GSH. Thiamine is involved in the
transketolase reaction of the pentose phosphate shunt yielding
NADPH (GSSG is reduced back to GSH by NADPH; GSH reductase).
Vitamin ~ (riboflavin) is also an advantageous addition.
Flavin mononucleotide (FMN) and flavin adenin dinucleotide (FAD)
are synthetized sequentially from riboflavin and are involved in
GSH reductase.
Some milks, especially those from New Zealand, are low in
selenium. Selenium is contained in GSH peroxidase. Mammals
deficient in selenium have markedly decreased peroxidase
activity. Therefore glutathione formation which is advantageous
for its anticancer effect requires an adequate level of selenium.
If we assume a dosage level of 60 grams undenatured whey
protein as a daily intake, the rec~mmended levels of Vitamin B
Vitamin ~ and selenium are as follows:
Vitamin Bl 1.5 - 2.0 mg
Vitamin ~ 1.7 - 2.2 mg
Selenium Methionine 40 mcg. - 60 mcg.
The effects of a small increment in cellular GSH may be
greater than expected. For example, there are many reports of
human and murine tumour cell lines selected in vitro for
resistance to a variety of chemotherapeutic agents. In a number
of these cell lines cellular GSH is increased consistently by
2-fold compared to the drug sensitive parental cell line, despite
the fact that the level of drug resistance is often much greater,
e.g. as much as 30-fold (24, 25, 26). In these cell lines,
depletion of cellular GSH by selective inhibition of synthesis
restores drug sensitivity to the resistant cells. This is
effective only if the GSH depletion is maintained throughout the
drug-treatment period.
Given the fact that cellular GSH is very tightly regulated,
that a 2- fold increase may be maximal, and that the effect of
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small increments in GSH may be amplified by a variety of
GSH-utilizing enzymes (e.g. glutathione peroxida6e,
glutathione-S-transfera6e), .the reproducible change in GSH
concentration observed in animals fed the whey-rich diet is
likely to have biological importance. The chronic nature of thi~
augmentation may contribute significantly to this effect.
Our findings show that in mice fed a casein diet the number
and size of DMH induced colon carcinoma were reduced by a factor
of 0.3 and 0.4 respectively in comparison to Purina fed controls
(Table 3, below). However, in mice fed the whey protein diet with
similar nutritional efficiency the number and size of DMH-induced
colon carcinoma were reduced four fold in comparison to the
Purina fed controls (Table 3, below). DMH- induced colon tumours
appear to be similar to those found in humans as far as type of
lesions and chemotherapeutic response characteristics are
concerned (27, 28). The superiority of the anti-cancer effect of
whey protein in comparison to casein has been reported in our
previous study (1). About 80% of the proteins in bovine milk are
caseins and the remaining 20% are whey proteins (29, 30). In
addition, using the traditional process of preparing casein, the
amount of whey protein co-precipitated along with the casein
varies from about 40 to 60% of the total amount of whey protein
present in the milk (31). Therefore it is conceivable that the
minor anti-cancer effect seen with casein could be due to the
relatively (to caseins) small amount of whey protein co-
precipitated with it. It is apparent from the above described
studies that the antitumour activity of the dairy products is
in the protein fraction and more specifically, as our invention
demonstrates, in the whey protein component of milk.
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Table 3
Effect of dietary milk protein on animal growth and tumour
development in A/J mice treated with the carcinogen
1,2-Dimethylhydrazine.
Whey Prot. Casein Purina Pur/Whey Pur/Cas
28 Weeks' 28 Weeks' 28 Week^ 20~8 Weeksb 20/8 Weeksb
Initial
WeightC (g) 21.7+0.5 21.5+0.7 21.9+0.8 21.9+0.4 22.0+0.7
Final
WeightC (g) 21.5+0.3 21.8+0.4 19.7~0.7 21.3+1.0 21.0+0.6
Number of
TumoursC 8.4+1.5 24.7+3.0 35.9+2.6 15.1+3.2 21.7+4.3
Tumour AreaC 38.8+6.4 90.9+10.6 160.0+11.4 47.9+10.4 77.7+10.9
a) Mice treated with DMH for 24 weeks, and then
sacrificed 4 weeks later.
b) Mice treated with DMH for 24 weeks, and then
sacrificed 4 weeks later. They were maintained on
Purina Mouse Chow for 20 week6 and then switched to
either Whey Protein or Casein diet for the remaining
8 weeks.
c) Mean + SEM.
__________________________________
ANOVA: solid line(s) connect those means not
significantly different (p<0.05).
Group Whey Pur/Whey Pur/Casein Casein
Number of
Tumours
Tumour
Area
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SURVIVAL STVDIES: THE BIOLOGICAL ACTIVITY
IS DEPENDENT ON THE UNDENATURED CONFORNATION OF WPC
(a) Survival of Old Nice Durina a Limited Time Period:
Our study shows that the mean survival time, over a limited
observation period of 6-7 months ending when 55% of male
C57BL/6NLA mice were dead, iB increased by about 30% in mice
commenced on the undenatured whey protein (U-Lacp) diet at the
onset of senescence (age 21 months) in comparison with Hcontrol6"
fed the nutritionally equivalent Purina mouse chow. The survival
curve of Purina fed mice was very similar to that of casein
diet-fed mice. However, in the 6ubsequent four months, mice on
undenatured whey protein diet were switched to a denatured whey
protein concentrate (D-Lacp) diet. During this period, the time
of death of the remaining whey protein diet-fed mice became
similar to that of their casein diet or Purina-fed counterparts.
Throughout the study repeat bioassays of PFC formation confirmed
the correlation between host immunoenhancement and undenatured
state of WPC in diet as indicated in Figure 3. In the second
part of the ~tudy, when the difference between survival curves
began to narrow, the immunoenhancing property of WPC wa6 absent
although its nutritional quality was preserved (D-Lacp).
Throughout the entire study no significant intergroup difference
was seen in calorie intake, and body weight. Since longevity is
dependent primarily upon the genome of the individual it is
unlikely that delayed mortality over a limited period of time
would have influenced overall longevity. However, at least in
terms of the immunoenhancing effect of the diet, this study could
be regarded as a single direction cross-over from test (VLacp)
to control (D-Lacp) diet6, showing that the biological activity
of WPC on 6urvival of old mice is dependent upon its undenatured
state and correlating directly with the PFC as6ay used in our
study (as illustrated in Figure 3).
" 2090377
- 15 -
(b) Short and Lon~ Term Survival of Mlce with DMH-Induced Colon
Cancer: -
In DNH treated mice we noticed a di$ference between
mortality by the 28 weeks end point and the survival time to the
end of the experiment in relation to dietary protein type. During
the first seven months of study, the mice fed undenatured whey
protein (U-Lacp) had no death as compared to a 33% mortality
observed towards the end of this period in the casein and Purina
groups. In the subsequent four months mice on whey protein were
fed denatured whey protein (D-Lacp). During this latter period
the D-Lacp diet appeared to have no favourable ef$ect on survival
in comparison to the casein diet (Table 4, below). Throughout
the study repeat bioassays of spleen PFC were done to document
the physlologic effects of the diets on i~mune function as
reported previously and the stability of these effects. The
immunoenhancing effect of the U-Lacp diet was consistently
confirmed for the first 7 months of the study; however, in the
following four months (D-Lacp), the immunoenhancing effect
~previously observed in mice fed the U-Lacp diet was absent. The
values o$ PFC response in relation to either the U-Lacp diet or
the DLacp diet were consistent with those presented in Figure 3.
This study therefore confirms the hypothesis that the biological
activity of WPC on survival of tumour bearing mice is dependent
upon its undenatured state correlating directly with the PFC
assay used in our study.
20~37~
.
- 16 -
T~bl~ ~
Effect of dietary milk protein on short and long term
survival in A/J mice treated with the carcinogen 1,2-
Dimethylhydrazine for 24 weeks.
DIETARY GROUPb
Whey Protein~ Casein purina
Mortality~ at 28 weeks 0% 33% 33
Survival timeC in weeks 40 41 30
a) significant by Chi Square analysis: Whey Protein vs.
Purina vs. Casein p<0.05.
b) Originally 12 mice per group.
c) Survival time in week6 from the first dose of
carcinogen. Whey protein and Casein differ
significantly from Purina, Mantel-Cox test p<0.01.
d) Undenatured Whey Protein used from weeks 3 to 28.
Denatured Whey Protein used from weeks 28 until end.
The experiment referred to above which are summarized in
Tables 3 and 4 used Lacprodan 80 as a source of undenatured WPC:
We now prefer to use a whey protein concentrate (WPC)
in undenatured form prepared from milk treated in the most
lenient way compatible with accepted standards of safety
with regard to bacterial contamination. The extremely high
solubility index indicated that the proteins present are
essentially undenatured, hence demonstrating the leniency
of the ultrafiltration process t31]- Although the proteins
contained in the concentrates from the other commercially
available sources examined were mostly in undenatured form,
as indicated by the relatively high solubility of the
concentrates, the content of serum albumin and
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immunoglobulins ~n these mixtures is below the level
~otivity [31]. These very thermolabile proteins are
denatured, hence precipitated and partially lost from whey
when high pasteurization temperaturee are utilized.
Our studies also showed that admini~tration of S(n-butyl)
homocysteine sulfoximine, which reduce~ splenic glutathione in
half, significantly reduced the humoral immune response of whey
protein-fed mice. This vas taken as further evidence for the
important role of glutathione in the immunoenhancing effect of
dietary whey protein (32).
Tissue glutathione concentration may be increased by
administration of gamma-glutamyl-cysteine. Glutathione increased
in the kidney by about 50%, 40-60 minutes after subcutaneous
(s.c.) in~ection in mice, returning to control values 2 hours
later (33). The administered gamma-glutamylcysteine is
transported intact and serves as a substrate for glutathione
synthetase (33).
Advances in amino acid sequencing of food proteins allowed
us to investigate the occurrence of glutamylcysteine groups in
whey protein and the possible relation to glutathione promotion.
Indeed, whey protein concentrate from bovine milk contains
substantial amounts of glutamylcysteine groups, unlike casein,
which does not increase tissue glutathione when fed to mice (35).
The glutamylcysteine groups are located primarily in the serum
albumin fraction (six groups/molecule). Glutamylcysteine groups
are extremely rare in animal and plant edible proteins. Extensive
search of all available data on amino acid sequencing of edible
proteins reveals that the Gly-Cys group with a disulfide link is
indeed limited to some of the whey protein, and to the ovomucoid
fraction of egg white which contains 2 of these groups in a
30,000 mol.wt.molecule (31).
Our recent (31) data further indicate that the humoral
immune response is highest in mice fed a dietary whey protein
concentrate exhibiting the highest solubility (undenatured
conformation) and, more importantly, a greater relative
concentration of the thermolabile bovine serum albumin (210%) and
immunoglobulins. In addition, the mice fed this type of whey
20~0377
- 18 -
protein concentrate exhlbit hlgher levels o~ tissue G8H. The
glutamylcyateine groups ~rare in ~ood protein) and the specific
intramolecular bond as related to the undenatured con~ormation
of the molecule are considered to be key factors in the
glutathione-promoting activity of the protein mixture.
Recent experiments in Japan t36] showed that spleen cell~
of BALB/c male mice fed 25g of our undenatured whey protein
concentrate (WPC) (for which the Trademark name "Immunocal~ ha~
been applied) per lOOg diet for 4 weeks had an increased immune
r-sponse to SRBC in vitro and a high content of L3T4+ cells
(12.58 x 106+ 1.36) than mice fed an isocaloric diet with 25g.
pure casein/lOOg. diet (3.69 X lo6_ 0.50). Similarly, the speen
L3T4+ /LYt - 2+ ratio was 1.36 + 0.07 in undenatured WPC fed mice
and 0.55 _ 0.07 in casein-fed controls (P<O,OOl). Conversely,
the relatively high concentrations ~f the thermosensitive serum
albu~in and immunoglobulins resulting from the low degree of
pasteurization of milk in our WPC, may reflect more closely the
pattern of raw milk. These data lend support to the hypothesis
~that the thermolabile Gly-Cys containing proteins such as serum
albumin in undenatured conformation are crucial elements for the
biological activity of whey protein concentrate.
8uitable bovine whey protein concentrate (WPC) has been
prepared by the ~Service de recherche sur les aliments du
Ninistere de l'agriculture du Quebec" in St-Hyacinthe, Quebec,
Canada, with the following characteristics: pure protein content
75% (the rest mostly lactose, some fat and moisture); solubility
index: (pH 4.6); 99.5%. Protein composition as of total whey
~protein m asured by polyacrylamide gel electrophoresis (31) was:
~beta-lactoglobulin 59.1 + 4.0; alpha-lactalbumin: 6.6 + 0.7;
serum albumin: 9.7 + 1.0; immunoglobulin 24.6 + 2.6 (mean + SD).
The solubility index should preferably be above 99%.
The serum albumin of about 10% of the total whey protein was
almost twice the corresponding value found in other commercially
available whey protein concentrates that have been examined. It
is believed that a serum albumin level 2 10% is highly
advantageous to improving the immune system.
20~37~
Serum albumin includes a substantial amount of glutamyl
cysteine which is a substrate for glutathione synthesis in the
body. The role of glutathione is discussed in detail in "The
Biological Activity of Undenatured Dietary Whey Proteins: Role
of GlutathioneN, Clin. Inve~t Med 14: 296 - 309, 1991, which is
incorporated by reference in its entirety.
Immunoglobulin in the range of about 25 to 30% of total whey
protein is also important. Pasteurization at 720c for 13 seconds
resulted in an immunoglobulin level of 28 + 2%. We have found
it possible to achieve a serum albumin level as high as 14 + 1%
with milk pasteurized at 72C for 13 seconds.
Upon bacteriological analysis no staph, salmonella, B
cereus, or E coli were isolated in either the WPC prepared by the
~Service de recherche sur les aliments du Ministere de
l'agriculture du Quebec" or in the sample pasteurized at 72C for
13 seconds.
2090377
- 20 -
The method u6ed to prepare the reference WPC is
schematically described below in Table 5.
TAP~E S
SCHEMATIC REPRESENTATION OF THE PROCESS TO PRODUCE
OUR UNDENATURED WPC
Ra~ milk
Skimmed at 35C - cream
.
Skimmed milk pasteurized at 63C for 30 minute6.
At 38C: Addition of rennet (20 ml/100 kilos),
allowing the agitation to resolve at low speed.
~ -~ curd
Whev
Filtered with cheese cotton to remove debris.
At 40C: Ultrafiltration (Romecon UFSI,
polysulphone membrane, cut off 50,000, pore
diameter 0.06 inch, surface 2-3 m').
: Diafiltration to wash out salts and
lactose
WheY Protein Concentrate
At 40C: Lyophylization.
Whey Protein Concentrate Powder
2090377
- 21 -
30ml of heparinized blood may be used to determine the
glutathione content of blood mononucleated phosphate buffered
saline adjusted so that there are 107 cells per tube. After
centrifugation 900 ul of water i8 added to the pellet to lyse all
the cells. To each aliquot is added 30% sulfosalicylic acid for
a final concentration of 3% in 1 ml. After 15 minutes
incubation, the samples are centrifuged, and the clear
supernatant is used for the biochemical assay according to the
method of Anderson [37]. Values are expressed as nanomol (nMol)
per GSH/107 cells. Blood lymphocyte subsets may be determined by
flow-cytometry.
The total serum protein, including the albumins and the
immunoglobulins may be determined by the Biuret method. The
level of Immunoglobulin A (IgA), Immunoglobulin G (IgG) and
Immunoglobulin M (lgM) may be measured by immunonephlometry.
The presence of glutamylcysteine groups in the serum albumin
component of the whey protein concentrate is considered to be a
key factor in the glutathione-promoting and immunoenhancing
activity of the protein mixture of the undenatured WPC. Our
laboratory studies indicate that whey protein concentrates from
other sources, did not produce significant biological activities
while exhibitîng similar nutritional efficiency. The percent
serum albumin concentration in these products is (as mean + SD)
respectively: 4+ 1 in Promod (Ross laboratories), 4+ in Alacen
855 (New Zealand Dairy), 4.8+ in Lacprodan - 80 (produced from
1989 by Danmark Protein), 4+ 0.1 in Sapro (Sapro, Montreal), 4+
1 in Savorpro - 75 (Golden Cheese, CA), 5+ 1 in Bioisolate
(Lesueur, Isolates, Minneapolis) [8] and 4.3+ 1 in Promix (Dumex,
Quebec). Similarly, the content of the other thermolabile
protein, immunoglobulin, was about half the value of the
undenatured WPC used in this study.
The results indicate that undenatured whey proteins by
providing specific fuel for glutathione replenishment in the
immunocytes could represent an adjuvant to other forms of
therapy.
209~37 1
- 22 -
Historically, and up until now, bacteria and spores in milk
were reduced by thermal treatment (pasteurization). In order to
be effective, that method inevitably produced denaturation, and
hence subsequent precipitation and 1088 in the curd of a
substantial amount of the most thermolabile and presumed
biologically active fractions of serum albumin and
immunoglobulin.
Our objective is to obtain a whey protein concentrate
(w.p.c) containing the proteins in proportion and conformation
as close as possible to that of raw milk, compatible with
accepted safety standards of bacterial content. Up until now we
have utilized the lowest acceptable level of heat treatment of
milk in order to preserve thermolabile whey protein. From now on
we will achieve this objective with a new method based on
membrane microfiltration.
Utilizing Bactocatch (Alfa-Laval Ltd. Scarborough, Ontario)
we can obtain a permeate by special membrane microfiltration of
the skimmed milk whose bacteria content has been reduced to less
than 0.5% of original input levels.
This permeate is then treated with rennet and the proteins
in the whey supernatant concentrated by a lenient procedure to
obtain the desired undenatured whey protein concentrate. We
believe that the membrane microfiltration concept replacing heat
treatment of milk will provide in the future the appropriate way
to preserve heat labile whey proteins, although techniques and
equipment may be improved in time.
Table 6 illustrates schematically a process to produce an
improved undenatured WPC which we have referred to under the
tradename Immunocal. Table 7 is a comparative chart showing the
characteristics of Immunocal in comparison with the sources of
WPC and showing also the consequences of 3 weeks dietary
treatment.
209~377
-- 23 --
TA:BL'E5 6
A 8C~EMATIC RgPR~8ENTATION OF T~ PROCE88 TO PRODUCE
T~ ~PC ~IC~ ~ R~F~R TO A8 IM~UNOCAL
Ra~ ~11~
Skimmed at 3 5 C --i cream
8~ ed uil~ pasteurized at 63 C for 30 minutes.
t 38 C: Addition of rennet (20 ml/100 kilos), allowing the
agitation to resolve at low speed.
curd
~hey
iltered with cheese cotton to remove debris (45 minutes):
t 40 C: Ultrafiltration (Romecon UFSI, polysulphone membrane,
cut off 50,000, pore diameter 60/1000 of an inch,
surface 2-3 m~).
: Diafiltration to wash out 6alts and lacto6e.
~h-v Protein ConcQntrat-
~I
Pasteurized at 63 C for 30 minutes.
At 40 C: Lyophylization (16 hours).
~hoy Proteia Concentrate. Pow~er:
Polyacrylamide Serum Albumin B-Lactoglobulin
gel electrophoresis 10+1% 57.8+0.9%
a-Lactalbumin Immunoglobulin
11. 4+0 . 6% 22+0 . 7%
209037 ~
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J :~ .i ~ ,~:
~1 o ~1 ~o o Ul
o ~ ~0
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u l .~ o o 1~
.~ .C
I ~' ~ O
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,I co u~ ~
X ,~
It, N
.
I ~ O ~ ~ .~
O _l +l +l +l +l +l +l +l I ~ O
C c~ r ~ ~ ~
~ t~ O
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a c ~ ~ +-~+1+l+l+l+l+
a ~ n .
~o
e ~ I ~ ~ ~ ~
_ ~~ .~ O ~1 ~ N ~ ~1 1 tJ~ ~ IU
p ~1 -- , ~ N N ~ ~1 .~ ,1
~ a~ a 8
o
+l +l ~ +l O +~ ~0 +1,~
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~ ç ~ ~ ~
~ I~ U ~1 9 ~
c c ~ 8 ~ ~ ~ o ~ ~
O ~ ~ 0 bq
~ O IU h 0 ~ O ~1 ~
~ O P~ '01 ~ :~
H ~ I¢ ~ C.q U~ m c~ ~
20~377
- 25 -
We have concluded as a result of our work that undenatured
whey protein concentrate is of value both in the prophylaxis
chemically induces cancer, such of colon cancer which is promoted
by carcinogens such as dimethylhydrazine. It also is useful for
the treatment of patients having chemically induced cancer cells
to inhibit the replication of such cells. An approximate dosage
for humans is in the range of about 8 to 40 grams daily and
preferably 30 to 40 grams daily. It has been established that
it i6 particularly advantageous to use WPC having a serum albumin
in concentration of at least 10%.
Tumors of the colon induced by dimethylhydrazine in
experiments with mice are similar to cancer of the colon in
humans in terms of the type of lesion and response to
chemotherapy. (27,28).
