CA2090377A1 - Anti-cancer therapeutic compositions for prophylaxis or for treatment of chemically induced cancer - Google Patents
Anti-cancer therapeutic compositions for prophylaxis or for treatment of chemically induced cancerInfo
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Abstract
ABSTRACT
This invention relates to a method of treatment of patients having lesions resulting from cancer cells and to an application as an anti-cancer composition of undenatured whey protein concentrate.
This invention relates to a method of treatment of patients having lesions resulting from cancer cells and to an application as an anti-cancer composition of undenatured whey protein concentrate.
Description
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 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).
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 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 20~377 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.
20~377 (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.
209~37~
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 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 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 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.
209~377 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.
20~377 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 20~377 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 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 209~377 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.
209~377 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 - 209~377 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 (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~
.
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 20~377 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 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.
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 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 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 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 ~
~ _ ~ o ~ l l l O L
J :~ .i ~ ,~:
~1 o ~1 ~o o Ul o ~ ~0 ~+1+~+
u l .~ o o 1~
.~ .C
I ~' ~ O
O +l +l +l +l +l +l ~ +l O
,I co u~ ~
X ,~
It, N
.
I ~ O ~ ~ .~
O _l +l +l +l +l +l +l +l I ~ O
C c~ r ~ ~ ~
~ t~ O
~;1 ,, ~ O
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,~
P * ~ ~ o ~ 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 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).
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 20~377 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.
20~377 (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.
209~37~
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 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 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 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.
209~377 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.
20~377 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 20~377 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 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 209~377 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.
209~377 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 - 209~377 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 (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~
.
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 20~377 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 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.
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 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 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 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 ~
~ _ ~ o ~ l l l O L
J :~ .i ~ ,~:
~1 o ~1 ~o o Ul o ~ ~0 ~+1+~+
u l .~ o o 1~
.~ .C
I ~' ~ O
O +l +l +l +l +l +l ~ +l O
,I co u~ ~
X ,~
It, N
.
I ~ O ~ ~ .~
O _l +l +l +l +l +l +l +l I ~ O
C c~ r ~ ~ ~
~ t~ O
~;1 ,, ~ O
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,~
P * ~ ~ o ~ 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 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).
Claims (10)
1. The application as an anti-cancer composition of a biologically active whey protein composition comprising a suitable concentration of whey protein concentrate wherein the whey protein concentrate contains the proteins which 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 small peptides patterns resulting from the contribution of all its protein components.
2. The application of claim 1 wherein the anti-cancer composition is used in the prophylaxis of chemically induced colon cancer in mammals.
3. The application of claim 2 wherein the colon cancer is induced by dimethylhydrazine.
4. 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 cell .
5. A method as in claim 4 in which the undenatured whey protein concentrate includes serum albumin at a level of about 10% or more.
6. A method as in claim 4 in which said lesions are lesions of the colon similar to those promoted by dimethylhydrazine.
7. A method as in claim 4, in which selenium is administered in the amount of about 40 - 60 mcg (calculated as selenium methionine) per 60 grams of undenatured whey protein concentrate.
8. A method as in claim 4, in which Vitamin B1 is administered in the amount of about 1.5 - 2.0 mg per 60 grams of undenatured whey protein concentrate.
9. A method as in claim 4, in which Vitamin B2 is administered in the amount of about 1.7 - 2.2 mg per 60 grams of undenatured whey protein concentrate.
10. A method as in claim 4, in which the following are administered per 60 grams of undenatured whey protein concentrate:
Vitamin B1 about 1.5 - 2.0 mg Vitamin B2 about 1.7 - 2.2 mg Selenium 40 - 60 mcg calculated as selenium methionine.
Vitamin B1 about 1.5 - 2.0 mg Vitamin B2 about 1.7 - 2.2 mg Selenium 40 - 60 mcg calculated as selenium methionine.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/188,276 US4842267A (en) | 1985-11-23 | 1988-04-29 | Amusement park device |
| US188,276 | 1992-04-10 | ||
| US92934792A | 1992-08-13 | 1992-08-13 | |
| US929,347 | 1992-08-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2090377A1 true CA2090377A1 (en) | 1993-10-11 |
Family
ID=26883901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2090377 Abandoned CA2090377A1 (en) | 1988-04-29 | 1993-02-25 | Anti-cancer therapeutic compositions for prophylaxis or for treatment of chemically induced cancer |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2090377A1 (en) |
-
1993
- 1993-02-25 CA CA 2090377 patent/CA2090377A1/en not_active Abandoned
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