DR. LUC MONTAGNIER is the co-discoverer of the AIDS VIRUS.

Nutriceutical Modulation of Glutathione with a Humanized Native Milk Serum Protein Isolate, IMMUNOCAL ^TM :
Application in AIDS and Cancer

Sylvain Baruchel and Ginette Viau
McGill University-Montreal Children's Hospital Research Institute, Montreal, Quebec, Canada.

Rene Olivier
Pasteur, Institute Paris, France

Gustavo Bounous
Montreal General Hospital, Montreal, Quebec, Canada

Mark A. Wainberg
Jewish General Hospital, Lady Davis Institute, Montreal, Quebec, Canada

NUTRITIONAL IMMUNOMODULATION AND ITS RELATION TO GLUTATHIONE SYNTHESIS

In 1981 it was discovered that normal mice fed a milk serum protein concentrate (specially prepared under mild nondenaturing conditions) exhibited a marked increase in the humoral immune response to a T helper cell-dependent antigen (1). In the following years, numerous experiments confirmed the consistency of this phenomenon (2-10). Over a period of 12 years and based on these findings a humanized native milk serum protein isolate (HNMPI) named Immunocal  was developed (Immunotec Research Corporation Ltd., Montreal, Quebec, Canada).

This property was found to be related, at least in part to a greater production of splenic glutathione (L-.-glutamylcysteinylglycine) (GSH) during the oxygen-requiring antigen-driven clonal expansion of the lymphocyte pool in animals fed with this bioactive HNMPI (9). Adequate levels of GSH are necessary for lymphocyte proliferation in the development of the immune response (11,12). Moderate but sustained elevation of cellular GSH as also found in the liver and the heart of healthy, old mice fed with this HNMPI for a prolonged period. In addition, HNMPI markedly increased their life expectancy in comparison to control animals fed nutritionally equivalent diets (13).

Glutathione is of major significance in cellular antioxidant activity in what Meister called the "GSH antioxidant system" because it participates directly in the destruction of reactive oxygen compounds and also because it maintains in reduced form ascorbate (vitamin C) and a-tocopherol (vitamin E), which also exerts a. antioxidant effect (14).

FUNCTION OF HNMPI AS A CYSTEINE DELIVERY SYSTEM

Systemic availability of oral GSH is negligible in man (I 5) and there is no evidence for transport of GSH into cells (16). Thus, it has to be synthesized intracellularly. This occurs in two steps: (a) glutamylcysteine synthesis; (b) glutathione synthesis. Even though the inflow of cysteine, glutamate, and glycine might prove somewhat limiting under selected circumstances, numerous observations have shown that it is the transport of cysteine (or cysteine, which usually is promptly reduced to cysteine on cell entry) which tends to be the ate-limiting event in GSH synthesis. whereas free cysteine does not represent an ideal delivery system (17) because it is toxic and is spontaneously oxidized. Cysteine present as the disulfide cysteine released during digestion in the gastrointestinal tract is more stable than free amino acid. GSH synthesis is submitted to negative feedback inhibition by the end-product GSH. The disulfide bond is pepsin- and trypsin-resistant, but may be split by heat and mechanical stress (9). Cysteine accounts for about 90% of the low-molecular-mass cysteine in the blood plasma, while reduced cysteine is present only at extremely low concentration (18).

In a comparative study, we found that commercial milk serum concentrates exhibiting far less bioactivity, including less GSH promoting activity, contain about half the amount of serum albumin (9) and 4 times less lactoferrin than HNMPI, expressed as percentage of total milk serum protein. I..UNOCALTM is produced in a proprietary lenient process which results in the preservation of the most thermolabile proteins in their native conformation.

In the serum albumin, there are 17 cysteine residues per 66 kDa molecule and 6 GluCys dipeptides (19); in lactoferrin there are 17 per 77kDa molecule and 4 Glu-Cys dipeptides (20); and in the @-lactalbumin there are 4 cysteines in a 14,000 kDa molecule

Table 1.

(19). On the other hand, P Lactoglobulin. has only 2 cysteines in a 18,4OOkDa molecule (19), and IGGI, the predominant immunoglobulin in cow whey, has only 4 disulfide bridges in a 166,OOOkDa molecule (Table 1). In addition, Meister and colleagues (16) have demonstrated that the y-glutamylcysteine (Glu-Cys) precursors of GSH can easily enter the cell and there be synthesized into GSH. It thus become noteworthy that the most labile milk proteins-, serum albumin and lactoferrin-are those which contain these putative GSH-promoting peptide components.

Finally, the bioavailibility of the presumed active component (cysteine and Glu-Cys group) may be influenced by the coexistence of the other proteins throughout the digestive-absorptive process.

This newly discovered property of HNMPI was found to be independent of its nutritional value, as other proteins of similar nutritional efficiency do not exhibit this unique property (1-10). The concept that a specific biological activity can exist in addition to and independent of the systemic effect Of IMMUNOCAL as a good protein source is further substantiated by recent in vitro assays (21).

The dietary provision of cysteine is particularly relevant to the immune system. The coordinated response of macrophages and lymphocytes in the T cell-mediated immune response is regulated, in part, by macrophage cysteine uptake and subsequent release of reduced cysteine into the local environment for uptake by lymphocytes. When the antigenpresenting macrophages come into close contact with antigen-specific T cells, they supply these cells with additional amounts of cysteine and thereby raise their intracellular GSH level (18).

The validity of this assumption is confirmed by the demonstration that the immunoenhancing and GSH-promoting (data not shown) effect of Immunocal is abolished by buthionine suIfoximine, which inhibits y-glutamylcysteine synthetase, the initial step in GSH synthesis (17).

IN VITRO MODULATION OF INTRACELLULAR GLUTATHIONE BY IMMUNOCAL

We demonstrated that normal human lymphocytes cultured for 3 days with HNMPI 100@g/ml show an increase in intracellular GSH content from 4.5 ± 0.4 to 10.5 ± 3.4nmol/10 6 cells, p <0.01 (Figure 1). This increase in GSH correlates with an increase in cellular proliferation measured by thymidine incorporation (data not shown). The

Table 2 Presence of Cytopathic Effects in MT-4 Cells
	TCID50/well3
IMMUNOCAL (ug/ml)	2000	200	20	2
0	+++	++	+	-
I	+++	++	+	-
10	++	+	+	-
100	-	-	-	-
500	-	-	-	-
1000	-	-	-	-
3+ Presence of cytopathic effects; - absence of cytopathic effects.

increase in GSH is dose-dependent and has not been found for casein or for any commercially available milk serum protein concentrate (Figure 2).

GSH depletion, a consequence of chronic oxidative stress, is part of the spectrum of HIV infection. GSH has, in addition, a crucial role in lymphocyte function and cell survival.

IMMUNOCAL functioning as a cysteine delivery system can enhance GSH synthesis in vitro (Figure 1) and inhibits HIV replication on a cord mononuclear cell system infected by HTL V-IIIB (Figure 3). IMMUNOCAL also inhibits the formation of syncitium between infected and noninfected cells. The inhibition of syncitium formation occurred at the same concentration as inhibition of HIV replication(Table 2). This viral inhibition was not associated with any cytotoxicity. IMMUNOCAL, via its GSH-promoting activity, educes apoptosis in HIV-infected cells. Apoptosis was evaluated by flow cytometry on PBMC from HIV-infected individuals (Dr. R. Olivier, AIDS and Retrovirus., Department, Pasteur Institute). HIV-infected PBMC cultured at concentrations of IMMUNOCAL' of I 00 ug/ml or higher were less prone to die of apoptosis than untreated cells: 15% ± 2.6% vs. 37% ± 2.4, p<0.001 (Figure 4).

IMMUNOCAL was administered twice a day as a powder diluted in water. In some patients, IMNUNOCAL,TM was administered via nasogastric tube when necessary. The administered starting dose was based on 20% of the total daily protein requirement and was increased by 5% each month over 4 months to reach 35% of the total protein intake at the end of the study. The total duration of the study was 6 months.

Weight, height, triceps skinfold and mid-arm muscle circumferences, CD4/CD8 counts, and peripheral lymphocyte GSH concentrations (measured by spectrophotometric assay) were measured monthly. Energy intake was assessed by the use of two independent 2-day food records with , 2-3 week period between the food records. Each food record included a weekday and a weekend, and the average of these records was calculated to reflect the daily nutritional intake. Out of 14 patients enrolled, 10 were evaluable. The ages of the patient were from 8 months to 15 years. The 10 patients studied were enrolled in four different centers across Canada: Montreal Children's Hospital (Dr. S. Baruchel), The Hospital For Sick Children Toronto (Dr. S. King), Children's Hospital for Eastern Ontario (Dr. U. Allen), and Centre Hospitalier Laval Quebec (Dr. F. Boucher). Of the 4 remaining patients, 2 lacked compliance after 2 months while the other 2 died of AIDS progressive disease within the first 2 months f entry into the study. None of the deaths was related to the tested product.

None of the patients experienced any major toxicity such as diarrhea or vomiting or manifestation of milk intolerance. One patient had to Stop IMMUNOCAL transiently for minor digestive intolerance such as nausea and vomiting (, twice/day) at month 3 and was subsequently able to restart the treatment without any problem.

At the end of the study, all patients experienced a weight gain in the range of 3.2% to 22% from their starting weight. The mean weight gain for the group was 8.4% ± 5.7%. On analysis of the mean percentage of requirement nutrient intake (RNI) per month for all

Table 3 Change from  Baseline (expressed as a Percentage) at Weeks 24
 and 36 in Weight, Anthropometric and GSH Patients Treated with IMMUNOCAL

the patients, no correlation was found between the weight gain and any significant increase in the mean percentage of RNI, suggesting educed catabolism rather than an anabolic effect of IMMUNOCAL. Six of ten patients have demonstrated an improvement in their anthropometric parameters such as triceps skinfold or mid-arm muscle circumference independently of an increase in energy intake (Table 3).

Two groups of patients were identified in terms of GSH modulation: responders and nonresponders. The responders were those who started the study with a low GSH level.

The nonresponders were those who stated with a normal GSH level. A positive correlation was found between increase in weight and increase in GSH (Figures 5,6,7). No changes were found in terms of blood lymphocyte CD4 cell count, but 2 patients exhibited an increase in the percentage of their CD8 cells and 4 patients showed a trend toward an increase in the number of NK cells.

In conclusion, this pilot study demonstrates that IMMUNOCAL is very well tolerated in children with AIDS and wasting syndrome and is associated FIGUREF an amelioration of the nutritional status of the patient as reflected by weight and antrhopometric parameters. Moreover, the GSH-promoting activity Of I.MUNOCAITM in vivo seem to be validated in 6 out of 10 patients. An international multicenter double-blind randomized study is currently under way in France and Canada in adults patients with AIDS and wasting syndrome.

SELECTIVE GLUTATHIONE MODULATION OF BREAST CANCER CELLS AND IMPACT ON CANCER CELL GROWTH

Recently, a direct inhibitory effect of HNMPI in human cancer cell replication was confirmed (21,29,30). In other human cancer cell studies, the inhibitory effect ,as found to be related to the serum albumin component of milk serum (3 1) and most recently to @-lactalbumin (32). Feeding lactoferrin to mice inhibited the growth of solid tumors and in addition reduced lung colonization by melanomas (33). Unlike other proteins, serum albumin ,as found to exhibit a strong antimutagenic effect in an in vitro assay using hamster cells (34). It is therefore noteworthy that in this HNMPI we have succeeded in concentrating serum albumin, @-lactalbumin, and lactoferrin, all containing a significant number of GSH precursors. A possible explanation for these newly discovered properties of dietary milk serum protein may be found in recent findings on the role of GSH in tumor biology (35).

The search for ways to inhibit cancer cells without injuring normal cells has been based over the years on a vain effort to identify the metabolic parameters in which cancer cells are at variance with normal cells. One such function could well be the all-important synthesis of cellular GSH.

Recent experimental evidence has revealed an intriguing response of tumor versus normal cells to GSH synthesis-promoting compounds. Cellular GSH levels have been found to be several times higher in human cancer cells than in adjacent normal cells (35). This finding is presumably related to their proliferative activity. In fact, cancer is the only condition in which elevation of such a tightly regulated system as GSH has been reported. However, when a cysteine- and GSH-promoting compound such as 2-l-oxothiazolidine-4-carboxylate (OTZ) as added to cultured human lung cancer cells exhibiting very high levels of GSH at the outset, no intracellular increase was noted, whereas GSH increased substantially in normal cells (35). This differential response is even more pronounced in vivo. We demonstrated that in tumor-bearing rats, OTZ treatment was actually found to deplete GSH in the tumors (36).

More specifically, an in vitro assay showed that, at concentrations that induce GSH synthesis and proliferation in normal human cells (Figure 1), IMMUNOCAL caused GSH depletion and inhibition of proliferation of cells in a rat mammary carcinoma (Figure 8) and Jurkat T cells (Figure 9) (21).

The selectivity demonstrated in these experiments may be explained by the fact that GSH synthesis is negatively inhibited by its own synthesis and since, as mentioned, baseline intracellular GSH in tumor cells is much higher than in normal cells, 'It is easier to reach the level at which negative feedback inhibition occurs in this cellular system than in a nontumor cellular system.

HNMPI IN CANCER CLINICAL TRIALS

A major problem in the use of chemotherapeutic agents in cancer therapy is the protection offered by the defense mechanisms of cancer cells. An important element of protection is represented by GSH, which is an effective detoxification agent that is relatively abundant in tumor cells. Indeed, when GSH synthesis is inhibited by buthionine sulfoximine (BSO), the activity of several chemotherapeutic agents such as alkylating agents is increased and drug resistance can be reversed (36-38). However, the concomitant depletion of GSH in normal cells greatly limits the practical usefulness of this modality of treatment.

We recently demonstrated that a selective GSH prodrug such as OTZ protects some normal tissue (36) but also potentiates the activity of some alkylating agents (38). The apparently selective depletion of tumor GSH levels by provision of a natural precursor of GSH as contained in IMUNNOCAL,Tm seems to be associated with inhibition of proliferation of cancer cells in vitro. This natural precursor of GSH favorably influences the GSH synthesis in normal cells. These in vitro and preliminary clinical results indicate that this newly discovered property of HNMPI may be a promising adjunct to the nutritional management of cancer patients undergoing chemotherapy. We are currently developing a phase 11 study in breast carcinoma, attempting to confirm that this selective depletion of GSH may, in fact, render tumor cells more vulnerable to chemotherapy and eventually protect normal tissue against the deleterious effect of chemotherapy.

ANALOGY BETWEEN HNMPI IMMUNOCAL AND HUMAN MILK

Scientific data based on the similarity between the bioactive components of this native milk protein isolate (HNMPI) of cow milk, IMMUNOCAL, and human whey protein appear to substantiate this theory, as will now be discussed in more detail.

It is well known that breast feeding is superior to the use of cow milk-based formulas of similar nutritional efficiency for the health of human babies. Breast feeding protect against otitis media, and pneumonia (40,41). Mothers milk also has a protective effect on the incidence of several types of childhood cancer including leukemia, lymphomas, bone tumors, and brain tumors (42). Children who are artificially fed or are breast fed for only a shot period of time are more at risk for developing several types of cancer before the age of 15 years as compared to long-term breast feeders (43). Thus, the concept of a biological activity in addition to but independent of the nutritional efficiency, formulated to describe the immunoenhancing and GSH-promoting activity of the HNMPI IMMUNOCAL, may indeed apply to the breast feeding of neonates and infants. Glutathione synthesis appears to be the crucial factor in the health benefit of HNMPI.

It may then be appropriate to 'Identify the features common to HNMPI and human whey proteins that are capable of influencing GSH synthesis in the host. Cysteine, a crucial limiting factor in the synthesis of GSH, is about as abundant in cow's whey protein as it is in whole human milk proteins and several times more abundant than in cow's whole milk (39), since most caseins contain either no cysteine or one or two cysteine residues(19). As mentioned earlier, our studies showed that the most thermolabile milk proteins, namely, serum albumin, a-lactalbumin, and lactoferrin, are crucial to expression of the bioactivity of HNMPI. As shown in Table 1, these proteins are rich in cysteine and glutamylcysteine residues, natural precursors of GSH. The presence of these dipeptides in the product IMMUNOCAL is a characteristic shared with human milk (Table 4).

Traditionally, it has been advocated that "humanized" cow milk should contain more a-lactalbumin because this protein is twice as abundant in human milk. On the basis of our experimental findings, we propose instead that the principal health factor in human milk,

Table 4. Protein Composition of Cow and Human Milk Composition (g/liter)

not denaturated by heat pasteurization, is due to the predominance of the thermolabile proteins rich in cysteine and containing the Glu-Cys dipeptide which are characteristic of the bioactive HNMPI, namely, serum albumin, a-lactalbumin, and lactoferrin. This HNMPI differs from other commercially available milk serum protein concentrates in having a relatively high content of serum albumin (about I 0%), lactoferrin (about 0.65 %),

CONCLUSION

This article has addressed the central role of GSH in providing protection against endogenous oxiradicals and foreign pollutants. As an antioxidant, GSH is essential for allowing the lymphocyte to express its full potential, without being hampered by oxiradical accumulation during the oxygen-requiring development of the immune response. In a similar fashion, GSH delays the muscular fatigue induced by oxiradicals during the aerobic phase of strenuous muscular contraction.

It is, however, the second function of GSH-that of detoxification of chemical pollutants, carcinogens and ultraviolet radiation-that may well be of greater concern to medical science today, because of the ever-increasing demand on GSH as the major detoxifying agent. Under normal circumstances, a nutritionally balanced diet should provide sufficient precursors of GSH to allow for intracellular synthesis of adequate amounts of GSH. But in our current polluted environment, trace amounts of precursors found in an otherwise adequate diet may not be sufficient to allow for full GSH replenishment. This results in highly undesirable competition for GSH precursors developing amongst different systems. Cysteine prodrugs have helped clarify the essential role of GSH in athletic performance, immune function, AIDS, etc., but their effect is short-lived and their long-term use is not without adverse effects.

Using modern technology, it has been possible to obtain and consistently preserve, in their native form, the specific cow's milk proteins which share with predominant human milk proteins the same extremely rare GSH-promoting components. This product-the patented WPC-differs from most commercial WPCs in that it contains the active ingredients-notably cysteine and glutamylcysteine-in undenatured form and an amount sufficient to exhibit its potency when given as a dietary supplement, without overloading the system with excessive nitrogen intake.

It is therefore possible to obtain, with the patented milk serum protein concentrate, long-term moderate but sustained intracellular elevation of GSH and GSH precursors so that, when the challenge occurs, an efficient cellular response can be achieved.

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