Pharmaceutical compositions containing undenatured whey protein concentrate

Pharmaceutical compos ו tionsc contain! ngI jindenatured־.. whey protein concentrate

IMMUNOTEC RESEARCH CORPORATION LTD.

C. 89299

Background

Reference.is made to twelve articles listed at the end of this specification, the contents of which are incorporated by reference herein.

Our studies showed that the humoral immune response (number of plaque-forming cells formed in response to sheep red blood cells) is significantly higher in mice fed a 20g whey protein concentrate/100 g diet than in mice fed formula diets of similar nutritional efficiency containing 20g/100 diet of any . . other type of commercially available semipurified . food .protein, such as casein, soy, wheat, corn, egg white, fish, beef protein, Spirulina maxima, Scenedesmus algae ..protein, or Purina mouse chow [1].

We have further shown that the immunoenhancing activity of dietary whey protein concentrate (WPC) is related to greater production of splenic glutathione (GSH) in the whey protein-fed animals during the oxygenrequiring antigen-driven clonal expansion of the lymphocyte [2]. It was then theorized that this might reflect the ability of the lymphocytes of whey protein diet-fed mice to offset potential oxidative damage, thus responding more fully to the antigenic challenge [3,4]. In fact, the capacity of a cell to recover from an oxidative insult is considered to be represented by its ability to regenerate intracellular stores of glutathione [5].

Our studies also showed that administration of S(n-butyl) homocysteine sulfoximine, which reduces splenic glutathione in half, significantly reduced the humoral immune response of whey protein-fed mice. This was taken as further evidence for the important role of glutathione in the immunoenhancing effect of dietary whey protein

[2]-

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.) injection in mice, returning to control values 2 hours later [6]. The -. administered gamma-glutamylcysteine is transported intact and serves as a substrate for glutathione synthetase [7], 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 [1], 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 GlU-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 [8].

Our recent [8] 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 (>10%) and immunoglobulins. In addition, the mice fed this type of whey protein concentrate exhibit higher levels of tissue glutathione. The presence in the serum albumin fraction of glutamylcysteine groups (rare in food protein) and the specific intramolecular bond as related to the undenatured conformation of the molecule are considered to be key factors in the glutathione-promoting activity of the protein mixture.

Recent experiments in Japan [9] showed that 10 spleen cells of BALB/c male mice fed 25g of our undenatured whey protein concentrate (WPC) (which we call ״Immunocal״) per 100g diet for 4 weeks had an increased immune response to SRBC in vitro and a higher content of L3T4⁺ cells (12.58 x 10⁶± 0.50) than mice fed an 15 isocaloric diet with 25g. pure casein/lOOg. diet (3.69 X

10⁶ + 0.50). Similarly, the spleen L3T4⁺ /LYt - 2⁺ ratio . was 1.36 ± 0.07 in undenatured WPC fed mice and 0.55 ± 0.07 in casein-fed controls (P <0,00!). Materials and Methods . The whey protein concentrate (WPC) used in the .examples was ־ 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 25 indicates that the proteins present are essentially undenatured, hence demonstrating the leniency of the ultrafiltration process [8]. Although the proteins contained in the concentrates from the other commercially available sources examined were mostly in undenatured 30 form, as indicated by the relatively high solubility of the concentrates, the content of serum albumin and immunoglobulins in these mixtures is below the level apparently necessary to produce a significant biological activity [8]. These very thermolabile proteins are 35 denatured, hence precipitated and partially lost from whey when high pasteurization temperatures are utilized.

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Conversely, the relatively high concentrations of the thermosensitive serum albumin 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 G1 u.-Cys containing proteins such as serum a T bum ח יו in undenatured conformation are crucial elements for the biological activity of whey protein concentrate.

The bovine whey protein concentrate (WPC) was especially prepared by the Service de recherche sur. les aliments du Ministere de.!'agriculture du Quebec in -StHyacinthe, 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, measured. by polyacrylamide gel electrophoresis [8] was: beta-lactoglobulin 59.1 ± 4.0; alpha-lactalbumin: 22± 0.7; serum albumin:. 9.7 ± 1.0; immunoglobulin 9.0 ± 1.0 (mean ± SD) . The solubility index should preferably be above 98%.

. -. . . 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 > . 10% is highly advantageous to improving the immune system.

Serumalbumin 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 Glutathione, Clin. Invest Med 14 : 296 - 309, 1991 [8],which is incorporated by reference in.its entirety.

We have found it possible to achieve a serum albumin level as high as 11 ± 1% with milk pasteurized at 72°C for 13 seconds.

Upon bacteriological analysis no staph, salmonella, B cereus, or the WPC prepared by the Aliments due Ministere de the sample pasteurized at !θ The method used examples is schematically

E coll were isolated m either Service de recherche sur les !^,agriculture du Quebec or in 72’C. for 13 seconds.

to prepare the WPC used in'the described below in Table 1.

TABLE SCHEMATIC REPRESENTATION OF THE PROCESS TO PRODUCE OUR UNDENATURED WPC PRODUCT Raw milk

4 . Skimmed at- 35’ C - . ־* cream

Skimmed milk pasteurized at 63⁴ C for 30 minutes. 10 .4 . .

At 38°C: Addition of rennet (20 ml/100 kilos), allowing the agitation to resolve at low speed.

-+ curd

Whey .־ 'I ' ' -.4 .J.. . . . ־ .

. Filtered;with' cheese cotton to remove debris. ׳ ' . . . _r . .. ... . . . .

־״. At 40°C:L- -Ultraf iltration (Romecon UFSI--, - polysulphone-־ .. membrane, cut off 50,000, pore diameter 0.06 inch, 20 surface 2-3 m²).

: Diafiltration to wash out salts and lactose. Whey Protein Concentrate

At 40°C: Lyophylization.

4

Whey Protein Concentrate Powder ml of heparinized blood are used to determine the glutathione content of blood mononucleated cells. Counted mononuclear cells are resuspended in phosphate buffered saline adjusted so that there are 10?

cells per tube. After centrifugation 900 μί of water is 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 10 supernatant is used, for the biochemical assay according ; .. . to, the method of Anderson [11]. Values are expressed as:.

nanomol (nMol) of GSH/10⁷ cells. Blood lymphocyte subsets are determined by.flow-cytometry.

The total serum protein,, including the albumins 15 and the immunoglobulins is : determined by the Biuret method. The level of Immunoglobulin A (IgA), Immunoglobulin G (IgG) and Immunoglobulin M (IgM) are measured by immunonephlometry. . ... ..

Description of the invention and examples

20- There are two important subsets of lymphocytes in _the blood; (1) CD4, also called T-helper cells, because they help in the immune response and (2) CD8, also called T-suppressors because they have the opposite effect, I.e. they suppress the immune response. In HIV25 seropositive individuals the number of CD4 (helper) cells is low, i.e. the ratio CD4/CD8 is down.

The object of this invention is to elevate the ratio of T helper cells to T suppressor cells. This is 30 accomplished by the administration of undenatured whey protein concentrate.

The amount administered should be in the range of about 8 to 40 grams daily and preferably 20 to 40 grams daily. It is particularly beneficial to the 35 glutathione level to administer 30 to 40 grams daily.

As more fully described in U.S. Patent No., 5,250,902 which is incorporated in its entirety by reference, raising the glutathione level is beneficial to the immune system.

EXAMPLE 1

A whey protein concentrate as previously described was administered to 3 male HIV-seropositive individuals identified in Table 2 as A, B and C. The product was drunk cold daily in a liquid chosen by the patient. Originally a fourth patient D was included but he took the WPC sporadically for lack of discipline and none during the final days of the study. He therefore has been classified in Table 2 as a control. the other controls are E and F.

The daily intake of WPC was increased stepwise. During the first three weeks 8.4 grams were prescribed daily, in the following three weeks 19.6 g, in the next three weeks 28 grams, and 39.2 grams in the final three weeks.

The observations made are shown below in Table

2.

WHEY י TABLE.2

<td> Patient</td><td> Weeks</td><td> Energy</td><td> Ideal</td><td> Body CD4% Helper</td><td> £D4</td><td> GSH</td><td> Serum Proteins</td><td> in</td><td> %</td>

<td> Initials</td><td> on</td><td> (KCal)</td><td> Body</td><td> Weight (2) Absolute</td><td> CD8</td><td> n moI-10³ cells</td><td> Alb IgG</td><td> IgA</td><td> IgM</td>

<td> (v)</td><td> Whey</td><td> Protein g</td><td> Weight</td><td> (Kg): 1 (3)׳.</td><td> (4)</td><td> (5)</td><td> Total</td><td></td><td></td>

(1) Μ

<td></td><td> 0</td><td> if</td><td> 86</td><td> 1015</td><td> 23</td><td> 368</td><td> 0.38</td><td> 9.75 _:</td><td> 77</td><td> 42</td><td> 20.8</td><td> 154</td><td> 1.14</td>

<td> A</td><td> 6</td><td> 1800</td><td></td><td> 105</td><td> ,26</td><td> 546'</td><td> 0.45</td><td> 1034</td><td> ־74</td><td> 39</td><td> 19.4</td><td> 156</td><td> 1.16</td>

<td></td><td></td><td> 106</td><td></td><td></td><td> ׳</td><td> ׳ . it . '</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> (35)</td><td> 12</td><td> 2111</td><td></td><td> 108</td><td> ,24 </td><td> 480. ׳</td><td> 0.41</td><td> 13.9</td><td> 80 .</td><td> 42</td><td> 20.1</td><td> 1.61</td><td> 1.05</td>

I

<td></td><td> 0 .</td><td></td><td> 75.2</td><td> 73.9</td><td colspan="2"> '435 15 ' J יי</td><td> '־ 0.22</td><td> 1022</td><td> 82</td><td> 50</td><td> 123</td><td> 438</td><td> 0.75</td><td> 1</td>

<td> B (32)</td><td> 6</td><td> 203$ 140</td><td></td><td> 74.5</td><td> 19</td><td> 532'</td><td> 028 _:</td><td> 9.6 ׳</td><td> 79</td><td> 45</td><td> 143</td><td> 453</td><td> 0.76</td><td></td>

<td></td><td> 12</td><td> 138</td><td></td><td> 76</td><td> 17 &lt; -»</td><td> 442</td><td> 024</td><td> 17.04</td><td> 72</td><td> 50</td><td> 16.9</td><td> 5.81</td><td> 0.95</td><td></td>

<td></td><td> 0 t</td><td> 100</td><td> 78</td><td> 76</td><td> 24</td><td> 672!</td><td> 039</td><td> 1038</td><td> 82</td><td> 52</td><td> 12.4</td><td> 359</td><td> 0.76</td>

<td> c</td><td> 6</td><td> 22QQ</td><td></td><td> 775</td><td> 27</td><td> &gt; 864</td><td> 0.46 .</td><td> 1255</td><td> 81</td><td> 49</td><td> 12.9</td><td> 3.99</td><td> 058</td>

<td></td><td></td><td> 116</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> (20)</td><td> 12</td><td> 14QQ</td><td></td><td> 78</td><td> 26</td><td> ’ 676)</td><td> 0.45 '</td><td> 7.06</td><td> 80</td><td> 50</td><td> 123</td><td> 33</td><td> 0.7</td>

CONTROL ! Table . 2, Continued .

<td> Patient Initials (1¢)</td><td> Weeks</td><td> Energy iKCal) Protein g (1)</td><td> Ideal Body Weight M</td><td> Body CD4% Helper Weight (2), ,Absolute M ; .(1). ' V '1 ׳׳I‘,'׳ </td><td> £04 CD8 w .</td><td> GSH n mol-10⁵ cells (5) 1.</td><td> Total</td><td colspan="2"> Strain Proteins Alb IgG</td><td> in IgA</td><td> % IgM</td>

<td></td><td> 0</td><td></td><td></td><td> 955 27 ;540</td><td> 0.47 .</td><td> 1236</td><td> 75</td><td> 47</td><td> 15.6</td><td> 1.63</td><td> 134</td>

<td> D</td><td> 6</td><td> . 25QQ</td><td></td><td> 85 •25 , .,650.</td><td> 0.42</td><td> 859’ ' .</td><td> 73</td><td> 42</td><td> 15</td><td> 15</td><td> 1.19</td>

<td> 0</td><td> 2$ 98</td><td> 70</td><td> 695 17 ''420 1 ' ' 'ί '</td><td> 103'</td><td> 81 '144</td>

<td> E 6</td><td> 2600</td><td></td><td> 685 '21 371</td><td> 11</td><td> ן 44 82</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td>

<td> (38)</td><td></td><td></td><td> 1</td><td> ! '1</td><td> H 0</td>

<td> 12</td><td> 248Q</td><td></td><td> 68 .18 359;.</td><td> 9.8 .</td><td> 81 44</td>

<td></td><td> 108</td><td></td><td></td><td></td><td> 1</td>

<td></td><td> 0</td><td> 222) 108</td><td> 74</td><td> 74</td><td> '16</td><td> 263 : f</td><td> 11.08</td><td> 80</td><td> 43</td>

<td> F</td><td> 6</td><td> no</td><td></td><td> 73</td><td> ׳12</td><td> &gt; 222׳</td><td> 103</td><td> 83</td><td> 43</td>

<td> (37)</td><td> 12</td><td></td><td></td><td> 72 </td><td> 125</td><td> ־'230</td><td> 9</td><td> • 80</td><td> 42</td>

no . The energy and protein intake indicated represents the mean value for the preceding weeks.

2.Normalrange:35-55 ¹ 7; , . Normal range: 580-1250 . Normal range: 1.42-3.56 . GSH: Glutathione content of mononuclear blood cells. Normal value in healthy HlV.seronegative individual of corresponding age: 17.05*2.40 (mcan'SD)

In Table 2:

CD 4% means the percentage of CD4 (helper cells) of the total lymphocytes in the blood.

Helper absolute refers to the real number of CD4 cells per unit of blood (X 10^ per microliter (μΐ))

CD4/CD8 refers to the ratio of the two types of lymphocytes in the blood, i,.e. the T-helper cells and the T-suppressor cells.

Total refers to the total amount of protein in the 10 serum which includes the albumins and the imunoglobulins.

IgA means Immunoglobulin A IgG means Immunoglobulin G IgM means Immunoglobulin M״~ The data in Table 2 and other information gathered 15 during the course of the work indicates the following.

Three patients took the undenatured WPC daily for .the 3 ׳-month period without any adverse side effects. In . all these patients body weight increased progressively (from 2 to 7 kilos); and in fact two of them (C and B) ־ ״20־-reached ideal body weight. Serum proteins, including - .' albumin, remained unchanged and within normal range;' indicating that protein replenishment per se was not likely the cause of increased body weight.

In all three of these patients the blood T25 helper cells concentration and the T-helper/suppressor ratios were moderately but consistently higher during the study than before undenatured WPC administration.

The blood mononuclear cells glutathione content was, as expected [10], below normal values in all 30 patients at the onset of the study. Over the three-month period, however, glutathione levels increased and in one case (B) rose by 70% to reach normal value.

Systemic glutathione deficiency in symptom-free HIV-seropositive individuals [10] at page 1297 presents 35 the re-establishment of normal extracellular concentrations of glutathione as an unsolved problem, ־12because GSH administered intravenously has a half life of only 1-6 minutes. This problem is however solved in accordance with this invention by the administration of undenatured WPC.

These objective changes were accompanied by a markedly improved sense of well being in all three patients.

It is noteworthy that one patient overconcerned that the . beneficial increase in body weight 10 could hamper his lean appearance, drastically reduced energy and undenatured WPC intake during the second period of study. (C, Table 2). During this time body weight increase was.reduced, and glutathione and T-helper cells failed to rise.

As indicated earlier, patient D. considered as _ -control for his total lack of submission to the protocol, .. exhibited a drop in body weight, T-helper/suppressor . ratio and glutathione on the 6-week end point and did not show up for. the 12-week appointment. The other two ²⁰ j J controls (E. and E.) exhibited weight loss and ho change :. .in GSH״levels־ .־t־־: u'.. ״./: . . . -:_____The example indicates that whenever patients . . .essentially maintained their, ,energy , intake at pre-study levels with only minor reductions in the prescribed WPC 25 intake, body weight increased and specific HIV indicators such as a blood cell glutathione, T-helper cells concentrations and T-helper/suppressor ratios were all moderately higher during the WPC administration.

The positive effects of undenatured WPC 30 observed in this very limited number of HIV-seropositive individuals acquires significance if viewed on the background of a large number of animal experiments showing increased cellular GSH and immune response by our WPC [1,2,8,9]. Animal studies emphasize the fact that 35 the immunoenhancing effect of undenatured WPC is not related to a greater systemic nutritional efficiency, when compared to several other protein sources with similar nutritional efficiency but no significant biological activity. Mice fed undenatured WPC did not exhibit increased body growth nor any changes in serum 5 protein levels. Similarly in our patients undenatured WPC did not produce any change in serum proteins which remained constant throughout the study. The increase in body weight observed in our patients did not correlate with increase in energy or .protein intake throughout the 10 study period but rather with improved sense of well being and HIV specific blood parameters. The extra protein intake through the undenatured WPC was generally compensated by reduced intake of protein from other sources. ־

The presence of glutamylcysteine groups in the serum albumin component of the whey protein concentrate is considered to be a key factor in the glutathionepromoting and immunoenhancing activity of the protein mixture of the undenatured WPC. Our laboratory studies 20 indicate that whey protein concentrates from other , sources,\ did’. . not ־ produce: ־־ significant'. biological ^r .-־. activities־ while exhibiting similar nutritional ~ efficiency. The percent serum albumin concentration in these products is (as mean ± SD) respectively: 4± 1 in 25 Promod (Ross Laboratories), 4± in Alacen 855 (New Zealand

Dairy), 4.8± in Lacprodan - 80 (produced from 1989 by Danmark Protein), 4.8+ 0.1 in Sapro (Saputo, Montreal), 4+ 1 in Savorpro - 75 (Golden Cheese, CA), 5± 1 in Bioisolate (Lesueur Isolates, Minneapolis) [8] and 4.3+ 1 30 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 35 proteins by providing specific fuel for glutathione replenishment in the immunocytes could represent an adjuvant to other forms of therapy.

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 loss 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.

0_u. . - Scarborough,. Ontario.) we can obtain by special ;membrane . ._r. . ^,micrpfil.tration. of ...the skim milk a permeate¹ whose bacteria content has been reduced to less than 0.5% of original input levels.

This permeate is then treated with rennet and ' 25 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 30 preserve heat labile whey proteins, although techniques and equipment may be improved in time.

REFERENCES

1) Bounous G, Kongshavn PAL, Gold. P: The immunoenhancing property of dietary whey protein concentrate. Clin Invest Med 11:271- 8, 1988.

2) Bounous G, Batist G, Gold P: Immunoenhancing property of dietary whey protein in mice: Role of glutathione. Clin Invest Med 12:154-61, 1989.

3) Fidelus RK, Tsan MF: Enhancement on intracellular glutathione promotes lymphocyte activation by mitogen. Cell Immunol 97:155-63, 1986.

4) Gougerot-Pocidalo MA, Fay M, Roche S: Mechanisms by which oxidative injury inhibits the proliferative response of. human lymphocytes to PHA, Effect of the thiol compound 2-mercaptoethanol. Immunology 64:281-8, 1988.

5) Noelle RJ, Lawrence DA: Determination of glutathione in lymphocytes and possible association of redox state and proliferative capacity of lymphocytes. Biochem J 198:571-9, 1981.

6) Anderson ME, Meister A: Transport and direct utilization of gamma-glutamylcyst(e)ine for glutathione synthesis. Proc Natl Acad Sci 80:707-11, 1983.

7) .; Meister A: 5-0xoprolinuria and other 3disorders, of glutathione biosynthesis. In:Stranbury JB, Wymgaarden JB, Frederikson DS, eds. Metabolic basis of inherited diseases 4th edn. McGraw Hill, 1978:328-35

8) Bounous G, Gold P: The biological activity of undenatured dietary whey proteins: role of glutathione. Clin Invest Med 14:296-309, 1991.

9) Hirai Y, Nakay S, Kikuishi H, Kawai K; Report: Evaluation of the immunological enhancement activities of Immunocal. Otsuka Pharmaceutical Co. Ltd: Cellular Technology Institute: December 13, 1990, Osaka, Japan.

10) Buhl R, Holroyd KJ, Mastrangeli A, Cantin AM, Jaffe HA, Wells c, Saltini C, Crystal RG:Systemic glutathione deficiency in symptom-free HIV-seropositive individuals. Lancet(December 2): 1294-7, 1989.

11) Anderson ME: Tissue glutathione: In:C.R.C. Handbook of methods for oxygen radical research. Boca Raton, Florida:CRC Press, Inc., 1985:317-29