DK158138B - METHOD OF PREPARING IMMUNOGLOBULIN-IGG-CONTAINING MILK OR FROM DERIVED PRODUCT TO TREAT RHEUMATOID ARTHRITIS - Google Patents

Description

in

DK 158138 B

The present invention relates to a method for producing immunoglobulin IgG-containing milk or derived product for the treatment of rheumatoid arthritis, using a vaccine prepared from cultures of bacterial strains grown in appropriate culture media, after which the bacteria have been killed, harvested, washed, lyophilized and suspended in a vehicle suitable for injection.

A number of years ago, it was widely believed that rheumatoid 10 arthritis had an infectious etiology. This view is not well considered today, although the inflammatory aspects and essential manifestations of rheumatoid arthritis - synovitis and granulomatous lesions, fever, tachycardia, leukocytosis, lymphadenopathy and occasional splenomegaly, the accelerated erythrocyte duct and other sedation disorders "acute-phase reactants" - all compatible with an infectious process. Competent and repeated bacteriological studies have not been able to recover in a consistent manner a single infectious agent from blood, synovial fluid (articular fluid), synovial tissue (articular tissue) or subcutaneous nodules (subcutaneous nodules). Attempts to transmit the disease by injection of joint fluid from patients with rheumatoid arthritis into the joints of other human subjects have been unsuccessful. Subcutaneous nodules have been unable to survive after a homologous transplant (Bauer, W., Clark, W.S. and Dienes, L, The Practitioner, 166: 25 (1961)).

An infectious process seems to promote the onset of the disease in rheumatoid arthritis in a significant number of patients and can exert a detrimental effect on the course of the disease when it has already occurred. There is statistical probability of this clinical impression (Lewis-Fanning, E., Ann. Rheum. Dis. Supplement 9 (1950)).

Many attempts have been made to produce a disease in animals similar to rheumatoid arthritis. Although a number of different bacteria can produce arthritis in animals, they are unable to reproduce the clinical and pathological features of rheumatoid arthritis, in particular the persistent characteristics of proliferative arthritis. An arthritis that bears some resemblance to the human disorder has been produced in mice with pleuro-pneumonia-like

DK 158138 B

2 organisms (Sabin, AB, Science, 89: 228 (1939)) and in pigs with Erysipelothrix rhusiopathiae (Sikes, D., Neher, GM, and Doyle, LP, Am. J. Vet. Res. 16 (1955) ). It has been postulated that these organisms initiate a hypersensitivity mechanism (Sikes, 5 D., Neher, G.M., and Doyle, L.P., Am. J. Vet. Res. 16 (1955)).

Researchers of rheumatoid arthritis nonetheless continue to be captured by certain reappearing themes that presume links between infections and joint disorders. Eg. is gonorrhea capable of producing not only typical gonorrheal arthritis but also occasionally initiating a chronic arthritis that develops into typical rheumatoid arthritis. There are no statistics on the frequency with which this occurs, so you cannot know how much weight you can put on the context. Tonsillitis (inflammation of the 15 tonsils) or pharyngitis (inflammation of the pharynx) may also be followed by a polyarthritis, which at first appears to be rheumatic fever, but which develops into rheumatoid arthritis. Acute viral infections, especially rubella in young women, may be followed by persistent polyarthritides involving the small joints as well as the large ones; These arthritides usually have several months of persistent joint resemblance to rheumatoid arthritis before gradually subsiding.

Although chronic infection with an unknown agent remains a popular assumption as the etiology of rheumatoid arthritis, no published data exists to support this presumption. Some of the researchers of the disease have the presumption that if infection is a factor, it may not be an infection with a specific type of microorganism, but rather an infection with a broad spectrum of banal microorganisms in connection with a changed host response produced by the infections. who are responsible for the disease. The present invention is based on this theory of the origin of rheumatoid arthritis.

Although an infectious etiology has never been established, 35 of several recent discoveries seem relevant in support of this theory.

Some of these are the following:

DK 158138 B

3 1. Patients with rheumatoid arthritis have lower IgA values than normal; IgA is the class of immunoglobulins found in the secretions of the gastrointestinal tract.

2. Immunoglobulin A produced in response to immunization via the salivary glands is found in serum colostrum and milk as well as in saliva. It is believed that IgA is transported to these various fluids via the gastrointestinal tract and the lymphatic system (Michelok, S.M., Rolman, A.F.R., and McGhee, J.R., Proc. Soc.

10 Exptl. Biol. Med .. 148; 1114 (1975)).

3. Following a bowel resection operation in morbus obesicas, some patients develop symptoms that are actually identical to rheumatoid arthritis. The onset of arthritis is accompanied by the emergence in blood serum of circulating cryoproteins, which are proteins that precipitate or gel upon standing at refrigerator temperature, sometimes even at somewhat higher temperatures, and again dissolve at 37 ° C composed of IgG, IgM, IgA, complement components C3, C1, C5 and IgG antibody to E. coli and 20 B. fragile ice. Removal of the intestinal tract results in the complete disappearance of the symptoms (Woods, J.R., La Mont, J.T.,

Maur, E., Isselbacher, K. J., New Enol. J. Med .. 294: 121 (1976)).

4. A type of Diplostreptococcus agalactiae belonging to the streptococ group B has been implicated as an etiologic agent in rheumatoid arthritis (Svartz, N., Acta. Med. Scand., 192: 231 (1972)). This streptococ is present in most commercially available pasteurized milk but not in immune milk.

30 5. A predisposition to rheumatic disorders appears to be hereditary via the histocompatible antigens (HL-A). These antigens are likely to determine the host organism's response to infectious agents.

On the basis of these indications, it is concluded that rheumatoid arthritis has an infectious origin, that the infection takes place in the digestive tract, that a number of different bacterial strains are involved in the infection, that the infection is likely to occur as a result of an infection.

DK 158138 B

4 defects in the host organism's immune defense mechanism, and that the most effective way to treat the disease is to reestablish immune protection against the infectious agent in the digestive tract.

Basically, there are two ways that can be used to treat infection: the immune method, which involves either active or passive immunization against the infectious pathogen, and the use of antibiotics such as penicillin, tetracycline, ampicillin, and the like. Antibiotics are not specific in their effect and they kill a wide spectrum of 10 beneficial as well as harmful bacteria. On the other hand, the immune method is highly specific. Bactericidal antibodies produced against a specific bacterial strain react only with this strain and have no deleterious effects on other types of bacteria. In addition, antibodies - as opposed to antibiotics - are natural body products that do not have 15 known side effects. Since the object of the invention is to control infection with a specific bacterial group without damaging beneficial bacteria in the digestive tract, the immune method is the method of choice.

There are two different methods of obtaining immune protection.

Active immunization is a method by which the host organism is actively immunized with a vaccine that stimulates the host organism's immune system to produce protective antibodies against factors contained in the vaccine. Active immunization occurs under natural conditions when the host organism is exposed to infectious pathogens.

Passive immunization is a method by which antibodies obtained from one subject that has been actively immunized are given to another subject. In this method, the protective antibodies are transferred from the immune host organism to the recipient. Passive immune protection is temporary and only lasts as long as the passively acquired antibodies continue to be present in the recipient's organism.

Eg. For example, antibodies obtained from horses that have been immunized against tetanus toxin can be given to humans infected with tetanus to provide a temporary immune response to the toxin produced by tetanus bacteria.

In the disclosure of a previous patent (U.S. Patent No. 3,626,057), a process for the preparation of tetanus antitoxin in milk is described. This patent teaches that the cow can be actively immunized against

DK 158138 B

5 tetanus toxin, that antibodies produced by the cow against the toxin can be obtained from the cow's milk, and that these antibodies can be used to treat animals that have been infected with tetanus bacteria in such a way that the antibodies neutralize the toxin. The patent teaches that the passive administration of antibodies neutralizes the life-threatening toxin produced by the bacteria, thereby providing temporary immunity to the toxin.

Passive immunization differs from active immunization in that the immune protection is temporary and only lasts as long as the protective antibodies are present. Active immunization is more permanent because the host organism's immune system continues to produce protective antibodies in the presence of the stimulating antigen.

15

Recent studies in the field of digestive tract immunology have shown the existence of a local immune mechanism in the digestive tract. This immune system in the digestive tract produces a special type of antibody that acts to control bacterial 2o attacks in the digestive tract cavity. The antibody, called secretion immunoglobulin or IgA, is produced in response to local, active immunization of the digestive tract mucosa with the antigen. The secretory immune system of the digestive tract prevents the colonization and propagation of harmful bacterial species in this environment. It is believed that failure of the local immune system in the digestive tract allows unknown, harmful bacteria to establish and that these bacteria cause rheumatoid arthritis. According to this theory, rheumatoid arthritis arises from a failing ability of the local immune system in the digestive tract to produce and 3Q secrete protective antibodies against harmful bacteria. Thus, the inability of the host organism to respond to active immunization precludes this method as a method of treating rheumatoid arthritis.

Thus, the present invention relates to a method of immunoglobulin IgG-containing milk or derived product for the treatment of rheumatoid arthritis, using a vaccine prepared from cultures of bacterial strains grown in appropriate culture media, after which

DK 158138 B

6 the bacteria have been killed, harvested, washed, lyophilized and suspended in a vehicle suitable for injection, the method being characterized in that the vaccine prepared from bacterial cultures of the following microorganisms: 5 ORGANISM ATCC NO.

Staphylococcus Aureus 11631

Staphylococcus epidermidis 155

Streptococcus pyogenes, A. type 1 8671 10 - type 3 10389 type 5 12347 type 8 12349 type 12 11434 type 14 12972 15 - type 18 12357 type 22 10403

Aerobacter aerogenes 884

Escherichia coli 26

Salmonella enteritidis 13076 20 Pseudomonas aeruginosa 7700

Klebsiella pneumoniae 9590

Salmonella typhimurium 13311

Haemophilus influenzae 9333

Streptococcus viridans 6249 25 Proteus vulgaris 13315

Shigella dysenteriae 11835

Streptococcus, group B Diplococcus pneumoniae Streptococcus mutans 30 Corynebacterium, Acne, types 1 and 2, are injected intramuscularly weekly into healthy cows for four consecutive weeks and then twice monthly for each injection.

ISLAND

20 x 10 bacterial cells comprise the milk from the immunized cows 35 from the beginning of the fourth week collected and tested for titers to ensure that the minimum titer of antibody to each of the vaccine-containing bacteria is 1-500 determined by the reagent gias agglutination test for antibody titers .

DK 158138 B

7

The immunoglobulin type is important to consider for the patentability of the invention, as there are five known immunogl obul in classes designated IgG, IgM, IgA, IgD and IgE (Nisonoff, A., Molecules of Immunity in Immunobiology. (Editors Good RA and 5 Fisher, W.) Sinauer Associates, Stamford, Connecticut (1971)). Each immunoglobulin type differs structurally (Waldman, T., Blaese, R. M. and Strobe, W., Plasma Protein Metabolism. (Editors Rothschild, M. and Waldman, Academic Press, p. 269 (1970)) and has a different biological function in body (Waldman, T., Immune Mechanisms on 10 Secretory Surfaces, Post Grad. Med. 50: 78 (1971), and Franklin, EC, Proo. there is a striking variation in the location of immunoglobulin in the bodies of the body, for example, the distribution clearly separates immunoglobulin in the classes IgA and IgG, the most striking feature of IgA is its high concentration in the body's external secretions, including the gastrointestinal tract. It has been clearly demonstrated that the immune system that supplies IgA to the gastrointestinal tract juice is a distinct system different from that which produces IgG.

In humans, IgG is found primarily in the vascular and intracellular compartments of the body (Waldman et al., 1970 (see above)), and very little IgG finds its way into the gastrointestinal tract fluids. Another important difference between the immunoglobulin classes relates to their metabolic rate.

Regardless of its localization in the body, the degradation of each 25 immunoglobulin class appears to be under separate control. The functional catabolic rate ranges from as low as 6.5% for IgG to as high as 90% for IgE and with other immunogl obul in n classes located therebetween (Waldman et al, 1970 (see above)). In addition, the different immunoglobulin classes also differ in their eagerness to bind to antigens and in their ability to combine with the compliment, which is one of the elements necessary to kill living bacterial cells (Heremans, JF, Les Golbulenes Serioues du Svsteme Gamma Leur Nature et Leur Patholoqie.

Arscia, Brussels & Masson, Paris (1960)). It is important to emphasize these differences in antibody types, as immune effects may vary depending on the type of antibody involved.

The most commonly accepted theory is that the various immunoglobulin classes have evolved to function in different environments 8

DK 158 138 B

in the body. It is e.g. It is known that a special and distinct immune system exists for the production of antibodies that act in the digestive tract environment. Furthermore, there is general agreement that the immune functions of the digestive tract are controlled specifically by IgA-5 antibodies and not by IgG. Therefore, under natural conditions, IgA is the n-class immunoglobulin that regulates the immune control of bacterial infections that occur in the gastrointestinal tract of man. Since IgG, IgM, IgD and IgE are not normally found in the intestinal secretions, it is not logical to expect that these antibody types would be effective in treating infections in the digestive tract environment.

The major immunoglobulin in cows' milk is IgG and not IgA (Sullivan, AL, Pendergast, RA, Antunes, LJ, Silverstein, AS, 15 and Tomasi, Jr., TB Jour, of Immunol., Volume 2, page 103, ( 1969)).

Therefore, bovine milk is not a nearby source of antibody to treat bacterial infections of the human digestive tract due to its high concentrations of IgG and low concentrations of IgA.

20

The immunization method is another important parameter to consider in the various immunoglobulin classes. It is well known to those skilled in the art that various immunization methods result in the preferential production of various antibody types. Local immunization of secretory tissue obtained by exposing the tissue to antigens stimulates, e.g. the preferential production and secretion of IgA-type immunoglobulins. The technique of intra-mammal perfusion, as described in U.S. Patent No. 3,376,198, is an example of local immunization. This method stimulates the production and secretion of IgA antibodies and is not an effective method for producing IgG.

According to the present invention, intramuscular injection is used in the method of the invention, since IgG is the main immunoglobulin in cow's milk, not IgA, and in the cow, and systemic immunization is the preferred method for producing IgG-type antibodies in milk. This distinction between immunoglobulin of IgG and IgA type is important as it teaches that systemic immunization and not local immunization is the preferred method of obtaining milk.

DK 158138 B

9 with high antibody titer. This distinction further teaches that the immunoproducts produced by the mammalian perfusion of a vaccine are distinctly different from the immune product produced by intra-muscular injection with the identical vaccine. Thus, the product (IgG antibodies) produced by the method of the present invention is clearly different from the product obtained by the method of the above-mentioned United States patent.

The immune product produced by the method of the present invention is an improvement over the product of the invention in the above-mentioned United States patent, since the concentration of IgG type antibodies is substantially higher than the IgA type antibody concentration. There is no evidence in the literature to support the claim that IgG antibodies can be produced by intramammal perfusion of antigens. Furthermore, since the extent of IgA immunoglobulins is either non-existent or extremely low in cow's milk, it is absurd to suggest that the teachings of the above-mentioned United States patent have any relevance to the claim of the present invention. In contrast, the teachings of the aforementioned United States patent do not depart from the discovery of the present invention since it assumes that IgA is a biologically active factor in cow's milk with potential therapeutic utility.

In the method of the present invention, a vaccine prepared from a unique combination of bacterial species is used which is given to healthy dairy cows. The immunoglobulins obtained from the milk from the immunized cows can be used for passive immunization of a patient by oral administration of the immunoglobulin which passively immunizes against a mixed spectrum of infectious bacteria present in the gastrointestinal tract. This treatment eliminates conditions of the gastrointestinal tract which cause rheumatoid arthritis.

The following is a brief description of the drawing, 35 in which:

FIG. 1 is a copy of a questionnaire discussed in the specification,

DK 158138 B

10

FIG. 1a is a continuation of the questionnaire of FIG. 1, and

FIG. 2 are curves showing the results of the test expressed by RF titers plotted against time over a 12 month period, 5 6 months on immune milk and 6 months on placebo.

In one embodiment of the method of the invention, a low-fat powdered milk is prepared containing a population of natural IgG-type antibodies that react with the bacterial species listed in Table 1.

TABLE 1

Bacterial Antigens ORGANISM ^ ATCC NO.

Staphylococcus aureus 11631

Staphylococcus epidermidis 155

Streptococcus pyogenes, A. type 1 8671 20 - type 3 10389 type 5 12347 type 8 12349 type 12 11434 type 14 12972 25 - type 18 12357 type 22 10403

Aerobacter aerogenes 884

Escherichia coli 26

Salmonella enteritidis 13076 30 Pseudomonas aeruginosa 7700

Klebsiella pneumoniae 9590

Salmonella typhimurium 13311

Haemophilus influenzae 9333

Streptococcus viridans 6249 35 Proteus Vulgaris 13315

Shigella dysenteriae 11835

Streptococcus, group B Diplococcus pneumoniae Streptococcus mutans

DK 158138 B

π

Corynebacterium, Acne, Type 1 and 2 x) American Type Culture Collection, 12301 Parklawn Dr., Rockville, Md. 20,852th

5

The antibacterial milk contains all the substances normally found in low-fat powdered milk. The main constituents constituting the antibacterial milk are shown in Table 2.

TABLE 2

Quantitative and qualitative analysis of antibacterial milk

Proteins 35.6% Fat 1.0%

Carbohydrates 52%

Minerals 7.8%

Humidity 3.5%

Each redissolved liter of 90-120 grams of 20 non-fat dry milk contains approximately: 1200 mg calcium 157% 935 mg phosphorus 125% 0.3 mg thiamine 32% 1.78 mg riboflavin 140% 25 M4 m9 niacin 10% 324 calories

Antibacterial milk and normal cow's milk contain about the same constituent concentrations by weight. In addition, the IgG-type immunoglobulin concentration in antibacterial and normal milk is identical. Therefore, it is only the specificity of the antibodies contained in the antibacterial milk that differentiates it from normal milk. By the specificity of the immunoglobulin is meant the spectrum of bacterial species with which the antibodies react.

Antibacterial milk contains no drug additives or any other components that are not natural cow food products.

35 n DK 158138 Β

The immune milk produced by the method of the present invention is also useful for controlling autoimmune disorders, e.g. lupus erythematosus o.1., which is caused or exacerbated by bacterial invasions of the gastrointestinal tract.

5

The polyvalent antigen used to produce antibacterial milk is prepared as follows:

Preparation of the vaccine 10

The bacterial strains listed in Table 1 were obtained from the American Type Culture Collection, which ensures the authenticity of the bacterial strains and the highest standard of purity available. Upon receipt, each bacterial strain was grown on a bl od agar plate to study the viability of the culture and to determine whether the growth pattern was typical or atypical of the bacterium in question.

A single colony from each of the study cultures was sampled for histological examination to further ensure the authenticity and purity of the culture. A single colony from each culture was used to inoculate 500 ml of standard culture medium. Recommended media were used by the American Type Culture Collection to grow each of the specific bacteria listed in Table 1.

All organisms were incubated as static cultures except 25 of the 12th, 13th, 14th and 16th bacterial strains of Table 1 which were incubated in a shaker to provide shaking. The identification of the bacterial strains and the American Type Culture Collection catalog numbers are shown in Table 1. Each culture was grown for 48 hours at 37 ° C. After the incubation, the cultures were killed by heating 30 to 60 ° C for two hours. Samples of the killed bacteria were used to inoculate fresh culture medium, which was then incubated for 24 hours at 37 ° C to determine the killing process was complete.

Only cultures that had been sterile in this procedure were used for further treatment. Sterile cultures were then washed 5 times in distilled water and the cells recovered by centrifugation. The bacterial cells were frozen by immersion in liquid nitrogen and lyophilized by the lyophilization method. Lyophilized cells were stored in sterile vials until used to produce the polyvalent vaccine. The polyvalent

13 DK 158138B

vaccine was prepared by weighing 1 gram of each bacterial strain. The dry cells were mixed and the mixture was suspended in sterile physiological saline (20 grams of bacteria per 500 ml of saline).

5

A sample of the concentrated solution was serially diluted with saline to determine the dilution yielding a concentration of 4x10 cm. The concentrated polyvalent vaccine was distributed in a number of containers and stored frozen.

10 A sufficient amount of concentrated antigen was included in each container to immunize 50 cows. The final dilution of the concentrate was performed just prior to immunization.

The preferred method is to remove a sufficient number of small bottles to immunize the number of cows to be treated. For example, the 15 small bottles are removed. 24 hours prior to the scheduled immunization time; a sample of the concentrate is then diluted in one

Q

sterile container to a final concentration of 4x10 cells per cell. ml.

ISLAND

The maximum response in cows is achieved by injection of 20x10 bacterial cells or 5 cm of the sterile preparation, which is 4x10 20 cells per cell. ml, according to the immunization method described below.

Preferred method for immunizing cows

The antibody product prepared by the method of the invention 25 is produced by immunizing cows with the polyvalent antigen 3 prepared as described above. The cows are injected 5 cm

ISLAND

valent antigen containing 20x10 bacterial cells. The injection is done intramuscularly into the gluteus maximum muscle of the hind leg. This procedure is repeated at 1 week intervals for 4 consecutive 30 weeks starting 2-3 weeks prior to the predicted calving date.

After the primary immunization, booster injections are given every 14 days using the same antigen concentration. This immunization method yields maximum antibody titers.

35 Collection, transport and treatment of the milk The milk from immunized cows is collected in a modern milking room. A fully automated milking system collects and stores the milk under perfectly hygienic conditions. The milking system consists of

DK 158138 B

14 automated machines connected to a refrigerated storage tank with a closed piping system. The entire system is cleansed and sterilized after each milking to ensure maximum hygienic conditions. It is important in the treatment of immune milk to take careful precautions to prevent bacterial growth, as these bacteria can decrease the antibody titers of the milk.

The milk is transported daily with milk transport trucks from the refrigerated storage tanks to a milk processing plant. At the milk processing plant, a high temperature short-time system is used to pasteurize the antibacterial milk. In particular, milk processing machinery provides the flash heating of a continuous flow of milk to 68.3 ° C for a period of not more than 15 seconds. Temperature and time are critical since antibody is heat-degrading sensitive. Milk antibody is destroyed at temperatures above 73.9 ° C if kept for more than 1 minute. After pasteurization, whole milk is immediately cooled, the fat is removed by centrifugation and the foamed antibacterial whole milk is transferred into powder form by a spray drying process. The spray drying process is carried out in a large drying chamber in which hot air (176.7 ° C) is blown at high speed. The foamed milk is atomized into the chamber, where the finely divided milk particles are immediately dried while falling to the bottom of the chamber. The dried milk is automatically removed by mechanical tools and the milk powder is packaged under hygienic conditions. Prior to atomization, the skimmed milk is concentrated by boiling in a chamber under vacuum (37.8-43.3 ° C). At each stage, it is critical to keep the milk free of bacteria contamination as this decreases the antibody titre.

30

testing procedure

Immune milk was prepared with inbred Holstein cows. The cows were immunized by intramuscular injection with a mixture of the bacterial antigens identified in Table 1. The vaccine was prepared by the method described above. The cows' immunological response was accelerated by two weekly injections with the vaccine. The milk from these cows was collected, the fat removed and the non-fat milk pasteurized by exposure to 71.1 ° C for 16 seconds.

DK 158138 B

15 followed by a spray drying process at which the temperature of the milk did not exceed 29.4 ° C. The milk was packaged in "quarter" polyethylene containers. Control milk (placebo) was non-fat powdered milk purchased from a local manufacturer.

5

Erythrocyte sedimentation rates were determined on freshly collected blood by the method of Westergren and corrected for hematocrit according to the method of Wintrobe, M.M. and Langsberg, J.W., Am. J. Sci., 189: 102 (1935). Rheumatoid factor titer values were determined by Singer, 10 J.M. and Plotz, C.M., Am. J. Med .. 21: 888 (1966) macroscopic reagent tube tests.

Patients for the study were accepted on the basis of an elevated erythrocyte sedimentation rate and a positive rheumatoid 15 factor titer. Nine patients were studied for 12 months and 11 patients were studied for 18 months. The patient group was composed of 13 women of the Caucasian race with an age of 32 to 69 years with an average age of 50.4 years and of 7 men of the Caucasian race - with an age of 43 to 70 years and with an average age of 58, 1 year.

20 The mean duration of arthritis was 10.8 years for the women and 11.0 for the men. Patients were randomly treated with either immune or non-immune milk (a commercial product purchased in the Dayton area served as a placebo). Both milk products were packaged in identical containers and were identified as having 25 immune or placebo by a blue and red pressure-sensitive label, respectively, which was attached to each container at the time it was filled. The labels were removed just prior to delivery of the milk to the patients. Thus, at no time did patients know whether they were receiving immune milk or placebo. Patients were randomly selected (30 by coin toss) to receive either immuno-milk or placebo during the first 6-month period. At the end of this period, those receiving immune milk were placed on placebo, and those receiving placebo were placed on immune milk for the next 6-month period.

35

At the end of this second 6-month period, 11 patients volunteered to continue the study for another 6 months. At this point, the milk type (immune or placebo) was again changed and observations continued. The study included

DK 158138 B

16 thus three 6-month periods with 11 patients participating for three periods and nine participating for two periods.

Patients were consulted at monthly intervals, at which time 5 months of one month's milk supply was provided, an evaluation questionnaire was completed, and a blood sample was taken to determine rheumatoid factor titer, erythrocyte sedimentation rate and hematocrit.

Patients were instructed to take a quantity of non-fat milk solids equivalent to one liter of milk after food twice daily. The milk solids were freshly dissolved in ½ liter of tap water immediately before ingestion shortly after waking in the morning and again just before bedtime in the evening. They were instructed to consult their physician as usual and to follow the prescribed treatment regimens prescribed by him. Medications could be taken ad libitum or as prescribed by their general practitioner. They were only asked to report the amount of medication taken.

20 A questionnaire was completed by each patient at monthly intervals.

It was divided into six sections which dealt with: 1) Duration of morning stiffness, 25 2) The degree of pain occurring in each of eight joints, 3} Type and amount of short-acting medication taken, 4) Type and amount of medication taken with long-term effects, 5) the patient's ability to perform his normal activities, and 6) the degree of symptoms of rheumatoid arthritis.

35

The numbers shown in the boxes after each answer indicate the value added to the answer when evaluating the questionnaires. In the point allocation, it was emphasized that the sections dealing with the medication reflected the relative anti-inflammatory and

DK 158138 B

17 analgesic effect of the various drugs used. A 324 mg aspirin tablet was added to the value one. All other drugs (with the exception of gold, Plaquenil® (trademark of hydroxychloroquinolyl sulfate) and cortisone injections rated 5 separately) were arbitrarily added to aspirin values. Thus, all salicylate preparations, Tylenol®, Darvon, Motrin®, etc. were considered equivalent to a 324 mg aspirin tablet and were also assigned the value one. The number of mg Prednisone was multiplied by four, the number of Indocin capsules taken was multiplied by 10 2.5. For each 64.8 mg of codeine, multiplied by two and the number of butazoladine tablets taken was multiplied by seven.

The average of the points awarded in each category was calculated for 15 every 6-month period. The difference between the mean values was then calculated by subtracting the mean values obtained during administration of immune milk from those obtained during the administration of placebo. When the results were calculated in this way, an improvement in the patient's condition during the period he received immune milk was indicated by negative values for questions 1 and 6 and by positive values for all other questions. Mean corrected erythrocyte sedimentation rates (ESR) and rheumatoid factor titers (RF) were shown in a similar fashion, respectively. They were calculated in such a way that positive values reflected a lower erythrocyte sedimentation rate or rheumatoid factor titer during administration of immune milk. The data were processed statistically using Goodnight et al's "Statistical Analysis System" (SAS Institute, Raleigh, N.C.). The calculations were performed using an IBM model 370/155 computer.

30

results

Immune milk was well tolerated by all patients except one who had pernicious anemia. This patient complained of diarrhea and 35 were left out of the study. Some patients reported a weight gain during the study. This may be due to the increased caloric intake from the milk or may reflect a general improvement in their physical condition.

18

DK 158138 B

«—I oi — i o · —i w in r>. o m co oo

CM »-) i I pH C 1 i-I CM pH O hn CM

o «3-ΙΛΟΟΟΟ · —IO M- CM O

0-1 O OOOOOOOO O O O

«S Λ Λ V» O OOOOOOOO o o o <u cj c · - * id) rv. co cn in a co <—i cm σι oo * - <o, - S_ -M- COOOOO'M-OCMCMp — i "M" CO m

cu CU ΓΟ CM r — i CM CM CO CM * - * CM «- <O CO

T5 M- * Λ .. ·. · * «. ·.«. · - * · * "~

X) <+ _ O OOOOOOOO O O

-Γ - Ι— i + + + + + + + + + + + +

ZE XJ

. co cm cn co in o o i— σι co co p— <> lo olom-colocdloo i — i m o o co cocor ^ incoincoin o i— cm · C i — I '—i' - * 3

E

£ i— <(- cu cn locotnwsnoioi m m- m- s_ xj r-. I — ii — i co cm cm co m cm * -h m- f ~ - co rs -O co i-vpioincocMiocor- '. M cm co

_ I W ZEL O OOOOOOOO CO o p-H

lu σ 1-1 co c: <C ·> - I— r—

"ISLAND

co x r-. i — irH.cor ^ cO' — icni ^ p · - <i— (¾ O · «s« * »* **

r— dj> LD r ^ COCOM-O-M-COCO «d- O CO

cu o · cn 10001--1001--1010 o m- co CO <T3 O p-i i — i SI «—i r— cm 'iCMctnfO'iHCO co m Μα; co inincMt -'- coocOM- co cm cm ό co or-.ooomor - o co co cm "O - --------- - - -

-r- o ooo · —lOOOO o o CM

s: cm

3 M-M-M-M-M-M-M-CMCM µ- µ- ΜΕ CM CM CM CM CM CM CM CM CM CM CM CM

in E

as S- t — t> cu

S- XJ

O) o CO -i— i— JO i- o o <D S- r- »t-— C-. Γ ~ »I—. S CO CO I— I ''

Q + J CM CM CM CM CM CM CM CM CM CM CM CM

i— on c ccf c o + -> o c -r- C + J xs £ = cu -1- J = o> s- z:

• I- CU XJ

+ J + J <y

<Λ S- S- E

c: cu cu S- ί-

αι EXJCU4-> CU I— cu CU C X

σ Ml— 3C0X3 + J CU XJ r— CU XI

s_ xioo ^ c't-a ^ x> - XJ —j

O cu-Χι— S- · Π3 O C COl ·! - £ = Q

s: um <> xijc: <u- ci- <* = c, -h CM COJO OT3 CU <+ I cn £ CO M- in

DK 158138 B

19

CO

CSJ «3- 10 CO OO

«D * CM i — I l ~ - CO

O O O CO UO

o o o r · ^ cn CCS

"^ Fs c r * I-I

ooooo cn • i— U-> · <-} »d · CTl CO r-1 tO I— <i (O cn so ns ro cm i — i co * -> + fooooo <e III +> + - * C * 1-

ISLAND

CL

i-i «d- CO cu r ~ ^ oo„ c 'd- cm ro CO — i <ut— (i— (* —t CO "ί- 00 ρ | g, c σ> oo ^ I— cm" d · oo ry t ~ - «d · r ^. cm ro S-

0 ^ d * LO «—t cn 00 <U

LL- CM CM 1—1 ~ Ό li__ Λ a * »LO LO L- CM CM CM CO 3 co -g

_I CD

LU

CO + ->

<C 00 00 CD I— on CCS

1 n a i> i · ii l — L

i — i oo t— cn lo CM i — li — I CM "d" cj U- ro tn "d * co oo Ό O 00 CM CD cn 3

CD CD CD CM CD

r> fi r * c »c% + J

i-I 1 (i-i CO CO 01

CO C

cn

CD

s-

CD JO

* d "M" "d * ro 00

CM CM CM CM CM

+ -> tu

C TO

(U c • r— • ΙΟ </) • i—

Cfj i— I — i LO h "« 4-CD

C CM CM CM CM CM M- + -> -r- CU · ι- S- o> -O ^ -r- C 00 + -> to c: .x

S- O CO

o · <“<4- + -> CJ> <0 '·! - D" Γ- 1—

• I- S- T5 (U S- CD

i— (U CU 00 Λ CCS

Ό + -> J = i—> Q

<U S-> tu

C CU · * -> CC \ II II

c (d ε 4- »ft) CO I Lul

s: co co n: lu | α: In

. . . c C c X

CO nj JO O t'-. 00 XX

DK 158138 B

20

As shown in Table 3, patients were observed over a total of 27 control periods (6-month periods during which they received placebo) and 24 test periods (6-month periods during which they received immune milk). One patient had a physical lesion of one ankle and foot. The pain 5 of these joints was not evaluated, which explains the smaller number of evaluation periods for these joints. The erythrocyte sedimentation rates of one patient were so extremely abnormal (deviating by more than two standard deviations from the average of the values of the other patients) that they were not included. This omission explains the 10 smaller number of reported observations for this variable.

The mean values and coefficients of variation (C.V.) for each variable are listed in the table. The differences between the mean values were calculated by subtracting the mean values obtained during the periods in which patients received immune milk from those obtained during the periods when they received placebo. A favorable response to immune milk is indicated by negative values for morning stiffness (question 1) and measurable change (questions 6a, b and c) and by positive values for all other variables. An effective response to 20 immune milk was obtained for all the data obtained from the questionnaires. The probabilities (P) indicate a high degree of statistical significance in each case. The small mean difference obtained for erythrocyte sedimentation rate and rheumatoid factor titer was not significant. However, when the erythrocyte sedimentation rates were assessed on an individual basis, four of the 20 patients studied had statistically significant reductions while receiving immune milk.

Although immune milk had no significant effect on the mean values for rheumatoid factor titers, a study of the individual patients revealed interesting responses. Seven of the 20 patients studied had negative rheumatoid factor titers on at least one occasion during the period when they received immune milk. Four of them became negative during the period in which they received immune milk and their titers did not become positive again during the following 6-month period during which they received the control milk (placebo) as shown in FIG. 2. Continuing the study past this reporting period reveals that of 13 of 25 patients lost the rheumatoid factor in their blood.

DK 158138 B

21

Discussion

The scientist who chaired this study personally interviewed each patient at monthly intervals and wrote down their 5 answers to the questions. Every effort was made not to influence patients' responses. Patients were initially informed and were often reminded that during certain periods of the study they would receive a placebo. It was assumed that this knowledge would serve as an incentive for patients to answer the 10 questions objectively and without tendentious bias. Patients were not informed at any time whether they were receiving immune milk or placebo.

The question related to the medication "yesterday" (Q. 3) and the question regarding gold, plaquenil and cortisone injections (Q. 4) are objective and are of primary importance in the assessment of the answers given to the other questions. These issues are important for two reasons: 20 1) If the immune milk is effective in relieving the symptoms of the disease, it is to be expected that the patient will take fewer of the drugs that were allowed ad libitum. On average, patients reported taking four fewer aspirins or equivalents per day. per day during the periods when they received 25 immune milk. They also reported that they received fewer gold injections, plaquenil and cortisone injections during these periods, and 2) if patients took smaller amounts of analgesics and other 30 medications useful in the treatment of rheumatoid arthritis, one would expect that they reported increased discomfort unless the immune milk affected the disorder favorably.

35 As indicated in Table 2, significantly less joint complications were reported during the periods when patients received immune milk, even though they were taking fewer medications for their arthritis.

Patients began the study at monthly intervals i

DK 158138 B

22 over a 1-year period, and the type of milk product (immune milk or placebo) that they initially received was given at random. The observation that positive response or improvement was obtained for all questionnaire parameters, and that these mean responses were statistically significant, strongly indicates that immune milk had a beneficial effect on patients. This finding is supported by the observation that 20% of patients showed a statistically significant (p <0.05) decrease in erythrocyte sedimentation rate while receiving immune milk.

10

The results of the rheumatoid factor titers are difficult to judge. This is due, at least in part, to the fact that the origin and role of the rheumatoid factors in the etiology and prognosis of rheumatoid arthritis are not understood. Rose, H.W., Rogan, C., Pearce, E.

15 and Lipman, M.O., Proc. Soc. Exo. Biol. Hed. 68: 1 (1948) showed that red sheep blood cells that had been sensitized to rabbit antibody underwent agglutination in the presence of blood serum from rheumatoid arthritis patients. The test depends on the specific reaction between normal immunoglobulin (either rabbit or human IgG) and 20 rheumatoid factors. The specificities exhibited by rheumatoid factor are like those; that would be expected from antibody to IgG (Epstein, W., Johnson, A.M., and Rogan, C., Proc. Soc. Exd. Biol. Med. 91: 235 (1956)). The incidence of rheumatoid factors has been correlated with disease severity in rheumatoid arthritis and can be identified in 25 proteins that are precipitated in tissues in rheumatoid arthritis patients. Although a small percentage of rheumatoid arthritis patients do not have positive rheumatoid factor titers, most rheumatologists generally agree that positive agglutination reactions do not return to negative, even when the disease is in remission. DeForrest, G.K., Mucci, M.B. and Biosvert, P.L., Arth. & Rheum. When an outbreak of the disease occurred, the test became positive again. Aho, K., Kirpila, J.

35 and Wayner, 0., Ann. Exd. Fenn. 37: 377 (1959), however, noted that most patients whose disease had become inactive remained serologically positive. The fact that negative titers were observed in 60% of patients in the present study and that in half of these titers remained negative for six months,

DK 158138 B

23 evidence that immune milk affects a primary etiologic factor responsible for rheumatoid arthritis.

The soothing effect of the immune milk on the symptoms of rheumatoid arthritis is particularly relevant when considered in light of the recently described link between the histocompatibility anti-genes (HL-A) and susceptibility to rheumatic disorder (Brewerton, DA, Arth & Rheum .. 19: 656 (1976) Histocompatibility antigens are genetically determined antigens found on all human cells. The 10 genes that control their inheritance are called histocompatibility genes.

More than 40 of these genetically determined antigens are now known. They are responsible for rejection for tissue transplants performed between individuals other than identical twins. Superficially, the HL-A antigens are similar to the ABO-b1 types in that they are inherited for 15 lifetimes. Their function is not yet known, except in the highly artificial situation produced by transplantation. However, it is known that the histocompatibility genes are closely related to the immune response genes on the sixth chromosome. In this connection, they can determine the individual's immune response to a foreign invader, such as a bacterium.

People with HLAB27 appear to be particularly susceptible to a number of rheumatic disorders. It is postulated that this histocompatibility antigen dictates a type of immune response that, in the presence of other pre-disposing factors, leads to rheumatoid arthritis. After a bowel infection with versi nia enterocolitica, some patients develop acute peripheral arthritis (Ahvonen, P., Sievers, K. and Aho, K.,

Acta. Rheum. Scand., 15: 232 (1969)). Similarly, approx. 2% of patients after a Salmonella infection acute peripheral arthritis 30 (Warren, C.P.W., Ann. Rheum. Dis. 29: 484 (1970)). HLAB27 was found in 43 of 49 patients with yersinia arthritis and in 15 of 16 with salmonella arthritis (Aho, K., Ahvonen, P., Lassus, A. et al., Arth. & Rheum .. 17: 521 ( 1974)). The possibility is attractive that infectious agents can possibly thrive in the intestinal tract without giving rise to local symptoms. In patients with HLAB27, a host response results in arthritis. Thus, it is not necessary for the infectious agent to access the joints. Immune milk is beneficial in patients with rheumatoid arthritis because it contains antibodies that effectively

DK 15 813 8 B

24 inactivates or neutralizes harmful bacteria and / or their metabolic products.

10 15 20 25 30 35

Claims (6)

A process for the preparation of immunoglobulin IgG-containing milk or derived product for the treatment of rheumatoid arthritis, using a vaccine prepared from cultures of bacterial strains grown in appropriate culture media, whereupon the bacteria are have been killed, harvested, washed, lyophilized and suspended in a vehicle suitable for injection, characterized in that the vaccine prepared from bacterial cultures of the following microorganisms: ORGANISM ATCC NO. Staphylococcus Aureus 11631 Staphylococcus epidermidis 155 15 Streptococcus pyogenes, A. type 1 8671 type 3 10389 type 5 12347 type 8 12349 type 12 11434 20. type 14 12972 type 18 12357 type 22 10403 Aerobacter aerogenes 884 Escherichia coli 26 Salmonella enteritidis 13076 Pseudomonas aeruginosa 7700 Klebsiella pneumoniae 9590 Salmonella typhimurium 13311 Haemophilus influenzae 9333 30 Streptococcus viridans 6249 Proteus vulgaris 13315 Shigella dysentery ae 11835 Streptococcus, group B Diplococcus pneumoniae 35 Streptococcus mutans Corynebacterium, Acne, types 1 and 2 are injected intramuscularly into healthy cows weekly for four consecutive weeks and then twice monthly DK 158138 BO comprises 20x10 bacterial cells that the milk from the immunized cows from the beginning of the fourth week is collected and tested for titers to ensure that the minimum titer of antibody to each of the bacteria included in the vaccine is 1-500 determined by the reagent gias agglutination method of antibody titre assay. Process according to claim 1, characterized in that the collected immunoglobulin IgG-containing milk is converted into milk powder. 10 The method of claim 1, characterized in that the collected immunoglobulin IgG-containing milk is converted into a non-fat milk powder. Method according to claim 2 or 3, characterized in that the immunoglobulin in the milk or milk powder is recovered as pure antibody. Method according to one of claims 1-4, characterized in that the milk, milk powder or pure antibody is taken up in an orally administered vehicle which is not harmful to the antibody and which helps prevent the destruction of the immunoglobulin in gastrointestinal tract one due to the action of proteolytic enzymes and pH changes. 25 A method according to any one of the preceding claims, characterized in that the bacteria used in the vaccine have been killed by heat killing and have been recovered by centrifugation. 30 35