HU217541B - Pharmaceologically active material called bpc protein and process for producing moreover pharmaceutical composition containing them - Google Patents

Abstract

The present invention relates to a process for the preparation of a protein called BPC having a molecular weight of 40,000 ± 5,000 Da, an ultraviolet and an infrared spectrum as defined in Figures 1 and 2, and having a partial N-terminal amino acid sequence as follows: H2N-Gly-Glü Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leû-Val -...COOH, human or animal monoclonal fluid. The process of the present invention is characterized by: 1. centrifuging and collecting separated into supernatant; 2. the supernatant is dialyzed against distilled water and lyophilized; 3. the dry residue is greased with water or a weakly acidic acetate buffer and purified with a low-base or alkaline-bodied cornflower-filled chromium powder. 4. concentrate the precursor pump, re-dialyze it against distilled water and lyophilize; 5. the dry residue is reprocessed in a weakly acidic acetate buffer and purified by gel chromium chromatography and the azeula is dialyzed and lyophilized. The invention also relates to a protein obtained by the above process and to a pharmaceutical composition containing it. ŕ

Description

The scope of the description is 16 pages (including 2 tabs)

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Figure 2

EN 217,541 B

EN 217.541

H ₂ N-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-AspAla-Gly-Leu-Val -... COOH, human or animal weed.

The process according to the invention is characterized in that

1. the collected and homogenized animal or human weeds are separated by centrifugation into solids and supernatants;

2. the supernatant liquid is dialyzed against lyophilized water and lyophilized;

3. dissolve the dry residue in water or weakly acidic acetate buffer and purify on a chromatography column filled with a weak basic or basic ion-exchange resin;

4. concentrate the eluate, re-dialyse and concentrate the concentrate against distilled water;

5. The dry residue is redissolved in weak acid acetate buffer and purified by gel chromatography and the eluate is dialyzed and lyophilized.

The invention also relates to the protein obtained by the above process and to the pharmaceutical composition containing it.

BACKGROUND OF THE INVENTION The present invention relates to a novel protein called BPC and its production from human and animal gastric juice; and a pharmaceutical composition comprising this protein.

Gastric juice, which can be considered as a gastric and secretory secretory product, contains a wide variety of electrolytic components, hydrochloric acid and a series of enzymes, namely pepsin, other proteinases, rennin, lipase, urease, and lysozyme. There are various peptides and peptide fragments present, such as gastrin peptide hormones, which are highly potent gastric secretion stimulants first discovered by Edkins JS [Proc. Roy. Soc. L. 76B, 376 (1905)].

Glycoproteins (mucins) are also present in normal gastric juice, as described by Hanrowitz F. Chem. and Biology of Proteins 199 (1950)] and an "intrinsic factor" (IF), which is a heat-disruptive mucoprotein of about 60,000 molecules. This factor promotes B ₁₂ [Castle et al., Am. J. Med. Sci. 178: 748 (1929)].

So far no substance has been found in the stomach that has very strong anti-stress effects and other body protection activities.

The process of the invention is described below.

So far, the gastrointestinal system, especially the stomach, has been seen as the target organ of stress. It has never been suggested that the gastrointestinal system, preferably the stomach, can be an organ that can initiate the entire organism's defensive response. This response, which is actually directed against stress, can be realized in the formation and release of a new endogenous agent. Our attempt was to isolate this drug from the stomach. Finally, we expected to isolate the expected material from the weeds of 542 patients (healthy or pancreatic or mammalian animals). This material was named BPC, which derives from the English name of the "body protection compound" (herodic Proteiction Compound).

The structure of this material is very complex and after our investigations we can only characterize it as a "folded" (folded, non-linear) protein with the following partial sequence at the N-terminus: H ₂ N-Gly-Glu-Pro-Pro-Pro-Pro-GlyLys-Ala-Asp-Asp-Ala-Gly-Leu-Val -... COOH. Its molecular weight is about 40,000 ± 5,000 daltons, determined by gel filtration.

As described above, BPC is produced from human or animal weeds. This weed was first homogenized, centrifuged, the supernatant was purified by dialysis, ion exchange chromatography, re-dialysis, and then lyophilized to finally obtain BPC by gel chromatography, dialysis and lyophilization. More specifically, according to the present invention, it is

(1) separating the collected and homogenized animal or human stomach by centrifugation into solid and supernatant;

2. dialysis and lyophilizing the supernatant liquid against distilled water;

3. Dissolve the dry residue in water or weakly acidic acetate buffer and purify on a chromatography column filled with a weak basic or basic ion-exchange resin;

4. concentrating the eluate, dialysis and lyophilizing the concentrate against distilled water;

5. The dry residue is redissolved in weakly acidic acetate buffer and purified by gel chromatography and the eluate is dialyzed and lyophilized.

The above process steps may be carried out by methods well known to those skilled in the art.

In step 3, a weakly acidic acetate buffer is preferably used as a buffer, the pH of which will, of course, depend on the buffer material and its concentration. Preferably, sodium acetate buffer is used, for example the pH of the 1M sodium acetate solution is 5.22.

The gel used in Step 5 is well known in the art. Such a gel is preferably dextran, polystyrene, polyacrylamide, agarose, polyvinyl or other matrix based and has a fractionation capacity in the range of 20,000 to 100,000 Da. Examples include Sephacryl S-200, Sephadex G-100 and Sephadex 50/80 gels (Pharmacia Fine Chemicals).

Until now, this material has never been described or otherwise published. It was also not used as a medicine at any time.

Testing the biological effects of BP using a variety of in vitro and in vitro methods shows the following.

It is known that many diseases and pathological conditions can be triggered by stress or trauma or disease

It can induce stress itself. These results therefore suggest that BPC is produced by stress, regardless of the origin of the stress.

The BPC protein of the present invention was tested in vitro and in vivo (rat, mouse, rabbit and guinea pig) and has the following pharmacological properties.

1. Acute toxicity

Acute toxicology was determined in both sex mice (18-25 g body weight) (animals were killed after a 30-day experimental period); BPC was administered intraperitoneally (i.p.), intravenously (iv), and gastrointestinal (ig.). The lethal dose could not be achieved even at very high doses, for example 100 mg / kg body weight, which is at least 10 ³ - 10 ⁵ more than the minimum effective dose. In a long-term test session, BPC is ip., Iv. and ig. injected (30 mg / kg) once daily for 30 days. No pathological (morphological or physiological) signs were recorded. In addition, the average effective dose is between 0.01 and 50 pg / kg ip. at least 1000.

2. Stress-induced ulcer by immersion or imprisonment in water

Male Wistar rats (180-240 g) were used. Animals were placed in cold water (26 ° C) for 3 hours or held for 48 hours. At the end of the experimental period, the stomachs were examined for the presence of "stress ulcer". BPC ip. and injected via the gastrointestinal tract (0.1-10 g / kg) 1 hour or 24 hours prior to induction of damage. The strong protective effect of BPC was demonstrated for gastrointestinal damage.

3. An ulcer induced by binding the bile duct and the liver artery or by administering cysteamine

Binding experiments were performed on male Wistar rats (160-250 g). Binding of bile duct and liver artery was performed. This session also induces ulcers of the stomach and duodenum within 6-24 hours, in addition to severe ischemic gonadal damage to the liver tissues.

Cysteamine was also administered subcutaneously (400 mg / kg) in a similar manner to induce duodenal ulcer within 24 hours in male rats.

BPC ip. used at doses between 0.1 and 10 pg / kg. The formation of large ulcers induced by the described binding in the stomach and duodenum was severely restricted in the BPC treated animals. Similarly, BPC pretreatment also prevented the development of cysteamine-induced ulcers.

4. Effect on myocardial induced infarction

Wistar rats (180-230 g) were immobilized for 24 hours. Animals were treated with indomethacin (25 mg / kg, respectively) at time 0 of fixation and 12 hours later. BPC (30 pg / kg) was applied 1 hour before the start of the session and at 6 and 18 hours of fixation.

In the second group, the animals were sacrificed for 48 hours. Following these sessions, the animals were sacrificed and enzyme values determined.

The protective effects of BPC were recorded.

The protective effects were quite outstanding. In contrast, there was no protection against isoprenaline-induced cardiac disorders. Isoprenaline (30 mg / kg ip.) Was used at zero time and 24 hours later. BPC (50 pg / kg, ip.) Was used 1 hour before the use of isoprenaline. The animals were sacrificed 24 hours after the second isoprenaline administration and examined.

5. Effect on arterial blood pressure

Male rats (180-220 g) were used which were previously anesthetized with urethane. A cannula was introduced into the cervical artery and blood pressure was recorded on a dinograph. BPC iv. was administered via the cervical vein at a dose of 10 pg / kg to 5 mg / kg. The results showed no change in the arterial blood pressure of the animals in the group of rats prepared in the above-mentioned session. Noradrenaline, adrenaline, 5-HT, acetylcholine or isoprenaline iv. administration resulted in a brief change in arterial blood pressure. In animals pretreated with BPC doses of 10 pg / kg to 5 mg / kg, the arterial pressure changes induced by vascular agents were not altered.

6. Effect on damaged liver

The experiments were performed on male Wistar rats (180-250 g). Binding of the single bile duct and liver artery was performed. This intervention induced severe ischemic and fatal lesions in the liver tissues over a period of 6 to 24 hours. BPC ip. were administered at doses between 0.1 and 10 pg / kg. This has severely prevented (for example, one hour before bleeding) or has completely eliminated the damage (for example, injected directly after binding). In addition, we have demonstrated ip at 10 pg / kg body weight. the protective effect of the administered BPC liver in cases where it was administered one hour before the poisoning of rats, poisoning up to 0.1-10 ml / kg. or ip. induced carbon tetrachloride. BPC (0.01-10 pg / kg, ip./ig.) Prevented changes in liver fat composition induced by 48-hour tight fixation stress.

7. Effect on damaged pancreas

Male Wistar rats (170-240 g) were used. Damage to the pancreas was caused by binding the bile duct to the entry point of the duodenum. This session led to powerful pancreatic death in 24 hours and approximately 50% of the rats were killed within the experimental period. Within the range of 0.1-10 pg / kg ip. BPC was administered with a strong and dose-dependent protective effect. Significantly beneficial effects on survival were found (about 82%).

8. Effect on damaged kidneys

a) Unilateral kidney disease

Experiments were carried out on Wistar albino rats of both sexes (190-250 g body weight). One-sided kidney transplantation was performed. The increase in residual kidney weight was determined in control and BPC groups (10.0 pg / kg body weight, ip, 1 hour before the start of the session) 24 hours after surgery. Kimutat3

21 pins that BPC strongly reduces growth in the remaining kidney organ mass.

Biochemical parameters are similar in both groups. Therefore, BPC seems to improve the function of remaining kidney tissue.

b) Gentamicin administration

Experiments were performed on male Wistar rats. Damage to the tubules was caused by the administration of gentamicin (once a day) at 40 mg / kg i.p. for 30 days. After the last treatment, all rats were sacrificed. In the group of control animals treated with gentamicin and saline saline, tubular damage was observed. In the BPC-treated group, the pathohistological manifestations were significantly smaller.

9. Effect on experimental diabetes

Male Wistar rats (175-230 g) were induced by streptozocin or aloxane-induced diabetes mellitus. BPC at doses of 10-100 pg / kg ip. It was in a direction that delayed streptozocin-induced diabetes. In addition, BPC significantly prolonged the survival of the animals.

10. Effect on Fever

BPC antipyretic activity was tested in male Wistar rats (180-220 g) using beer yeast injection. Pre-treatment with BPC at a single dose (5-20 pg / kg; ip.) 1 hour before the yeast injection significantly reduced the temperature increase for at least 3 hours at 30 minutes intervals.

11. Effect on carrageenan-induced edema

Experimental edema was induced in male mice (18-24 g) in both sexes by injecting carrageenan into the hind paws. BPC at doses of 10 pg / kg ip. was effective when injected 1 hour before the experimental session; the effect was to reduce the volume of edema induced by said agent. Otherwise, BPC was inactive at the same doses in hot plate studies (53.5 ° C) in mice, unlike opioid agonists.

12. Effects on experimental rhinitis (rhinitis)

Wistar rats of both sexes were exposed to 10% formaldehyde once daily for 15 days. BPC was introduced into the experimental group at doses of 10 pg / kg ip. daily 1 hour before formaldehyde treatment. Animals were sacrificed 13-15 days after treatment. In the BPC treated animals, the oropharyngeal edema was significantly smaller and epithelial cells were retained, unlike the comparator control group. The secretion (secretion) was loopy but not purulent.

13. Effect on experimental fracture

We examined the effect of BPC (10 pg / kg; ip.) On experimental fracture in the rat fracture source process. Considering the controls, the histological examination showed significantly increased boiling time in all rats treated with BPC afterwards. In addition, these animals had significantly less local haemorrhage than controls.

14. Effects on experimental burns

The experimental procedure, i.e., the examination of skin or nasal mucosa burn wounds, was performed in mice (20-25 g). In comparison with the control group (treated with physiological saline solution) no purulent precipitation (secretion) was observed and consistently the increased healing rate was observed for all BPC pretreated (10 pg / kg; ip, one hour before the experimental session) ) in animals.

In addition, swelling of the nose in the nasal cavity was only observed after a very moderate accident. Subsequently, normal nasal breathing was only poorly damaged and survival was smooth. These data are clearly in contradiction with the data received from the control group.

15. Effects on skin wounds

The experimental procedure was performed in Wistar rats (170-220 g). Skin wounds are caused by a large incision of the skin. BPC ip. at a single dose of 10 pg / kg prior to incision. Contrary to the control group, there was a consistent observation of the lack of edema and edema in the treated group, poor pork production and increased healing rates.

16. Neuropharmacological activity

a) The neuropharmacological study carried out by Irwing showed that BPC, even when used at a dose of a thousand times more than was proven to be therapeutically effective on several different models, ranged from 20.0 to 40.0 g / kg ip. po. (oral) - did not induce any significant change in behavior compared to control mice.

b) In the isolated rat and rabbit heart, BPC at 30 1 / ml Ringer-Locke solution dose did not induce any change in intensity and frequency of contraction (using Langendorf procedure).

c) In the isolated hip lining model (rat, guinea pig, or rabbit) (which is performed by Magnus), BPC at the same dose did not have any effect on the bowel, nor on acetylcholine, histamine or serotonin-induced contraction.

17. Effects of BPC on delayed type hypersensitivity (DNFB) induced by DNFB.

As a model, the DTH reaction to DNFB was used on ears and paws of mice bearing the NMRI strain. Animals pretreated with two sensitizing doses of DNFB had significantly thicker ears / paws compared to the control group. With BPC ip. pretreated mice, where pretreatment was 0.1 g / 10 g body weight one hour prior to the DNFB test dose, showed significantly less difference between treated and untreated ears / paws.

18. Influence on pupil size

In these experiments, in which both sex mice (18-22 g) were used, BPC was csexed.

21 217 541 Β left in the left eye. The size of the pupil was measured with the help of a hemispherical lens. In addition, a group of animals ip. BPC was administered. The results showed that locally-instilled BPC caused fibroids. The ip. injected BPC also produced about 20% pupil contraction. This effect lasted at least 30 minutes.

19. Effect of BPC on Colon Disorders

The contact sensitivity of the mice was induced using DNFB (2,4-dinitrofluorobenzene). Mice were given two sensitizing doses [25-25 μ-25 0.5% DNFB (acetone: olive oil in 4: 1 mixture)] on the previously shaved skin of the abdominal wall for two consecutive days. 72 hours after the last sensitizing dose, mice were challenged with a 20 µáló 0.2% DNFB challenge dose in the ether during anesthesia with ether. Twenty-four hours later, the animals were sacrificed and colonies examined. Pathological changes in the rectum were characterized by severe internal bleeding and ulceration. In some animals, these lesions completely captured all parts of the colon so that they could have been pierced.

When BPC (0.1 pg / 10 g) was used one hour after challenge with DNFB, the incidence of lesions in the colon was significantly reduced. The most common damage was point-point bleeding.

20. Effects on tumor cells

a) Two cell lines, L-924 and B-16 melanoma lines were cultured in vitro under standard conditions. In vitro, we examined the activity of BPC in the growth of said cell cultures. BPC was administered at 3% and 0.3% (0.1 ml) two days after starting the cell cultures. Three days later, the cell count was determined by cell counts. The results showed that BPC has an inhibitory effect on both cell lines, L-924 and B-16 melanoma.

b) The Ehrlich ascites tumor (EAT) is the tumor that can grow in all mouse strains. This can grow in ascites or in solid form, depending on how the tumor cells are introduced. We attempted to determine whether the incubation of EAT cells in BPC would alter the survival time of mice in EAT cells treated in this way. or ip. after injection.

The animals were observed for a period of 45 days. 15 control groups of male NMRI mice from 0.4 to 10 ⁶ EAT was injected. Before injection, the tumor cells were incubated for one hour at 4 ° C in physiological saline. The volume of injection was 0.2 ml. The average survival time of the animals in this group was 36 days, and only 3 of the 15 animals survived 45 days.

The 15 NMRI male mouse experimental group received the same amount of tumor cells in the same volume, but these tumor cells were previously incubated in BPC solution (2 μg / ml). During the observation period (45 days), only 2 of the 15 mice died, the others survived for 45 days and even more. The difference between the control and the experimental group was significant (p <0.01).

If the same session was performed but EAT cells were ip. (and not.), there was no difference between the groups.

We have come to the conclusion that BPC prolongs the survival of tumorous animals, ie has anti-tumor effects.

21. Influence on radiation-induced injuries

The experiments were carried out in both mice (22-28 g) of the NMR-Y strain (64).

The first control group (16 mice) was not treated and irradiated.

The second control group (16 mice) was treated with 0.2 ml of physiological saline and irradiated with a dose of 9 Gy supraletal (i.e., lethal) (Co-60).

Both experimental groups (16 + 16 mice) were irradiated with the same supraletal dose (9 Gy). One hour after the irradiation of the first group of mice, BPC was administered at a dose of 20 pg / kg. The second group received the same dose of BPC, but one hour before the irradiation.

BPC administered after irradiation had no effect on the survival of mice.

The BPC administered before irradiation after 12 days increased the survival rate by 68.75% compared to other study groups.

22. Influence on the hematopoietic system

Cytostatics (Endoxan, Vincristin, Adriablastine, Cytosin Arabinoside) were used on albino mice in LD _5o dose ip. In the test group, BPC was administered one hour prior to administration of the cytostatics at a dose of 10 pg / kg, while in the control group only physiological saline was injected. Animals were sacrificed from each group of animals on days 3, 5, 7, and 11 of the experiment. The following tests were performed: blood test (E; Hb; Htc; L; Tb; absolute number of neutrophils); bone marrow; cytological and histological examination of liver and spleen. On day 3 of the experiment, there was no difference in L, E, Tb and neutrophil values between the experimental group and the control group. However, BPC treated animals had hematopoietic cells intact in the bone marrow while control animals had aplasia. In the experimental group on day 5, the neutrophils and the L number increased significantly and these values returned to normal on day 7 of the experiment. In the control group, this normalization never occurred. before the day.

23. BPC and fertilization ability - influence on oligo-syndrome

The experiment was carried out on 10 men aged 30 to 40 who suffered from oligoastenospermis. From these patients, ejaculate was taken after 3-4 days of self-restraint. After liquefaction (30 minutes), 0.5 ml of medium was added to the 0.5 ml ejaculate. The control medium consisted of HAM-FI 0 with 10% inactivated umbilical cord serum. In addition, the experimental medium also contained 2 pg / ml or 4 pg / ml BPC. After 90 minutes of waiting time at 37 ° C, an atmosphere5

HU 217 541, which is 5% CO ₂ In the Horwell Fertility Testing Chamber, the number of progressively moving, locally mobile and immobile spermatozoa in 1 ml ejaculate was determined. Preliminary results showed no effect on sperm motility at lower BPC concentrations. However, at a concentration of 4 pg BPC / ml, we found that the percentage of mobility was significantly higher than that of the control.

24. The effect of BPC on reproductive processes

The effect of BPC on reproduction was studied in mice that had three pregnancies in their history. Twenty days after the end of the last lactation, the animals were mated. BPC was applied at 10 g / kg ip. once a day during the entire period of gestation (19-21) and breastfeeding (3 weeks later). At the same time, the control group received an equal volume of physiological saline. We fixed it per female! number of offspring and number of offspring.

Careful statistical analysis consistently revealed an increased number of female offspring, but surprisingly there was no difference in body weight between the periods studied.

All female animals were allowed to regenerate for 25 days after completion of breastfeeding. We then mated them again to examine the effect of the fifth pregnancy.

Compared to the control group, increased fertility rates and increased milk production capacity were recorded in all BPC treated animals.

It should be noted that neither pathological changes nor changes in fertility rates were recorded in the offspring of mice treated with BPC (independent of the number of maternal pregnancies).

Accordingly, it seems likely that BPC can improve fertility rates even in old mice. It is clear from the above that the results obtained are consistent with the overall protective effect of BPC and with in vitro data obtained using human sperm.

25. The protective effect of BPC on dental root lesions

The purpose of this study was to investigate the effect of BPC on the uptake of soft root roots by the mechanical injury of the first barrier of albino rats.

A four-week-old rat was divided into a control group treated with physiological saline and an experimental group treated with BPC. All animals were treated with a needle-like tool (0.6 mm wide and 1.9 mm long) to cause mechanical damage, inserted into the middle jaw for 1 second at a depth of 1 mm from the edge of the middle gums. with its middle surface.

The experimental group was treated with a BPC solution after mechanical damage (0.02 ml / 200 g). The rats divided into 12 groups were treated with 1, 3, 5, 6, and 6

We killed on the 14th or 21st day.

The right upper jaw (maxilla) of all control and experimental rats was removed, fixed in 10% neutral buffered formalin solution, mineralized with formic acid, embedded in paraffin, and 6 μηι serial sections were made on mesodist microtome. The sections were stained with hemotoxylin and eosin and examined under a light microscope.

The histological findings of the first group of rats in the control group were as follows. The presence of a large number of inflammatory cells was recorded. 1. and

Between day 3, a small resorption hole (lacuna) was observed at a portion of the root surface, at the level of the alveolar tar. On day 5, the resorption hole was extended in the coronal direction. The hole contained more toothpicks. On day 7, the resorption hole spread further in the coronal direction. On the 14th and 21st days, the resorption hole spread even more coronally, and a portion of the hole was close to the tooth enamel fit.

Experimental rats underwent a similar change between days 1 and 5, but the root resorption did not advance between days 14 and 21. Thus, it can be said that BPC exerts a protective effect on the mechanical damage of the rat tooth root tissues.

26. The influence of BPC on nucleus basal and spinal cord lesions

Alzheimer's type of dementia (DAT) is characterized by generalized damage to the cerebral cortex, which affects memory and perception. Post mortem examinations in patients with DAT have shown that nucleus basal (NB) neurons undergo profound and selective degeneration. These neurons are the main source of cholinergic innervation from the outside to the cerebral cortex. The purpose of this study was to examine the influence of BPC on passive depletion behavior in rats suffering from bilateral electrolytic damage to NB.

This means that rats suffering from NB damage may be the experimental model of DAT.

The study was carried out in albino rats. Each group of rats (damaged and not damaged in NB) was subjected to the passive emptying test according to the procedure of Ashford and Jones. Animals injured in NB received physiological saline (control group) or BPC solution (10 pg / kg ip.).

The test substances were injected a) once only, immediately after induction of NB lesions, or b) once a day for four consecutive days after induction of NB lesions. BPC was also administered once a day during the four study days one hour before the passive emptying studies started. All animals were subjected to the described behavioral test after a 20-day postoperative recovery period. The statistical significance is calculated by a variance analysis of the Duncan test with multiple comparisons (p <0.05). The test results show that

EN 217.541

- bilateral electrolyte damage adversely affects passive emptying in rats;

- in the dose used, BPC does not affect the passive emptying behavior of otherwise intact, undamaged animals;

- BPC significantly improves passive emptying behavior in NB damaged rats.

27. The influence of BPC on the motor activity of crushed rabbits

The purpose of this experimental work was to examine whether BPC could modify the consequences of a crushed spinal cord. In this study, adult rabbits of both sexes were used. Middle Dorsal L, -L ₆ laminectomy was performed with pentobarbital anesthesia. Using the technique of Albin et al., We created a measurable, severe injury to the spinal cord. Motor activity was checked on the hind legs according to Tarlov's system once a day for nine days. The underlying estimates were: 1. total bilateral limb paralysis; 2. Minimal deliberate movements; 3. the animals are able to stand up but unable to run; 4. the animals are able to run, but with some spasticity and somewhat uncoordinated; and 5. normal motor activity.

In the control group, the animals received physiological saline, BPC in the experimental group at 10 pg / kg iv, once a day after 9 operations.

Spinal cord trauma caused almost total bilateral extremity paralysis. The average of the total motor activity studied (during the 9 days of surgery) was 1.3 Tarlov units (N = 4) in the physiological saline. In animals receiving BPC, this value was 2.5 Tarlov units.

Thus, it is obvious that BPC improves the motor activity of vertebrate rabbits.

From the results presented, it can be assumed that PBC can be a promising drug for the treatment of impaired nervous function (brain or spinal cord trauma) and that BPC should be given as early as possible after nerve damage.

28. Influence of BPC on intestinal motility, basal temperature and urine excretion

The use of BPC had no influence on urine excretion, lower basal temperature in in vivo, and intestinal smooth muscle contraction in guinea pigs.

29. Activity against Trichinella spiral

Applying BPC Concentrate ip. At 10 pg / kg body weight, experimental mice infected with Trichinella spiral larvae had a significant influence on the survival of the animals.

30. Application in Large Animal Breeding

1419 healthy pigs (out of a total of 4,306 pigs) received BPC (10 pg / kg body weight im.) Immediately after birth, on the 13th day of life of another 1440 pigs, one day before transplantation, while the remaining 1477 pigs received the same. received traditional Fe therapy. After 4 weeks, we examined the injuries, scraps, mortality, weight and feed consumption. The BPC only significantly reduced the discard rate (once in the group that was treated with BPC immediately after birth) and the injury rate (in both BPC treated groups) only once. We received the same body weight in BPC-treated animals as in control but with significantly lower food intake.

Influence on Infection: We examined 40 28-day-old piglets infected with E. coli enterocolitis. In 20 animals, BPC was used (10 pg / kg body weight; im.). One month later, the mortality rate was significantly reduced in BPC treated animals.

In another experiment, the mortality rate was measured after the first week of life for the total amount of 1200 healthy pigs. 400 animals received BPC immediately after birth in addition to conventional Fe therapy. Significantly lower mortality was recorded in BPC treated animals (10 pg / kg body weight; im.)

31. Antiviral Activity

Antiviral activity was tested in in vitro and in vitro (in vivo mice). BPC was applied at a dose of 10 pg / kg body weight ip. or We have found significant activity against enteroviruses (Echo type: 6; 9; 11 and 16; Coxsackie type: A9; B3 and B4), CNS viruses (tick-borne encephalitis, LCM choromeningitis) and ARBO (Thayna, Bhanja and Calovo). In infected mice, increased survival (72 hours survival time) was recorded.

In the following section, certain aspects of the invention are described in other words.

The technical problem

The technical problem is whether it is possible or not to interact with pharmacotherapy at the same time:

I

1. Acute hepatitis (viral, drug-induced, toxic, acute, irrespective of pathogen);

2. chronic hepatitis;

3. cirrhosis of the liver;

4. bleeding;

5th gallstones;

6. bile duct inflammation;

7. Liver transplant - therapy before and after transplantation;

8. therapy prior to or after other surgical treatment of the liver (e.g., congenital distortion, benign and malignant bile duct);

II.

9. prevention of pancreatitis;

10. improving the course of pancreatitis;

11. reduction in the incidence of post-surgical pancreatitis;

12. preventing complications and mortality from acute pancreatitis;

13. reducing the frequency of chronic pancreatitis;

III.

14. Acute bleeding in the upper gastrointestinal tract;

EN 217.541

15. destructive gastritis;

16. Acute ulcer, gastric ulcer and duodenal ulcer ("stress ulcer");

17. chronic peptic ulcer;

18. non-peptic ulcer of the gastrointestinal tract;

ARC.

19. shock (irrespective of its origin);

V

20. protecting kidney parenchyma from developing acute and chronic ischemic and haemorrhagic necrosis;

21. Acute and chronic renal failure as well as all causes capable of inducing it;

VI.

22th osteoarthritis (fractures);

23. therapeutic actions on the skeleton (bone marrow);

24. bone transplantation (scarring);

25. pseudoarthritis;

26. joint stiffening;

27. wounds (increased healing rate, healing "per prime");

28. burns (skin and mucous membranes; increased healing rate);

VII.

29. antitumor activity;

VIII.

30. anti-inflammatory, anti-edema, anti-rheumatic activity;

31. anti-fever activity;

32. rheumatoid arthritis;

33. bone and joint inflammation;

34. erectile dysfunction;

35. Gouty arthritis;

36. non-articular rheumatic diseases (mucositis, tendonitis; arthritis; upper arm shoulder periarthritis);

37. inflammation and edema after surgical and non-surgical treatment, fracture and tear;

IX.

38. acute myocardial infarction;

39. myocardial inflammation;

40. halting ischemic changes in tissues;

X.

41. less food consumption;

XI.

43. Parkinson's disease, parkinsonism;

XIII.

44. green algae (reduction of intraocular pressure dopamine and pilocarpine-like activity);

XIV.

45. diabetes;

XV.

46. Carbite poisoning;

XVI.

47. stress and any pathological event that develops as a result of stress.

State of the art

Until now, there is not yet a complete special pharmacotherapy for these, and the treatment of complications is beyond the scope of pharmacotherapy.

Description of the solution to the technical problem

The digestive organs, and the stomach in particular, are currently considered to be the target of stress. The possibility of suggesting that the gastrointestinal tract, and especially the stomach, may be a body from which the body's defensive response may be started has never been considered. We isolated a substance - later called BPC - that triggers, mediates and regulates the body's defensive response; this material was isolated from 542 patient weeds and animal sources by dialysis, ion exchange filtration and gel chromatography. Thus, the BPC protein (although its structure is not yet fully elucidated) represents a different model to that of human patients in both in vitro and in vitro. On the basis of the results obtained, it is quite possible to use this spectrum in pharmacotherapy.

The substance that we have isolated and called BPC has not been known so far, so it has not been used in pharmacotherapy so far.

The resulting BPC protein was studied in several different experimental species (e.g., rat, mouse, guinea pig) and was studied on human cells in vitro.

The PBC protein was designated by the abbreviation "5ody, Protecting Compound".

Induction of damage to liver, pancreas, stomach and duodenum

In addition to mild ether anesthesia, we performed middle abdominal surgery. The bile duct is then ligated from the duodenum distally at the point where the liver artery (which is also tied with the bile duct by the same surgical operation) enters the left side, somewhat beneath the liver artery, which remains intact above the binding. Then the wound was stitched and the animals were no longer disturbed until they were killed or killed.

In addition to mild ether anesthesia, a central abdominal incision was performed and the bile duct was bound just before entering the duodenum. The wound was then stitched and the animals were not disturbed until they were killed or killed.

The animals were immersed in water at 26 ° C, 20 cm deep for 3 hours. Immediately thereafter, they were killed. 24 hours before dipping into the water, the animals were not allowed to eat, but they had free access to water.

In addition to mild ether anesthesia, central abdominal distension was performed and the lower stomach was bound. The wound was stitched and the animals were not disturbed until they were killed; killing was done 15 minutes after the lower stomach mouth was tied.

The animals were tightly fastened for 24 hours. Indomethacin was used with the fixation and 12 hours later. After 24 hours the animals were killed.

Induction of fractures, wounds, skin and mucous membranes

In addition to mild ether anesthesia, tibia fracture, hypomochlion was induced. Animals were killed on day 5, 8, 9, 12, and 30 after fracture.

EN 217.541

In addition to mild ether anesthesia, a large skin pocket was dropped. We killed the animals every day for the first five days, and then every three days for the 15th day and then for five days at the 30th day after the wound.

In addition to mild anesthesia, a burning wound was produced on the back of the animal (15x30 mm) with a 15 second incubation of a cauterizing needle. Animals were killed on days 5, 9, and 30 after injury.

In addition to mild anesthesia, the mucous membrane was burned on two sides by incubating the cauterizing needle for 5 seconds. The animals were injured from injury 5,

We killed on the 9th and 30th days.

To cause kidney damage

After mild anesthesia, the kidney cavity was bound. The wound was stitched and the animals were not disturbed until they were killed or killed. Animals were killed 24 hours later.

Induction of anti-inflammatory, anti-edema and anti-fever effects

Carrageenin (1%) was used (0.05 mL; se) in the paw as an anti-inflammatory agent; the paw volume was measured 3 hours later with a standard session.

Siccoferment (2.5 g / kg; ip.) Was used to raise body temperature. The temperature was then measured in the rectum for 3 hours.

Induction of heart damage

The animals were tightly fastened for 24 hours. Indomethacin was used at the start of the fixation (0 h) and 12 h later. At the end of the 24-hour period, the animals were killed.

Isoprenaline (30.0 mg / kg; ip.) Was used at 0 and 24 h and the animals were sacrificed at the end of the 48 h period.

Induction of effects on CNS (central nervous system)

Investigating effects on spontaneous behavior of animals (mice).

To determine the presence of dopamine neurons in the activity of BPC, apomorphine, a conventional dopamine agonist (20 mg / kg; se.), As well as serotonin (5 mg / kg ip.) (Comparative control) or physiological saline (control). ). The behavior of the animals was investigated after application. The behavior was evaluated according to Malick's (1983) procedure.

- Examining the effects on the iris muscle.

The test solution was applied topically or intraperitoneally. The pupil diameter was measured at appropriate intervals (5; 10; 15; 30; 60; 90 and 120 minutes) according to standard sessions.

- Investigation of the effects of barbiturate-induced depressive activity

By using Na-pentobarbital (50 mg / kg, iv), narcosis was induced. Barbiturate sleep was measured by the absence or presence of the so-called "righting" reflex.

Induction of effects causing diabetes

Diabetes was induced using alloxane (100 mg / kg; ip.) And streptozotocin (100 mg / kg; ip.).

Glucose content was measured by standard procedures in both urine and blood. The animals were not disturbed until their destruction.

Impact assessment

dispersion

THE ^l25 Studies with BPC labeled with I indicate accumulation of BPC in a dose-dependent and time-dependent manner, especially in the stomach, but distributed throughout the body after oral and parenteral administration.

Clinical observations

In studies of liver, pancreas, stomach, duodenum, kidney and heart, fractures, burns (skin, mucous membranes), diabetes, the general condition of animals treated with BPC was significantly better than in the control group. The complete absence or severe mortality of the control group was consistently observed.

Macroscopic and pathological observations

In control animals in which the bile duct and liver artery were bound, large yellow-green liver necrosis developed, sometimes spreading to the entire liver lobe; pancreatic necrosis and haemorrhagic lesions in the stomach and duodenum have also developed. The use of BPC successfully and dose-dependent prevented the damage described above. In the control animals, these lesions appeared about 6 hours after the binding, while in the treated animals they did not disappear even 48 hours after the binding. The patohistological examination supported the macroscopically observed changes and differences.

In control animals in which the bile duct was bound to the duodenal entry site, severe necrosis appeared in the pancreas and haemorrhagic lesions were observed in the stomach and duodenum. The administration of BPC prevented the development of the above-described lesions in a dose-dependent manner. The patohistological examination supported the macroscopically observed changes and differences.

In control animals subjected to water immersion stress, significant stomach damage was observed in control animals undergoing stress-induced treatment with indomethacin and in animals under short-term (15 min) gastric lavage. The administration of BPC has drastically reduced (or completely prevented) gastric damage following the above-described adverse intervention.

Serious lesions of the kidneys were detected in the control animals undergoing renal cortex. In the BPC-treated rats, these lesions were significantly smaller. The patohistological study strongly supported macroscopic changes and differences.

Compared to the tibia control animals, the development of post-traumatic hematoma in BPC treated animals was significantly less pronounced and the injured leg became operational earlier. Qualitative pathohistological studies using conventional methods in the treated animals are scars9

EN 217 541 Β increased and elevated velocity of the drug in each examined period.

In the control animals with the wound, there was a high rate of development of the purulent wound on the first days of injury. In contrast to the control group, the BPC-treated animals had dry wounds, without sweat and without visible pustules (i.e., healed prime). The patohistological examination strongly supported macroscopic changes and differences in all the examined periods.

Typical myocardial lesions were observed in control animals treated with tight fixation + indomethacin and CPK values increased in serum. Administration of BPC significantly reduced myocardial damage, and CPK values were also lower. The pathohistological examination has confirmed the macroscopic differences and changes observed.

In animals with experimentally induced diabetes, BPC eliminated transient glucose clearance in the aloxane-treated group. In streptozotacin-treated animals, BPC administration nearly tripled survival. All animals treated with BPC were less stressed by thirsty thirst and sickness.

Anti-inflammatory, anti-edema and antipyretic effects

In animals treated with BPC, a marked decrease in carrageenan-induced edema was observed relative to control animals in each test period.

In siccoferment-treated animals, administration of BPC significantly reduced body temperature relative to the results recorded in the control group.

Data on in vitro effects

Two days after the start of culture of the L-929 human fibroblast cell line and the B-16 melanoma cell line, BPC was added to the culture. Three days later, the cell count was determined in a cell counting device. BPC protein showed dose-dependent toxic effects on melanoma cells. Fibroblast cells had no observable effect.

Compared to the burned control animals, accelerated scar formation was observed in treated animals. In the first two days of burnt mucosal control (e.g., burned mammary glands), severe edema of the injured tissue was observed followed by partial obstruction of unwanted secretion and nasal breathing. The development of the above described changes was not observed in BPC-treated animals. The pathohistological examination strongly confirmed the macroscopic differences and changes observed.

Effects on CNS (ie CNS)

The effects of BPC on CNS were studied by observing the behavior of animals (mice).

5 minutes after application, there was an increase in motion activity. The restlessness of the animals increased from minute to minute, reaching its peak after 30 minutes. Then the animals stood on their hind legs, upward like a candle, and sniffed. The Straub symptom was obvious, the animals ran around the cage, and the piloing was also prominent. After Apomorphine, the change occurred after 45 minutes and was partially associated with the hind legs. After the use of serotonin, only a very short period of normal behavior was observed (increased motion activity, sniffing, purification with less intensity).

Local administration on the eye (or ip application) led to pupillary constriction (about 20%).

The use of BPC shortened the duration of barbiturate-induced sleep; this was determined by shortening the time without a straightening (.uighting) reflex (about 60%).

Effects on blood pressure, bowel movement, basal temperature and urine excretion

The use of BPC had no effect on adrenaline, noradrenaline, isoprenaline, acetylcholine and serotonin induced changes in cardiac pressure, nor on baseline blood pressure values.

The use of BPC did not reduce the baseline temperature.

The use of BPC did not lead to increased urine excretion.

Mechanism of action

It can be assumed that the effect of BPC, like other exogenous proteins, is due to one of the following mechanisms: the direct interaction of the protein or fragments with the cell receptors; stimulating or inhibiting the release of biologically active (stimulating or inhibiting) proteins or non-proteins by a receptor-bound or receptor-bound mechanism; interaction with enzymes that break down endogenous, biologically active peptides.

Protective effects on otherwise non-related symptoms can only be explained if they are caused by a common cause - stress that triggers them in the body. Thus, it seems likely that the BPC protein represents the primary common response (which starts from the stomach) to any stressful event that is dangerous to the body. Therefore, the mechanism of action of BPC represents the primary defense mechanism against various stress-related attacks.

From the above it can be concluded that it is possible to exert a pharmacologically advantageous effect on the course of the following diseases using BPC:

I

1. Acute hepatitis (virus-induced, medical intervention, toxic, acute - independent of pathogenesis)

2. Chronic hepatitis

3rd cirrhosis

4. epigestion

5th gallstones

6. Bile duct inflammation

EN 217.541

Ί. liver transplantation therapy before and after transplantation

8. Pre-operative and post-operative therapy for other surgical procedures in the liver (eg congenital distortion, benign and malignant constriction of bile duct)

II.

9. prevention of pancreatitis

10. alleviating the course of pancreatitis

11. reducing the incidence of post-surgical pancreatitis

12. preventing the complications and mortality of acute pancreatitis

13. reducing the frequency of chronic pancreatitis

III.

14. Acute bleeding in the upper gastrointestinal tract

15. erosive gastritis

16. Acute ulcers, gastric and duodenal ulcer ("stress ulcers")

17. chronic peptic ulcers

18. non-peptic ulcers of the gastrointestinal tract

ARC.

19th shock (any pathologist)

V

20. kidney protection against ischemic and bleeding necrosis (acute and chronic)

21. Acute and chronic kidney disease, and all diseases that result from it

VI.

22. Bone system trauma (fractures)

23. bone surgery operations (bone marrow)

24. bone transplantation

25. pseudoarthritis

26. joint stiffness

27th wounds (faster source, source "per prime")

28th burns (skin, mucous membrane: faster source)

VII.

29. antitumor effect

VIII.

30. anti-inflammatory, anti-edema, anti-rheumatic effect

31. anti-fever effect

32. rheumatoid arthritis

33. arthritis

34. erectile dysfunction

Gout 35

36. Non-articular rheumatic diseases (mucositis, tendonitis, arthritis, arteriosclerosis)

37. inflammations and edema after surgical and neurosurgical interventions associated with fractures and sprains

IX.

38. acute myocardial infarction

39. Myocardial Inflammation

40. prolongation of ischemic tissue changes

X.

41. Decrease in food intake (loss of appetite)

XI.

42. hyperprolactinemia

43. Parkinson's disease

XIII.

44. glaucoma (decrease in intraocular pressure dopamine and polycarpine)

XIV.

45. diabetes

XV.

46. barbiturate poisoning

XVI.

47. stress and all stress-induced damage in the body

The substance may be administered orally or parenterally in the form of pills, capsules, dragees, tablets, suppositories, suppositories, injection solutions, nasal sprays, balms, creams, gels, sprays, eye drops, drops, or nasal drops.

Examples of technical exports

Example 1

Production of BPC protein from human gastric juice

The starting material from which the BPC protein was produced formed 542 patient weeds, which were obtained by gastric catheterization by aspiration of gastric juice. The collected weed was stored at + (2-4) ° C. The material was allowed to warm to room temperature and then homogenized by stirring. An aliquot (200 mL) was centrifuged (12,000 G, 30 min); this results in supernatant liquid and a sub-bottom layer. The liquid was dialyzed at + 4 ° C in 1:25 volume deionized water until 100 pS conductivity. The solution is then lyophilized. The dry residue (200 mg of a green substance containing about 80% protein as determined by the Louvry method) is dissolved in 10 ml of distilled water and applied to a chromatographic column having a size of 25 A 250 mm and modified with a DEAE modified ion exchange gel (e.g. France) produced by Trisacryl M-DEAE gel).

For the elution, 0.01 M, pH 5.2 sodium acetate buffer is used. The concentration of (10-10 ml) fractions was determined with a UV detector at 280 nm. Fractions between 40 and 120 ml of eluate were collected, concentrated using Aquacide-3 (Calbiochem) and lyophilized. The resulting 25 mg dry matter is a white amorphous powder. The protein compound content is about 95% as determined by the Louvry assay and exhibits biological activity at about 1-10 μ / kg body weight according to the standard stress-flow model for ulcer.

The material is dissolved in 10 ml of 0.2 M pH 7.4 buffer and chromatographed on a 25 x 450 ml Sephacryl S-200 (Pharmacia Fine Chemicals) gel column. The recorded flow rate is 1.2 ml / min. 20-20 ml fractions were collected, UV absorbance was examined at 280 nm and the fractions between 80 and 200 ml were collected; the material

It is dialyzed against distilled water, concentrated using Aquacid-3 and lyophilized. A white solid (2.0 mg, 98%) was obtained.

The resulting BPC protein is highly soluble in water; in the following, biological and pharmacological studies were performed. It was found to decompose between 180 and 200 ° C.

Molecular weight by gel chromatography was determined to be 40,000 ± 5000 dalion. The UV spectrum (in water) is

Figure 1 shows absorption in the ranges shown in Figure 1.

The IR spectrum (KBr) shows absorption bands in the regions shown in Figure 2.

The material is a folded protein having a partial amino acid sequence starting from the N-terminal according to the Edman method:

H ₂ N-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-AspAsp-Ala-Gly-Leu-Val -... -COOH.

Based on the described pharmacological properties, the BPC protein can be used to treat a number of diseases and disorders, especially in those cases where the normal formation of these agents in the body is insufficient or completely absent.

They can be used to handle:

a) All stress-induced diseases and disorders

b) Circulatory disorders

Ulcers (of different origin), shock (various pathogens), ischemia and infarction (in different organs).

c) Inflammations and edema

Inflammation and edema of all kinds, hepatitis (acute and chronic), cirrhosis, bile duct inflammation, pancreatitis (various pathogens), gastric and duodenal ulcer, renal inflammation and kidney disease, myocarditis, cardiac inflammation, cardiovascular inflammation, degenerative arthritis and other arthritis ( various diseases), rhinitis, rheumatic disorders, diseases caused by a combination of different pathogens, fever, free radical damage.

d) Traumas and surgical interventions

Injuries (pre-injury and post-injury treatments), transplants, wounds, burns, fractures, post-surgical complications

e) Malignant diseases

Certain types of tumors

f) Sugar]

g) Dopaminergic or similar pathological disorders

Obesity, Parkinson's disease, hyperprolactinemia, glaucoma, barbiturate-induced sleep disturbances (barbiturate poisoning)

h) Diseases and disorders of the nervous system

Alzheimer's Disease, Nervous System And Traumatic Injuries, Delay Of Old Age Degenerative Changes

i) Dentistry

Diseases and disorders of the oral mucosa, surgical treatment of gingival cysts, inhibition of root absorption

j) Hematopoietic system

Neutropenia, Aplasic Anemia, Myelodysplastic Syndrome, Bone Marrow Transplant

k) Injuries caused by irradiation

Acute irradiation syndrome, protection against irradiation injuries.

l) Immunology

Hypersensitivity reactions, allergies, transplants

m) Fertility

Increase in fertility, therapy of oligostenospermia, promotion of milk formation

n) Veterinary use

Mortality, stunts and injuries in commercial breeding, improved feed processing, weight gain

o) Virology

Antiviral therapy

P) Mixed

Changes in fat composition in the liver, bile duct inflammation, gallbladder, chronic kidney failure, kidney and liver toxicity.

The BPC protein can be used as a medicament with enteral, topical, rectal or parenteral administration. The pharmaceutical compositions are formed by the addition of organic and inorganic adjuvants. Examples of tablets and dragees include talc, lactose, starch, stearates, and the like. water, alcohols, glycerol, and the like are added to solutions and suspensions. For example, natural oils, hardened oils, waxes or polyethylene glycols are added to the suppositories. The formulations may generally contain suitable preservatives, buffers, stabilizers, surfactants, dissolving intermediates, sweeteners and colorants.

The appropriate daily dose of BPC is between 5 and 50 pg / kg of body weight and can be administered in a single dose or in divided doses.

Example 2

Production of BPC protein from human gastric juice

Homogenized human weed from the same source as in Example 1 was centrifuged, dialyzed, and lyophilized according to the procedure described in Example 1.

The dry residue (80.4 mg) was dissolved in 0.1 M acetate buffer (3 mL; pH 5.22) and applied to a chromatographic column (10 x 50 mm) previously equilibrated with the same acetate buffer using a Fractogel-TSK DE A (Merck) ion exchanger. filled with resin. Flow rate 0.7 ml / min. The 20-20 mles fractions were collected according to Example 1 and the absorbance was monitored by UV spectrophotometry at 280 nm. The first strong UV adsorption fraction (from 60 ml of eluate) was treated by dialysis against distilled water and lyophilized (47 mg, 89% protein content).

This dry residue was redissolved in the same acetate buffer (1 mL) and applied to a chromatography column (16x560 mm) which had been previously equilibrated with the same acetate buffer.

HU 217 was filled with 541 x dex-G-100 (Pharmacia Fine Chemicals). The flow rate is 0.3 ml / min. The 10-10 ml fractions between 66 and 108 ml were collected, dialyzed against distilled water and lyophilized. 0.85 mg of a white powder is the same as that described in the previous example.

Example 3

Preparation of BPC protein from porcine stomach

Homogenized porcine weeds (85 mL) from healthy animals were centrifuged, dialyzed, and lyophilized according to the procedure described in the previous examples; 440 mg of dry residue are obtained. 110 mg of this residue was then dissolved in 0.1 mol / L acetate buffer (6 mL, pH 5.22) and applied to a chromatographic column (10x50 mm) filled with Fractogel-TSK ion exchange resin and followed by the same procedure. described in the previous example. 44 mg of a yellowish concentrate were obtained.

Part of this concentrate (24 mg) was dissolved in 1 ml of the above acetate buffer and applied to a chromatographic column (16x565 mm) previously filled with Sephadex-50/80 gel balanced with acetate buffer. The eluates between fractions 65 and 108 were collected, dialyzed against distilled water and lyophilized. The resulting white powder (10 mg) has the same molecular weight within the range of precision and reproducibility of the analytical method used as the product described in the previous examples and has the same properties.

Example 4 Tablets components mg / tablet BPC 0.3 lactose 37.7 starch 6.0 talc 3.0 tragacanth 2.5 magnesium stearate Example 5 Capsules 0.5 to 50.0 mg components mg / capsule BPC 0.3 lactose 99.0 magnesium stearate Example 6 Tablet sublingual 0.7-100.0 mg components mg / tablet BPC 0.3 mannitol 100.0 starch 5.0 poly (vinyl pyrrolidone) 1.5 talc 3.2 110.0 mg

Example 7 Suppositories Components mg / suppository BPC 0.5 polyethylene glycol 300 478.0 polyethylene glycol 1500 529.0 polyethylene glycol 6000 38.0 emulsifier 14.5 1060.0 mg Example 8 Cream components g / 25g BPC protein 0.05 emulsifier 25.00 hydrogenated peanut oil 50.00 Tween 60 120.00 propylene glycol 30.00 methyl hydroxybenzoate 0.16 propyl hydroxybenzoate 0.17 250.00 g Example 9 Injection Solution Components mg / ml BPC protein 0.3 sodium carboxymethyl cellulose 1.5 poly (vinyl pyrrolidone) 5.8 lecithin 2.7 benzyl alcohol 0.01 buffer as required twice distilled water Up to 1 ml Example 10 Solution components g / 25 ml BPC protein 0.05 glycerol 15.00 benzyl alcohol 0.01 buffer as required twice distilled water 25 ml Example 11 Dry Injection Components weight (mg) BPC protein 0.3 mannitol 10.0 10.3 mg Example 12 Nasal Spray Components mg per dose BPC protein 0.5 benzyl alcohol 0.15 Uliglyol-812 14.00 Freon 12/114 60.00

74.65 mg

EN 217.541

Example 13

Eye drops, Fill drop, Nose drops Components mg / 5 ml

BPC Protein Stabilizer Sodium Hydrogen Phosphate

116 sodium dihydrogen phosphate benzalkonium chloride

0.5 ml of distilled water per ml

Example 14

pessary

Ingredients mg / inch

BPC protein

0.3 polyethylene glycol 300

150.0 polyethylene glycol 1500

1290.0 propylene glycol

200.0

Tween 40

59.7 lactose

100.0

2500.0 mg

Example 75

eye Balm

Components mg / 3.5 mg

BPC protein paraffin oil

1000 emulsifiers

100 White Vaseline

2355

3500 mg

Claims (5)

PATIENT INDIVIDUAL POINTS A method for the production of a protein called BPC having a molecular weight of 40,000 ± 5000 Da, an ultraviolet and infrared spectrum as defined in Figures 1 and 2 and a partial N-terminal amino acid sequence as follows: H ₂ N-Gly-Glu-Pro- Pro-Pro-Gly-Lys-Pro-AlaAsp-Asp-Ala-Gly-Leu-Val -... COOH, human or animal gastric juice, characterized in that (1) separating the collected and homogenized animal or human stomach by centrifugation into solid and supernatant; 2. dialysis and lyophilizing the supernatant liquid against distilled water; 3. dissolving the dry residue in water or weakly acidic acetate buffer and purifying on a chromatography column filled with a weak basic or basic ion-exchange resin; 4. concentrating the eluate, dialysis and lyophilizing the concentrate against distilled water; 5. The dry residue is redissolved in weak acid acetate buffer and purified by gel chromatography and the eluate is dialyzed and lyophilized. A BPC protein obtainable according to claim 1 having a molecular weight of 40,000 ± 5000 Da, an ultraviolet and an infrared spectrum as defined in Figures 1 and 2 and a partial N-terminal sequence as follows: H ₂ N-Gly-Glu -Pro-Pro-Pro-Gly-Lys-ProAla-Asp-Asp-Ala-Gly-Leu-Val- .COOH. A pharmaceutical composition for use in human and veterinary medicine comprising as active ingredient the BPC protein of claim 2 and the usual excipients in a pharmaceutical formulation suitable for enteral, oral, rectal or topical administration. EN 217 541 Β Int.Cl. ⁷ : C 07 K 2/00 240 260 280 300 320 340 350 EN 217 541 Β Int Cl. ⁷ : C07K 2/00 Figure 2 Published by the Hungarian Patent Office, Budapest Responsible for release: Zsuzsanna Törőcsik Head of Department Windor Bt., Budapest