METHOD FOR PRODUCING A MAMMALIAN BRAIN PROTEIN HYDROLYSATE

Abstract

A method for producing a mammalian brain protein hydrolysate composition, by providing a mammalian brain protein hydrolysate, adding Zn ions to the mammalian brain protein hydrolysate to form a complex with the Zn ions and the mammalian brain protein hydrolysate, and obtaining the complex with the Zn ions and the mammalian brain protein hydrolysate in a mammalian brain protein hydrolysate composition. A composition with a mammalian brain protein hydrolysate. A nutritional supplement with a mammalian brain protein hydrolysate. A pharmaceutical composition with a mammalian brain protein hydrolysate.

Claims

1. A method for producing a mammalian brain protein hydrolysate composition, comprising: providing a mammalian brain protein hydrolysate; adding Zn ions to the mammalian brain protein hydrolysate to form a complex comprising the Zn ions and the mammalian brain protein hydrolysate; and obtaining the complex comprising the Zn ions and the mammalian brain protein hydrolysate in a mammalian brain protein hydrolysate composition.

2. The method according to claim 1, wherein the mammalian brain protein hydrolysate is a hydrolysate of porcine or bovine brain tissue.

3. The method according to claim 1, wherein the Zn ions are added as Zn gluconate, Zn sulfate or Zn acetate, in an amount of 0.1 to 500 mg/mL.

4. The method according to claim 1, wherein the mammalian brain protein hydrolysate is an enzymatically hydrolysated mammalian brain protein hydrolysate.

5. The method according to claim 1, wherein a pH of the mammalian brain protein hydrolysate composition is adjusted upon formation of the complex comprising the Zn ions and the mammalian brain protein hydrolysate to a pH of 3.0 to 9.0.

6. The method according to claim 1, wherein the Zn ions are added to the mammalian brain protein hydrolysate in a relative amount of 0.01 to 50 mg Zn per mg mammalian brain protein hydrolysate (as mg N.sub.2).

7. The method according to claim 1, wherein the Zn complexed mammalian brain protein hydrolysate is adjusted to a concentration of 0.05 to 50 mg/mL.

8. The method according to claim 1, wherein the Zn complexed mammalian brain protein hydrolysate is finished to a nutritional supplement or to a pharmaceutically acceptable preparation.

9. The method according to claim 1, wherein the Zn complexed mammalian brain protein hydrolysate is dried.

10. A composition comprising a mammalian brain protein hydrolysate composition produced by the method according to claim 1.

11. A nutritional supplement comprising a mammalian brain protein hydrolysatecomposition produced by the method according to claim 1.

12. The nutritional supplement according to claim 11, wherein the nutritional supplement is in dose form.

13. The nutritional supplement according to claim 11, wherein the nutritional supplement comprises 5 to 1000 mgof the Zn complexed mammalian brain protein hydrolysate per mL in aqueous solution.

14. A pharmaceutical composition comprising a mammalian brain protein hydrolysate composition produced by the method according to claim 1, and a pharmaceutically acceptable excipient.

15. A therapeutic treatment method, comprising: administering the pharmaceutical composition according to claim 14 to a patient, wherein the composition comprises the mammalian brain protein hydrolysate.

Description

[0062] The present invention is further described in the following examples and the figures, yet without being limited thereto.

[0063] FIG. 1 shows size exclusion chromatograms of mammalian brain protein hydrolysate and water-soluble protein hydrolysate - Zn complexes formed by addition of 2 or 4 mg zinc gluconate (ZnGlu) /mg N.sub.2 in the mammalian brain protein hydrolysate.

[0064] FIG. 2 shows the enlarged part of the SE-chromatograms showing, upon complexation, a shift in the peak positions to shorter retention times, i.e. higher molecular weights, resulting from the binding of zinc ions to the peptide molecules. In addition, when using 4 mg rather than 2 mg ZnGlu/mg N.sub.2 in the mammalian brain protein hydrolysate, the peak at RT ~ 52.5 min loses in intensity and an additional small peak appears at RT ~ 48 min. Amino acids elute in the peak at RT ~ 58 min. The minor shift to lower RT shows the formation of amino acid-zinc complexes.

[0065] FIG. 3 shows a dose response of NPEP-Z on BBB permeability.

[0066] FIG. 4 shows Western Blot data of NPEP-Z on endothelial proteins.

[0067] FIG. 5 shows Western Blot data of NPEP-Z on endothelial proteins under tPA condition.

[0068] FIG. 6 shows Western Blot data of NPEP-Z on endothelial proteins under fibrin condition.

[0069] FIG. 7 shows the percentage of p75NTR expression on surfaces of differently treated PC12 Cells relative to medium treated Cells. PC12 Cells were cultivated for 24 hours with 100 pl/ml N-PEP-Z or Zn only dilutions as indicated and analysed for the expression of p75NTR using flow cytometry.

EXAMPLES

Preparation of Mammalian Brain Protein Hydrolysate - Zinc Complexes

Lab Experiment

[0070] An aqueous solution of zinc gluconate (10 ml, 350 - 750 mg) was added with stirring to a mammalian brain protein hydrolysate solution (20 ml, 7-15 mg/ml nitrogen (N.sub.2), pH 2-7). The pH changed, indicative of complex formation. After adjustment to neutral, the suspension was used for analysis.

Pilot Experiment

[0071] Mammalian brain protein hydrolysate solution (40 1) was adjusted to pH 3 using 6N HCl. Zinc gluconate (858 g) was added with stirring. The obtained clear solution was sterile filtered. 40 ml of the solution was adjusted to pH 7.2 and then centrifuged before analysis.

Analytical Characterization

[0072] To determine the percentage of zinc forming water soluble complexes with peptide and amino acids, the supernatant was subjected to size exclusion chromatography. The collected fractions were analyzed for zinc using microwave digestion/ICP-MS.

[0073] To determine the percentage of peptides and free amino acids in the water-soluble fraction and in the precipitate, total nitrogen content and free amino acid concentration were determined both in the supernatant and in the mammalian brain protein hydrolizate.

[0074] For size exclusion chromatography, a Superdex Peptide 10/300 column was used. 50 .Math.L of the filtered supernatant was eluted with 100 mM ammonium acetate in 30% acetonitrile at a flow rate of 0.4 ml/min. UV detection was at 214 nm. The eluate was collected in three fractions and analyzed for zinc. Ribonuclease A, somatostatin and serine were used for column calibration.

[0075] For nitrogen analysis, the Flash Dynamic Combustion method was used. The determination of the concentration of the individual free amino acids was based on pre-column derivatization RP-HPLC.

[0076] Nitrogen content of peptides, calculated by subtracting the nitrogen content of all amino acids from the total nitrogen content, was used as a surrogate for peptide content.

[0077] FIG. 1 shows the size exclusion chromatograms of mammalian brain protein hydrolysate and the water-soluble fraction of protein hydrolysate - Zn complexes formed by addition of 2 or 4 mg Zn gluconate (ZnGlu)/mg nitrogen (N2) in the mammalian brain protein hydrolysate.

[0078] FIG. 2 shows the enlarged part of the SE-chromatograms showing, upon complexation, a tiny shift in the peak positions to shorter retention times, i.e. higher molecular weights, resulting from the binding of zinc ions to the peptide molecules. In addition, when using 4 mg rather than 2 mg Zn gluconate (ZnGlu)/mg N.sub.2 in the protein hydrolysate, the peak at RT ~ 52.5 min loses in intensity and an additional small peak appears at RT ~ 48 min. Amino acids elute in the peak at RT ~ 58 min. The minor shift to lower RT shows the formation of amino acid-zinc complexes.

TABLE-US-00001 Percentage of zinc found in the individual SEC fractions of water-soluble mammalian brain protein hydrolysate - zinc-complexes when using 2, 4 (both lab experiments) or 2.5 (pilot experiment) mg ZnGlu/mg N.sub.2 in the protein hydrolysate lab experiment pilot experiment # no. composition RT (min) % Zn (2 mg ZnGlu/mg N.sub.2) % Zn (4 mg ZnGlu/mg N.sub.2 ) % Zn (2.5 mg ZnGlu/mg N.sub.2) 1 peptides 30 - 50 15.2 27.8 27.3 2 peptides 50 - 55 24.4 26.8 27.7 3 amino acids 55 - 60 4.4 3.1 3.0 soluble Zn 44.0 57.7 58.0

TABLE-US-00002 Percentage of amino acids and peptides found in the individual SEC fractions of water-soluble mammalian brain protein hydrolysate -zinc complexes when using 2, 4 (both lab experiments) or 2.5 (pilot experiment) mg ZnGlu/mg N.sub.2 in the protein hydrolysate lab experiment pilot experiment (2 mg ZnGlu/mg N.sub.2) (4 mg ZnGlu/mg N.sub.2) (2.5 mg ZnGlu/mg N.sub.2) % amino acids 92.9 85.4 87.3 % peptides 89.1 85.5 81.2

Effects of Peptide-Zinc Complex on Human Neuroendothelial Cells in Vitro.

[0079] To investigate a dose response of N-PEP-Z on transendothelial permeability, human cerebral endothelial cells were seeded in the insert of a transwell and FITC-dextran was added into the inserted well (Li et al., Stroke 50 (2019), 2547-2554). The cerebral endothelial cells were treated with tissue plasminogen activator (tPA) (10.Math.l/ml), or fibrin (1.5.Math.l/ml) in the presence or absence of N-PEP-Z or controls. FITC-dextran signal in the main well were quantified as an index of transendothelial permeability. Doses of N-PEP-Z were adapted from preclinical study with N-PEP-12 (Windisch et al., J. Neural Transm. 112 (2004), 1331-1343). FITC signals were measured in the individual main well after transwell removal. The FITC signals in the control group were then normalized to 1 and any changes in the individual intervention groups were calculated and compared to the control group. Data in FIGS. 3 to 6 are presented as fold changes of BBB permeability vs the control (y-axis).

Effect of N-PEP-Z on Inflammatory Cytokines and Tight Junction Proteins

[0080] Cerebral endothelial cells were treated with tPA and fibrin in the presence or absence of N-PEP-Z for 24 h. After treatment, total proteins were extracted from the treated endothelial cells and Western blot analysis was performed to quantify changes of proteins. Proteins to induce thrombosis, inflammation and vascular injury were examined (including TNFα, intercellular adhesion molecule 1 (ICAM1), high mobility group protein B1 (HMGB1) and NFκB) as well as proteins mediating functional integrity of the blood brain barrier such as, Such as Zonula Occludens 1, (ZO1), Occludin, and Claudin 5.

Results

[0081] It was found that all tested doses of N-PEP-Z significantly reduced endothelial permeability induced by tPA or Fibrin (FIG. 3) whereas in unstressed condition 1.6 mg/ml of N-PEP-Z significantly reinforced endothel integrity and reduced inflammation.

[0082] In addition, it was found that compared with saline, NPEP-non-Z (no Zinc) and NPEP-low pH (no Peptide-Zinc complex formation) did not significantly alter pro-inflammatory proteins without the tPA or Fibrin stress. However, NPEP-Zinc significantly reduced pro-inflammatory proteins of ICAM1, HMGB1, and active NFκB compared with saline, NPEP-non-Zinc and NPEP-low pH whereas markers of Blood Brain Barrier integrity remained unaffected (FIG. 4). tPA (FIG. 5) or fibrin (FIG. 6) significantly increased ICAM1, HMGB1, TNFα, and active NFκB and reduced tight junction proteins, ZO1, Occludin, and Claudin 5. NPEP-Z (lowest concentration tested in FIG. 3) significantly reversed tPA- (FIG. 5) or fibrin-altered (FIG. 6) proteins.

[0083] The observed positive impact of N-PEP zinc according to the present invention on the basal state of inflammation (no tPA or fibrin stress) shows the use of this composition in non-pathological conditions having a strong contribution of inflammatory components. Such changes occur during normal aging and are associated with cognitive decline in the elderly (Simen et al. Ther. Adv. Chronic. Dis. (2011), 175-195) but also during stress induced damage of the hippocampal formation (Gulyaeva et al., Biochemistry, (2019), 1306-1328).

Effects of N-PEP-Z on Surface Expression of p75NTR in PC12 Cells

[0084] The neurotrophin receptor p75NTR is a member of the TNF receptor superfamily and involved in the regulation of survival of neurons. The receptor is known to be upregulated under pathologic conditions mediating apoptosis (for review see Dechant G. et al., Nature Neuroscience 2002). Many studies show the upregulation of p75NTR during Stroke or Traumatic Brain Injury (Grade S. et al., PlosOne 2013; Irmady K. et al., J. of Neurosci. 2014; Shi J. et al., Stem Cell 2013), as well as in Alzheimer’s disease (Y Hu et al., Cell and Disease 2013; S. Ito et al., Neurochemistry International 2016; B. Charkravathy et al., Journal of Alzheimer’s disease 2009) and several other neurodegenerative diseases (for review see Meeker R. et al., J Neuroimmune Pharmacol. 2014).

Method

[0085] To investigate a putative effect of N-PEP-Z on p75NTR-receptor expression, PC12 Cells were seeded into 6-well plates and treated with N-PEP-Z comprising Zn-concentrations from 0 to 0.12 relative to N-PEP-Z stock solution (31.84 mM Zn). The dilutions were prepared as described in Table 1 (a Zn-gluconate solution was diluted the same way in PBS, serving as “Zn only control”). The PC12 Cells were treated with 100 .Math.l/ml of the respective dilutions for 24 h. After treatment, PC12 cells were collected for surface staining with Alexa-647 conjugated anti-p75NTR antibody. Alexa-647 signals were measured using flow cytometry and normalized to medium treated PC12 Cells (% expression). Data in FIG. 7 is presented as % expression of p75NTR receptor (y-axis).

TABLE-US-00003 Used dilutions for the N-PEP-Z treatment 1x N-PEP [.Math.L] 1x N-PEP-Z [.Math.L] N-PEP 0.12x Zn 880 120 N-PEP 0.10x Zn 900 100 N-PEP 0.08x Zn 920 80 N-PEP 0.06x Zn 940 60 N-PEP 0.04x Zn 960 40 N-PEP 0.02x Zn 980 20 N-PEP 0.00x Zn 1000 0

Results

[0086] Dose response curve of N-PEP-Z showed a significant reduction of p75NTR surface expression compared to Zn only treatment. The inflection point of the dose response curve was determined at 0.06x Zn (FIG. 7).

[0087] In this study the downregulation of the p75NTR receptor by N-PEP-Z complexes on PC12 cell surfaces was investigated. These data show the protective activity of N-PEP-Z complexes according to the present invention during neurodegenerative disease progression by regulating elevated p75NTR levels.

[0088] The present invention therefore discloses the following preferred embodiments:

[0089] 1. Method for producing a mammalian brain protein hydrolysate composition, comprising the following steps: [0090] providing a mammalian brain protein hydrolysate, [0091] adding Zn ions to the mammalian brain protein hydrolysate to form a complex comprising the Zn ions and the mammalian brain protein hydrolysate, [0092] obtaining the complex comprising the Zn ions and the mammalian brain protein hydrolysate in a mammalian brain protein hydrolysate composition.

[0093] 2. Method according to embodiment 1, wherein the mammalian brain protein hydrolysate is a hydrolysate of porcine or bovine brain tissue, preferably an intermediate of the brain protein hydrolysate manufacturing process, especially a peptide-enriched intermediate product.

[0094] 3. Method according to embodiment 1 or 2, wherein the Zn ions are added as such as Zn gluconate, Zn sulfate or Zn acetate, preferably as Zn gluconate, and preferably in amount of 0.1 to 500 mg/ml, preferably from 0.5 to 100 mg/ml, especially from 1 to 70 mg/ml.

[0095] 4. Method according to any one of embodiments 1 to 3, wherein the Zn ions are added to the mammalian brain protein hydrolysate at a pH of 2.5 to 7.5. preferably of 3.0 to 6.5.

[0096] 5. Method according to any one of embodiments 1 to 4, wherein the mammalian brain protein hydrolysate is an enzymatically hydrolysated mammalian brain protein hydrolysate, preferably hydrolysated by pancreatin, trypsin, pepsin, chymotrypsin, papain, carboxypeptidase, or mixtures thereof.

[0097] 6. Method according to any one of embodiments 1 to 5, wherein the pH of the mammalian brain protein hydrolysate composition is adjusted upon formation of the complex comprising the Zn ions and the mammalian brain protein hydrolysate to a pH of 3.0 to 9.0, preferably [0098] (a) to a pH of 5.0 to 9.0, more preferred to a pH of 6.0 to 8.0, especially to a pH of 7.0 to 7.5; or [0099] (b) to a pH of 3.0 to 6.0, more preferred to a pH of 3.0 to 5.0, especially to a pH of 4.0 to 5.0 and/or to a pH of about 4.7, namely of 4.5 to 4.9.

[0100] 7. Method according to any one of embodiments 1 to 7, wherein the mammalian brain protein hydrolysate provided contains from 0.5 to 100 mg/ml nitrogen (N.sub.2), preferably 1 to 50 mg/ml, especially 5 to 20 mg/ml.

[0101] 8. Method according to any one of embodiments 1 to 7, wherein the Zn ions are added to the mammalian brain protein hydrolysate in a relative amount of 0.01 to 50 mg Zn per mg mammalian brain protein hydrolysate (as mg N.sub.2), preferably of 0.05 mg to 20 mg Zn per mg mammalian brain protein hydrolysate (as mg N.sub.2), especially 0.1 mg to 4 mg Zn per mg mammalian brain protein hydrolysate (as mg N.sub.2) .

[0102] 9. Method according to any one of embodiments 1 to 8, wherein the obtained complex is centrifuged and the supernatant with the Zn complexed mammalian brain protein hydrolysate is separated from a precipitate formed during centrifugation.

[0103] 10. Method according to any one of embodiments 1 to 8, wherein the Zn complexed mammalian brain protein hydrolysate is adjusted to a concentration of 0.05 to 50 mg/ml, preferably from 0.1 to 25 mg/ml, especially from 0.2 to 20 mg/ml.

[0104] 11. Method according to any one of embodiments 1 to 10, wherein the Zn complexed mammalian brain protein hydrolysate is finished to a final preparation, preferably to a nutritional supplement or to a pharmaceutically acceptable preparation, especially by filling the Zn complexed protein hydrolysate into a nutritionally acceptable dosage form or into a pharmaceutically acceptable container.

[0105] 12. Method according to any one of embodiments 1 to 11, wherein the Zn complexed mammalian brain protein hydrolysate is dried, preferably spray-dried, fluid bed dried or lyophilised.

[0106] 13. Method according to any one of embodiments 1 to 12, wherein additional substances are admixed, preferably formulation additives, stabilizers, nutritional additives, pharmaceutically acceptable excipients, or mixtures thereof, especially carbohydrates, such as lactose, mannitol, maltodextrin, sucrose, dextran, trehalose, cellulose, hydrolysed starch, and mixtures thereof.

[0107] 14. Composition comprising a Zn complexed mammalian brain protein hydrolysate, preferably obtainable according to any one of embodiments 1 to 13.

[0108] 15. Nutritional supplement comprising a Zn complexed mammalian brain protein hydrolysate, preferably obtainable according to any one of embodiments 1 to 13.

[0109] 16. Nutritional supplement according to embodiment 15 in dose form.

[0110] 17. Nutritional supplement according to embodiment 16, wherein the does form is provided as capsules, pastilles, tablets, pills and other similar forms, sachets of powder, ampoules of liquids, drop dispensing bottles, and other similar forms of liquids and powders designed to be taken in measured small unit quantities.

[0111] 18. Nutritional supplement according to embodiment 15 to 17, wherein the composition contains 5 to 1000 mg, preferably 7.5 to 500 mg, especially 10 to 300 mg, of Zn complexed mammalian brain protein hydrolysate per ml in aqueous solution, or may be reconstituted to such an aqueous solution.

[0112] 19. Nutritional supplement according to any one of embodiments 15 to 18, wherein composition is in dose form wherein the dose contains 0.1 to 100 ml preferably 1 to 50 ml of Zn complexed mammalian brain protein hydrolysate, corresponding to 21.5 to 21.520 mg of Zn complexed mammalian brain protein hydrolysate in aqueous solution, or may be reconstituted to such an aqueous solution.

[0113] 20. Nutritional supplement according to any one of embodiments 15 to 19, having a pH in aqueous solution of 3.0 to 9.0, preferably [0114] (a) a pH of 5.0 to 9.0, more preferred a pH of 6.0 to 8.0, especially a pH of 7.0 to 7.5; or [0115] (b) a pH of 3.0 to 6.0, more preferred to a pH of 3.0 to 5.0, especially a pH of 4.0 to 5.0 and/or a pH of about 4.7, namely a pH of 4.5 to 4.9.

[0116] 21. Nutritional supplement according to any one of embodiments 15 to 20, wherein the Zn complexed mammalian brain protein hydrolysate has in aqueous solution a concentration of 0.05 to 50 mg/ml, preferably from 0.1 to 25 mg/ml, especially from 0.2 to 20 mg/ml, or wherein the Zn complexed mammalian brain protein hydrolysate is reconstitutable to an aqueous solution a concentration of 0.05 to 50 mg/ml, preferably from 0.1 to 25 mg/ml, especially from 0.2 to 20 mg/ml.

[0117] 22. Nutritional supplement according to any one of embodiments 15 to 21, in dose form, wherein the dose contains 5 to 1000 mg, preferably 7.5 to 500 mg, especially 10 to 300 mg, of Zn complexed mammalian brain protein hydrolysate.

[0118] 23. Nutritional supplement for use according to any one of embodiments 15 to 22, wherein the Zn complexed mammalian brain protein hydrolysate contains at least one peptide selected from the group NMVPFPR, ASAFQGIGSTHWVYDGVGNS, and DLHW.

[0119] 24. Nutritional supplement according to any one of embodiments 15 to 23, in dry form, preferably as capsule, pastille, tablet, pill, or sachet of powder, especially as coated tablet or capsule, for example a coating comprising methyl acrylate-methacrylic acid copolymers, gelatine, collagen, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose acetate trimellitate, sodium alginate, zein, enteric coating aqueous solution (ethylcellulose, medium chain triglycerides (coconut), oleic acid, sodium alginate, stearic acid) (coated softgels), or mixtures thereof.

[0120] 25. Nutritional supplement according to any one of embodiments 15 to 24, comprising, per dose, 10 mg to 5 g, preferably 50 mg to 1 g, especially 100 mg to 500 mg, of a carbohydrate as an additional substance, preferably a disaccharide, especially lactose, trehalose, or mixtures thereof.

[0121] 26. Nutritional supplement according to any one of embodiments 15 to 25, wherein the nutritional supplement is in dose form and contains a total amount of Zn from 0.05 to 25 mg, preferably from 0.1 to 15 mg, especially from 0.5 to 10 mg.

[0122] 27. Nutritional supplement according to any one of embodiments 15 to 26, wherein the nutritional supplement further comprises vitamins, such as vitamin A, different vitamins of the B group, vitamin C, vitamin D, E and/or K.

[0123] 28. Nutritional supplement according to any one of embodiments 15 to 27, wherein the nutritional supplement further comprises mineral substances and/or trace elements, such as calcium, magnesium, iron, copper, sodium, manganese, iodine, potassium, selenium, chromium, molybdenium, fluorine, chlorine, and/or phosphorous.

[0124] 29. Nutritional supplement according to any one of embodiments 15 to 28, wherein the nutritional supplement further comprises caffeine, taurine, fatty acids, such as Q-fatty acids, alpha lipoic acid, phospholipids, phosphatidylserines, and/or plant extracts.

[0125] 30. Nutritional supplement according to any one of embodiments 15 to 29, wherein the nutritional supplement further comprises flavouring substances, colorants, like titanium dioxide or ferric oxides, and/or preserving agents.

[0126] 31. Nutritional supplement according to any one of embodiments 15 to 30, wherein the nutritional supplement further comprises preserving agents selected from the group consisting of ethylparaben (p-Hydroxybenzoic acid ethyl ester), benzalkonium chloride, benzethonium chloride, benzoic acid, butylparaben (p-Hydroxybenzoic acid butyl ester), methylparaben (p-Hydroxybenzoic acid methyl ester), potassium sorbate, propionic acid, propylparaben (p-Hydroxybenzoic acid propyl ester), sodium benzoate, sodium propionate, sorbic acid, and/or mixtures thereof.

[0127] 32. Nutritional supplement according to any one of embodiments 15 to 31, wherein the nutritional supplement is provided for use to prevent, ameliorate, and/or counteract deficiencies related with the aging process preferably in humans, especially in elderly humans.

[0128] 33. Pharmaceutical composition comprising a Zn complexed mammalian brain protein hydrolysate, preferably obtainable according to any one of embodiments 1 to 13, and a pharmaceutically acceptable excipient.

[0129] 34. Pharmaceutical composition according to embodiment 33, wherein the composition contains 50 to 1000 mg, preferably 75 to 500 mg, especially 100 to 300 mg, of Zn complexed mammalian brain protein hydrolysate per ml in aqueous solution.

[0130] 35. Pharmaceutical composition according to embodiment 33 or 34, wherein composition is in dose form wherein the dose contains 0.1 to 100 ml preferably 1 to 50 ml of Zn complexed mammalian brain protein hydrolysate, corresponding to 21.5 to 21.520 mg of Zn complexed mammalian brain protein hydrolysate.

[0131] 36. Pharmaceutical composition according to any one of embodiments 33 to 35, for use in a therapeutic treatment, wherein the composition preferably contains Zn complexed mammalian brain protein hydrolysate.

[0132] 37. Pharmaceutical composition for use according to any one of embodiments 33 to 36, wherein the treatment is performed with an intramuscularly administered dose of 0.1 to 10 ml, preferably 0.5 to 5 ml, corresponding to 108 mg to 1076 mg Zn complexed mammalian brain protein hydrolysate.

[0133] 38. Pharmaceutical composition for use according to any one of embodiments 33 to 37, wherein the treatment is performed with an intravenously administered dose of 0.1 to 100 ml, preferably 1 to 50 ml, corresponding to 215.2 to 21.520 mg Zn complexed mammalian brain protein hydrolysate.

[0134] 39. Pharmaceutical composition according to any one of embodiments 33 to 38, wherein the nutritional supplement is in dose form and contains a total amount of Zn from 0.1 to 25 mg, preferably from 0.5 to 10 mg, especially from 0.5 to 5 mg.

[0135] 40. Pharmaceutical composition for use according to any one of embodiments 33 to 39, wherein by continuously infusing Zn complexed mammalian brain protein hydrolysate.

[0136] 41. Pharmaceutical composition for use according to embodiment 40, wherein infusion is performed for an infusion duration of 5 min to 4 h, preferably 10 min to 2 h, especially 15 to 60 min; and/or wherein infusion is performed for 1 to 100 d, preferably from 5 to 50 d, especially from 10 to 30 d.

[0137] 42. Pharmaceutical composition for use according to embodiment 41, wherein infusion is performed once per day.

[0138] 43. Pharmaceutical composition for use according to any one of embodiments 40 to 42, wherein infusion is performed by dilution of Zn complexed mammalian brain protein hydrolysate with 0.9% sodium chloride solution, Ringer’s solution, or 5 % glucose.

[0139] 44. Pharmaceutical composition for use according to any one of embodiments 33 to 43, wherein the composition contains a Zn complexed mammalian brain protein hydrolysate and sodium hydroxide.

[0140] 45. Pharmaceutical composition for use according to any one of embodiments 33 to 44, wherein the treatment is performed in treatment cycles which are repeated after a treatment-free period of 1 to 6, preferably of 1 to 3, especially of 2 to 3, months.

[0141] 46. Pharmaceutical composition for use according to any one of embodiments 33 to 45, in treating and preventing cerebrovascular disease, preferably cerebral Ischemia (8B10, 8B11, 8B1Y/Z), especially intracranial haemorrhage (8B00-3), cerebrovascular disease with no acute cerebral symptom (8B21), certain specified cerebrovascular diseases (8B22), hypoxic-ischaemic encephalopathy (8B24), late effects of cerebrovascular disease (8B25), vascular syndromes of brain in cerebrovascular diseases (8B26), cerebrovascular diseases, unspecified (8B2Z); disorders with neurocognitive impairment as a major feature, preferably Alzheimer disease (8A20), progressive focal atrophies (8A21), Lewy body disease (8A22), frontotemporal lobar degeneration (8A23), other specified disorders with neurocognitive impairment as a major feature (8A2Y), disorders with neurocognitive impairment as a major feature, unspecified (8A2Z); headache disorders (8A80-85), preferably other specified headache disorders (8A8Y) and headache disorders, unspecified (8A8Z); motor neuron diseases or related disorders, preferably motor neuron disease (8B60), spinal muscular atrophy (8B61), post-polio progressive muscular atrophy (8B62), other specified motor neuron diseases or related disorders (8B6Y), and motor neuron diseases or related disorders, unspecified (8B6Z); injuries of the nervous system, especially injury of cranial nerves (NA04), intracranial injury (NA07), and crushing injury of head (NA08); hypoxic ischaemic encephalopathy of newborn (8B24KB04), spinal cord disorders excluding trauma, disorders of nerve root, plexus or peripheral nerves, diseases of neuromuscular junction or muscle, disorders of nerve root, plexus or peripheral nerves, cerebral palsy, disorders of autonomic nervous system, and postprocedural disorders of the nervous system; associated memory impairment, age associated cognitive decline, mental agility support, compromised age associated microvascular patency, protection of cell constituents (DNA, Proteins and Lipids) from oxidative damage, normalization of immune system and vascular function, improvement of neuron vitality and recovery, support of nerve growth and connection, enhancing cognitive function and activating bioelectrical activity, preferably in healthy subjects, especially in healthy elderly subjects (age: 51+), protecting against age and disease associated lesions in cortical neurons.

[0142] 47. Use of a composition as defined in any one of the embodiments 14 to 46 for non-therapeutic treatment, preferably for ameliorating age associated memory impairment and activating bioelectrical activity in healthy subjects, especially in healthy elderly subjects (age: 51+).