Animal plasma or fractions thereof for use in treating cognitive impairment disorders in humans and companion animals

Abstract

The present application relates to the administration of plasma, fractions thereof, or mixtures thereof to humans or animals to treat or otherwise improve cognitive impairment disorders, including dementia (e.g., vascular dementia, dementia with Lewy bodies, dementia resulting from Alzheimer's disease, dementia resulting from Parkinson's disease, frontotemporal dementia, and dementia resulting from normal pressure hydrocephalus in humans, and cognitive dysfunction syndrome in companion animals), concussion, and traumatic brain injury. In certain embodiments, the invention comprises a method of treating a cognitive impairment disorder in a human or companion animal subject, said method comprising: administering to said subject one or more cognitive functioning tests to identify a subject suffering from a cognitive impairment disorder; and administering to said subject a therapeutically effective amount of an animal plasma composition; wherein said administration provides an improvement in said subject's results in said one or more cognitive impairment tests.

Claims

1. A method of treating a cognitive impairment disorder in a human or companion animal subject, said method comprising: a. administering to said subject one or more cognitive functioning tests to identify a subject suffering from a cognitive impairment disorder; and b. orally administering to said subject a therapeutically effective amount of an animal plasma composition; wherein said administration provides an improvement in said subject's results in said one or more cognitive impairment tests.

2. The method of claim 1, wherein said animal plasma composition is derived from animal plasma from one or more animals selected from the group consisting of porcine, bovine, ovine, equine, and avian animals.

3. The method of claim 1, wherein said animal plasma composition is administered at a dose of 5 mg to 100 g per day.

4. The method of claim 1, wherein said animal plasma composition is administered at a dose of 500 mg to 5 g per day.

5. The method of claim 1, wherein said animal plasma composition is administered at a dose of 10 mg to 1 g per kg of body weight of the animal to be treated per day.

6. The method of claim 1, wherein said animal plasma composition is administered at a dose of 25-100 mg per kg of body weight of the animal to be treated per day.

7. The method of claim 1, wherein said animal plasma composition is administered for at least 3 months.

8. The method of claim 1, wherein said animal plasma composition is administered for at least 9 months.

9. The method of claim 1, wherein said animal plasma composition is administered for at least 12 months.

10. The method of claim 1, wherein said cognitive impairment disorder is selected from the group consisting of dementia disorders, concussion, and traumatic brain injury.

11. The method of claim 1, wherein said cognitive impairment disorder is a concussion.

12. The method of claim 1, wherein said cognitive impairment disorder is a traumatic brain injury.

13. The method of claim 1, wherein said cognitive impairment disorder is a dementia disorder.

14. The method of claim 1, wherein said cognitive impairment disorder is dementia resulting from Alzheimer's disease.

15. The method of claim 1, wherein said cognitive impairment disorder is dementia resulting from Parkinson's disease.

16. The method of claim 1, wherein said cognitive impairment disorder is vascular dementia.

17. The method of claim 1, wherein said subject is a companion animal selected from the group consisting of canine, feline, or equine.

18. The method of claim 1, wherein said subject is a human.

19. The method of claim 1, wherein said animal plasma composition is administered in food.

20. The method of claim 1, wherein said improvement in said subject's results in said one or more cognitive impairment tests comprises improved short term memory.

21. The method of claim 1, wherein said improvement in said subject's results in said one or more cognitive impairment tests comprises improved long term memory.

22. The method of claim 1, wherein said animal plasma composition comprises 70-90% by weight protein.

23. The method of claim 22, wherein said animal plasma composition comprises: i. about 1.0-6.0% by weight alpha-2 macroglobulin; j. about 1.0-5.5% by weight transferrin; k. about 0.1-1.0% by weight vitamin-D binding protein; l. about 0.1-2.0% by weight alpha-1-glycoprotein; m. about 5-30% by weight IgG; n. about 1.0-6.0% by weight IgA; o. about 0.5-5.0% by weight of IgM; and p. about 25-80%-by weight albumin.

24. The method of claim 23, wherein said animal plasma composition comprises: i. 2.0-3.5% by weight alpha-2 macroglobulin; j. 2.0-3.0% by weight transferrin; k. 0.3-0.45% by weight vitamin-D binding protein; l. 0.5-1.2% by weight alpha-1-glycoprotein; m. 10-20% by weight IgG; n. 1.5-3.25% by weight IgA; o. 0.75-3.0% by weight of IgM; and p. 40-50% by weight albumin.

25. The method of claim 1, said animal plasma composition comprises 90-95% by weight protein.

26. The method of claim 25, wherein said animal plasma composition comprises: i. about 1.0-9.0% by weight alpha-2 macroglobulin; j. about 2-18% by weight transferrin; k. about 0.1-1.0% by weight vitamin-D binding protein; l. about 0.1-2.0% by weight alpha-1-glycoprotein; m. about 25-75% by weight IgG; n. about 1.0-6.0% by weight IgA; o. about 2.5-10% by weight of IgM; and p. about 2.5-25% by weight albumin.

27. The method of claim 26, wherein said animal plasma composition comprises: i. 3.0-6.0% by weight alpha-2 macroglobulin; j. 4-10% by weight transferrin; k. 0.25-0.6% by weight vitamin-D binding protein; l. 0.4-1.3% by weight alpha-1-glycoprotein; m. 40-65% by weight IgG; n. 1.5-3.25% by weight IgA; o. 3.5-7% by weight of IgM; and p. 7-15% by weight albumin.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Other advantages and features of the invention can be seen from the following description in which preferred non-limiting embodiments of the invention are described in reference to the attached drawings, where:

(2) FIG. 1 provides the evolution of body weights of control (SAMR1) and experimental mice (SAMP8), with or without SDP supplementation.

(3) FIG. 2 provides the food intake of control and experimental mice. Results are expressed as means±SEM (n=20-30 mice).

(4) FIG. 3 provides a motor activity dendrogram.

(5) FIG. 4 provides the experimental design and groups used to study the cognitive function.

(6) FIG. 5 provides an image of an NOR Test maze.

(7) FIG. 6 provides the food intake of control and experimental mice. Results are expressed as means±SEM (n=34-40 mice).

(8) FIG. 7 provides evolution of body weights of SAMP8 mice, with or without SDP supplementation. Results are expressed as means±SEM (n=9-19 mice). Means with a common letter differ, P<0.1.

(9) FIG. 8 provides the explorative preference of the experimental groups. Results are expressed as means±SEM (n=10-13 mice).

(10) FIG. 9 provides the explorative index at 5 min of the acquisition trial. Results are expressed as means±SEM (n=10-13 mice). Means with a common letter differ, P<0.1.

(11) FIG. 10 provides the discrimination index for each of the experimental groups in the first retention trial, which evaluated short-term memory retention. Results are expressed as means±SEM (n=10-13 mice). Means with a common letter differ, P<0.1.

(12) FIG. 11 provides the discrimination index for each of the experimental groups in the second retention trial, which evaluated long-term memory retention. Results are expressed as means±SEM (n=10-13 mice). Means with a common letter differ, P<0.1.

(13) FIG. 12 provides the percentage of mice determined to possess and lack a sufficient short-term memory in each of the experimental groups in the first retention trial (n=10-13 mice).

(14) FIG. 13 provides the percentage of mice determined to possess and lack a sufficient long-term memory in each of the experimental groups in the second retention trial (n=10-13 mice).

(15) FIG. 14 provides the expression of synaptophysin in cortex (panel A) and in hippocampus (panel B) for mice in the 2-month, 6-month control, and 6-month SDP groups. Results are expressed as means±SEM (n=6-8 mice). Means with a common letter differ, P<0.01.

(16) FIG. 15 provides the expression (Real-Time PCR) and abundance (Western Blot) of synaptophysis in brain tissue.

(17) FIG. 16 provides the expression (Real-Time PCR) and abundance (Western Blot) of PSD-95 in brain tissue.

(18) FIG. 17 provides the abundance (immunohistochemistry) of GFAP in brain cells.

(19) FIG. 18 provides the abundance (immunohistochemistry) of IBA1 in brain cells.

(20) FIG. 19 provides the expression (Real-Time PCR) and abundance (Western Blot) of total Tau in brain tissue.

(21) FIG. 20 provides the expression (Real-Time PCR) and abundance (Western Blot) of p-Tau in brain tissue.

(22) FIG. 21 provides the expression (Real-Time PCR) and abundance (Western Blot) of sAPP-β in brain tissue.

(23) FIG. 22 provides the expression (Real-Time PCR) and abundance (Western Blot) of sAPP-α in brain tissue.

(24) FIG. 23 provides the expression (Real-Time PCR) of BACE1 in brain tissue.

(25) FIG. 24 provides the expression (Real-Time PCR) of ADAM10 in brain tissue.

DETAILED DESCRIPTION OF THE INVENTION

(26) Embodiments may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that the embodiments are not bound by any theory presented.

(27) Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

(28) The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include plural forms as well, unless the context clearly indicates otherwise. Thus, for example, reference to “a dose” or “the dose” also includes a plurality of doses. Additionally, as used herein, the term “comprises” is intended to indicate a non-exhaustive list of components or steps, thus indicating that the given composition or method includes the listed components or steps and may also include additional components or steps not specifically listed. As an example, a core weight “comprising SBI” may also include additional components, such as flavorants, colorants, etc. The term “comprising” is also intended to encompass embodiments “consisting essentially of” and “consisting of” the listed components or steps. Similarly, the term “consisting essentially of” is also intended to encompass embodiments “consisting of” the listed components or steps. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

(29) Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(30) The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of 10% of the referenced value. In other embodiments, the term “about” indicates that the number differs from the given number by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

(31) Plasma Protein Compositions:

(32) The present application relates to the effects of the administration of plasma protein compositions, such as SDP, on cognitive impairment. Plasma protein compositions useful in the present invention can be obtained from any suitable animal source. Preferably the animal plasma, fractions thereof or mixtures thereof are derived from the group of animals formed by porcine, bovine, ovine, equine and avian.

(33) Various protein fractions and/or components can be purified from plasma using methods that are well known and commonly practiced by those of ordinary skill in the art. For example, globulin concentrates can be obtained by spray drying, lyophilization, or any other drying method. One preferred method involves spray-drying of plasma separated from animal blood to produce a dried composition of plasma proteins, referred to herein as spray-dried plasma (SDP). Spray-dried plasma consists primarily of albumin and globulins, along with lesser quantities of other proteins or peptides. As used herein, unless otherwise indicated, the terms “plasma,” “plasma proteins,” and “SDP” can be used interchangeably and encompass blood plasma and/or any protein fractions and/or components which may be further purified therefrom.

(34) Plasma proteins used of the current application can be used in any suitable form, including both dried and liquid forms. In certain embodiments, the compositions used in this application can be in the form of tablets, capsules, ampoules for oral use, granulate powder, cream, both as a unique ingredient and associated with other excipients or active compounds, or even as a feed additive. In certain embodiments, the plasma protein compositions described herein can be provided or administered in powder form, in some instances suspended or dissolved in a suitable liquid, such as water, saline, or milk. The plasma protein composition can be combined with a pharmaceutically acceptable carrier such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solutions of dextrose, and the like. In general, in addition to the active compounds, the compositions of this invention may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. In certain embodiments, the plasma proteins may also be microencapsulated, thereby protecting and stabilizing them from high temperature, oxidants, pH-like humidity, etc.

(35) In one embodiment of the invention, the plasma protein composition contains at least about 10% by weight Ig. In other embodiments, the plasma protein composition contains from about 10% to about 80% by weight Ig. In further embodiments, the plasma protein composition contains from about 12% to about 75%, from about 15% to about 70%, from about 18% to about 65%, from about 20% to about 60%, from about 22% to about 65%, or from about 25% to about 50% by weight Ig. In other embodiments of the invention, the plasma protein composition contains at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%0, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, or 80% by weight Ig. In further embodiments of the invention, the plasma protein composition contains less than about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, or 80% by weight Ig.

(36) In one embodiment of the invention, the plasma protein composition contains at least about 10% by weight IgG. In other embodiments, the plasma protein composition contains from about 10% to about 80% by weight IgG. In further embodiments, the plasma protein composition contains from about 12% to about 75%, from about 15% to about 70%, from about 18% to about 65%, from about 20% to about 60%, from about 22% to about 65%, or from about 25% to about 50% by weight IgG. In other embodiments of the invention, the plasma protein composition contains at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 280, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, or 80% by weight IgG. In further embodiments of the invention, the plasma protein composition contains less than about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 240, 25%, 260, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, or 80% by weight IgG.

(37) In one embodiment of the invention, the plasma protein composition contains from about 1% to about 10% by weight IgA. In other embodiments of the invention, plasma protein composition contains about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight IgA. In further embodiments of the invention, plasma protein composition contains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight IgA. In still further embodiments of the invention, plasma protein composition contains less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight IgA. In further embodiments, the plasma protein composition contains from about 1% to about 2%, from about 1% to about 3%, from about 1% to about 4%, from about 1% to about 5%, from about 1% to about 6%, from about 1% to about 7%, from about 1% to about 8%, from about 1% to about 9%, from about 2% to about 40%, from about 2% to about 6%, from about 2% to about 8%, from about 5% to about 10%, from about 3% to about 6%, from about 3% to about 9%, or from about 8% to about 10% by weight IgA. In a further embodiment, the plasma protein composition contains about 1% by weight IgA. In a particular embodiment, the plasma protein composition contains 2% by weight or less of IgA

(38) In one embodiment of the invention, the plasma protein composition contains from about 1% to about 10% by weight IgM. In other embodiments of the invention, the plasma protein composition contains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight IgM. In further embodiments of the invention, the plasma protein composition contains less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by weight IgM. In other embodiments of the invention, the plasma protein composition contains about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight IgM. In further embodiments the plasma protein composition contains from about 1% to about 2%, from about 1% to about 3%, from about 1% to about 4%, from about 1% to about 5%, from about 1% to about 6%, from about 1% to about 7%, from about 1% to about 8%, from about 1% to about 9%, from about 2% to about 4%, from about 2% to about 6%, from about 2% to about 8%, from about 5% to about 10%, from about 3% to about 6%, from about 3% to about 9%, or from about 8% to about 10% by weight IgM. In a further embodiment, the plasma protein composition contains about 5% by weight IgM.

(39) In one embodiment of the invention, the plasma protein composition contains from about 15% to about 80% by weight albumin. In other embodiments of the invention, the plasma protein composition contains at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% by weight albumin. In other embodiments of the invention, the plasma protein composition contains less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% by weight albumin. In further embodiments the plasma protein composition contains from about 20% to about 75%, from about 25% to about 75%, from about 30% to about 70%, from about 35% to about 65%, from about 40% to about 60%, or from about 45% to about 55% by weight albumin. In particular embodiments, the plasma protein composition comprises about 45-55% albumin by weight.

(40) In other embodiments, the plasma protein composition comprises 65-95% by weight protein, and in further embodiments the plasma protein composition comprises 70-90% by weight protein. In certain embodiments, the protein component of the plama protein composition comprises a. about 1.0-6.0%, about 1.4-5.6%, about 2.0-3.5%, about 2.5-3.0%, about 2.7-2.9%, or about 2.8% by weight alpha-2 macroglobulin; b. about 1.0-5.5%, about 1.3-5.2%, about 1.8-3.3%, about 2.0-3.0%, about 2.5-2.7%, or about 2.6% by weight transferrin: c. about 0.1-1.0%, about 0.18-0.76%, about 0.2-0.55%, about 0.3-0.45%, about 0.36-0.40%, or about 0.38% by weight vitamin-D binding protein: d. about 0.1-2.0%, about 0.4-1.6%, about 0.5-1.2%, about 0.7-0.9%, or about 0.8% by weight alpha-1-glycoprotein; e. about 5-30%, about 7-28%, about 10-20%, about 12-16%, or about 14% by weight IgG; f. about 1.0-6.0%, about 1.25-5.0%, about 1.5-3.25%, about 2.0-3.0%, about 2.3-2.7%, or about 2.5% by weight IgA; g. about 0.5-5.0%, about 0.6-4.0%, about 0.75-3.0%, about 1.0-2.0%, about 1.3-1.7%, or about 1.5% by weight of IgM; and h. about 25-80%, about 30-70%, about 35-60%, about 40-50%, about 42-48%, or about 45% by weight albumin.

(41) In other embodiments, the plasma protein composition comprises 80-100% by weight protein, and in further embodiments the plasma protein composition comprises 90-95% by weight protein. In certain embodiments, the protein component of the plama protein composition comprises a. about 1.0-9.0%, about 2.0-8.0%, about 3.0-6.0%, about 3.5-5.0%, about 3.7-4.3%, or about 4.0% by weight alpha-2 macroglobulin; b. about 2-18%, about 3-15%, about 3.5-12%, about 4-10%, about 5-9%, about 6-8%, or about 7% by weight transferrin: c. about 0.1-1.0%, about 0.2-0.8%, about 0.25-0.6%, about 0.3-0.5%, about 0.38-0.42%, or about 0.4% by weight vitamin-D binding protein; d. about 0.1-2.0%, about 0.35-1.5%, about 0.4-1.3%, about 0.5-1.1%, about 0.6-0.8%, or about 0.7% by weight alpha-1-glycoprotein; e. about 25-75%, about 30-75%, about 35-70%, about 40-65%, about 45-62%, about 50-60%, or about 55% by weight IgG; f. about 1.0-6.0%, about 1.25-5.0%, about 1.5-3.25%, about 2.0-3.0%, about 2.3-2.7%, or about 2.5% by weight IgA; g. about 2.5-10%, about 3-9%, about 3.25-8%, about 3.5-7%, about 4-6%, about 4.5-5.5, or about 5% by weight of IgM; and h. about 2.5-25%, about 5-20%, about 7-15%, about 8-12%, about 9-11%, or about 10% by weight albumin.

(42) The compositions for use in the present invention are manufactured in a manner which is itself well known in the art. For example, the preparations may be made by means of conventional mixing, granulating, dragee-making, dissolving, and/or lyophilizing processes. The processes to be used will depend ultimately on the physical properties of the ingredients used and the desired form of the end product.

(43) Suitable excipients are, in particular, fillers such as sugars for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are flow-regulating agents and lubricants, for examples, such as silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate and/or polyethylene glycol. Dragee cores may be provided with suitable coatings which, if desired, may be resistant to gastric juices.

(44) For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, dyestuffs and pigments may be added to the tablet or dragee coatings, for example, for identification or in order to characterize different combination of compound doses.

(45) Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. In addition to administration with conventional carriers, active ingredients may be administered by a variety of specialized delivery drug techniques which are known to those of skill in the art.

(46) Dosage, Duration, and Subjects:

(47) In certain embodiments, the subjects to be treated are humans and/or companion animals. In certain embodiments, the companion animals are selected from the group consisting of canine, feline, or equine animals.

(48) “Administering” a composition may be accomplished by oral administration, injection, infusion, parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal, intranasal, intraperitoneal, intraarterial, subcutaneous absorption or by any method in combination with other known techniques. In one embodiment of the invention, the serum-derived immunoglobulin concentrate is administered orally. In certain embodiments, the animal plasma, fractions thereof or mixtures thereof according to the invention is administered in a pharmaceutically acceptable aerosol, through a nebulizer or is orally administered (preferably in food).

(49) A “therapeutically effective amount” can be, but is not limited, to an amount of plasma protein composition that is sufficient to provide an improvement in the subject's results in one or more cognitive impairment tests.

(50) The dosage and number of doses (e.g., single or multiple dose) administered to the subject will vary depending upon a variety of factors, including the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired, and the like. These parameters can be determined for each system by well-established procedures and analyses, e.g., in phase I, II, and III clinical trials. In one embodiment of the invention, plasma protein composition is administered once daily. In another embodiment of the invention, plasma protein composition is administered twice daily. In another embodiment of the invention, plasma protein composition is administered three times a day. In another embodiment of the invention, plasma protein composition is administered four times daily. In one embodiment of the invention, plasma protein composition is administered from about 1 week to about 25 weeks. In a further embodiment, plasma protein composition is administered from about 1-4 weeks, 1-5 weeks, 1-10 weeks, 1-15 weeks, 1-20 weeks, 5-10 weeks, 5-15 weeks, 5-20 weeks, 5-25 weeks, 10-15 weeks, 10-20 weeks, 10-25 weeks, 15-20 weeks, or 15-25 weeks. In another embodiment of the invention, plasma protein composition is administered for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months. In another embodiment of the invention, plasma protein composition is administered from about 1-2 months, 1-3 months, 1-4 months, 1-5 months, 1-6 months, 1-7 months, 1-8 months, 1-9 months, 1-10 months, 2-3 months, 2-4 months 2-5 months. In a further embodiment, plasma protein composition is administered for about 1 week. In a further embodiment, plasma protein composition is administered for about 2 weeks. In a further embodiment, SBI is administered for about 3 weeks. In a further embodiment, plasma protein composition is administered for about 4 weeks. In a further embodiment, plasma protein composition is administered for about 24 weeks.

(51) In one embodiment of the invention, plasma protein composition is administered in an amount from about 5 mg to about 100 g to the subject daily. In a further embodiment of the invention, the amount of plasma protein composition administered to the subject daily is from about 10 mg to about 90 g, from about 20 mg to about 80 g, from about 50 mg to about 70 g, from about 100 mg to about 60 g, from about 250 mg to about 50 g, from about 500 mg g to about 50 g. from about 1 g to about 50 g, from about 5 g to about 50 g, or from about 10 g to about 45 g.

(52) In one embodiment of the invention, plasma protein composition is administered in an amount from about 10 mg to about 1 g per kg body weight of the subject per day. In a further embodiment of the invention, the amount of plasma protein composition administered to the subject daily is from about 20 mg to about 900 mg per kg body weight, from about 30 mg to about 800 mg per kg body weight, from about 40 mg to about 750 mg per kg body weight, from about 50 mg to about 700 mg per kg body weight, from about 60 mg to about 650 mg per kg body weight, from about 70 mg to about 600 mg per kg body weight, from about 80 mg to about 550 mg per kg body weight, from about 90 mg to about 500 mg per kg body weight, from about 100 mg to about 500 mg per kg body weight, from about 150 mg to about 450 mg per kg body weight, from about 200 mg to about 400 mg per kg body weight per day.

(53) In certain embodiments, dosages used for a model animal, such as a mouse, rat, dog, or pig, can be converted to an appropriate dose for a human subject using conversion factors that are well known and readily available to those skilled in the art. See, e.g., “Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers,” U.S. Dept. of Health and Human Services, July 2005.

(54) In one embodiment of the invention, plasma protein composition is administered in an amount from about 0.05% to about 5% by weight of the subject's total daily dietary intake. In a further embodiment of the invention, plasma protein composition is administered in an amount from about 0.05% to about 0.1%, from about 0.05% to about 0.2%, from about 0.05% to about 0.5%, from about 0.05% to about 1% from about 0.05% to about 2%, from about 0.1% to about 0.2%, from about 0.1% to about 0.5%, from about 0.1% to about 1% from about 0.1% to about 2% from about 0.1% to about 5%, from about 0.5% to about 1%, from about 0.5% to about 2%, from about 0.5% to about 5%, from about 1% to about 2%, from about 1% to about 5%, or from about 2% to about 5% by weight of the subject's total daily dietary intake. In another embodiment, plasma protein composition is administered in an amount of about 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, or 5% by weight of the subject's total daily dietary intake. In a further embodiment, the amount of plasma protein composition administered is about 0.2% by weight of the subject's total daily dietary intake. In a further embodiment, the amount of plasma protein composition administered is about 0.4% by weight of the subject' total daily dietary intake.

(55) Having described the invention with reference to particular compositions, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates to the contrary.

REFERENCES

(56) All publications cited herein are incorporated herein by reference in their entirety for all purposes. Bene E R. Sepulveda A M, 2014. Clinical test instrument development to identify and track recovery from concussion. Semin. Speech Lang. 35:173-185 Brodaty H. Low L-F, Gibson L, Burns K. 2006. What is the best dementia screening instrument for general practitioners to use?. Am J Geriatr Psychiatry 14(5):391-400 Coffey R D, Cromwell G L. 2001. Use of spray-dried animal plasma in diets for weanling pigs. Pig News Info. 22:39N-48N. Gao Y Y, Jiang Z Y, Lin Y C, Zheng C T, Zhou G L, Chen F. 2010. Effects of spray-dried animal plasma on serous and intestinal redox status and cytokines of neonatal pigs. J. Anim. Sci. doi:10.2527/jas.2010-2967. Gisbert E, Skalli A, Campbell J. Solovyev M M, Rodriguez C, Dias J. Polo J. 2015. Spray-dried plasma promotes growth, modulates the activity of antioxidant defenses, and enhances the immune status of gilthead sea bream (Sparus aurata) fingerlings. J. Anim. Sci. 93:278-286. doi:10.2527/jas2014-7491 Heckler M C T, Tranquilim M V, Svicero D J, Barbosa L, Amorim R M. 2014. Clinical feasibility of cognitive testing in dogs (Canis lupus familiaris). J. Vet. Behavior 9:6-12 Kiraly M A and Kiraly S J. 2007 Traumatic brain injury and delayed sequelae: a review—traumatic brain injury and mild traumatic brain injury (concussion) are precursors to later-onset brain disorders, including early-onset dementia. TheScientificWorldJOURNAL 7, 1768-1776. DOI 10.1100/tsw.2007.269. Maijó M. Miró L, Polo J, Campbell J. Russell L, Crenshaw J, Weaver E, Moretó M, Pérez-Bosque A. 2011. Dietary plasma proteins attenuate the innate immunity response in a mouse model of acute lung injury. Br J. Nutr. doi:10.1017/S0007114511003655. Maijó M, Miró L, Polo J, Campbell J, Russell L, Crenshaw J. Weaver E, Moretó M, Pérez-Bosque A. 2012. Dietary plasma proteins modulate the adaptive immune response in mice with acute lung inflammation. JNutr. 142: 264-70. Moretó M, Pérez-Bosque A. 2009. Dietary plasma proteins, the intestinal immune system, and the barrier functions of the intestinal mucosa. J. Anim. Sci. 87:E92-E100. Peace R M, Campbell J, Polo J, Crenshaw J. Russell L, Moeser A. 2011. Spray-dried porcine plasma influences intestinal barrier function, inflammation and diarrhea in weaned pigs. J Nutr. 141:1312-1317. Petschow B W, Burnett B, Shaw A L, Weaver E M, Klein G L. 2014. Serum-derived bovine immunoglobulin/protein isolate: postulated mechanism of action for management of enteropathy. Clin Exp Gastroenterol 7:181-190 [PMID: 24904221 doi: 10.2147/CEG.S628231]. Shively S, Scher A I, Perl D P, Diaz-Arrastia R. 2012. Dementia resulting from traumatic brain injury: what is the pathology? Arch. Neurol. 69(10):1245-1251. doi:10.1001/archneurol.2011.3747. Torrallardona D. 2010. Spray-dried animal plasma as an alternative to antibiotics in weanling pigs: a review. Asian-Aust. J. Anim. Sci. 32:131-148. Van Dijk A J, Everts H, Nabuurs M J A, Margry R J C F, Beynen A C. 2001. Growth performance of weanling pigs fed spray-dried animal plasma: a review. Livest. Prod. Sci. 68:263-274. WHO (World Health Organization). Fact sheet 2016. http://www.who.int/mediacentre/factsheets/fs362/en/ Accessed 24 Apr. 2017. Xiong Y. Mahmood A, Chopp M. 2013. Animal models of traumatic brain injury. Nature reviews Neuroscience. 14(2):128-142. doi:10.1038/nrn3407.

EXAMPLES

(57) To study the effect of the administration of animal plasma proteins on cognitive impairment disorders, the inventors used the SAMP8 mouse model of dementia. The SAMP8 mouse model is a group of related inbred strains consisting of senescence-prone inbred strain (SAMP8) and senescence-resistant inbred strain (SAMR1), which have been successfully developed by genetic selection. The characteristic feature of aging common to the SAMP8 and SAMR1 is accelerated senescence and normal aging, respectively.

(58) To that end, SAMP8 mice can be used as a model for dementia in humans and other animals as these mice show age-related behavioral deterioration, such as deficits in learning and memory, emotional disorders (reduced anxiety-like behavior and depressive behavior) and altered circadian rhythm associated. SAMP8 mice have severely impaired acquisition and retention of the passive avoidance response and show impairment in spatial memory tasks, in which the mice learn by escaping from the aversive situation. Brains of SAMP8 mice show similar neuropathological changes to those of Alzheimer's disease brains from humans, that is, deposition of β-amyloid protein. Also SAMP8 mice have spongiform degeneration related with vacuoles of various sizes in the brain while no vacuoles were evident in the brain of SAMR1 mice.

(59) The inventors unexpectedly observed that supplement based on animal plasma proteins, fractions thereof, or mixtures of plasma proteins and fractions thereof fed to SAMP8 mice reduced the nocturnal motor activity of the SAMP8 mice, making the results closer to the pattern shown by the Control-SAMR1 mice, indicating improvements in orientation of these aged animals with markers of cognitive impairment disorders. The inventors also observed that variables of both short-term and long-term memory were deteriorated in aged (6 month-old) mice as compared with younger (2 month-old). The older mice showed lower explorative behaviors and memory retention. Feeding SDP for 4 months reduced the effects of aging on memory indicators, suggesting that SDP might delay the progressive deterioration of cognitive functions. The test used, novel object recognition (NOR) test, is a “pure” recognition memory test and a valid task to assess working memory. The test does not involve positive or negative reinforces and this makes NOR comparable to memory tests currently used in humans. In summary, the inventors surprisingly found that administration of plasma proteins prevents the deterioration of cognitive functions associated with aging.

Example 1—Effects of Plasma Protein Administration on Nocturnal Motor Activity in SAMP8 Mice

(60) Materials and Methods:

(61) Animals:

(62) Experiments have been done using a senescence-accelerated prone strain of mice (SAMP8). At 9 months of age, these animals have fully developed senescence. Mice of the senescence resistant strain (SAMR1) were used as controls. Mice were maintained in conventional housing (3-4 animals per cage) for 5 months (except if they showed an aggressive behavior in which case they were separated and housed individually). Mice were monitored for food intake and body weight throughout the experimental period.

(63) Diets:

(64) Mice were fed with the experimental diets for up to 4 months. The composition of experimental diets is detailed in Table 1. Food intake and body weight were monitored throughout the experimental period.

(65) TABLE-US-00001 TABLE 1 Composition of experimental diets. Diets were produced by APC-Europe, S. A. Ingredient (g/kg) Control SDP SDP — 80 Corn starch 199.3 335.7 Skim milk 530.7 370.1 Sucrose 94.5 102.7 Soybean oil 70 76.1 Cellulose 50 54.4 AIN-93-G-MX (94046)* 35 38 AIN-93 VX (94047)* 15 16.3 DL-Methionine 2.5 3.5 Choline bitartrate 3 3.3

(66) In this study, the average mouse weight was 32 g and the average daily feed intake was 4.5 g. This provided an average daily plasma intake of 11.25 g SDP/kg body weight. Based on composition of plasma being approximately 20% IgG, the average daily IgG intake was 2.25 g IgG/kg body weight.

(67) Experimental Design:

(68) Animals received the experimental diet for 3 months (from 6 to 9 months of age). The 6 month-old animals were distributed into three groups: SAMR1 (SAMR1 mice fed the Control diet), SAMP8 (SAMP8 mice fed the Control diet), and SAMP8-SDP (SAMP8 mice fed the SDP-supplemented diet). Body weight was recorded weekly. Animals were sacrificed when were 9 month-old and samples were taken.

(69) To study the effects of the different strains and the effects of dietary supplementation, data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test, using SPSS-20.0 software (SPSS Inc., Chicago, Ill.). In both analyses, differences were considered significant at P<0.05.

(70) Motor Activity: Five days before sacrifice, animals were housed individually Pattern of motor activity was studied for five days under a 12-12 hours light (LL)-dark (DD) cycle

(71) Behavioral Analysis: Motor activity was recorded through activity meters Registration of the times that the mouse crosses one of the infrared beams Data recorded every 15 minutes Calculations done with an integrated package for chronobiology

(72) Results:

(73) Body Weight Evolution:

(74) The body weight of the SAMP8 population was about a 10% lower than the SAMPR1 mice during the period studied, starting at week 26 (6 months-old) until week 39 (9 months-old; P<0.05; FIG. 1). Supplementation with SDP did not modify this pattern. Results are expressed as means±SEM (n=20-30 mice). * Indicates differences between SAMR1 and SAMP8. P<0.05.

(75) Food Consumption:

(76) Food intake was measured three times per week during the feeding period. FIG. 2 summarizes the daily mean food consumption. Food intake was the same in all groups.

(77) Motor Activity:

(78) The SAMP8 strain shows a higher nocturnal motor activity than the SAMR1 strain (FIG. 3). This would indicate that the aging pattern of this animal model is associated with increased nocturnal motor activity.

(79) The control SAMP8 mice show larger nocturnal activity than resistant mice (Control-SAMR1), indicating that there are basal activity level differences between both strains. This higher increase on motor activity in SAMP8 strains can be related with the fact that these animals had reduced memory and learning capacity, therefore these animals may have had lower orientation, as is typical in Alzheimer's disease, which can explain the higher activity of this strain compared with the resistant SAMR1 strain.

(80) Analysis of the DD/LL ratios support the view that SDP fed animals have an intermediate pattern between the values observed by the Control-SAMR1 group and the Control-SAMP8 group. The results indicated that SAMP8 mice fed with SDP supplementation had reduced dark activity compared with SAMP8 mice fed control feed. This may indicate that supplementation with SDP helps to improve orientation and memory of these animals and reduce negative effects associated with dementia.

(81) Conclusions:

(82) Results from motor activity analysis, though preliminary, suggest that SDP supplementation reduced the nocturnal motor activity of the SAMP8 mice making it closer to the pattern shown by the Control-SAMR1 mice. This suggests that administration of the animal plasma, fractions thereof, or mixtures thereof can be used in treating cognitive impairment disorders, such as dementia.

Example 2—Effects of Plasma Protein Administration on Learning and Memory Functions in SAMP8 Mice

(83) Materials and Methods:

(84) Animals:

(85) Experiments have been done using SAMP8 mice. Mice were maintained in conventional housing (3-4 animals per cage) until the age of 2 months (except if they showed an aggressive behavior in which case they were separated and housed individually).

(86) Diets:

(87) Mice were fed with the experimental diets for up to 4 months. The composition of experimental diets is detailed in Table 1 of Example 1. Food intake and body weight were monitored throughout the experimental period.

(88) Experimental Design:

(89) In this set of the experiments, the inventors aimed to know the effect of plasma protein administration on short and long-term memory in SAMP8 mice by performing the Novel Object Recognition (NOR) Test. The following experimental groups were used (FIG. 4): 2M: mice of 2-month-old, young reference group. 4M-CTL: mice of 4-month-old, fed with control diet for 2 months. 4M-SDP: mice of 4-month-old, fed with SDP supplemented diet for 2 months. 6M-CTL: mice of 6-month-old (senescent), fed with control diet for 4 months. 6M-SDP: mice of 6-month-old (senescent), fed with SDP supplemented diet for 4 months.

(90) Food intake and evolution of the body weight were evaluated from 2 to 6 months of age. To study the effect of aging and the effect of dietary supplementation, data were analyzed by one-way analysis of variance (ANOVA) followed by the Fisher post hoc test using the GraphPad Prism® software ver. 6 (GraphPad Software, Inc., USA). Body weight has been analyzed by two-way ANOVA followed by the Fisher post hoc test. Differences were considered significant at P<0.1.

(91) Novel Object Recognition Test (NORT):

(92) Mice were placed in a 90°, two-arm (25-cm-long, 20-cm-high, 5-cm-wide) black maze (FIG. 5). Light intensity in the middle of the maze was 30 lx. The objects to be discriminated were made of plastic. Mice (one at a time) were individually put in the maze for 10 min during three consecutive days. On the fourth day, animals were submitted to a 10 min acquisition trial, in which they were placed in the maze in the presence of two identical objects (A+A or B+B) situated at the end of each arm. During this trial, the explorative preference (the percentage of time exploring each object) and the explorative index (defined as the time exploring both objects/time on the maze) were measured. A 10 min retention trial was carried out 2 h later that evaluates the short-time memory. During this trial, one of the objects was replaced by a novel one (A+B or B+A) and the behavior of the mice was recorded with a camera. The time spent exploring the novel object (TN) and the time exploring the old object (TO) were measured. The discrimination index (DI) was calculated by (TN−TO)/(TN+TO). Mice with a DI equal or higher than 0.2 have been considered as animals “with memory.” In order to avoid object preference biases, objects A and B were counterbalanced so that one half of the animals in each experimental group were exposed first to object A and then to object B, whereas the remaining half saw object B first and then object A. The maze and the objects were cleaned with 96% ethanol after each test in order to eliminate olfactory cues. Twenty-four hours later, a second retention trial was performed to evaluate the long-time memory. During this second trial, the formerly novel object was replaced by another one (A+C or B+C) and, again, the behavior of the mice was recorded with a camera and DI was calculated.

(93) Results:

(94) Food Intake and Body Weight:

(95) Food intake was measured three times per week during the feeding period. FIG. 6 summarizes the daily mean food intake during the 4 months that mice have received the experimental diets. No differences have been observed between mice fed with control and SDP diet.

(96) The body weight of SAMP8 mice was measured once a month. At 2-month-old, SAMP8 mice have a similar body weight. After 1 month feeding with SDP diet, SAMP8 mice have an increased body weight gain than those fed with control diet (FIG. 7), and this difference was maintained along the experimental period.

(97) Novel Object Recognition Test (NORT):

(98) Mice did not show any preference for one of the identical objects during the acquisition trial as shown in FIG. 8, where all the groups presented an explorative preference nearly to 50%. This result indicates that no group preferred one of the arms of the maze.

(99) FIG. 9 shows the explorative index calculated during the first 5 min of the acquisition trial. At 2-month-old, mice explored the objects 3.3±0.4% of the time spent in the maze. In dementia-model mice, this index decreased over time, eventually reaching 2.1±0.4% (P<0.1) at 6 months of age, indicating a lower explorative capacity. Administration of plasma protein compositions does not prevent this effect associated to age.

(100) On the first retention trial (evaluates the short-term memory; FIG. 10), the discrimination index decreased at 6-month-old during the first 5 min of the test, indicating a reduction on the short-term memory in the dementia-model mice. However, 6-month-old mice supplemented with SDP diet maintained the same results seen in 2-month-old mice and had a significantly improved discrimination index compared to 6-month-old mice fed the control diet. The same pattern is observed on the second retention trial (24 h after the first retention trial) in dementia-model mice that is associated with the long-term memory (FIG. 11). At 4-month-old, the long-term memory is already significantly impaired in dementia-model mice fed the control diet with a similar level of impairment observed in 6-month-old control animals. Dementia-model mice supplemented with SDP showed an increased discrimination index (suggesting improved long term memory) at 4 months, although not significantly different from control animals. However, at 6 months, dementia-model mice supplemented with SDP showed an increased discrimination index that was statistically greater than 6-month-old SAMP8 mice fed the control diet and similar to the discrimination index observed at 2 months.

(101) On FIG. 12 is represented the percentage of mice “with memory,” understood as a DI higher than 0.2; during the first retention trial. This value is reached when the mouse spends twice the amount of time exploring the novel object in comparison with the old object. When mice were 2-months-old, nearly 90% of the population conserved the short-term memory, while at 6-months of age, this percentage decreased to about 40% in control mice. However, when mice were administered plasma protein compositions, the percentage of mice “with memory” at 4- and 6-months of age is similar to that found in the younger group.

(102) In the second retention trial, approximately 70% of the 2-month-old mice conserved the long-term memory (FIG. 13). This percentage decreased with age, taking values of 56% for the 4-month-old mice and 36% for the 6-month-old mice. However, when mice were administered plasma protein compositions, the percentage of mice “with long-term memory” at 4- and 6-months of age is similar to, or even better than, that found in the younger group.

(103) Conclusions:

(104) The Novel Object Recognition Test (NORT) evaluates recognition memory and is useful to study short-term and long-term memory. NORT is a “pure” recognition memory test and a valid task to assess working memory. The test does not involve positive or negative reinforces and this makes NOR comparable to memory tests currently used in humans for dementia diseases, such as Alzheimer's disease. Using this test, it was observed that variables of both short-term and long-term memory were deteriorated in aged (6 month-old) mice as compared with younger (2 month-old) mice in the SAMP8 dementia model. The older mice showed lower explorative behaviors and memory retention. Unexpectedly, feeding a diet supplemented with plasma protein compositions (e.g., SDP) for 4 months reduced the severity of memory loss in the dementia model mice, suggesting that this might delay the progressive deterioration of cognitive functions.

(105) In summary, administration of a diet supplemented with plasma protein compositions prevented the deterioration of cognitive functions associated with dementia, as demonstrated by the Novel Object Recognition test.

Example 3—Effects of Plasma Protein Administration on Expression of Synaptophysin, a Neural Function Marker in SAMP8 Mice

(106) Aim:

(107) Six-month-old SAMP8 mice show a reduced expression of neural function markers, such as synaptophysin-1, as compared to control mice, suggesting a decrease in the number of synaptic connections in the SAMP8 mice. In this set of experiments the inventors aimed to determine whether SDP supplementation for 4 months could prevent the reduction in the synaptophysin-1 neural function markers, thereby suggesting a reduction in neural degeneration in 6-month-old SAMP8 mice.

(108) Materials and Methods:

(109) Animals:

(110) Experiments were again performed using a senescence-accelerated prone strain of mice (SAMP8), AND THE MICE FROM Example 2 were used for this experiment, as well. The experimental groups employed were: 2M: mice of 2-month-old, young reference group; 6M-CTL: mice of 6-month-old (senescent), fed with control diet for 4 months; and 6M-SDP: mice of 6-month-old (senescent), fed with SDP supplemented diet for 4 months.

(111) Sample Collection and Homogenization:

(112) At the end of the experiment of example 2, mice were anaesthetized with xilacin/ketamine. The brain was removed and samples of cortex and hippocampus were quickly frozen at −80° C. for further uses. Samples of cortex and hippocampus were homogenized with a Polytron (PRO Scientific Inc. USA) at 20,000 rpm in a lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton, 200 mM PMSF, 1 mM DTT and 2% (v/v) inhibitor protease cocktail. The homogenate was centrifuged at 4° C. at 955 g for 20 min.

(113) Determining Protein Abundance by Western Blot:

(114) Samples (50 μg of protein from cortex and hippocampus) were denatured and separated on 10-12.5% SDS-PAGE polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked by incubation for 90 min at room temperature in Tris buffered saline containing 0.1% Tween 20 (TBST) and 5% dry milk, and then incubated overnight with a mouse anti-synaptophysin (obtained from Dako) (1/3000 dilution) at 4° C. Membranes were washed several times with TBST and incubated for 2 h with an anti-mouse horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, USA). After washing with TBST, protein bands were visualized using the Clarity chemiluminescence detection kit (Bio-Rad, USA). The assay was carried out in accordance with the manufacturer's instructions. After their detection, bands were quantified using ImageLab (Bio-Rad).

(115) Statistical Analysis:

(116) Data were analyzed by one-way analysis of variance (ANOVA) followed by the Fisher post hoc test using the GraphPad Prism® software ver. 6 (GraphPad Software, Inc., USA). Protein abundance has been analyzed by two-way ANOVA followed by the Fisher post hoc test. Differences were considered significant at P<0.1.

(117) Results:

(118) FIG. 14 shows the expression of the synaptophysin, which is a protein involved in the neuronal synapsis. The abundance of this protein was reduced in the cortex and hippocampus of aged mice with markers of cognitive impairment disorders (P<0.05 and P=0.053, respectively; FIGS. 14A and 14B, respectively). SDP supplemented diet showed different effects regarding brain tissue, since there was no dietary effect on cortex, but SDP prevented aged-associated reduction in synaptophysin in hippocampus (P<0.05). Therefore, the animal plasma, fractions thereof, or mixtures thereof according to the invention is effective in treating the reduced expression of synaptophysin protein in the hippocampus in senescent animals.

Example 4—Effects of Plasma Protein Administration on Expression of Markers Associated with Microgliosis, Astrogliosis and Amyloid Plaque Formation in SAMP8 Mice

(119) Aim:

(120) In this set of experiments, the inventors aimed to determine whether SDP supplementation for 4 months could affect microgliosis and astrogliosis associated to senescence, as well as amyloid plaques formation.

(121) Altered synaptic morphology, progressive loss of synapses and glial (astrocyte and microglial) cell activation are considered as characteristic hallmarks of certain cognitive dysfunction disorders, including those associated with aging. Neuronal loss during senescence decreases the expression of the presynaptic protein synaptophysin and the post-synaptic protein PSD-95. Thus, analysis of synaptophysin and PSD-95 in brain tissue provides information about neuronal integrity.

(122) Emerging evidence implies that glial dysregulation may have a significant impact on the disease onset of AD. Reactive astrogliosis is a universally acknowledged feature of AD. Moreover, the degree of astrogliosis is correlated with cognitive decline (Frost and Li, 2017). Microglial cells are able to undergo directed changes in immunophenotype in regulating the functions of various brain structures and in the process of development of age-related and mental pathology. Thus, analysis of GFAP and IBA1 in brain tissue provide information about the microgliosis and astrogliosis in senescent mice. β-amyloid plaques and tau hyperphosphorylation have been considered the principal mechanisms associated with the development of AD. Quantification of Tau, p-Tau and amyloid precursor protein in brain tissue provide information about principal markers of AD in brain tissue of senescent mice.

(123) Materials and Methods:

(124) Animals:

(125) Experiments were again performed using a senescence-accelerated prone strain of mice (SAMP8), which were taken and grown in the animal facility of the Facultat de Farmácia i Ciéncies de l'Alimentació of the Universitat de Barcelona (UB). Protocols used in this example were approved by the Animal Experimentation Ethics Committee (CEEA) of the UB, in accordance with the Generalitat de Catalunya's guidelines for the Care and Use of Laboratory Animals (DAAM: 7939 and 9272).

(126) Diets:

(127) Mice were fed with the experimental diets for 2 or 4 months. The composition of experimental diets is detailed in Table 2.

(128) TABLE-US-00002 TABLE 2 Composition of experimental diets. SDP was provided by APC- Europe, S. A.. AIN-93 VX is a vitamin mix and AIN-93-G-MX is a mineral mix, both of which were provided by Envigo, Italy. Ingredient (g/kg) Control SDP SDP — 80 Corn starch 199.3 308.8 Skim milk 530.7 340.5 Sucrose 94.5 94.5 Soybean oil 70 70 Cellulose 50 50 AIN-93-G-MX (94046).sup.2 35 35 AIN-93 VX (94047).sup.2 15 15 Choline bitartrate 3 3 DL-Methionine 2.5 3.2

(129) Experimental Design:

(130) Mice were maintained in conventional housing (3-4 animals per cage) until the age of 2 months feeding the commercial standard feed. After 2 months, the animals were fed experimental diets (Control or SDP) for 4 months. The experimental design of this study consisted in 3 groups of 9-11 animals/group: 2M: mice of 2-months-old, young reference group; 6M-CTL: mice of 6-months-old, fed with Control diet for 4 months; and 6M-SDP: mice of 6-months-old, fed with SDP supplemented diet for 4 months.

(131) Sample Collection:

(132) At the end of the experiment, mice were anaesthetized with xilacin/ketamine. Blood was directly collected from the heart and then killed by exsanguination. The brain was removed and was quickly frozen at −80° C. for further uses.

(133) Perfusion Mice:

(134) Mice were anaesthetized and transcardially perfused thoroughly with PBS to remove all of the intravascularly distributed dye. Afterwards, mice were perfused with 4% paraformaldehyde. The brain was removed and samples of hemispheres were quickly frozen at −80° C. for further uses.

(135) Sample Homogenization:

(136) Samples of brain hemisphere were homogenized with a Polytron (PRO Scientific Inc, USA) at 20,000 rpm in a lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton, 1 mM PMSF, 1 mM DTT and 2% (v/v) inhibitor protease cocktail. The homogenate was centrifuged at 4° C. at 955 g for 20 min.

(137) Immunohistochemistry:

(138) Perfuse brains were embedded in a drop of Tissue-Tek O.C.T. Compound (Miles) and immediately submerged into isopentane. They were then stored at −80° C. Brains were cut with a cryostat CM3050S (Leica Microsystems). Brain sections were permeabilized with a solution containing 10 g/L bovine serum albumin, glycine 20 mM (both, Sigma-Aldrich-Aldrich, USA) and Triton X-100 1% (v:v; Fluka. USA) in PBS at room temperature for 30 min. The slices were incubated overnight at 4° C. with a solution containing 10 giL bovine serum albumin, glycine 20 mM and the corresponding primary mouse monoclonal antibody in a humidified chamber. The primary antibodies used were Iba-1 (Wako Chemicals, USA), GFAP (Abcam, United Kingdom), NeuN and CD11b (Millipore, USA). Sections were washed with PBS and incubated with Alexa Fluor secondary antibody for 2 h at room temperature in a humidity chamber. Afterwards, the samples were washed in PBS and counterstained with nuclear marker Hoechst 33258 (Calbiochem, USA) for 20 min at room temperature. They were then washed again with PBS and mounted in Mowiol-488 (Calbiochem. USA). Negative controls were performed without the primary antibodies. The samples were stored at 4 C until observation by confocal microscopy.

(139) Confocal Scanning Laser Microscope and Image Processing:

(140) Digital fluorescence images were acquired by confocal scanning laser microscope SPII (Leica Microsystems) (images not shown). Five different fields for each cellular staining were analyzed in a blinded fashion, resulting in a minimum of 25 images per animal. Images were analyzed using Fiji (Schindelin et al., 2012). Results expressed as percentage positive cells of total cells/field.

(141) Western Blot:

(142) Western blot procedures were performed according to a previous study (Pérez-Bosque et al., 2016). Samples of brain hemisphere were homogenized and protein concentration was determined using the Bradford method (Bio-Rad, Hercules, Calif., USA). Equal amounts of protein (100 μg) were separated on 10-18% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, USA). Membranes were blocked by incubation for 90 min at room temperature in Tris buffered saline containing 0.1% Tween 20 (TBST) and 5% dry milk. Afterwards, membranes were incubated overnight at 4° C. with diluted primary antibodies Table 2. Membranes were washed and incubated with HRP-conjugated secondary antibodies (Sigma-Aldrich, St Louis, Mich., USA) for 2 h at room temperature. Protein bands were visualized using a chemiluminescence detection kit Clarity and ChemiDoc XRS+ instrument (both from Bio-Rad, Hercules, Calif., USA). After their detection, hybridization bands were quantified using ImageJ gel analyzer software.

(143) TABLE-US-00003 TABLE 3 List of antibodies used for Western Blot. Antibody Host Dilution Source NeuN Mouse 1/750 Millipore p-Tau Mouse 1/1000 Invitrogen PSD-95 Mouse 1/1000 Abcam sAPPα Rabbit 1/1000 Covance sAPPβ Rabbit 1/1000 Covance Synaptophysin Mouse 1/3000 Dako Tau Mouse 1/1000 Millipore β-actin Mouse 1/30000 Sigma-Aldrich

(144) Real-Time PCR:

(145) RNA extraction and reverse transcription were carried out as described previously (Pérez-Bosque et al., 2016). RNA quality and quantity were assessed by spectrophotometry (NanoDrop ND-1000; ThermoFisher Scientific, Waltham, Mass., USA). Total RNA was reverse-transcribed using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, Calif., USA). Real-time PCR was performed on a MiniOpticon Real-Time PCR System (Bio-Rad, Hercules, Calif., USA). TaqMan gene expression assays (Applied Biosystems, N.Y., USA) were used for the following genes: β-secretase (Bace1, Mm00478664_ml) and Disintegrin and Metalloproteinase 10 (Adam10, Mm00545742_ml) following the manufacturer's instruction. Each PCR run included duplicates of reverse transcription for each sample and negative controls (reverse transcription-free samples, RNA-free sample). Quantification of the target gene transcripts was done using hypoxanthine phosphoribosyltransferase 1 (Hprt1, Mm00446968_ml) gene expression as reference, and was carried out with the 2-ΔΔCT method (Schmittgen & Livak, 2008). Product fidelity was confirmed by melt-curve analysis.

(146) Statistical Analysis:

(147) Results are expressed as means±standard error of the mean (SEM). To study the effects of aging and the dietary supplementation, data were analyzed by one-way analysis of variance (ANOVA) followed by the Fisher post hoc test using the GraphPad Prism® software ver. 6 (GraphPad Software, Inc., USA). Differences in tests were considered statistically significant when P<0.05. A value of P between 0.05 and 0.1 was considered to establish a significant trend (Curran-Everett & Benos, 2004).

(148) Results:

(149) FIG. 15 shows the expression and abundance in brain tissue of synaptophysin, which is a major integral membrane glycoprotein in presynaptic vesicles of neurons. Results are expressed as means±SEM (n=6-7 mice). FIG. 15A shows the results of Real-Time PCR. Means without a common letter differ, P<0.1. FIG. 15B shows representative images of the Western Blot of synaptophysin and D-actin (control). The abundance of synaptophysin decreased in senescent mice and SDP supplementation preserves the neuronal integrity in older mice.

(150) FIG. 16 shows the expression and abundance in brain tissue of PSD-95, which has an important role in the molecular organization of the postsynaptic density. Results are expressed as means±SEM (n=6-7 mice). FIG. 16A shows the results of Real-Time PCR. Means without a common letter differ, P<0.1. FIG. 16B shows representative images of the Western Blot of PSD-95 and β-actin (control). The abundance of PSD-95 decreased in senescent mice and SDP supplementation preserves the neuronal integrity in older mice.

(151) FIG. 17 shows the abundance in brain tissue of Glial fibrillary acidic protein (GFAP), which is the main intermediate filament protein in mature astrocytes and also an important component of the cytoskeleton in astrocytes during development. Abundance of GFAP positive cells (B) in brain tissue was determined through immunohistochemistry on perfuse brain tissue. Results are expressed as means±SEM (n=3-4 mice). Results expressed as percentage of total cells/field. Means without a common letter differ, P<0.1. Senescent mice had more abundance of GFAP.sup.+ cells and SDP supplementation attenuates astrogliosis in aged mice with markers of cognitive impairment disorders.

(152) FIG. 18 shows the abundance in brain tissue of Ionized Calcium Binding Adaptor Molecule 1 (IBA1), which is a microglia/macrophage-specific calcium-binding protein and has the actin-bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. Abundance of IBA1 positive cells in brain tissue was determined through immunohistochemistry on perfuse brain tissue. Results are expressed as means±SEM (n=3-4 mice). Results expressed as percentage of total cells/field. Means without a common letter differ, P<0.1. Senescent mice presented an activation of the microglia and SDP supplementation prevented it.

(153) FIG. 19 shows the expression and abundance in brain tissue of total Tau, which are microtubule-associated proteins (MAPs) and are abundant in neurons of the CNS. Several studies indicate that Tau protein plays an important role in the neurodegeneration observed in AD patients. Results are expressed as means±SEM (n=6-7 mice). FIG. 19A shows the results of Real-Time PCR. FIG. 19B shows representative images of the Western Blot of total Tau and β-actin (control). FIG. 20 shows the expression and abundance in brain tissue of hyperphosphorylation of Tau (p-Tau), which disrupts normal neuronal function in the regulation of axonal transport and leads to the accumulation of neurofibrillary tangles and toxic species of soluble tau and p-Tau also correlates with a cognitive decline. Results are expressed as means±SEM (n=6-7 mice). FIG. 20A shows the results of Real-Time PCR. Means without a common letter differ, P<0.1. FIG. 20B shows representative images of the Western Blot of p-Tau and total Tau. Aged SAMP8 mice showed increased abundance of the phosphorylated form of Tau (p-Tau), which is related to cognitive disorders. SDP supplementation reduced p-Tau formation during aging.

(154) FIGS. 21 and 22 show the expression and abundance in brain tissue of sAPP-β and sAPP-α. Aβ42 is a peptide of β-amyloid protein that has high aggregative capacity and therefore forms senile aggregates and plaques in brain tissue that are responsible for the neuron degeneration observed in AD patients. This peptide originates by the cleavage of the amyloid precursor protein (APP) by the β-secretases. Senescent SAMP8 mice present an increase in APP production. Results are expressed as means±SEM (n=4-5 mice). FIGS. 21A and 22A show the results of Real-Time PCR in relation to sAPP-β and sAPP-α, respectively. FIGS. 21B and 22B show representative images of the Western Blot of β-actin (control) and sAPP-β and sAPP-α, respectively. The abundance of sAPP-R was increased in senescent mice. SDP supplementation prevented the accumulation of this protein. This observation suggests that this supplement has neuroprotective properties.

(155) FIG. 23 shows the expression in brain tissue of BACE1, which is the p-secretase that participates in the production of the β-amyloid (Aβ42) peptide that is widely considered to have a crucial early role in the etiology of AD. Results are expressed as means±SEM (n=6-7 mice). Results of Real-Time PCR are shown. Means without a common letter differ, P<0.1. The expression of BACE1 was not affected by aging and SDP reduced its expression.

(156) FIG. 24 shows the expression in brain tissue of ADAM10, which is a protein that has been found in increased amounts in the hippocampal neurons of AD patients. Results are expressed as means±SEM (n=6-7 mice). Results of Real-Time PCR are shown. Means without a common letter differ, P<0.1. ADAM10 expression increased during aging and SDP prevented this age-dependent changes.

(157) Conclusions:

(158) Senescence is characterized by a reduction of neuronal dysfunction. SDP maintains neuronal integrity because it prevented the loss of pre- and post-synaptic connections associated with aging. In the CNS of aged SAMP8 mice there is an activation of microglia and astrogliosis. SDP supplementation prevented the activation of the microglia and astroglia. Aged SAMP8 mice showed increased abundance of the phosphorylated form of Tau (p-Tau) and the formation of the sAPP-β. Both proteins are related to cognitive disorders. SDP supplementation reduced p-Tau and sAPP-β during aging of SAMP8 mice.

(159) SDP presents neuroprotective properties improving the cognitive functions of aged animals with markers of cognitive impairment disorders.

Example 5 (Prophetic)—Reduction in Severity of Cognitive Impairment Following Mild Traumatic Brain Injury in Individuals Consuming Plasma Proteins/Fractions

(160) The consumption of plasma, or plasma fractions, for 1-2 months prior to a TBI episode will lessen the severity of neurological and physiological changes resulting from TBI. The severity of neurological and physiological changes resulting from TBI includes both the magnitude of the absolute change in a measure and/or the time required for healing and subsequent improvement in neurological and physiological changes resultant from the TBI. Examples of animal models of TBI include the fluid percussion injury, cortical impact injury, weight drop-impact acceleration injury, and blast injury (Xiong Y, Mahmood A, Chopp M. Animal models of traumatic brain injury. Nature reviews Neuroscience. 2013; 14(2):128-142. doi:10.1038/nrn3407). Neurological processes such as motor function, alertness, seeking behavior, and recovery of short-term and long-term memory function would be improved for subjects consuming plasma proteins. Physiological parameters that would be improved include blood-brain barrier integrity, swelling, intracranial pressure, and reduced changes in specific biochemical markers of brain injury.

(161) Having described the invention with reference to particular compositions and methods, theories of effectiveness, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates to the contrary.