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Title:
PHARMACEUTICAL COMPOSITION COMPRISING PORCINE BRAIN EXTRACT
Document Type and Number:
WIPO Patent Application WO/2010/007620
Kind Code:
A1
Abstract:
The present invention relates to pharmaceutical compositions comprising an extract of fetal porcine brain tissue, and use thereof for treatment of a variety of diseases and conditions, in particular neurodegenerative diseases, stroke, cardiac ischemia and burns.

Inventors:
SHIKHOV, Sergey (44/6, Nikolaeva Str.Elektrosta, Moscow 3, 14400, RU)
FOMIN, Grigory (7/9 Smolnaya Str, Moscow, 3, 12549, RU)
Application Number:
IL2009/000698
Publication Date:
January 21, 2010
Filing Date:
July 13, 2009
Export Citation:
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Assignee:
DELAPHARM LTD. (42 Herzl Street, Kfar-Sava, 44444, IL)
SHIKHOV, Sergey (44/6, Nikolaeva Str.Elektrosta, Moscow 3, 14400, RU)
FOMIN, Grigory (7/9 Smolnaya Str, Moscow, 3, 12549, RU)
International Classes:
A61K35/30
Attorney, Agent or Firm:
WEBB & ASSOCIATES et al. (P.O. Box 2189, Rehovot, 76121, IL)
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Claims:
CLAIMS

1. A pharmaceutical composition comprising as an active ingredient an extract of fetal porcine brain proteins, wherein the proteins in the extract are of molecular weight of at least about 5,000 Da, and wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.

2. The pharmaceutical composition according to claim 1, wherein the proteins in the extract are of molecular weight of at least about 10,000 Da.

3. The pharmaceutical composition according to claim 1, wherein the proteins in the extract are of molecular weight in the range from about 5,000 to about 150,000 Da. 4. The pharmaceutical composition according to claim 1, wherein the proteins in the extract are of molecular weight in the range from about 10,000 to about 140,000 Da.

5. The pharmaceutical composition according to claim 1, wherein the proteins in the extract are of molecular weight in the range from about 20,000 to about 120,000 Da.

6. The pharmaceutical composition according to claim 1, wherein the proteins in the extract have a molecular weight distribution substantially as depicted in Fig. 1.

7. The pharmaceutical composition according to claim 1, wherein the brain proteins are derived from a porcine fetus of gestational age of about 3 to about 15 weeks.

8. The pharmaceutical composition according to claim 7, wherein the gestational age is about 6 to about 12 weeks. 9. The pharmaceutical composition according to claim 1, wherein the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 5.0 milligrams per milliliter (mg/ml).

10. The pharmaceutical composition according to claim 9, wherein the concentration is in the range from about 0.05 to about 0.5 mg/ml, or in the range from about 0.5 to about 3.0 mg/ml.

11. The pharmaceutical composition according to claim 1, wherein the concentration of fetal porcine brain proteins in the extract is in the range from about 0.1 to about 10 mg/ml.

12. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier or diluent is selected from an aqueous carrier or diluent and a lipid carrier or diluent.

13. The pharmaceutical composition according to claim 1, in a form selected from the group consisting of a liquid, a powder, an aerosol, a capsule, a tablet, a suppository, a cream, a gel and an ointment.

14. The pharmaceutical composition according to claim 13, wherein the liquid is selected from the group consisting of an aqueous solution, a lotion, a suspension, a spray, an emulsion, and a microemulsion. 15. The pharmaceutical composition according to claim 1, formulated for administration by a route selected from the group consisting of topical, transdermal, intranasal, oral, buccal, parenteral, inhalation, transmucosal, rectal and vaginal.

16. The pharmaceutical composition according to claim 1, formulated for administration as a spray or as an aerosol. 17. The pharmaceutical composition according to claim 1, wherein the composition is a solution formulation for parenteral administration and the concentration of the extract of fetal porcine brain proteins in the solution is in the range from about 0.1 to about 3.0 mg/ml.

18. The pharmaceutical composition according to claim 17, wherein the concentration of the extract of fetal porcine brain proteins in the solution is about 1.5 mg/ml.

19. The pharmaceutical composition according to claim 1, wherein the composition is a liquid formulation for intranasal administration and the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml. 20. The pharmaceutical composition according to claim 1, wherein the composition is a formulation for topical administration selected from the group consisting of a cream, a gel, an ointment and a combination thereof, and the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml. 21. The pharmaceutical composition according to claim 1, wherein the composition further comprises a mixture of polynucleotides.

22. The pharmaceutical composition according to claim 21, wherein the polynucleotides have a length in the range from about 1,000 to about 7,000 nucleotides.

23. The pharmaceutical composition according to claim 21, wherein the mixture of polynucleotides comprises fish sperm DNA. 24. The pharmaceutical composition according to claim 23, wherein the fish sperm DNA is selected from the group consisting of salmon sperm DNA, herring sperm DNA, sturgeon sperm DNA and combinations thereof.

25. The pharmaceutical composition according to claim 21, wherein the concentration of the mixture of polynucleotides in the composition is in the range from about 0.1 to about 3.0 milligrams per milliliter (mg/ml).

26. The pharmaceutical composition according to claim 25, wherein the concentration of the mixture of polynucleotides in the composition is about 1.0 mg/ml.

27. The pharmaceutical composition according to claim 1, further comprising at least one of a preservative and an antioxidant. 28. The pharmaceutical composition according to claim 1, wherein the composition is a formulation for topical administration selected from the group consisting of a cream, a gel and an ointment, and wherein the formulation comprises: the extract of fetal porcine brain proteins at a concentration of about 0.1 to about 0.5 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 0.5 to about 2.0 mg/ml, and lecithin at a concentration of about 1.0 to about 3.0% (w/v).

29. The pharmaceutical composition according to claim 28, further comprising DL-α- tocopherol acetate at a concentration of about 0.05 to about 0.5% (w/v).

30. The pharmaceutical composition according to claim 1, wherein the composition is a liquid formulation for intranasal administration, wherein the formulation comprises: the extract of fetal porcine brain proteins at a concentration of about 0.1 to about 0.5 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 0.5 to about 2.0 mg/ml, and lecithin at a concentration of about 1.0 to about 3.0% (w/v).

31. The pharmaceutical composition according to claim 30, further comprisimg DL-α- tocopherol acetate at a concentration of about 0.05 to about 0.5% (w/v).

32. The pharmaceutical composition according to claim 1, substantially devoid of porcine brain tissue proteins of molecular weight less than about 5,000 Da.

33. A process for prepared a fetal porcine brain protein extract, the process comprising: (i) harvesting brain tissue from at least one porcine fetus; (ii) mechanically homogenizing the brain tissue from (i) in an appropriate buffer;

(iii) obtaining a soluble fraction from the homogenized brain tissue of (ii);

(iv) subjecting the soluble fraction of (iii) to anion exchange chromatography so as to obtain a bound and eluted fraction, and

(v) subjecting the bound and eluted fraction of (iv) to molecular weight separation so as to obtain a purified fraction of proteins having a molecular weight in the range from about 5,000 to about 150,000 Da.

34. The process according to claim 33, wherein the at least one porcine fetus is of gestational age of about 3 to about 15 weeks.

35. The process according to claim 34, wherein the porcine fetus is of gestational age of about 6 to about 12 weeks.

36. A method for treating a neurological or cardiovascular disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a native extract of fetal porcine brain proteins, wherein the proteins in the extract are of molecular weight of at least about 5,000 Da. 37. The method according to claim 36, wherein the extract is substantially devoid of porcine brain tissue proteins of molecular weight less than about 5,000 Da.

38. The method according to claim 36, wherein the neurological disorder is selected from the group consisting of ischemic stroke, cerebral infarction, brain ischemia, brain hemorrhage, traumatic brain injury, cerebral palsy, pre-senile dementia, Alzheimer's disease, vascular encephalopathy, and chronic fatigue syndrome.

39. The method according to claim 36, wherein the cardiovascular disorder is selected from the group consisting of hypertension, cardiac ischemia, myocardial infarction, angina, and post-infarction cardiosclerosis.

40. The method according to claim 36, wherein the proteins in the extract are of molecular weight of at least about 10,000 Da.

41. The method according to claim 40, wherein the proteins in the extract are of molecular weight in the range from about 5,000 to about 150,000 Da. 42. The method according to claim 36, wherein the proteins in the extract are of molecular weight in the range from about 10,000 to about 140,000 Da.

43. The method according to claim 36, wherein the proteins in the extract have a molecular weight distribution substantially as depicted in Fig. 1.

44. The method according to claim 36, wherein the administering comprises administering a pharmaceutical composition comprising the extract of fetal porcine brain proteins, wherein the pharmaceutical composition is in a form selected from the group consisting of a liquid, a powder, an aerosol, a capsule, a tablet, a suppository, a cream, a gel and an ointment.

45. The method according to claim 36, wherein the administering is carried out topically, transdermally, intranasally, orally, buccally, parenterally, by inhalation, transmucosally, rectally, intralumbarly or vaginally.

46. The method according to claim 36, wherein the disorder is a neurological disorder and the administering comprises administering an initial dose intravenously or intralubarly and thereafter administering subsequent doses intranasally. 47. The method according to claim 36, wherein the administering is in an amount sufficient to provide a daily dose of about 0.05 to about 5 mg of the extract of fetal porcine brain proteins.

48. The method according to claim 36, wherein the administering is carried out intranasally, in an amount sufficient to provide a daily dose of about 0.05 to about 0.5 mg of the extract of fetal porcine brain proteins.

49. The method according to claim 36, wherein the administering is carried out topically, in an amount sufficient to provide a daily dose of about 0.05 to about 0.5 mg of the extract of fetal porcine brain proteins.

50. The method according to claim 36, wherein the administering is carried out in an amount sufficient to provide a daily dose of about 0.05-50 μg per kg body weight.

51. The method according to claim 44, wherein the pharmaceutical composition further comprises a mixture of polynucleotides selected from the group consisting of salmon sperm DNA, herring sperm DNA, sturgeon sperm DNA and combinations thereof.

52. A method for treating a skin wound or disorder, comprising topically administering to a subject in need thereof a therapeutically effective amount of a native extract of fetal porcine brain proteins, wherein the proteins in the extract are of molecular weight of at least about 5,000 Da, thereby treating the skin wound or disorder.

53. The method according to claim 52, wherein the topically administering comprises administering a formulation selected from the group consisting of a cream, a gel, an ointment and a combination thereof, and wherein the concentration of the extract of fetal porcine brain proteins in the formulation is in the range from about 0.05 to about 0.5 mg/ml.

54. The method according to claim 52, wherein the skin disorder is selected from the group consisting of psoriasis, eczema, contact dermatitis and seborrheic dermatitis. 55. The method according to claim 52, wherein the skin disorder is selected from the group consisting of a burn, a traumatic skin injury and a decubitus ulcer.

56. The method according to claim 52, wherein the proteins are of molecular weight of at least about 10,000 Da.

57 The method according to claim 52, wherein the proteins in the extract are of molecular weight in the range from about 5,000 to about 150,000 Da.

58. The method according to claim 57, wherein the proteins in the extract are of molecular weight in the range from about 10,000 to about 140,000 Da.

59. The method according to claim 52, wherein the in the extract proteins have a molecular weight distribution substantially as depicted in Fig. 1. 60. The method according to claim 52, wherein the topically administering is in an amount sufficient to provide a daily dose of about 0.05 to about 0.5 mg of the extract of fetal porcine brain proteins.

61. The method according to claim 52, wherein the topically administering is in an amount sufficient to provide a daily dose of about 0.05-50 μg per kg body weight.

62. Use of a native extract of fetal porcine brain proteins, wherein the proteins in the extract are of molecular weight of at least about 5,000 Da for the preparation of a medicament for treatment of a disorder selected from the group consisting of a neurological disorder, a cardiovascular disorder and a skin disorder.

63. Use of the pharmaceutical composition according to any of claims 1-32 for treating a disorder selected from the group consisting of a neurological disorder, a cardiovascular disorder and a skin disorder.

Description:
PHARMACEUTICAL COMPOSITION COMPRISING PORCINE BRAIN EXTRACT

FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions comprising an extract of fetal porcine brain tissue, and use thereof for treatment of a variety of diseases and conditions, in particular neurodegenerative diseases, stroke, cardiac ischemia and burns.

BACKGROUND OF THE INVENTION Peptide products derived from bovine or porcine brain have been disclosed for treating a variety of diseases and conditions in humans. The peptide-based drug referred to under the trade name Cerebrolysin™ (Ebewe Pharma, , Austria) is one such product presently in preclinical trials (phase III) for use in treating neurological and cognitive impairment. Cerebrolysin™ is produced by enzymatic hydrolysis of proteins derived from adult pig brain, and the active peptide ingredients have a molecular weight of less than 10 kDa (see Wong et al. "Effect of cerebrolysin on moderate and severe head injury: Preliminary result of a cohort study"; Annals of the College of Surgeons of Hong Kong. 8(l):22-24, February 2004). European Patent No. 0452,299 discloses that Cerebrolysin™ comprises 5% of peptides of molecular weight of 10,000 Da or less, and 85% of free amino acids. According to this disclosure, the product exhibits activity in enhancing neuroaxon production in ganglion cells and may be used for prophylaxis or treatment of dementia, including Alzheimer's disease. Other disclosed uses of Cerebrolysin™ include treatment of vascular and ischemic disorders, as described in Russian patent application Nos. RU 2152039 and RU 2145895; wound healing, as described in Ukranian patent application No. UA 002322OU; treatment of spastic diplegia in infantile cerebral paralysis, as described in Russian patent application No. RU 2281091; treatment of anovulation in obese women, as described in Russian patent application No. RU 2141361; and treatment of partial optic nerve atrophy, as described in Russian patent application No. RU 2261714.

A disadvantage of Cerebrolysin™ is that it has a relatively low therapeutic efficacy in certain applications, and therefore high doses are often required. Accordingly, the daily dose of Cerebrolysin™ may require more then 1Og of solid substance per day or even more, making this treatment regimen very expensive. Additional peptide drugs derived xrυm porcine and bovine brain tissue include Cortexin™ (Geropharm, Russia; see Russian Patent No. RU 2,275,924), and Cerebrocurin™ (NIR, Ukraine).

U.S. Patent Nos. 6,140,116 and 6,258,353 disclose isolated populations of porcine neural cells, such as cerebral cortical and lateral ganglionic cells, which are derived from embryonic pigs of a gestational age of between 20 and 50 days, and use thereof for treatment of human neurodegenerative diseases by transplantation into an area of neurodegeneration in the brain of a human subject.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprising as an active ingredient a fetal pig brain tissue extract containing a mixture of native peptides and proteins having molecular weights of at least about 5,000 Da, and preferably at least about 10,000 Da. It is now disclosed for the first time that such a composition exerts a variety of beneficial biological effects, including but not limited to protection of cardiac muscle cells and neurons against hypoxia and apoptosis. Further, the compositions of the invention have been shown to be effective in treatment of stroke and cardiac ischemia, and in enhancing burn wound healing in epithelial tissue.

Surprisingly, the present invention requires the presence of conformationally intact fetal brain proteins having molecular weight of at least about 5,000 Da, and preferably at least about 10,000 Da. In particular embodiments, the fetal brain proteins are of molecular weight in the range from about 5,000 to about 150,000 Da, or from about 10,000 to about 140,000 Da. In contrast, prior art compositions derived from porcine brain contain only proteolytic hydrolysis peptide products, such as in the case of Cerebrolysin™ and Cerebrocurine™; and/or are derived from brains of adult or juvenile animals, such as in the case of Cerebrolysin™ and Cortexin™. Furthermore, these prior art products are of a different molecular weight range, generally less than 10,000 Da.

The composition of the invention may further optionally comprise at least one polynucleotide or a mixture of polynucleotides which functions as a stabilizing agent for the protein extract. Examples of suitable polynucleotide mixtures include salmon sperm DNA and herring sperm DNA.

Without wishing to be bound by any theory or mechanism of action, the activity of the fetal brain tissue extract of the present invention requires a heterogeneous mixture of proteins. An illustrative mixture is shown in ngure 1. At least some of the protein species present therein may be in the form of a complex and/or have synergistic activity. Furthermore, the formation and stability of such complexes may be promoted and/or enhanced by the inclusion in the composition of a polynucleotide stabilizing agent. That is, when in the form of a complex, the protein extract may be relatively more stable against denaturating conditions and the activity of proteolytic enzymes, as compared to the non- complexed state. Enhanced stability of the active fraction may enable a reduction in the minimal effective concentration and a corresponding reduction or elimination of any possible side effects. The protein fraction of the invention has been shown to be effective in animal models and tissue culture systems for treatment of stroke and cardiac ischemia, in protection against cardiomyocyte apoptosis, and in protection against neuronal hypoxia. Neuronal protection was demonstrated by functional, histological and behavioral studies in animals. Further, the invention has been shown in extensive animal studies to be safe, well tolerated and non-allergenic, even at doses that are 100- or 1000-fold greater than the recommended therapeutic human dose. hi a first aspect, the invention provides a pharmaceutical composition comprising as an active ingredient an extract of fetal porcine brain proteins, wherein the proteins in the extract are of molecular weight of at least about 5,000 Da, and wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.

It is to be specifically understood that the present invention does not encompass known compositions or extracts prepared by proteolytic digestion or chemical hydrolysis of porcine brain tissue, or extracts prepared using any of detergents, organic solvents or zinc salts such as zinc chloride. In a particular embodiment, the proteins in the extract are of molecular weight of at least about 10,000 Da. In a particular embodiment, the proteins in the extract are of molecular weight in the range from about 5,000 to about 150,000 Da. In a particular embodiment, the proteins in the extract are of molecular weight in the range from about 10,000 to about 140,000 Da. hi a particular embodiment, the proteins in the extract are of molecular weight in the range from about 20,000 to about 120,000 Da. hi a particular embodiment, the proteins in the extract have a molecular weight distribution substantially as depicted in Fig. 1. In a particular embodiment, the proteins are substantially native or intact. In a particular embodiment, the extract is derived from a tissue homogenate. In a particular embodiment, the CAUUUI is substantially devoid of proteins of molecular weight less than about 5,000 Da. In a particular embodiment, the extract is substantially devoid of proteins of molecular weight less than about 10,000 Da. hi a particular embodiment, the extract is substantially devoid of proteins of molecular weight less than about 15,000 Da. hi a particular embodiment, the composition is substantially devoid of porcine brain tissue proteins of molecular weight less than about 5,000 Da. hi a particular embodiment, the composition is substantially devoid of porcine brain tissue proteins of molecular weight less than about 10,000 Da. m a particular embodiment, the composition is substantially devoid of porcine brain tissue proteins of molecular weight less than about 15 ,000 Da. hi a particular embodiment, the brain proteins are derived from a porcine fetus of gestational age 3 to 15 weeks. In a particular embodiment, the gestational age is 6 to 12 weeks. hi particular embodiments, the concentration of fetal porcine brain proteins in the extract is in the range from about 0.1 to about 10.0 milligrams per milliliter (mg/ml), such as in the range from about 1 to about 5 mg/ml. hi particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 5.0 mg/ml, such as in the range from about 0.1 to about 5.0 mg/ml, or in the range from about 0.05 to about 0.5 mg/ml. hi particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.5 to about 3.0 mg/ml, or in the range from about 1.0 to about 3.0 mg/ml. hi particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml, or in the range from about 0.1 to about 0.5 mg/ml. hi particular embodiments, the concentration of the extract of fetal porcine brain proteins in a liquid composition is in the range from about 0.05 to about 5.0 mg/ml. hi particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.005 to about 0.5% (w/v). hi particular embodiments, the pharmaceutically acceptable carrier is selected from an aqueous carrier and a lipid carrier. In particular embodiments, the extraui υi xetal porcine brain proteins is in the form of a liquid or in the form of a dry powder.

In particular embodiments, the composition is in a form selected from the group consisting of a liquid, a powder, an aerosol, a capsule, a tablet, a suppository, a cream, a gel and an ointment. In one embodiment, the liquid is selected from the group consisting of an aqueous solution, a lotion, a suspension, a spray, an emulsion, and a microemulsion. In particular embodiments, the composition is formulated for administration as a spray or as an aerosol.

In particular embodiments, the composition is formulated for administration by a route selected from the group consisting of topical, transdermal, intranasal, oral, buccal, parenteral, inhalation, transmucosal, rectal, intralumbar and vaginal.

In particular embodiments, the composition is a solution formulation for parenteral administration and the concentration of the extract of fetal porcine brain proteins in the solution is in the range from about 0.1 to about 3.0 mg/ml, such as or in the range from about 0.5 to about 3.0 mg/ml, or in the range from about 1.0 to about 3.0 mg/ml. In a particular embodiment, the concentration of the extract of fetal porcine brain proteins in the solution is about 1.5 mg/ml.

In particular embodiments, the composition is a liquid formulation for intranasal administration and the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml, or in the range from about 0.1 to about 0.5 mg/ml. In particular embodiments, the composition is a formulation for topical administration selected from the group consisting of a cream, a gel and an ointment, and the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml, or in the range from about 0.1 to about 0.5 mg/ml.

In particular embodiments, the composition further comprises a mixture of polynucleotides. In a particular embodiment, the polynucleotides are polydeoxyribonucleotides. In a particular embodiment, the polynucleotides have a length in the range from about 1,000 to about 7,000 nucleotides, hi a particular embodiment, the polynucleotides have a length in the range from about 2,000 to about 5,000 nucleotides. In a particular embodiment, the mixture of polynucleotides comprises fish sperm DNA. hi a particular embodiment, the fish sperm DNA is selected from the group consisting of salmon sperm DNA, herring sperm DNA, siurgeυn sperm DNA and combinations thereof. In a particular embodiment, the fish sperm DNA is hydrolyzed or sheared, hi particular embodiments, the concentration of the mixture of polynucleotides in the composition is in the range from about 0.1 to about 3.0 milligrams per milliliter (mg/ml). In particular embodiments, the concentration of the mixture of polynucleotides in the composition is about 1.0 mg/ml. hi particular embodiments, the concentration of the mixture of polynucleotides in a liquid composition is in the range from about 0.1 to about 3.0 milligrams per milliliter (mg/ml). In particular embodiments, the concentration of the mixture of polynucleotides in a liquid composition is about 1.0 mg/ml. As used herein, the term "Cognitone" refers to a pharmaceutical composition comprising a fetal porcine brain tissue extract as described herein, and further comprising hydrolyzed salmon sperm DNA (or the equivalent) as a stabilizing agent.

In particular embodiments, the compositions further comprise at least one of a preservative and an antioxidant. hi particular embodiments, the composition is a formulation for topical administration selected from the group consisting of a cream, a gel and an ointment, and wherein the formulation comprises: the extract of fetal porcine brain proteins at a concentration of about 0.05 to about 0.5 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 0.5 to about 2.0 mg/ml, and lecithin at a concentration of about 1.0 to about 3.0% (w/v). In particular embodiments, the formulation for topical administration further comprises DL-α-tocopherol acetate at a concentration of about 0.05 to about 0.5% (w/v). hi particular embodiments, the formulation for topical administration comprises: the extract of fetal porcine brain proteins at a concentration of about 0.1 to about 0.2 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 1.0 mg/ml; and lecithin at a concentration of about 2.0% (w/v). hi particular embodiments, the composition is a liquid formulation for intranasal administration, wherein the formulation comprises: the extract of fetal porcine brain proteins at a concentration of about 0.05 to about 0.5 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 0.5 to about 2.0 mg/ml, and lecithin at a concentration of about 1.0 to about 3.0% (w/v). hi particular embodiments, the formulation for intranasal administration further comprises DL-α-tocopherol acetate at a concentration of about 0.05 to about 0.5% (w/v). In particular embodiments, the liquid formulation for intranasal administration comprises: the extract of feiai porcine brain proteins at a concentration of about 0.1 to about 0.2 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 1.0 mg/ml; and lecithin at a concentration of about 2.0% (w/v). In particular embodiments, the liquid formulation for intranasal administration further comprises saline or phosphate buffered saline.

In particular embodiments, the extract of fetal porcine brain proteins is obtained by a process comprising: (i) harvesting brain tissue from porcine fetuses; (ii) mechanically homogenizing the brain tissue from (i) in an appropriate buffer; (iii) obtaining a soluble fraction from the homogenized brain tissue of (ii); (iv) subjecting the soluble fraction of (iii) to anion exchange chromatography so as to obtain a bound and eluted fraction, and (v) subjecting the bound and eluted fraction of (iv) to molecular weight separation so as to obtain a purified fraction of proteins having a desired molecular weight range.

In other aspects, the invention provides methods for treating a disorder selected from the group consisting of a neurological disorder and a cardiovascular disorder. These methods comprise administering to a patient in need thereof a therapeutically effective amount of the composition of the invention, thereby treating the disorder. Neurological disorders include ischemic stroke, cerebral infarction, brain ischemia, brain hemorrhage, traumatic brain injury, cerebral palsy, pre-senile dementia, Alzheimer's disease, vascular encephalopathy, and chronic fatigue syndrome. Cardiovascular disorders include hypertension, cardiac ischemia, myocardial infarction, angina, and post-infarction cardiosclerosis.

In other aspects, the invention provides use of the composition of the invention for preparation of a medicament for treatment of a neurological disorder or a cardiovascular disorder. In other aspects, the invention provides the composition of the invention for treating a neurological disorder or a cardiac disorder.

In other aspects, the invention provides methods for treating a skin wound or a skin disorder, comprising topically administering to a patient in need thereof a therapeutically effective amount of the composition of the invention, thereby treating the skin wound or skin disorder. Skin disorders include psoriasis, eczema, contact dermatitis and seborrheic dermatitis. Skin wounds include thermal and chemical burns, traumatic skin injury and decubitus ulcer. In other aspects, the invention provides use of the composition of the invention for preparation of a medicament for treatment of a skin wound or a skin disorder. In other aspects, the invention provides the composition of the invention for treating a skin wound or a skin disorder. In particular embodiments, the composition is administered topically, transdermally, intranasally, orally, buccally, parenterally, by inhalation, transmucosally, rectally, intralumbarly or vaginally.

In a particular embodiment, the subject is suffering from a neurological disorder and the composition is initially administered intravenously or intralubarly and thereafter the composition is administered intranasally. That is, an initial dose is administered intravenously or intralubarly, and thereafter subsequent doses are administered intranasally. hi a particular embodiment, the subject is suffering from a burn wound and the composition is administered topically. hi particular embodiments, the composition is administered in an amount sufficient to provide a daily dose of about 0.005 to about 5 mg of the extract of fetal porcine brain proteins. In particular embodiments, the composition is administered in an amount sufficient to provide a daily dose of about 0.05 to about 5 mg of the extract of fetal porcine brain proteins. In particular embodiments, the composition is administered parenterally, intranasally or intranasally in an amount sufficient to provide a daily dose of about 0.005 to about 5 mg of the extract of fetal porcine brain proteins. hi particular embodiments, the composition is administered parenterally, in an amount sufficient to provide a daily dose of about 0.05 to about 5 mg of the extract of fetal porcine brain proteins. hi particular embodiments, the composition is administered intranasally, in an amount sufficient to provide a daily dose of about 0.005 to about 0.05 mg of the extract of fetal porcine brain proteins. hi particular embodiments, the composition is administered topically, in an amount sufficient to provide a daily dose of about 0.05 to about 1.0 mg of the extract of fetal porcine brain proteins. In particular embodiments, the composition is administered to the subject in an . amount sufficient to provide a daily dose of about 0.05 to about 50 μg per kg body weight. In particular embodiments, the composition is administered to the subject in an amount sufficient to provide a daily dose of about 1-40 μg per kg body weight. Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an illustrative size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) profile of the fetal porcine brain tissue extract of the invention. Arrows indicate the elution volume of the stated molecular weight standards.

Figure 2 shows the effect of compositions of the invention on viability of cardiomyocyte primary cultures subjected to hypoxia, as assessed by release of lactate dehydrogenase (LDH). Systems shown are control (empty bar); treated with Cognitone (black bar); treated with fetal porcine brain tissue extract (gray bar), and treated with hydrolyzed salmon sperm (hss) DNA (cross-hatched bar).

Figure 3 shows the effect of compositionsof the invention on viability of cardiomyocyte primary cultures subjected to hypoxia, as assessed by cell count. Systems shown are control (empty bar); treated with Cognitone (black bar); treated with fetal porcine brain tissue extract (gray bar), and treated with hss DNA (cross-hatched bar). Figure 4 shows the effect of compositions of the invention on apoptosis of cardiomyocyte primary cultures subjected to hypoxia, as assessed by caspase 3 activity. Systems shown are control (empty bar); treated with Cognitone (black bar); treated with fetal porcine brain tissue extract (gray bar), and treated with hss DNA (cross-hatched bar).

Figure 5 shows the effect of compositions of the invention on viability of neuron primary cultures subjected to hypoxia, as assessed by release of lactate dehydrogenase (LDH). Systems shown are control (empty bar); treated with Cognitone (black bar); treated with fetal porcine brain tissue extract (gray bar), and treated with hss DNA (cross-hatched bar).

Figure 6 shows the effect of compositions of the invention on viability of neuron primary cultures subjected to hypoxia, as assessed by cell count. Systems shown are control (empty bar); treated with Cognitone (black bar); treated with fetal porcine brain tissue extract (gray bar), and treated with hss DNA (cross-hatched bar).

Figure 7 shows the effect of compositions of the invention on apoptosis of neuron primary cultures subjected to hypoxia, as assessed by caspase 3 activity. Systems shown are control (empty bar); treated with Cognitone (black bar); treated with fetal porcine brain tissue extract (gray bar), and treated with hss DNA (cross-hatched bar).

Figure 8 shows the effects of intranasal (LN.) and intraperitoneal (LP.) administration of compositions of the invention on neurological status in an animal model of stroke ischemia. Systems shown are: sham operated animals (diamond symbols); animals subjected to middle cerebral artery occlusion (MCAo) (square symbols); MCAo followed by LP. administration of Cognitone (filled triangle symbols); MCAo followed by intranasal I.N. administration of purified brain tissue extract of the invention (empty curcle symbols); MCAo followed by LN. administration of Cognitone (empty triangle symbols), and MCAo followed by LP. administration of purified brain tissue extract of the invention (filled circle symbols).

Figure 9 shows the effects of LN. and LP. administration of compositions of the invention on the size of infarction area in an animal model of stroke ischemia. Systems shown are: animals subjected to MCAo followed by saline administration (empty bar); MCAo followed by LP. administration of Cognitone (checked bar); MCAo followed by LN. administration of Cognitone (black bar); MCAo followed by LP. administration of purified brain tissue extract of the invention (diagonally striped bar), MCAo followed by LN. administration of purified brain tissue extract of the invention (gray bar), and sham operated animals. Figure 10 shows the effects of LN. and LP. administration of compositions of the invention on white blood cell count in an animal model of stroke ischemia. Systems shown are: sham operated animals (diamond symbols); animals subjected to MCAo (square symbols); MCAo followed by LP. administration of Cognitone (filled triangle symbols); MCAo followed by LN. administration of Cognitone (empty triangle symbols); MCAo followed by LP. administration of purified brain tissue extract of the invention (filled curcle symbols), and MCAo followed by intranasal LN. administration of purified brain tissue extract of the invention (empty circle symbols).

Figure 11 shows the effects of LN. and LP. administration of compositions of the invention on the level of IL-6 in an animal model of stroke ischemia. Systems shown are: sham operated animals (diamond symbols); animals subjected to MCAo (square symbols); MCAo followed by LP. administration of Cognitone (filled triangle symbols); MCAo followed by LN. administration of Cognitone (empty triangle symbols); MCAo followed by LP. administration of purified brain tissue extract of the invention (filled circle symbols), and MCAo followed by intranasal LN. administration of purified brain tissue extract of the invention (empty circle symbols).

Figure 12 shows a comparison of the effect of compositions of the invention versus that of prior art porcine brain tissue extract products on the size of infarction area in an animal model of stroke ischemia. Systems shown are: animals subjected to MCAo followed by saline administration (empty bar); MCAo followed by LP. administration of Cognitone (checked bar); MCAo followed by I.N. administration of Cognitone (black bar); MCAo followed by LP. administration of purified brain tissue extract of the invention (diagonally striped bar); MCAo followed by LP. administration of Cerebrolysin™ (stippled bar), and MCAo followed by LP. administration of Cortexin™ (diamond filled bar). Figure 13 shows a comparison of the effect of compositions of the invention versus that of prior art porcine brain extract products on neurological status in an animal model of stroke ischemia. Systems shown are: sham operated animals (diamond symbols); animals subjected to MCAo (square symbols); MCAo followed by LP. administration of Cognitone (filled triangle symbols); MCAo followed by LN. administration of Cognitone (empty triangle symbols); MCAo followed by LP. administration of purified brain tissue extract of the invention (filled circle symbols); MCAo followed by LP. administration of Cerebrolysin™ (empty circle symbols), and MCAo followed by LP. administration of Cortexin™ (asterisk symbols).

Figure 14 shows a comparison of the effect of compositions of the invention versus that of prior art brain extract products on mortality rate in an animal model of stroke ischemia. Systems shown are: animals subjected to MCAo followed by saline administration (empty bar); MCAo followed by LP. administration of Cognitone (checked bar); MCAo followed by LN. administration of Cognitone (black bar); MCAo followed by LP. administration of purified brain tissue extract of the invention (diagonally striped bar); MCAo followed by LP. administration of Cerebrolysin™ (stippled bar), and MCAo followed by LP. administration of Cortexin™ (diamond filled bar).

Figure 15 shows the effect of intraperitoneal administration of compositions of the invention on the size of cardiac infarction in an animal model of cardiac ischemia. Systems shown are: sham operated animals (gray bar); animals subjected to cardiac ischemia (empty bar); cardiac ischemia followed by LP. administration of purified brain tissue extract of the invention (diagonally striped bar); cardiac ischemia followed by LP. administration of Cognitone (checked bar); cardiac ischemia followed by LP. administration of vehicle (stippled bar); and cardiac ischemia followed by LP. administration of vehicle plus hssDNA (cross-hatched bar). Figure 16 shows the effect of administration of the fetal porcine brain tissue extract of the invention on cognitive ability in an animal behavior model. Systems shown are: control animals not operated upon (1); sham operated animals (2); photothrombosis group treated with saline (3), and photothrombosis group treated with Cognitone (4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention features a purified protein extract derived from fetal porcine brain tissue, comprising a heterogenous mixture of proteins having a characteristic molecular weight range and distribution, such as that depicted in Figure 1 herein. The fetal porcine protein extract has been shown to exhibit a range of beneficial biological activities, and may be effectively and safely used in the treatment of a variety of serious neurological and cardiovascular conditions in humans which are of high morbidity and mortality in the Western world. Definitions In the present invention, the terms "fraction", "extract", "purified protein extract" and the like are used interchangeably to refer to a protein preparation derived from fetal porcine brain tissue, which has undergone mechanical homogenization and a purification process to remove molecules smaller than a predetermined molecular weight. Typically, the molecular weight cutoff used for purification is 5 kDa, so that molecules below this size are excluded. Accordingly, the lower limit of the molecular weight range of the protein extract will be 5 kDa. In particular embodiments, the lower limit of the molecular weight range may be a higher value, in particular, 10,000 Da or 15,000 Da or 20,000 Da.

As used herein "substantially devoid" in reference to proteins of a specified molecular weight range, means that the subject composition or extract contains less than about 5%, preferably less than about 2%, more preferably less than about 1%, and even more preferably less than about 0.1% of proteins having the stated molecular weight range. Thus for example, an "extract substantially devoid of proteins of molecular weight less than about 10,000 Da", could contain less than about 1% of proteins of molecular weight less than about 10,000 Da.

It is to specifically understood that the present invention does not encompass porcine brain tissue compositions or extracts prepared by methods which include proteoloytic i.e. enzymatic digestion or chemical hydrolysis of the proteins. Furthermore, in certain embodiments, the extract of the invention is has not been contacted with detergents, organic solvents, zinc salts or chaotropic agents during the course of preparation. Accordingly, in certain embodiments, the proteins in the exxraci substantially retain their native conformation and are essentially intact.

As used herein, "native conformation" refers to the three-dimensional structure of a protein. As used herein, "an appropriate buffer" refers to a buffer which may be used to prepare the fetal porcine protein extract of the invention without substantial loss of its biological activity. Accordingly, an appropriate buffer preferably has pH in the range from 7 to 8, and is free of denaturing agents such as detergents, organic solvents, zinc salts or chaotropic agents. As used herein, the terms "porcine", "pig" and "swine" are used interchangeably to refer to the ungulate mammal known as Sus domestica.

As used herein, the terms "fetal" and "embryonic" are used interchangeably to refer to a porcine fetus of gestational age less than the full-term gestational period of 112 to 114 days, i.e. gestational age less than about 16.3 weeks. In particular embodiments, the fetal porcine brain tissue extract is derived from porcine fetuses of gestational age of about 3 to about 15 weeks, such as about 6 to about 12 weeks. The fetuses may be of either males or females.

As used herein, the term "Cognitone" refers to a pharmaceutical composition comprising as the active ingredient a fetal porcine brain tissue extract as described herein, and further comprising hydrolyzed salmon sperm DNA (or the equivalent) as a stabilizing agent. Preparation of Cognitone is described in Example 1, although it is to be understood that other protocols which yield a substantially similar extract with regard to degree of purity, molecular weight range and distribution, and biological activity can be used.

As used herein, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise. As used herein, a "subject in need thereof refers to a subject who exhibits at least one clinical sign or symptom of a disease or condition which may be treated using the composition and methods of the invention, such as for example stroke ischemia, cardiac ischemia or Alzheimer's disease.

As used herein, a "therapeutically effective amount" refers to that amount or dosage of the composition according to the invention which is sufficient to substantially inhibit, slow, or reverse the progression of a disease or condition intended for treatment using the composition. The description herein of concentrations, amounts and so forth of various components of the invention in a range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as 1.0 to 5.0 mg/ml should be considered to have specifically disclosed subranges such as 1 to 2 mg/ml, 2 to 3 mg/ml, 3 to 4 mg/ml, 4 to 5 mg/ml, as well as individual numbers within that range. This applies regardless of the breadth of the range and in all contexts throughout this application. As used herein, the term "about" in reference to a numerical value means +/- 10% of the stated numerical value.

Preparation and characterization of the protein extract

Extracts of fetal brain tissue may be prepared by first mechanically shearing and homogenizing fresh or frozen and thawed tissues harvested from fetuses. Homogenizatin is carried out in the presence of an appropriate homogenization buffer, as described in Example 1. Prior to homogenization, the tissue may be dissected, chopped and/or minced. Tissue homogenization may be carried out using various motorized instruments, either those adapted from the food industry or dedicated tissue homogenization instruments developed for biological experimentation. For example, a Potter-El vehj em glass-Teflon™ homogenizer and a Waring™ Blendor™ are well known in the art for such a purpose, but many additional homogenizers, including flow-through homogenizers and high pressure homogenizers may be used.

Preferably, the buffer is one which provides physiological buffering capacity and is devoid of agents which denature proteins such as detergents, chaotropic agents, organic solvents, and cross-linking reagents. Further, the extraction and purification process is preferably carried out while maintaining the tissue or fraction thereof at cold temperatures i.e.

4-8 0 C at all stages.

The rate at which the tissue is homogenized, the ratio of buffer to tissue, the pressure applied, the number of homogenization cycles and other parameters are readily determined by one of average skill in the art.

The homogenate may then be centrifuged to isolate the solubilized fraction of the homogenate, for example, at 10,000 to 20,000 g for 30 to 60 min. The supernatant thus obtained may then be subjected to ion exchange cnromatography, for example anion exchange chromatography as described in Example 1. The choice of anion exchange resin may be readily made by one of average skill in the art, and include for example quarternary (Q)- amine based and diethylaminoethane (DEAE) based resins. Conveniently, the anion exchange chromatography is run in a buffer (also termed "mobile phase buffer") which is substantially the same as that as the homogenization buffer. If required however, a different buffer may be used, in which case the solubilized fraction to be loaded onto the column may first be subjected to buffer exchange, for example by dialysis. Following loading of the sample onto the appropriately equilibrated chromatography column, the column is washed, and the proteins bound to the anion exchange resin are eluted with an elution buffer of increased ionic strength. Such procedures are well known in the art. Elution may be carried out either in a "single step" e.g. the elution buffer contains the mobile phase buffer and 0.1 M NaCl, or with a linear gradient e.g. the elution buffer contains the mobile phase buffer and 1 M NaCl. A suitable buffer for anion exchange chromatography may be readily determined by one of skill in the art, depending on the resin used and other considerations, hi general however, it may be preferable to use a system in which the pH is as close as possible to the physiological range so as to minimize protein denaturation. Buffers suitable for anion exchange cnromatography include without limitation, L-histidine, bis-Tris, triethanolamine, Tris, N-methyl- diethanolamine, diethanolamine, diaminopropane and ethanolamine. In certain embodiments, the ion exchange cnromatography may be cation exchange cnromatography, in which case the fraction of interest may be that which does not bind to the column and which is found in the "flow-through". The choice of cation exchange media may be readily made by one of average skill in the art, and include for example sulfate and carboxylate based resins. Buffers suitable for cation exchange chromatography include without limitation, malonic acid, phosphate, HEPES and BICINE.

Following the step of ion exchange cnromatography, the fraction of interest may be subjected to molecular weight separation so as to obtain a protein fraction having a desired lower molecular weight limit for example, about 5,000 Da, or about 10,000 Da, or about 15,000 Da, or about 20,000 Da. That is, proteins of molecular weight of less than about 5,000 Da, or less than about 10,000 Da, or less than about 15,000 Da, or less than about 20,000 Da are specifically excluded by a molecular weight separation step. The molecular weight separation may be carried out by any means known in the art for example, membrane dialsysis using tubing of appropriate molecular weight cutoff, or diafϊltration or cross-flow ultrafiltration using a membrane of appropnaic molecular weight cutoff, or size exclusion chromatography. Size exclusion chromatography may further be used to obtain a fraction also having a desired upper molecular weight limit for example, about 100,000 Da, or about 120,000 Da, or about 140,000 Da 5 or about 150,000 Da. In particular embodiments, the proteins in the purified extract are of molecular weight in the range from about 5,000 to about 150,000 Da, or in the range from about 10,000 to about 140,000 Da, or in the range from about 20,000 to about 120,000 Da. In an illutrative embodiment, the proteins have a molecular weight distribution substantially as depicted in Fig. 1. In a particular embodiment, the proteins are substantially intact proteins. In a particular embodiment, the proteins are substantially in a native state. Methods for determining conformation of proteins are known in the art, including for example circular dichroism.

Protein concentration may be monitored at any stage during the purification process and particularly at the completion of the process using methods well known in the art, such as the Bradford assay (Bradford (1976) Anal. Biochem. 72, 248); the Lowry assay (Lowry et al (1951) J. Biol. Chem. 193: 265; a modified Lowry assay for example as disclosed in Hartree (1972). Anal. Biochem. 48:422; or in Dulley et al (1975) Anal. Biochem. 64:136; or a micro volumetric method such as that disclosed in in Desjardins et al (2009) Current Protocols in Protein Science Unit Number: UNIT 3.10 DOI: 10.1002/0471140864.PS0310S55.

The porcine fetal protein extract may be sterilized, for example by passage through a 0.22 micron filter membrane.

Pharmaceutical compositions

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein, or physiologically acceptable salts thereof, with other chemical components such as physiologically acceptable carriers and diluents. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to an organism.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration and/or stability of the active ingredient(s).

Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

As used herein "formulation" refers to the physical state of a pharmaceutical composition that includes a carrier. and at leasi υue pharmaceutically active agent. In general, the carrier is selected to produce the desired type of formulation, for example, a gel, a lotion, or a spray. A single formulation may fit into more than one category or description, for example, a gel formulation may be formulated as a spray. Pharmaceutical compositions may also include one or more additional active ingredients, which are widely known to be effective to treat similar conditions.

The pharmaceutical composition may be in the form of a liquid, a powder, an aerosol, a capsule, a tablet, a suppository, a cream, a gel and an ointment. Exemplary types of liquid include an aqueous solution, a lotion, a suspension, a spray, an emulsion, and a microemulsion. In particular embodiments, the composition is formulated for administration as a spray or as an aerosol.

The pharmaceutical composition may be formulated for administration by a route selected from the group consisting of topical, transdermal, intranasal, oral, buccal, parenteral, inhalation, transmucosal, rectal and vaginal. Different formulations of the composition of the invention are envisioned for different indications.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the composition of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example DMSO, or polyethylene glycol are generally known in the art. For oral administration, the composition can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for orai use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In addition enterocoating are useful as it is desirable to prevent exposure of the active ingredients of the invention to the gastric environment.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compounds doses.

Pharmaceutical compositions, 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 may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers, hi soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler orάnsufflator may be formulated cotuamiπg a powder mix of the active ingredient and a suitable powder base such as lactose or starch.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The pharmaceutical compositions herein described may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

As used herein, the term "gel" refers to a semisolid consisting of large particles interpenetrated by a liquid (see for example, Physical Pharmacy: Physical Chemical Principles In The Pharmaceutical Sciences, A. Martin, J. Swarbrick, A. Cammarata, Eds., Lea & Febiger, Philadelphia, 4th Edition, 1993). In the present invention, the gel compositions are gnerally single-phase gels, meaning that no discernable boundary exists between the polymers and the liquid, since the polymers or other large particles are substantially uniformly distributed throughout the liquid. The polymer particles may be referred to as the dispersed phase, while the liquid may be referred to as the continuous phase.

The carrier of the present invention may further include, for example, a thickener, an emollient, an emulsifier, a humectant, a suspending agent, a film forming agent, a foam building agent, a preservative, an antifoaming agent, a fragrance, a lower monoalcoholic polyol, a high boiling point solvent, a propellant, a colorant, a pigment or mixtures thereof. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC 50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al. (1975) in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Acute and chronic toxicity studies of the fetal porcine brain protein extract of the invention are disclosed herein in Examples 13 and 14 respectively. Acute and chronic toxicity studies of a gel ointment formulation of Cognitone, comprising the fetal porcine brain protein extract and hydrolyzed salmon sperm DNA, are disclosed herein in Examples 9 and 10 respectively. Studies of the allergenic properties of the aforementioned are disclosed herein in Examples 11 and 15. These studies show that the active ingredient of the compositions of the invention, and various formulations thereof are non-toxic even at very high doses, free of allergy-inducing properties and safe for therapeutic use.

Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, withcourse of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.

In particular embodiments, the concentration of fetal porcine brain proteins in the extract is in the range from about 0.1 to about 10.0 milligrams per milliliter (mg/ml), such as in the range from about 1 to about 5 mg/ml.

In particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 5.0 mg/ml, such as in the range from about 0.1 to about 5.0 mg/ml, or in the range from about 0.05 to about 0.5 mg/ml. In particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.5 to about 3.0 mg/ml, or in the range from about 1.0 to about 3.0 mg/ml. In particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml, or in the range from about 0.1 to about 0.5 mg/ml. hi particular embodiments, the concentration of the extract of fetal porcine brain proteins in a liquid composition is in the range from about 0.05 to about 5.0 mg/ml.

In particular embodiments, the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.005 to about 0.5% (w/v).

In particular embodiments, the composition is a solution formulation for parenteral administration and the concentration of the extract of fetal porcine brain proteins in the solution is in the range from about 0.5 to about 3.0 mg/ml, or in the range from about 1.0 to about 3.0 mg/ml. In aparticular embodiment, the concentration of the extract of fetal porcine brain proteins in the solution is about 1.5 mg/ml.

In particular embodiments, the composition is a liquid formulation for intranasal administration and the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml, such as in the range from about 0.1 to about 0.5 mg/ml. In particular embodiments, the composition is a formulation for topical administration selected from the group consisting of a cream, a gel and an ointment, and the concentration of the extract of fetal porcine brain proteins in the composition is in the range from about 0.05 to about 0.5 mg/ml, such as in the range from about 0.1 to about 0.5 mg/ml. In particular embodiments, the tυuijμυsition further comprises a mixture of polynucleotides. In a particular embodiment, the polynucleotides are polydeoxyribonucleotides. In a particular embodiment, the polynucleotides have a length in the range from about 1,000 to about 7,000 nucleotides. In a particular embodiment, the polynucleotides have a length in the range from about 2,000 to about 5,000 nucleotides, hi a particular embodiment, the mixture of polynucleotides comprises fish sperm DNA. hi a particular embodiment, the fish sperm DNA is selected from the group consisting of salmon sperm DNA, herring sperm DNA, sturgeon sperm DNA and combinations thereof, hi a particular embodiment, the fish sperm DNA is hydrolyzed or sheared, hi particular embodiments, the concentration of the mixture of polynucleotides in the composition is in the range from about 0.1 to about 3.0 mg/ml. hi particular embodiments, the concentration of the mixture of polynucleotides in the composition is about 1.0 mg/ml. hi particular embodiments, the concentration of the mixture of polynucleotides in a liquid composition is in the range from about 0.1 to about 3.0 mg/ml. hi particular embodiments, the concentration of the mixture of polynucleotides in a liquid composition is about 1.0 mg/ml.

In particular embodiments, the composition is a formulation for topical administration selected from the group consisting of a cream, a gel and an ointment, and wherein the formulation comprises: the extract of fetal porcine brain proteins at a concentration of about 0.05 to about 0.5 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 0.5 to about 2.0 mg/ml, and lecithin at a concentration of about 1.0 to about 3.0% (w/v). hi particular embodiments, the formulation for topical administration comprises: the extract of fetal porcine brain proteins at a concentration of about 0.1 to about 0.2 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 1.0 mg/ml; and lecithin at a concentration of about 2.0% (w/v). In particular embodiments, the composition is a liquid formulation for intranasal administration, wherein the formulation comprises: the extract of fetal porcine brain proteins at a concentration of about 0.05 to about 0.5 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 0.5 to about 2.0 mg/ml, and lecithin at a concentration of about 1.0 to about 3.0% (w/v). hi particular embodiments, the liquid formulation for intranasal administration comprises: the extract of fetal porcine brain proteins at a concentration of about 0.1 to about 0.2 mg/ml; hydrolyzed salmon sperm DNA at a concentration of about 1.0 mg/ml; and lecithin at a concentration of about 2.0% (w/v). Therapeutic indications

Disorders which may be effectively treated in accordance with the invention include brain disorders, neurological disorders and cardiovascular disorders. These methods comprise administering to a patient in need thereof a therapeutically effective amount of the composition of the invention, thereby treating the disorder. Brain disorders include ischemic stroke, cerebral infarction, brain ischemia, brain hemorrhage and traumatic brain injury. Neurological disorders include cerebral palsy, pre-senile dementia, Alzheimer's disease, vascular encephalopathy, and chronic fatigue syndrome. Cardiovascular disorders include hypertension, cardiac ischemia, angina, and post-infarction cardiosclerosis. In other aspects, the invention provides methods for treating a skin wound or a skin disorder, comprising topically administering to a patient in need thereof a therapeutically effective amount of the composition of the invention, thereby treating the skin wound or skin disorder. Skin disorders include psoriasis, eczema, contact dermatitis and seborrheic dermatitis. Skin wounds include thermal and chemical burns, and decubitus ulcer. In particular embodiments, the composition is administered in an amount sufficient to provide a daily dose of about 0.005 to about 5 mg of the extract of fetal porcine brain proteins. hi particular embodiments, the composition is administered parenterally, topically or intranasally, in an amount sufficient to provide a daily dose of about 0.05 to about 3 mg of the extract of fetal porcine brain proteins. hi particular embodiments, the composition is administered to the subject in an amount sufficient to provide a daily dose of about 0.05 to about 50 μg per kg body weight, or a daily dose of about 1 to about 40 μg per kg body weight.

Brain ischemia, also known as cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand. This leads to poor oxygen supply or cerebral hypoxia and thus to the death of brain tissue or cerebral infarction/ischemic stroke. It is a sub-type of stroke along with subarachnoid hemorrhage and intracerebral hemorrhage. Ischemia leads to alterations in brain metabolism, reduction in metabolic rates, and energy crisis. The broad term, "stroke" can encompasses brain ischemia, subarachnoid hemorrhage and intracerebral hemorrhage. Brain ischemia encompasses focal ischemia, which is confined to a specific region of the brain, and global ischemia, which encompasses wide areas of brain tissue. Focal brain ischemia occurs when a blood clot has occluded a cerebral vessel. Focal brain ischemia reduces blood flow to a specific brain region, increasing the risk of cell death to that particular area. It can be caused by thrombosis or embolism. Global brain ischemia occurs when blood flow to the brain is halted or drastically reduced. This is commonly caused by cardiac arrest. If sufficient circulation is restored in a short period of time symptoms may be transient. However if a significant amount of time passes before the restoration defects will be permanent. Reperfusion, even though it is essential to save as much brain tissue as possible may lead to a reperfusion injury. A reperfusion injury is the damage that is done to the tissue when blood supply returns after a period of ischemia. Brain ischemia can be further subdivided by cause into thrombotic, embolic, and hypoperfusion.The symptoms that occur with brain ischemia depend on which part of the brain is being deprived of blood and oxygen. Arteries that branch from the internal carotid artery that experience brain ischemia may cause symptoms such as blindness in one eye, weakness in one arm or leg, or weakness in one entire side of the body. Arteries that branch from the vertebral arteries in the back of the brain may cause the experience of symptoms such as dizziness, vertigo, double vision, or weakness on both sides of the body, during brain ischemia in that part of the brain. Other symptoms that may be experienced include difficulty speaking, slurred speech, and the loss of coordination. The symptoms of brain ischemia can be apparent for a very short amount of time, from a few seconds to a few minutes, or they may persist for largely extended periods of time. If the brain becomes damaged irreversibly and infarction occurs, the symptoms will remain constant.

Similarly to cerebral hypoxia, severe or prolonged brain ischemia will result in unconsciousness, brain damage or death, mediated by the ischemic cascade.

Multiple cerebral ischemic events may lead to subcortical ischemic depression, also known as vascular depression. This condition is most commonly seen in elderly depressed patients. Late onset depression is increasingly seen as a distinct variety of depression, and is commonly detected with an MRI.

Brain ischemia has been linked to a variety of diseases including sickle cell anemia, the compression of blood vessels, ventricular tachycardia, plaque buildup in the arteries, blood clots, extremely low blood pressure as a result of heart attack, and congenital heart defects. Ventricular tachycardia is a series of irregular heartbeats that may cause the heart to completely shut down and cause all oxygen flow to cease. Also the irregular heartbeat can cause blood clots to be released to the brain or other parts of the body. Therefore, all of the major organs could undergo ischemia. Blockage of the arteries as a result of plaque buildup is also a cause of ischemia. Even a small amount of plaque being built, up causes the narrowing of passageways for blood to flow, causing that area to become more prone to blood clots. Large blood clots can block blood flow to a major organ including the brain, resulting in brain ischemia. A heart attack can also cause brain ischemia due to the correlation that exists between heart attack and low blood pressure. Extremely low blood pressure usually represents the inadequate oxygenation of tissues. Untreated heart attacks may slow blood flow enough that blood may start to clot and prevent the flow of blood to the brain or other major organs. Extremely low blood pressure can also result from drug overdose and reactions to drugs. Congenital heart defects may also cause brain ischemia due to the lack of appropriate artery formation and connection.

Traumatic brain injury (TBI) is damage to the brain resulting from external mechanical force, such as rapid acceleration or deceleration, impact, blast waves, or penetration by a projectile. Brain function is temporarily or permanently impaired and structural damage may or may not be detectable with current technology. TBI is one of two subsets of acquired brain injury (brain damage that occurs after birth); the other subset is nontraumatic brain injury, which does not involve external mechanical force (examples include stroke and infection). All traumatic brain injuries are head injuries, but the latter term may also refer to injury to other parts of the head. However, the terms head injury and brain injury are often used interchangeably. TBI is usually classified based on severity, anatomical features of the injury, and the mechanism (the causative forces). Mechanism-related classification divides TBI into closed and penetrating head injury. A closed (also called nonpenetrating, or blunt) injury occurs when the brain is not exposed. A penetrating, or open, head injury occurs when an object pierces the skull and breaches the dura mater, the outermost membrane surrounding the brain. The Glasgow Coma Scale (GCS), a universal system for classifying TBI severity, grades a person's level of consciousness on a scale of 3-15 based on verbal, motor, and eye-opening reactions to stimuli, wherein a TBI with a GCS of 13 or above is mild, 9-12 is moderate, and 8 or below is severe. Other classification systems determine severity based on the GCS after resuscitation, the duration of post-traumatic amnesia, loss of consciousness, or combinations thereof.

Damage from TBI can be focal or diffuse, confined to specific areas or distributed in a more general manner, respectively. Diffuse injury manifests with little apparent damage in neuroimaging studies, but lesions can be seen with microscopy techniques post-mortem. Types of injuries considered diffuse include concussion and diffuse axonal injury, widespread damage to axons in areas including white matter and the cerebral hemispheres. Focal injuries often produce symptoms related to the functions of the damaged area, manifesting in symptoms like hemiparesis or aphasia when motor or language areas are respectively damaged. Cerebral palsy (CP) is an umbrella term encompassing a group of non-progressive, motor, non-contagious conditions that cause physical disability in human development.

CP is caused by damage to the motor control centers of the developing brain and can occur during pregnancy (about 75 percent), during childbirth (about 5 percent) or after birth (about 15 percent) up to about age three. Cerebral palsy describes a group of permanent disorders of the development of movement and posture, causing activity limitation, that are attributed to nonprogressive disturbances that occurred in the developing fetal or infant brain. The motor disorders of cerebral palsy are often accompanied by disturbances of sensation, perception, cognition, communication, and behaviour, by epilepsy, and by secondary musculoskeletal problems. There is no known cure for CP, and medical intervention is limited to the treatment and prevention of complications arising from its effects.

CP is divided into three major classifications to describe different movement impairments i.e. spastic, ataxic and athetoid/diskinetic.

Spastic CP is the most common type, occurring in 70% to 80% of all cases. Moreover, spastic CP accompanies one of the other types in 30% of all cases. Individuals with spastic CP are hypertonic and have a neuromuscular condition stemming from damage to the corticospinal tract or the motor cortex that affects the nervous system's ability to receive gamma amino butyric acid in the area(s) affected by the disability. Spastic CP is further classified according to the region of the body affected, namely: spastic hemiplegia (one side affected); spastic diplegia (the lower extremities are affected with little to no upper-body spasticity), and spastic quadriplegia (all four limbs affected equally).

Spastic hemiplegia is the result of injury to one side of the brain resulting in motor deficit on the opposite side of the body. Thus, muscle-nerves controlled by the brain's left side will cause a right body deficit, and vice versa. Typically, individuals with spastic hemiplegia are the most ambulatory, although they generally have dynamic equinus on the affected side. Individuals with spastic diplegia are fully ambulatory and have a scissors gait. Flexed knees and hips to varying degrees are common. Hip problems, dislocations, and in three- quarters of spastic diplegics, also strabismus (crossed eyes), can be present as well. In addition, these individuals are often nearsighted.

Individuals with spastic quadriplegia are the least likely to be able to walk. Some children with quadriplegia also have hemiparetic tremors, an uncontrollable shaking, that affects the limbs on one side of the body and impairs normal movement.

Ataxia type symptoms can be caused by damage to the cerebellum. Forms of ataxia are less common types of cerebral palsy, occurring in at most 10% of all cases. Some of these individuals have hypotonia and tremors. Motor skills may be affected, as well as balance.

Commonly, affected individuals have difficulty with visual and/or auditory processing of objects.

Athetoid or dyskinetic is mixed muscle tone, and most affected individuals have difficulty holding themselves in an upright, steady position for sitting or walking, and often show involuntary motions. The damage occurs to the extrapyramidal motor system and/or pyramidal tract and to the basal ganglia.

Alzheimer's disease (AD), is the most common form of dementia. This incurable, degenerative, and terminal disease is usually diagnosed in people over 65 years of age, although the less-prevalent early-onset Alzheimer's can occur much earlier. In the early stages, the most commonly recognised symptom is memory loss, such as difficulty in remembering recently learned facts. The diagnosis of AD is usually confirmed with behavioural assessments and cognitive tests, often followed by a brain scan. As the disease advances, symptoms include confusion, irritability and aggression, mood swings, language breakdown, long-term memory loss, and general withdrawal of the sufferer as the senses decline. Gradually, bodily functions are lost, ultimately leading to death. Individual prognosis is difficult to assess, as the duration of the disease varies. AD develops for an indeterminate period of time before becoming fully apparent, and it can progress undiagnosed for years. The mean life expectancy following diagnosis is approximately seven years, and fewer than three percent of individuals live more than fourteen years after diagnosis. The cause and progression of Alzheimer's disease are not well understood, and a large body of research indicates that the disease is associated with abnormal protein plaques and tangles in the brain.

Vascular encephalopathy, also termed subcortical vascular encephalopathy (SVE) is a progressive disability with immobilisation because of gait- and postural disturbances and with a progressive subcortical vascular dementia which is composed of cognitive slowing, loss of initiative and forgetfulness. The pathophysiological basis is a cerebral microangiopathy leading to lacunar infarcts and to diffuse ischemic white matter lesions. Such lesions lead to an interruption of parallel functional prefrontal-subcortical circuits, which are essential for psychomotor function. Neuroradiological methods like computed tomography (CT) and magnetic resonance imaging (MRI) are required for its accurate diagnosis.

Chronic fatigue syndrome (CFS), also known as myalgic encephalomyelitis, is a poorly understood, variably debilitating disorder or disorders characterized by medically unexplained symptoms. Symptoms of CFS include widespread muscle and joint pain, cognitive difficulties, chronic, often severe mental and physical exhaustion and other characteristic symptoms in a previously healthy and active person. Diagnosis requires a number of features the most common being severe mental and physical exhaustion which is "unrelieved by rest", is worsened by exertion and is present for at least six months. All diagnostic criteria require that the symptoms must not be caused by other medical conditions. Additional symptoms include muscle weakness, cognitive dysfunction, hypersensitivity, orthostatic intolerance, digestive disturbances, depression, poor immune response, cardiac and respiratory problems. It is unclear if these symptoms represent co-morbid conditions or are produced by an underlying etiology of CFS. Hypertension (HTN), also known as high blood pressure, is a medical condition in which the blood pressure is chronically elevated. The term "hypertension" without a qualifier normally refers to systemic, arterial hypertension, while pulmonary hypertension involves lung circulation.

Hypertension can be either essential (primary) or secondary. Essential hypertension (about 90-95% of cases) indicates that no specific medical cause can be found to explain the condition. Secondary hypertension indicates that the high blood pressure is a result of another condition, such as kidney disease or tumors.

Persistent hypertension is one of the risk factors for strokes, heart attacks, heart failure and arterial aneurysm, and is a leading cause of chronic renal failure. Even moderate elevation of arterial blood pressure leads to shortened life expectancy. At severely high pressures, defined as mean arterial pressures 50% or more above average, a person can expect to live no more than a few years unless appropriately treated. Beginning at a systolic pressure (of 115 mmHg and diastolic pressure of 75 mrnHg (115/75 mmHg), cardiovascular disease risk doubles for each increment of 20/10 mmHg. Hypertension may be classifed as prehypertension, hypertension (stages I and II), and isolated systolic hypertension, which is a common occurrence among the elderly. These readings are based on the average of seated blood pressure readings that were properly measured during 2 or more office visits. In individuals older than 50 years, hypertension is considered to be present when a person's blood pressure is consistently at least 140 mmHg systolic or 90 mmHg diastolic. Resistant hypertension is defined as the failure to reduce blood pressure to the appropriate level after taking a three-drug regimen (include thiazide diuretic).

Headaches are a common symptom of hypertension, although mild to moderate essential hypertension is largely asymptomatic. Accelerated hypertension is associated with somnolence, confusion, visual disturbances, and nausea and vomiting (hypertensive encephalopathy). Retinas are affected with narrowing of arterial diameter to less than 50% of venous diameter, copper or silver wire appearance, exudates, hemorrhages, or papilledema. Signs and symptoms of hypertension in infants and neonates includes failure to thrive, seizure, irritability or lethargy, and respiratory distress, hi children hypertension may cause headache, fatigue, blurred vision, epistaxis, and bell palsy.

Cardiac ischemia occurs when blood flow to the heart muscle (myocardium) is decreased by a partial or complete blockage of a coronary artery. A sudden, severe blockage may lead to a heart attack (myocardial infarction; MI), or arrhythmia, which can cause sudden death. Signs and symptoms of cardiac ischemia include chest pain (angina pectoris), pain in neck, jaw or arm, clammy skin, shortness of breath, nausea and vomiting. Diagnosis of cardiac ischemia is based on a combination of medical history, physical examination, electrocardiogram, cardiac enzyme assay, stress test, magnetocardiogreaphy and coronary angiogram.

Shortness of breath occurs when the damage to the heart limits the output of the left ventricle, causing left ventricular failure and consequent pulmonary edema. The symptoms of diaphoresis (an excessive form of sweating), weakness, lightheadedness, nausea, vomiting, and palpitations are likely induced by a massive surge of catecholamines from the sympathetic nervous system which occurs in response to pain and the hemodynamic abnormalities that result from cardiac dysfunction. The two basic types of acute MI are transmural MI, associated with atherosclerosis involving major coronary artery, and subendocasdial MI which involves a small area, e.g. the subendocardial wall of the left ventricle, ventricular septum, orpapillary muscles. Transmural MI is further subclassified into anterior, posterior or inferior.

Approximately one fourth of all MI cases are silent, without chest pain or other symptoms. These cases can be discovered later on electrocardiograms or at autopsy without a prior history of related complaints. A silent course is more common in the elderly, in patients with diabetes mellitus and after heart transplantation. In diabetics, differences in pain threshold, autonomic neuropathy,

MI rates are higher in association with intense exertion, either psychological stress or physical exertion, especially if the exertion is more intense than the individual usually performs.Acute severe infection, such as pneumonia, can trigger myocardial infarction. Risk factors for atherosclerosis are generally risk factors for myocardial infarction, and include diabetes (with or without insulin resistance), smoking, hyperlipoproteinemia, hypertension, familiy history of ischemic heart disease (IHD), obesity (defined by a body mass index of more than 30 kg/m 2 , or alternatively by waist circumference or waist-hip ratio), old age, hyperhomocysteinernia; occupational stress, use of combined oral contraceptive pills, especially in the presence of other risk factors, such as smoking. Inflammation is an important step in the process of atherosclerotic plaque formation. C-reactive protein (CRP) is a sensitive but non-specific marker for inflammation. Elevated CRP blood levels, especially measured with high sensitivity assays, can predict the risk of MI. Inflammation in periodontal disease may be linked to coronary heart disease.

Acute MI refers to two subtypes of acute coronary syndrome, namely non-ST-elevated myocardial infarction and ST-elevated myocardial infarction, which are most frequently a manifestation of coronary artery disease. The most common triggering event is the disruption of an atherosclerotic plaque in an epicardial coronary artery, which leads to a clotting cascade, sometimes resulting in total occlusion of the artery. Atherosclerosis is the gradual buildup of cholesterol and fibrous tissue in plaques in the wall of arteries (in this case, .the coronary arteries), typically over decades. Blood stream column irregularities visible on angiography reflect artery lumen narrowing as a result of decades of advancing atherosclerosis. Plaques can become unstable, rupture, and additionally promote a thrombus (blood clot) that occludes the artery; this can occur in minutes. When a severe enough plaque rupture occurs in the coronary vasculature, it leads to MI (necrosis of downstream myocardium).

If impaired blood flow to the heart lasts long enough, it triggers a process called the ischemic cascade; the heart cells in the territory of the occluded coronary artery die (chiefly through necrosis) and do not grow back. A collagen scar forms in its place. Apoptosis also plays a role in the process of tissue damage subsequent to myocardial infarction. As a result, the patient's heart will be permanently damaged. Myocardial scarring puts the patient at risk for potentially life threatening arrhythmias, and may result in the formation of a ventricular aneurysm that can rupture with catastrophic consequences. Myocardial scarring following MI is also termed post-infarction cardiosclerosis.

Injured heart tissue conducts electrical impulses more slowly than normal heart tissue. The difference in conduction velocity between injured and uninjured tissue can trigger reentry or a feedback loop that is believed to be the cause of many lethal arrhythmias. The most serious of these arrhythmias is ventricular fibrillation (V-Fib/VF), an extremely fast and chaotic heart rhythm that is the leading cause of sudden cardiac death. Another life threatening arrhythmia is ventricular tachycardia (V-Tach/VT), which may or may not cause sudden cardiac death. However, ventricular tachycardia usually results in rapid heart rates that prevent the heart from pumping blood effectively. Cardiac output and blood pressure may fall to dangerous levels, which can lead to further coronary ischemia and extension of the infarct.

Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

Example 1. Preparation of the Pharmaceutical Composition termed "Cognitone"

A. Homogenization and Extraction of Porcine Tissue Active Fraction

1. Frozen (-20 0 C) brain tissue of pig embryos (6-11 weeks of gestation) were defrosted at 4-8 C and transferred into cooled isolation buffer (0.05M Tris-glycine, ImM EDTA, pH 7.6-7.8). Approximately 300-400 ml of buffer was added for each 100 g of tissue.

AU subsequent procedures were performed at 4-8 0 C:

2. The defrosted tissue in isolation buffer was minced and homogenized in a blender (e.g. "Kitchen Aid" Model 5KSB3EWH3, USA) for 4-5 min.

3. The homogenate was centrifuged for 40 min. at 10000-1 lOOOg (approximately 14000 rpm, at an approximate centrifuge radius of 5 cm).

4. The supernatant was filtered through a glass microfiber filter paper (grade GFfB, Whatman International Ltd). The filtrate was collected for the further purification.

5. The filtrate was fractionated by anion-exchange chromatography on a glass column (2.5x25cm) packed with the anion exchanger Toyopearl DEAE-650M (Tosoh Bioscience, Pennsylvania USA), previously equilibrated with mobile phase buffer

(0.05M Tris-glycine, 0.1 mM EDTA pH 7.8). The mobile phase buffer was delivered to the column using a peristaltic pump (Microperpex model 2132 manufactured by LKB, Bromma, Sweden), at the flow rate 3-5 ml/min. The optical density was monitored with a UV detector at wavelength 280 nm. After equilibration the tissue extract was passed through the column at a flow rate of 2-3 ml/min. Then the column was washed with mobile phase buffer at the flow rate of 3-5 ml/min until E 280 become less then 0.1 OD.

6. The fraction of interest was eluted with elution buffer (0.1M NaCl, O.lmM EDTA and 0.05M Tris-glycine pH 7.8). 7/ The collected fraction was diafiltrated through an Ultracell PL ultrafiltration membrane,- having a molecular weight cut off (MWCO) of 5 kDa (Millipore Corp) using an excess amount of PBS (0.14 M NaCl 5 0.01 M sodium phosphate buffer, pH 7.2-7.4). 8. The protein concentration in the collected fraction was determined according to the method of Lowry (Lowry OH, et al., J. Biol. Chem. 1951, Nov; 193(l):265-75), with

BSA as standard. The fraction was diluted with PBS to the final protein concentration of 2 mg/ml. 8. The solution was sterilized with filtration through a Millipore™ GSWP membrane

(0.22μ pore size). 9. The fraction was stored at -20 0 C until use.

Analysis of the purified porcine brain tissue extract (120 μg of protein) by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) was carried out using a TSK gel G2000SW column (7.5 x 300 mm) run in 0.2 M NaCl, 25 niM Na phosphate buffer, pH 7.2-7.3 at a flow rate of 1.0 ml/min and UV detection at 280 nm. Protein molecular weight calibration standards were human IgG (150 kDa); bovine serum albumin (BSA), dimer (134 kDa); BSA, monomer (67,000 Da); ovalbumin (46,000 Da); cytochrome C (12,300 Da) and aprotinin (6500 Da). The chromatogram obtained from analysis of the porcine brain tissue extract (Fig. 1) indicates a mixture of peptides and proteins ranging in molecular weight between about 5,000 and about 140,000 Da.

B. Hydrolyzed Salmon Sperm DNA (hssDNA) preparation.

Salmon or herring sperm DNA was degraded by alkaline hydrolysis to fragments of 2000-5000 nucleotide base pairs according to the following procedure:

1. Salmon sperm DNA (Calbiochem or Sigma Aldrich, Cat. No. Dl 626) was dissolved in buffer containing 0.15M NaCl, 0.015M citric acid trisodium salt, 0.001M EDTA

(pH 7.4).

2. 1OM NaOH was added to the DNA solution to attain a final concentration of IM NaOH. The hydrolysis of DNA was performed for 24-36 hours at 45-50 0 C.

3. The DNA hydrolysis reaction was terminated by cooling of the alkaline DNA solution to room temperature and adding acetic acid to achieve pH 9.0.

4. The solution of hydrolyzed DNA (hssDNA) was mixed with cold ethanol at a ratio of 1 :2 and incubated for 1 -1.5 hours at 4-8 0 C.

5. The hssDNA was centrifuged for 20 min at 150Og. The sediment was dissolved with buffer containing 0.15 NaCl, 0.015M citric acid trisodium salt, 0.001M EDTA (pH 7.4). 6. The hssDNA was filtered through a glass microfiber filter (grade GF/B, Whatman International Ltd).

7. Steps 4 and 5 were repeated.

8. The pH of hssDNA solution was adjusted to 7.4-7.6 with 0.1 M NaOH.

9. The hssDNA concentration was determined at wavelength 260nm with the extinction coefficient of E 26 o 1%= 2OO.

10. The hssDNA was diluted to a final concentration of 0.01 g/ml and sterilized by filtration through Millipore™ GSWP membrane (0.22 μ pore size).

11. The hssDNA preparation was stored at -20 0 C until use.

C. Cognitone Preparation (Purified Brain Tissue Extract + Hydrolyzed Salmon Sperm DNA) To prepare 100 ml Cognitone:

1. hssDNA, prepared as in step B, was defrosted and heated at 30-35 0 C for 20-30 min.

2. 10 ml of phosphate buffered saline, 10-fold concentrate (PBS 10x ; 1.5M NaCl, 0.1 M Na phosphate, pH 7.2-7.4 of 1 x solution)was added to 70 ml of distilled H 2 O, and mixed thoroughly.

3. hssDNA was added to a final concentration of 1.0 mg/ml, and mixed thoroughly.

4. Purified brain tissue extract fraction, prepared as in step A, was added to a final protein concentration of 0.1 mg/ml, and mixed thoroughly. 5. The volume of preparation was adjusted up to 100 ml with distilled H 2 O and mixed thoroughly.

6. The solution was sterilized by filtration through a Millipore™ GSWP membrane

(0.22μ pore size).

D. Preparation of a lipidic formulation of Cognitone

1. 2.Og of lecithin (AppliChem GmbH, Germany, Cat. No. A-0893,0100) and 100 mg of Vitamin E acetate (Sigma Aldrich, Cat. No. T-3376) were dissolved in 1 ml of 20% (w/vol) methyl paraben (Sigma Aldrich, Cat. No. H-5501)solution in 96% ethanol at 35-40 0 C.

2. The lecithin solution was transferred into the glass tube of a homogenizer, the glass pestle was inserted and the solution was distributed over the walls of the tube. 100 ml of

Cognitone solution, prepared as in step C 5 was poured into the homogenizer.

3. The Cognitone solution was dispersed in the lecithin solution using several pestle strokes.

4. The solution was tested for sterility by observing the amount of bacterial growth after 2 days of incubation at 37 0 C. The solution was sealed in sterile vials. The composition of the lipidic formulation of Cognitone is shown in Table 1.

Table 1.

Example 2. Biological effects of Cognitone and brain tissue extract on primary cultures of cardiomyocvtes under conditions of hypoxia.

Study design and methods.

A primary culture of cardiomyocytes was prepared from rat hearts (1-2 days old). 0.1 ml of either Cognitone, or of brain tissue extract of the invention, were added to the cardiomyocyte culture 5 min before the hypoxia treatment. The cells were subjected to hypoxia for 120 min in a hypoxic incubator in which the atmosphere was replaced by the inert gas argon (100%) in glucose-free PBS. A control culture was treated with hss DNA in the absence of brain tissue extract.

Twenty four hours after administration of hypoxia treatment, the level of cell death (necrotic and apoptotic death) was determined. Lactate dehydrogenase (LDH) level and cell count were used as indicators of necrotic death. Caspase-3 activity was used as an indicator of apoptosis.

Cell death was assessed using an LDH Cytotoxicity Detection Kit (Clontech Laboratories, Inc.) which measures damage to the plasma membrane, based on release of lactate dehydrogenase (LDH). Twenty four hours following hypoxia treatment, culture supernatants were incubated with the kit reagents for 30 min, and the absorbance was measured at 492 nm with a multiwell plate reader. Study groups were: 1) untreated control (no hypoxia); 2) control with hypoxia; 3) hypoxia and treated with 0.1 ml of Cognitone; 4) hypoxia and treated with brain tissue extract, 5) hypoxia and treated with hssDNA. The untreated control culture (no hypoxia and no tested substances) was used as a baseline, in order to estimate the amount of cell damage. All treated groups were compared to this control which was assumed as zero cell death.

For cell counting, cardiomyocyte cultures were washed, cells were collected and counted.

Caspase-3 activity was measured using the Caspase-3 Colorimetric Assay Kit (ApoAlert, Clontech Laboratories, Inc.), based on spectrophotometric detection of the chromophore p-nitroanilide (pNA). Study groups were: 1) untreated control (no hypoxia); 2) control with hypoxia, 3) hypoxia and treated with 0.1 ml of Cognitone; 4) hypoxia and treated with brain tissue extract, 5) hypoxia and treated with hssDNA. The level of caspase-3 activity in the untreated control was used as a baseline, and all treated groups were compared to this control which was assumed as zero cell death. Results

As shown in Fig. 2, treatment with Cognitone (0.1 ml) protected cardiomyocytes from damage caused by hypoxia. Cell damage was only 17±4% as compared to untreated culture, where cell damage after hypoxia reached 76±23%. The difference between control cultures and those treated with Cognitone was found to be significant (*; PO.001).

Brain tissue extract of the invention also exhibited significant protective effect

(38±12%; ***; PO.01), however the effect was significantly lower than that exerted by Cognitone (**; P<0.01). No significant difference was observed between control and hssDNA.As shown in Fig. 3, cultures treated with Cognitone had about 0.7±0.17xl0 6 cells while in the untreated control, the cell count was only about 0.3±0.1xl0 6 cells. The difference between control and Cognitone was significant (n=10; *; PO.01).

Additionally, brain tissue extract had a significant positive effect on the cell count, compared to the control. The cell count for brain tissue extract was 0.5±0.12xl0 6 cells as compared to the control (n=10; **; PO.01).

In contrast, the difference between Cognitone and brain tissue extract was not significant. Further, no significant difference was observed between control cultures and those treated with hssDNA. As shown in Fig. 4, caspase-3 activity in cardiomyocytes treated with 0.1 ml

Cognitone was significantly lower (10±2%) than that in the untreated control (31±7% ; n=10;

*; PO.001). Similarly, cardiomyocytes treated with brain tissue extract exhibited caspase 3 activity significantly lower (16±3%) than that of the untreated control (31±7%; n=10; ***; - P<0.001).In addition, a significant difference was observed between cultures treated with Cognitone and those treated with brain tissue extract (n=10;**; P<0.01).No significant difference was observed between control cultures and those treated with hssDNA alone.

Example 3. Biological effects of Cognitone and brain tissue extract on primary cultures of neurons under conditions of hypoxia. Study design and methods

Primary cultures of neurons were prepared from mouse brain (1-2 days old). Cognitone (0.1 ml), brain tissue extract alone or hssDNA were added to tissue cultures 5 min before hypoxia treatment. Twenty fours after hypoxia cell death was assessed according to LDH release, cell count and caspase-3 activity, essentially as described in Example 2. Results

As shown in Fig. 5, treatment with Cognitone (0.1 ml) protected primary cultures of neuronal cells from hypoxia-induced damage, with such cultures exhibiting only 10±3% of cell damage, as compared to 54±17% of cell damage in control cells. The difference between control and Cognitone was found to be significant (n=10; *; P<0.001).

Brain tissue extract of the invention also exerted a significant protective effect, resulting in 21±8% of cell damage as compared to the control (n=10; ***; P<0.01). However, this protective effect is significantly lower than that of Cognitone (P<0.01). No significant difference was observed between control cultures and those treated with hssDNA

As shown in Fig. 6, cultures treated with Cognitone contained about 0.8±0.21xl0 6 cells., in contrast to the hypoxia control with about 0.2±0.15xl0 6 cells. The difference between control and Cognitone was found to be significant (n=10, *P<0.01).

Brain tissue extract of the invention, had significant positive effect on the cell count, 0.6±0.18xl0 6 cells, compared to control (n=10, **P<0.01).

No significant difference was found between Cognitone and brain tissue extract of the invention, nor between control and hssDNA.

As shown in Fig. 7, caspase 3 activity in neuronal culture cells treated with 0.1 ml Cognitione was significantly lower (7±3%) than that in the untreated control (44±12%, n=10, PO.001).

Similarly, neuronal cells treated with tissue extract of the invention exhibited caspase- 3 activity significantly lower (13±6%) than that of untreated control (44±12%, n=10, ***P<0.001).In addition, a significant difference was observed between cultures treated with Cognitone and and those treated with tissue extract of the invention (n=10, **P<0.01). No significant difference was observed between control and hss DNA

As apparent from Example 2 and Example 3, Cognitone protects both types of cell cultures examined (primary neuronal and cardiomyocyte cultures) from necrotic and apoptotic death in stress conditions (hypoxia). Reduction of cell death was observed upon addition of either Cognitone or purified tissue extract. Cognitone (purified extract + hss DNA) resulted in improved cell protection, as compared to purified extract alone. A control treatment of hss DNA without purified tissue extract, showed no protective effect.

Example 4. Neuroprotective Effect of Cognitone in an Animal Model of Stroke Ischemia; Intranasal Versus Intraperitoneal Administration. Study design and methods Middle cerebral artery occlusion (MCAo) was performed on various groups of rats, followed by neurological evaluation of the animals.

Rats (Wistar strain, weighing 250 - 330 g), were selected and divided into groups of 14 animals per group. Treatment was administered 1 hour after onset of occlusion and then daily (every 24 hours) for a period of 7 days. Treatment groups are described in Table 2.

Table 2.

Induction of permanent Middle Cerebral Artery occlusion (MCAo) was performed by inserting a 4-0 nylon suture in the left internal carotid artery via external carotid artery, until the suture reached and occluded the ostium of the left middle cerebral artery. Intranasal Administration

For I.N. administration, the nasal lipidic formulation prepared as described in Example 1, was administered via a PE 10 tube attached to a microliter syringe inserted 1 cm into each nostril of the rat. A dose of 0.1 mg/kg was delivered.The administration protocol was as described in (Almeida AJ, Alpar HO, Brown MR., "Immune response to nasal delivery of antigenically intact tetanus toxoid associated with poly(L-lactic acid) microspheres in rats, rabbits and guinea-pigs"; J Pharm Pharmacol. 1993 Mar;45(3): 198-203.)

Inflammatory mediator

For measurement of IL-6 production in serum, blood was collected into sterile tubes containing trisodium citrate and a proteinase inhibitor mixture (125 mmol/ml citrate, 5000 mmol/ml EDTA, 6000 mmol/ml N-ethylmaleimide, and 500 kIU/ml aprotinin). The blood samples were immediately centrifuged at 2,000 x g for 10 min at 4 °C and stored at -80 °C until analysis (performed within 1 month). Quantitative determination of rat cytokines in serum was done by enzyme-linked immunosorbent assays (ELISA), using specific kits for IL- 6 (R&D Systems Europe, Abingdon, United Kingdom). The lower detection limit was 10 pg/ml for IL-6. All samples were analyzed in duplicate and were assayed at optimal concentrations, according to the manufacturer's instructions. Protocol was as described by Tavares et al. in "Circulating Inflammatory Mediators during Start of Fever in Differential Diagnosis of Gram-Negative and Gram-Positive Infections in Leukopenic Rats", in Clin Diagn Lab Irnmunol. 2005 September; 12(9): 1085-1093.

For hematological studies, whole blood for cell counts was collected into sterile tubes containing K3EDTA, and cells were counted by an automatic hematological analyzer equipped with veterinary software (Cell Dyne 3500; Abbot, Allentown, PA), which uses a laser beam and measures the light scattered by the cells to give the total and differential leukocyte counts. Total peripheral leukocytes, lymphocytes, monocytes, and neutrophils counts were determined at 1, 2, 4 and 12 h after treatment was administered on day one.This method was described by Tavares et al. in "Circulating Inflammatory Mediators during Start of Fever in Differential Diagnosis of Gram-Negative and Gram-Positive Infections in Leukopenic Rats", in Clin Diagn Lab Immunol. 2005 September; 12(9):1085-1093. Neurological Evaluation

Neurological evaluation of rats was assessed before and after MCAo. The total score was calculated according to Table 3. This model and method were described in De Ryck M, Reempts JV, Borgers M 5 Wauquier A, Janssen PAJ. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke. 1989;20:1383-1390.

Table 3.

Item Normal Score Deficit

Postural reflex ("hang test")* 0 2

Placing test^ (performed on each side)

Forward 0 2

Sideways 0 2

Tactile placing

Dorsal surface of paw 0 2

Lateral surface of paw 0 2

Proprioceptive placing 0 2

Total score 0 12 * Scores are as follows: 0, no observable deficit; 1, limb flexion during hand test; 2, deficit on lateral push.

* Scores are as follows: O 5 complete immediate placing; 1, incomplete and/or delayed placing (<2 s); 2, absence of placing.

The significant changes in neurological deficit in animals could be decisively recognized as stroke-induced abnormalities. Infarction area analysis

Seven days after induction of ischemia, the rats were anesthetized using chloral hydrate and perfused transcardially with 2% paraformaldehyde. The brains were removed, embedded in paraffin, and cut into 4 mm thick coronal sections at 400 mm intervals. The brain slices were stained with hematoxylin and eosin and Ladewig's trichrome stain for detection of fibrin. The infarct areas were assessed planimetrically (OPTIMAS 5.1, BioScan Inc). For each brain, 24 slices were measured encompassing the entire infarct. Areas of pannecrosis were examined, in such areas there is no affinity for hematoxylin. Infarct volume was calculated by multiplying the infarct area of each slice by the distance (400 mm) between successive slices. The infarct size was expressed as the percentage of the intact zone in sham operated animal brains. Results Neurological status evaluation

As shown in Fig. 8, animals treated with the active ingredients of the invention recovered significantly faster (*; p<0.05) from MCAo impairment compared to non-treated control animals.

More specifically, ischemic untreated animals (MCAo, square symbols) remained neurologically impaired at day 7. Sham operated animals, in contrast, received a neurological score of zero, representing the baseline, and plotted on the x-axis. Purified brain tissue extract, when administered LP. , resulted in the best neurological scores (circle symbols), as shown by the rapid advance to the neurological score of 8 on day 2, and to a score of 1 on day 7. The results were found to be significant (p<0.05).

The same purified brain tissue extract when administered LN., resulted in a neurological score of 2 on day 7.

Cognitone (i.e. purified brain tissue extract plus hss DNA), when administered LP. or I.N., resulted in neurological improvement as well, reaching a neurological score of approximately 3 on day 7.

Thus, brain tissue extract, prepared according to the invention, with or without hssDNA, resulted in significant neurological recovery after an ischemic incident, as shown by Fig. 8. The improvement was significant over non-treated animals, which exhibited only slight neurological improvement at day 7. Infarction area quantification

The results of the infarction area analysis are shown in Fig. 9, and indicate that treatment with either purified brain tissue extract or Cognitone (both LP. and LN. administration), significantly decreased (*; p<0.05) the infarction area as compared to the saline treated control. More specifically, LP. administration of purified brain tissue extract (diagonally striped bar) was most efficient in limiting the infarct area, as compared to the saline treated control (open bar). Similar results were obtained by intranasal administration of the same purified extract (gray bar). LP. administration of Cognitone, (checked bar) resulted in a certain degree of limitation of the infarct area, and intranasal administration of Cognitone (black bar) were the least effective, although the infarct area was nevertheless significantly smaller than in animals that received saline. Hematological parameters White blood cell count in peripheral blood samples was measured as an indication of the level of inflammatory response occurring at a given time.

Fig. 10 demonstrates that a specific inflammatory response occurs after intraperitoneal administration of Cognitone. This inflammatory response is significantly greater (*; p<0.05) than that induced by intranasal administration of Cognitone during the first four hours following administration. However, this difference between administration methods was diminished during the first 12 hours. Thus, although Cognitone causes a transient inflammatory response this fact does not affect the neuroprotective properties it provides. The level of the cytokine IL-6 was measured as an estimate of the extent of the inflammatory response. As shown in Fig. 11, intraperitoneal injection of Cognitone (filled triangle symbols) causes significantly greater immune system response as compared to the other treatment and control groups. The difference as compared to the control groups was found to be significant (*; p<0.05). Conclusion

Figs. 8-11 conclusively demonstrate the protective effect of both Cognitone (purified porcine brain tissue extract plus hssDNA), and of purified porcine brain extract on its own, when administered in an animal model of stroke ischemia. Both intranasal and intraperitoneal administration were shown to be effective.

Cognitone, when injected intraperitoneally, caused a temporary inflammatory response, which did not negate the neuroprotective effect.

Example 5. Cognitone and brain tissue extract of the invention are more effective than prior art brain tissue peptide extracts in neuroprotection in an animal model of stroke ischemia

The efficacy of Cognitone in protecting against stroke ischemia, was compared to that of the prior art brain tissue peptide extracts, Cerebrolysin™ and Cortexin™.

Middle cerebral artery occlusion, and neurological evaluation were performed essentially as described in Example 4, but with each group of rats containing 17 animals. Cerebrolysin™ (Ebewe Pharma, Austria) was administered (LP.) at a dosage of 2.5 ml/kg/day (approximately

80 mg/kg of peptides), according to the manufacturer's instructions.Cortexin™ (Geropharm,

Russia) was administered (LP.) at a dosage of 0.07 mg/kg, at a final volume of 1 ml according to the manufacturer's instructions.

Cognitone or purified tissue extract of the invention, were administered at a dosage of 0.01 ml (0.003-0.004 mg/kg of active proteins), diluted with saline to the final volume of 1ml, as described in Example 4. All other aspects of the Experiment are as described in Example 4.

Results

Stroke area quantification

As shown in Fig. 12, animals treated by LP. or I.N. administration of Cognitone exhibited stroke infarction areas that were significantly decreased (*; p<0.05) as compared to animals treated with the prior art brain tissue peptide extracts Cortexin™ and Cerebrolysin™. Neurological status evaluation The neurological status of the treated rats was assessed as in Example 4. As shown in Fig. 13, animals which received Cognitone or purified extract of the invention (both by LP. or LN. administration) had a more complete and significantly faster recovery (*; p<0.05) as compared to animals that received Cerebrolysin™ or Cortexin™ (LP. ). Mortality rate comparison

The mortality rate of the animals used in this example was evaluated. As shown in Fig. 14, animals treated with Cognitone or with purified tissue extract of the invention had a significantly lower mortality rate as compared to animals treated with either of the prior art peptide extracts Cerebrolysin™ and Cortexin™. Conclusion

Figs. 12-14 demonstrate that Cognitone or purified extract of the invention, in either administration form (LP. and I.N.), resulted in the highest recovery rate, as seen both by neurological evaluation and by mortality rate. The treatments of the invention resulted in a significantly reduced area of injury, compared to animals treated with prior art tissue extracts, and to control groups.

Example 6. Effect of Cognitone administration., in an animal model of transient cardiac ischemia Methods

Rats (Wistar strain, weighing 350-430 g), were selected and divided into groups with 12 animals per group. Study groups were: 1) sham (no cardiac ischemia); 2) induced cardiac ischemia (CI); 3) CI and vehicle administration [PBS 10x - 1/10 vol.; distilled H 2 O up to 9/10 vol.; Lecithin 2%; methyl paraben 0.2%; Vitamin E acetate 0.1% (0.1 ml solution diluted with saline to the final volume of 1 ml)]; 4) CI and Cognitone administration (0.01 ml of Cognitone diluted with saline to the final volume of 1 ml); 5) CI and purified protein extract (0.01 ml of extract diluted with saline to the final volume of 1 ml); 6) CI and vehicle and hssDNA (0.1 ml solution diluted with saline to the final volume of 1 ml).

For treatment, each group was treated daily by LP injection with a volume of 1 ml, for a period of 7 days

Cardiac Ischemia Animal Model

Male Wistar rats, were anesthetized with pentobarbital sodium (60 mg/kg I.P.). The trachea was intubated through a cervical incision. Mechanical ventilation was achieved with a positive-pressure respirator (MD Industries, Mobile, AL) using 100% OZ, a tidal volume of 5 ml, and a rate of 50 breaths/min. The respiratory rate was subsequently adjusted if necessary to keep the blood pH within a normal range. The heart was exposed through a left thoracotomy in the third intercostal space, and the pericardium was opened. A 2-0 silk thread with a taper needle, was passed around a prominent branch of the left main coronary artery, close to its origin, and the ends of the thread were passed through a small vinyl tube to form a snare. The coronary artery was occluded by pulling on the snare. The snare was then fixed by clamping the tube with a hemostat. Myocardial ischemia was confirmed by regional cyanosis. Reperfusion was achieved by releasing the snare.

All groups of rats, except sham operated animals, received a 30-min coronary occlusion. After a period of 7 days, all rats were sacrificed and the hearts were removed for analysis. Measurement of infarct size, ischemic (risk) area

At the end of the study the hearts were quickly removed and mounted on a Langendorff apparatus, where they were perfused with room temperature saline for ,i min. The snare was again tightened, and 1-10 pm zinc-cadmium fluorescent particles (Duke Scientific, Palo Alto, CA) were infused into the perfusate to mark the risk zone as a nonfluorescent perfusion defect. The hearts were weighed, frozen, and then sliced into 2-mni-thick sections. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in pH 7.4 buffer for 20 min. The areas of infarct (TTC negative) and risk zone (nonfluorescent under ultraviolet light) were determined by planimetry for each slice. The infarct zone and the risk zone volumes were calculated by multiplying each area by the slice thickness, and summing the products. The infarct size was expressed as the percentage of mfarcted risk zone.

This cardiac ischemia model was previously described by Liu et al. ("Ischemic preconditioning protects against infarction in rat heart"; Am. J. Physiol. 263 Heart Circ. Physiol. 32: H1107-H1112, 1992).

Results

The results shown in Table 4 and Fig. 15 indicate that purified extract of the invention and Cognitone each exert a significant protective effect of after ischemia-induced cardiac dysfunction. It is clearly seen that both Cognitone and purified tissue extract, protect cardiomyocyte tissue from loss of viability in response to a cardiac ischemic event. Table 4.

(*) p < 0.05 vs. control

Values are SE=standard error of the mean; n= no. of hearts; RA =risk zone area (cm 3 ); IA= infarct area (cm 3 ); I/R= % of risk zone which underwent infarction.

Example 7. Cognitone administration preserves cognitive and behavioral capabilities in animals under experimental brain ischemia conditions.

Experiments were conducted in a rodent model of stroke, in order to evaluate the effect of treatment with the active compositions of the invention. Methods

Animal research experiments were carried out in accordance with the National Institutes of Health Guidelines. A bilateral focal photothrombosis in prefrontal cortex was performed on 210-26Og in-house bred rats. All animals were deeply anesthetized with chlorhydrate via intraperitoneal administration (300 mg/kg). Animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). A midline scalp incision was made and the scalp was retracted laterally. The photosensitive dye rose bengal (Sigma, St. Louis, MO), was dissolved in 0.9% saline and injected for over 2 min through a catheter placed in the left femoral vein at a dose of 40 mg/kg of animal body weight. The brain was concurrently illuminated through the intact skull overlying the sensorimotor cortex for 20 min by an argon laser-activated light beam (Lexel ion laser, model 75, Evergreen Laser Corporation) at a power of 250 mW.

This results in formation of crystals originating in the photosensitive dye. Such crystals form vascular occlusions which simulate stroke-induced ischemia. Sham-operated animals underwent the same experimental procedure, without rose bengal injection. Body temperature was kept constant throughout surgery at 37 0 C by use of a thermo-regulated pad. After illumination, the wounds were sutured, and animals were returned to their cages in an environmentally controlled room (23 + 2 °C; 12 hr light /12 hr dark cycle) with free access to food and water.

The study groups were: 1) control animals (not operated upon); 2) sham operated animals; 3) animals with photothrombosis which received saline injections for the period of 7 days; 4) animals with photothrombosis which received Cognitone saline injections at a daily dose of 0.5 ml for the period of 7 days. All groups underwent a set of behavioral evaluations before and after the photothrombosis induction. A. Passive Avoidance Protocol hi general, the passive avoidance task is a trial for fear-motivated avoidance, in which the rat learns to refrain from stepping through a door to an apparently safer but previously punished dark compartment. The latency to refrain from crossing into the punished compartment serves as an index of the ability to avoid, and allows memory to be assessed. Apparatus used in the Passive-Avoidance Protocol

The training chamber consists of a rectangular chamber divided into 2 compartments. One compartment is lighted by an overhead stimulus light and the other remains dark. The two compartments are separated by an automatic guillotine door and each has a grid floor placed through which a foot shock can be delivered.

Procedure used in Passive- Avoidance Protocol

Habituation

On habituation day, the rat is placed in the lighted compartment, facing away from the dark compartment and allowed to explore for 30 seconds. After 30 seconds the door is raised and the rat is allowed to explore freely. When the rat enters the dark compartment with all four paws, the guillotine door is closed, and the latency to enter is recorded (from the time the door is lifted). Once the rat crosses to the darkened chamber the door closed and the rat is immediately removed and returned to the home cage.

Training On training day, the rat is placed in the lighted compartment, facing away from the dark compartment and allowed to explore for 30 seconds. After 30 seconds the guillotine door is lifted. When the rat enters the dark compartment with all four paws, the guillotine door is closed, and the latency to enter is recorded (from the. time the door is lifted). Three seconds after the door is closed a foot shock (0.5 mA, 2 seconds duration) is delivered. Thirty seconds after the foot shock, the rat is removed to its home cage.

Testing On the test day (24 hours after training), the rat is returned to the lighted compartment, facing away from the dark compartment. After 5 seconds, the guillotine door is lifted. When the rat enters the dark compartment with all four paws, the guillotine door is closed, and the latency to enter the dark compartment is recorded (from the time the door is lifted). The rat is removed and returned to the home cage. B. Open Field Protocol

The open field test is an assessment used to determine the general activity levels, gross locomotive activity, and exploration habits in rodents. Assessment over 2-4 days allows evaluation of the habituation to the environment.

Apparatus for Open Field Protocol Assessment takes place either on a circular platform arena (0=61 cm, wall height 24 cm), or on one of two square arenas (either 20.3 x 20.3cm or 40.6 x 40.6cm). The square arenas are mounted within specially designed sound attenuating shells constructed of polypropylene, regular and expanded PVC.

Procedure Procedures are identical on all days and are as follows: A rat is placed in the center of the open field arena and allowed to freely move about for 20-60 minutes while being tracked by an automated tracking system. At the conclusion of each trial the surface of the arena is cleaned with 90% ethanol. Results Damage caused to prefrontal cortical layers may lead to impairment of short term memory processes. The open field performance scores before and after a bilateral cortical photothrombosis induction are shown in Table 5. The data clearly show that administration of Congitone not only stops propagation of the damage, but significantly restores (*; p<0.05) cognitive abilities. Table 5

The results of passive avoidance behavior are shown in Fig. 16, which indicates a significant difference (*; p<0.005) between groups 3 (photothrombosis treated with saline) and 1 (control, not operated); a significant difference ( Λ ; p<0.007) between groups 3 and 2 (sham operated); and a significant difference (#; p<0.05) between groups 3 and 4 (photothrombosis treated with Cognitone). Associative learning tested by a passive avoidance behavior model demonstrates that Cognitone treatment after bilateral photothrombosis allows preservation of memory formation as well as memory retrieval. These results support a finding that Cognitone has activity in restoring the tissue metabolism and plasticity.

Example 9. Acute toxicity studies of a gel ointment formulation of Cognitone

Acute toxicity studies were carried out in experimental rodents and dogs, in which an ointment formulation of Cognitone, containing the active drug substance from porcine fetal brain (0.2 g/1), hssDNA (1 g/1), lecithin (2%) in a gel base (referred to herein as "the Test Article") was administered cutaneously or intragastrically. Materials and methods

The study was conducted on unbred white rats (age 13-14 weeks; 160-180 g), white mice (age 8-9 weeks; 18-20 g) and dogs (age 4-6 years; 8-13 kg) of both genders. Quarantine (acclimatization period) for all animals lasted 14 days. During the quarantine each animal was examined daily for behavior and general status, and twice a day the animals were watched in their cages for incidence and mortality rate. Before the study the animals meeting study inclusion criteria were randomized into groups. Animals not meeting the criteria were excluded from the study during the quarantine.

For mice and rats, the animal cages were placed in separate rooms, with a light regimen: of 12 hours with lights on, and 12 hours with lights off. Air temperature was maintained at 19-25 0 C, and relative humidity rate 50-70%. Temperature and air humidity were recorded daily. In case of temperature changes the air exchange in the facility was controlled using anemometer and measuring carbonic acid and ammonia. The set ventilation regimen provided about 15 facility volumes with CO 2 concentration not higher than 0.15 volume % and ammonia concentration not higher than 0.001 mg/1. Dogs were contained in cabins and open-air cages. Temperature, .air humidity and air exchange conditions were not monitored. During the study each animal was examined daily, including general behavior and general status assessment. During the drug application period animals were examined 2 hours after manipulations. AU animal groups were examined daily immediately after the Test Article introduction and at the end of the day.

For acute toxicity testing, the Test Article was applied to white mice and rats (10 animals each) of both genders (5 males and 5 females per animal group).

One day before the study, the back hair was shaved (4x4 cm for rats and 2x2 cm for mice; 5-8% if back surface).

Cutaneous (CU) administration of the Test Article was carried out using increasing doses (6300 to 40,000 mg/kg) as per Litchfield-Wilcoxon method. Control animals were treated with a similar volume of white petroleum jelly. Following application, animals were placed in a plexiglass container for 6 hours, followed by placement in cages for daily observation.

Intragastric (IG) administration of the Test Article (2500-15,000 mg/kg for mice; 2000-20000 mg/kg for rats) or white petroleum jelly as a control (both preheated in a water bath) was carried out via atraumatic metal probe. The Tested Article was administered intragastrically to two dogs (male and female) at a dose of 10 g/kg.

Body weight, behavior and food and water consumption were recorded throughout the study. The study was carried out for a period of 2 weeks, after which study animals were euthanized by ether inhalation. Following sacrifice, morphological studies were conducted on all animals treated with the top dose, and on all control animals. The morphological study included necropsy, macroscopy, weight and histology of internal organs. Results

The dose dependent lethal effects following cutaneous or intragastric administration of the ointment formulation of Cognitone to experimental rodents of both genders at high dosages are shown in Tables 6-10. No deaths were recorded among the animals. Table 6. Cognitone ointment formulation toxicity following IG administration to male mice

Animals treated by IG administration of doses of greater than 6000 mg/kg exhibited languor, stupefaction, decreased food and water consumption on the first day following administration, and diarrhea on the second day. Since control animals exhibited the same signs, these effects were attributed to mechanical impaction.

Body weights of experimental animals during the course of the study are shown in Tables 11 and 12. hi comparison with controls, there were no significant differences in body weight of treated animals during the course of the study.

Table 11. Body weight of animals following IG administration of Cognitone ointment formulation

Table 12. Body weight of animals following CU administration of Cognitone ointment formulation

Body weight (g)

Mice Rats

Observation period

M F M F

Baseline 21 ±1 20 + 2 160 ±4 162 + 3

Day 7 22 + 2 21 + 1 167 ±5 166 + 6

Day 14 23 + 2 23 ±2 173 ±4 172 ± 5

Organ weight coefficients of experimental animals, expressed as g/kg of body weight, are shown in Tables 13-16.

Table 13. Organ weight coefficients (g/kg of body weight) in white mice following IG administration

Group

Organ Control Cognitone treated

M F M F

Heart 3.9 ± 0.4 4.1 ±0.6 4.0 ± 0.2 4.1 ± 0.6

Lungs with trachea 6.3 + 0.5 6.6 ± 0.4 6.5 ± 0.3 6.4 ± 0.4

Thymus 0.92 ± 0.06 0.86 ± 0.04 0.86 ±0.04 0.88 ± 0.05

Liver 38.6 + 0.9 37.5 + 1.5 40.1+2.8 j 38.3 ±2.6

Spleen 3.2 + 0.2 3.6 ±0.3 3.4 ±0.8 3.2 ± 0.6

Kidneys 4.5 ± 0.5 4.4 ± 0.6 4.3 ± 0.7 4.6 ± 0.2

Adrenals 0.20 + 0.05 0.18 ±0.07 0.20 ± 0.05 0.17 ±0.03

Brain 16.0 + 0.8 15.6 ±0.5 16.3 + 0.8 16.0 ±0.8

Table 14. Organ weight coefficients (g/kg of body weight) in white rats following IG administration

Group

Organ Control Cognitone treated

M F M F

Heart 3.8 ±0.4 3.7 + 0.2 3.5 ±0.1 3.4 + 0.2

Lungs with trachea 6.4 ±0.1 6.2 + 0.2 6.0 ±0.1 6.2 ± 0.3

Thymus 1.4 + 0.2 1.6 ±0.4 1.4 ±0.3 1.5 ±0.3

Liver 28.6+1.2 30.4 ±0.8 30.2 ±2.4 30.1 ±2.1

Spleen 5.1 ±0.3 5.1 ±0.2 5.0 + 0.1 5.1 ±0.2

Kidneys 8.0 ± 0.3 7.8 ±0.1 7.9 ± 0.2 _j 8.0 ±0.1

Adrenals 0.10 ±0.03 0.10 ±0.04 0.10 ±0.02 0.10 ±0.03

Brain 9.9 + 0.6 9.8 ±0.8 9.6 ± 0.4 9.3 ± 0.3 Table 15. Organ weight coefficients (g/kg of body weight) in white mice following CU administration

Group

Organ Control Cognitone treated

M F M F

Heart 3.8 + 0.2 3.9 ± 0.6 4.0 ± 0.4 3.9 ± 0.4

Lungs with trachea 6.8 + 0.2 6.6 ± 0.4 6.6 ± 0.6 6.5 + 0.5

Thymus 0.91 + 0.07 0.89 ± 0.06 0.86 ± 0.06 0.90 ± 0.08

Liver 39.8 + 1.1 38.8 ± 1.2 39.1 ± 1.1 39.1 ± 0.7

Spleen 3.4 ± 0.4 3.2 ± 0.4 3.2 ± 0.4 3.0 ± 0.5

Kidneys 4.2 ± 0.2 4.0 ± 0.2 4.0 ± 0.1 4.1 + 0.2

Adrenals 0.19 ± 0.02 0.20 ± 0.04 0.18 ± 0.04 0.19 ± 0.03

Brain 16.0 ± 0.5 16.1 ± 0.6 15.8 ± 0.6 15.9 ± 0.3

Table 16. Organ weight coefficients (g/kg of body weight) in white rats following CU administration

Group

Organ Control Cognitone treated

M F M F

Heart 3.6 ± 0.2 3.7 + 0.1 3.6 + 0.3 3.6 ± 0.1

Lungs with trachea 6.2 + 0.2 6.3 ± 0.2 6.1 + 0.3 6.2 ± 0.2

Thymus 1.5 ± 0.5 1.6 + 0.3 1.4 + 0.4 1.6 ± 0.2

Liver 29.3 ± 1.4 28.6 ± 1.6 30.0 ± 1.5 29.6 ± 0.8

Spleen 5.0 ± 0.1 5.1 ± 0.2 5.4 ± 0.3 5.0 + 0.5

Kidneys 8.0 + 0.3 8.2 ± 0.4 7.9 + 0.4 8.2 ± 0.6

Adrenals 0.09 ± 0.03 0.10 + 0.04 0.10 ± 0.04 0.11 ± 0.05

Brain 9.2 + 0.5 9.4 + 0.6 9.5 + 0.5 9.6 ± 0.6

As shown, in comparison with controls there were no significant differences in organ weight coefficients in the treated animals, indicating an absence of internal pathological changes.

Similarly, macroscopic examination of internal and endocrine organs, brain, skin and intestinal mucosa, did not reveal any differences among the treated groups with either administration route and the corresponding control groups. hi summary, these studies showed that cutaneous or intragastric administration of acute doses of the ointment formulation of Cognitone to animals had substantially no toxic or irritating effects, and did not induce any macroscopic, clinical or behavioral abnormalities.

Accordingly, the ointment formulation of Cognitone corresponds to a class V (virtually non-toxic) compound (Gosselin et al. Clinical Toxicology of Commercial Products: Acute Poisoning. 4 th ed. Williams and Wilkins, Baltimore, 1976). Example 10. Subacute and chronic toxicity studies of a gel ointment formulation of Cognitone

Studies were carried out on experimental rats (both genders, 15 animals per group) and dogs (both genders, 3 animals per group), maintained under conditions as described in Example 9. The ointment formulation of Cognitone, as described in Example 9 (referred to herein as "the Test Article") was administered cutaneously (CU) on a daily basis for 180 days, either at 400 mg/kg (the maximal therapeutic dose), or at 4000 mg/kg (the suggested maximal dose), corresponding respectively to 10 and 100 times the therapeutic dose. Control animals were treated by cutaneous administration of the same volume of white petroleum jelly. Indices recorded on days 30, 60, 90 and 180 included body weight, water and food consumption, rectal temperature, physiological tests, and cardiological tests (electrocardiography, heart rate and systolic blood pressure). Ophthamological tests (eye mucous membranes, corneal reflexes, pupil size and palpebral fissure width); spontaneous motor activity (SMA); behavior test (open field) were carried out in rats. Laboratory tests included hematology (hematocrit; hemoglobulin; erythrocyte count; ESR; leukocyte count; leukogram; platelet count; reticulocyte count); blood serum chemistry (total protein; aspartate aminotransferase (AST); alanine aminotransferase (ALT); lactate dehydrogenase (LDH); alkaline phosphatase; urea; creatinine; glucose; total lipids; cholesterol; bilirubin and fractions; sodium and potassium); urinalysis; myelogram; hexenal test; phenol red test (renal secretory capacity test). Narcotic (hexenal) sleep duration was used to assess liver detoxifying function in rats. The experiment was conducted in warm and quiet room. Animals were weighed and 2% hexenal solution was introduced intraperitoneally at a dose of 90 mg/kg with 0.9% NaCl solution as a dissolvent. Narcosis onset time (lying sidelong) and emergence time (turning) are recorded as minutes. The open field utilizes a rectangular chamber having wooden sides and back and a glass front panel (60x60x60 cm). The chamber floor is elevated and divided onto 9 squares to record investigational activity. There is a light source above the chamber. Type and number of movements is recorded by the researcher. Six behavioral components are registered during 4- minute observation: latent exit period, vertical and horizontal activities, number of peepings into holes, grooming and boluses. Testing is conducted as baseline and after drug introduction completion. To determine the effect intensity the ratio of movements in test/control animals is used.

For the spontaneous motor activity (SMA) test, a rat is placed for 5 min into a chamber having an automatic motion recording system to obtain baseline data. For the next 30 min SMA is recorded for each 5 minute interval. Results are analyzed by comparing number of motions at 30 minute period at baseline in tested and control groups. Testing is conducted as baseline and after drug introduction completion.

Morphological studies were conducted at the end of the study on all animals treated with the top dose, and on all control animals. Results

A. Studies in rats

In comparison with controls, there were no significant differences in any of body weight, food consumption, water consumption, rectal temperature, narcotic (hexenal) sleep duration of treated animals during the entire study period. There were neither gender-related nor other differences associated with the dose value.

The additional tests carried out showed that the cutaneous administration toirats of the ointment formulation of Cognitone did cause significant changes compared to control treated animals in any of: renal function, ophthamological function, cardiological function, motor activity, behavior, blood chemistry, hematological function, hemopoiesis, metabolism or electrolyte status. Further, autopsy and histological and macroscopic examination did not reveal any differences in the studied organs among the groups by the end of the study. hi conclusion, daily cutaneous administration of the ointment formulation of Cognitone at a dose of 400 or 4000 mg/kg for 180 days to rats of both genders does not cause irritation, inflammation or local damage at the application site, nor does it result in any adverse physiological effects, or adverse effects in internal organs, parenchyma and stroma..

B. Studies in dogs

Over the course of the 180 day study, daily examination of the animals of the test and control groups did not reveal any changes in general status, as assessed with respect to body position, build, constitution, temperament, and nourishment. In addition, no changes were noted in upper respiratory tract and thoracic organ status, oral cavity mucosa, ears and hearing acuity.

Studies of weight, rectal temperature, and food and water consumption at baseline, and at 30, 90 and 180 days showed that during the 180 day observation period there were no significant differences of these parameters among the test and control groups. The animals did not gain any substantial weight, rectal temperatures were not above the physiological normal limits, and appetite and food intake varied within the background index range.

Further, over the course of the study period there were no significant changes in any of renal function, cardiological function, blood chemistry, hematology or hematopoiesis among the control and test groups. Autopsy and histological and macroscopic examination did αiot reveal any differences in the studied organs among the groups by the end of the study.

In conclusion, daily cutaneous administration of the ointment formulation of Cognitone at a dose of 400 or 4000 mg/kg for 180 days to dogs of both genders does not cause irritation, inflammation or local damage at the application site, nor does it result in any adverse physiological effects, or adverse effects in internal organs, parenchyma and stroma.

Example 11. Investigation of allergenic properties of a gel ointment formulation of Cognitone Methods Studies were carried out on CBA mice, white unbred rats and guinea pigs of both genders. Cages with animals of different species maintained in separate rooms, with a light regimen of 12 hours with lights on, and 12 hours with lights off. Air temperature was maintained at 18-20 0 C, relative humidity 50-70%. Room aeration and quartz-lamp sterilization were conducted daily. Each animal was examined daily during the study. On days of administration of the

Test Article (as described in Example 9), examinations were conducted before application, after application and 2 hours later. The examination included general behavior and general status assessment.

Guinea pigs and CBS mice were distributed into groups as follows: Group 1, female, control (white petroleum jelly); Group 2, female, ointment formulation of cognitone (40 mg/kg); Group 3, female, ointment formulation of Cognitone (400 mg/kg); Group 4, male, control (white petroleum jelly); Group 5, male, ointment formulation of Cognitone (40 mg/kg); Group 6, male, ointment formulation of Cognitone (400 mg/kg).

During a 30 day sensitization period each guinea pig was treated by a daily cutaneous administration of the ointment formulation of Cognitone (40 or 400 mg/kg; also referred to as the "Test Article"), or petroleum jelly.

For studies of anaphylactic shock, 21 days after the end of the sensitization period, an intracardiac challenge dose equal to the sum of the sensitizing dose was introduced to the animals. A similar dose was introduced to the control guinea pigs. Anaphylaxis shock intensity was recorded in Weigle index points:

++++ Shock with lethal outcome.

+++ Severe shock (general convulsions, asphyxia, animal cannot stand, falls down but does not die).

++ Moderate shock (minor convulsions, considerable bronchospasm signs). + Light shock (some anxiety signs, higher breathing rate, face scratching, involuntary urination, defecation, disheveled hair).

0 No shock has developed, no shock signs are present.

To study immune complex formation, 10 days after the last sensitizing dose a challenge dose of 0.5 ml was cutaneously administered. A similar volume of white petroleum jelly was used in the control groups. The challenge Test Article dose, determined on intact animals, was considered as the highest antigen titer which does not cause visible skin changes 1 hour after administration. Skin reactions were visually assessed and scored, wherein a score of 0 indicates a negative skin reaction.

For studies of indirect mast cell degranulation reaction, mast cells were obtained from white unbred rats by introducing sterile normal saline (6 ml, pH = 7.4) pre- warmed to 37 °C into the abdominal cavity of exsanguinated animals. After massage of the abdominal wall (1 min) a 1.5 cm incision was made at the median peritoneal line, the body was turned over and exudate flowing along intestine loop was collected into a sterile test tube.

Samples were prepared on plates pre-stained with 0.3% alcohol solution of neutral red and dried at room temperature. Equal volumes (30 μl) of the mast cell suspension and serum of sensitized guinea pigs (according to the groups described above) were mixed, followed by addition of the Test Article in an amount in which mast cell degranulation did not exceed 5% in the control. Prepared samples were covered with cover glass, and incubated in a thermostat at 37 °C for 15 min. Samples were observed under microscope (2OX magnification), and results were assessed with a differential recording method to calculate mast cell degranulation rate (MCDR) according to the formula: MCDR = (1-a + 2-b + 3-c + 3-d) / 100, where a, b, c, d are the amount (mean of three repetitions) of degranulated cells corresponding to degranulation rate (low, moderate, severe and complete cell degranulation). hi each sample 100 cells were estimated, and reaction was considered positive if MCDR was above 0.2.

For conjunctiva tests, 10 days after the last sensitizing dose 50 μg of the Test Article at a dose of 10 mg/kg was introduced under the upper eyelid of control and test group guinea pigs. Water was introduced under the upper eyelid in some controls. Reactions were recorded at 15 min, 24 and 48 hrs after application. For studies of specific leukocyte lysis reaction (SLLR), 24 h and 14 days after Test

Agent application 0.1 ml of blood was incubated with 3.8% Na citrate and agent. The mix was incubated at 37 °C for 24 h, after which 0.2 ml was transferred to another tube containing 0.4 ml of 3% acetic acid. The leukocyte count was conducted in Goraev chamber. SLLR was calculated according to the formula:

Lk- Lv

SLLR = 100

Lk Wherine L k = Control leukocytes; and

Ly = Test leukocytes.

Study results were processed with statistical methods using Student's t-test criteria. Results In the analysis for anaphylactic shock, a Weigle index score of 0 was obtained for all groups, indicative of no shock.

In the indirect mast cell degranulation reaction, the MCDR obtained among the groups ranged from 0.01 to 0.07. Since the obtained values were less than 0.2, in all the cases the reaction was negative. In the immune complex reaction, scores of 0 were obtained for all groups at all time points, indicative of a negative skin reaction. hi the conjunctiva test, none of the test group animals exhibited reactions following administration of the Test Article. hi the SLLR test, all samples were negative. In conclusion, daily cutaneous administration of the ointment formulation of

Cognitone at a dose of 40 or 400 mg/kg for 30 days, does not cause a general anaphylaxis reaction and does not induce allergenic effects.

Example 12. Comparative study of the therapeutic efficacy of Cognitone in a thermal burn animal model The gel ointment formulation of Cognitone, as described in Example 9, was investigated for clinical efficacy in the treatment of thermal burns and compared to the efficacy of the prior art burn medication Dermazin™ (sulfadiazinum cream formulation; Lek Pharmaceutical and Chemical Co., Slovenia). Methods

Thermal skin burns of area 2.5 x 2 cm were inflicted on the backs of previously shaved unbred white male rats (2 burns per animal), using a dedicated device. Total surface area of the burns was 10 cm 2 , corresponding to 4% of the body surface area. The depth grade of the burns was 3a and b. The day following burn infliction, the tested medications were topically administered to the burn sites at a dose of 150 mg/10 cm 2 or 800-900 mg/kg. Treatment with the medications was continued throughout the healing process. Control animals were not treated with any medication, and all animals were bandaged at the burn sites. AU manipulations were carried out under ketamine narcosis. Each study group contained 30 animals. AU animals were assessed for general condition, size of burn site and epithelization rate. Results

The results obtained show that while both tested medications have a positive influence on the skin regeneration process, the ointment formulation of Cognitone according to the invention was significantly more effective than Dermazin™. Table 17 shows that the animals treated with Cognitone ointment exhibited higher rates of perifocal liquidation, eschar removal, granulation and epithelization, as compared to animals treated with Dermazin™. Table 18 shows that a higher percentage of animals treated with Cognitone ointment reached full epithelization at days 25 and 30, as compared to animals treated with Dermazin™. Furthermore, keloid formation occurred in 50% of animals treated with Dermazin™, but none of the animals treated with Cognitone ointment.

Table 17. Effect of burn treatments on skin re eneration arameters da s, M±m

significant difference from Control (p < 0.05) Table 18. Effect of burn treatments on fall e ithelization

The obtained results were supported by pathomorphologic data. The back skin was cut, fixed in 10-15% formalin 1 and embedded into celloidin or paraffin. Internal sections were stained with hematoxylin-eosin and using van-Gieson's method.

Microscopic evaluation indicated that skin regeneration started at day 5 in animals treated with Cognitone ointment, as compared to days 8-10 and days 12-15 in the Dermazin™ and control groups, respectively. Further, full epithelization occurred by day 15 in animals treated with Cognitone ointment, as compared to days 17-20 and 25-30 in the Dermazin lM and control groups, respectively.

By day 25, the skin of animals treated with Cognitone ointment showed several layers of cells and developed hair bulbs.

At days 35-37 animals in the control group showed scar development. hi conclusion, these studies show that an ointment formulation of Cognitone is highly effective in promoting skin regeneration without keloid development following burn injury. Furthermore, the ointment formulation of Cognitone was found to be more effective than sulfadiazinum.

Example 13. Acute toxicity studies of the fetal porcine brain protein active drug substance of Cognitone

Acute toxicity studies of the fetal porcine protein active drug substance Cognitone, prepared essentially as described in Example 1 steps A to C (referred to herein as "the Test Substance") were carried out in experimental rodents and dogs. Materials and methods Animals and maintenance conditions were essentially as described in Example 9. The

Test Substance was administered intragastrically (IG) to white mice and rats of both genders (5 animals per group) via atraumatic metal probe in increasing doses as per Litchfield- Wilcoxon method. The average lethal doses were calculated according to Prosorovsky (Pharmacology and Toxicology, 1978, No. 4, p. 497-502). For higher drug doses the Test Substance was introduced repeatedly every 20-30 minutes for 4 hours. Control animals were . introduced with similar volumes of the dissolvent distilled water.

The Test Substance was administered intravenously by injection into the tail vein at a rate of 1-2 ml per minute. The Test Substance was administered IG to male and female dogs (n=2) via atraumatic metal probe with short time intervals over 6 hours for a total dose of 5000 mg/kg. The control male was administered with similar volume of the water.

Body weight, behavior and food and water consumption were recorded throughout the study. The study was carried out for a period of 14 days, after which study animals were euthanized by ether inhalation. Following sacrifice, morphological studies were conducted on all animals treated with the top dose, and on all control animals. The morphological study included necropsy, macroscopy, weight and histology, of internal organs. Results

No deaths were recorded among any of the test group animals, indicating that intragastric or intravenous administration of the active drug substance of Cognitone does not exert lethal effects at acute doses.

With the exception of the occurrence of ataxia, dormancy, flaccidity, decrease in water and food consumption in the rodents on Day 1 following administration, the general status and behavior of the experimental animals did not differ from that of the controls throughout the remainder of the study.

The autopsy results indicated that in comparison with controls, there were no significant differences in organ weight coefficients in any of the treatment group animals, indicating an absence of internal pathological changes.

Similarly, macroscopic examination of internal and endocrine organs, brain, skin and intestinal mucosa, did not reveal any differences among the treated groups with either administration route and the corresponding control groups. hi summary, these studies showed that intragastric or intravenous administration of acute doses of the active drug substance of Cognitone to animals had substantially no toxic or irritating effects, and did not induce any macroscopic, clinical or behavioral abnormalities. Accordingly, the active drug substance of Cognitone corresponds to a class V

(virtually non-toxic) compound (Gosselin et al. Clinical Toxicology of Commercial Products: Acute Poisoning. 4 th ed. Williams and Wilkins, Baltimore, 1976). Example 14. Subacute and chronic toxicity studies of the fetal porcine brain protein active drug substance of Coenitone

Studies were carried out in experimental rats (both genders, 20 animals per group) and dogs (both genders, 3 animals per group), maintained under conditions as described in Example 9. Periods of 1 month and 3 months were used to assess subacute toxicity and chronic toxicity, respectively. Methods and materials

The active drug substance of Cognitone, as described in Example 13 (referred to herein as the "Test Substance") was administered intragastrically (IG) to rats, or orally to dogs (solution dropped under the tongue), once a day for 90 days, either at 30 μg/kg (the maximal therapeutic dose for humans); or at 3000 μg/kg (the suggested maximal dose); or at 500 μg/kg (an intermediate dose). Control animals were administered similar volumes of the dissolvent distilled water.

In all animals, general behavior was assessed daily. In rats, water and food consumption were assessed at days 7, 30, 60, 90 and 120; body weight was assessed at days 7, 30, 60 and 90; rectal temperature, cardiological tests, ophthamological tests, spontaneous motor activity (SMA); behavior tests and laboratory tests (renal function, hematological and blood chemistry) were carried out on days 30 and 90, all essentially as described in Example 10. In dogs, water and food consumption, body weight and rectal temperature were assessed at days 7, 30, 60 and 90; cardiological tests, ophthamological tests, spontaneous motor activity (SMA), behavior tests and laboratory tests (renal function, hematological and blood chemistry) were carried out on days 30 and 90, all essentially as described in Example 10. Autopsy was carried out at the end of the studies. Results A. Studies in rats.

In comparison with controls, there were no significant differences in any of body weight, food consumption, water consumption, rectal temperature, narcotic (hexenal) sleep duration, renal function, ophthamological parameters, hematological function or hemopoiesis of treated animals during the study period. Further, autopsy and histological and macroscopic examination did not reveal any differences in the studied organs among the groups by the end of the study.

Animals treated with the 30 or 500 μg/kg of the Test Substance also showed no differences compared to controls with respect to the motor, behavior or cardiological tests carried out. However, animals treated with the highest dose (3000 μg/kg) exhibited decreased SMA at 30 and 90 days, compared to control animals. Similarly, in open-field behavior tests these animals exhibited significantly increased latent period and significantly decreased vertical standings, crossings and peepings, compared to the baseline and control. Animals treated with the highest dose further exhibited significant increase in subthreshold impulse summation and significant increase in blood pressure.

Animals treated with the 30 μg/kg of the Test Substance showed no differences compared to controls with respect to blood chemistry, while animals in the higher dosage test groups exhibited significantly decreased levels of transaminases, and animals in the highest dosage group exhibited significantly decreased levels of calcium. The observed changes were transient, as the relevant parameters returned to normal levels 1-3 weeks after cessation of administration of the Test Substance.

In conclusion, daily- intragastric administration of the active substance of Cognitone at a dose of 30, 500 or 3000 μg/kg for 90 days to rats of both genders does not have a major negative impact on most physiological functions, blood chemistry parameters and internal organs and mucosa, indicating that the drug is generally well tolerated and relatively safe B. Studies in dogs

Over the course of the 90 day study, daily examination of the animals of the test and control groups did not reveal any significant changes in general status, as assessed with respect to body position, build, constitution, temperament, and nourishment, m addition, no changes were noted in upper respiratory tract and thoracic organ status, oral cavity mucosa, ears and hearing acuity.

Studies of weight, rectal temperature, and food and water consumption at baseline, and at 7, 30, 60 and 90 days showed that during the observation period there were no significant differences of these parameters among the test and control groups. The animals did not gain any substantial weight, rectal temperatures were not above the physiological normal limits, and appetite and food intake varied within the background index range.

Further, over the course of the study period there were no significant changes in any of renal function, cardiological function, blood chemistry, hematology or hemopoiesis among the control and test groups. Autopsy and histological and macroscopic examination did not reveal any differences in the studied organs among the groups by the end of the study. hi conclusion, daily oral administration of the active substance of Cognitone at doses of 30, 500 or 3000 μg/kg for 90 days to dogs of both genders does not cause any pathologic effects on the general status and behavior of the animals, nor does it have toxic impact on any of renal function, ophthamological parameters, cardiovascular function, hematology parameters, blood chemistry, hematopoiesis, liver function and electrolyte metabolism, indicating that the Test Substance has a high safety profile.

Example 15. Investigation of allergenic properties of the active drug substance of Cognitone The active drug substance of Cognitone as described in Example 13 (referred to herein as the "Test Substance") was tested for allergenic properties, as assessed by induction of anaphylaxis reaction, immune complex reaction, conjunctiva test and indirect mast cell degranulation reaction.

Methods Guinea pigs and CBS mice were distributed into groups (6-8 animals per group)as follows: Group 1, female, control (distilled water); Group 2, female, active drug substance of

Cognitone (30 μg/kg); Group 3, female, active drug substance of Cognitone (500 μg/kg);

Group 4, male, control (distilled water); Group 5, male, active drug substance of Cognitone

(30 μg/kg); Group 6, male, active drug substance of Cognitone (500 μg/kg). During a 5 day sensitization period each guinea pig was treated by daily intragastric administration of the active drug substance of Cognitone (30 or 400 μg/kg; also referred to as the "Test Article"), or distilled water.

For studies of anaphylactic shock, 21 days after the end of the sensitization period, an intracardiac challenge dose equal to the sum of the sensitizing dose was introduced to the animals. A similar dose was introduced to the control guinea pigs. Anaphylaxis shock intensity was assessed as described in Example 11.

For studies of immune complex formation, 10 days after the last sensitizing dose, guinea pigs were challenged by intracutaneous adminstration of 500 μg/kg (0.5 ml) of Test

Substance. Similar volumes of distilled water was used in the control groups. Immune complex formation was assessed as described in Example 11.

Studies of indirect mast cell degranulation reaction and conjunctiva tests (using 1 drop of the Test Substance as a challenge dose), were carried out essentially as described in

Example 11.

Results In the analysis for anaphylactic shock, a Weigle index score of 0 was obtained for all groups, indicative of no shock.

In the indirect mast cell degranulation reaction, the MCDR obtained among the groups ranged from 0.09 to 0.18. Since the obtained values were less than 0.2, in all the cases the reaction was negative. In the immune complex reaction, scores of 0 were obtained for all groups at all time points, indicative of a negative skin reaction.

In the conjunctiva test, none of the test group animals exhibited reactions following administration of the Test Substance. In conclusion, intragastric administration of the active drug substance of Cognitone at a dose of 30 or 500 μg/kg for 5 days does not cause a general anaphylaxis reaction and does not induce allergenic effects.

Example 16. Investigation of immunomodulating properties of the fetal porcine brain protein active drug substance of Cognitone

The purpose of the current study was to determine the immunomodulating properties of the active drug substance of Cognitone (the "Test Substance"). Methods and materials

The Test Substance was prepared as a 0.01% solution in water. To induce emotional stress, mice (C57B1/6; Fl(CBAxC57Bl), or CBA of both genders; 18-23 g body weight) were subjected to overcrowding (40 mice per cage of dimensions 17x25x8 cm) for 10 days. Test animals were treated by intraperitoneal (IP) or intragastric (IG) administration of Test Substance (30 or 500 μg/kg) Control animals were administered a similar volume of distilled water. Quantitation of antibody producing cells (APCs) in mice spleen at 7 days post- immunization was carried with optimal (1x10 8 ) and suboptimal (2x10 7 ) doses of sheep erythrocytes (SE), as described in Jerne et al. Plague formation in agar antibody-producting cells Science, 1963, v. 140, No 3565, p. 405.

Hemagglutinin (HA) and SE titers were determined at 7 and 14 days post- immunization with optimal and suboptimal antigen doses in 96-well round-bottom plates, as described in Zigle Hemagglutination reaction. In: Immunologic methods, ed. H. Frimel 1979, p. 108-112.

Delayed hypersensitivity (DTH) reaction was tested using intravenous immunization of mice using a dose of 2x10 5 SE. The SE resolving dose (1x10 8 cells in 0.04 ml of normal saline) was introduced into posterior leg cushion at the 7 th day post-immunization. Local inflammatory reaction was recorded at 24 hours as weight difference of tested (Po) and control (Pk) pads. Reaction index (RI) was calculated for each mouse using the formula: Po - Pc RI = x 100%,

Pc as described in Kimatura K. A food weight assay method to avaluate DTH in the mice. J.Immunol.Meth.1980, v. 39, No. 2, p. 277-283.

Graft vs. host reaction (GVH) was induced by subcutaneous administration into the right posterior pad cushion of FI (CBAxC57BI) mice of a dose of 2xlO 6 of lymph node cells of parent genotype (CBA). Syngeneic lymphocytes were introduced in the left pad. At day 9 the number of cells in 5 ml of homogenate of the left (control) and the right (experiment) lymph node was determined .

Phagocyte activity of mice peritoneal macrophages was assessed as described in Argyris B.F. Role of macrophages in antibody production. J.Immunol. 1967 v. 99, No. 4, p.744-750. Macrophages were attracted to the abdominal cavity using 4% hydrolyzed starch solution. At the 3 rd -4 th day 0.5 ml of 5% SE suspension was introduced intragastrically. The cell suspension was washed out. Phagocyting cell percentage was determined in the Romanovsky-Gimsa-stained sample. To determine phagocytosis index (PI 1 ) number of captured SEs was calculated with spectrophotometry by hemoglobin concentration using calibrating curve. After 3-hour incubation at 37 0 C phagocytosis index was determined again (PI 2 ). Complete phagocytosis index (CPI) was determined using the following formula:

PIi - PI 2 CPI = x 100 %.

PIi Phagocytosis activity in mice in vivo was determined introducing to mice 0.5 ml of 25% ink solution intravenously. During 15 minutes every 3 minutes a blood sample was taken from an orbital sinus (0.02 ml) and introduced in 1 ml of 3% acetic acid. After taking the blood samples the animals were euthanized, and the body, liver and spleen weights were determined. Hemolyzed blood portion optical density was determined at SF-26 with the wavelength of 610 nm and the f(D) graph was made afterwards. Points Ig 0' (zero time) and Ig 10' (at the graph curve) were found. Phagocytosis constant (C) was calculated with the formula:

Ig O' - Ig 10' C = ,

10 True phagocytosis index (α) was calculated using the formula: body weight

(X = x K liver weight + spleen weight

Complement activity was determined with the titer method using activation of rabbit erythrocyte (RE) treated with hyperimmune guinea pig antiserum, as described in Tanaka et al. Assay of classical and alternative pathway activities of murine complement using antibody sensitized rabbit erythrocytes. J.Immunol.Meth., 1986, v. 86, p.161-170, for the direct pathway; and RE treated with potassium iodide solution for the alternative pathway, as described in Van Dijk et al. Study of the optimal reaction condition for assay of the mouse alternative complement pathway activities. J. Immunol. Meth. 1985, v. 85, p. 233-243. The activity was expressed as 50% lysis units per serum ml (CH50).

Result statistical processing was done using the applied software set. Student's criterion and Wilcoxon-Mann- Whitney method were used. When APC count and HA titer values were processed, the value logarithms were used. Results and Conclusions Effect of active drug substance of Cognitone on antibody immune response

The results of studies on APC production in mice spleen and serum HA titers in mice of different lineages immunized with optimal and suboptimal SE doses are shown in Tables

19 and 20 respectively. The data indicate that in C57BI/6 mice, IP administration of the Test

Substance increases the rate of APC production in spleen by about 2-fold (Table 19), while a relatively low response rate to either antigen dose in HA titer was observed (Table 20). In FI mice, Test Substance doses of 30 and 500 μg/kg were associated with a strong response rate after SE immunization (Table 20).

Ln C57BI/6 mice, 7-day IG administration of the Test Substance administration at a dose of 500 μg/kg caused an increase in APC count for optimal and suboptimal SE immunization regimens (Table 19), while HA titers increased only in the case of the optimal SE antigen dose (Table 20). A dose-dependent inhibition of APC production was observed in FI mice spleen after administration of optimal SE dose (Table 19), as well as diminution of HA titer in serum after IP and IG administration of the Test substance at a dose of 500 μg/kg. Such an event may be associated with the simultaneous introduction of SE and a xenobiotic agent (the Test Substance), resulting in the activation of both respiratory chain enzymes (Trufakin Immunomorphologic aspects of autoimmune processes.Novosibirsk: Nauka, 1983, p. 177) and monooxigenase activity, particularly of cytochrome P-450 (Kovalev et al Antibodies to physiologically active compounds. M., Medizina, 1981, p. 126) The latter is associated with functional association of immune system and metabolism resulting in enhanced erythrocyte degradation in phagocytosing cells, decrease of SE antigenic properties and, therefore, diminished antibody immune response intensity in the intact animals with normal physiological parameters.

Table 20 further shows that HA titer decrease has a phase pattern and by the 14th day post-immunization the active and control groups have no significant differences.

Table 19. Antibod - roducin cell APC in mice s leen.

Effect on Cell Immunity Response Development

After a single IP administration the Test Substance does not alter cell type reaction intensity. IG administration of the Test Substance (500 μg/kg) for 7 days induces DTH in C57BI/6 mice that is 2-2.5 times higher than that induced in CBA mice. The Test Substance has no- direct impact on development of the GVH reaction after IG or IP administration. Effects on phagocyte macrophage activity

The study results show that the Test Substance, upon a single IP adminstration does not change either phagocyte cell count in peritoneal exudate or phagocyte ability to capture opsonized SE 3 however, it increases by 1.5-2 times macrophage digesting capacity. IG administration of the Test Substance at a dose of 500 μg/kg increases the amount of phagocyte cells in exudate but it does not change PI and CPI. The Tested Article ability to stimulate phagocytosis completeness after IP introduction can be associated with its local activity.

After IP and IG introduction substance does not change intensity of inert ink particle capture and does not alter blood clearance rate.

Effect on complement activity

The study results show that the Test Substance does not influence the complement activity in mice serum.

Effects on immune system in stressed animals

The study results of the effects of the Test Substance on immunodeficiency severity induced with emotional stress are presented in Table 21.

Overcrowded (stress) conditions of animals for 10 days causes antibody immunity suppression (APC count and HA titer), complete phagocytosis suppression and blood clearance rate from inert ink particles. IG administration of the Test Substance for 10 days at a dose of 500 μg/kg prevents development of such abnormalities.

Thus, the active drug substance of Cognitone protects against stress-induced immunosuppressive effects. Table 21. Effect of the active drug substance of Cognitone on immune system of stressed mice.

The conclusions of this study are as follows:

1. The active drug substance of Cognitone at doses of 30 and 500 μg/kg was found to stimulate the antibody immune response in low-responding C57B1/6 mice and in high-responding Fl(CBA χ C57Bl) mice immunized with suboptimal antigen doses. The Test Substance at doses of 30 and 500 μg/kg (intraperitoneal administration) and at a dose of 500 μg/kg (intragastric administration) in Fl(CBAxC57Bl) mice immunized with optimal antigen dose suppresses the antibody immune response.

2. Intraperitoneal administration of the Test Substance does not influence DTH reaction severity, and after 7 days of oral administration at a dose of 500 μg/kg it suppresses DTH reaction development in high-responding C57B1/6 mice.

3. The Test Substance does not influence the GVH reaction course.

4. The Test Substance has the capacity to stimulate phagocytosis ability of peritoneal macrophages after intraperitoneal administration at doses of 30 and 500 μg/kg. The dose of 500 μg/kg intragastrically administered was found to increase phagocyte cell count in peritoneal exudate.

5. The Test Substance does not affect blood clearance rate from ink particles and serum complement activity in mice blood. 6. Following intragastric administration of a dose of 500 μg/kg for 10 days the Test Substance prevents diminution of antibody immune responses and macrophage function alteration associated with emotional stress.

Example 16. Pharmacokinetic studies of intravenous and intranasal formulations of Cognitone

Studies were carried out in male Chinchilla rabbits, body weight 2.95 ± 0.08 kg, 6 animals per group. The animals were maintained in standard cages according to a 12 hour on, 12 hour off light regimen and free access to food and water. The duration of the quarantine (acclimatization period) for animals was 14 days.

During the quarantine each animal was examined daily for behavior and general status.

An intravenous solution formulation of teh fetal porcine brain, protein extract was prepared substantially as described in Example 1, A-C, followed by dilution in sterile saline for a final concentration of 1.5 g/1. For intravenous administration, 1.5 ml of the formulation, was further diluted in 20 ml of saline and intravenously administered. The dose of the active drug substance was about 0.77±0.02500 mg/kg. For determination of the active drug substance in blood a 3 H-labeled form of the drug (specific activity 10 mCurie /mg) was intravenously administered.

An intranasal solution formulation of Cognitone containing fetal porcine brain tissue extract (0.1 g/1); hssDNA (1 g/1) and lecithin (2%) in a saline solution was intranasally administered to rabbits (0.15 ml per animal). The dose of the active drug substance was

0.05±0.01 mg/kg. For determination of the active drug substance in blood the aforementioned

3 H-labeled form of the drug was intranasally administered.

Animals received standard feed 2 hours after the onset of each experiment. Blood samples were taken from the accentric auricular vein of rabbits at the following time intervals following drug administration: 0.5; 1.0; 2.0; 3.0; 4.0; 6.0; 8.0; 12.0; 24.0; 32.0 and 48.0 hours.

Blood samples were centrifuged (15 min, 3000 rpm), and plasma aliquots of 1.5-2.0 ml were frozen and stored at -20 0 C until analysis. Liquid scintillation counting was performed on 0.3 ml aliquots. Calculations of pharmacokinetic parameters were made individually for each animal.

The following pharmacokinetic parameters were determined: β Constant of decreasing on distribution tail area (hr "1 )

T max time of intensity maximum (hr)

C max maximum activity of index (μg/ml) AUC 48 area under curve in interval 0-48 hi .;. (hr-μg/ml)

AUCoo full area under curve

(hr-μg/ml)

MRT mean retention time (hr)

Cl total clearance

(ml/hr/kg) v ss steady-state volume of distribution (ml/kg)

T 172 = 0.693 / β half-life period (hr)

Teff = AUC 00 / C max effective duration (hr) Parameters T max , C max and β were estimated from the concentration curve.

Formulas used for calculation of additional parameters were:

C1 = D / AUC

V SS = C1 - MRT where D — injected dose of the drug.

Results

The accuracy of the results of quantification of the active drug substance of Cognitone in blood plasma of experimental animals was estimated by calculation of results obtained from 5 parallel measurements in three plasma samples. Representative results are shown in Table 22.

The results show that the content of the drug in the samples is within the calculated confidence limits, and the method of quantitation of the active drug substance in blood plasma does not depend on system errors. Calculated pharmacokinetic parameters following intravenous administration of the solution formulation of fetal porcine brain tissue extract are presented in Table 23. The maximum concentration of the drug substance (7.25±0.26 μg/ml) was attained at 30 min. The plasma half-time was about 9.5 hours. The total mean time of the drug (MRT) was about 14 hr. The apparent fixed volume of distribution (V ss) was about 100 ml/kg.

Table 23. Pharmacokinetic parameters of an intravenously administered solution of fetal porcine brain protein extract

Calculated pharmacokinetic parameters following intranasal administration of the intranasal formulation of Cognitone are presented in Table 24. The maximum concentration of the drug substance (0.39±0.04 μg/ml) was attained at 60 min. The plasma half-time was about 10 hours. The total mean time of the drug (MRT) was about 16 hr. The apparent fixed volume of distribution (V SS) was about 123 ml/kg.

Example 17. Clinical effects of Cognitone and purified protein extract of the invention Several clinical cases were treated using the compositions of the invention.

Case l

Diagnosis: A 47 year old male suffering from blood flow impairment of the middle cerebral artery region, and resultant left sided hemiplegia.

Treatment: Purified protein extract as described in Example 1 (A-C) was administrated LV. on the 5 th day after symptom manifestation. One ml (protein concentration 1.5 mg/ml) of purified protein extract was diluted with 200 ml of glucose solution (5%) and infused intravenously to the patient at a dosage of 0.02 mg per kg body weight. Subsequently during a period of 3 weeks after infusion, the patient was treated with Cognitone lipidic formulation as described in Example 1(D) 5 administered as in intranasal drop form, daily, at a dosage of 0.4 μg per kg body weight.

Therapeutic effect: On the 3 rd day of treatment, the symptoms of left sided hemiplegia disappeared. Case 2

Diagnosis: A 75 year old male suffering from right sided hemorrhagic insult was diagnosed with coma (Stage 1), hypertonia and post-infarction cardiosclerosis. Treatment: On the lst th day after symptom manifestation the patient was treated with 1 ml (protein concentration 1.5 mg/ml) of Purified protein extract as described in Example 1 (A-C) as an intralumbar injection at a dose of 0.02 mg/kg body weight. Simultaneously he was administered with dexamethasone (4 mg in 1 ml). For the next 3 days, the patient was infused (i.v.) with 2 ml of the purified protein extract (1.5 mg/ml) diluted with 200 ml glucose solution (5%) daily at the dosage of 0.04 mg per kg body weight. For the next 3 months, the patient was treated with Cognitone lipidic formulation as described in Example 1 (D), administered in an intranasal emulsion drop form, daily, at the dosage of 0.4 μg per kg body weight.

Therapeutic effect: The patient recovered from coma 48 hours after treatment onset. The symptoms of left sided hemiplegia disappeared after 7 days of treatment. During the course of treatment a gradual reduction of peripheral neurological symptoms occurred.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.