WO2008062420A2

WO2008062420A2 - Use of glucagon and insulin in methods and compositions for the treatment of acute brain injury and neurodegenerative disorders

Info

Publication number: WO2008062420A2
Application number: PCT/IL2007/001458
Authority: WO
Inventors: Abd Higazi; Nuha Hijazi
Original assignee: Hadasit Medical Research Services & Development Ltd.
Priority date: 2006-11-26
Filing date: 2007-11-26
Publication date: 2008-05-29
Also published as: IL183009A0; WO2008062420A3

Abstract

The invention relates to the use of a substance which reduces the circulating levels of lactate and neurotoxic excitatory amino acids, for the treatment or prevention of a pathologic condition involving neurological injury. Such substance may be glucagon, insulin or any combination or mixture thereof. The invention further provides methods and compositions for protecting the central nervous system (CNS) from injury and death, which may result from, for example, a head trauma, a stroke or a neurodegenerative disorder, using glucagon, insulin or any combination or mixture thereof which reduce the levels of circulating lactate and neurotoxic excitatory amino acids.

Description

USE OF GLUCAGON AND INSULIN IN METHODS AND COMPOSITIONS FOR THE TREATMENT OF ACUTE BRAIN INJURY AND NEURODEGENERATIVE DISORDERS

Field of the Invention

The invention relates to methods and compositions for protecting the central nervous system (CNS) from injury and death, which may result from, for example, a head trauma, a stroke or a neurodegenerative disorder. More particularly, the invention relates to the use of a substance which reduces the levels of circulating lactate and neurotoxic excitatory amino acids, for preventing and treating conditions involving neuronal damage. Such substance may be glucagon, insulin or any combination or mixture thereof.

Background of the Invention

All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Stroke, head trauma and dementia are among the leading causes of death and disability in the world and constitute a significant financial burden. Although stroke and head trauma are caused by different mechanisms, they share a common pathway of lactate production that causes neuronal injury.

Gluconeogenesis is the process that converts non-hexose sugars, such as pyruvate/lactate, glycerol, oxaloacetate and glucogenic amino acids to glucose. Gluconeogenesis provides substrates for glucose-dependent organs like the brain [Nelson, D. and Cox, M. Principles of Biochemistry, fourth edition, Freeman, New York. Chapter 14:543 (2005)], by conversion of non- hexose sugars, such as pyruvate/lactate, glycerol, oxaloacetate and glucogenic amino acids to glucose. Glucose is generated from lactate produced by anaerobic glycolysis (e.g. muscle during vigorous exercise), as well as by tissues lacking mitochondria (e.g. red blood cells) and by cells under hypoxic conditions [Wasserman, D. et al. Am. J. Physiol. 257:E1O8 (1989)]. Gluconeogenesis thus prevents the accumulation of lactate and protects against a decrease in tissue pH. Elevations of lactate have been reported in several pathological conditions, including head trauma, where increased concentrations in the cerebrospinal fluid (CSF) portend a poor outcome [Bakay, R. et al. Neurosurgery 18:234-243 (1986)].

Without being bound by any theory, the inventors hypothesized that gluconeogenesis may protect against cerebral hypoxia by enhancing the hepatic clearance of lactate and its metabolites. Since gluconeogenic amino acids are removed from the circulation during gluconeogenesis [Nelson (2005) ibid.; Brockman, R.P. et al. American Journal of Physiology 229:1344 (1975)], neuroprotection may be further facilitated by the clearance of neurotoxic excitatory amino acids, including glutamate and its precursor glutamine, aspartate and alanine (present in equilibrium with pyruvate and lactate), which are also elevated in the CSF of patients with acute head injuries [Yamamoto, T. et al. Acta Neurochirurgica. Supplementum 75:17-19 (1999); Zhang, H. et al. Clin. Chem. 47:1458-1462 (2001)].

Gluconeogenesis may also provide neuroprotection through the preconditioning response. The mammalian brain institutes a protective phenotype in response to stress, such as short periods of hypoxia [Schurr, A. et al. Brain Research 374:244-248 (1986); Dahl, N. et al. American Journal of Physiology 207:452-456 (1964)]. These changes promote cell survival in the face of subsequent cerebral injury [Yellon, D. et al. Circulation Research 87:543 (2000); Gidday, J. Nature Rev. Nuroscience 437:437 (2006)]. Since gluconeogenesis is triggered by stress in general [McGuinness O. P. et al. Am. J. Physiol. Endocrinol. Metab. 265:E314-E322 (1993)] and specifically by hypoxia through the combined effects of epinephrine- [Wright, P. et al. J. Exp. Biol. 147:169 (1989)], Cortisol [Fujiwara, T. et al. Metabolism: Clinical and Experimental 45:571-578 (1996)] and glucagons [Wasserman (1989) ibid.], activation of this pathway prior to neurological insult may contribute to the protective "preconditioning" response.

Gluconeogenesis is not the only pathway that mediates clearance of lactate and amino acids from the circulation. Although it may appear paradoxical, it is well known that insulin has similar effects on the levels of amino acids [Luck, J.M. et al. J. Biol. Chem., 151 (1928)] and lactate in the circulation [Brockman (1975) ibid.]. However, in contrast to glucagon, which utilizes lactate and amino acids to synthesize glucose, insulin decreases their concentrations by stimulating their incorporation into peripheral tissues [Brockman (1975) ibid.]. Several groups have shown that the use of insulin to decrease blood glucose in diabetics immediately after a brain insult, such as stroke [Lees, K.R. and Walters, M.R., Cerebrovasc. Dis. 20 (1):9 (2005)] or head trauma, improves the clinical outcome [Jeremitsky, E. et al. J Trauma. 58:47 (2005)]. The inventors therefore postulated that the neuroprotective effect seen in insulin treated diabetics might be due, at least in part, to the capacity of insulin to reduce the concentration of neurotoxic metabolites, such as lactate and excitatory amino acids in the circulation. Lactic acid or lactate is the end-product of anaerobic glycolysis. Tissues depend on anaerobic glycolysis during oxygen deprivation, like vascular obstruction or insufficient oxygen supply.

The production lactate ions is considered as deleterious to tissues, since it decreases the tissue pH and may thereby increase directly the hypoxic damage. Furthermore, increased lactate production contributes to secondary damage by inducing vasodilation in cases of head injury, vasodilation can thereby affect brain hemodynamics and cause a further increase of brain damage [Bakay, R.A. et al. Neurosurgery 18:234-243 (1986)].

Lactate levels were shown to increase in the cerebrospinal fluid of patients with various grades of head injury. Furthermore, elevated CSF lactate levels are significantly correlated to the degree of brain injury. CSF lactate levels were found to be closely correlated with poor prognosis (an increase above 50 mg/dl indicate a critical life threatening condition). Therefore, it has been proposed that lactic acidosis, and the attendant decrease in pH, may be a major cause of severe ischemic brain injury.

In addition to acute brain injury, many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, human immunodeficiency virus (HIV) -associated dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and glaucoma, may share final common pathways leading to neuronal injury and therefore may benefit from the reduction of lactate levels.

Considering these findings the inventors hypothesized that decreasing the levels of lactate may improve the outcome in patients with hyperlacticacidemia due to stroke or head trauma associated with vascular obstruction and hypoxia.

The approach used by the present invention is based on decreasing the levels of lactate by inducing gluconeogenesis. More particularly, administration of the hyperglycemic hormone glucagon, which induces gluconeogenesis from circulating lactate, induces the production of glucose and reduces circulating lactate levels and thereby ameliorates neuronal damage.

Glucagon is a peptidic hormone that mediates the regulation of glucose levels in the blood. Glucagon is primarily used for the emergency treatment of severe hypoglycemia.

Glucagon is synthesized as proglucagon and proteolytically processed to yield glucagon within alpha cells of the pancreatic islets. Proglucagon is also expressed within the intestinal tract, where it is processed not into glucagon, but to a family of glucagon-like peptides (enteroglucagon). As indicated above, the major effect of glucagon is to stimulate an increase in blood concentration of glucose. Moreover, the brain in particular has an absolute dependence on glucose as a fuel, because neurons cannot utilize alternative energy sources like fatty acids to any significant extent. When blood levels of glucose begin to fall below the normal range, it is imperative to find and pump additional glucose into blood. Glucagon exerts control over two pivotal metabolic pathways within the liver, leading that organ to dispense glucose to the rest of the body, by stimulating breakdown of glycogen stored in the liver and activating Hepatic-gluconeogenesis. The inventors now show that administration of a single dose of glucagon or insulin to normal or diabetic mice 10 min after head trauma, reduced significantly lesion size, cellular apoptosis and accelerated neurological recovery. The neuroprotective effect of insulin and glucagon was independent of the blood glucose levels. The neuroprotective effect of glucagon was related to its capacity to activate gluconeogenesis, decreasing lactate and neuroexcitatory amino acids, whereas insulin stimulates the clearance of lactate and the same neurotoxic amino acids from the circulation to peripheral tissues.

It is therefore one object of the invention to provide the use of a substance which reduces the levels of circulating lactate and neurotoxic excitatory amino acids, for the treatment and prevention of neuronal injury and death. Such substance may be preferably glucagon, insulin or any combination or mixture thereof.

Another object of the invention is to provide methods and compositions for the treatment and prevention of acute brain injury, head trauma, stroke and neurodegenerative disorders using a substance which reduces the levels of circulating lactate and neurotoxic excitatory amino acids glucagon. Such substance may be preferably glucagon, insulin or any combination or mixture thereof.

These and other objects of the invention will become apparent as the description proceeds. Summary of the Invention

In a first aspect, the invention relates to the use of a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, in preparation of a pharmaceutical composition for the treatment or prevention of a pathologic condition involving neurological injury or damage.

According to one embodiment, a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids used by the invention may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

In a second aspect, the invention relates to a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, for the treatment or prevention of a pathologic condition involving neurological injury.

According to one specifically preferred embodiment, such substance may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

The invention further provides glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, for the treatment or prevention of a pathologic condition involving neurological injury.

Still further, the invention provides insulin, any functional fragment or derivative thereof, any combination or mixture thereof, for the treatment or prevention of a pathologic condition involving neurological injury.

In another aspect, the invention relates to a method for the treatment or prevention of a pathologic condition involving neuronal injury or damage. The method of the invention therefore comprises the step of administering to a subject suffering from such pathological condition, a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids.

In another aspect, the invention relates to a pharmaceutical composition for the treatment of a pathologic condition involving neurological injury or damage. The composition of the invention comprises as an active ingredient a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, and optionally any further pharmaceutically acceptable carrier, diluent, excipient and/or additive.

A further aspect of the invention relates to a kit for achieving a therapeutic effect in a subject in need thereof. The kit of the invention may comprise: (a) at least one of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a first unit dosage form; (b) at least one of insulin, any functional fragment or derivative thereof, or any combination or mixture thereof and a pharmaceutically acceptable carrier or diluent in a second unit dosage form; and (c) container means for containing said first and second dosage forms.

The invention further provides a method for making a medicament for the treatment of a pathologic condition involving neurological injury or damage. Accordingly, the method of the invention comprises the step of: (a) providing a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids; (b) admixing said substance with at least one of a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Brief Description of the Figures

Figure 1A-1B Effect of pretreatment with glucagon on pre and post

CHT neurological recovery

Fig. IA. Effect of pretreatment with glucagon on pre CHT neurological recovery

Ip injections with glucagon significantly improved neurological severity score as exhibited by extent of recovery (dNSS) in mice pretreated with glucagon

(10 min before induction of trauma) compared to control group receiving saline alone. Fig. IB Effect of pretreatment with glucagon on post CHT neurological recovery

Effect of treatment with glucagon on post CHT neurological recovery

Ip injections with glucagon significantly improved neurological severity score

(as exhibited by extent of recovery (dNSS) in mice treated with glucagon (10 min after induction of trauma) compared to control group receiving saline alone. Abbreviations: control (cont.)

Figure 2A-2B: The effect of glucagon on post-CHI neurobehavioral function

Fig. 2A. Ten minutes post CHI mice were injected IP with 5 μg glucagon in normal saline (open squares) or saline alone (filled squares) and the neurological severity score (NSS) was measured. A total of 24 animals in each cohort were studied.

Figure 2B. The mean + SE of the NSS on day 28 is shown (n=24 for each group). The difference between the NSS in the glucagon and saline treated groups was significant (p = 0.0026). Abbreviations: Tre. (treated), cont.

(control), D. Po. CHI (days post closed head injury).

Figure 3A-3C Appearance of the brain lesion post CHI

Fig. 3A. Representative brains of mice treated with glucagon or saline at 28 days post CHI.

Fig. 3B. Slices from representative brains showing area of damage (missing portions) 28 days post CHI.

Fig. 3C. Volume of the damaged area after glucagon vs. saline. The mean and SE of the lesion size from 12 animals/group, treated with glucagon

(treated) vs. saline (Control) is shown. The difference between the NSS in the glucagon and saline treated groups was significant at p = 0.0003. Abbreviations: Tre. (treated), cont. (control), Dam. (damaged), Zo. (zone).

Figure 4A-4C Effect of Glucagon on the levels of glucose, alanin and glutamate in the circulation

Fig. 4A Mice were injected with ip glucagon (G) or saline (S). Blood glucose was determined 15 min before (Pr-S or Pr-G) and 15 min after glucagon or saline injection (Po-G or Po-S).

Fig. 4B Mice were injected ip with saline (Cont) or glucagon (Glucagon). 15 min after injection, blood was withdrawn and alanine concentration was determined.

Fig. 4C Ip injections with glucagon significantly reduced plasma glutamate levels when treated mice were compared to control group receiving saline alone. Abbreviations: Tre. (treated), cont. (control),

Figure 5A-5B: The effect of glucagon on the development of post CHI apoptosis

Ten minutes after CHI mice were injected IP with saline (Control) or saline containing 5 μg glucagon (Treated). Twenty-four hours later, brains were extracted sliced and TUNEL stained; the extent of apoptosis was determined in equivalent regions in both groups of animals at the edge of the injury cavity (see Methods).

Fig. 5A: Representative brain sections from control and treated animals showing apoptosis.

Fig. 5B: The % of apoptotic cells in each area was calculated. The data show the mean ± SE of 12 mice per group. The difference between the fraction of apoptotic cells in the glucagon and saline treated groups was significant at p=

0.0028. Abbreviations: Tre. (treated), cont. (control). Figure 6A-6C. Therapeutic window for protection by glucagon

Fig. 6A. Mice were injected with saline (Cont) or saline containing 5 μg glucagon IP 10 min, 3 hours, or 10 min and 3 hours post CHI and the NSS was evaluated periodically; the mean of the NSS ± SE of 10 mice/group on day 28 is shown.

Fig. 6B. Mice were injected with saline containing glucagon (open squares) or with saline alone (filled squares) 10 min prior to CHI and the NSS was evaluated periodically, as indicated. The mean ± SE of 10 mice/group is shown.

Fig. 6C. The NSS in glucagon-pretreated and control mice on day 28. The difference between treated and control groups was significant at p= 0.006.

Abbreviations: Tre. (treated), pre. Tre. (pretreated), cont. (control), min.

(minutes), D. (days), Po. (post).

Figure 7A- ZE Comparison of the effect of glucagon and insulin on post CHI neurobehavioral function

10 minutes post CHI, normal (Fig. 7A) or diabetic mice (Fig. 7D) were injected IP with saline (Controls), saline containing 5 μg glucagon or saline containing 1 u/kg of insulin and the NSS was measured as indicated. The data show the mean ± SE in 24 animals per group.

Figs 7B and 7E: The mean ± SE of the NSS on day 28. The difference between each of the treated groups and the controls was significant at p <

0.001.

Fig. 7C: Therapeutic window for protection by insulin. Mice were injected with saline (Cont) or saline containing 1 u/kg insulin IP 10 min, 3 hours, or

10 min and 3 hours post CHI and the NSS was evaluated periodically; the mean of the NSS ± SE of 10 mice/group on day 28 is shown. Abbreviations: Tre. (treated), pre. Tre. (pretreated), cont. (control), min. (minutes), D. (days), Po. (post).

Figure 8 Representative regions at the edge of the injury cavity selected for TUNEL evaluation

The number of TUNEL-positive cells in the lesion was determined from up to 12 sites (depending on the size of the lesion) around the injury.

Detailed Description of the Invention

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

In view of the deleterious effect of lactate, particularly on brain tissue and the need of novel approaches to the treatment of pathological conditions involving neuronal injury, head trauma and neurodegenerative disorders, the present inventors have examined whether excess lactate and the pathologic conditions caused thereby, may be eliminated by induction of gluconeogenesis by a gluconeogenesis inducing agent, particularly, glucagon.

The data disclosed by the present invention reveal a previously undescribed role for glucagon as a neuroprotective agent in experimental head trauma. The neuroprotective effect of glucagon was associated with a decrease in the plasma concentrations of lactate and glucogenic amino acids due to activation of gluconeogenesis. The finding that insulin, the physiological antagonist of glucagon, has the same neuroprotective effect and shares its "therapeutic window" indicates that the neuroprotective effect of both hormones is due to their capacity to reduce the concentration of lactate and excitatory neurotoxic amino acids in the circulation, rather than an effect on plasma glucose concentration.

The lack of association between blood glucose and neuro-protection shown in the present invention mitigates concerns about the deleterious effects of hypoglycemia, which for many years was the major reason for avoiding insulin administration in hyperglycemic stroke patients [Lees (2005) ibid.; Adams, H. et al. Stroke 38:1655 (2007)] and suggests its utility in normo- or hyper- glycemic patients at doses that do not induce significant hypoglycemia.

Thus, as a first aspect, the invention relates to the use of a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, in preparation of a pharmaceutical composition for the treatment or prevention of a pathologic condition involving neurological injury or damage.

It should be noted that the substance of the invention decreases the levels of at least one of lactate and neurotoxic excitatory amino acids in the circulation. As shown by the following Examples, in blood samples obtained from treated subjects, measurements of lactate concentrations as well as of different excitatory amino acids, clearly indicated a decrease or reduction of about 40% to 50% as compared to blood samples obtained from control untreated subjects (see for Example Table 1). Therefore, it should be appreciated that the term "reduction", "reduces" as used herein is meant a decrease of about 10% to 90% of the lactate or excitatory amino acid blood levels, compared to untreated control. Preferably, a decrease of about 20% to 80%, 30% to 70%, more preferably a decrease of about 40% to 60%. In yet another specifically preferred embodiment, the substance of the invention leads to decrease of about 40% to 50% in at least on of lactate and excitatory amino acids. Non-limiting examples for neurotoxic excretory amino acids or gluconeogenic amino acids may include glutamate and its precursor glutamine, aspartate and alanine (present in equilibrium with pyruvate and lactate).

According to one embodiment, a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids is used for treating and preventing pathologic condition involving neurological injury or damage. Such pathologic condition may be any one of acute or traumatic brain injury, brain anoxia, stroke, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgery intervention, hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

In yet another embodiment, the use of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, according to the invention may be suitable for a subject suffering of a neurodegenerative disorder such as Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, human immunodeficiency virus (HIV)- associated dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and glaucoma.

According to another embodiment, the use according to the invention may be for preventive preoperative neurosurgery intervention, where the use may be immediately before operation.

According to a specifically preferred embodiment, the substance used by the invention may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof.

Glucagon is a key hormonal agent that, in co-operation with insulin, mediates homeostatic regulation of the amount of glucose in the blood. Glucagon primarily acts by stimulating certain cells (mostly liver cells) to release glucose when blood glucose levels fall. The action of glucagon is opposite to that of insulin, which stimulates cells to take up and store glucose whenever blood glucose levels rise. Both glucagon and insulin are peptide hormones. Glucagon is produced in the alpha islet cells of the pancreas and insulin in the beta islet cells. Glucagon exerts its action by binding to and activating its receptor, which is part of the Glucagon- Secretin branch of the 7-transmembrane G-protein coupled receptor family. The receptor functions by activating the adenylyl cyclase second messenger system and the result is an increase in cAMP levels.

Glucagon is a linear peptide of 29 amino acids. Its primary sequence is almost perfectly conserved among vertebrates, and it is structurally related to the secretin family of peptide hormones. The polypeptide has a molecular weight of 3485 daltons, its primary structure is: NrΪ2-His-Ser-Gln-Gly-Thr- Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Ghi-Asp-Phe- Val-Gln-Trp-Leu- Met-Asn-Thr-COOH as also denoted by SEQ ID NO. 1, and its empirical formula is C153H225N43O49S.

It should be noted that the glucagon as represented by SEQ ID NO. 1, is the human glucagon, however, the invention encompasses the use of any other species of glucagon. The term "species" such as in "glucagon species" is used according to its art accepted meaning and refers to those mammalian glucagon proteins having a biological activity such as human glucagon and glucagon from non-human mammals as well as variants of human glucagon.

It should be further noted that the invention encompasses the use of all forms of glucagon, including, but not limited to naturally occurring glucagon, glucagon produced by recombinant DNA technology, or glucagon produced by or derived from any other means. Glucagon components also include fragments, agonists, and analogs of glucagon, which have the same or similar pharmacological activity as glucagon. It should be further appreciated that any commercial form of glucagon may also be used by the invention, for example, glucagon is commercially available in the North American market is human glucagon of rDNA origin produced by Eh Lilly & Co or Bedford Labs (Novo). Several brand names are known, these being: Glucagon Diagnostic Kit (Lilly); Glucagon Emergency Kit (Lilly); Glucagon Emergency Kit for Low Blood Sugar (Lilly); GlucaGen^® Diagnostic Kit, Bedford; and most recently, GlucaGen HypoKit^® Novo Nordisk A/S). Novo produces glucagon under its own name outside of North America. Novo produces its glucagon in yeast and Lilly produces its glucagon in E. coli. The following examples illustrate practice of the methods of the present invention using such commercially available glucagon preparations.

It should be appreciated that as used herein, glucagon includes, but is not limited to, the glucagon, glucagon formulations, and routes of administration described in U.S. patent application publication No. 2002114829 and U.S. Pat. Nos. 6,197,333 and 6,348,214, which describe liposome formulations of glucagon that provide for reduced dosage effect and are long acting; PCT patent publication No. WO0243566, which describes the delivery of glucagon via trans-dermal patch; U.S. Pat. No. 5,445,832, which describes a long- acting glucagon formulation in polymeric microspheres; PCT patent publication No. WO0222154, which describes a slow-release glucagon that can have a duration of action measured in weeks; and U.S. Pat. No. 3,897,551 and Great Britain Patent No. 1,363,954, which describes the prolongation of glucagon duration by iodination. In an embodiment, the glucagon is administered as a slow-release or depot formulation (e.g., comprising polyethylene glycol).

European patent publication EP1224929 and U.S. Pat. No. 6,004,574, which describe an inhaled glucagon with melezitose diluent; U.S. Pat. No. 5,942,242. According to one embodiment, the glucagon used by the invention should be in therapeutically effective amount, which preferably being in a dosage unit form comprising from about O.lμg to about lmg glucagon per IKg of body weight, preferably, 250μg/kg.

According to another alternative embodiment, the substance used by the method of the invention for reducing the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, may be any on of insulin, any functional fragment or derivative thereof, or any combination or mixture thereof.

Accordingly, the therapeutically effective amount of insulin may preferably be in a dosage unit form comprising from about 0.1U to about 1OU insulin per IKg of body weight, preferably, lU/kg.

Insulin is a peptide hormone composed of 51 amino acid residues and has a molecular weight of 5808 Da. It is produced in the Islets of Langerhans in the pancreas. In beta cells, insulin is synthesized from the proinsulin precursor molecule by the action of proteolytic enzymes, known as prohormone convertases (PCl and PC2), as well as the exoprotease carboxypeptidase E. These modifications of proinsulin remove the center portion of the molecule, or C-peptide, from the C- and N- terminal ends of the proinsulin. The remaining polypeptides (51 amino acids in total), the B- and A- chains, are bound together by disulfide bonds. A properly cross-linked insulin contains three disulfide bridges: one between position 7 of the A-chain and position 7 of the B-chain, a second between position 20 of the A-chain and position 19 of the B-chain, and a third between positions 6 and 11 of the A-chain. Insulin is essential for proper metabolism in humans. In addition to its familiar role as the chief regulator of blood sugar levels in humans, it is essential for carbohydrate, lipid, and protein metabolism.

It should be appreciated that insulin of any species, any type, or any analogue of insulin may be used as a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, by the methods and compositions of the invention.

The term "insulin analog" means proteins that have an A-chain and a B- chain that have substantially the same amino acid sequences as the A-chain and B-chain of human insulin, respectively, but differ from the A-chain and B-chain of human insulin by having one or more amino acid deletions, one or more amino acid replacements, and/or one or more amino acid additions that do not destroy the insulin activity of the insulin analog.

More specifically, "analog" refers to a molecule that is structurally similar or shares similar or corresponding attributes with another molecule (e.g. an insulin variant capable of forming an insulin/insulin dimer complex). Eor example, insulin LISPRO is an analog of human insulin where the B28 Proline and B29 Lysine are interchanged. The effects of this change are to fundamentally decrease the propensity to form the hexameric insulin structure and to increase the relative amount of insulin monomer present in solution.

The term "species" such as in "insulin species" is used according to its art accepted meaning and refers to those mammalian insulin proteins having a biological activity that allows them to be used in the treatment of diabetes such as human insulin and insulins from non-human mammals as well as variants of human insulin (e.g. porcine insulin and LISPRO insulin). Such animal insulin molecules are for example rabbit, pork, beef, and sheep insulin. Non-human insulin species generally share at least about 90% or more amino acid homology with human insulin (e.g. using BLAST criteria). A review of the research, development, and recombinant production of human insulin may be found in U.S. Pat. Nos. 4,652,525 (rat insulin) and 4,431,740 (human insulin).

Typical regular insulin formulations include insulin in an un-buffered or phosphate buffered solution containing glycerin for isotonicity, zinc ions for stability and a phenolic preservative such as phenol or m-cresol. These formulations are generally not sufficiently stable, either chemically or physically, for use in implantable pumps with the exception being insulin in a formulation of Tris (tris-hydroxymethyl amino methane) buffer and Genapol, a polyoxyethylene, polyoxypropylene copolymer non-ionic surfactant. In addition to using regular insulin in such formulations, insulin analogs such as LISPRO can also be used (see e.g. U.S. Pat. No. 6,034,054).

Another type of insulin analog, "monomelic insulin analog" is well-known in the art. Monomeric insulin analogs are structurally very similar to human insulin, and have activity similar or equal to human insulin, but have one or more amino acid deletions, replacements or additions that tend to disrupt the contacts involved in dimerization and hexamerization which results in their having less tendency to associate to higher aggregation states.

As indicated herein before, glucagon and insulin and any fragment analogue or derivatives thereof may be used for reducing the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, according to the invention. By "analogs and fragments" is meant the "variants", "analogs" or "derivatives" of said peptide molecule. A "fragment" of a molecule, such as any of the amino acid sequences of the present invention, is meant to refer to any amino acid subset of the glucagon or insulin peptidic molecule. A "variant" of such molecule is meant to refer a naturally occurring molecule substantially similar to either the entire molecule or a fragment thereof. An "analog" of a molecule can be without limitation a paralogous or orthologous molecule, e.g. a homologous molecule from the same species or from different species, respectively, i.e., glucagon or insulin amino acid sequence of different species, which therefore may have slight changes in the sequence. By "functional" is meant having same biological function, for example, required for reducing levels of at least one of lactate and excitatory amino acids in the circulation. The terms derivatives and functional derivatives as used herein mean peptides comprising the amino acid sequence of for example, glucagon, as denoted by SEQ ID NO. 1, or alternatively, the amino acid sequence of insulin, with any insertions, deletions, substitutions and modifications to the peptide that do not interfere with the ability of said peptide to reduce lactate and or excitatory amino acids levels in the circulation (hereafter referred to as "derivative/s"). A derivative should maintain a minimal homology to said amino acid sequence, e.g. even less than 30%, preferably, homology of between 30% to 100%, 40% to 90%, 50% to 80%, 60% to 70%, preferably, homology of about 75%. It should be appreciated that by the term "insertions", "deletions", "substitutions" and "modifications", as used herein it is meant any addition, deletion, or replacement, respectively, of amino acid residues to the peptides of the invention, of between 1 to 100 or 1 to 50 amino acid residues, preferably between 20 to 1 amino acid residues, and most preferably, between 1 to 10 amino acid residues. More specifically, "insertions", "deletions", "substitutions" and "modifications", as used herein it is meant any addition, deletion, or replacement, respectively, of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90 and 100, identical or different amino acid residues.

It should be further appreciated that the peptides glucagon or insulin used by the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different amino acid residues. As an example for such extension, the peptide may be extended at the N-terminus and/or C- terminus thereof with identical or different hydrophobic amino acid residue/s which may be naturally occurring or synthetic amino acid residue/s. One example for a synthetic amino acid residue is D-alanine.

An additional and preferred example for such an extension may be provided by peptides extended both at the N-terminus and/or C-terminus thereof with a cysteine residue. Naturally, such an extension may lead to a constrained conformation due to Cys-Cys cyclization resulting from the formation of a disulfide bond.

Another example may be the incorporation of an N-terminal lysyl-palmitoyl tail, the lysine serving as linker and the palmitic acid as a hydrophobic anchor.

In addition, the glucagon, insulin or any similar peptide may be extended by aromatic amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s. It should be appreciated that the invention encompasses any modification of glucagon or insulin made in order to prolog its half life in circulation or increase its efficacy. Nonetheless, according to the invention, the glucagon or insulin peptides used by the methods of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different organic moieties which are not naturally occurring or synthetic amino acids. As an example for such extension, the peptide may be extended at the N-terminus and/or C- terminus thereof with an N- acetyl group.

The lack of structure of linear peptides renders them vulnerable to proteases in human serum and acts to reduce their affinity for target sites, because only few of the possible conformations may be active. Therefore, it is desirable to optimize the peptide structure, for example by creating different derivatives of the glucagon, insulin or any similar peptides used by the invention.

In order to improve peptide structure, the peptides glucagon or insulin used by the invention, or any derivative or fragment thereof, can be coupled through their N-terminus to a lauryl-cysteine (LC) residue and/or through their C-terminus to a cysteine (C) residue, or to other residue/s suitable for linking the peptide to adjuvant/s for immunization, as will be described in more detail hereafter.

The peptides glucagon or insulin used by the invention, as well as derivatives thereof may all be positively charged, negatively charged or neutral and may be in the form of a dimer, a multimer or in a constrained conformation. A constrained conformation can be attained by internal bridges, short-range cyclizations, extension or other chemical modification. For the glucagon or insulin peptide sequences used by the invention and disclosed herein, this invention includes the corresponding retro-inverso sequence wherein the direction of the peptide chain has been inverted and wherein all the amino acids belong to the D-series.

Peptides longer than the 29 amino acid residues of glucagon or longer than the 51 amino acid residues of insulin may also be a result of a tandem repetition, in which the basic peptidic sequence (of the peptide of the invention) is repeated from about 2 to about 100 times.

Regarding derivatives and fragments of glucagon peptide, it should be noted that a single glucagon gene encodes a larger biosynthetic precursor, proglucagon, in mammals. Tissue-specific processing of proglucagon gives rise to glucagon in the brain, and glicentin, oxyntomodulin, Glucagon-like peptide-1 (GLP-I), and Glucagon-like peptide-2 (GLP-2) in the intestine, with an intermediate profile of peptides liberated in the central nervous system. GLP-I is synthesized in intestinal endocrine cells in two principal major molecular forms, as GLP-l(7-36)amide and GLP-l(7-37), and regulates blood glucose via stimulation of glucose-dependent insulin secretion, inhibition of gastric emptying, and inhibition of glucagon secretion. GLP-2 is a 33 amino acid peptide, co-secreted along with GLP-I from intestinal endocrine cells in the small and large intestine and has recently been shown to display intestinal growth factor activity in rodents. It should be therefore appreciated that according to one specifically preferred embodiment, the invention relates to the use of glucagon and any fragments or derivatives thereof, provided that these derivatives are not Glucagon-like peptides, e.g. GLP-I or GLP-2 or any derivative or variant thereof. Thus, any derivatives or functional fragments of glucagon, and particularly the human glucagon peptide of SEQ ID NO. 1, used by the present invention, are with the proviso that they do not consist the sequences of GLP-I or GLP-2.

According to one embodiment, the dosage unit form of glucagon or insulin used by the invention, may be used for either a single or alternatively, repeated administration. According to another embodiment, administration of such dosage unit form may be repeated every one to eight hours for a therapeutically sufficient period of time. Alternatively, the active ingredient, glucagon or insulin may be in a sustained-released dosage form enabling continues release of the preferred effective amount of glucagon, insulin or any mixture or combination thereof for a therapeutically sufficient period of time.

It should be noted that the amount of glucagon or insulin used in for preparing a composition according to the present invention is an amount effective to treat the target indication. However, the amount can be less than that amount when the composition is used in a dosage unit form because the dosage unit form may contain a plurality of delivery agent glucagon, glucagon agonist or insulin compositions or may contain a divided effective amount. The total effective amount can then be administered in cumulative units containing, in total, an effective amount of a glucagon or insulin component. Moreover, those skilled in the filed will recognize that an effective amount of glucagon or insulin component will vary with many factors including the age and weight of the patient, the patient's physical condition.

Another aspect of the invention relates to a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, for the treatment or prevention of a pathologic condition involving neurological injury. According to one specifically preferred embodiment, such substance may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

Still further, the invention provides insulin, any functional fragment analogue or derivative thereof, any combination or mixture thereof, for the treatment or prevention of a pathologic condition involving neurological injury.

According to a specifically preferred embodiment, the glucagon or insulin or any substance provided by the invention are particularly intended for the treatment and prevention of a pathologic condition involving neurological injury. Such condition may be any one of acute or traumatic brain injury, stroke, brain anoxia, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgery intervention, hypoxia-induced nerve cell damage such as hi cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

As shown by the following Examples, glucagon significantly induces gluconeogenesis and thereby reduction of circulating levels of lactate and excitatory amino acids levels. Without being bound by any theory, reduction of lactate levels may lead, at least in part, to the observed protective effect of glucagon on head trauma and conditions involving neuronal damage. It should be therefore appreciated that the present invention further provides the use of a therapeutically effective amount of a gluconeogenesis inducing agent, preferably, glucagon, in the preparation of a pharmaceutical composition for the treatment or prevention of a pathologic condition involving neurological injury or damage.

The invention further provides the use of a therapeutically effective amount of a gluconeogenesis inducing agent in the preparation of a pharmaceutical composition for reducing levels of circulating lactate in a subject in need thereof.

According to one embodiment, the use of a gluconeogenesis inducing agent may be for the preparation of a composition for the treatment of a subject suffering of pathologic condition involving neurological injury or damage, and also for reducing levels of circulating lactate in said subject.

According to a specifically preferred embodiment, the invention relates to the use of gluconeogenesis inducing agent, preferably, glucagon, any functional fragment or derivative thereof, glucagon agonist or any combination and mixture thereof.

Gluconeogenesis is the pathway by which non-hexose substrates such as lactate, glycerol and amino acids are converted to glucose. As such, it provides another source of glucose for blood. Gluconeogenesis occurs mainly in the liver with a small amount also occurring in the cortex of the kidney. The starting point of gluconeogenesis is pyruvic acid, although oxaloacetic acid and dihydroxyacetone phosphate also provide entry points. The source of pyruvate and oxaloacetate for gluconeogenesis is mainly lactate and catabolism of certain amino acid. Some amino acids are catabolized to pyruvate, oxaloacetate, or their precursors, and in some conditions such as fasting, diabetes or starvation, even muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs. Therefore, the inventors hypothesized that induction of gluconeogenesis may recruit lactate and reduce its levels in the circulation and thereby may prevent pathologic conditions caused or enhanced by accumulation of lactate.

Global Control in liver cells includes reciprocal effects of a cyclic AMP cascade, triggered by the hormone glucagon when blood glucose is low. Phosphorylation of enzymes and regulatory proteins in liver by Protein Kinase A (cAMP-Dependent Protein Kinase) results in inhibition of glycolysis and stimulation of gluconeogenesis, making glucose available for release to the blood.

According to one embodiment, the substance used by the method of the invention may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

According to one preferred embodiment, pathologic condition involving neurological injury or damage may be any one of acute or traumatic brain injury, neurosurgical intervention, brain anoxia, stroke, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

More particularly, the method of the invention may be specifically applicable for the treatment and prevention of ischemic damage such as stroke. It is well established that tissue damage results from ischemia (stoppage of blood flow to the tissue) followed by reperfusion of the tissue. The ischemic injury with the consecutive reperfusion is responsible for the disturbance of microcirculation with ensuing tissue damage and organ dysfunction. One well-known example of ischemia and its effects is stroke, which is a condition resulting from a reduction or blockage of blood flow to the brain (cerebral ischemia). Symptoms of stroke include weakness, numbness or paralysis of the face, arm or leg, sudden loss or dimness of vision, loss of speech or difficulty using or understanding language, sudden, severe unexplained headache, or unexplained dizziness, coma unsteadiness or sudden falls.

According to another preferred embodiment, the method of the invention may be applicable for the treatment of any neurological disease or neurodegenerative disorder such as for example, Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, human immunodeficiency virus (HIV) -associated dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and glaucoma.

A "neurological disorder" is a disease or disorder characterized by an abnormality or malfunction of neuronal cells or neuronal support cells. The disorder can affect the central and/or peripheral nervous system. Exemplary neurological diseases include neuropathies, skeletal muscle atrophy and neurodegenerative diseases.

"Neurodegenerative disorders" are complex and pernicious diseases, their onset is followed by progressive deterioration. Clinical manifestations are determined by the location and seriousness of the disorder. Although the causes may differ, patients with neurodegenerative disorders are likely to show localized to generalized atrophy of brain cells, leading to compromises in both mental and physical function. Exemplary neurodegenerative diseases include: Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), Huntington's disease, taupathies such as Pick's disease, fronto temporal dementia, cortico-basal degeneration and progressive supranuclear palsy and Spongiform encephalopathies such as Scrapie, mad cow disease and Bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Fatal Familial Insomnia, Gerstmann-Straussler-Scheinker syndrome and Kuru.

Mentally, patients may exhibit forgetfulness, poor memory, decrease in mental capacities, emotional disturbances, and/or poor speech. Physically, patients may exhibit partial to complete incontinence, aspiration of food particles, tremor, poor balance, muscle rigidity, and/or muscle paralysis. In neurosurgury interventions, administration of the glucagon or insulin prior and/or post to operation can be utilized to substantially reduce brain lactate levels thereby substantially reducing the risk of its deleterious effects on brain tissue. As shown by Figure 6B, glucagon exhibited efficient preventing effect when administered 10 minutes prior to trauma induction. Furthermore, glucagon exert a therapeutic effect when is given after the brain insult. Therefore, it should be appreciated that the method of the invention may be applicable also as a preventive treatment in conditions such as open-heart surgery and coronary artery bypass surgery grafting and neurosurgery interventions.

According to one specifically preferred embodiment, the substance used by the method of the invention may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof.

In yet another embodiment, preferred therapeutically effective amount of glucagon administered by the method of the invention may range from about O.lμg/kg to about lmg/Kg body weight, preferably, 250μg/kg. This effective amount of glucagon is preferably comprised within a dosage unit form.

In yet another alternative embodiment, the substance used by the method of the invention may be any on of insulin, any functional fragment, analogue or derivative thereof, or any combination or mixture thereof.

Therefore, according to one specifically preferred embodiment, the therapeutically effective amount of insulin used by the method of the invention may be in a dosage unit form comprising from about 0.1U to about 1OU insulin per IKg of body weight, preferably, lU/kg.

According to another preferred embodiment, the dosage unit form used by the method of the invention may be either for a single or for repeated administration. According to one preferred embodiment, administration of said dosage unit form is repeated every one to eight hours for a therapeutically sufficient period of time. According to another preferred embodiment, the dosage unit form is a sustained-released dosage unit form which provides a continues pH independent drug release for a considerable period of time after administration.

The efficacy and safety profile of glucagon recommends its use, rather than that of insulin, by first aid providers in cases of brain insult such as TBI or stroke. Concerning the therapeutic window, in humans, cerebral hyperglycolysis (the source of lactate) persists for approximately one week after brain injury [Bergsneider, M. et al. J Neurosurg. 87:803 (1997)] compared to the 30 min described in rodents [Yoshino, A. et al. Brain Res. 561:106 (1991)]. The elevation in the extracellular concentrations of excitotoxic amino acids in human TBI and stroke is also more sustained (6 h [Davalos, A. et al. Stroke 28:708 (1997)] to 6 days [Bullock, R. et al. Stroke 25:(1995)]) than in rodents (70 min) [Takagi (1993) ibid.]). Therefore, the inventors anticipate that the therapeutic window of glucagon in clinical practice will be longer (6 h to one week) than in the experimental model used by the present invention (< 3 h). This may permit a far greater number of patients to be treated with few, if any, side effects. Therefore, it should be noted that administration of a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, may be performed within a period of between about 1 minute to about two weeks post neurological injury. More specifically, the substance used by the method of the invention, specifically, glucagon, insulin or any combination or mixture thereof may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 and 60 minutes after neurological injury occurred. In yet another embodiment, the substance, glucagon or insulin used for the method of the invention may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hr after occurrence of the neurological injury. According to another embodiment, the substance, e.g. glucagon or insulin used by the method of the invention may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days post neurological injury.

According to a specifically preferred embodiment, such substance, e.g., glucagon or insulin, may be administered within a period of between abut 10 minutes to about 3 hours post neurological injury.

According to another specifically preferred embodiment, specifically were the treated subject is a human subject, the substance , e.g., glucagon or insulin, may be administered within a period of between about 6 hours to about 6 days post neurological injury.

According to another embodiment, the method of the invention may include any mode of administration, for example, intravenous, intraarterial, oral, intraoral, intramuscular, intracerebral, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical or rectal administration, or any combination thereof.

As described herein above, the invention provides methods for the treatment of pathologic condition. The terms "treat, treating, treatment" as used herein and in the claims mean ameliorating one or more clinical indicia of disease activity in a patient having a pathologic condition involving neuronal injury or a neurodegenerative disease.

"Treatment" refers to therapeutic treatment. Those in need of treatment are mammalian subjects suffering from any pathologic condition involving neuronal injury or a neurodegenerative disorder. By "patient" or "subject in need" is meant any mammal for which administration of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, specifically any one of glucagon, insulin or any mixture and combination thereof, or any pharmaceutical composition comprising this compound or derivatives thereof is desired, in order to prevent, overcome or slow down such infliction. "Mammal" or "mammalian" for purposes of treatment refers to any animal classified as a mammal including, human, research animals, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In a particular embodiment said mammalian subject is a human subject.

As shown by Example 5, the beneficial effect of glucagon and insulin is due to reduction of circulating levels of lactate and excitatory amino acids. Moreover, it has been shown by the present invention that the neuroprotective beneficial effect is not related to blood glucose levels. Therefore, it should be appreciated that the method of the invention which uses either insulin, glucagon or any combination or mixture thereof, may be applicable for the treatment and prevention of a pathologic condition involving neurological injury, in a diabetic subject as well as in non-diabetic subject in need thereof. According to one specific embodiment, the invention relates to the use of glucagon and or insulin for the treatment and prevention of a pathologic condition involving neurological injury, in a mammalian subject suffering of diabetes. According to another specifically preferred embodiment, the invention relates to methods and compositions using glucagon and or insulin for the treatment and prevention of a pathologic condition involving neurological injury, in a mammalian subject which is not suffering of diabetes, more specifically, a non-diabetic subject.

To provide a "preventive treatment" or "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease. It should be noted that the preventive effect of glucagon, is clearly demonstrated in Example 4 and Figure 6B. This Example shows that treatment with glucagon prior to induction of head trauma, significantly improves neurological recovery.

The terms "effective amount" or "sufficient amount" of the substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids e.g., glucagon or insulin used by the method of the invention, mean an amount necessary to achieve a selected result. The "effective treatment amount" is determined by the severity of the disease in conjunction with the preventive or therapeutic objectives, the route of administration and the patient's general condition (age, sex, weight and other considerations known to the attending physician). The method of the invention should be applied to a subject suffering from a pathologic condition involving neuronal injury or a neurodegenerative disorder. As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein.

Without being bound by any theory, glucagon may exert it's beneficial effect on neuronal damage through induction of gluconeo genesis, which leads to reduction of lactate levels. Thus, the invention further provides a method for the treatment and for the prevention of a pathologic condition involving neurological injury or damage by increasing and enhancing gluconeogenesis. The method of the invention comprises the step of administering to a subject in need thereof, a therapeutically effective amount of a gluconeogenesis inducing agent, thereby reducing levels of blood lactate levels, as well as the circulating levels of excitatory amino acids.

According to another specifically preferred embodiment, the method of the invention may use as a gluconeogenesis inducing agent, glucagon, any functional analog, variant, derivative or fragment thereof, any glucagon agonist or any combinations or mixtures thereof. Therefore, according to another specifically preferred embodiment, the gluconeogenesis inducing agent used by the method of the invention may be glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof or adrenalin. The invention preferably relates to the use of glucagon as a preferred gluconeogenesis inducing agent.

Preferred embodiment relates to the use of a therapeutically effective amount which preferably being in a dosage unit form comprising from about O.lμg to about lmg glucagon per IKg of body weight, preferably, 250μg/kg. According to another embodiment, administration of such dosage unit form may be repeated every one to eight hours for a therapeutically sufficient period of time. Alternatively, the active ingredient, glucagon may be in a sustained- released dosage form enabling continues release of the preferred effective amount of glucagon for a therapeutically sufficient period of time.

Still further, the administration step in any of the methods provided by the invention may include any mode of administration, including intravenous, oral, intramuscular, intracerebral, subcutaneous, intraperitoneal administration, or any combination thereof.

The agent, preferably glucagon, utilized by the method of the present invention may be administered to an individual subject per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier to create a pharmaceutical composition.

Likewise, the present invention also provides a method for reducing levels of circulating lactate and/or excitatory amino acids. This method comprises the step of administering to a subject in need thereof, a therapeutically effective amount of a substance which, reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, preferably of glucagon or insulin, thereby reducing levels of circulating lactate and/or excitatory amino acids.

According to one embodiment, the method of the invention is intended for reducing levels of circulating lactate in a subject suffering of a pathologic condition involving neuronal injury or damage. Such pathologic condition may be any one of acute or traumatic brain injury, brain anoxia, stroke, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgury intervention, hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

According to another particular embodiment, the method of the invention may be used for reducing levels of circulating lactate in a subjects suffering of a neurodegenerative disorder such as Alzheimer's Disease, Parkinson's Disease, human immunodeficiency virus (HIV)-associated dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and glaucoma.

In yet another embodiment, the gluconeogenesis inducing agent may be glucagon, any functional fragment or derivative thereof, any glucagon agonist, any mixture or combination thereof.

In a further aspect, the invention relates to a pharmaceutical composition for the treatment of a pathologic condition involving neurological injury or damage. The composition of the invention comprises as an active ingredient a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, and optionally any further pharmaceutically acceptable carrier, diluent, excipient and/or additive.

According to one specifically preferred embodiment, the substance used for the composition of the invention may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

It will be appreciated that the pharmaceutical compositions and administration routes described hereinabove are preferably used in treating stroke, brain anoxia, brain ischemia, perinatal brain damage, traumatic head injury, bacterial meningitis, subarachoid haemorhage, migraine, stress, hemorrhagic shock, epilepsy, open heart surgery, aneurysm surgery, coronary artery bypass grafting.

According to another specific embodiment, the compositions of the invention may be applicable for subjects suffering of a neurodegenerative disorder such as Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, human immunodeficiency virus (HIV) -associated dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and glaucoma.

It should be appreciated that the invention further encompasses the combination of glucagon, insulin or any combination or mixture thereof, with any other known therapeutic agent that is used for treating pathological conditions involved in neuronal damage. As indicated herein above, the pharmaceutical composition of the invention may comprise any pharmaceutically acceptable carrier, diluent, excipient and/or additive. Hereinafter, the phrase "pharmaceutically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under this phrase. One of the ingredients included in the pharmaceutically acceptable career can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media

Herein the term "excipient" refers to an inert material added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

Suitable routes of administration of the pharmaceutical composition of the present invention may, for example, include intravenous, intra cerebral, oral, rectal, transmucosal, transnasal, intraartial, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections. Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body or by direct administration to brain tissues (e.g. topical) during, for example, open brain surgery.

When continuous administration is required a continuous drug release is preferred provided that endogenous production in the depleted organ does not occur. Therefore, as indicated above, the composition of the invention may comprise a sustained-release dosage unit form.

The advantages of sustained release formulations are well known in the pharmaceutical field. These include the ability of the given pharmaceutical preparation to maintain a desired therapeutic effect over a comparatively longer period of time, reduced side effects, etc. Moreover, for drugs having a short elimination half-life, less frequent administration and better patient compliance may be obtained with sustained release preparations as compared to the conventional dosage forms.

As used herein, the term "formulations" refers to compounds, compositions, and dosage unit forms, such as, for example, immediate release and sustained release dosage forms.

It should be appreciated that the present invention further provides methods and processes of administering a dosage form, compound or composition of the present invention to a mammal, e.g., to a human. In some embodiments, the dosage unit forms, compounds, or compositions may be orally administered. In one aspect, they are orally administered once every 1 to 8 hours. In some particularly preferred embodiments, the dosage unit forms are sustained-released forms which enable release of the active ingredient for a considerable period of time.

It should be noted that the present invention further provides a composition (e.g., a pharmaceutical composition) comprising at least one delivery agent compound and glucagon, insulin or both. Preferably, the composition includes a therapeutically effective amount of glucagon, insulin or both and a delivery agent compound which enables a sustained-release of glucagon for a considerable period of time. The composition of the present invention may facilitate the delivery of glucagon or insulin and increases its bioavailability compared to administration without the delivery agent. An "effective amount of delivery agent" is an amount of the delivery agent which enables and/or facilitates the absorption of a desired amount of glucagon or insulin via any route of administration (such as those discussed in this application including, but not limited to, intravenous, intra cerebral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections, oral (e.g., across a biological membrane in the gastrointestinal tract), nasal, pulmonary, dermal, intraoral, vaginal, ocular route and any combinations thereof).

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, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. As indicated above, pharmaceutical compositions for use in accordance with the present invention 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 active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Pharmaceutical compositions for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

For oral administration, the compounds can be formulated readily by combining the active compounds 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 oral 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). Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

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.

Accordingly, the invention further encompasses glucagon or insulin dosage forms that can be intraorally administered. The terms "intraoral administration" and "intraorally administering" include administration by adsorption through any surface inside the mouth or upper throat (such as the cheek (e.g., the inner cheek lining), gums, palate, tongue, tonsils, periodontal tissue, lips, and the mucosa of the mouth and pharynx). These terms, for example, include sublingual and buccal administration.

The administration compositions may alternately be in the form of a solid, such as a tablet, capsule or particle, such as a powder or sachet. Solid dosage forms may be prepared by manually or physically blending the solid form of the delivery agent compound with the solid form of glucagon or insulin. Alternately, a solid may be obtained from a solution of the delivery agent compound and glucagon or insulin by methods known in the art, such as freeze- drying (lyophilization), precipitation, crystallization, air drying and solid dispersion. For administration by nasal inhalation, the active ingredient for use according to the present invention, glucagon or insulin, may 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 a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based 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 active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition 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.

Thus, the pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self- emulsifying solids and self-emulsifying semisolids.

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 active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those-skilled in the art. For any pharmaceutical composition used by the treatment method of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from the 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.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state or symptoms is achieved.

The amount of the pharmaceutical 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, etc.

As indicated above, said therapeutic effective amount, or dosing, is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual peptide such as glucagon or insulin, and can generally be estimated in in vitro as well as in in υiυo animal models.

According to one specific embodiment, where the active ingredient is glucagon, dosage is from O.lμg/kg to about lmg/Kg body weight, preferably, 250μg/kg. According to another specific embodiment, where the active ingredient is insulin, dosage is from 0.1U/kg to about 10U/Kg body weight, preferably, lU/kg.

According to another embodiment, the dosage unit form may be for a single or for repeated administration. In general, the dose may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years, preferably, repeated every one to eight hours or more (up to bout two weeks) for a therapeutically sufficient period of time. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of glucagon or insulin, in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state. Wherein the glucagon is administered in maintenance doses, ranging from about O.lng/kg to about lmg/Kg body weight, preferably, 250μg/kg, of body weight, once or more daily. Alternatively or additionally, wherein the insulin is administered in maintenance doses, ranging from about 0.01U/kg to about 10U/Kg body weight, preferably, 0.1U/kg, of body weight, once or more daily.

It should be noted that the invention further provides a pharmaceutical composition for the treatment of a pathologic condition involving neurological injury or damage. The composition of the invention comprises as an active ingredient a therapeutically effective amount of a gluconeogenesis inducing agent and optionally any further pharmaceutically acceptable carrier, diluent, excipient and/or additive.

Still further, the invention provides a pharmaceutical composition for reducing levels of circulating lactate in a subject in need thereof, comprising as an active ingredient a therapeutically effective amount of a gluconeogenesis inducing agent, and optionally any further pharmaceutically acceptable carrier, diluent, excipient and/or additive.

According to one embodiment, the compositions of the invention are intended for prevention, treatment and reducing lactate levels in a subject suffering of pathologic condition involving neurological injury or damage.

In another preferred embodiment, the gluconeogenesis inducing agent used by the compositions of the invention may be glucagon, any functional fragment or derivative thereof, glucagon agonist or any combination or mixture thereof.

As indicated herein before, the invention further encompasses the use of a combination of at least two different substances which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, specifically, glucagon and insulin, for preparing a medicament for treating or preventing a pathologic condition involving neurological injury.

The combined substances of the present invention (e.g., glucagon and insulin) may generally administered in the form of a pharmaceutical composition comprising both substances of this invention together with a pharmaceutically acceptable carrier or diluent. Thus, the substances used by this invention can be administered either individually in a kit or together in any conventional oral, parenteral or transdermal dosage form.

More particularly, since the present invention relates to the treatment of diseases and conditions with a combination of active ingredients which may be administered separately, the invention also relates as a further aspect, to combining separate pharmaceutical compositions in kit form.

The kit according to the invention includes two separate pharmaceutical compositions: at least one of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a first unit dosage form and at least one of insulin, any functional fragment or derivative thereof, or any combination or mixture thereof and a pharmaceutically acceptable carrier or diluent in a second unit dosage form. The kit further includes container means for containing both separate compositions; such as a divided bottle or a divided foil packet however, the separate compositions may also be contained within a single, undivided container. Typically the kit includes directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual oomponents of the combination is desired by the prescribing physician.

Achieving a therapeutic effect is meant for example, where the kit is intended for the treatment of pathologic condition involving neurological injury, the therapeutic effect may be for example slowing the progression of such condition.

It should be noted that combination or mixture of at least two substances which reducing the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, e.g., glucagon and insulin, may be either synergistic or additive. It should be noted that such combinations may also be used for the treatment of subjects presenting with symptoms or signs of such disorders.

By synergic combination is meant that the effect of both glucagon and insulin is greater than the sum of the therapeutic effects of administration of any of these substances separately, as a sole treatment.

According to one embodiment, the kit is particularly intended for the treatment of a subject suffering from a pathologic condition involving neurological injury selected from acute or traumatic brain injury, stroke, brain anoxia, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgery intervention, hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder. Accordingly, the invention further provides a method for the treatment of a pathologic condition involving neurological injury comprising the step of administering to a subject in need thereof a therapeutically effective amount of a first and a second unit dosage forms comprised in the kit of the invention.

Still further, the invention provides a method for preventing or reducing the risk of developing a pathologic condition involving neurological injury, comprising the administration of a prophylactically effective amount of a first and a second unit dosage forms comprised in the kit of the invention, to a person at risk of developing said pathologic condition.

It should be appreciated that both components of the kit, the glucagon in the first dosage form and the insulin in the second dosage form may be administered simultaneously.

Alternatively, said first compound or dosage form and said second compound or dosage form are administered sequentially in either order.

The invention further provides a method for making a medicament for the treatment of a pathologic condition involving neurological injury or damage. Accordingly, the method of the invention comprises the step of: (a) providing a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids; (b) admixing said substance with at least one of a pharmaceutically acceptable carrier, diluent, excipient and/or additive. According to a specifically preferred embodiment, the substance used by the method of the invention may be any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

According to another embodiment, the composition made by the method of the invention may be used by a subject suffering of a pathologic condition involving neurological injury or damage, for example, acute or traumatic brain injury, brain anoxia, stroke, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgery intervention, hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder. More particularly, such neurodegenerative disorder may be any one of Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, human immunodeficiency virus (HFV)-associated dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and glaucoma.

According to one embodiment, where glucagon is used, preferred therapeutically effective amount used may be from about O.lμg/kg to about lmg/Kg body weight, preferably, 250μg/kg, preferably, said effective amount being encompassed in a dosage unit form. Alternatively or additionally, wherein the insulin is used, preferred therapeutically effective amount used may be from about O.lU/kg to about 10U/Kg body weight, preferably, lU/kg, of body weight, once or more daily. According to another preferred embodiment, administration of such dosage unit form may be repeated every one to eight hours for a therapeutically sufficient period of time. Alternatively, said effective amount, comprised within a sustained-release dosage unit form which enables continues release of the preferred effective amount of the active ingredient for a sufficiently therapeutically period of time after administration.

The present invention is defined by the claims, the contents of which are to be read as included within the disclosure of the specification.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The following Examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the intended scope of the invention.

Examples

Experimental Procedures

Trauma Model, Closed Head Injury (CHI)

All studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee of the Hebrew University. C57 black male mice (12- weeks old) were fed on regular chow, allowed free access to drinking water and kept under controlled temperature and lighting conditions. CHI was induced using a weight-drop device, as described in detail elsewhere [Chen, Y. et al. Neurotrauma 13:557 (1996)]. Briefly, after induction of anesthesia, a midline longitudinal incision was performed, the skin was retracted and the skull was exposed. The left anterior frontal area was identified and a Teflon tipped cone (2- mm diameter) was placed 1 mm lateral to the midline in the midcoronal plane. The head was held in place manually and a 75-g weight was dropped on the cone from a height of 18 cm, resulting in focal injury to the left hemisphere. Saline or saline containing 5 μg of glucagon or insulin 1 u/kg was injected IP before or after the CHI, as indicated. Neurobehaυioral Evaluation

The neurological severity score (NSS) is a 10-point scale that assesses the functional neurological status of mice based on the presence of various reflexes and the ability to perform motor and behavioral tasks such as beam walking, beam balance, and spontaneous locomotion [Beni-Adani, L. J. Pharmacol. Exp. Ther. 296:57-63 (2001)]. Animals were awarded one point for failure to perform one item, so that scores can range from zero (healthy uninjured animals) to a maximum of 10, indicating severe neurological dysfunction, with failure to perform all tasks. The NSS obtained 1 h after trauma reflects the initial severity of injury and is inversely correlated with the neurological outcome. Animals were evaluated 1 h after CHI, as well on average of every two days until 28 days later. Each animal was assessed by an observer who was blinded to the treatment. The extent of recovery (dNSS) was calculated as the difference between the NSS at 1 h and at any subsequent time point. Thus, a positive dNSS reflects recovery, zero reflects no change, and a negative dNSS reflects neurological deterioration. Animals were evaluated 3 h after CHI, and periodically thereafter, as indicated in the figures, up to 28 days.

Lesion volume

After the last neurobehavioral evaluation on day 28, the mice were euthanized with Isoflurane (Rhodia organique fine Ltd., Bristol, UK.) and the brains were removed and sectioned into 1-mm coronal segments. Brain slices were immersed in 4% formalin/PBS overnight. The sections were photographed and the area of each section was determined using the NIH computer image analysis program. Any missing area was estimated by overlaying the affected section with images from the contralateral side from the same animal. The volume of the missing portion of the brain was defined as the sum of the missing areas in all sections multiplied by their thickness expressed in cubic millimeters (mm³). The volume of each hemisphere was determined in a similar manner. Data are presented as mean ± SE. Differences were analyzed by the t-test and the level of significance was corrected using a post-hoc analysis with the Bonferroni test. Statistical significance was set at P<0.05.

Quantification of apoptosis

Detection and quantification of apoptosis at the single cell level was performed using a commercial kit (Roche Diagnostics, Mannheim, Germany, Catalogue No. 11684 795 910). Brains were removed 24 hours after CHI, fixed in 4% paraformaldehyde overnight, dehydrated, and embedded in paraffin (Fisher Scientific, Montreal, QC, Canada). To perform the TUNEL assay, paraffin-embedded brain sections (4 μm) were soaked in toluene to deparaffinize, then rehydrated by sequential addition of alcohol in graded concentrations (100% to 70%). To enzymatically digest formalin-fixed tissue, the sections were preincubated for 30 min at RT in 15 μg/ml proteinase K/10 mM Tris/HCl. Terminal deoxynucleotidyl transferase (Boehringer Mannheim, QC, Canada) diluted in TUNEL reaction mixture was added to the slides for 10 min at 37⁰C. After rinsing with PBS, histological examinations were conducted using fluorescence microscopy. The estimation of TUNEL-positive cells was carried out by cell counts from up to 14 sites around the lesion (Figure 8). These sites were viewed at 10x and then magnified to 2Ox and captured and counted with Image pro plus 6.0 Software. Results are shown as the ratio of TUNEL-positive cells to the total number of cells per area sampled. Data are presented as mean ± SE. Differences were analyzed using the t-test and the level of significance was corrected using a post-hoc analysis with the Bonferroni test. Statistical significance was set at P<0.05. Activation of gluconeogenesis by glucagon

Mice were anesthetized Isoflurane (Rhodia organique fine Ltd., Bristol, UK.) A blood sample was taken from the tail of 20 naϊve animals to determine basal glucose levels using a manual glucometer. The animals were then randomized into two groups that were given an IP injection of saline or saline containing glucagon (5 μg). Fifteen min later, plasma glucose, lactate and amino acids were measured on a second blood sample taken by cardiac puncture.

Blood lactate and amino acids

Mice were anesthetized as above. Fifteen min after injection of glucagon, blood was withdrawn to measure plasma glutamate, glutamine, aspartate, alanine and lactate. The plasma was separated immediately in a refrigerated centrifuge and deproteinized with sulfosacylic acid. Amino acid concentrations were measured on a Bio-Chrom 20 amino acid analyzer. Lactate and glucose concentrations were asayed on a Kodak analyzer. Lactate is in equilibrium with pyruvate through the activity of lactate dehydrogenase and pyruvate is in equilibrium with alanine through the activity of alanine aminotransferase. Therefore, where specified, changes in the concentration of plasma lactate were confirmed by following the changes in the concentration of alanine.

More particularly, the concentrations of glucose were determined using a manual glucometer. To determine the basal levels of glucose, blood was withdrawn from the tail 15 min before i.p. injection of glucagon. To evaluate the effect of glucagon, another blood sample was withdrawn 15 min after glucagon administration for analysis of plasma glucose and lactate. To determine alanine concentration the plasma was immediately separated in a refrigerated centrifuge and deproteinized with sulfosacylic acid. Concentration of amino acids was determined on a Bio-Chrom 20 amino acid analyzer.

Diabetic mice

Six-week-old male C57BL mice were used in this study. Animals were purchased from Hebrew university animal facility, and housed in a temperature-controlled room on a 12-h light-dark cycle. Mice were randomly placed on a normal fat (NF) (n=30) or a HF diet (n=30), corresponding to 10% or 55% of the calories from fat (Harlan Teklad, Madison, WI), for six weeks and provided with water ad libitum; this HF diet has been shown to induce diabetes in C57BL/6 mice [Park, S. et al. Diabetes 3530-3540 (2005)]. Food intake monitored throughout the study was almost identical in the two groups. Body weights were measured weekly. At the end of 6 weeks the group fed the HF diet had gained more weight than the controls (27.5±0.63 vs. 24.8+0.39 grams, respectively) (P < 0.01). Fasting blood glucose was monitored weekly using an Elite Glucometer (Bayer, Mishawaka, IN). At the end of the 6 weeks the group fed the HF diet had showed significant increased fasting blood glucose levels compared the controls (5.28±3.33 vs. 8.5±6.11 m mol/L respectively) (P < 0.01).

Glucose tolerance tests: To further characterization of the diabetic mice a glucose tolerance tests were performed as described by others [Sandu, O. et al. Diabetes 54: 2314-2319 (2005)]. The inventors found that HF mice displayed impaired glucose response during glucose tolerance tests (not shown), which was markedly different from the control NF group (P < 0.01). Example 1

Neuroprotective effect of glucagon after head trauma

The inventors examined the potential neuroprotective effect of glucagon on neurological damage caused for example by head trauma. Therefore, experimental closed head trauma was induced as described by experimental procedures and mice were injected (IP) with 5 μg glucagon 10 minutes before (Figure IA) or 10 minutes after (Figure IB) induction of head trauma. Neurological evaluation was performed for each group. As clearly demonstrated by both Figures IA and IB, injection of glucagon improved the neurological recovery in mice, compared to animals treated with saline alone (controls). The significant beneficial effect of glucagon was demonstrated even when glucagon was administered prior to induction of trauma. It should be further noted that the neurological severity score (NSS) was significantly lower in glucagon treated animals. These preliminary results clearly demonstrate the protective and preventive as well as the beneficial effect of glucagon on neurological recovery after head trauma, and therefore illustrates that glucagon may be used for the treatment of stroke, head trauma and neurodegenerative disorders.

Encouraged by these results, the inventors next performed detailed analysis examining the neuroprotective effect of glucagon for a longer period of time, and for additional parameters. Therefore, saline samples or saline containing 5μg glucagon samples were injected IP 10 minutes after subjecting mice to controlled closed head injury (CHI). Three hours later, there was no significant difference in the neurological severity score (NSS) between the glucagon and saline treated mice (Figure 2). However, when the mice were repetitively tested over the succeeding 4 weeks, recovery was more rapid and more complete in the glucagon-treated animals. As shown by Figure 2 A, the difference was apparent by 3 days and reached statistical significance by one week post-CHI. The better neurological function stabilized at about 3 weeks and remained so for the duration of the observation period (4 weeks), by which time the NSS was 2.38 in the treated group compared to 5 in the controls (p = 0.0026, Figure 2B).

To further investigate the beneficial neuroprotective effect of glucagon, the inventors next examined the effect of glucagon on the extent of brain damage induced by CHI. Four weeks after CHI, after the last neurological evaluation, the animals were sacrificed, brains were extracted, evaluated macroscopically (Figure 3A), cut into slices (Figure 3B) and the volume of each hemisphere was measured. Volume lost as a result of trauma and cell death was calculated using these results as illustrated by Figure 3C. As shown by the histogram, brain damage was less extensive in glucagon-treated animals than in controls by about 57% (P = 0.0003; Figure 3C). Similar results were seen when the areas of the two hemispheres were compared where the size of the affected hemisphere was smaller by about 15.7% (P=COl).

Example 2

Gluconeogenic effect of glucagon

Without being bound by any theory, the inventors hypothesized that the neuroprotective effect of glucagon may be due to a reduction in the levels of lactate and excitatory amino acids that cause apoptosis after cerebral trauma [Hara, M.R. and Snyder, S.H., Annu. Rev. Pharmocol. Toxicol 47:117 (2007)]. To test this hypothesis, the effect of glucagon on gluconeogenesis was studied. The effect of glucagon on gluconeogenesis has been examined by measuring circulating glucose levels and clearance of gluconeogenesis precursors from the circulation.

As clearly shown by Figure 4A, IP injection of 5μg glucagon to mice caused a significant increase in the level of glucose in the circulation. Examination of circulating alanine levels showed clear decrease (demonstrated by Figure 4B), which reflects similar decrease in the levels of circulating lactate. Figure 4C demonstrate a decrease in the level of glutamate in the circulation, in response to IP injection of 5μg glucagon to mice.

More detailed analysis of glucagon effect on gluconeogenesis is represented by the results of Table 1. Ten minutes after IP injection of saline or saline containing 5μg of glucagon, blood glucose increased from 4.94 ± 0.372 mmol/L to 7.61 ± 0.88 (P < 0.001, Table 1), concomitantly, the single injection of glucagon significantly decreased the plasma concentrations of glutamate, glutamine, aspartate, alanine, and lactate (Table 1), compared with animals given saline alone, which had no significant effect (not shown).

Table 1: effect of glucagon on gluconeogenesis

Example 3

Effect of glucagon on post CHI appoptosis

In view of these results, and the observed effect of glucagon in reducing brain damage in response to CHI (Figure 3), the inventors next examined the effect of glucagon on post CHI apoptosis. Animals were treated with glucagon (5 μg) or with saline 10 min after CHI and the brains were extracted 24 hours later to quantify apoptosis, measured by using a TUNEL assay (Figure 5A). As clearly shown by Figure 5A and illustrated by the histogram of Figure 5B, injection of glucagon decreased apoptosis by more than 70% (P= 0.0001).

Example 4

Temporal window of glucagon-mediated neuroprotection post-CHI

The metabolic changes that appear in the brain after trauma are transient. For example, after occlusion of the middle cerebral artery in rats, extracellular levels of glutamate increase, reach a peak about 30 min after the onset of ischemia and return to near baseline levels by 70 min, notwithstanding persistent vascular occlusion [Takagi, K. et al. J Cereb Blood Flow Metab. 13:575 (1993)]. Furthermore, rats have increased local cerebral metabolic rates for glucose (LCMRgIc), the source of lactate, immediately after head trauma, which revert to normal by 30 min [Yoshino, A. et al. Brain Res. 561:106 (1991)]. Therefore, the inventors next examined the relationship between the effect of glucagon and the time curve of increased glucose utilization by the brain, and the release of the excitatory amino acid glutamate. Glucagon was administered at 10 min (as in Figure 2) or at 3 hours after CHI. In contrast to the neuroprotective effect seen when glucagon was given 10 min after CHI (Figure 2 and 6A), no effect on neurological outcome was seen when glucagon was given 3 hours after CHI, as clearly demonstrated by the histogram of Figure 6A. To exclude the possibility that the lack of effect of glucagon at 3 h results from a deleterious effect that offsets a beneficial effect, glucagon was given 10 minutes and 3 hours after CHI. The injection at 3 hours did not reduce the beneficial effect of the glucagon injection at 10 min post CHI (Figure 6A).

Preconditioning effect of glucagon

To examine the possibility that activation of gluconeogenesis participates in the preconditioning response to head trauma, saline or saline containing 5 μg glucagon was injected IP 10 min prior to CHI. Repeated neurological testing over the succeeding 4 weeks showed that the neurological recovery in animals treated 10 min before head trauma was significantly faster and more complete than in control mice (Figures 6B and 6C).

Example 5

Neuroprotective effect of insulin

The data of the present invention strongly suggest that the capacity of glucagon to decrease the concentrations of excitatory amino acids and lactate in head trauma is neuroprotective. Insulin which antagonizes the effect of glucagon on gluconeogenesis has for a long time been known to have similar effects on the clearance of amino acids in diabetic subjects [Luck (1928) ibid.] and lactate from the circulation [Brockman (1975) ibid.]. The inventors have therefore examined the possibility that insulin, like glucagon, has a neuroprotective effect.

To examine the potential neuroprotective effect of insulin, non-diabetic mice were given insulin (lu/kg), glucagon (5 μg) or saline IP 10 min after CHI. The mice were tested repetitively over the succeeding 4 weeks. As clearly shown by Figure 7, similarly to treatment with glucagon (Figure 2), more rapid recovery was apparent in the insulin or glucagon treated animals by 3 days and the difference reached statistical significance by one week post- CHI (Figure 7A); the pattern of the improvement in neurological function in both groups was almost the same, stabilized at about 3 weeks and remained so for the duration of the observation period, by which time the NSS was 1.71 (SE + 0.62) in insulin- and 1.9 (SE ± 0.57) in the glucagon- treated groups, compared to 4.2 (SE ± 0.71) in the controls (p = 0.0007) (Figure 7B).

To further evaluate the neuroprotective effect of insulin, the temporal window of its neuroprotective activity was determined. As clearly shown by Figure 7C, similar to glucagon effect (Figure 6A), insulin given 3 hours after CHI had no effect on the neurological outcome.

Tight control of blood glucose has been reported to improve the outcome after traumatic brain injury [Jeremitsky, E. et al. J. Trauma. 58:47 (2005)]. This finding led to the conclusion that hyperglycemia contributes to secondary brain injury and that insulin exerts a neuroprotective effect by decreasing blood glucose. To examine the hypothesis, that increased clearance of lactate and neurotoxic amino acids, rather than its hypoglycemic activity, is involved in the beneficial effect of insulin after CHI, the possible neuroprotective effect of insulin and glucagon in diabetic mice with high basal glycemia was next examined. As shown by Figure 7D, IP injection of saline containing 5 μg glucagon or lu/kg of insulin 10 min after CHI, induced a significant improvement in NSS of diabetic mice compared to saline alone. Furthermore, Figure 7D shows that the pattern and intensity of neurological recovery were similar in diabetic mice injected with either hormone; at the end of the observation period (4 weeks) the NSS was 1 (SE ± 0.4), 1.04 (SE + 0.5 ) and 4.3 (SE ± 0.7) in the insulin, glucagon or saline groups, respectively (Figure 7E). The better NSS seen in Figure 7D did not correlate with glucose levels, which were affected by insulin and glucagon in opposite directions; injection of insulin decreased blood glucose from 10.33±1.55 to 5.05±4.1 m mol/L (p= 0.005), whereas glucagon increased blood glucose from 9.67+1.23 to 13.11±18.86 m mol/L (p=0.009). In contrast to the lack of correlation between plasma glucose levels and the improvement of the NSS, the inventors found that insulin, like glucagon (Table 1) decreased plasma glutamate and lactate; injection of 1 u/kg insulin decreased the concentrations of glutamate and lactate from 270.3+43.0 to 88.6 7+36.0 (p< 0.001) and from 1319.9+119.6 to 461.1+85.2 μmol/L (p< 0.001), respectively. Significant reductions in the concentrations of alanine, glutamine and aspartate were also found in insulin treated mice; furthermore, a similar pattern for the measured amino acids and lactate was found in non diabetic mice treated with insulin (data not shown).

Claims

Claims:

1. Use of a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, in the preparation of a pharmaceutical composition for the treatment or prevention of a pathologic condition involving neurological injury.

2. The use according to claim 1, wherein said pathologic condition involving neurological injury is any one of acute or traumatic brain injury, stroke, brain anoxia, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgery intervention, hypoxia -induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

3. The use according to claim 1, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

4. The use according to claim 3, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof.

5. The use according to claims 1 and 4, wherein said therapeutically effective amount is in a dosage unit form comprising from about O.lμg to about lmg glucagon per IKg of body weight, preferably, 250μg/kg.

6. The use according to claim 3, wherein said substance is any on of insulin, any functional fragment or derivative thereof, or any combination or mixture thereof.

7. The use according to claims 1 and 6, wherein said therapeutically effective amount is in a dosage unit form comprising from about 0.1U to about 1OU insulin per IKg of body weight, preferably, lU/kg.

8. The use according to any one of claims 5 and 7, wherein said dosage unit form is for any one of a single and repeated administration.

9. The use according to claim 8, wherein said dosage unit form is for repeated administration every one to eight hours for a therapeutically sufficient period of time.

10. The use according to any one of claims 5 and 7, wherein said dosage unit form is a sustained-released dosage form.

11. A substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, for the treatment or prevention of a pathologic condition involving neurological injury.

12. The substance according to claim 11, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

13. Glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, for the treatment or prevention of a pathologic condition involving neurological injury.

14. Insulin, any functional fragment or derivative thereof, any combination or mixture thereof, for the treatment or prevention of a pathologic condition involving neurological injury.

15. A method for the treatment or prevention of a pathologic condition involving neurological injury, said method comprises the step of administering to a subject in need thereof, a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids.

16. The method according to claim 15, wherein said pathologic condition involving neurological injury is any one of acute or traumatic brain injury, stroke, brain anoxia, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgery intervention hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

17. The method according to claim 16, wherein prevention of a pathologic condition involving neurological injury comprising preoperative and/or postoperative administration of a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, to a subject in need of a neurosurgery intervention and said administration is prior to said intervention.

18. The method according to claim 15, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

19. The method according to claim 18, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof.

20. The method according to claims 15 and 19, wherein said therapeutically effective amount is in a dosage unit form comprising from about 0.1 μg to about lmg glucagon per IKg of body weight, preferably, 250μg/kg.

21. The method according to claim 18, wherein said substance is any on of insulin, any functional fragment or derivative thereof, or any combination or mixture thereof.

22. The method according to claims 15 and 21, wherein said therapeutically effective amount is in a dosage unit form comprising from about 0.1U to about 1OU insulin per IKg of body weight, preferably, lU/kg.

23. The method according to any one of claims 20 and 22, wherein said dosage unit form is for any one of single and repeated administration.

24. The method according to claim 23, wherein administration of said dosage unit form is repeated every one to eight hours for a therapeutically sufficient period of time.

25. The method according to any one of claims 20 and 22, wherein said dosage unit form is a sustained-released dosage form.

26. The method according to claim 15, wherein administration of a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, is performed within a period of between about 1 minute to about two weeks post neurological injury.

27. The method according to claim 26, wherein said substance is administered within a period of between abut 10 minutes to about 3 hours post neurological injury.

28. The method according to claim 26, wherein said substance is administered within a period of between about 6 hours to about 6 days post neurological injury.

29. The method according to claims 20 and 22, wherein said administration step comprises intravenous, intraarterial, oral, intraoral, intramuscular, intracerebral, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical or rectal administration, or any combination thereof.

30. A pharmaceutical composition for the treatment of a pathologic condition involving neurological injury, said composition comprises as an active ingredient a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids, and optionally any further pharmaceutically acceptable carrier, diluent, excipient and/or additive.

31. The composition according to claim 30, wherein said pathologic condition involving neurological injury is any one of acute or traumatic brain injury, stroke, brain anoxia, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, hypoxia- induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

32. The composition according to claim 31, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.

33. The composition according to claim 32, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof.

34. The composition according to claims 30 and 33, wherein said therapeutically effective amount is in a dosage unit form comprising from about O.lμg to about lmg glucagon per IKg of body weight, preferably, 250μg/kg.

35. The composition according to claim 32, wherein said substance is any on of insulin, any functional fragment or derivative thereof, or any combination or mixture thereof.

36. The composition according to claims 30 and 35, wherein said therapeutically effective amount is in a dosage unit form comprising from about 0.1U to about 1OU insulin per IKg of body weight, preferably, lU/kg.

37. The composition according to any one of claims 34 and 36, wherein said dosage unit form is for any one of a single and repeated administration.

38. The composition according to claim 37, wherein said dosage unit form is for repeated administration every one to eight hours for a therapeutically sufficient period of time.

39. The composition according to claim 38, wherein said dosage unit form is a sustained-released dosage unit form.

40. A kit for achieving a therapeutic effect in a subject in need thereof comprising: a. at least one of glucagon, any functional fragment or derivative thereof, glucagon agonist, or any combination or mixture thereof or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent in a first unit dosage form; b. at least one of insulin, any functional fragment or derivative thereof, or any combination or mixture thereof and a pharmaceutically acceptable carrier or diluent in a second unit dosage form; and c. container means for containing said first and second dosage forms.

41. The kit according to claim 40, wherein said subject is suffering from a pathologic condition involving neurological injury selected from acute or traumatic brain injury, stroke, brain anoxia, perinatal brain damage, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, neurosurgery intervention, hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, anxiety, and a neurodegenerative disorder.

42. A method of treatment of a pathologic condition involving neurological injury comprising the step of administering to a subject in need thereof a therapeutically effective amount of a first and a second unit dosage forms comprised in a kit according to claim 42.

43. A method for preventing or reducing the risk of developing a pathologic condition involving neurological injury, comprising the administration of a prophylactically effective amount of a first and a second unit dosage forms comprised in a kit according to claim 40, to a person at risk of developing said pathologic condition.

44. A method for making a medicament for the treatment of a pathologic condition involving neurological injury, said method comprises the step of: a. providing a therapeutically effective amount of a substance which reduces the circulating levels of at least one of lactate and neurotoxic excitatory amino acids; b. admixing said substance with at least one of a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

45. The method according to claim 44, wherein said substance is any on of glucagon, any functional fragment or derivative thereof, glucagon agonist, any combination or mixture thereof, insulin, any functional fragment or derivative thereof, any combination or mixture thereof or any combination or mixture of glucagon and insulin.