Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4853—Kallikrein (3.4.21.34 or 3.4.21.35)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

• the present invention provides methods of treating psychiatric disorders including schizophrenia, associated conditions of the schizophrenic spectrum and bipolar disorder, comprising administering tissue kallikrein (KLKl), variants or active fragments thereof.
• tissue kallikrein KLKl
• Schizophrenia is a debilitating mental illness that affects approximately 1 % of the world population at large (Lang et al, Cell Physiol Biochem., 2007, 20:687-702). This complex, chronic brain disorder displays a variety of symptoms that are classified as positive, negative, or cognitive impairment type and begin to appear in adolescence or early adulthood.
• the first drugs developed to treat schizophrenia termed typical antipsychotics, are high affinity dopamine D2 receptor antagonists which prevent excessive levels of dopamine primarily thought to be responsible for the psychotic positive symptoms of the disease.
• This group of drugs exhibits reduced extrapyramidal symptom side effects, but is hampered by other side effects, including increased weight gain and onset of diabetes (Leiberman et al., 2005). In addition, atypical antipsychotics are unable to adequately alleviate negative and cognitive impairment symptoms (Leiberman et ah, 2005).
• Mood-stabilizers treat the mania (a positive symptom) and depression (a negative symptom) of the disease.
• lithium must be used at a dose that is almost toxic to the body. As such, constant monitoring is required to prevent kidney toxicity, dehydration, convulsions, and tremors that can be fatal.
• lithium also has common undesirable side effects, such as numbness, dazed feeling, and drowsiness.
• An extensive review has shown that lithium treatment alone has no effect for schizophrenia (Leucht et al. , Cochrane Database Sy st Rev. , 2007 3 :CD001258).
• Bipolar disorder is believed to affect 2.6% of the population in the United States (Muzina et al, Ann Clin Psychiatry 2007, 19(4):305-12). However, the actual number of individuals affected by this disorder may be greater due to inconsistent diagnosis. Bipolar disorder is characterized by periods of depression and periods of mania which both greatly disrupt everyday life. Bipolar disorder can be classified in two groups (either bipolar I disorder or bipolar II disorder), and both of these groups include symptoms of depression and mania. Mania can be defined as racing thoughts, rapid speech, elevated levels of activity and agitation as well as an inflated sense of self-esteem while the depression phase includes feeling a lack of self worth, isolation, sadness, feeling overwhelmed and may include suicidal thoughts. This debilitating condition requires medical intervention, often including the use of pharmaceuticals for regulation of the symptoms.
• Typical treatments include mood stabilizers, antimanic agents, anticonvulsants and antiepileptics which can include lithium, chlorpromazine, aminophenylpyridone, aripiprazole, olanzapine and fluoxetine, just to name a few.
• these drugs can have several undesirable side effects. In particular lithium is known to cause nausea, vomiting, tremor and diarrhea. Hospitalization is often required during the treatment of manic periods (Muzina Prim Care 2007, 34(3):521 -50).
• the current treatments available for schizophrenia and bipolar disorder are far from satisfactory. Issues of undesirable side effects and inadequate alleviation of symptoms still persist and thus, better therapies are required (Javitt et al, Biol Psychiatry. 1999, 45:668-679).
• phencyclidine an N-methyl D-aspartate (NMDA) receptor (NMDAR) antagonist
• PCP phencyclidine
• N-methyl D-aspartate NNDAR
• Kynurenic acid a naturally occurring NMDA receptor antagonist
• NMDA receptor antagonism has revealed decreased phosphorylation of Akt and GSK-3 ⁇ in the PI3K/Akt/GSK-3 ⁇ pathway (Lei et al, Neuropsychopharmacology, 2007, doi: 10.1038/sj.npp.130151 1).
• Schizophrenia patients exhibit decreased Akt activity compared to normal individuals (Emamian et al, Nat Med., 2004, 36: 131 -137), such that its inhibitory regulation of GSK-3 ⁇ is likely impaired.
• GSK-3 ⁇ is a serine-threonine kinase implicated in neuronal degeneration and apoptosis, which may lead to the loss of neuronal synaptic connectivity and volume.
• neurotrophins such as nerve growth factor (NGF)
• NGF nerve growth factor
• Mature neurotrophins usually activate the PI3K/Akt/GSK-3 ⁇ pathway by binding to their appropriate Trk receptor, which also leads to their increased expression.
• Neurotrophins are noted for their ability to provide neuroprotection, induce axonal growth and associated synaptic connectivity, and neurogenesis.
• Treatment strategies disclosed herein will address a variety of symptoms of schizophrenia and bipolar disorder that are not found in current treatments for the disease.
• Figures 1 -3 show schematic forms of the biochemical pathways thought to be implicated in schizophrenia and bipolar disorder, and KLKl 's possible route of action.
• the present invention includes methods of treating a psychiatric disorder that are affected by the P13K/Akt/GSK-3 ⁇ pathway comprising administering tissue kallikrein (KLKl), variants or active fragments thereof.
• a psychiatric disorder can be schizophrenia, associated conditions of the schizophrenic spectrum, or bipolar disorder.
• One aspect of the invention provides a method to treat symptoms of schizophrenia and associated conditions comprising administering tissue kallikrein, variants or active fragments thereof.
• a symptom of schizophrenia can be a positive symptom, a negative symptom, or a cognitive symptom.
• Positive symptoms can include, but are not limited to, delusions, hallucinations, and catatonic behavior.
• Negative symptoms can include, but are not limited to, lack of emotion, inability to enjoy activities, low energy, lack of interest in life, alogia, inappropriate social skills, inability to make friends, and social isolation.
• Cognitive symptoms include, but are not limited to, impairment of attention/information processing, sensory gating, problem solving, processing speed, verbal and visual learning and memory, and working memory.
• the invention includes a method of treating the prodomal stage of first onset or relapse of schizophrenia, or bipolar disorder comprising administering KLKl , or a variant or an active fragment thereof.
• An embodiment of the invention includes treating a symptom of bipolar disorder comprising administering KLKl , or a variant or an active fragment thereof.
• a symptom of bipolar disorder can be continuous mood disruption, which includes both periods of depression and mania.
• the invention includes a method of increasing neuroprotection of brain cells by administering KLKl , or a variant or an active fragment thereof.
• Neuroprotection can include, but is not limited to, an increase of neurotrophins (e.g., NGF) and/or a decrease in GSK-3 ⁇ activity.
• the invention includes a method of preventing apoptosis of cells in the brain by administering KLKl , or a variant or an active fragment thereof.
• a method of preventing apoptosis includes, but is but not limited to, increasing neurotrophins such as NGF, and/or decreasing GSK-3 ⁇ activity.
• the invention provides a method of preventing neurodegeneration in the brain by administering KLKl , or a variant or an active fragment thereof.
• a method of preventing neurodegeneration includes, but is not limited to, increasing neurotrophins such as NGF, and/or a decrease in GSK-3 ⁇ activity.
• KLKl , or a variant or an active fragment thereof can be administered orally.
• Oral administration may be an enteral administration.
• Oral formulations can be liquids, pills, solution, tablets, sustained release capsules, enteric coated capsules, or syrups.
• An oral therapeutic dose can be a maximum dose range of about 1 to about 1000 International Units (IU) per day.
• KLKl , or a variant or an active fragment thereof can be administered intranasally.
• Formulations for intranasal administration can be ointments, creams, lotions, pastes, gels, sprays, aerosols, oils, and the like.
• a nasal therapeutic dose is a maximum dose of about 1 to about 5000 IU per day.
• Another aspect of the present invention includes treatment and prevention methods as described herein, further comprising concurrent administration of a therapeutic compound useful for treating schizophrenia or bipolar disorder.
• Therapeutic compounds useful for treating schizophrenia and/or bipolar disorder include, but are not limited to, typical and atypical antipsychotics, and mood stabilizers such as lithium and valproic acid.
• Another aspect of the present invention includes a pharmaceutical composition formulated for oral administration comprising about 1 to about 1000 IU of KLKl, or a variant or an active fragment thereof, optionally further comprising a pharmaceutically acceptable excipient, and optionally further comprising an additional therapeutic compound as described above.
• Another aspect of the present invention includes a pharmaceutical composition formulated for intranasal administration comprising about 1 to about 5000 IU of KLKl, or a variant or an active fragment thereof, optionally comprising a pharmaceutically acceptable excipient.
• Tissue kallikrein or "KLKl” is a serine protease that is primarily noted for its role in controlling hypertension through its cleavage of kininogen into lysyl- bradykinin (kallidin) (Yousef et al., Endocrine Rev., 2001 ; 22: 184-204).
• KLKl appears to be a ubiquitous or multiple target acting enzyme.
• tissue kallikrein is synonymous with the following terms: callicrein, glumorin, padreatin, padutin, kallidinogenase, bradykininogenase, pancreatic kallikrein, onokrein P, dilminal D, depot-Padutin, urokallikrein, or urinary kallikrein.
• Tissue kallikrein polypeptide has the following sequence: NP 001001911 GI:50054435 Sus scrofa 1 - 1 7 s i gr al pept ide 1 8- 24 propept ide 25- 26 3 rrature pept ide
• CKGDSGGPLICNGKWQGITSWGHTPCGSANKPSIYTKLIFYLDWINDTITENP SEQ ID NO : 1
• Another embodiment includes:
• VGDSGGPLKCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENS SEQ ID NO: 2
• active fragment refers to smaller portions of the KLKl polypeptide that retain the activity of the full-length KLKl polypeptide.
• a “variant” or “mutant” of a starting or reference polypeptide is a polypeptide that 1 ) has an amino acid sequence different from that of the starting or reference polypeptide and 2) was derived from the starting or reference polypeptide through either natural or artificial (manmade) mutagenesis.
• Such variants can include an amino acid deletion, insertion, substituting, or combinations thereof.
• a variant amino acid in this context, refers to an amino acid different from the amino acid at the corresponding position in a starting or reference polypeptide sequence (such as that of a source antibody or antigen binding fragment). Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics.
• amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites.
• Methods for generating amino acid sequence variants of polypeptides are described in U.S. Patent No. 5,534,615, expressly incorporated herein by reference.
• a “wild type” or “reference” sequence or the sequence of a "wild type” or “reference” protein/polypeptide maybe the reference sequence from which variant polypeptides are derived through the introduction of mutations.
• a "wild type” sequence for a given protein is a sequence that is most common in nature.
• a "wild type” gene sequence is the sequence for that gene which is most commonly found in nature. Mutations may be introduced into a "wild type” gene (and thus the protein it encodes) either through natural processes or through man induced means. The products of such processes are “variant” or “mutant” forms of the original "wild type” protein or gene.
• Percent (%) amino acid sequence identity with respect to the polypeptides identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
• Percent (%) nucleic acid sequence identity is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a reference polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
• % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
• amino acid is used in its broadest sense and is meant to include the naturally occurring L ⁇ -amino acids or residues.
• the commonly used one and three letter abbreviations for naturally occurring amino acids are used herein
• the term includes all D-amino acids as well as chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not usually incorporated into proteins such as Norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid.
• analogs or mimetics of phenylalanine or proline which allow the same conformational restriction of the peptide compounds as natural Phe or Pro are included within the definition of amino acid.
• Such analogs and mimetics are referred to herein as "functional equivalents" of an amino acid.
• protein has an amino acid sequence that is longer than a peptide.
• a “peptide” contains 2 to about 50 amino acid residues.
• polypeptide includes proteins and peptides. Examples of proteins include, but are not limited to, antibodies, enzymes, lectins and receptors; lipoproteins and lipopolypeptides; and glycoproteins and glycopolypeptides.
• a “fusion protein” and a “fusion polypeptide” refer to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property.
• a property may be a biological property, such as activity in vitro or in vivo.
• a property may also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc.
• the two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other.
• the two portions of the fusion polypeptide are obtained from heterologous or different polypeptides.
• terapéuticaally effective amount refers to an amount of a composition effective to "alleviate” or “treat” a disease or disorder in a subject or mammal. Generally, alleviation or treatment of a disease or disorder involves the lessening of one or more symptoms or medical problems associated with the disease or disorder. In an embodiment, a “therapeutically effective amount” is an amount that treats schizophrenia and the associated symptoms of schizophrenia, the prodomal stage of first onset, relapse of schizophrenia, associated conditions of schizophrenic spectrum and bipolar disorder.
• treatment refers to inhibiting, alleviating, and healing a disease and conditions or symptoms thereof.
• Treating or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
• Treatment includes administering KLKl , or a variant or an active fragment thereof to a patient with schizophrenia or bipolar disorder.
• Administration of KLKl , or a variant or active fragment thereof includes a "therapeutically effective amount” which includes a prophylactic amount (e.g., an amount effective for alleviating or healing the above mentioned diseases or symptoms thereof).
• Successful treatment and improvement of the disease can be assessed by psychiatric evaluation.
• prevention and “prevent” refers prophylaxis, i.e., to keep a disease, disease condition, disease pathology, and/or disease symptoms from happening.
• Schizophrenia and bipolar disease can be prevented by administering a therapeutically effective amount of KLKl , or a variant or an active fragment thereof.
• a "therapeutically effective amount” as used herein includes a prophylactic amount (e.g., an amount effective for preventing the above mentioned diseases or symptoms thereof).
• Successful treatment and improvement of the disease can be assessed by psychiatric evaluation.
• schizophrenia refers to a chronic psychological/mental disorder which can be characterized by psychosis (loss of contact with reality), hallucinations (false perceptions), delusions (false beliefs), disorganized speech and behavior, flattened effect (restricted range of emotions), cognitive deficits (impaired reasoning and problem solving) and occupational and social dysfunction. Symptoms of schizophrenia may be classified by a skilled physician based on medical history, interview consultation, physical examination and lab tests.
• the term “schizophrenia” for use herein encompasses all subtypes of schizophrenia, including, but not limited to disorganized type, catatonic type, paranoid type, residual type and undifferentiated type (The Merck Manual of Diagnosis and Therapy, 2006)
• Disorders related to schizophrenia include: a) brief psychotic disorder, b) delusional disorder and c) schizoaffective disorder.
• BP bipolar disorder
• manic depressive disorder or “manic depressive illness” refers to a chronic psychological/mood disorder which can be characterized by significant mood changes including periods of depression and euphoric manic periods.
• BP is diagnosed by a skilled physician based on personal and medical history, interview consultation and physical examinations.
• mania or "manic periods” refers to periods where an individual exhibits some or all of the following characteristics: racing thoughts, rapid speech, elevated levels of activity and agitation as well as an inflated sense of self-esteem, euphoria, poor judgment, insomnia, impaired concentration and aggression.
• depression refers to periods where an individual with a depressed mood meaning a mental state that produces feelings including but not limited to sadness and discouragement, which may result in destructive behaviors.
• prodomal stage of first onset refers to early symptoms and signs of an illness that occur before characteristic manifestations are seen in a fully developed illness. In schizophrenia and bipolar disorder, this represents a period of prepsychotic disturbance or an interval between onset of usual behaviour disturbance and first signs of prominent psychotic symptoms. A prodomal period can be before the first behavioural episode of a schizophrenic or individual with bipolar disorder or a period before the relapse of the episode.
• relapse of schizophrenia refers to regression of symptoms of a healthy individual formerly affected with schizophrenia back to a state at which the were upon schizophrenia affection as diagnosed by a psychiatrist.
• relapse of bipolar disorder refers to regression of symptoms of a healthy individual formerly affected with bipolar disorder back to a state at which they were upon bipolar disorder affection as diagnosed by a psychiatrist.
• increase neuroprotection or “increased neuroprotection” refers to inhibition of neuronal damage and death.
• a pharmaceutical composition can be separate compounds or separate pharmaceutical compositions administered consecutively, simultaneously, or at different times.
• KLKl and a therapeutic compound are administered simultaneously.
• additional therapeutic compound refers to a therapeutic used for treating schizophrenia or bipolar disorder other than KLKl , a variant or a fragment thereof.
• An additional therapeutic compound for treating schizophrenia or bipolar disorder includes, but is not limited to, typical and atypical antipsychotics and mood stabilizers such as lithium and valproic acid.
• schizophrenia spectrum refers to closely related psychotic conditions that share a variety of symptoms in common yet differ in severity ranging from mild to severe.
• Schizoid personality disorder is located in the mild range of the spectrum, schizotypal personality falls in the middle, and schizophrenia is at the severe end of the spectrum.
• Common symptoms and cognitive impairments found across the whole spectrum are due to shared genetic and environmental events which lead to abnormal temporal brain structures in comparison to normal individuals.
• the more severe end of the spectrum, chronic schizophrenia displays additional abnormalities in brain structure like the frontal lobe deficits accounting for worse symptoms while the milder end of the spectrum, schizoid personality disorder, have no such additional abnormalities and can perhaps better compensate for the temporal deficit thus milder symptoms.
• positive symptom refers to, but is not limited to, delusions, hallucinations, disorganized thinking, and catatonic motor behaviors.
• negative symptom refers to, but is not limited to, lack of emotion, inability to enjoy activities, low energy, lack of interest in life, alogia, avolition, inappropriate social skills, inability to make friends and social isolation.
• cognitive symptom refers to, but is not limited to, abilities of attention/information processing, sensory gating, problem solving, processing speed, verbal and visual learning, and memory and working memory.
• the present invention provides methods for treating schizophrenia, associated conditions of the schizophrenic spectrum, and bipolar disorder.
• One embodiment includes a method of treating schizophrenia, associated conditions of the schizophrenic spectrum, or bipolar disorder in a mammal by administering tissue kallikrein, a variant or active fragment thereof to the mammal. Administration can be oral or intranasal.
• the pathology of schizophrenia, associated conditions of the schizophrenic spectrum, and bipolar disorder includes apoptosis of brian cells and other neural tissues.
• Administration of KLKl provides neuroprotection, i.e., KLKl inhibits apoptosis of brain cells.
• KLKl activates the bradykinin B2 receptor, leading to activation of the PI3K/Akt/GSK-3 ⁇ pathway. This activation of Akt prevents apoptosis and, thereby producing a neuroprotective effect.
• Activation of bradykinin B2 receptor by KLKl also activates the regulatory inhibition of GSK-3 ⁇ . This process is represented diagrammatically in Figure 2.
• inhibition of GSK-3 ⁇ prevents/inhibits ⁇ -catenin phosphorylation, which then prevents/inhibits ubiqitin-dependent breakdown of ⁇ -catenin.
• Activity of ⁇ -catenin can improve mood- stabilization ability achieved by the administration of lithium. This process is represented diagrammatically in Figure 1. Administering KLKl , a variant or active fragment thereof, concurrently with lithium further improves mood stabilization.
• KLKl can also interact with neurotrophins to produce a neuroprotection from apoptosis and neurodegeneration, promote axonal growth and improve neuronal synaptic connectivity.
• Administering KLKl , or a variant or active fragment thereof can modify neurotrophins, for example NGF, to provide a neuroprotective effect and thereby treat schizophrenia, conditions of the schizophrenia spectrum, and bipolar disease.
• the precursor form of NGF is post-transitionally modified by KLKl cleavage into its mature form such that NGF is then able to activate the PI3K/Akt/GSK-3 ⁇ pathway through Trk A. This process is represented diagrammatically in Figure 3.
• KLKl or a variant or active fragment thereof, also treats schizophrenia, conditions of the schizophrenia spectrum, and bipolar disease through KLKl modifications of neurotrophins.
• compositions of the invention include formulations to be administered orally or intranasally.
• Formulations suitable for intranasal administration include powder, granules, solution, drops, ointments, creams, lotions, pastes, gels, sprays, aerosols, oils and the like.
• Solutions or suspensions of the invention can be applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette or spray.
• Formulations may be provided in a single or multidose form.
• a solution may be sterile, isotonic or hypotonic, and otherwise suitable for administration by injection or other means and may contain appropriate adjuvants, buffers, preservatives and salts. Solutions such as nose drops may contain antioxidants, buffers, and the like.
• Powder or granular forms of a pharmaceutical composition can be combined with a solution and with diluting, dispersing and/or surface active agents.
• Formulations for aerosol administration include formulations designed for intranasal administration.
• An active ingredient can be provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) (e.g., dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane), carbon dioxide, or other suitable gas.
• CFC chlorofluorocarbon
• An aerosol may also contain a surfactant such as lecithin.
• a dose of drug may be controlled by a metered valve.
• active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP).
• a powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatine or blister packs from which the powder may be administered by means of an inhaler.
• a pharmaceutical composition formulated for intranasal administration comprises about 1 to about 5000 IU of KLKl , or a variant, or an active fragment thereof, optionally, further comprising a pharmaceutically acceptable excipient.
• An intranasal dose of KLKI, variant, or active fragment thereof can be a dose of about 1 to about 5000 IU per day; about 1 to about 4000 IU per day; about 1 to about 3000 IU per day; about 1 to about 2500 IU per day; about 1 to about 2000 IU per day; about 1 to about 1000 IU per day; about 1 to about 750 IU per day; about 1 to about 500 IU per day; about 1 to about 400 IU per day; about 1 to about 300 IU per day; about 1 to about 250 IU per day; about 1 to about 200 IU per day; about 1 to about 150 IU per day; about 1 to about 100 IU per day; about 1 to about 75 IU per day; about 1 to about 50 IU per day; about 1 to about 50 IU per day;
• Formulations suitable for oral administration include liquids, pills, solution, tablets, sustained release capsules, enteric coated capsules or syrups.
• a pharmaceutical composition formulated for oral administration comprises about 1 to 1000 IU of KLKl , or a variant or an active fragment thereof, optionally further comprising a pharmaceutically acceptable excipient.
• An oral dose of KLKI, variant, or active fragment thereof can be a dose of about 1 to about 1000 IU per day; about 1 to about 750 IU per day; about 1 to about 500 IU per day; about 1 to about 400 IU per day; about 1 to about 300 IU per day; about 1 to about 250 IU per day; about 1 to about 200 IU per day; about 1 to about 150 IU per day; about 1 to about 100 IU per day; about 1 to about 75 IU per day; about 1 to about 50 IU per day; about 1 to about 50 IU per day; about 1 to about 25 IU per day; about 1 to about 20 IU per day; about 1 to about 15 IU per day; about 1 to about 10 IU per day; about 1 to about 5 IU per day; about 5 to about 1000 IU per day; about 10 to about 1000 IU per day; about 15 to about 1000 IU per day; about 20 to about 1000 IU per day; about 25 to about 1000 IU per day; about 50 to about 1000 IU per day; about 75 to
• Intravenous (i.v.) administration may require trained medical professionals, which is time consuming, costly to the health care system, and may result in patient compliance issues. Risks associated with intravenous administration are also present (e.g., infection at the injection site).
• Intranasal administration allows a medicament to be 'fast acting' since it reaches the brain by a more direct route. Intranasal administration is convenient and virtually eliminates issues of patient compliance. Mucosal and submucosal epithelial cells are selectively permeable. Thus, proteins such as KLKl pass through and bypass the blood-brain-barrier via an intranasal route. Intranasally administered KLKl can produce its effects directly on the brain, thereby minimizing peripheral effects due to involvement of the olfactory region in the upper portion of the nasal pathway.
• a substance administered intranasally may follow two possible routes — intraneuronal or extraneuronal.
• Uptake of peptides into olfactory neurons where the peptides travel along axons to bypass the blood-brain-barrier is an intraneuronal route.
• Passage through unique intercellular clefts in epithelia of the olfactory region is an extraneuronal route that allows peptides to diffuse into the subarachnoid space.
• An extraneuronal route is more preferable due to rapid passage time to the brain, avoidance of proteolytic degradation involved in intraneuronal pathways (Born et ah, Nat. Neurosci., 2002, 5(6):514-6), and rapid eliciting of biological effects at multiple sites of the brain (Throne et al, Neuroscience, 2004, 127(2):481 -96).
• compositions may be administered orally or intranasally.
• Formulations suitable for intranasal administration include ointments, creams, lotions, pastes, gels, sprays, aerosols, oils and the like. Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette or spray. Formulations may be provided in a single or multidose form. For a dropper or pipette, a patient can administer an appropriate, predetermined volume of a solution or suspension. A spray can be administered by metering atomizing spray pump.
• Formulations for aerosol administration, particularly to the respiratory tract, include intranasal administration.
• An active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas.
• a suitable propellant such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas.
• An aerosol may also contain a surfactant such as lecithin.
• a dose of drug may be controlled by a metered valve.
• active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP).
• a powder carrier can form a gel in the nasal cavity.
• a powder composition may be presented in a unit dose form, for example in capsules, cartridges of gelatine, or blister packs.
• a powder can be administered by means of an inhaler.
• a method of the invention includes delivering compounds to affected areas of the brain through transneuronal retrograde and anterograde transport mechanisms. Delivery of neurologic agents to the brain by that transport system may be achieved in several ways. One technique comprises delivering KLKl alone to the nasal cavity.
• KLKl can facilitate its transport to diseased neurons in the brain.
• Auxiliary substances are capable of delivering KLKl to peripheral sensory neurons and/or along neural pathways to malfunctioning areas of the brain.
• Peripheral nerve cells of the olfactory neural pathway can be utilized in order to deliver KLKl to damaged neurons in those regions of the brain that are connected to the olfactory bulb.
• a method of the invention delivers KLKl to the nasal cavity of a mammal. It is preferred that KLKl be delivered to the olfactory area in the upper third of the nasal cavity and particularly to the olfactory epithelium in order to promote transport of the agent into the peripheral olfactory neurons rather than the capillaries within the respiratory epithelium. Transport of KLKl to the brain by means of the nervous system instead of the circulatory system so that KLKl can be delivered to damaged neurons in the brain.
• KLKl alone or in combination with other substances as a pharmaceutical composition may be administered to the olfactory area located in the upper third of the nasal cavity.
• the composition may be dispensed intranasally as a powdered or liquid nasal spray, nose drops, a gel or ointment, through a tube or catheter, by syringe, by packtail, by pledget, or by submucosal infusion.
• Oral administration includes enteral administration of solution, tablets, sustained release capsules, enteric coated capsules, and syrups.
• KLKl can be combined with a carrier and/or other adjuvants to form a pharmaceutical composition.
• Potential adjuvants include, but are not limited to, GM- 1 , phosphatidylserine (PS), and emulsifiers such as polysorbate 80.
• Further supplementary substances include, but are not limited to, lipophilic substances such as gangliosides and phosphatidylserine (PS).
• KLKl can be administered to the nasal cavity alone or in combination with a second therapeutic compound useful in treating schizophrenia or bipolar disorder.
• a second therapeutic compound useful for treating schizophrenia includes, but is not limited to trifluoperazine, fluanxol, loxapac, loxitane, etrafon, trilafon, thorazine, halopoidol, fluphenazine decanoate, aripiprazole, clozapine, ziprasidone, resperidone, questiapire, olanzapine, iloperidone (Titan/Novartis), DTA 20 IA (Knoll), DV 127090 (Solvayl Lundbeck), ORG 5222 (Organon), Osanetant (Sanofi-Synthelabo), and MEM 3454 (Memoray Pharmaceuticals Corp.).
• a second therapeutic compound useful for treating bipolar disorder includes, but is not limited to aripiprazole, asenapine (investigational), carbamazepine, fluoxetine and alanzapine, lamotrigine, lithium, olanzapine, oxcarbazepine, quetiapine, risperidone, topirimate, valproic acid, valproic acid, divalproex sodium, and ziprasidone.
• KLKl can be combined with micelles comprising of lipophilic substances.
• Micelles may modify the permeability of the nasal membrane and enhance absorption of the agent.
• Lipophilic micelles can include gangliosides, particularly GM-I ganglioside, and phosphatidylserine (PS).
• KLKl Once KLKl has crossed the nasal epithelium, KLKl is transported along the olfactory neural pathway. KLKl may be capable of movement within the olfactory system. In particular, neurotrophic and neuritogenic substances have demonstrated ready incorporation into nerve cell membranes and an affinity for nerve cell receptor sites.
• the doses used are: PCP 0.5 to 3.0 mg/kg, Clozapine 1 to 15 mg/kg and KLKl 0.01 to 1000 IU.
• KLKl is given one hour before pre-pulse inhibition testing.
• Clozapine is given -30 min before pre-pulse inhibition testing.
• PCP is given -15 minutes before pre-pulse inhibition testing.
• Pre-pulse inhibition is defined by the percent reduction in startle amplitude in the presence of a pre-pulse compare to the amplitude in the absence of a pre-pulse ( 100-( 1OO x mean of pre-pulse Q OV , m ⁇ ⁇ or h i ah ) + startle /mean of startle).
• the PCP treated-vehicle only group show a statistically significant decrease in percent pre-pulse inhibition compared non-PCP treated group.
• the clozapine treatment group shows a statistically significant increase in percentage pre-pulse inhibition compared to the PCP treated-vehicle group.
• Treatment with KLKl restores pre-pulse inhibition after PCP treatment, such that there is a statistically significant increase in percent pre-pulse inhibition compared to the PCP treated-vehicle only treatment group.
• Example 2 PCP Enhanced Immobility Time in a Forced Swim Test Decreased by KLKl
• control and treatment groups 3 groups.
• Two treatment groups each receive 10 mg/kg/day PCP injected intraperitoneally for 14 days while the control receives saline.
• On day 15 one of the treatment groups receives 0.01-1000 IU of KLKl for 30-60 minutes.
• Rats are subjected to a forced swim test by placement into a Plexiglas® cylinder (60.5 cm high and 29 cm in diameter) filled with 30 cm of water at 25 0 C, for six minutes. Immobility time is recorded to determine the mean of the group.
• KLKl is able to decrease the immobility time of mice treated with PCP.
• the PCP + KLKl group shows an unexpected and significant decrease in mean immobility time in comparison to the PCP only treatment group.
• the Novel Objection Recognition (NOR) task relies on an animal's natural tendency to explore a novel object instead of a familiar object. Exploration of the novel objects indicates learning and memory (cognition). Less exploration time with a novel object compared to an unaffected control indicates a cognitive impairment.
• Acclimatized female Hooded-Lister rats are divided into control and treatment groups. Two treatment groups receive 10 mg/kg/day PCP injected intraperitoneally for 7 days while the control groups receives saline, followed by 7 days without treatment for all groups. On day 15 one of the PCP treatment groups receives 0.01- 1000 IU of KLKl for 30-60 minutes.
• Each rat is placed in an open Plexiglas® box with black walls (52 cm L; 52 cm W; 31 cm H) in which two objects of similar height are placed. Rats are then removed from the box for a short period of time ( 1 minute) and returned to find that one of the objects has been replaced with a new object. Exploration time of the animal to each of the objects is determined. Exploration time includes the time spent sniffing, licking, touching, standing or sitting on the object. Prior to this test rats are introduced to the box so that they may become familiar with this environment and this stimulus will therefore not affect test results.
• the PCP only treatment group is unable to discriminate between the familiar object and the novel object due to PCP-induced cognitive impairment. Also, there is no statistically significant difference found in mean exploration time between the familiar and novel objects.
• the PCP + KLKl treatment group shows that KLKl is able to inhibit PCP-induced cognitive impairment due the observation of an unexpected and significant increase in mean exploration time towards the novel object compared to the familiar object.
• KLKl is able to reduce cognitive deficit-like symptoms thought to replicate cognitive deficits of psychiatric disorders.
• Sprague-Dawley rat embryonic forebrain cells (E18-E19) are dissociated using cold Hanks balanced salt solution (HBSS) without Mg 2+ or Ca 2+ , and re-plated in polylysine (5 mg/ml) coated multi-well plates at 10 x 10 "* cell/ml and grown in Neurobasal Medium (Invitrogen, Carlsbad, CA) supplemented with 0.05 mM L- glutamine and 10% B27 (Invitrogen) at 37 0 C in 5% CO 2 . The culture media is replaced every four days. After 14 days, cells are treated with 1 ⁇ M PCP over various time frames: 0, 1 ,
• each time point is Western blotted to determine the presence and for quantification of phosphorylated GSK-3 ⁇ (inactive) and activated caspase-3 ( 17kDa). The relative band density of each is compared to the control (non-PCP treated cell lysate) at each time point and plotted on a density graph as a percentage of the control.
• the graph shows that over time in PCP only treated cells, the level of phosphorylated GSK-3 ⁇ S9 decreases while the level of activated capase-3 ( 17 kDa) increases up to the 24 hour time point.
• PCP treatment leads to GSK-3 ⁇ activation, which results in activation of casapse-3 downstream by active GSK-3 ⁇ .
• This trend is indicative of neuronal cell death via caspase-3 activation, which is a hallmark of the neurodegeneration observed in psychiatric disorders.
• Example 5 Prevention of pre- pulse inhibition of startle deficit after treatment with KLKl in mice Acclimatized male mice that have ad libitum access to food and water at room temperature are divided into five treatment groups:
• the doses used are: PCP 1.0 to 10.0 mg/kg, Clozapine 1 to 15 mg/kg and KLKl 0.01 to 1000 IU.
• KLKl is given one hour before pre-pulse inhibition testing.
• Clozapine is given -30 min before pre-pulse inhibition testing.
• PCP is given -15 minutes before pre-pulse inhibition testing.
• Pre-pulse inhibition is defined by the percent reduction in startle amplitude in the presence of a pre-pulse compare to the amplitude in the absence of a pre-pulse ( 100-( 1OO x mean of pre-pulse ( ⁇ m m ⁇ or hlgh) + startle /mean of startle).
• the PCP treated-vehicle only group show a statistically significant decrease in percent pre-pulse inhibition compared non-PCP treated group.
• the clozapine treatment group shows a statistically significant increase in percentage pre-pulse inhibition compared to the PCP treated-vehicle group.
• Treatment with KLKl restores pre-pulse inhibition after PCP treatment, such that there is a statistically significant increase in percent pre- pulse inhibition compared to the PCP treated-vehicle only treatment group.

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