[go: up one dir, main page]

WO2001054729A1 - Traitement des maladies neuropsychiatriques au moyen d'inhibiteurs de protease et de neuraminidase - Google Patents

Traitement des maladies neuropsychiatriques au moyen d'inhibiteurs de protease et de neuraminidase Download PDF

Info

Publication number
WO2001054729A1
WO2001054729A1 PCT/US2001/002417 US0102417W WO0154729A1 WO 2001054729 A1 WO2001054729 A1 WO 2001054729A1 US 0102417 W US0102417 W US 0102417W WO 0154729 A1 WO0154729 A1 WO 0154729A1
Authority
WO
WIPO (PCT)
Prior art keywords
cam
disorder
brain
compound
neuraminidase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2001/002417
Other languages
English (en)
Inventor
Marquis P. Vawter
William J. Freed
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
Original Assignee
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Priority to AU2001231137A priority Critical patent/AU2001231137A1/en
Priority to US10/182,162 priority patent/US20030211545A1/en
Publication of WO2001054729A1 publication Critical patent/WO2001054729A1/fr
Anticipated expiration legal-status Critical
Priority to US11/355,257 priority patent/US20060205650A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A61K31/245—Amino benzoic acid types, e.g. procaine, novocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K38/55—Protease inhibitors
    • 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
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of therapy of neuropsychiatric diseases in human subjects.
  • the present invention relates to a method of treating neuropsychiatric diseases associated with an abnormally elevated level of a fragment of a neural cell adhesion molecule (N-CAM) in brain or cerebrospinal fluid, comprising administering a protease inhibitor, a neuraminidase inhibitor or a combination of both with or without a neuroleptic medication.
  • N-CAM neural cell adhesion molecule
  • Schizophrenia is a life-long illness with multiple features that are defined by DSM-IV criteria and with additional features accepted by those skilled in the art: increased ventricle size of the brain, thinning of the cortical grey matter, and cognitive decline.
  • Schizophrenia has been treated with a variety of antipsychotic drugs such as phenothiazines, butyrphenones, xithioxanthenes, and newer atypical drugs, such as clozapine which are known to act as blockers of dopamine, serotonin, and cholinergic receptor sites.
  • antipsychotic drugs such as phenothiazines, butyrphenones, xithioxanthenes, and newer atypical drugs, such as clozapine which are known to act as blockers of dopamine, serotonin, and cholinergic receptor sites.
  • the modulation of a neurotransmitter receptor is the standard pharmacologic approach to treatment of schizophrenia.
  • Bipolar disorder has been treated with anti-epileptic drugs and lithium which also block certain neurotransmitter receptors with limited efficacy.
  • the present invention overcomes the previous limitations and shortcomings in the art by providing a novel way of treating the diseases by providing a method of inhibiting endogenous proteases and neuraminidases which convert N-CAM into cN-CAM and other breakdown fragments in the brain and CSF.
  • An unexpected discovery of the present invention is that the breakdown product of N-CAM assayed in brain and CSF is found in synapses and can be reduced with protease inhibitors and that neura inidase treatment of brain induces less resistance to protease inhibition. Therapy that reduces the N-CAM breakdown products is useful in treating the neuropsychiatric disorders named herein. Also provided are methods of monitoring the efficacy of treatment and screening for effective therapeutic compounds which can be used alone or in combination with standard antipsychotic therapy.
  • the present invention provides a method of treating a neuropsychiatric disease in a human subject comprising administering a therapeutically effective amount of at least one compound selected from the group consisting of protease inhibitors and ' neuraminidase inhibitors, whereby administering the compound to the subject treats the neuropsychiatric disease.
  • the present mvention provides a method of reducing breakdown of N- CAM into a fragment in a brain of a human subject, comprising administering a therapeutically effective amount of a composition comprising at least one compound selected from the group consisting of protease inhibitors and neuraminidase inhibitors, whereby administering the composition to the subject reduces the breakdown of N- CAM into the fragment in the brain of the subject.
  • the present invention provides a method of treating a neuropsychiatric disease in a human subject comprising administering a therapeutically effective amount of a compound which reduces breakdown of endogenous protease inhibitors, whereby administering the compound to the subject treats the neuropsychiatric disease.
  • the present invention provides a method of monitoring efficacy of treatment of a neuropsychiatric disease in a human subject, comprising detecting a reduction in concentration of a fragment of a neural cell adhesion molecule in cerebrospinal fluid of the subject, whereby the reduction in concentration of the fragment of the neural cell adhesion molecule in cerebrospinal fluid of the subject indicates efficacy of treatment of the neuropsychiatric disease.
  • the present invention provides a method of screening for a compound effective in treating a neuropsychiatric disease associated with the presence of an abnormally elevated level of a fragment of a neural cell adhesion molecule in cerebrospinal fluid of a human subject, comprising the following steps: a) contacting a sample of human brain cortex with the compound and b) detecting a reduction of breakdown of the neural cell adhesion molecule into the fragment, whereby the reduction of breakdown of the neural cell adhesion molecule into the fragment indicates that the compound is effective in treating the neuropsychiatric disease.
  • a fragment of N-CAM wherein the fragment is selected from the group consisting of dN-CAM, VASE N-CAM 155, VASE N-CAM 165 and VASE N-CAM 200.
  • the present invention also provides a method of treating a neuropsychiatric disease in a human subject comprising administering a therapeutically effective amount of a compound which reduces breakdown of endogenous protease inhibitors, whereby administering the compound to the subject treats the neuropsychiatric disease.
  • the present invention also provides a method of screening for a compound effective in reducing the breakdown of endogenous protease inhibitors in human brain cortex, comprising the following steps: a) contacting a sample of brain cortex with the compound; and b) detecting a rise in level or maintenance of a steady-state level of endogenous protease inhibitor, whereby the rise in the level or maintenance of a steady- state level of endogenous protease inhibitor indicates that the compound is effective in reducing the breakdown of endogenous protease inhibitors in the brain.
  • a "steady-state level” means that the level of protease inhibitor does not fall from the initial baseline measurement.
  • the present mvention provides a composition comprising a protease inhibitor and a neuraminidase inhibitor in a pharmaceutically acceptable carrier.
  • FIG. 1 The effect of protease inhibitors on cortical synaptosome preparation from human brain.
  • the synatposomes were identified by synaptophysin immunoreactivity and also analyzed for N-CAM by SDS-PAGE and immunob lotting. The protease inhibitor was removed from the synaptosomal preparation prior to denaturing and running on a gel. The results show a decrease in N-CAM 180 and increase in cN-CAM. Lane numbers refer to the sucrose density gradient fractions.
  • B Quantification of the immunodensity results show the protease inhibitors decrease the amount of cN-CAM relative to N-CAM 180 in each synaptosomal brain fraction.
  • FIG. 2 The effect of protease on proteo lysis of N-CAM 180 in human cortex.
  • A Cytosolic extracts of cortical samples were left at room temperature for 4 or 48 hr and treated with protease inhibitors (+) or no protease inhibitor (-). Membrane extracts of brain samples were similarly treated. The results show that membrane N- CAM 180 is predominantly converted to cytosolic N-CAM at 48 hrs. Without protease inhibitors the proteolysis is not halted and all N-CAM 180 isoforms are degraded by proteolysis.
  • B The effect of protease on proteo lysis of N-CAM 180 in human cortex.
  • the quantification shows that the cN-CAM and N-CAM 180 are unchanged in frozen samples; however, as the proteolysis reaction proceeds at 4 hr and 48 hr there is a progressive loss of N-CAM 180 and build-up of cytosolic N-CAM (cN- CAM). The maximally effective action of the protease inhibitor appears at the 4 hr time point in this experiment.
  • FIG. 3 High density particles from sucrose density ultracentrifugation of cytosolic brain were obtained at a sucrose density of 1.194 g/L (Fig. 3N) and showed ⁇ -CAM immunoreactivity. The step gradient also showed synaptophysin immunoreactivity that co-migrated with c ⁇ -CAM (Fig. 3B).
  • Pretreatment of the brain cytosol with ⁇ P-40 prior to ultracentrifugation altered the migration of N-CAM in sucrose density gradient (Fig. 3C). Approximately 50% of cN-CAM stayed at the top of the gradient following NP-40 pretreatment and the remaining sedimented to the 1.237 g/L density.
  • the NP-40 pretreatment disrupted the migration of synaptophysin in the sucrose gradient (Fig. 3D) so that synaptophysin was seen in low concentration in all sucrose fractions, due to membrane fragmentation.
  • FIG. 4 The control fraction from the brain sucrose density preparations (PBS treatment only) was visualized by fluorescent immunocytochemistry for N-CAM (Fig. 4A) and synaptophysin (Fig. 4B).
  • FIG. 6 The correlation of cN-CAM and changes in ventricular volume as measured by repeat MRI scans at a two-year interval. Patients with higher initial cN- CAM concentration showed higher ventricular enlargement. Ventricular enlargement is the most widely replicable indicator of brain differences between patients with schizophrenia and age-matched controls. Ventricular enlargement is believed to be an indicator of a progressive illness and a more severe illness often refractory to standard treatment.
  • CAM CAM.
  • A Neuraminidase enzyme treatment (+) of membrane and cytosolic human brain extracts is shown to be permissive for conversion of N-CAM 180 to a low molecular weight N-CAM isoform (dN-CAM).
  • cN-CAM is partially glycosylated as shown in a small band migrating ahead of the majority of cN-CAM.
  • cN-CAM neuraminidase treatment also produced a small amount of dN-CAM that is visible on the original film.
  • N-CAM 75 kDa is also shown to be glycosylated and converted to dN-CAM.
  • N-CAM 180 The main effect of neuraminidase treatment is that the deglycosylation of N-CAM 180 permits proteolytic processing of N-CAM 180 to cN-CAM and dN- CAM isoforms.
  • Figure 9 Neuroserpin gene expression in postmortem brain tissue specimens from the prefrontal cortex in schizophrenia, expressed as a z-ratio.
  • Z-ratio values are based on the distribution of z-score differences among a series of 1128 genes evaluated by microarray analysis.
  • a z-ratio of 1 indicates that the difference is one standard deviation above the mean difference between patients with schizophrenia and controls.
  • a z-ratio of negative 1 indicates a decrease of one standard deviation below the mean difference between schizophrenia and the matched control group.
  • a z-ratio change of negative 2 represents approximately an 88% reduction in relative gene expression levels between groups.
  • Each pool of samples contains a separate matched group of five patients with schizophrenia and five controls. A total of 20 patients and 20 controls is represented by the average z-ratio.
  • an may mean one or more.
  • an inhibitor may mean one inhibitor or more than one inhibitor.
  • the” fragment may mean one fragment or more than one fragment.
  • the present invention provides a method of treating a neuropsychiatric disease in a human subject comprising administering a therapeutically effective amount of at least one compound selected from the group consisting of protease inhibitors and neuraminidase inhibitors, whereby administering the compound to the subject treats the neuropsychiatric disease.
  • a "neuropsychiatric" disease is one which affects the neurologic system or the mind of a human subject.
  • a neuropsychiatric disease may cause any of the following symptoms of psychosis: delusions, hallucinations, disorganized speech, grossly disorganized or catatonic behavior, negative symptoms. Other symptoms of neuropsychiatric disease are known to a person skilled in the art.
  • Examples of a neuropsychiatric disease which are treatable with the present invention include but are not limited to Schizophrenia, Bipolar Disorder I, Bipolar Disorder I with Psychotic Features, Bipolar Disorder II, Bipolar Disorder II with Psychotic Features, Psychotic Disorder Not Otherwise Specified, Schizophreniform Disorder, Schizoaffective Disorder, Unipolar Disorder, Unipolar Disorder with Psychotic
  • a chronic disease is one which is present in a subject and manifests its signs and symptoms for at least 3 to 6 months.
  • schizophrenia is a life-long illness associated with increased ventricle size of the brain, thinning of the cortical grey matter and cognitive decline.
  • Symptoms are grouped into three categories: positive or expressive symptomatology, negative or deficit symptomatology and disorganized symptomatology further defined in DSM-IV.
  • the disease may be diagnosed after a period of time known as the prodrome, which may last from a few months up to two years.
  • the prodrome of schizophrenia the subject manifests early symptoms suggesting the presence of a neuropsychiatric disease.
  • a conclusive diagnosis of schizophrenia is not usually made for several months.
  • a subject will manifest positive, negative or disorganized symptomatology for a significant portion of time during a 1 month period.
  • the present invention is directed to the treatment of subjects with chronic schizophrenia who have manifested some symptoms of the disease for approximately six months.
  • neural cell adhesion molecule is one found in a synaptosome in the synaptic region of a neuron in the brain and functions to facilitate neurite growth, neuronal migration, signaling and transmission of impulses across the synaptic junction between neurons in the brain.
  • the present invention treats neuropsychiatric diseases characterized by the presence of an elevated level of proteolytic breadkdown products of an isoform of N- CAM.
  • the breakdown products are fragments of the isoform of N-CAM and are present at abnormally elevated levels in bipolar disorder which is characterized clinically by abnormal affect related to excessive swings in mood between depression, euthymia and mania.
  • Fragments of N-CAM elevated in bipolar disorder are VASE N- CAM and SEC N-CAM in the brain. 7
  • cN-CAM is elevated in bipolar disorder I.
  • the present invention provides a method of reducing breakdown of N-
  • CAM into a fragment in a brain of a human subject comprising administering a therapeutically effective amount of a composition comprising at least one compound selected from the group consisting of protease inhibitors and neuraminidase inhibitors, whereby administering the composition to the subject reduces the breakdown of N- CAM into the fragment in the brain of the subject. Reducing the breakdown of N- CAM into fragments in the brain or CSF thereby treats the neuropsychiatric disease.
  • the present invention is based on the unexpected and surprising discovery that reducing the breakdown of a neural cell adhesion molecule in a brain of a subject affected with a neuropsychiatric disease improves the cognitive and psychiatric functions of the subject.
  • the present invention provides a method of reducing the proteolytic breakdown of N-CAM, a neural cell adhesion molecule, into fragments associated with the presence of a neuropsychiatric disease.
  • N-CAM In normal brain metabolism, endogenous proteases and neuraminidases degrade an isoform of N-CAM and release fragments of the isoform into CSF.
  • Isoforms of N- CAM are N-CAM 180, N-CAM 140, N-CAM 120, SEC N-CAM 115 and VASE N- CAM 200.
  • Fragments produced by the proteolytic breakdown of the isoforms of N- CAM are cN-CAM, dN-CAM, SEC N-CAM 108,VASE N-CAM 165 and VASE N- CAM 155.
  • the present invention provides methods to treat neuropsychiatric diseases characterized by symptoms described above for "neuropsychiatric" disorders similar to schizophrenia but include Psychotic Disorder, Schizophreniform Disorder, Schizoaffective Disorder, Mood Disorder with Psychotic Features, Substance Induced Psychotic Disorder and Schizotypal Personality Disorder.
  • neuropsychiatric diseases characterized by symptoms described above for "neuropsychiatric" disorders similar to schizophrenia but include Psychotic Disorder, Schizophreniform Disorder, Schizoaffective Disorder, Mood Disorder with Psychotic Features, Substance Induced Psychotic Disorder and Schizotypal Personality Disorder.
  • Elevated means characterized by an increase in the amount present compared to an age-matched control or a pre-disease state in the subject.
  • the present invention provides a method of treating a neuropsychiatric disease characterized as a psychosis.
  • the psychosis is usually chronic and is characterized by the presence of one or more of the following: delusions, hallucinations, disorganized speech, and grossly disorganized or catatonic behavior.
  • an acute drug-induced exacerbation of a chronic psychosis caused by the subject's long-term drug abuse may also be treated by the method of the present invention.
  • these disorders there is an abnormally elevated level of fragments of an isoform of N-CAM in the CSF of affected subjects.
  • the present invention is directed at reducing the proteolytic breakdown in the synaptosome of an isoform of a neural cell adhesion molecule known as N-CAM by inhibiting endogenous proteases and neuraminidases. It is the excessive breakdown of N-CAM into fragments that is associated with the presence of the neuropsychiatric diseases.
  • endogenous proteases include but are not limited to serine proteases, aspartyl proteases, tissue plasminogen activator, metalloproteinases, aminopeptidases and cysteine proteases.
  • examples of endogenous neuraminidases include but are not limited to neuraminidase 1, neuraminidase 2 and neuraminidase 3.
  • the present invention discloses a method of reducing the proteolytic breakdown of N-CAM, comprising either administering a compound which inhibits the enzymes responsible for degrading N-CAM or administering a compound which either elevates the level and activity of endogenous protease inhibitors and neuraminidase inhibitors or which reduces the breakdown of the endogenous protease inhibitors and neuramimdase inhibitors.
  • the main protease inhibitor target in the brain is tissue type plasminogen activator (t-PA), a serine protease. Because t-PA degrades N-CAM, t-PA is a prime target for treatment with protease inhibitors and compounds of the invention.
  • t-PA activity in the central nervous system is regulated by the endogenous protease inhibitors known as serpins.
  • serpins are plasminogen activator inhibitor (PAI-I), protease nexin-1 (PN-1), and neuroserpin (NSP) 1 .
  • PKI-I plasminogen activator inhibitor
  • PN-1 protease nexin-1
  • NSP neuroserpin 1 .
  • Tt is contemplated in the present invention that a serpin will be administered to a subject with neuropsychiatric disease to reduce the proteolytic breakdown of N-CAM by endogenous proteases.
  • a compound which increases the level of a serpin or reduces breakdown of a serpin, thereby inhibiting breakdown of N-CAM can be administered to a subject with a neuropsychiatric disease.
  • nucleic acids encoding serpins can be administered to a subject with neuropsychiatric disease, as can nucleic acids that encode compounds that reduce the breakdown of serpins or nucleic acids that themselves interfere with the breakdown of serpins (e.g., an antisense nucleic acid directed to block a nucleic acid which encodes a protease).
  • protease inhibitors There are several classes of protease inhibitors. See Table 1. Examples of aspartic protease inhibitors (A) include Nelfinavir, Saquinavir, Indinavir, Amprenavir, Ritonavir, Pepstatin, AG1776, ABT-387 and ⁇ -secretase inhibitors.
  • Serine protease inhibitors (B) examples include Aprotinin, AEBSF, Leupeptin (Acetyl-leucyl-leucyl-arginal), Elastatinal (Leu-(Cap)-Gln-Ala-al, N-[(S)-lcarboxy- isopentyl)-carbamoyl-alpha-(2-iminohexahydro-4(S)-pyrimidyl]-L-glycyl-L- glutaminyl-L-alaninal), Serpins, Antipain ( [(S)-l-Carboxy-2-Phenyl]-carbamoyl-Arg- Val-arginal), APMSF ((4-Amidino-Phenyl)-Methane-Sulfonyl Fluoride) and PMSF.
  • Cysteine protease inhibitors (C) examples include AG7088, Leupeptin and E-64 ( L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane or N-[N-(L-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine ).
  • Aminopeptidase inhibitors (D) examples include Bestatin ([(2S, 2R)-3-Amino- 2-hydroxy-4-Phenylbutanoyl]-L-Leucine) and Amastatin ([(2S, 2R)]-3-Amino-2- hydroxy-5-methylhexanoyl]-Val-Val-Asp-OH) .
  • Table 1 shows examples of neuraminidase inhibitors (E) including GS 4071, GS 4104, oseltamivir, zanamivir and RWJ-270201. Table 1 shows which compounds are currently approved by the FDA and which are not yet approved. Moreover, the underlined compounds are components of the protease inhibitor cocktail.
  • E neuraminidase inhibitors
  • the present invention provides a "protease inhibitor cocktail” comprising the following compounds:
  • the present patent provides numerous examples of protease inhibitors and neuraminidase inhibitors for use in the compositions and methods of the invention. It is understood, however, that any protease inhibitor or neuraminidase inhibitor now known or later developed can be routinely screened for efficacy in the present methods or can be included in the present compositions.
  • antipain and leupeptin are the two protease inhibitors most effective in preventing the breakdown of N-CAM 180 and the accumulation of cN-CAM ( Figure 8). These compounds inhibit both serine and cysteine proteases. When assayed in human brain, antipain and leupeptin are the most potent compounds compared to the protease inhibitor cocktail. See Example 9.
  • the protease inhibitor or neuramimdase inhibitor can be admimstered to a subject in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a carrier that is not biologically or otherwise undesirable, i.e., the carrier may be administered to a subject, along with the protease inhibitor or neuramimdase inhibitor, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any breakdown of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • the protease inhibitor or neuraminidase inhibitor may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, mtrarectally, intrathecally, by inhalation into the respiratory tract, topically or the like, although oral administration is typically preferred.
  • parenterally e.g., intravenously
  • intramuscular injection by intraperitoneal injection
  • transdermally e.g., extracorporeally, mtrarectally, intrathecally, by inhalation into the respiratory tract, topically or the like
  • the exact amount of the protease inhibitor or neuraminidase inhibitor required will vary from subject to subject, depending on the age, weight and general condition of the subject, the severity of the disease or condition being treated, the particular protease inhibitor or neuraminidase inhibitor used, its mode of administration and the like. Thus, it is not possible to specify an exact amount of protease inhibitor or neuraminidase inhibitor to administer. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein 2 . Examples of dosage ranges and preferred dosages and routes of administration for FDA approved protease inhibitors and neuraminidase inhibitors are shown in Table 2.
  • Parenteral administration of the protease inhibitor or neuraminidase inhibitor of the present invention is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is inco ⁇ orated by reference herein.
  • Suitable carriers for use in the present invention include, but are not limited to, pyrogen-free saline.
  • a sterile solution or suspension is prepared in saline that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected, for example, into subcutaneous or intramuscular tissues.
  • Suitable carriers for oral administration of a protease inhibitor or neuraminidase inhibitor include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants.
  • Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans.
  • Suitable liquid carriers may be water, pharmaceutically accepted oils, or a mixture of both. The liquid can also contain other suitable pharmaceutical additions such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents. Examples of suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a pH-regulated gel.
  • the protease inhibitor or neuraminidase inhibitor may be microencapsulated with either a natural or a synthetic polymer into microparticles 4-8 ⁇ m in diameter, which target intestinal lymphoid tissues and produce a sustained release for up to four weeks.
  • TTD investigational new drugs
  • Tables 4 and 5 show compounds which are in pre-clinical testing and the dosages and routes of administration for those drugs.
  • the present invention comprises administering a protease inhibitor or a neuraminidase inhibitor to a subject diagnosed with a neuropsychiatric disease.
  • the present mvention includes administering a protease inhibitor and a neuraminidase inhibitor in combination to a subject diagnosed with a neuropsychiatric disease.
  • either a protease inhibitor or a neuraminidase inhibitor or a combination of both may be administered with a neuroleptic compound to a subject with neuropsychiatric disease.
  • a "neuroleptic" compound is an antipsychotic medication and includes typical neuroleptics such as dopamine antagonists, for example Haldol® and other drugs which block dopamine receptors in the brain.
  • An example of an atypical neuroleptic compound is clozapine which blocks other receptors as well as some dopamine receptors in the brain.
  • Neuroleptics have a beneficial effect on the positive and negative symptomatology of chronic schizophrenia but do not improve the cognitive function of affected subjects.
  • a benefit of the present invention is the improvement in cognitive function of an affected subject.
  • the present invention provides a method of monitoring the efficacy of treatment of a neuropsychiatric disease in a human subject comprising detecting a reduction in concentration (level) of a fragment of a neural cell adhesion molecule in CSF of the subj ect, whereby the reduction in concentration of the fragment of the neural cell adhesion molecule in cerebrospinal fluid of the subject indicates efficacy of treatment of the neuropsychiatric disease.
  • a person with chronic schizophrenia may be noted to have fewer delusions, fewer hallucinations, fewer negative symptoms, less disorganized speech, and less grossly disorganized or catatonic behavior, as measured by psychometric tests known and used routinely by persons skilled in the art.
  • the present invention provides a laboratory method of monitoring the efficacy of treatment of the present invention.
  • a baseline measurement of the level of fragments in the CSF of the subject is made.
  • the fragments to be measured comprise cN-CAM, dN-CAM, SEC N-CAM 108, VASE N-CAM 165 and VASE N-CAM 155.
  • a repeat lumbar puncture is performed to obtain the subject's CSF to measure the level of fragments in it.
  • Clinical improvement in the subject correlates with a significant reduction in the level of fragments in the CSF.
  • a subject with a baseline pretreatment level of fragments 135% of normal would be expected to show a reduction of fragments to no more than 117% of no ⁇ nal.
  • a subject with more severe clinical disease correlated with a baseline pretreatment level of fragments of 200%> of normal, would be expected to show a reduction in fragment level in the CSF to approximately 140% to 160% of normal.
  • a method of monitoring the efficacy of treatment is disclosed in Example 7. For patients with intermediate levels of elevation, the reduction will range from about 40-60%.
  • the present invention also provides a method of screening for a compound effective in treating a neuropsychiatric disease associated with the presence of an elevated level of a fragment of a neural cell adhesion molecule in brain or CSF of a human subject, comprising the following steps: a) contacting a sample of brain cortex with the compound, and b) detecting a reduction of breakdown of the neural cell adhesion molecule into the fragment, whereby the reduction of breakdown of the neural cell adhesion molecule into the fragment indicates that the compound is effective in treating the neuropsychiatric disease.
  • the brain tissue may be pulverized, sonicated, homogenized or dissolved in solution prior to contacting with the compound.
  • any physical process that allows brain cortex to come into contact with a compound for in vitro testing of N-CAM breakdown into fragments is contemplated by the present invention. See Example 1.
  • cN-CAM is present in sub-human primate (for example, squirrel monkey) brain and cerebrospinal fluid.
  • sub-human primate for example, squirrel monkey
  • primates can be used as a model for studies on the effects of protease inhibitors on cN-CAM and N-CAM 180 kDa levels. See Example 8.
  • the present invention provides a method of screening for compounds which are effective in treating neuropsychiatric diseases characterized by the abnormal elevation of fragments of N-CAM in the brain or CSF of a human subject.
  • the present screening methods can be used to screen for the neuropsychiatric diseases described herein. See Example 6.
  • Examples of chronic neuropsychiatric diseases which are characterized by abnormally high levels of fragments of an isoform of the neural cell adhesion molecule N-CAM are Schizophrenia, Bipolar Disorder I, Bipolar Disorder I with Psychotic Features, Bipolar Disorder II, Bipolar Disorder II with Psychotic Features, Psychotic Disorder Not Otherwise Specified, Schizophreniform Disorder, Schizoaffective Disorder, Unipolar Disorder, Unipolar Disorder with Psychotic Features, Substance Induced Psychotic Disorder, Schizotypal Personality Disorder and Mood Disorder with Psychotic Features.
  • Isoforms of N-CAM comprise N-CAM 180, N-CAM 140, N-CAM 120, SEC N-CAM 115 and VASE N-CAM 200.
  • a subject with a history of a chronic psychosis caused by long-term drug abuse may be found to have an abnormally elevated level of fragments in the CSF during an acute psychotic reaction from a drug overdose.
  • an acute exacerbation of a dementia associated with delusions and hallucinations is a disease which can be diagnosed by finding abnormally elevated levels of fragments of N-CAM.
  • the fragments include cN-CAM, dN-CAM, SEC N-CAM 108, VASE N-CAM 165 and VASE N-CAM 155.
  • a person skilled in the art has the knowledge to perform a lumbar puncture to obtain CSF for laboratory analysis. The procedure is performed routinely by medical practitioners to diagnose diseases of the brain and spinal cord.
  • the present invention provides a fragment of a neural cell adhesion molecule selected from the group consisting of dN-CAM, VASE N-CAM 155, VASE N-CAM 165 and VASE N-CAM 200.
  • the present invention also provides a method of treating a neuropsychiatric disease in a human subject comprising administering a therapeutically effective amount of a compound which reduces breakdown of an endogenous protease inhibitor in brain, whereby administering the compound to the subject treats the neuropsychiatric disease.
  • a compound that reduces the breakdown of endogenous protease inhibitors will cause the level of endogenous protease inhibitors in brain to rise or remain steady. Thus, the breakdown of N-CAM into fragments associated with the neuropsychiatric diseases described herein will be reduced. Therefore, the neuropsychiatric disease is treated.
  • mRNA messenger RNA for the endogenous serine protease inhibitor neuroserpin (gene product PI12, Genbank Accession # Z81326) is decreased in the prefrontal cortex of patients with schizophrenia, as compared to controls ( Figure 9). It has also been found that expression of neuroserpin mRNA is decreased by approximately 88% in patients with schizophrenia, as measured in four separate cohorts of patients. See Example 10. Thus, the methods of the present invention which counteract the defects in protease inhibition will decrease the buildup in the brain of cN-CAM and other breakdown products of N-CAM that are associated with schizophrenia.
  • Also provided is a method of screening for a compound effective in reducing the breakdown of endogenous protease inlnbitors in human brain cortex comprising the following steps: a) contacting a sample of brain cortex with the compound, and b) detecting a rise in level or maintenance of a steady-state level of an endogenous protease inhibitor, whereby the rise in the level or maintenance of the steady-state level of the endogenous protease inhibitor indicates that the compound is effective in reducing the breakdown of endogenous protease inhibitors in the brain.
  • baseline levels of endogenous protease inhibitors are determined in samples of brain cortex, some brain samples are contacted with a test compound and other brain samples are controls. Finding elevated or maintained levels of endogenous protease inhibitors in the samples of brain contacted with the compound compared to controls indicates that the compound reduces the breakdown of endogenous protease inhibitors and is effective in treating neuropsychiatric diseases.
  • the present invention provides a composition comprising a protease inhibitor and a neuraminidase inhibitor in a pharmaceutically acceptable carrier.
  • the composition can include one or more protease inhibitors selected from the group of aspartic protease inhibitors, serine protease inhibitors, cysteine protease inhibitors and aminopeptidase inhibitors.
  • protease inhibitors examples include but are not limited to Nelfinavir, Saquinavir, Indinavir, Amprenavir, Ritonavir, Aprotinin, Pepstatin, AG1776, ABT-387, Beta-secretase inhibitors, AEBSF, Leupeptin, Elastatinal, Serpins, Antipain, APMSF, PMSF, AG7088, E-64, Betastatin and Amastatin.
  • the composition can include one or more neuramimdase inhibitors.
  • neuraminidase inhibitors examples include but are not limited to Zanamivir, Oseltamivir, RWJ-270201, GS 4071 and GS 4104. Suggested dosages and routes of administration of the protease inhibitors and neuraminidase inhibitors are shown in Tables 2, 3 and 4.
  • EXAMPLE 1 cN-CAM localizes in human brain synaptosomes.
  • a section from the right human occipital cortex (49.4 g) was obtained 12 hours post-mortem and stored overnight at 4°C, diluted 20% wt/vol in solution A (0.32 M sucrose solution with lmM of NaHCO3, 1 mM MgC12, and 0.05 mM of CaC12 with protease inhibitors) and homogenized by 12 strokes with a glass-Teflon homogemzer. The homogenate was filtered with a nylon mesh screen sieve and centrifuged at a low speed 1475 x g. The supernatant was saved and the resultant pellet (PI) was resuspended with solution A and centrifuged at 755 x g.
  • solution A 0.32 M sucrose solution with lmM of NaHCO3, 1 mM MgC12, and 0.05 mM of CaC12 with protease inhibitors
  • This supernatant was combined with the first supernatant and the combined supernatants (SI A) were centrifuged at 17,500 x g.
  • the resultant pellet (P2) containing synaptosomes and mitochondria was resuspended in solution B (solution B is 0.32M sucrose solution with lmM NaHCO3) and loaded into a sucrose density gradient of 0.85, 1.0, 1.2M and centrifuged for 2 hours at 100,000 x g.
  • the band between 1.0 and 1.2 M sucrose which contains synaptosomes (SX) was saved as well as the other sucrose fractions S3 (top), S4 (0.85M), S5 (1.0M), and S6 (1.2M).
  • the synaptosome band (SX) was combined with 4 volumes of solution B and centrifuged at 48,200 x g for 20 min.
  • the pellet (P3) was resuspended with solution C (0.16M sucrose with 6mM Tris-HCl containing 0.5%> Triton X-100) and centrifuged at 48,200 x g.
  • the supernatant was saved (S7) and the pellet (P3) was resuspended in solution B (S8) and loaded on another sucrose gradient density with density steps of 1.0 (S10), 1.5 (SI 1) and 2.0 M (S12), and centrifuged for 2 hours at 275,000 x g.
  • the band between 1.5 and 2.0 M contains the post synaptic densities, and the floating band at the top contains the synaptosomal membranes (S9).
  • Rotors are Sorvall SS-34 (17,500 g) and Beckman T40I rotors(l 00,000 x g and 275,000 x g)- N-CAM 180 is found in both non-synaptosomal and synaptosomal fractions (lanes 3 versus 4,5,6 shown in Figures 1 A and IB). The synaptosome fraction is further delineated by synaptophysin reactivity in lanes 4,5,6.
  • N-CAM 180 the breakdown fragment of N-CAM is also found in the synaptosome in both unconcentrated and concentrated samples, while the preponderance of N-CAM 180 disappears.
  • the disappearance of N-CAM 180 can be quantified from the film images by calculating a ratio between the N-CAM 180 and N-CAM 105-115.
  • enzyme breakdown is apparent in the synaptosomes as there is a decrease in the N-CAM 180 / N-CAM 105- 115 ratio following concentration in an Amicon filter (MW cut-off 3,000) and removal of the protease inhibitors.
  • EXAMPLE 2 cN-CAM occurs in brain via proteolytic mechanism converting N-CAM 180 to cN- CAM as shown above in the synaptosome.
  • EXAMPLE 3 cN-CAM is colocalized to particles in the brain that correspond to synapses without a particular limitation to a type of synapse.
  • Schizophrenia is associated with a high level of "immature" synapses defined as an elevated ratio of cN-CAM /synaptophysin levels 3 .
  • Cytosolic sample of brain homogenate (described below in Example 5) was ultracentrifuged for 120 min at 38,000 rpm in a Beckman SW40Ti rotor at 4 °C through a 0.5 M, 1.0 M and 1.2M sucrose density step gradient and analyzed for N- CAM and synaptophysin by Western immunoblot. A pellet was also obtained and analysed for N-CAM immunoreactivity by immunoblot. The pellet in 1.2 M sucrose was resuspended and centrifuged for 60 min at 38,000 rpm at 4 °C in the Beckman SW40Ti rotor.
  • the overlay of PBS was removed except approximately 0.5 ml of PBS and the resulting pellet was resuspended by pipetting up and down.
  • a 100 microliter aliquot was treated with 1.9 ml of NP-40 detergent (5% solution in PBS, pH 7.4 with protease inhibitors) or control treatment consisted of 1.9 ml of PBS (phosphate buffered saline) with protease inhibitors.
  • the tubes were then incubated at 4 °C on a rocker for 30 min. Each treated aliquot was overlaid on a step sucrose density gradient (4M, 3M, 2M, 1.5 M, IM sucrose) with protease inhibitors in PBS.
  • the tubes were topped off with 0.3 M sucrose and centrifuged at 30,000 rpm at 4°C for 60 min. One ml fractions from each tube were removed and the absorbance at 280 nm measured against a IM sucrose blank. Each sucrose fraction was run on a gel (70 ⁇ l sample and 30 ⁇ l 2x denaturing solution) and analyzed for N-CAM and synaptophysin.
  • the high density particles identified by ultracentrifugation of cytosolic brain were immunostained in chamber slides with antibodies to N-CAM and synaptophysin. All steps were performed at room temperature in an eight well chamber slide precoated with 0.1% BSA for 15 min, washed, and allowed to air dry in a dust free environment. An aliquot of the N-CAM enriched sucrose gradient sample was diluted 1 : 10 in PBS and 2 ⁇ l was spotted into the center of each chamber. The slide was fixed with 20 ⁇ l of 5% paraformaldedyde in PBS pH 7.4 for 10 min and washed with 200 ⁇ l of PBS three times.
  • N-CAM primary antibody (1:1000) and synaptophysin (1:1000) were added for 45 min at room temperature followed by three PBS washes and a final incubation with 100 ⁇ l of secondary antibody (1 : 250 anti-rabbit IgG FITC and anti-mouse rhodamine, Boehringer Mannheim Corp.) for 40 min followed by three PBS washes.
  • the chambers were removed and an anti-fade solution (Molecular Probes, OR) was added prior to coverslip.
  • the immunostained particles were imaged by confocal laser scanning microscopy (Zeiss Model 410, Germany).
  • a suspension of particles from sucrose density gradient ultracentrifugation was fixed with 2% glutaraldehyde in PBS on ice for 20 min.
  • the particles were pelleted at 10,000 g for 1 hour and the pellet was resuspended in 25 ⁇ l PBS and then mixed with 25 ⁇ l of 3%> agarose at 60 °C.
  • a 1 ⁇ l droplet of agarose-particle mixture was solidified on parafilm, and cut into 1 - 2 mm cubes. The cubes were rinsed for 15 minutes three times in PBS and then fixed with 1% osmium tetroxide in PBS for 1 hour, and rinsed 3 x lOmin in PBS.
  • the cubes were en block stained with 2% uranyl acetate for 1 hour, dehydrated through a series of 30%, 50%, 70%, 80%, 95%, and 100% ethanol solutions for 5 min each.
  • the dehydrated cubes were infiltrated overnight at 55 °C with unaccelerated Durcupan ACM resin (Electron Microscopy Sciences, Fort Washington, PA), embedded into a flat mold with accelerated Durcupan resin and polymerized at 55°C for 72 hours.
  • Ultrathin sections of 90 nm from two different cubes were prepared on an Ultracut E ultramicrotome. The sections were stained with uranyl acetate and lead citrate, and viewed with Zeiss 600 EM10A transmission electron microscope at 60 kV.
  • EXAMPLE 4 cN-CAM is strongly related to progressive ventricular enlargement in patients with schizophrenia.
  • CSF was collected in the lateral decubitus position in 1 ml aliquots, which were immediately (at bedside) immersed in dry ice, with subsequent maintenance at -70 °C (or in dry ice during shipping) until the time of assay.
  • the eighth 1 ml sample of CSF was used for the assessment of CSF N-CAM.
  • the results showed a decrease in CSF N-CAM in first episode patients 5 .
  • the patients were also given a Magnetic Resonance Imaging brain scan and then followed up for a period of 2 years and underwent a second scan using the same methodology 6 .
  • Modulation of polysialic acid on N-CAM decreases protease resistance to breakdown.
  • Membrane "extracts" and "cytosolic" tissue extracts of human occipital cortex were prepared as described 8 with slight modifications 9 .
  • Frozen pulverized occipital cortex ⁇ 50 g was suspended in 1000 ml of cold 0.05 M Tris buffered saline (TBS, pH 7.4) with protease inhibitors: antipain (4 ⁇ g/ml), pepstatin A (2 ⁇ g/ml), aprotinin (2 ⁇ g/ml), leupeptin (2 ⁇ g/ml), and phenyl methyl sulfonyl fluoride (0.1 ⁇ g/ml).
  • the solution was homogemzed (Tissumizer; Tekmar, Cincinnati OH) for 5 x 10 sec pulses in a 4°C ice bath and 30 sec cooling interval between pulses.
  • the homogenate was centrifuged for 30 min at 42,000 x g at 4°C.
  • the clear supernatant with visible lipid removed was the "cytosolic" fraction.
  • the pellet was resuspended and washed with cold TBS-protease inhibitor cocktail and re-centrifuged at 42,000 x g for 30 min at 4°C. The supernatant was discarded and the pellet extracted in cold TBS + protease inhibitor + 1% NP-40 detergent.
  • Neuramimdase treatment of cytosolic and membrane brain extracts was accomplished by using neuraminidase enzyme from Vibrio cholerae ( ⁇ 2.49 Units/ ml, Fluka) in pH 5.5, 0.15 M NaCI, 4 mM CaCl 2 that was diluted with 50 mM sodium acetate, 4 mM CaCl 2 and 0.2 mM EDTA, (pH 5.0; Buffer A) to 1.25 U / ml. Aliquots of cytosolic or membrane extracts of brain (100 ⁇ l) were mixed with 100 ⁇ l of diluted neuramimdase and incubated for 18 hrs at 37 °C. Control aliquots of cytosolic and membrane extracts were treated with 100 ⁇ l of Buffer A. Additional controls were refrozen for 18 hrs. Samples were analyzed by immunoblot for N-CAM with antibody 3732.
  • the procedure used previously for detection of N-CAM, synaptophysin, and actin was used as detailed 9 . Briefly, the cytosolic and membrane extracted fractions were diluted (1 volume sample : 1 volume loading buffer, SepraSol, Integrated Separation Systems, Natick, MA), denatured at 95 °C and 40 - 80 ⁇ g of protein separated by 7.5% SDS-PAGE 10 . The immunoblots were probed with N-CAM antisera, actin, or synaptophysin. Primary antibody binding to the blotted membrane was visualized with a secondary antibody (goat anti-rabbit IgG horseradish peroxidase conjugate, Sigma, St.
  • Neuraminidase treatment produces a minor alteration of cN-CAM resulting in a faster migrating immunoreactive band indicating partial glycosylation of cN-CAM (Fig 7A), while neuraminidase incubation with membrane extracts resulted in removal of higher MW N-CAM 180.
  • This result in which we might expect a preservation of N- CAM 180 and only a minor modification from adult brain, indicates that upon removal of the polysialic acid residues in N-CAM 180 parent molecules the resistance to protease breakdown is lost.
  • cN-CAM as expected from breakdown of the N-CAM 180 there is only a partial conversion to cN-CAM with the majority converted to a novel dN-CAM ( ⁇ 51 kDa peptide).
  • N-CAM 180 appears to degrade via endogenous neuraminidase to N-CAM 105-115, but upon first removing polysialic acid the breakdown continues to a fragment now identified as dN-CAM (-51 kDa peptide).
  • the conversion product of N-CAM 180 to N-CAM 105-115 is found in higher abundance in neuraminidase treated cytosolic extracts of brain, while the breakdown of.N-CAM 180 via removal of polysialic acid and subsequent breakdown to dN-CAM ( «51 kDa peptide) occurs largely in membrane bound N-CAM isoforms (Figure 7B).
  • Fresh or frozen human brain cortex is dissected free of pia mater layer and any obvious blood vessels on the surface of the cortex. Pulverize the cortex in a sufficient quantity of liquid Nitrogen to a friable state with mortar and pestle, and accurately weigh out replicate samples of pulverized brain sample while keeping all vessels frozen in dry ice. Place the quantity of protease inhibitor or neuramimdase inhibitor in a PBS buffer of final pH 7.4 and maintain buffer on ice. Add the varying concentrations and combinations of protease inhibitors and/or neuraminidase inhibitors to separate pulverized brain in tubes in a final volume to yield a 20% weight of brain tissue / buffer volume. Allow the tubes to adjust to room temperature, and incubate at standard room temperature conditions for varying time points.
  • the end-point of the assay is accomplished by addition of a 2x denaturing reducing sample buffer such as commercially available and previously published, and heat 3 min at 95 degrees C. Proceed to standard SDS-PAGE and immunoblot conditions to determine the breakdown products of parent molecule N-CAM. Use appropriate control incubations for temperature breakdown and buffer breakdown without protease inhibitors and/or neuraminidase inhibitors. The results can be quantitatively assessed by densitometry of immunoblots as previously published which yields a linear response within a loading range of proteins ( ⁇ 1 - 100 ⁇ g of protein) depending on gel capacity used and concentrations of antibody and detection substrates which are empirically determined with standard methods.
  • the modulation of N-CAM fragments occurs in synaptosomes in the brain.
  • the assay used above for screening protease inhibitors can be applied to the synaptosomal preparation from human brain as a model system for determining partial therapeutic efficacy in preclinical trials of neuraminidase inhibitors and protease inhibitors for treatment of the neuropsychiatric diseases described herein. Further, as described below, in vivo monitoring of therapeutic effect can be made by monitoring breakdown fragments of N-CAM in CSF. See Figures IA and IB.
  • a reduction in the progression of chronic schizophrenia is demonstrated with serine-protease inhibitors, aspartic-protease inhibitors, cysteine-protease inhibitors, aminopeptidase-protease inhibitors and neuraminidase inhibitors which are compounds shown to reduce N-CAM breakdown in vivo and in vitro. Breakdown products of N- CAM in humans have been shown to correlate with positive symptoms (psychosis) and ventricular enlargement (accelerated brain aging) in patients with schizophrenia. Serine protease inhibitors reduce the breakdown of N-CAM in rodent brain 11 . Patients are afforded the opportunity to halt and reverse the progressive features of schizophrenia: positive symptom (psychosis), ventricular enlargement (accelerated brain aging), and cognitive decline in a controlled clinical trial.
  • Therapeutically appropriate dosages for (A) aspartic protease inhibitors, (B) serine protease inhibitors, (C) cysteine protease inhibitors, (D) aminopeptidase inhibitors and (E) neuraminidase inhibitors are listed in Tables 2, 3 and 4.
  • the compounds or combinations of compounds from these classes of compounds (A, B, C, D and E) are administered in dosages that are effective in reducing the breakdown products of N-CAM in CSF as measured prior to treatment and during treatment.
  • CSF N-CAM breakdown products are correlated with improvements in cognitive functioning (4 weeks and later), positive and negative symptom reduction (4 weeks and later), and longer term MRI changes in CSF ventricular space at the two year clinical trial time point.
  • the present treatment is applicable to any patient, usually between the ages of 18 and 65 years old who has a history of a Psychotic Disorder, NOS; Schizophrenia; Schizoaffective Disorders; or other Neuropsychiatric Disorders as defined herein.
  • the patient will preferably be abstinent from alcohol and all illicit drugs for at least 30 days at the time of initial lumbar puncture and 16 week lumbar puncture.
  • a patient can be stabilized on a "typical” or “atypical” neuroleptic treatment regimen for 30 days prior to treatment according to the present invention. If the patient has never been treated with neuroleptics, under the discretion of medical responsible psychiatrist, neuroleptics may be prescribed as part of the present protocol.
  • the patient should exhibit no uncorrectable loss of hearing or eyesight that precludes psychometric testing and should have the ability to comprehend instructions or respond to test items of the Repeatable Battery for the Assessment of Dementia (RBAD) during baseline administration and Mini Mental Status Examination.
  • RBAD Repeatable Battery for the Assessment of Dementia
  • Aprotinin data from clinical trials indicate that it is generally well tolerated in humans, 12 with few adverse events. Hypersensitivity reactions occur in ⁇ 0.1 to 0.6%> of patients receiving aprotinin for the first time. Clinical evidence to date supports the use of aprotinin over its competitors in patients at high risk of hemorrhage, in those for whom transfusion is unavailable or in patients who refuse allogeneic transfusions.
  • Amprenavir may be associated with acute hemolytic anemia, diabetes mellitus, and hyperglycemia, but the drug's effects on patients lipid profiles at this point appears clinically insignificant.
  • the most frequently reported adverse events in clinical trials were nausea, diarrhea, vomiting, rash, and perioral paresthesia. Severe and life-threatening skin reactions, including Stevens- Johnson syndrome, occurred in 1% of patients treated with amprenavir.
  • Amprenavir is taken twice a day, with or without food, but it should not be taken with a high-fat meal, as that would decrease the absorption of the drug.
  • Drug route doses, frequency, duration
  • Doses are adjusted to within IND and FDA recommended levels (e.g., see Tables 2, 3, 4 and 5) in an ascending phase for 2 weeks prior to achieving therapeutic dosage levels.
  • Patients receive a protease inhibitor or a neuraminidase inhibitor or a combination of a protease inhibitor and a neuraminidase inhibitor in a safely administered dosage according to physician recommendation.
  • Lumbar taps can cause headaches in 30-40% of patients within the first few days after a lumbar puncture. Usually the headaches disappear without treatment beyond a mild pain reliever. Prolonged headaches, lasting longer than seven day, occur in about 0.5 to 2 percent of patients. These prolonged headaches usually taper off within two weeks of the lumbar puncture. For prolonged headache, a blood patch, that is a small injection of blood into the area of the back where the lumbar puncture was performed is sufficient to seal any CSF leak and cause the headaches to disappear. The only other risk to CSF lumbar tap is temporary double vision and infection.
  • Magnetic Resonance Imaging (MRI) of the brain uses a magnetic field and radio waves and is more sensitive to structural changes than X-ray and carries no radiation risk.
  • a patient is placed in a cylinder for up to one hour with monitoring and asked to remain still for 10-15 minutes at a time.
  • Evaluations prior to drug administration can include the following: Brain MRI scan, CSF lumbar tap (10 ml), Neuropsychological instruments to include Repeatable Battery for Assessment of Dementia, Mini Mental Status Exam, Wechsler Memory Scale, Wisconsin Card Sort, Trails A and Trails B, Finger Tapping, Rey Auditory Verbal Learning Test, Olfactory Identification Test, Subtests of the Wechsler Adult Intelligence Scale (Digit Symbol, Block Design, Arithmetic, Similarities, Vocabulary), and the Wide Range Achievement Test - Reading, as detailed in Handbook of Neuropsychological Testing (Muriel Lezak). Psychiatric symptom assessment scale (Scale for Assessment of Negative Symptoms (SANS) and Scale for Assessment of Positive Symptoms (SAPS) by Nancy Andreasen, Univ. of Iowa.
  • SANS Scale for Assessment of Negative Symptoms
  • SAPS Scale for Assessment of Positive Symptoms
  • Evaluations after 4 and 8 weeks of drug admimstration can include the following: Repeatable Battery for Assessment of Dementia, Mini Mental Status Exam, Finger Tapping, Olfactory Identification Test, Digit Symbol, Block Design, Scale for Assessment of Negative Symptoms (SANS) and Scale for Assessment of Positive Symptoms (SAPS). Evaluations after 12 weeks of drug administration can include the following: CSF lumbar tap, optional (10 ml)
  • Evaluations after 16 weeks of drug administration can include the following: Neuropsychological instruments to include Repeatable Battery for Assessment of Dementia, Mini Mental Status Exam, Wechsler Memory Scale, Wisconsin Card Sort, Trails A and Trails B, Finger Tapping, Rey Auditory Verbal Learning Test, Olfactory Identification Test, Subtests of the Wechsler Adult Intelligence Scale (Digit Symbol, Block Design, Arithmetic, Similarities, Vocabulary), and the Wide Range Achievement Test - Reading.
  • Psychiatric symptom assessment scale Scale for Assessment of Negative Symptoms (SANS) and Scale for Assessment of Positive Symptoms (SAPS).
  • Evaluations after 52 weeks of drug administration can include the following: (optional) CSF lumbar tap, 10 ml (optional) MRI scan
  • Evaluations after 104 weeks of drug administration can include the following: (optional) CSF lumbar tap, 10 ml (optional) MRI scan
  • Occipital cortex obtained from squirrel monkey was extracted by the same method for cytosolic and membrane fractions of human brain described herein, and for example, see Example 9.
  • N-CAM, c-N-CAM or other fragments of N-CAM can be measured in CSF pre- and post- injection of protease inhibitor to determine the degree of inhibition of release of cN-CAM, N-CAM or other fragments into CSF.
  • Protease inhibitors that were assayed individually were: Aprotinin, AEBSF, Antipain, Bestatin, Amastatin, Elastatinal, AMPSF, Leupeptin, Pepstatin A, PMSF, L-trans- epoxysuccinyl-leucylamide-(4-guanido)-butane, and Amastatin ([(2S, 2R)]-3-Amino-2- hydroxy-5-methylhexanoyl]-Val-Val-Asp-OH). All protease inhibitors were obtained from Sigma.
  • protease inhibitors that showed the most activity in prevention of the breakdown of N-CAM 180 and subsequent release of cN-CAM were leupeptin (Figure 8B) and antipain (Figure 8C).
  • antipain 4 ⁇ g/ml
  • pepstatin A 2 ⁇ g/ml
  • aprotinin 2 ⁇ g/ml
  • leupeptin 2 ⁇ g/ml
  • phenyl methyl sulfonyl fluoride 0.1 ⁇ g/ml.
  • the brain tissue was placed in the protease inhibitor solution and homogenized (Tissumizer; Tekmar, Cincinnati OH) for 5 x 10 sec pulses in a 4 °C ice bath with a 30 sec cooling interval between pulses.
  • the homogenate was centrifuged for 30 min at 42,000 x g at 4°C.
  • the clear supernatant with visible lipid removed was the "cytosolic" fraction.
  • the pellet was resuspended and washed with cold TBS-protease inhibitor cocktail and re- centrifuged at 42,000 x g for 30 min at 4°C. The supernatant was discarded and the pellet extracted in cold TBS + protease inhibitor + 1% NP-40 detergent.
  • FIG. 8 A shows the standard protease inhibitor cocktail effects
  • Figure 8B shows the addition of leupeptin (10 ⁇ M concentration)
  • Figure 8C shows the addition of antipain to a 10 ⁇ M concentration.
  • EXAMPLE 10 Measurement of neuroserpin gene by microarray in patients with schizophrenia was used to screen 1128 human genes relevant to brain function. Two brain regions were investigated using pools of total RNA from patients with schizophrenia and controls: dorsolateral prefrontal cortex (Brodmann area (BA) 46 and prefrontal cortex (PFC), (BA) 9). A neuroarray was developed and the details of the development of the NTA-Neuroarray are available at URL htto://www.grc.nia.nih.gov/branches/rrb/dna.htm) and are reported (Vawter, et al., Brain Research Bulletin, 2001).
  • the 15 samples of the PFC obtained from controls formed 3 pools of 5 samples and similarly 3 matched pools of patients with schizophrenia were formed.
  • the total RNA was extracted from each brain sample by first pulverizing the frozen tissue in liquid N 2 with a mortar and pestle to a fine powder.
  • 0.1 - 0.2 g of the brain powder was homogenized in ice-cold Trizol (Life Technologies Inc, Rockville MD) using a Tissumizer (Tekmar, Cincinnati, OH) at #40 speed for 3 x 30 sec pulses and 30 sec cooling.
  • the samples were processed by the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987) according to the procedure recommended for Trizol extraction, i.e. 0.2 vol chloroform was added to the Trizol-brain homogenate, samples hand shaken for 30 sec vigorously, and centrifuged at 12,000 x g for 20 min at 4 °C.
  • the supernatant was transferred to a fresh tube and the RNA precipitated with 0.5 vol isopropyl alcohol, centrifuged as above, and the pellet was extracted with 75% ethanol.
  • the ethanol mixture was centrifuged at 7,500 x g for 20 min at 4 °C.
  • the supernatant was decanted and the pellet was resuspended in 75% ethanol, and recentrifuged at 10,000 x g for 10 min at 4 °C.
  • the resulting pellet was left slightly wet and partially dried at room temperature under vacuum.
  • the pellet was resuspended in TE buffer, pH 7.5 and a 1:100 dilution was made in DEPC -treated H 2 0, and the A 260 and A 260 /A 280 ratio obtained in a Beckman spectrophotometer (DU-64, Fullerton CA).
  • the yield of total RNA was in the range between 20 - 90 ⁇ g per 100 mg of brain tissue for all brain regions.
  • a sample of the total RNA was diluted to 1 ⁇ g/ ⁇ l with a 10X RNA sample buffer (Quality Biologicals, Gaithersburg MD) and run on a denaturing 1.2% formaldehyde agarose gel. The resulting 28S and 18S ribosomal bands were visualized as well as any streaking of DNA or degradation of samples.
  • a 260 /A 280 ratio was usually > 1.9 or the sample was considered for re-extraction.
  • a pool of total RNA (20 ⁇ g) was formed for each group using equal amounts of total RNA from each individual.
  • the [33]" P-dCTP-cDNA is purified through a spin column by size separation (BioSpin, Bio-Rad, CA) from [33]" P-dCTP and the heat denatured probe ( ⁇ 5 x 10 6 cpm) is diluted in 4 ml of Microhyb solution (Research Genetics) and hybridized to the neuroarray for l6 - 18 h at 50 °C with rotation. Two washes with 2X SSC at room temperature are carried out to remove unhybridized probe. The neuroarray is placed under saran wrap and exposed to a low energy phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 1 - 5 days and scanned in a Phosphorimager 860 (Molecular Dynamics) at 50 ⁇ m resolution.
  • a z-score normalization method was devised and applied to each hybridization image. This method involves calculating a distribution of z-scores for all genes in each array, and employing the differences of the z-scores (z-ratio) between the two conditions to search for genes for which expression is changed in schizophrenia.
  • the mean z-score difference for all genes on two neuroarrays (S - C) is 0; however, the standard deviation of the z differences distribution ranges between ⁇ 0.2 - 0.4 for each neuroarray.
  • Neuroserpin gene has the largest average down-regulation of gene expression among all 1128 genes surveyed ( Figure 2).
  • Neuroserpin is an endogenous serine protease inhibitor.
  • This deficiency in expression of the gene for an endogenous serine protease inhibitor in the PFC of patients with schizophrenia can be responsible for elevation in cN-CAM.
  • the effect is most noticeable in pools of patients that have the greatest male composition, in that pool 3 has the fewest male patients and showed the smallest change in neuroserpin.
  • VASE-containing N-CAM isoforms are increased in the hippocampus in bipolar disorder but not schizoplirenia. Exp Neurol, 154(1), 1-11.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Emergency Medicine (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Psychiatry (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention porte sur un procédé de traitement d'une maladie neuropsychiatrique caractérisée par un taux anormalement élevé d'un fragment d'une isoforme d'une molécule d'adhésion cellulaire neuronale, N-CAM, dans le cerveau ou dans le fluide cérébro-spinal du sujet affecté. Ce procédé consiste à administrer une quantité efficace d'un point de vue thérapeutique d'au moins un composé sélectionné dans le groupe comprenant des inhibiteurs de protéase et de neuraminidase. L'invention porte également sur un procédé efficace de surveillance du traitement, ainsi que sur un procédé de criblage de composés efficaces dans le traitement d'une maladie neuropsychiatrique associée à un taux anormalement élevé d'un fragment d'une molécule d'adhésion cellulaire neuronale du fluide cérébro-spinal du sujet affecté. Des fragments d'une isoforme de N-CAM du fluide cérébro-spinal de sujets affectés sont également décrits.
PCT/US2001/002417 2000-01-25 2001-01-25 Traitement des maladies neuropsychiatriques au moyen d'inhibiteurs de protease et de neuraminidase Ceased WO2001054729A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2001231137A AU2001231137A1 (en) 2000-01-25 2001-01-25 Treatment of neuropsychiatric disease with protease and neuraminidase inhibitors
US10/182,162 US20030211545A1 (en) 2001-01-25 2001-01-25 Treatment of neuropsychiatric disease with protease and neurominidase inhibitors
US11/355,257 US20060205650A1 (en) 2000-01-25 2006-02-14 Treatment of neuropsychiatric disease with protease and neuraminidase inhibitors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17797100P 2000-01-25 2000-01-25
US60/177,971 2000-01-25

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/355,257 Continuation US20060205650A1 (en) 2000-01-25 2006-02-14 Treatment of neuropsychiatric disease with protease and neuraminidase inhibitors

Publications (1)

Publication Number Publication Date
WO2001054729A1 true WO2001054729A1 (fr) 2001-08-02

Family

ID=22650670

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/002417 Ceased WO2001054729A1 (fr) 2000-01-25 2001-01-25 Traitement des maladies neuropsychiatriques au moyen d'inhibiteurs de protease et de neuraminidase

Country Status (2)

Country Link
AU (1) AU2001231137A1 (fr)
WO (1) WO2001054729A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004113280A1 (fr) * 2003-06-20 2004-12-29 Glaxo Group Limited Inhibiteurs de transporteurs de glyt1 et leurs utilisations dans le traitement de troubles neurologiques et neuropsychiatriques
DE102006036373A1 (de) * 2006-08-02 2008-02-07 Johannes-Gutenberg-Universität Mainz Arzneimittel gegen LCT-Vergiftungen
WO2024255128A1 (fr) * 2023-06-15 2024-12-19 苏州志恒生物科技有限公司 Utilisation du ritonavir dans la préparation d'un médicament pour le traitement d'un trouble psychologique provoqué par un événement traumatique

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07145161A (ja) * 1991-09-18 1995-06-06 Mercian Corp 新規セスキテルペン誘導体
US5854036A (en) * 1994-03-25 1998-12-29 Gruppo Lepetit S.P.A. Sesquiteterpenic dervatives
WO1999034789A1 (fr) * 1998-01-08 1999-07-15 Hoffman Keith B Utilisation d'inhibiteurs de serine protease pour inhiber la pathophysiologie et la neuropathologie chez un hote
WO1999040183A1 (fr) * 1998-02-06 1999-08-12 Human Genome Sciences, Inc. Serine-protease humaine et polypeptides serpin
CA2226919A1 (fr) * 1998-02-13 1999-08-13 Peter Sonderegger Neuroserpine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07145161A (ja) * 1991-09-18 1995-06-06 Mercian Corp 新規セスキテルペン誘導体
US5854036A (en) * 1994-03-25 1998-12-29 Gruppo Lepetit S.P.A. Sesquiteterpenic dervatives
WO1999034789A1 (fr) * 1998-01-08 1999-07-15 Hoffman Keith B Utilisation d'inhibiteurs de serine protease pour inhiber la pathophysiologie et la neuropathologie chez un hote
WO1999040183A1 (fr) * 1998-02-06 1999-08-12 Human Genome Sciences, Inc. Serine-protease humaine et polypeptides serpin
CA2226919A1 (fr) * 1998-02-13 1999-08-13 Peter Sonderegger Neuroserpine

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BARBEAU DAVID ET AL: "Altered level of a neural cell adhesion molecule isoform in schizophrenia.", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 20, no. 1-2, 1994, 24th Annual Meeting of the Society for Neuroscience;Miami Beach, Florida, USA; November 13-18, 1994, pages 623, XP000995551, ISSN: 0190-5295 *
DATABASE WPI Section Ch Week 199531, Derwent World Patents Index; Class B02, AN 1995-237182, XP002166070 *
VAWTER MARQUIS P ET AL: "CSF N-CAM in neuroleptic-naive first-episode patients with schizophrenia.", SCHIZOPHRENIA RESEARCH, vol. 34, no. 3, 30 November 1998 (1998-11-30), pages 123 - 131, XP000995532, ISSN: 0920-9964 *
VAWTER MARQUIS P ET AL: "Elevated concentration of N-CAM VASE isoforms in schizophrenia.", JOURNAL OF PSYCHIATRIC RESEARCH., vol. 34, no. 1, January 2000 (2000-01-01), pages 25 - 34, XP000995557, ISSN: 0022-3956 *
VAWTER MARQUIS P ET AL: "VASE-containing N-CAM isoforms are increased in the hippocampus in bipolar disorder but not schizophrenia.", EXPERIMENTAL NEUROLOGY, vol. 154, no. 1, November 1998 (1998-11-01), pages 1 - 11, XP000985978, ISSN: 0014-4886 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004113280A1 (fr) * 2003-06-20 2004-12-29 Glaxo Group Limited Inhibiteurs de transporteurs de glyt1 et leurs utilisations dans le traitement de troubles neurologiques et neuropsychiatriques
DE102006036373A1 (de) * 2006-08-02 2008-02-07 Johannes-Gutenberg-Universität Mainz Arzneimittel gegen LCT-Vergiftungen
WO2024255128A1 (fr) * 2023-06-15 2024-12-19 苏州志恒生物科技有限公司 Utilisation du ritonavir dans la préparation d'un médicament pour le traitement d'un trouble psychologique provoqué par un événement traumatique

Also Published As

Publication number Publication date
AU2001231137A1 (en) 2001-08-07

Similar Documents

Publication Publication Date Title
Ingebrigtsen et al. Increased serum concentrations of protein S-100 after minor head injury: a biochemical serum marker with prognostic value?
Alvarez et al. Blood levels of histamine, IL-1β, and TNF-α in patients with mild to moderate Alzheimer disease
Roy et al. Cerebrospinal fluid monoamine and monoamine metabolite concentrations in melancholia
Rubinow et al. CSF somatostatin in affective illness
Leung et al. Multiple symmetric lipomatosis (Launois‐Bensaude syndrome): effect of oral salbutamol
Duffau et al. Intraoperative unmasking of brain redundant motor sites during resection of a precentral angioma: evidence using direct cortical stimulation
Guerrini et al. A nonsense mutation of the ATRX gene causing mild mental retardation and epilepsy
Lauria et al. Clinical and magnetic resonance imaging findings in chronic sensory ganglionopathies
JPH05506990A (ja) プロテアーゼ　ネキシン―２の精製、検出、及び使用方法
WO1996003153A1 (fr) Procede de diagnostic de la maladie d'alzheimer
Angelini et al. Insulin-like growth factor-1 (IGF-1): relation with cognitive functioning and neuroimaging marker of brain damage in a sample of hypertensive elderly subjects
Brod et al. Combination therapy with glatiramer acetate (copolymer‐1) and a type I interferon (IFN‐α) does not improve experimental autoimmune encephalomyelitis
JP4908514B2 (ja) アルツハイマー病に特異的なｅｒｋ１／ｅｒｋ２リン酸化比の変化−アルツハイマー病に特異的な分子生物マーカー（ａｄｓｍｂ）
US5213962A (en) Purification, detection and methods of use of protease Nexin-2
Gerner et al. Cerebrospinal fluid cholecystokinin, bombesin and somatostatin in schizophrenia and normals
US11160788B2 (en) Caspase-1 inhibition and uses thereof for prevention and treatment of neurological conditions
Numakura et al. Hemizygous mutation of the peripheral myelin protein 22 gene associated with Charcot‐Marie‐Tooth disease type 1
US20160274126A1 (en) Biomarkers for the diagnosis of interstitial cystitis
Kalman et al. Serum and cerebrospinal fluid cystatin C levels in vascular and Alzheimer's dementia
WO2001054729A1 (fr) Traitement des maladies neuropsychiatriques au moyen d'inhibiteurs de protease et de neuraminidase
US20240012012A1 (en) Biomarker and treatment for vascular cognitive impairment and related conditions
US20030211545A1 (en) Treatment of neuropsychiatric disease with protease and neurominidase inhibitors
US7365049B2 (en) Afamin-containing compositions and methods of use
Konkoy et al. Acute administration of neuroleptics decreases neurotensin metabolism on intact, regional rat brain slices.
Graf et al. Diffusion‐weighted magnetic resonance imaging in boys with neural cell adhesion molecule L1 mutations and congenital hydrocephalus

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 10182162

Country of ref document: US

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP