CN118251415A

CN118251415A - Methods and products for treating or diagnosing schizophrenia

Info

Publication number: CN118251415A
Application number: CN202280071071.3A
Authority: CN
Inventors: 鲁白; 管小明
Original assignee: Fubei Biomedical Technology Beijing Co ltd; Tsinghua University
Current assignee: Fubei Biomedical Technology Beijing Co ltd; Tsinghua University
Priority date: 2021-10-29
Filing date: 2022-10-28
Publication date: 2024-06-25
Also published as: WO2023072228A1

Abstract

The application provides a medicine for preventing, alleviating and/or treating schizophrenia, comprising a tropomyosin-receptor-kinase-B (TrkB) agonist and an inhibitor against an early-in-life stress-related factor.

Description

Methods and products for treating or diagnosing schizophrenia

Technical Field

The application relates to the field of biological medicine, in particular to a method and a product for treating or diagnosing schizophrenia.

Background

Schizophrenia (SCZ) is a serious mental disorder affecting 2100 tens of thousands worldwide, creating a huge social and economic burden. SCZ patients may present difficulties in thinking, perception, emotion and language. SCZ episodes generally occur in the late pubertal phase (16 to 25 years) or in the early adulthood, which is an important age group for social and professional life (Addington, 2007). The life expectancy of SCZ patients is about 10 to 25 years shorter than healthy individuals. SCZ includes three clinical symptoms: positive symptoms (e.g., hallucinations, delusions, and psychosis), negative symptoms (e.g., social withdrawal, affective disorders, speech deficits), and cognitive deficits (e.g., poor executive function, impaired working memory, and attention problems) (Hustig and Norrie, 1998). There are two classes of known antipsychotics that can alleviate to some extent positive or negative symptoms, including first generation (e.g. haloperidol and chlorpromazine) and second generation antipsychotics (e.g. risperidone and olanzapine) (Remington, 2003). However, these drugs have little effect in improving cognitive impairment and often cause significant side effects (Mintz and Kopelowicz, 2007). Therefore, there is an urgent need to develop new drugs that can simultaneously alleviate three clinical symptoms of SCZ with less side effects.

Based on the mouse model of the susceptibility gene mutation of the schizophrenia, only certain 'surface' characteristics of the schizophrenia can be simulated, no single gene mutation can induce all symptoms of the schizophrenia, and no single gene can explain the etiology of the schizophrenia. Much evidence suggests that the pathophysiology of schizophrenia involves several key neurotransmitters including dopamine, glutamate, serotonin and gamma-aminobutyric acid (GABA). However, drug-induced models can only mimic some aspects of the schizophrenic phenotype. Thus, there is a great impediment to the study of schizophrenia due to the lack of good animal models. Although the pathogenesis of schizophrenia involves both genetic and environmental factors, it is not yet determinable which genetic factors and what environmental factors interact with each other to cause schizophrenia.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal differentiation and synaptic development, affecting adult development, synaptic transmission and plasticity. However, the genomic structure of the Bdnf gene is quite complex. There are a total of 9 promoters located upstream of the 5 'untranslated region (5' UTR) of the 9 small exons, and each promoter is spliced onto a common exon, exon 10, which encodes the precursor protein of precursor BDNF (pre-proBDNF protein) (Pruunsild et al, 2007). There is a great deal of evidence that different promoters drive the expression of different Bdnf transcripts in different brain regions, different cell types (neurons/glia cells), different subcellular compartments (e.g. somatic cells/dendrites) and different developmental stages (Baj et al, 2011; greenberg et al, 2009). In addition, the 9 Bdnf promoters are regulated by different transcription factors and epigenetic factors and are involved in a variety of physiological functions (Pruunsild et al, 2007). For example, selective disruption of Bdnf promoter I or Bdnf promoter II can result in thermogenic defects and obesity (McAllan et al, 2018; you et al, 2020). In addition, bdnf promoter IV (Hong et al, 2008; sakata et al, 2009 a), which drives activity-dependent BDNF transcription, plays a key role in behavioral maintenance by regulating GABA energy transfer (Jiao et al, 2011; sakata et al, 2013; sakata et al, 2009 a). However, it is not clear which Bdnf promoter plays a key role in SCZ.

Thus, there is an urgent need to deepen understanding of SCZ mechanism, and there is also a need for better animal models.

Disclosure of Invention

The inventors found that the deficiency in BDNF expression driven by promoter VI, coupled with early life stress, together resulted in a schizophreniform-like internal phenotype. Promoter VI mutant mice (Bdnf-e 6-/-) that were postnatally exposed to stress scenes such as hypoxia or social isolation show defects in social interactions, spatial memory and sensorimotor gating such as prepulse inhibition (PPI). Neither early life stress nor Bdnf-e6 (Bdnf-e 6 mRNA and BDNF protein translated therefrom) alone would cause these abnormalities. In addition, postnatal stress causes elevated blood glucocorticoid (e.g., cortisol and/or corticosterone) levels in Wild Type (WT) mice; administration of glucocorticoids (e.g., cortisol and/or corticosterone) to Bdnf-e 6-/-mice experiencing stress early in life also results in pre-pulse suppression (PPI) deficits and social dysfunction. Furthermore, glucocorticoid (e.g., cortisol and/or corticosterone) antagonists or TrkB agonist treatment rescue PPI deficiency in hypoxic or socially isolated Bdnf-e 6-/-mice.

In one aspect, the present application provides a medicament for preventing, alleviating and/or treating schizophrenia. The pharmaceutical product may include a tropomyosin-receptor-kinase B (TrkB) agonist and a glucocorticoid (e.g., cortisol or corticosterone), any of which may also be referred to as a cor inhibitor in the present application.

In some embodiments, the TrkB agonist comprises a TrkB agonist antibody or antigen binding fragment thereof.

In some embodiments, the TrkB agonist is capable of specifically binding to human TrkB.

In some embodiments, the antigen binding fragment comprises a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb.

In some embodiments, the TrkB agonist comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and the LCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81, and 91.

In some embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92.

In some embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93.

In some embodiments, the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98.

In some embodiments, the TrkB agonistic antibody comprises a light chain constant region, and the light chain constant region is a human igκ constant region or a human igλ constant region.

In some embodiments, the TrkB agonist comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94.

In some embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95.

In some embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96.

In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97.

In some embodiments, the TrkB agonist antibody comprises a heavy chain constant region, and the heavy chain constant region is a human IgG constant region.

In some embodiments, the glucocorticoid inhibitor is capable of reducing the amount of glucocorticoid. For example, the glucocorticoid inhibitor may be a CORT inhibitor, possibly capable of reducing the amount of the CORT.

In some embodiments, the glucocorticoid inhibitor is capable of inhibiting the activity of a glucocorticoid. For example, the glucocorticoid inhibitor may be a CORT inhibitor, possibly capable of inhibiting the activity of the CORT.

In some embodiments, the glucocorticoid inhibitor (e.g., a CORT inhibitor) comprises mifepristone (RU-486) or a functional derivative thereof.

In another aspect, the present application provides a method for preventing, alleviating and/or treating schizophrenia in a subject in need thereof. The method may comprise administering a TrkB agonist and/or glucocorticoid (e.g., cortisol, corticosterone, or CORT) inhibitor to a subject, and the subject has a reduced level and/or activity of BDNF (e.g., BDNF-e 6) expression and an increased level and/or activity of glucocorticoid (e.g., cortisol, corticosterone, or CORT).

In some embodiments, the subject has a defect in promoter VI of the Bdnf gene.

In some embodiments, the subject has undergone post-natal stress.

In some embodiments, the post-natal stress comprises post-natal hypoxia and/or social isolation.

In some embodiments, the subject has reduced levels and/or activity of BDNF (e.g., BDNF-e 6) expression in the hippocampus, prefrontal cortex, and/or hypothalamus.

In some embodiments, the glucocorticoid (e.g., cortisol, corticosterone, or CORT) inhibitor is capable of reducing the amount of glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the glucocorticoid inhibitor is a CORT inhibitor.

In some embodiments, the glucocorticoid (e.g., cortisol, corticosterone, or CORT) inhibitor is capable of inhibiting the activity of a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the glucocorticoid inhibitor may be a CORT inhibitor.

In some embodiments, the CORT inhibitor comprises mifepristone (RU-486) or a derivative thereof.

In another aspect, the application provides a method for determining whether a subject has or is at risk of developing schizophrenia, comprising determining the level and/or activity of BDNF (e.g., BDNF-e 6) expression in the subject, and determining the level and/or activity of a glucocorticoid (e.g., cortisol, corticosterone or CORT) in the subject.

In some embodiments, the expression level and/or activity of BDNF (e.g., BDNF-e 6) is determined from a blood sample of the subject.

In some embodiments, the level and/or activity of the glucocorticoid (e.g., cortisol, corticosterone, or CORT) is determined from a blood sample of the subject.

In some embodiments, the method further comprises selecting a subject having a reduced level and/or activity of BDNF (e.g., BDNF-e 6) expression and an elevated level and/or activity of a glucocorticoid (e.g., cortisol, corticosterone, or CORT), the selected subject being determined to be predisposed to or at risk of developing schizophrenia.

In some embodiments, the method further comprises determining whether the subject has a defect in promoter VI of the Bdnf gene.

In some embodiments, the method further comprises determining whether the subject has experienced post-natal stress.

In some embodiments, the method comprises determining the expression level and/or activity of BDNF (e.g., BDNF-e 6) in the hippocampus, prefrontal cortex, and/or hypothalamus of the subject.

In some embodiments, the method further comprises administering to the selected subject a TrkB agonist and/or a glucocorticoid (e.g., cortisol, corticosterone, or CORT) inhibitor.

In another aspect, the application provides a system for determining whether a subject has or is at risk of developing schizophrenia, the system comprising: a first module for determining whether the subject has a reduced level and/or activity of BDNF (e.g., BDNF-e 6) expression, and a second module for determining whether the subject has an elevated level and/or activity of a glucocorticoid (e.g., cortisol, corticosterone, or CORT).

In some embodiments, the BDNF (e.g., BDNF-e 6) is from the blood of the subject.

In some embodiments, the glucocorticoid (e.g., cortisol, corticosterone, or CORT) is from the blood of the subject.

In another aspect, the application provides a medicament for preventing, alleviating and/or treating schizophrenia comprising an tropomyosin-receptor-kinase B (TrkB) agonistic antibody or antigen binding fragment thereof and an inhibitor against an early-in-life stress-related factor. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is capable of specifically binding to human TrkB. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is capable of inducing activation of a TrkB downstream signaling pathway. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is capable of inducing gene expression comparable to natural human TrkB ligand Brain Derived Neurotrophic Factor (BDNF). In some embodiments, the antigen binding fragment comprises a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb. In some embodiments, the TrkB agonist antibody or antigen binding fragment thereof comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 to LCDR3, and the LCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91. In some embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92. In some embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93. In some embodiments, the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98. In some embodiments, the TrkB agonistic antibody comprises a light chain constant region, and the light chain constant region is a human igκ constant region or a human igλ constant region. In some embodiments, the TrkB agonist antibody or antigen binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94. In some embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95. In some embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97. In some embodiments, the TrkB agonist antibody comprises a heavy chain constant region, and the heavy chain constant region is a human IgG constant region. In some embodiments, the early-life stress comprises viral infection, smoking, intelligence, social cognition, cannabis use, social frustration, childhood trauma, prenatal and postprenatal hypoxia and/or prenatal malnutrition. In some embodiments, the level of expression and/or activity of the early-life stress-related factor is increased due to early-life stress. In some embodiments, the early life stress-related factor comprises a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the inhibitor against an early-life stress-related factor is capable of inhibiting the expression and/or activity level of the early-life stress-related factor. In some embodiments, the inhibitor against an early-in-life stress-related factor is capable of inhibiting the content of a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the inhibitor against an early-life stress-related factor comprises mifepristone (RU-486).

In another aspect, the application also provides a method of preventing, alleviating and/or treating schizophrenia in a subject in need thereof, wherein the subject is administered a TrkB agonistic antibody or antigen binding fragment thereof and/or an inhibitor against an early-in-life stress-related factor. In some embodiments, a pharmaceutical product is administered to the subject. In some embodiments, the subject has reduced BDNF expression and/or activity levels compared to a healthy subject. In some embodiments, the subject's BDNF expression and/or activity level is reduced by at least 10% as compared to a healthy subject. In some embodiments, the expression and/or activity level of the stress-related factor is increased in the early-in-life of the subject as compared to a healthy subject. In some embodiments, the subject has an increased glucocorticoid (e.g., cortisol, corticosterone, or CORT) content as compared to a healthy subject. In some embodiments, the subject's cor content is increased by at least 10% as compared to a healthy subject. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is capable of specifically binding to human TrkB. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is capable of inducing activation of a TrkB downstream signaling pathway. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is capable of inducing gene expression comparable to natural human TrkB ligand Brain Derived Neurotrophic Factor (BDNF). In some embodiments, the antigen binding fragment comprises a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb. In some embodiments, the TrkB agonist antibody or antigen binding fragment thereof comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 to LCDR3, and the LCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91. In some embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92. In some embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93. In some embodiments, the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98. In some embodiments, the TrkB agonistic antibody comprises a light chain constant region, and the light chain constant region is a human igκ constant region or a human igλ constant region. In some embodiments, the TrkB agonist antibody or antigen binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94. In some embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95. In some embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97. In some embodiments, the TrkB agonist antibody comprises a heavy chain constant region, and the heavy chain constant region is a human IgG constant region. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is administered to the subject in need thereof if the subject's glucocorticoid (e.g., cortisol, corticosterone, or CORT) content is increased compared to a healthy subject. In some embodiments, the early-life stress comprises viral infection, smoking, intelligence, social cognition, cannabis use, social frustration, childhood trauma, prenatal and postprenatal hypoxia and/or prenatal malnutrition. In some embodiments, the level of expression and/or activity of the early-life stress-related factor is increased due to early-life stress. In some embodiments, the early life stress-related factor comprises a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the inhibitor against an early-life stress-related factor is capable of inhibiting the expression and/or activity level of the early-life stress-related factor. In some embodiments, the inhibitor against an early-in-life stress-related factor is capable of inhibiting the content of a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the inhibitor against an early-life stress-related factor comprises mifepristone (RU-486). In some embodiments, if the subject's BDNF expression and/or activity level is reduced compared to a healthy subject, an inhibitor against the glucocorticoid (e.g., cortisol, corticosterone, or CORT) is administered to the subject in need thereof.

In another aspect, the application also provides a method of diagnosing and/or clinically classifying a subject suffering from schizophrenia, comprising the steps of: measuring the level of BDNF expression and/or activity in the subject, and measuring the level of expression and/or activity of an early-in-life stress-related factor in the subject. In some embodiments, the BDNF expression and/or activity level of the subject is measured by a detection method capable of measuring the DNA, RNA, and/or protein expression and/or activity level of BDNF. In some embodiments, the Bdnf expression and/or activity level of the subject is measured by a primer pair capable of amplifying the Bdnf gene or portion thereof, a Bdnf (e.g., bdnf-e 6) transcript (e.g., mRNA or cDNA), and/or an agent (e.g., probe or antibody) capable of specifically binding Bdnf. In some embodiments, the early life stress-related factor comprises a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the glucocorticoid (e.g., cortisol, corticosterone, or CORT) content of the subject is measured by a detection method capable of measuring the CORT content. In some embodiments, the glucocorticoid (e.g., cortisol, corticosterone, or CORT) content of the subject is measured by a probe capable of specifically binding to a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the detection method uses a sample from the subject, including a blood sample. In some embodiments, the method further comprises the steps of: the expression and/or activity level of BDNF is compared to healthy subjects and the expression and/or activity level of stress-related factors early in life of the subject is compared to healthy subjects. In some embodiments, the subject is selected for suffering from schizophrenia if the subject's BDNF expression and/or activity level is reduced compared to healthy subjects and/or the subject's early-in-life stress-related factor expression and/or activity level is increased compared to healthy subjects. In some embodiments, the subject's BDNF expression and/or activity level is reduced by at least 10% as compared to a healthy subject. In some embodiments, the expression and/or activity level of the stress-related factor is increased in the early-in-life of the subject as compared to a healthy subject. In some embodiments, the subject has an increased glucocorticoid (e.g., cortisol, corticosterone, or CORT) content as compared to a healthy subject. In some embodiments, the method further comprises the steps of: administering to the selected subject a TrkB agonistic antibody or antigen binding fragment thereof and/or an inhibitor against an early-in-life stress-related factor. In some embodiments, the inhibitor against the early-life stress-related factor is administered to the selected subject if the BDNF expression and/or activity level of the selected subject is reduced compared to a healthy subject. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is administered to the selected subject if the expression and/or activity level of the subject's early-in-life stress-related factor is increased compared to a healthy subject. In some embodiments, the TrkB agonistic antibody or antigen binding fragment thereof is administered to the selected subject if the subject's glucocorticoid (e.g., cortisol, corticosterone, or CORT) content is increased compared to a healthy subject. In some embodiments, if the level of BDNF expression and/or activity in the subject is reduced compared to a healthy subject and the level of expression and/or activity of an early life stress-related factor in the subject is increased compared to a healthy subject, the TrkB agonistic antibody or antigen binding fragment thereof and/or an inhibitor against the early life stress-related factor is administered to the selected subject. In some embodiments, if the subject's BDNF expression and/or activity level is reduced compared to a healthy subject and the subject's glucocorticoid (e.g., cortisol, corticosterone, or CORT) content is increased compared to a healthy subject, then the TrkB agonistic antibody or antigen binding fragment thereof and/or an inhibitor against the glucocorticoid (e.g., cortisol, corticosterone, or CORT) is administered to the selected subject.

In another aspect, the application also provides a system for diagnosing and/or clinically classifying a subject suffering from schizophrenia, comprising: a first measurement module that measures BDNF expression and/or activity levels in a subject, and a second measurement module that measures expression and/or activity levels of stress related factors early in life in the subject. In some embodiments, the first measurement module is capable of measuring DNA, RNA, and/or protein expression and/or activity levels of BDNF. In some embodiments, the first measurement module comprises a primer pair capable of amplifying the Bdnf gene, and/or a probe capable of specifically binding Bdnf. In some embodiments, the early life stress-related factor comprises a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the second measurement module is capable of measuring the content of a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the second measurement module comprises a probe capable of specifically binding to a glucocorticoid (e.g., cortisol, corticosterone, or CORT). In some embodiments, the system comprises a sample collection module that collects a sample from the subject suffering from schizophrenia. In some embodiments, the sample comprises a blood sample. In some embodiments, the system comprises a determination module that compares the level of BDNF expression and/or activity in the subject with schizophrenia to a healthy subject and/or compares the level of expression and/or activity of a stress related factor early in life in the subject with schizophrenia to a healthy subject. In some embodiments, the determination module determines whether the BDNF expression and/or activity level of the subject with schizophrenia is reduced compared to a healthy subject. In some embodiments, the determination module determines whether the expression and/or activity level of the subject's early-life stress-related factor is increased compared to a healthy subject. In some embodiments, the determination module determines whether the glucocorticoid (e.g., cortisol, corticosterone, or CORT) content of the subject is increased compared to a healthy subject. In some embodiments, the system includes a advice module that provides advice regarding a treatment regimen to the subject suffering from schizophrenia based on the determination from the determination module.

Additional aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present application are shown and described. As will be realized, the application is capable of other and different embodiments and its several details are capable of modification in various obvious respects, all without departing from the application. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not as restrictive.

Incorporation of reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth exemplary embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures"), of which:

FIGS. 1A to 1J show behavioral profile analysis of Bdnf-e6 mutant mice. (FIGS. 1A-1B) three-chamber test. In the social ability test, bdnf-e 6-/-and WT mice were both longer in the room containing "strange mouse 1" (FIG. 1A), and were better in the social novelty preference test for "strange mouse 2" (FIG. 1B). (FIG. 1C) Morris water maze test (MWT). Schematic of Morris water navigation task. (FIG. 1D) in the study trial, the escape latency was the same for WT and Bdnf-e 6-/-mice except on day 6. (FIGS. 1E-1G) in the pilot trial, bdnf-E6-/-mice did not delay reaching the plateau (FIG. 1E), but stayed in the target quadrant for less time (FIG. 1F) and passed through the plateau position less times (FIG. 1G) than the WT mice. (FIGS. 1H-1J) PPI test. The startle response of Bdnf-e 6-/-mice to 120dB sound did not change (FIG. 1H) and the average PPI ratio for all intensities did not change (FIG. 1I). Schematic of the PPI assay (FIG. 1J). In this and all other figures, significant differences between groups are indicated. * p < 0.05, p <0.01, p <0.001, p < 0.0001. The number of animals used for each trial is shown in bar graph (FIGS. 1A-1I: unpaired Student t test).

FIGS. 2A-2I show social dysfunction induced by postnatal hypoxia in hypoxic e 6-/-mice. (FIG. 2A) depicts a schematic of the experimental design. WT and Bdnf-e 6-/-mice underwent a hypoxic or normoxic survival environment on day 4 (P4) after birth for 7 consecutive days, and then were bred to adulthood in normoxic environments. Open field, stick, social interaction, and PPI trials were performed in succession. (FIG. 2B) total range of movement of normoxic and hypoxic treated mice in open field experiments. (FIG. 2C) rod drop time of normoxic and hypoxic treated mice in the rod-rotating test. (FIG. 2D) experimental design of social interaction trial. (fig. 2E, fig. 2F) social ability (fig. 2E) and social novelty preference (fig. 2F) of normoxic and hypoxic treated mice. It should be noted that Bdnf-e 6-/-mice lost social capacity and social novelty preference after postnatal hypoxia treatment. (FIGS. 2G-2I) Effect of hypoxia on PPI. The average PPI ratio at all intensities was selectively reduced in postnatal hypoxic e 6-/-mice. (FIGS. 2B-2C, FIGS. 2G-2I: two-way ANOVA analysis and Tukey multiple comparison test. FIGS. 2E-2F: unpaired Student t test).

Figures 3A to 3E show juvenile social isolation-induced hyperactivity and social dysfunction. (FIG. 3A) depicts a schematic of the experimental design. On postnatal day 21, bdnf-e 6-/-and WT mice were kept on either isolated rearing (PIR) or social rearing (PSR) for four consecutive weeks (P21 to P49). As noted, different types of behavioral tests were performed according to a schedule. (FIG. 3B) total range of PIR and PSR mice in open field experiments. (FIG. 3C) residence time of PIR and PSR mice in the central region of open field. (fig. 3D-3E) social behavior of PIR and PSR mice measured by the time spent sniffing in social ability tests and social novelty preference tests. It should be noted that Bdnf-e 6-/-mice employing PIR showed defects in the social novelty bias test, but not in the social ability test. (FIGS. 3B-3C: two-way ANOVA analysis and Tukey multiple comparison test: FIGS. 3D-3E: unpaired Student t test).

FIGS. 4A-4H show that juvenile isolation induced PPI defects in Bdnf-e 6-/-and WT mice, but adult isolation induced PPI defects only in Bdnf-e 6-/-mice. (FIG. 4A, FIG. 4E) experimental design. The young social isolation paradigm is the same as fig. 3 (fig. 4A). For adult social isolation, bdnf-E6-/-and WT adult mice were kept for isolation (AIR) or social rearing (ASR) for four consecutive weeks (P70 to P98) on postnatal day 70, and PPI experiments were performed at P100 to P103 (FIG. 4E). (FIGS. 4B-4D) PIR mice have reduced PPI ratios at 71dB, 81dB and 90 dB. (FIGS. 4B-4D: two-way ANOVA analysis and Tukey multiple comparison test). The pre-pulse suppression for all intensities (71 dB, 81dB, 90 dB) is shown (fig. 4F to 4H), respectively. It should be noted that AIR treatment did not induce PPI defects at any pre-pulse intensity in WT mice, but significantly reduced PPI in Bdnf-e 6-/-mice.

FIGS. 5A-5C show the increase in plasma corticosterone levels due to environmental stress in WT mice. In all figures, the experimental design schematic is shown at the top, while plasma corticosterone levels are shown at the bottom. (FIG. 5A) WT mice on postnatal day 4 were exposed to a hypoxic or normoxic social environment for 7 consecutive days (P4 to P10). After environmental stress, mice were bred to adults in an normoxic social setting. It should be noted that postnatal hypoxia significantly increases adult plasma corticosterone levels (P60). (FIG. 5B) WT mice on postnatal day 21 were subjected to either isolated feeding (PIR) or social feeding (PSR) for 29 consecutive days (P21 to P49). After environmental stress, mice were socially bred until adulthood. It should be noted that juvenile social isolation causes a significant increase in adult plasma corticosterone levels (P60). (fig. 5C) social (ASR) or isolated (AIR) feeding was performed during adulthood (P70 to P98) and blood samples were collected at P100 using the same experimental design as (fig. 5B). It should be noted that pubertal social isolation still resulted in an increase in plasma corticosterone levels (FIGS. 5A-5C: unpaired Student t test).

FIGS. 6A-6H show that juvenile CORT exposure in Bdnf-e 6-/-mice results in a deficiency in social novelty. (FIG. 6A) experimental design. Corticosterone was given daily from P42 to P63 and behavioural tests were performed at the times indicated. (FIG. 6B, FIG. 6C) WT and Bdnf-e 6-/-mice were subjected to open field trials with or without juvenile CORT exposure. It should be noted that in WT and Bdnf-e 6-/-mice, the "total range of motion" is significantly reduced with young CORT exposure, while the "central zone residence time" is not reduced. The (FIG. 6D, FIG. 6E) CORT exposure paradigm is the same as (FIG. 6B, FIG. 6C), except that social behavior is measured by (FIG. 6D) social ability test and (FIG. 6E) social novelty preference test. It should be noted that Bdnf-E6-/-mice exposed to young CORT showed normal social ability (FIG. 6D), but were unable to distinguish between strange mouse 1 and strange mouse 2, indicating a deficiency in social novelty preference (FIG. 6E). The (fig. 6F to 6H) CORT exposure pattern is the same as (fig. 6B, 6C), and the measurement subject becomes PPI. Although both genotypes showed a decrease in PPI ratio after juvenile CORT exposure, the decrease in Bdnf-e 6-/-mice was more pronounced at the intensities of P81P120 (FIG. 6G), P90P120 (FIG. 6H). (FIG. 6B, FIG. 6C, FIG. 6F through FIG. 6H: two-way ANOVA analysis and Tukey multiple comparison test. FIG. 6D, FIG. 6E: unpaired Student t test).

Figures 7A to 7H show that TrkB agonistic antibodies rescue PPI defects in Bdnf-e 6-/-mice exposed to postnatal hypoxia or postweaning social isolation. (FIGS. 7A to 7D) effect on hypoxic Bdnf-e 6-/-mice. Mice were exposed to hypoxic conditions from P4 to P10, and TrkB agonistic antibody AbB (1 mg/kg) was given intravenously 48 hours prior to the PPI test (fig. 7A). Normoxic e 6-/-refer to the data of figure 2. It should be noted that the reduction in PPI ratio at 81dB, 90dB intensity in hypoxic e 6-/-mice was saved by AbB treatment. (FIGS. 7E-7H) effect on PIR Bdnf-E6-/-mice. Mice were given social isolation as young from P21 to P49 AbB901 in the same manner as in fig. 7A. PSR e6 refers to the data of fig. 3. It should be noted that, despite exhibiting an efficacy trend of AbB901 at 70dB and 81dB intensities, the PPI ratio reduction phenomenon of PIR Bdnf-e 6-/-mice at 90dB intensities was significantly rescued by AbB901 treatment. (FIGS. 7B-7D, FIGS. 7F-7H: unpaired Student t test).

Fig. 8A-8H show RU-486 rescued PPI defects in Bdnf-e 6-/-mice exposed to postnatal hypoxia or postweaning social isolation (fig. 8A-8C). (FIGS. 8A to 8D) effect on hypoxic Bdnf-e 6-/-mice. Mice were exposed to hypoxic conditions from P4 to P10, and RU-486 (40 mg/kg) was administered daily to P91 to P98. PPI was measured at P100 (fig. 8A). Normoxic e 6-/-refer to the data of figure 2. It should be noted that the phenomenon of reduced PPI ratio at 90dB intensity in hypoxic e 6-/-mice was rescued by RU-486 treatment. (FIGS. 8E-8H) effect on PIR Bdnf-E6-/-mice. From P21 to P49, mice were subjected to juvenile social isolation while RU-486 was administered, and PPI was measured at P100 (fig. 8A). PSR e6 refers to the data of fig. 3. It should be noted that the phenomenon of reduced PPI ratio at all intensities (70 dB, 81dB, 91 dB) in PIR Bdnf-e 6-/-mice was significantly rescued by RU-486 treatment. (FIGS. 8B-8D, FIGS. 8F-8H: unpaired Student t test).

FIGS. 9A to 9K show the characterization of Bdnf-e 6-/-mice. (fig. 9A) left: design of Bdnf-e6 mutant mice. The mouse Bdnf genome structure consists of ten exons, only one of which contains a protein coding region. Each non-coding exon is transcribed from its corresponding promoter and is alternatively spliced to the commonly encoded exon (exon IXa-BDNF). In Bdnf-e6 mutant mice, the eGFP-STOP expression cassette was inserted after exon VI and the PGK-Neo expression cassette was placed as the antisense sequence to eGFP. Subsequently, PGK-Neo was deleted by Cre recombinase expression. Thus, bdnf-eGFP fusion transcripts and eGFP were produced at the mRNA and protein levels, respectively. Right: representative results of genotyping. The WT allele band is longer than the mutant allele band. (FIG. 9B) relative expression of Bdnf-e 6mRNA in various brain regions (hippocampus, mPFC, cortex, thalamus, olfactory bulb) and peripheral organs (heart, lung, kidney, liver, spleen). (FIG. 9C, FIG. 9D) relative expression of Bdnf-e1/2/4/6mRNA in the hippocampus (FIG. 9C) and mPFC (FIG. 9D) of WT and Bdnf-e 6-/-mice. It should be noted that Bdnf-e4 mRNA expression was also reduced in the hippocampus of Bdnf-e 6-/-mice. (fig. 9E) body weight. Body weights of male WT and Bdnf-e 6-/-mice were recorded at different time points. Disruption of promoter VI did not cause a change in male mouse body weight. (FIG. 9F) rotating stick test. In this and subsequent experiments, male WT and Bdnf-e 6-/-mice (2 to 4 months old) were used. Bdnf-e 6-/-mice showed no motor dysfunction on the accelerated rotarod. (FIGS. 9G-9H) open field test. Bdnf-e 6-/-mice were active in locomotion (FIG. 9G), but were shorter in the central region (FIG. 9H). (FIG. 9I) GFP protein expression in various brain regions (hypothalamus, mPFC, hippocampus, striatum, olfactory bulb, cortex, thalamus, midbrain) of WT and Bdnf-e 6-/-mice. (FIG. 9J) GFP expression can be detected in various brain regions, especially in the hippocampus, by immunostaining. (FIG. 9K) images of the whole brain structures of WT and Bdnf-e 6-/-mice were acquired by 7.0T MRI. It should be noted that the lateral ventricle volumes of mice of both genotypes are similar. (FIGS. 9C-9H: unpaired Student t test).

Fig. 10A to 10D show the weight conditions affected by postnatal hypoxia. (FIG. 10A) depicts a schematic of the experimental design. (FIGS. 10B to 10D) weight of postnatal hypoxia treated WT and Bdnf-e 6-/-mice. 4 experimental groups were set up: WT: normoxic and anoxic; bdnf-e6: normoxic and anoxic. It should be noted that both genotypes significantly reduced body weight at 3 weeks of age and 5 weeks of age (fig. 10B, fig. 10C), and recovered to normal at 9 weeks of age (fig. 10D). Bdnf-e6 deficiency had no further effect on body weight, except at 3 weeks of age. (FIGS. 10B-10D: two-way ANOVA analysis and Tukey multiple comparison test).

Fig. 11A to 11E show nesting behavior and new object recognition ability affected by postnatal hypoxia. (FIG. 11A) shows a representative image of a nesting score rating. (FIG. 11B, FIG. 11C) 4 sets of nesting scores (FIG. 11B) and respective score composition ratios (FIG. 11C). Higher scores indicate better nesting quality. It should be noted that postnatal hypoxia treatment resulted in poor nesting quality in both genotypes, while Bdnf-e6 defects appeared to further deteriorate nesting quality. (fig. 11D, fig. 11E) search time (fig. 11D) and discrimination ratio (fig. 11E) of the new object recognition test of the 4 groups. It should be noted that postnatal hypoxia treatment resulted in Bdnf-e 6-/-mice lacking new object recognition capacity, whereas WT mice did not. (FIG. 11B, FIG. 11E: two-way ANOVA analysis and Tukey multiple comparison test. FIG. 11D: unpaired Student t test).

Fig. 12A-12H show that adult social isolation or postnatal hypoxia does not induce PPI deficiency in Bdnf-e 4-/-mice. (FIG. 12A, FIG. 12E) experimental design. Adult social isolation and postnatal hypoxia paradigms are the same as fig. 4A and 2A, respectively. The PPI test is performed at P100 or adulthood. (FIGS. 12B-12D) AIR Bdnf-e 4-/-mice were not reduced in PPI ratio at 71dB, 81dB and 90dB compared to WT mice. (FIGS. 12F-12H) there was no decrease in PPI ratio at 71dB, 81dB and 90dB in Bdnf-e 4-/-mice experiencing postnatal hypoxia compared to WT mice. (unpaired Student t test).

FIGS. 13A-13D show Bdnf expression and TrkB signaling affected by postnatal hypoxia and Bdnf-e6 deficiency. (FIG. 13A, FIG. 13B) Bdnf and Bdnf-e6mRNA expression in the mPFC (FIG. 13A) and hippocampus (FIG. 13B) of WT mice that underwent postnatal hypoxic or normoxic environments. It should be noted that postnatal hypoxia induced moderate but significant decrease in Bdnf-e6mRNA expression in the hippocampus. (FIG. 13C) the ratio pTrkB/TrkB in the hippocampus. WT and Bdnf-e 6-/-mice experience postnatal hypoxic or normoxic environments. It should be noted that pTrkB/TrkB was significantly down-regulated in Bdnf-e 6-/-mice that underwent postnatal hypoxia. (FIG. 13D) TrkB expression and phosphorylation levels in WT and Bdnf-e 6-/-mouse hippocampus subjected to postnatal hypoxic or normoxic environments. N: normal oxygen; h: hypoxia; WT: wild type; e6: bdnf-e6-/-. (FIGS. 13A-13C: unpaired Student t test).

FIGS. 14A-14D show the effect of RU-486 on Bdnf-e 6-/-mouse PPI of Adult Isolated Rearing (AIR). (fig. 14A) includes experimental design of time points of year isolation and RU-486 administration. (FIGS. 14B-14D) PPI at all intensities (71 dB, 81dB, 90 dB). RU-486 had no effect on PPI. (FIGS. 14B-14D: unpaired Student t test).

FIGS. 15A-15D show the effect of RU-486 on PPI in postnatal isolated feeding (PIR) WT mice. (FIG. 15A) experimental design. WT mice were fed postnatally in isolation (P21 to P49), and P42 to P49 were given RU-486. (FIGS. 15B-15D) effect on PPI in PIR model. PSR WT refers to the data of fig. 4. It should be noted that after PIR the PPI ratio at all intensities (71 dB, 81dB, 90 dB) was slightly reduced (compare fig. 1I), and pretreatment with RU-468 resulted in a small but significant increase in PPI ratio at 81dB and 90 dB. (FIGS. 15B-15D: unpaired Student t test).

FIGS. 16A-16D show the effect of pubertal administration of RU-486 on PPI in an MK801 model. (FIG. 16A) experimental design. RU-486 was administered from P91 to P98 and MK801 was injected at P100 to induce the SCZ endophenotype. (FIGS. 16B-16D) effect on PPI in MK801 model. It should be noted that after MK801 administration, the PPI ratio was greatly reduced at all intensities (71 dB, 81dB, 90 dB), pretreatment with RU-468 increased the PPI ratio at the 90dB intensity. (FIGS. 16B-16D: unpaired Student t test).

Detailed Description

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many changes, modifications and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Definition of terms

As used herein, the term "pharmaceutical product" generally refers to a formulation that exists in a form that allows for the biological activity of the active ingredient to be effective. For example, the pharmaceutical product may not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered. In some embodiments, these formulations may contain the active ingredient of the drug and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical product may include a pharmaceutical product that is administered parenterally, transdermally, intracavitary, intraarterially, intrathecally and/or intranasally or directly into tissue. The drug may be administered by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In the present application, the term may be a combination comprising at least two components. The components of the pharmaceutical product may be used together. Wherein, collocation can refer to simultaneous use or separate use. The components of the drug product may be mixed together or placed in different containers. In the present application, the pharmaceutical product may include a tropomyosin-receptor-kinase B (TrkB) agonist (e.g., an agonistic antibody or antigen binding fragment thereof) and an inhibitor (e.g., a cor inhibitor) against an early-in-life stress-related factor.

As used herein, the term "schizophrenia" also known as SCZ, generally refers to a mental disorder characterized by abnormal behavior and misunderstanding of reality. Schizophrenia may generally refer to a mental disorder characterized by a continuous onset or recurrence of psychosis. The principal symptoms may include hallucinations (typically hearing sounds), delusions, and confusion. Other symptoms may include social withdrawal, reduced emotional expression, and apathy.

As used herein, the term "tropomyosin-receptor-kinase B" generally refers to a protein encoded by the NTRK2 gene, also known as tyrosine-receptor-kinase B or TrkB or BDNF/NT-3 growth factor receptor or neurotrophic tyrosine kinase receptor type 2. TrkB is a receptor for brain-derived neurotrophic factor (BDNF), a member of the family of tyrosine kinase receptors including TrkA and TrkC. In the present application, trkB may be human TrkB, or may be rodent TrkB (e.g., mouse TrkB).

As used herein, the term "tropomyosin-receptor-kinase B agonist", also known as TrkB agonist, generally refers to a substance that is capable of eliciting a physiological response when bound to TrkB. TrkB agonists may encompass chemicals that activate TrkB to produce a biological response. TrkB agonists may include endogenous agonists (e.g., trkB ligands such as BDNF) or exogenous agonists (e.g., mimics of their ligands such as BDNF mimics). TrkB agonists may be small molecules, polypeptides/proteins (e.g., antibodies or antigen binding fragments thereof). TrkB agonists may be full agonists (e.g., a full agonist may bind to and activate a receptor, producing the greatest response that can be elicited at the receptor), or may be partial agonists. It may be a full agonist in some tissues/cells and a partial agonist in some other tissues/cells. In some cases, a TrkB agonist may be a co-agonist that acts with other co-agonists to co-produce a desired effect. The TrkB agonist may be a selective agonist selective for TrkB.

As used herein, the term "tropomyosin-receptor-kinase B-agonistic antibody", also known as TrkB-agonistic antibody, generally refers to an immunoglobulin or fragment or derivative thereof that is capable of activating TrkB, and encompasses any polypeptide that may comprise a TrkB binding site, whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. In the present application, the term may also include antibody fragments, such as Fab, fab ', F (ab) ₂, fv fragments, F (ab') ₂, scFv, di-scFv, dAb, and/or retain its antigen-binding function. In the present application, trkB agonistic antibodies may comprise light chains and/or heavy chains. In the present application, trkB agonistic antibodies may specifically bind to human TrkB. The NCBI accession number for human TrkB is AAB33109.1. The NCBI accession number of the gene encoding human TrkB is 4915.

As used herein, the term "antigen-binding fragment" generally refers to the region of an antibody that binds an antigen. Antigen binding fragment antibodies may consist of one constant and/or one variable domain per heavy and/or light chain. The variable domain may comprise a paratope (antigen binding site) at the amino terminus of the monomer, comprising a set of complementarity determining regions. In the present application, the antigen binding fragment may comprise a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb.

As used herein, the term "Brain Derived Neurotrophic Factor (BDNF)" generally refers to a protein encoded by the BDNF gene. BDNF is a member of the neurotrophin family of growth factors, which are associated with typical nerve growth factors. The neurotrophic factors are present in the brain and periphery. In the present application, BDNF may be human BDNF, or may be mouse BDNF. The NCBI accession number for human BDNF is P23560.1. The NCBI accession number of the gene encoding human BDNF was 627.

As used herein, the term "BDNF-e6" generally refers to exon 6 of the Bdnf gene. The Bdnf gene has 9 unique promoters that drive transcription of at least 20 different Bdnf transcripts encoding Bdnf proteins. Expression of BDNF-e6 is driven by promoter VI of the Bdnf gene.

As used herein, the term "light chain" generally refers to a small polypeptide subunit of an antibody. Each light chain may consist of two tandem immunoglobulin domains: a constant (C _L) domain and a variable (V _L) domain. The light chain protein may be about 211 to about 217 amino acids in length. In the present application, the light chain may be a human light chain. In the present application, the light chain may comprise a light chain variable region, which may comprise one or more of LCDR1, LCDR2 and LCDR 3.

As used herein, the term "heavy chain" generally refers to the large polypeptide subunit of an antibody. Each heavy chain may have two regions: constant (C _L) regions and variable (V _L) regions. The heavy chain protein may be about 450 to about 550 amino acids in length. In the present application, the heavy chain may be a human heavy chain. In the present application, the heavy chain may comprise a heavy chain variable region, which may comprise one or more of HCDR1, HCDR2 and HCDR 3.

As used herein, the term "early-life stress" generally refers to a experience that represents a departure from the intended environment and that requires adaptation. In the present application, early life stress may include exposure to childhood abuse, sexual attack, neglect, and chronic poverty. Early life stress is associated with an increased risk of many mental and physical health problems. In the present application, the term may include viral infection, smoking, intelligence, social cognition, cannabis use, social isolation, social frustration, childhood trauma, prenatal and postprenatal hypoxia and/or prenatal malnutrition.

As used herein, the term "CORT" also known as 17-deoxycortisol, corticosterone or 11 beta, 21-dihydroxyprogesterone, generally referred to as 21-carbon steroid hormones of the corticosteroid type, may be produced in the adrenal cortex. In some cases, "corticosterone" or "CORT" also encompasses the corresponding active substance in another species (e.g., in humans), and may encompass cortisol. Corticosterone (including CORT or cortisol) is the predominant glucocorticoid involved in energy regulation, immune response and stress response in many species including amphibians, reptiles, rodents and birds. In the present application, corticosterone may be an early life stress-related factor. The CAS number for CORT may be 50-22-6 or 50-23-7.

As used herein, the term "inhibitor against an early-life stress-related factor" generally refers to a molecule that can reduce the activity of an early-life stress-related factor. For example, inhibitors against early life stress related factors may bind to early life stress related factors. Binding of inhibitors may stop and/or prevent reactions from early life stress. The binding between the early life stress-related factor and its inhibitor may be reversible or irreversible. In the present application, the early life stress-related factor may be a glucocorticoid (e.g., cortisol, corticosterone or CORT), which term may also be mifepristone (RU-486). The CAS number for RU-486 may be 84371-65-3.

Detailed Description

In one aspect, the application provides a medicament for preventing, alleviating and/or treating schizophrenia. The pharmaceutical product may include a TrkB agonist and an inhibitor against an early-in-life stress related factor (such as a cor inhibitor).

In another aspect, the present application provides a method for preventing, alleviating and/or treating schizophrenia in a subject in need thereof. The method may comprise administering to the subject a TrkB agonist and/or an inhibitor against an early-in-life stress related factor (such as a cor inhibitor), and the subject has a reduced level and/or activity of BDNF-e6 expression and an increased level and/or activity of cor.

In another aspect, the application provides a method for determining whether a subject has or is at risk of developing schizophrenia. The method may comprise determining the BDNF-e6 expression level and/or activity of the subject, and determining the cor level and/or activity of the subject.

In another aspect, the application provides a system for determining whether a subject has or is at risk of developing schizophrenia. The system may include: a first module for determining whether the subject has a reduced level and/or activity of BDNF-e6 expression, and a second module for determining whether the subject has an elevated level and/or activity of CORT.

BDNF

Although BDNF deficiency has long been recognized as a risk factor for schizophrenia, its specific mechanism is not yet understood. Different Bdnf transcripts are expressed in different brain regions, cell types, developmental stages and cause different functions. Bdnf-e1 and Bdnf-e2 are involved in different aspects of obesity (thermogenesis and food intake), challenge, and serotonin signaling (Maynard et al, 2016; mcAllan et al, 2018; you et al, 2020). One of the main functions of Bdnf-e4 and Bdnf-e6 is to promote GABAergic neuron development and function (Jiao et al, 2011; maynad et al, 2016; sakata et al, 2013; sakata et al, 2009b; xu et al, 2021). The defect in gabaergic neurons is one of the pathological features of schizophrenia (Dienel and Lewis, 2019), and Bdnf-e4 and Bdnf-e6 are expressed in brain regions associated with schizophrenia, such as the hippocampus, prefrontal cortex, and hypothalamus (Maynard et al, 2016). The Bdnf-e4 or Bdnf-e6 deletion resulted in a decrease of about 50% in BDNF protein expression in hypothalamus, prefrontal cortex and sea horse at day 28 post-natal (PSD 28) and 50%, 30% and 30% in the 3 regions of adulthood, respectively (Maynard et al 2016).

However, no schizophreniform phenotype was observed in either Bdnf-e 4-/-or Bdnf-e 6-/-mice. Studies have found that early life stress in Bdnf-e6 deficient mice results in SCZ-like internal phenotypes such as social capacity and social cognition after adulthood, spatial memory and sensorimotor gating function (PPI) deficits; no similar situation was found in Bdnf-e4 deficient mice. Neither early life stress nor Bdnf-e6 deficiency alone causes these abnormalities. These results support the "double hit" hypothesis of schizophrenia and define a pair of genetic and environmental factor combinations that are critical to SCZ pathophysiology. Interestingly, postnatal stress also increased blood glucocorticoid (e.g. cortisol, corticosterone or CORT) levels in wild type mice, and administration of CORT to adult Bdnf-e 6-/-mice without early life stress history resulted in the same PPI deficiency and social dysfunction. In addition, PPI defects in hypoxic or Socially Isolated (SI) treated Bdnf-e 6-/-mice can be rescued by treatment with CORT inhibitors (such as CORT antagonist RU-486) or TrkB agonists (such as TrkB agonistic antibodies).

It has long been shown that BDNF is involved in SCZ development (Di Carlo et al, 2019), although the Bdnf gene was not located in the schizophrenia-associated locus based on GWAS PGC2 studies (schizophrenic working group of the psychogenomics alliance, 2014). BDNF is a powerful regulator of synaptic and neural circuits that are important in mood control and cognition, which are key components of SCZ (Chao, 2003; fig et al, 1996; ji et al, 2010; lohof et al, 1993; martinowich et al, 2007; molteni et al, 2001; xu et al, 2000). Postmortem brain analysis revealed defects in BDNF transcription and downstream signaling in the hippocampus and PFC of SCZ patients (Reinhart et al, 2015;Thompson Ray et al, 2011). (Emamian et al, 2004; green et al, 2011; issa et al, 2010; szamosi et al, 2012; weickert et al, 2005; wong et al, 2013; yoshimura et al, 2016; yuan et al, 2010). There are 9 major Bdnf transcripts in the brain, each with a short 3'UTR and a long 3' UTR, so it is necessary to define specific transcripts that are critical to SCZ. The association between SCZ and the C270T polymorphism located in Bdnf exon 6 provides some elicitations (Neves-Pereira et al, 2005; szekers et al, 2003). Interestingly, bdnf-e 6-/-mice do not show any schizophreniform-like internal phenotype by themselves.

It is believed that the combination of genetic factors and early (e.g., nutritional or maternal factors) or late (e.g., social stress or drug abuse) environmental risk factors may cause SCZ attacks. This is the basis of the "double hit" hypothesis (Freedman, 2003; maynard et al, 2001; mcGrath et al, 2003). However, it is not clear at what time, in what way, environmental stress factors and with which susceptibility genes act can lead to schizophrenia. In the present application, the pattern of schizophreniform-like internal phenotype is produced by: early developmental stage stress (postnatal hypoxia or juvenile isolation) interacts with Bdnf-e 6-/-mice, two conditions.

In the present application, the subject may have reduced plasma BDNF levels. For example, the subject's plasma BDNF level can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or more, as compared to a control subject (e.g., a subject not suffering from or at risk of suffering from schizophrenia).

In the present application, the subject may have an altered Bdnf gene, e.g., the subject may have one or more mutations at promoter VI of the Bdnf gene. In some cases, the subject may comprise a BDNF Val66 mutation. In some cases, the subject may comprise a BDNF Val66Met polymorphism (Hashimoto and Lewis, 2006).

Brain-derived neurotrophic factor (BDNF), one of the genetic factors, plays an important role in neuronal differentiation and synaptic development, affecting adult development, synaptic transmission and plasticity, and also plays an important role in behavior associated with schizophrenia. Abnormal expression of BDNF has been observed in the prefrontal cortex (PFC) and plasma of schizophrenic patients. In the present application, the mouse strain with specific disruption may comprise a defect of the Bdnf promoter VI. Furthermore, defects in Bdnf promoter VI can lead to reduced expression and/or activity levels of Bdnf, or significant impairment of gabaergic interneuron markers in PFC.

In the present application, the BDNF expression and/or activity level may be reduced in SCZ subjects compared to healthy subjects. For example, the expression and/or activity level of BDNF-e6 can be reduced by at least 10% (e.g., can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more as compared to a healthy subject).

In the present application, a healthy subject may be a subject who does not suffer from or is at risk of suffering from schizophrenia.

Trkb agonists

In the present application, trkB agonists may encompass any substance capable of eliciting a physiological response when bound to the receptor TrkB. The TrkB agonist may be an endogenous agonist (e.g., a TrkB ligand, e.g., BDNF) or an exogenous agonist (e.g., a mimetic of its ligand, e.g., a BDNF mimetic). TrkB agonists may be small molecules, polypeptides/proteins (e.g., antibodies or antigen binding fragments thereof), or any other functional entity (e.g., polymers, cells, etc.). The TrkB agonist may be a full agonist, a partial agonist, or a combination thereof. In some cases the TrkB agonist may be a full agonist, and in some other cases the TrkB agonist may be a partial agonist. In some cases, the TrkB agonist may be a co-agonist that acts with other co-agonists to co-produce the desired effect. The TrkB agonist may be a selective agonist selective for TrkB.

For example, the TrkB agonist may comprise a TrkB agonistic antibody or antigen binding fragment thereof capable of activating TrkB. The TrkB agonistic antibody or antigen binding fragment thereof may be capable of inducing gene expression comparable to natural human TrkB ligand Brain Derived Neurotrophic Factor (BDNF). For example, the TrkB agonistic antibody or antigen binding fragment may be capable of specifically binding to human TrkB. In the present application, the TrkB agonistic antibody or antigen binding fragment thereof may be capable of inducing activation of a TrkB downstream signaling pathway.

In the present application, the TrkB agonistic antibody or antigen binding fragment thereof may be capable of inducing gene expression comparable to that of natural human TrkB ligand Brain Derived Neurotrophic Factor (BDNF).

In the present application, the antigen-binding fragment of the antibody can comprise a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise a light chain variable region, which may comprise LCDR1, LCDR2 and LCDR3.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR1, and the LCDR1 may comprise an amino acid sequence of any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR2, which LCDR2 may comprise an amino acid sequence of any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR3, and the LCDR3 may comprise an amino acid sequence of any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR1 to LCDR3, the LCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91, the LCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92, and the LCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise a light chain variable region which may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98.

In the present application, the TrkB agonistic antibody may comprise a light chain constant region, which may be a human igκ constant region or a human igλ constant region.

In the present application, the TrkB agonistic antibody may comprise a light chain which may comprise the amino acid sequence shown in any one of SEQ ID NOs 90 and 100.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise a heavy chain variable region, wherein the heavy chain variable region may comprise HCDR1 to HCDR3.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise HCDR1, which HCDR1 may comprise an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise HCDR2, which HCDR2 may comprise an amino acid sequence of any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise HCDR3, which HCDR3 may comprise an amino acid sequence of any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise HCDR1 to HCDR3, the HCDR1 may comprise an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94, the HCDR2 may comprise an amino acid sequence of any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95, and the HCDR3 may comprise an amino acid sequence of any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise a heavy chain variable region which may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, the LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 9 to 11, respectively, and the HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 12 to 14, respectively.

In the present application, the TrkB agonist antibody or antigen-binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, wherein LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 15-17, respectively, and HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 18-20, respectively.

In the present application, the TrkB agonist antibody or antigen-binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, wherein LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 21-23, respectively, and wherein HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 24-26, respectively.

In the present application, the TrkB agonist antibody or antigen-binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, wherein LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 29-31, respectively, and wherein HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 32-34, respectively.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, the LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 37 to 39, respectively, and the HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 40 to 42, respectively.

In the present application, the TrkB agonist antibody or antigen-binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, wherein LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 45-47, respectively, and HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 48-50, respectively.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, the LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 53 to 55, respectively, and the HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 56 to 58, respectively.

In the present application, the TrkB agonist antibody or antigen binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, the LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 61 to 63, respectively, and the HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOs 64 to 66, respectively.

In the present application, the TrkB agonist antibody or antigen-binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, wherein LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 69-71, respectively, and wherein HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 72-74, respectively.

In the present application, the TrkB agonist antibody or antigen-binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, wherein LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 81-83, respectively, and wherein HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 84-86, respectively.

In the present application, the TrkB agonist antibody or antigen-binding fragment thereof may comprise LCDR1 to LCDR3 and HCDR1 to HCDR3, wherein LCDR1 to LCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 91-93, respectively, and wherein HCDR1 to HCDR3 may comprise the amino acid sequences shown in SEQ ID NOS: 94-96, respectively.

In the present application, the heavy chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97, and the light chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98.

In the present application, the TrkB agonist antibody may comprise a heavy chain constant region, which may be a human IgG constant region.

In the present application, the TrkB agonist antibody may comprise a heavy chain, which may comprise the amino acid sequence shown in any one of SEQ ID NOs 89 and 99.

In the present application, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 89, and the light chain may comprise the amino acid sequence shown in SEQ ID NO. 90; alternatively, the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO. 99, and the light chain may comprise the amino acid sequence set forth in SEQ ID NO. 100.

In the present application, the pharmaceutical product may comprise a tropomyosin-receptor-kinase B (TrkB) agonistic antibody or antigen-binding fragment thereof and an inhibitor against an early-in-life stress-related factor, and may optionally comprise a pharmaceutically acceptable carrier.

In the present application, the pharmaceutically acceptable carrier may include any of all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents, may be compatible with pharmaceutical administration, and may be generally safe and nontoxic.

The TrkB agonistic antibody may bind to an epitope comprised by one of the extracellular domains of TrkB and is capable of activating TrkB, wherein the extracellular domains may comprise an extracellular D1 domain having the amino acid sequence shown in SEQ ID NO. 2, a D2 domain having the amino acid sequence shown in SEQ ID NO. 3, a D3 domain having the amino acid sequence shown in SEQ ID NO. 4, a D4 domain having the amino acid sequence shown in SEQ ID NO.5, a D5 domain having the amino acid sequence shown in SEQ ID NO. 6 and a membrane proximal domain having the amino acid sequence shown in SEQ ID NO. 7.

In certain embodiments, the antibody binds to an epitope comprised by the membrane proximal domain and is capable of activating a truncated TrkB having the amino acid sequence shown in SEQ ID NO. 8.

In certain embodiments, the truncated TrkB lacks the D1 to D5 domains and has the amino acid sequence shown in SEQ ID NO. 8.

In the present application, the TrkB-ECD (extracellular domain) may comprise an amino acid sequence shown in SEQ ID NO. 1.

Early life stress related factor

In the present application, developmental stage stress, which is one of the environmental factors, can affect the development of schizophreniform-like phenotypes. In the present application, developmental stage stress may include early life stress. In the present application, early life stress may include viral infection, smoking, intelligence, social cognition, cannabis use, social frustration, childhood trauma, pre-and post-natal hypoxia and/or pre-natal malnutrition. For example, early life stress may include juvenile isolation and/or postnatal hypoxia. The expression and/or activity level of the early-life stress-related factor may be a result of early-life stress.

In the present application, the expression and/or activity level of the early life stress-related factor may be increased in an SCZ subject as compared to a healthy subject. For example, the early-in-life stress-related factor may include CORT, the content of which may be increased by at least 10% (e.g., may be increased by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% as compared to a healthy subject).

In the present application, the early life stress-related factor may be related to a glucocorticoid (e.g., cortisol, corticosterone or CORT) and/or a signal pathway thereof. For example, the early life related factor may include prednisone, methylprednisone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, and/or dexamethasone. The early life stress related factor may include: glucocorticoids (e.g. cortisol, corticosterone or CORT).

In the present application, the subject may have elevated plasma cor levels. For example, the subject's plasma cor level can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or more, as compared to a control subject (e.g., a subject not suffering from or at risk of suffering from schizophrenia).

In the present application, the inhibitor against the early-life stress-related factor (e.g., CORT) may be capable of inhibiting the expression and/or activity level of the early-life stress-related factor (e.g., CORT).

In the present application, the inhibitor against an early-life stress-related factor (for example, against CORT) may include mifepristone (RU-486) or a derivative thereof.

Therapeutic method

In another aspect, the application further provides a method of preventing, alleviating and/or treating schizophrenia in a subject in need thereof, wherein a TrkB agonist (e.g., trkB agonistic antibody or antigen binding fragment thereof) and/or an inhibitor against an early-in-life stress-related factor (e.g., a cor inhibitor) can be administered to the subject.

In the present application, there is provided a method of preventing, alleviating and/or treating schizophrenia in a subject in need thereof, wherein the medicament of the application may be administered to said subject.

In the present application, the TrkB agonistic antibody or antigen binding fragment thereof may be administered to a subject in need thereof if the subject's cor content is increased compared to healthy subjects. In the present application, the inhibitor against CORT may be administered to the subject in need thereof if the subject's CORT content is increased compared to healthy subjects.

In the present application, the inhibitor against CORT may be administered to the subject in need thereof if the subject's (e.g., in the subject's plasma) expression and/or activity level of BDNF (e.g., BDNF-e 6) is reduced compared to a healthy subject. In the present application, the TrkB agonist (e.g., trkB agonistic antibody or antigen binding fragment thereof) may be administered to the subject in need thereof if the subject's BDNF (e.g., BDNF-e 6) expression and/or activity level is reduced compared to healthy subjects.

In the present application, the TrkB agonist (e.g., trkB agonist antibody or antigen binding fragment thereof) and the inhibitor against CORT may be administered to the subject in need thereof if the subject's (e.g., in plasma) expression and/or activity level of BDNF (e.g., BDNF-e 6) is reduced compared to healthy subjects and the subject's (e.g., in plasma) CORT content is increased compared to healthy subjects.

For example, if the subject's (e.g., in plasma) BDNF (e.g., BDNF-e 6) expression and/or activity level is reduced by at least about 5% (e.g., can be reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least 60% or more as compared to a healthy subject), and the subject's CORT content is increased by at least about 5% (e.g., can be increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least 60% or more as compared to a healthy subject), a TrkB agonist (e.g., a TrkB agonist antibody or antigen-binding fragment of the application) and an inhibitor against CORT of the subject can be administered to the subject in need thereof.

In the present application, the medicines may be administered simultaneously. In the present application, the pharmaceutical product may be applied by mixing together or separately the components, i.e. the TrkB agonist, e.g. the TrkB agonist antibody or antigen binding fragment thereof of the application and the inhibitor of the application against CORT.

The drugs may be administered in the same manner, e.g., to the same vein or other blood vessel, or may be administered in different manners, e.g., intravenous administration and oral administration may be performed simultaneously.

In the present application, the TrkB agonist (e.g., the TrkB agonistic antibody or antigen binding fragment of the present application) and the inhibitor against the early-in-life stress-related factor may be administered sequentially. The order of administration may be, first, administration of the TrkB agonist (e.g., a TrkB agonistic antibody or antigen binding fragment of the application) followed by administration of the inhibitor against the early-in-life stress-related factor; alternatively, the inhibitor against the early-in-life stress-related factor may be administered first, followed by administration of the TrkB agonist (e.g., a TrkB agonistic antibody or antigen binding fragment of the application).

In the present application, the TrkB agonist (e.g., the TrkB agonistic antibody or antigen binding fragment of the present application) and the inhibitor against the early-in-life stress-related factor may be applied in the same manner or in different manners. Each component (i.e. a TrkB agonist (e.g. a TrkB agonistic antibody or antigen binding fragment of the application) and an inhibitor of the application against CORT) may be applied once or in multiple applications.

In the present application, sequential administration may be performed at any time interval, including minutes, hours, days, weeks, months, or years. Sequential administration in the present application may refer to administration that is separated by any time interval of at least 1 minute (e.g., at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 60 minutes, or longer).

Diagnostic method

In another aspect, the application provides a method for determining whether a subject has or is at risk of developing schizophrenia, comprising determining the level and/or activity of BDNF-e6 expression in the subject, and determining the level and/or activity of CORT in the subject.

In another aspect, the application also provides a method of diagnosing and/or clinically classifying a subject suffering from schizophrenia, comprising the steps of: measuring the level of BDNF (e.g., BDNF-e 6) expression and/or activity in the subject (e.g., in the subject's plasma), and measuring the level of expression and/or activity of an early life stress related factor (e.g., CORT) in the subject (e.g., in the subject's plasma).

In the present application, the level of BDNF (e.g., BDNF-e 6) expression and/or activity of the subject can be measured by a detection method that can measure the level of DNA, RNA, and/or protein expression and/or activity of BDNF (e.g., BDNF-e 6).

In the present application, the detection method of BDNF may include qPCR, qRT-PCR, southern blotting, northern blotting, western blotting and/or ELISA.

In the present application, the level of BDNF (e.g., BDNF-e 6) expression and/or activity of the subject can be measured by a primer pair that may be capable of amplifying the Bdnf gene (e.g., BDNF-e 6) and/or a probe that may be capable of specifically binding to BDNF (e.g., BDNF-e 6). For example, the above expression and/or activity levels may be measured by a primer pair that may be capable of specifically amplifying the Bdnf gene (e.g., BDNF-e 6).

In the present application, the cor content of the subject may be measured by a detection method that may be capable of measuring the cor content.

In the present application, the detection method of the CORT may include infrared spectroscopy, nuclear magnetic resonance spectroscopy, photochemical analysis (e.g., refraction, uv-vis spectrophotometry, fluorescence analysis) and/or electrochemical analysis (e.g., potentiometry, coulometry, polarography and/or voltammetry).

In the present application, the cor content of a subject in the method may be measured by a probe that may be capable of specifically binding to cor.

In the present application, the detection method may use a sample from the subject. For example, the sample may comprise a blood sample. For example, the blood sample may be derived from peripheral blood.

In the present application, the method may include the steps of: the expression and/or activity level of BDNF is compared to a healthy subject and the expression and/or activity level of the stress-related factor early in life of the subject is compared to a healthy subject.

In the present application, the selected subject suffering from schizophrenia may include the following cases: (1) The subject's BDNF (e.g., BDNF-e 6) expression and/or activity level may be reduced compared to a healthy subject; (2) The subject's cor content may be increased as compared to a healthy subject; (3) The BDNF expression and/or activity level of the subject may be reduced as compared to a healthy subject, and the cor content of the subject may be increased as compared to a healthy subject.

In the present application, the subject's BDNF (e.g., BDNF-e 6) expression and/or activity level may be decreased as compared to a healthy subject and/or the subject's early life stress-related factor expression and/or activity level may be increased as compared to a healthy subject, and the subject suffering from schizophrenia may be selected.

For example, if the subject's BDNF (e.g., BDNF-e 6) expression and/or activity level is reduced by at least 10% (e.g., can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% as compared to a healthy subject), and the subject's cor content is increased by at least 10% (e.g., can be increased by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% as compared to a healthy subject), then the subject with schizophrenia can be selected as a potential subject for further treatment (e.g., administration of a TrkB agonist, such as a TrkB agonist antibody or antigen-binding fragment of the application and an inhibitor against cor of the application).

In the present application, the method may include the steps of: a TrkB agonist, e.g., a TrkB agonistic antibody or antigen binding fragment thereof according to the application and/or an inhibitor against an early-in-life stress-related factor, may be administered to a selected subject.

In the present application, the inhibitor against the early life stress-related factor may be administered to a selected subject if the BDNF (e.g., BDNF-e 6) expression and/or activity level of the selected subject is reduced compared to a healthy subject (e.g., may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% compared to a healthy subject).

In the present application, the TrkB agonist, e.g., trkB agonist antibody or antigen binding fragment thereof, may be administered to a selected subject if the level of expression and/or activity of the early-life stress-related factor (e.g., CORT) in the subject is increased compared to a healthy subject (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% increased compared to a healthy subject).

In the present application, the TrkB agonist, e.g., trkB agonist antibody or antigen-binding fragment thereof, may be administered to a selected subject if the subject's cor content is increased as compared to a healthy subject (e.g., may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% as compared to a healthy subject).

In the present application, if the subject's BDNF (e.g., BDNF-e 6) expression and/or activity level is reduced compared to a healthy subject (e.g., can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% compared to a healthy subject), and the subject's early-life stress-related factor (e.g., CORT) expression and/or activity level is increased compared to a healthy subject (e.g., can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% compared to a healthy subject), then the TrkB agonist, e.g., trkB agonist antibody or antigen-binding fragment thereof and/or an inhibitor against the early-life stress-related factor, can be administered to the selected subject.

In the present application, the TrkB agonist, e.g., trkB agonist antibody or antigen binding fragment thereof and/or inhibitor against CORT, may be administered to a selected subject if the subject's BDNF (e.g., BDNF-e 6) expression and/or activity level is reduced compared to a healthy subject (e.g., can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% compared to a healthy subject) and the subject's CORT content is increased compared to a healthy subject (e.g., can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% compared to a healthy subject).

Diagnostic system

In another aspect, the application also provides a system for diagnosing and/or clinically classifying a subject suffering from schizophrenia, comprising: a first measurement module that measures the level of expression and/or activity of BDNF (e.g., BDNF-e 6) in the subject (e.g., in plasma), and a second measurement module that measures the level of expression and/or activity of an early-life stress-related factor (e.g., CORT) in the subject (e.g., in plasma).

In the present application, the first measurement module may be capable of measuring DNA, RNA and/or protein expression and/or activity levels of BDNF (e.g. BDNF-e 6).

For example, the first measurement module may include any reagents and/or devices formulated to measure DNA, RNA, and/or protein expression and/or activity levels of BDNF. For example, the first measurement module may include any reagents and/or equipment formulated for measuring BDNF by qPCR, qRT-PCR, southern blotting, northern blotting, western blotting, and/or ELISA methods.

In the present application, the first measurement module may include a primer pair that may be capable of amplifying the Bdnf gene, and/or a probe that may be capable of specifically binding Bdnf.

For example, the primer of BDNF may be capable of specifically amplifying the BDNF gene (e.g., promoter VI of the BDNF gene). For example, the probe may comprise a sequence that is at least partially complementary and/or at least partially identical to the sequence of BDNF.

In the present application, the first measurement module may include an apparatus for performing qPCR and/or qRT-PCR. In the present application, the first measurement module may include a device for displaying a measurement of BDNF expression and/or activity level of the subject. For example, the results may be displayed in the form of numbers, graphs, and/or tables.

In the present application, the second measurement module may be capable of measuring the plasma content of CORT.

For example, the second measurement module may include any reagent and/or device formulated to measure the content of CORT. For example, the second measurement module may include any reagent and/or device formulated for measuring CORT by methods of infrared spectroscopy, nuclear magnetic resonance spectroscopy, photochemical analysis (e.g., refraction, uv-vis spectrophotometry, fluorescence analysis), and/or electrochemical analysis (e.g., potentiometric analysis, coulometry, polarography, and/or voltammetry).

In the present application, the second measurement module may comprise a probe that may be capable of specifically binding to CORT. In the present application, the second measurement module may comprise a pure CORT compound as a control sample.

In the present application, the second measurement module may comprise a device for displaying a measurement of the cor content of the subject. For example, the results may be displayed in the form of numbers, graphs, and/or tables.

In the present application, the system may include a sample collection module that collects a sample from the subject suffering from schizophrenia.

In the present application, the sample in the system may comprise a blood sample. For example, the sample collection module may comprise a tube and/or a blood collection device.

In the present application, the system may include a judgment module that compares the BDNF expression and/or activity level of the subject with schizophrenia to a healthy subject and/or compares the expression and/or activity level of a stress-related factor early in life of the subject with schizophrenia to a healthy subject.

In the present application, the determination module may determine whether the BDNF expression and/or activity level of the subject suffering from schizophrenia is likely to be reduced as compared to a healthy subject. For example, the judgment module may display the result of a decrease in BDNF expression and/or activity level in the subject suffering from schizophrenia as compared to a healthy subject.

In the present application, the determination module may determine whether the expression and/or activity level of the stress-related factor in the early life of the subject is likely to be increased as compared to a healthy subject.

In the present application, the determination module may determine whether the subject's CORT content is likely to be increased as compared to a healthy subject. For example, the determination module may show the result of an increase in the cor content of the subject as compared to a healthy subject.

In the present application, the system may include a advice module that provides advice on a treatment regimen to the subject suffering from schizophrenia according to the judgment result from the judgment module.

For example, if the judgment module shows the result that BDNF expression and/or activity level of the subject suffering from schizophrenia is reduced compared to a healthy subject and the cor content of the subject is increased compared to a healthy subject, the suggestion module may suggest that the subject suffering from schizophrenia is suitable for administration of a TrkB agonist, such as a TrkB agonistic antibody or antigen-binding fragment thereof according to the application and/or an inhibitor against cor according to the application.

The application provides the following embodiments:

Embodiment 1. A medicament for preventing, alleviating and/or treating schizophrenia comprising an tropomyosin-receptor-kinase B (TrkB) agonistic antibody or antigen binding fragment thereof and an inhibitor against an early-in-life stress-related factor.

Embodiment 2. The pharmaceutical product according to embodiment 1, wherein said TrkB agonistic antibody or antigen binding fragment thereof is capable of specifically binding to human TrkB.

Embodiment 3. The pharmaceutical product of any one of embodiments 1-2, wherein said TrkB agonist antibody or antigen binding fragment thereof is capable of inducing activation of a TrkB downstream signaling pathway.

Embodiment 4. The pharmaceutical product of any one of embodiments 1 to 3, wherein the TrkB agonistic antibody or antigen binding fragment thereof is capable of inducing gene expression comparable to natural human TrkB ligand Brain Derived Neurotrophic Factor (BDNF).

Embodiment 5. The pharmaceutical product according to any one of embodiments 1 to 4, wherein the antigen binding fragment thereof comprises Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb.

Embodiment 6. The pharmaceutical product of any one of embodiments 1 to 5, wherein the TrkB agonist antibody or antigen binding fragment thereof comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 to LCDR3, and the LCDR1 comprises the amino acid sequence of any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91.

Embodiment 7. The pharmaceutical product according to embodiment 6, wherein the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92.

Embodiment 8 the pharmaceutical product according to any one of embodiments 6 to 7, wherein the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93.

Embodiment 9. The pharmaceutical product according to any one of embodiments 6 to 8, wherein the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98.

Embodiment 10. The pharmaceutical product of any one of embodiments 1 to 9, wherein the TrkB agonist antibody comprises a light chain constant region, and the light chain constant region is a human igκ constant region or a human igλ constant region.

Embodiment 11. The pharmaceutical product according to any one of embodiments 1 to 10, wherein the TrkB agonist antibody or antigen binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94.

Embodiment 12. The pharmaceutical product according to embodiment 11, wherein the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95.

Embodiment 13 the pharmaceutical product according to any one of embodiments 11 to 12, wherein the HCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96.

Embodiment 14. The pharmaceutical product according to any one of embodiments 11 to 13, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97.

Embodiment 15. The pharmaceutical product of any one of embodiments 1 to 14, wherein the TrkB agonist antibody comprises a heavy chain constant region, and the heavy chain constant region is a human IgG constant region.

Embodiment 16. The pharmaceutical product of any one of embodiments 1 to 15, wherein the early-life stress comprises viral infection, smoking, intelligence, social cognition, cannabis use, social frustration, childhood trauma, prenatal and postprenatal hypoxia and/or prenatal malnutrition.

Embodiment 17 the pharmaceutical product according to any one of embodiments 1 to 16, wherein the expression and/or activity level of the early-life stress-related factor is increased due to the early-life stress.

Embodiment 18 the pharmaceutical product of any one of embodiments 1-17, wherein the early-life stress-related factor comprises Corticosterone (CORT).

Embodiment 19 the pharmaceutical product according to any one of embodiments 1 to 18, wherein the inhibitor against the early-life stress-related factor is capable of inhibiting the expression and/or activity level of the early-life stress-related factor.

Embodiment 20 the pharmaceutical product according to any one of embodiments 1 to 19, wherein the inhibitor against an early-life stress-related factor is capable of inhibiting the content of CORT.

Embodiment 21 the pharmaceutical product of any one of embodiments 1-20, wherein the inhibitor against an early-life stress-related factor comprises mifepristone (RU-486).

Embodiment 22. A method of preventing, alleviating and/or treating schizophrenia in a subject in need thereof, wherein an TrkB agonistic antibody or antigen binding fragment thereof and/or an inhibitor against an early-in-life stress-related factor is administered to said subject.

Embodiment 23. A method of preventing, alleviating and/or treating schizophrenia in a subject in need thereof, wherein the subject is administered a pharmaceutical product according to any one of embodiments 1 to 21.

Embodiment 24 the method of any one of embodiments 22 to 23, wherein the subject's BDNF expression and/or activity level is reduced compared to a healthy subject.

Embodiment 25. The method of embodiment 24, wherein the subject's BDNF expression and/or activity level is reduced by at least 10% compared to a healthy subject.

Embodiment 26 the method of any one of embodiments 24 to 25, wherein the expression and/or activity level of the early life stress-related factor of the subject is increased compared to a healthy subject.

Embodiment 27. The method of embodiment 26, wherein the early-life stress-related factor comprises CORT and the subject's content of CORT is increased compared to a healthy subject.

Embodiment 28. The method of embodiment 27, wherein the subject's cor content is increased by at least 10% as compared to a healthy subject.

Embodiment 29. The method of any one of embodiments 22 to 28, wherein said TrkB agonist antibody or antigen binding fragment thereof is capable of specifically binding human TrkB.

Embodiment 30. The method of any one of embodiments 22 to 29, wherein said TrkB agonist antibody or antigen binding fragment thereof is capable of inducing activation of a TrkB downstream signaling pathway.

Embodiment 31 the method of any one of embodiments 22 to 30, wherein said TrkB agonistic antibody or antigen binding fragment thereof is capable of inducing gene expression comparable to that of natural human TrkB ligand Brain Derived Neurotrophic Factor (BDNF).

Embodiment 32. The method of any one of embodiments 22 to 31, wherein the antigen binding fragment thereof comprises a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb.

Embodiment 33. The method of any one of embodiments 22 to 32, wherein said TrkB agonist antibody or antigen binding fragment thereof comprises a light chain variable region, wherein said light chain variable region comprises LCDR1 to LCDR3, and said LCDR1 comprises the amino acid sequence of any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91.

Embodiment 34. The method of embodiment 33 wherein the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92.

Embodiment 35 the method of any one of embodiments 33 to 34 wherein the LCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93.

Embodiment 36. The method of any one of embodiments 33 to 35, wherein the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98.

Embodiment 37 the method of any one of embodiments 22 to 36, wherein said TrkB agonist antibody comprises a light chain constant region, and said light chain constant region is a human igκ constant region or a human igλ constant region.

Embodiment 38. The method of any one of embodiments 22 to 37, wherein said TrkB agonist antibody or antigen binding fragment thereof comprises a heavy chain variable region, wherein said heavy chain variable region comprises HCDR1 to HCDR3, and said HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94.

Embodiment 39. The method according to embodiment 38, wherein the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95.

Embodiment 40. The method according to any one of embodiments 38 to 39, wherein the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96.

Embodiment 41. The method of any one of embodiments 38 to 40, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97.

Embodiment 42. The method of any one of embodiments 22 to 41, wherein the TrkB agonist antibody comprises a heavy chain constant region, and the heavy chain constant region is a human IgG constant region.

Embodiment 43 the method of any one of embodiments 27 to 42, wherein said TrkB agonist antibody or antigen binding fragment thereof is administered to said subject in need thereof if the subject's cor content is increased compared to a healthy subject.

Embodiment 44. The method of any of embodiments 22 to 43, wherein the early-life stress comprises a viral infection, smoking, intelligence, social cognition, cannabis use, social frustration, childhood trauma, prenatal and postprenatal hypoxia and/or prenatal malnutrition.

Embodiment 45 the method of any one of embodiments 22-44, wherein the level of expression and/or activity of the early-life stress-related factor is increased due to the early-life stress.

Embodiment 46. The method of any one of embodiments 22 to 45, wherein the early-life stress-related factor comprises Corticosterone (CORT).

Embodiment 47 the method of any one of embodiments 22 to 46, wherein the inhibitor against the early-life stress-related factor is capable of inhibiting the expression and/or activity level of the early-life stress-related factor.

Embodiment 48 the method of any one of embodiments 22-47, wherein the inhibitor against an early-life stress-related factor is capable of inhibiting the content of CORT.

Embodiment 49 the method of any one of embodiments 22 to 48, wherein the inhibitor against an early-life stress-related factor comprises mifepristone (RU-486).

Embodiment 50 the method of any one of embodiments 22 to 49, wherein the inhibitor against CORT is administered to the subject in need thereof if the subject's BDNF expression and/or activity level is reduced compared to a healthy subject.

Embodiment 51 a method of diagnosing and/or clinically classifying a subject suffering from schizophrenia comprising the steps of: measuring the level of BDNF expression and/or activity in the subject, and measuring the level of expression and/or activity of an early-in-life stress-related factor in the subject.

Embodiment 52. The method of embodiment 51, wherein the level of BDNF expression and/or activity of the subject is measured by a detection method capable of measuring the level of DNA, RNA and/or protein expression and/or activity of BDNF.

Embodiment 53. The method of embodiment 52, wherein the level of Bdnf expression and/or activity of the subject is measured by a primer pair capable of amplifying the Bdnf gene and/or a probe capable of specifically binding to Bdnf.

Embodiment 54 the method of any one of embodiments 51 to 53, wherein the early-life stress-related factor comprises Corticosterone (CORT).

Embodiment 55. The method of embodiment 54, wherein the subject's cor content is measured by a detection method capable of measuring cor content.

Embodiment 56 the method of any of embodiments 54 to 55, wherein the subject's cor content is measured by a probe capable of specifically binding to cor.

Embodiment 57 the method of any one of embodiments 51-56, wherein the detection method uses a sample from the subject, and the sample comprises a blood sample.

Embodiment 58 the method of any one of embodiments 51 to 57, wherein the method further comprises the steps of: the expression and/or activity level of BDNF is compared to healthy subjects and the expression and/or activity level of stress-related factors early in life of the subject is compared to healthy subjects.

Embodiment 59 the method of embodiment 58, wherein the subject with schizophrenia is selected if the level of BDNF expression and/or activity in the subject is reduced compared to a healthy subject and/or the level of expression and/or activity of the early-life stress-related factor in the subject is increased compared to a healthy subject.

Embodiment 60 the method of any one of embodiments 58 to 59, wherein the subject's BDNF expression and/or activity level is reduced by at least 10% compared to a healthy subject.

Embodiment 61 the method of any one of embodiments 58-60, wherein the expression and/or activity level of the early life stress-related factor of the subject is increased compared to a healthy subject.

Embodiment 62 the method of any one of embodiments 54 to 61, wherein the subject's cor content is increased compared to a healthy subject.

Embodiment 63 the method of any one of embodiments 51 to 62, wherein the method further comprises the steps of: administering to the selected subject a TrkB agonistic antibody or antigen binding fragment thereof and/or an inhibitor against an early-in-life stress-related factor.

Embodiment 64 the method of embodiment 63, wherein the inhibitor against the early-life stress-related factor is administered to the selected subject if the BDNF expression and/or activity level of the selected subject is reduced compared to a healthy subject.

Embodiment 65 the method of any one of embodiments 63-64, wherein said TrkB agonistic antibody or antigen binding fragment thereof is administered to a selected subject if the expression and/or activity level of said early life stress related factor in said subject is increased compared to a healthy subject.

Embodiment 66 the method of any one of embodiments 54 to 65, wherein said TrkB agonist antibody or antigen binding fragment thereof is administered to the selected subject if the subject's cor content is increased compared to a healthy subject.

Embodiment 67 the method of any one of embodiments 51 to 66, wherein if the level of BDNF expression and/or activity in the subject is reduced compared to a healthy subject and the level of expression and/or activity of the early life stress-related factor in the subject is increased compared to a healthy subject, the TrkB agonistic antibody or antigen binding fragment thereof and/or the inhibitor against the early life stress-related factor is administered to the selected subject.

Embodiment 68 the method of any one of embodiments 54 to 67, wherein said TrkB agonistic antibody or antigen binding fragment thereof and/or said inhibitor against cor is administered to a selected subject if the level of BDNF expression and/or activity in said subject is reduced compared to a healthy subject and the cor content in said subject is increased compared to a healthy subject.

Embodiment 69. A system for diagnosing and/or clinically classifying a subject having schizophrenia, comprising: a first measurement module that measures BDNF expression and/or activity levels in the subject, and a second measurement module that measures expression and/or activity levels of stress-related factors early in life in the subject.

Embodiment 70. The system of embodiment 69, wherein the first measurement module is capable of measuring DNA, RNA, and/or protein expression and/or activity levels of BDNF.

Embodiment 71 the system of any one of embodiments 69 to 70 wherein the first measurement module comprises a primer pair capable of amplifying the Bdnf gene and/or a probe capable of specifically binding Bdnf.

Embodiment 72 the system of any one of embodiments 69-71 wherein the early-life stress-related factor comprises Corticosterone (CORT).

Embodiment 73 the system of any of embodiments 69-72 wherein the second measurement module is capable of measuring the content of CORT.

Embodiment 74 the system of any one of embodiments 69 to 73 wherein the second measurement module comprises a probe capable of specifically binding to CORT.

Embodiment 75 the system of any one of embodiments 69 to 74, wherein the system comprises a sample collection module that collects a sample from the subject having schizophrenia.

Embodiment 76. The system of embodiment 75, wherein the sample comprises a blood sample.

Embodiment 77 the system of any one of embodiments 69 to 76 wherein the system comprises a judgment module comparing the subject suffering from schizophrenia to a healthy subject's expression and/or activity level of BDNF and/or comparing the subject suffering from schizophrenia to a healthy subject's expression and/or activity level of the early-life stress-related factor.

Embodiment 78 the system of embodiment 77, wherein said determination module determines whether the level of BDNF expression and/or activity in said subject with schizophrenia is reduced compared to a healthy subject.

Embodiment 79 the system of any one of embodiments 77-78, wherein said determination module determines whether the expression and/or activity level of said early life stress-related factor of said subject is increased as compared to a healthy subject.

Embodiment 80 the system of any one of embodiments 77-79, wherein said determination module determines whether the subject's cor content is increased compared to a healthy subject.

Embodiment 81 the system of any of embodiments 69-80 comprising a advice module that provides advice regarding a therapeutic regimen to the subject suffering from schizophrenia based on the determination from the determination module.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp for base pairs, kb for kilobases, pl for picoliters, s or sec for seconds, min for minutes, h or hr for hours, aa for amino acids, nt for nucleotides, i.m. for intramuscular (ground), i.p. for intraperitoneal (ground), s.c. for subcutaneous (ground), and so on.

Materials and methods

Animals

Bdnf-e6 mutant mice were generated using methods previously reported (Maynard et al 2016). Briefly, in Bdnf-e6 mutant mice, the eGFP-STOP expression cassette was inserted into exon VI and the PGK-Neo expression cassette was placed as the antisense sequence to eGFP. Subsequently, PGK-Neo was deleted by Cre recombinase expression. Thus, bdnf-eGFP fusion transcripts and eGFP were produced at the mRNA and protein levels, respectively. The genotyping primers were designed to distinguish between WT (forward: 5'-AA TCGAAGCTCAACCGAAGA-3' (SEQ ID NO: 77), reverse: 5'-TTTTTCTCTCACACTGAAGGGATT-3' (SEQ ID NO: 78)) and mutant (GFP) alleles (forward: 5'-AA TCGAAGCTCAACCGAAGA-3' (SEQ ID NO: 79), reverse: 5'-TCCAGCTCGACCAGGATG-3' (SEQ ID NO: 80)).

All experimental mice were bred by heterozygotes (e.g., bdnf-e6+/-) male mice with heterozygote female mice.

Mice are raised in animal facilities without specific pathogens. Under standard conditions, at a temperature of 22 ℃ to 26 ℃, a 12 hour light/12 hour dark cycle was set to provide sterile pellet feed and water for free access by mice.

Genotyping analysis revealed that the WT mice had a 566 base pair WT fragment and Bdnf-e6 mutant mice had a 367 base pair mutant fragment, and Bdnf-e6 +/-heterozygote mice had both WT and mutant fragments.

In all experiments, mice (male only, 2 to 4 months of age) were housed with siblings, 5 to 6 mice of different genotypes per cage, except for the social isolation paradigm in which standard mouse cages were used to individually house each mouse for 4 weeks. All mice were treated continuously for at least 3 days to acclimatize prior to testing: i.e. transferred to the test chamber at least 1 hour before the start of the test. All behavioral tests were performed during the light phase of the circadian cycle, arranged between 09:00 and 17:00.

All animal experiments were approved by the institutional animal care committee (ACUC) and were conducted in compliance with government agency and university of bloom animal welfare guidelines.

Animal magnetic resonance imaging

Magnetic Resonance Imaging (MRI) images were obtained using a 7.0T scanner (Bruker, biospec 70/20USR 7). Prior to scanning, all animals were initially anesthetized in an anesthetic chamber with 5% isoflurane in combination with oxygen, and then maintained under anesthesia by mask administration of 1.5% -2% isoflurane in combination with oxygen. MRI data contained the following parameters: repetition Time (TR) =2500 ms, echo Time (TE) =136 ms, acquisition Number (NA) =512, number of samples=2048, spectral width=4006 hz.

Behavioral testing

All mice (male only, 2 to 4 months of age) were housed in groups of 5 to 6 mice of different genotypes per cage, except for the social isolation housing paradigm in which standard mouse cages were used for 4 weeks each alone. All mice were treated continuously for at least 3 days to acclimatize prior to testing: i.e. transferred to the test chamber at least 1 hour before the start of the test. All behavioral tests were performed during the light phase of the circadian cycle, arranged between 09:00 and 17:00.

Rotating rod test

The rod-turning test consists of a training phase and a exploratory test phase (UGO, 47650). Prior to the test, mice were placed on their respective walkways, and were allowed to fit on a roller bar for 1 minute at a constant speed of 10 rpm. In both phases, the bar rotating device was set to accelerate from 10rpm to 30rpm, with a test time of up to 80 seconds. Mice were tested three times daily with an interval (ITI) of 20 minutes. After three consecutive days of training, an exploration test was performed on day 4. In all experiments, the average rod drop time during rod rotation was recorded automatically.

Open field test

The open field device was made of a white acrylic tank (50 cm. Times.50 cm. Times.40 cm). The central region is defined as the 25cm x 25cm region in the center of the box. Animals were placed in the central area, allowing the animals to freely explore for 10 minutes. All measurements were recorded by an automated tracking system and analyzed using EthoVision XT software (Noldus, netherlands), including total range in open field and time of stay in the central area.

Social interaction test

Social attribution and social memory were assessed by a three-room apparatus (60 cm. Times.45 cm. Times.20 cm). The social interaction test consists of a social ability test and a social novelty preference test. The test mice were placed in the apparatus and left free to explore for 5 minutes before social ability testing was performed. The mice were then confined in the middle chamber and "stranger 1" was randomly introduced into one of the two identical cages located in the lateral chamber. After opening the door, the test mice were allowed to freely explore for 10 minutes. In the social novelty preference test, a new strange mouse named "strange mouse 2" was placed in an empty cage. Again, the same test mice were allowed to freely explore for 10 minutes. Note that all strange mice used herein have the same background, age and sex as the test mice. Throughout the process, the residence time of the mice in each chamber and the time spent sniffing were monitored automatically by tracking the video and analyzed manually.

Morris water maze test

The Morris Water Maze (MWM) consists of a circular trough (120 cm diameter. Times.40 cm depth) and a circular platform (11 cm diameter. Times.18 cm depth). The tank was equally divided into 4 quadrants, each quadrant marked with circle, square, triangle and cross marks on the wall. Each quadrant is named north-east (NE), north-west (NW), south-west (SW) and south-east (SE), respectively. The behavioral experiments consisted of three phases: (1) hint phase (day 1): titanium dioxide is added to the water to make the water milky. The water temperature was set at 22℃and the water depth was set at 25cm to 30cm. The platform is randomly placed in the center of one of the quadrants. The mice were then placed in water at the desired location (rather than throwing into the sink) at the level with the mice facing the sink wall. The computer tracking program is started when the animal is placed in water. After the mice reached the platform, the timing was stopped and the mice were wiped dry and returned to the home cage. Mice that cannot find the platform are considered to be potentially abnormal in swimming ability or vision and are eliminated. (2) acquisition phase (day 2 to day 7): after 24 hours of the cue phase (day 2), the mice were placed in water (water depth 30cm to 45 cm), the platform was placed in the SW quadrant, and the motion profile was recorded for 60 seconds. If the mouse cannot reach the platform within 60 seconds, the experimenter will guide it to the platform and stay on the platform for 15 to 20 seconds. The experimenter would also direct the mouse to stay on the platform for 10 seconds if it successfully reached the platform but was unable to stay on the platform. After the experiment, the mice were wiped dry and returned to the home cage. Each mouse was trained 4 times per day at intervals of 20 to 30 minutes. The acquisition phase lasts for 6 days. The starting position for each test is listed in the table below. Changing water every 2-3 days to eliminate peculiar smell interference. The average time taken to successfully reach the platform per day is calculated. Note that it takes a shorter time to successfully reach the platform, indicating better spatial learning ability. (3) exploration phase (day 8): the mice were placed in the NE quadrant of the sink, and no platform was placed in the sink. The motion profile was recorded for 60 seconds. The time the mice remained in each quadrant, the number of passes through the home platform location, the time it took to successfully locate the home platform for the first time, was analyzed. Note that the more dwell times in the quadrant (SW) where the original platform is located, the more passes through the original platform location and the shorter the time it takes to successfully locate the original platform, indicating that the spatial memory is better.

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Pre-pulse inhibition assay

Pre-pulse suppression (PPI) tests were performed using Xeye startle reflex system (beijing Tianming macro technology development limited, china). The present PPI test consists of three phases. During the adaptation phase, the test mice were confined to the outdoor cage and then placed in the laboratory for 15 minutes the day before the test without any surprise stimulus. On the day of the test, there was first a familiar time of 5 minutes, followed by two types of stimulation tests in a pseudo-random order: (1) monopulse: 120dB stimulation (40 ms duration), (2) pre-pulse: a pre-pulse of 71dB, 81dB or 90dB (for 20 ms) was added before 120dB stimulation (for 40 ms). Ten times each test was performed using variable test intervals (ITI; 15 seconds to 20 seconds). In addition, 65dB of background noise was provided throughout the test period to avoid noise from outside the startle chamber from affecting the mice. PPI was calculated as a percentage score for each pre-pulse trial type: % ppi= [ (startle amplitude for monopulse test-startle amplitude for prepulse-pulse test)/startle response for monopulse test ] ×100.

New object identification test

New object identification tests were performed as described previously. The experimental set-up consisted of a white acrylic tank (31 cm. Times.31 cm. Times.20 cm). The object recognition task consists of three phases. In the first phase, three days in succession, the mice tested were placed in open field, other than the living cage, once daily for a familiarity time of 5 minutes. During the learning phase, the test mice were free to explore two identical objects located 5cm from the wall for 5 minutes. Before the memory retention phase, the mice were returned to their home cages for a period of time (5 minutes or 1 hour) and the two objects were removed. In the memory retention phase, the test mice were again placed in the open field, allowing the mice to explore an object identical to the original object ("familiar" object) and a new object, also for 5 minutes. The exploration time of all experiments was recorded manually throughout the process, with specific treatment modes blinded. The discrimination ratio of the memory retention phase is calculated using the following equation: [ (time spent exploring new objects-time spent exploring familiar objects)/total exploration time ]. The NOR trial used four male mouse cohorts (about 12 weeks old).

Nesting

Nesting was measured in living cages. Prior to the experiment, all nesting material in the living cages, including hay, string, wood chips, was removed. One hour prior to the dark phase, each mouse was placed individually in a living cage provided with an absorbent sponge as nesting material. According to fig. 11A, the next morning, the nests of each mouse were rated in the range of 1 minute to 4 minutes.

Environmental stress

Postnatal hypoxia paradigm

The parental mice used for breeding were heterozygous mice (Bdnf-e 6+/-) or Bdnf-e4+/-). Littermates and their genetics mice were randomly housed under normoxic or hypoxic conditions and food and water were freely available. During hypoxia, young and adult mice were housed in Plexiglas chambers (#a-30274-P, bioSpherix ltd., lacona, NY) equipped with a nitrogen (N ₂)/compressed air gas delivery system that used a compact oxygen controller (ProOx P, bioSpherix ltd.) to mix N ₂ with indoor air. Mice were exposed to hypoxic (10% O ₂ level) or normoxic (21% O ₂ level) conditions for 6 days (P4 to P10 neonatal stage or P70 to P76 adult stage). At three weeks of age, male littermates were separated from females and their genotypes were identified by PCR. Bdnf-e6/e4 homozygous male mice and WT male littermate control mice were used for further experiments.

Juvenile social isolation

The parental mice used for breeding were heterozygous mice (Bdnf-e 6+/-) or Bdnf-e4+/-). Male mice were housed in separate ventilated cages (up to six mice per cage) barrier facilities at the university of Qinghua. At weaning (PND 21), bdnf-e 6-/-and WT mice were randomly assigned to either isolated feeding (IR) conditions (1 mouse per cage) or social feeding (SR) conditions (4 to 5 mice per cage), fed for 4 weeks (to PND 49), ensuring equal assignment of mice of different genotypes in the cages of each SR condition. All mice in the cages were kept under the same conditions (12 hours light/12 hours dark cycle, 22 ℃ to 26 ℃) and sterile pellet feed and water were freely available so that the IR mice could still see, hear and smell the other mice but without physical contact.

Blood corticosterone measurement

Orbital blood samples (500. Mu.L) were collected into 1.5mL Eppendorf tubes, allowed to stand at room temperature for 45 minutes, then centrifuged at 1500rpm at 4℃for 15 minutes, and the supernatant serum was transferred to fresh 1.5mL Eppendorf tubes. According to serum volume, pre-frozen HPLC grade methanol (-80 ℃) was added to the tube, 400. Mu.L methanol was added per 100. Mu.L serum. The mixture was shaken gently by hand for 1 minute and incubated at-80℃for 2 hours. After incubation at-80 ℃,14000g, centrifugation at 4 ℃ for 10 minutes, and further, freeze-drying of the supernatant with Speedvac (Thermo SAVANT SPD, 1010) to a powder. The dried samples were used for corticosterone measurements or stored in a-80 ℃ refrigerator. The ultra-high performance liquid chromatography system was coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher, CA) equipped with an APCI probe. For corticosterone analysis, the extract was isolated by 150X 2.1mm biphenyl column. A binary solvent system was used in which mobile phase a consisted of 100% H ₂ O, 0.1% FA and mobile phase B consisted of 100% CAN containing 0.1% FA. Gradient elution was performed for 10 minutes at a flow rate of 350. Mu.L/min. The column chamber and sample tray were maintained at 40℃and 10℃respectively. Data in the mass range of 300 to 400 m/z are obtained in the positive ion mode, and the data are acquired in a full scanning mode with 70000 resolution. The source parameters are as follows: a discharge current of 6 μA; capillary temperature: 320 ℃; heater temperature: 400 ℃; sheath air flow: 35Arb; auxiliary gas flow rate: 10Arb. To calculate the absolute value of the glucocorticoid content, 200. Mu.L of 100. Mu.g/mL corticosterone diluted with HPLC grade methanol was used as a standard sample. The software used for data analysis and quantification was xcalibur3.0.63 (Thermo Fisher, CA).

Drug treatment

Long-term treatment of corticosterone

The corticosterone was first dissolved with 100% ethanol and then further dissolved in mouse drinking water to give an ethanol concentration of 1% and a corticosterone content of 0.1mg/mL. The drinking water bottle was completely covered with silver paper, and water was changed every 3 days to prevent degradation of corticosterone over time. The drinking water of the mice of the control group contained only 1% ethanol.

Long term treatment of RU-486

RU-486 was dissolved to 10mg/mL with 100% Tween 80 and 0.9% saline, with 1% Tween 80 in the final working solution. RU-486 groups of mice were injected intraperitoneally once daily with 40mg/kg RU-486 (4. Mu.L/g working solution) for 8 consecutive days. Whereas control mice were intraperitoneally injected with 1% Tween 80 in 0.9% saline at 4. Mu.L/g.

Trkb agonistic antibody therapy

Adult Bdnf-e 6-/-mice underwent postnatal hypoxia or social isolation during their childhood, and the MK801 treated WT mouse model was treated with AbB901 (1 mg/kg) single tail intravenous injection 48 hours before the PPI trial began. Control mice were given saline or normal IgG (1 mg/kg) by tail vein injection. Details of the model treated with MK801 are set forth below. HCDR1 in AbB antibodies has the amino acid sequence shown as SEQ ID NO. 48, HCDR2 has the amino acid sequence shown as SEQ ID NO. 49, HCDR3 has the amino acid sequence shown as SEQ ID NO. 50, LCDR1 has the amino acid sequence shown as SEQ ID NO. 45, LCDR2 has the amino acid sequence shown as SEQ ID NO. 46, and LCDR3 has the amino acid sequence shown as SEQ ID NO. 47. The VH in AbB901,901 antibody has the amino acid sequence shown in SEQ ID NO. 52. VL in AbB-901 antibody has the amino acid sequence shown in SEQ ID NO. 51.

MK801 treatment

MK801 (Merck) was first dissolved in saline and diluted to give working solutions (concentration 10mM/mL to 4 mM/mL). MK801 (0.2 mg/kg) was administered intraperitoneally to adult WT mice 30 minutes prior to the initiation of the PPI assay.

RT-PCR

Total RNA was isolated and extracted from frozen target tissues using a cell/tissue total RNA isolation kit (Vazyme) according to the manufacturer's instructions. RNA concentration was determined using NanoDrop (Denovix) and then reverse transcribed into cDNA using HISCRIPT IIQ RT SuperMix (Vazyme) according to the manufacturer's instructions. Quantitative real-time RT-PCR was performed using AceQ qPCR SYBR GREEN MASTER Mix (Vazyme) according to the recommended protocol.

Western blotting

Milling was performed in 1mL of chilled RIPA lysis buffer to homogenize the dissected brain tissue from each mouse. After centrifugation at 17000g for 30 min at 4℃the supernatant was collected and then the protein concentration was measured by BCA protein assay kit (Thermo Scientific). Thereafter, the protein was denatured at 95℃for 10 minutes according to the total concentration. Proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred onto an activated PVDV membrane (Immobilon-P, millipore). The membranes were blocked for 1 hour at room temperature in 0.1M tris buffered saline (0.1% Tween 20, i.e., TBST) solution in 5% BSA. The membranes were then incubated overnight in primary anti-dilution buffer at 4 ℃. After washing with TBST, the membranes were incubated in secondary anti-dilution buffer for 2 hours at room temperature. After TBST washing, the membrane was detected by Tanon 5200 (software: tanon MP, v 1.02) using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and analyzed by Tanon Gis (v 4.2). The primary antibodies used were anti-GFP antibodies (1:500, genscript, A01694-40), anti-TrkB antibodies (1:1000, CST, 4603S), anti-pTrkB antibodies (1:1000, CST, 4168S), anti- β -actin antibodies (1:2500, cwbio, CW 0096M).

Immunofluorescent staining

Mice were deeply anesthetized with avermectin (Avertin), perfused with PBS (ph 7.4), and fixed using a PBS solution containing 4% Paraformaldehyde (PFA). Brains were dissected, post-fixed (post-fix) overnight in 4% PFA at 4 ℃ and then coronal sections 40 μm thick were cut with a vibrating microtome. The free floating sections were again fixed with 4% Paraformaldehyde (PFA) in PBS for 15 min, washed three times with PBS at room temperature (15 min), and blocked overnight at 4 ℃ in PBS solution containing 5% normal goat serum and 0.3% Triton X-100. The sections were then incubated overnight with primary antibody at 4 ℃. The next day, sections were washed three times with PBS at room temperature (15 min) and then treated with secondary antibody overnight at 4 ℃. Finally, the sections were washed three times (15 minutes) in PBS at room temperature and mounted on slides with mounting medium (with DAPI) and then coverslipped. The slide was scanned with an Axio scan.z1 (ZEISS) microscope to obtain images, which were analyzed with ZEISS Zen (v 2.1) and ImageJ (v 1.52 i). The primary antibody used for immunostaining was GFP (1:1000, AVES, GFP-1020) and the secondary antibody was donkey anti-chicken IgG H & L Alexa Fluor 488 (1:1000,Jackson ImmunoResearch,703-545-155).

Quantitative and statistical analysis

Unpaired Student t test was used in social interaction tests, morris water maze test, pre-pulse inhibition test, new object recognition test, measurement of corticosterone, and detection of relative mRNA expression. In open field tests, stick rotation tests, pre-pulse suppression tests, new object identification tests, nesting tests and weight measurements, two-way ANOVA analysis and Tukey multiple comparison tests were used. In the multi-column, statistical significance was determined by analysis of variance, post hoc inspection. The number of animals used for each trial is shown in the bar graph or embodied herein. The sample size is based on similar published studies to ensure adequate statistical certainty. Furthermore, the sample size of each experiment has been presented in detail in the illustration of the figures and statistically confirmed by appropriate examination. Genotyping data was not blinded, but was reviewed by different researchers. Western blotting was used and data were analyzed by different researchers. For morphological analysis, data collection was performed together in a common microscope, with each experimental condition blinded.

GRAPHPAD PRISM 8.0.0 software (GraphPad Software) was used for all statistical analyses. Data are expressed as mean ± SEM. * P is less than 0.05; * P < 0.01; * P < 0.001; * P < 0.0001; ns=insignificant.

Example 1 disruption of Bdnf-e6 alone does not cause schizophreniform behavior

A mouse knock-in line Bdnf-e 6-/-wasconstructed in which Bdnf transcription via promoter VI was selectively disrupted (Maynard et al 2016). In this line (Bdnf-e 6-/-), the eGFP expression cassette was inserted immediately downstream of Bdnf exon 6, followed by placement of multiple STOP codons. Thus, bdnf promoter VI drives the production of a transcript containing the 5' UTR-eGFP-Bdnf encoding exon IXa, which in turn is translated into eGFP, rather than BDNF protein (FIG. 9A).

Genotyping analysis revealed that WT and Bdnf-e6 mutant mice had a wild-type (WT) fragment of 566 base pairs and a mutant fragment of 367 base pairs, respectively (FIG. 9A). Quantitative PCR (qPCR) experiments demonstrated that Bdnf-e6 mRNA was highly enriched in brain in adult WT mice, whereas peripheral tissues were not seen (FIG. 9B) (Aid et al, 2007). In the WT mouse brain, hippocampus and mPFC were more enriched for Bdnf-e6 mRNA than the other regions (fig. 9B). In Bdnf-e 6-/-mouse brains, bdnf-e6 transcripts were not present at all, but levels of Bdnf-e1, bdnf-e2 and Bdnf-e4 transcripts were relatively normal in hippocampus and mPFC (except for a slight decrease in Bdnf-e4 transcript in hippocampus) (FIGS. 9C and 9D). GFP protein was detected in the various brain regions of Bdnf-e 6-/-mice, but not in the WT mouse brain using Western blotting (FIG. 9I). Immunohistochemistry showed that Bdnf-e6-eGFP was highly enriched in the hippocampus of mutant mice (FIG. 9J). In addition, bdnf-E6 deficiency did not lead to weight changes, motor impairment (FIGS. 9E-9F) and lateral ventricle enlargement (FIG. 9K).

A set of schizophrenia-related behavioral tests were performed in adult male Bdnf-e 6-/-mice. In open field trials, total range of motion was considered as an indicator of motor output, which is often elevated in animal models of schizophrenia (Seibenhener and Wooten,2015; tatem et al, 2014). As shown in fig. 9G and 9H, bdnf-e 6-/-mice showed normal motor activity, but the residence time in the central region was shorter, indicating a slight increase in anxiety (p=0.0253). Three-room trials evaluate social home feel and social memory, two internal phenotypes that are often impaired in schizophrenia. In the social ability test, both Bdnf-e 6-/-mice and WT mice exhibited normal social interactions, taking more time to sniff the cup containing the live mice (strange mouse 1) than the empty cup (FIG. 1A, WT mouse P=0.0002, bdnf-e 6-/-mouse P=0.0008). Similarly, in the social novelty bias test, these mice also biased to contact new mice (strange mouse 2) rather than familiar mice (strange mouse 1) (fig. 1b, wt mouse p=0.0229, bdnf-e 6-/-mouse p=0.0326), indicating that social memory is intact. In the Morris Water Maze (MWM) test (FIG. 1C), the amount of time it takes for WT mice and Bdnf-e 6-/-mice to learn the hidden platform position was similar (except for small differences on day 6) (FIG. 1D). There was no difference in the performance of WT mice and Bdnf-e 6-/-mice in terms of delayed arrival at the platform during the pilot trial (fig. 1e, p=0.3748). However, the time that the Bdnf-e 6-/-mice remained in the target quadrant (fig. 1f, p=0.0029) and the number of passes through the plateau positions in the target quadrant (fig. 1g, p=0.0271) did decrease slightly compared to WT mice, indicating impaired spatial memory. Finally, a pre-pulse inhibition (PPI) test was performed to examine sensorimotor gating function, another common schizophrenia-related internal phenotype. Bdnf-e 6-/-mice showed normal startle response (FIG. 1H, P= 0.3303) and PPI (FIG. 1I) at all pre-pulse intensity levels, indicating that Bdnf-e 6-/-mice have normal sensorimotor gating function.

EXAMPLE 2Bdnf-e6 disruption and postnatal hypoxia co-induce schizophreniform-like internal phenotype

Bdnf-e 6-/-pups and WT pups were kept in normoxic (21% O ₂) or hypoxic (10% O ₂) environments for 6 consecutive days starting at day 4 after birth and behavioural tests were performed in adulthood (2 to 3 months of age) (FIG. 10A). Thus, there are four experimental groups: normoxic WT, anoxic WT, normoxic e 6-/-and anoxic e6-/-. Early-life hypoxia resulted in a transient decrease in body weight during postnatal development in WT mice and Bdnf-e 6-/-mice (fig. 10B-10D). At 3 weeks (PND 20, fig. 10B) and 5 weeks (PND 36, fig. 10C), mice exposed to hypoxia were lighter than normoxic fed cohort control mice (WT), but no similar phenomenon was seen at 9 weeks (PND 63, fig. 10D). It was also noted that during development, bdnf-e 6-/-mice had slightly greater (3 weeks) or the same (5 weeks) body weight loss than WT mice, and that the body weight loss was completely restored in adulthood (9 weeks) (FIG. 10D).

In the open field trial, there was no difference in "total range of motion" between the 4 groups, indicating that early life stress did not cause movement defects (fig. 2B). Also, in the Bdnf-e 6-/-mouse rotarod test, motor coordination and motor learning skills appeared normal, whether or not the mice experienced early-life hypoxia (FIG. 2C).

To investigate whether this interaction of the gene (Bdnf-e 6-/-) and the environment (postnatal hypoxia) (GxE) also affects other schizophrenia-related behaviors, a three-chamber test was performed. In the social ability test, bdnf-E6-/-mice experiencing postnatal hypoxia spent the same amount of time to explore empty cups and cups containing live mice (stranger 1) (FIG. 2E). In contrast, the other three groups all prefer to spend more time exploring cups containing live mice (fig. 2E). Thus, bdnf-e6 transcriptional disruption and early life hypoxia may not have an impact on social activity as a whole. In the social novelty bias trial, WT mice were kept in contact with new mice (strange mouse 2, fig. 2F) longer than with familiar mice (strange mouse 1), whether or not hypoxia was experienced. Bdnf-e 6-/-mice that did not experience postnatal hypoxia also showed preference for new social strangers (FIG. 2F). In sharp contrast, hypoxic e 6-/-mice showed no significant preference (p= 0.3894) (fig. 2F). Thus, the ability of an animal to socialize with a new companion can also be affected by Bdnf-e6 mice experiencing stress early in life. Both social ability and social novelty preference tests together reveal a strong interaction between Bdnf-e 6-/-and postnatal hypoxia, suggesting a role for a particular GxE combination in social attribution and social memory.

Nesting is another inherent social behavior that is believed to be associated with mating and parent-child associations (Jirkof, 2014). After 24 hours, the average nesting score of hypoxic e6 mice was significantly reduced compared to normoxic WT mice or normoxic e6 mice (fig. 11B), indicating impaired nesting behavior in hypoxic e6 mice. However, in postnatal hypoxic mice, the impaired nesting behavior of Bdnf-e 6-/-mice appears to be more severe than WT mice, although not statistically significant (fig. 11A, fig. 3C). Nesting scores were almost 4 points in hypoxic e 6-/-mice (FIG. 11C). Thus, bdnf-e6 defects may cause further impaired nesting behavior under early life hypoxic conditions.

Since MWM trials have shown spatial memory defects in Bdnf-e 6-/-mice that have not undergone hypoxia (fig. 1D-1G), there is a need for a cognitive test that is more sensitive to reveal the effect of GxE combinations. The one-time new object identification test is based entirely on the ability of rodents to distinguish new objects from familiar objects. In the learning phase, four groups of mice had no significant difference in exploration time for two identical objects with a delay of 5 minutes (data not shown). In the memory-preserving phase, the subject mice were presented with new objects sideways to the familiar objects. Hypoxia negatively affects the ability to distinguish new and familiar objects (fig. 11D). In addition to hypoxia, bdnf-E6 deficiency also compromised the ability of mice to distinguish between "old" and "new" objects (FIG. 11E). These results indicate that Bdnf-e6 mice may also exhibit cognitive deficits if they experience early life stress.

PPI tests were then performed, a more direct behavioral test with schizophrenia (Carr et al 2016; papaleo et al 2016). At all pre-pulse intensities (71 dB, 81dB, 90 dB), PPI levels were greatly reduced for the hypoxia e6 group alone (fig. 2G-2I). Thus, disruption of Bdnf-e6 expression, as well as postnatal hypoxia, together trigger PPI defects associated with schizophrenia.

Example 3 social isolation of young animals likewise induces schizophreniform abnormalities

The dramatic impact of postnatal hypoxia on schizophrenic inner phenotype has prompted the inventors to continue to study whether other forms of early life stress such as social isolation also trigger schizophrenic behavior. Animals post weaning (21 days old) were randomly assigned to social rearing groups (PSR, multiple animal population rearing) or isolated rearing groups (PIR, each animal individually reared in a single cage) for 4 weeks, and behavioral tests were conducted in adulthood (fig. 3A). PIR caused an overall increase in locomotor activity (total range of movement) in both genotypes of mice compared to PSR in open field experiments (fig. 3B). Interestingly, disruption of Bdnf-e6 selectively reduced the residence time of PSR mice in the central region, whereas PIR mice were unaffected (FIG. 3C). PIR may mask the effects of Bdnf-e6 disruption, which is revealed under social rearing conditions.

In social ability experiments, the quarantine feeding had no effect on either Bdnf-e 6-/-mice or WT mice. Animals of both genotypes spent more time interacting with mice (stranger 1), whether PSI or PIR fed (fig. 3D), than smelling empty cups. In the social novelty test, animals raised with PSR interacted with new mice (stranger 2) for longer periods of time than with familiar peers (stranger 1), regardless of genotype (fig. 3D). In contrast, PIR substantially eliminates the preference for interaction with the new companion, and in both WT mice and Bdnf-E6 mice, the time spent interacting with stranger 1 and stranger 2 is the same (right side of fig. 3E), indicating that merely isolating this condition may be sufficient to induce a deficit in interaction behavior with the new companion.

In the PPI test, PIR as a whole inhibited PPI at all pre-pulse intensities (71 dB, 81dB, 90 dB) (FIGS. 4B-4D). Disruption of Bdnf-e6 expression appears to further reduce PPI ratio at 71dB and 81dB pre-pulse intensities, but this effect is not statistically significant. Thus, postweaning social isolation appears to greatly inhibit sensorimotor gating behavior, and so it is difficult to reveal the additional effects of Bdnf-e6 disruption. The idea is also corroborated by the fact that: adult isolation feeding (AIR, whose effect is considered weaker than PIR) can induce PPI deficiency in Bdnf-e 6-/-mice but not WT mice at all pre-pulse intensities (71 dB, 81dB, 90 dB) (fig. 4F-4H). Taken together, these results indicate that impairment of Bdnf-e6 expression can act as a genetic factor, in conjunction with environmental stress during postnatal development or adulthood, to induce schizophreniform-like internal phenotypes.

Similar to Bdnf-e6, bdnf-e4 is a major regulator of GABAergic function and is expressed in brain regions associated with schizophrenia, such as the hippocampus, prefrontal cortex, and hypothalamus (Maynard et al 2016). Therefore, it was examined whether Bdnf-e 4-/-mice also show a schizophreniform-like behavioral phenotype under early life environmental stress conditions. The data show that Bdnf-e 4-/-mice that underwent postnatal hypoxia or juvenile social isolation did not exhibit PPI deficiency (FIG. 12). These results indicate that animals with Bdnf-e6 deficiency are more likely to develop a schizophreniform phenotype upon exposure to environmental stress.

Example 4 increased blood corticosterone and disruption of Bdnf-e6 together lead to a schizophreniform-like internal phenotype

Plasma corticosterone is released in response to environmental stress and is used as an indicator of rodent and human body stress responses. Thus, it was investigated whether postnatal hypoxia or social isolation paradigms could increase corticosterone levels. Consistent with previous findings (Barlow et al, 1975; krishnan et al, 2007; zheng et al, 2019), plasma Corticosterone (CORT) levels increased in WT mice with exposure to environmental stresses (FIGS. 5A-5C), including postnatal hypoxia, puberty and adult social isolation.

Mice that were chronically treated with CORT (0.1 mg/kg,22 days) (FIG. 6A) were then tested for their ability to mimic stress patterns, resulting in schizophreniform-like internal phenotypes. Long-term CORT exposure (from P42 to P63) did not lead to anxiety-like behavior in WT mice or Bdnf-e 6-/-mice (fig. 6c, P > 0.05), although locomotor activity was significantly reduced (fig. 6B). Interestingly, chronic pubertal exposure to CORT did not impair the social ability of mice of either genotype, but impaired the social novelty preference of Bdnf-E6-/-mice, which WT mice were unaffected (FIGS. 6D and 6E). In addition, the same treatment also selectively reduced PPI in Bdnf-e 6-/-mice at 81dB and 90dB pre-pulse intensity (FIGS. 6G-6H). After the CORT treatment, the PPI at 71dB was so low that Bdnf-e6 disruption no longer induced a further reduction in PPI (FIG. 6F). These data support the notion that blood corticosterone levels can affect the susceptibility of mice to postnatal stress, and demonstrate that chronic administration of CORT mimics postnatal stress to induce a schizophrenic-like internal phenotype in Bdnf-e 6-/-mice.

Example 5 treatment with TrkB agonist antibodies or CORT antagonists to rescue PPI deficiency

One obvious result of Bdnf-e6 disruption is a decrease in BDNF signaling in the brain. The data indicate that there was no change in Bdnf mRNA in the prefrontal cortex and hippocampus after post-natal hypoxia. However, bdnf-e6 mRNA was slightly but significantly reduced in the hippocampus (FIG. 13B). In connection therewith, it was observed that pTrkB levels were also slightly decreased in the same region of the population (in the hippocampus of Bdnf-e 6-/-mice experiencing postnatal hypoxia) (FIGS. 13C-13D). Thus, experiments were conducted with PPI as an indicator, and it was investigated whether administration of an agonistic antibody AbB (Guo et al, 2019; han et al, 2019) that selectively activated the BDNF receptor TrkB could rescue the behavioral deficit in this GxE model (Bdnf-e 6-/- + postnatal hypoxia or social isolation).

Bdnf-E6-/-mice were postnatally exposed to hypoxia (FIG. 7A) or social isolation (FIG. 7E), and then single tail adult animals were given either normal IgG or AbB901 (1 mg/kg) intravenously. Notably, abB901 can rescue PPI damage at the intensity of P81P120 and P90P120, and not P71P120 in hypoxic Bdnf-e 6-/-mice (fig. 7B-7D). AbB901 was also able to significantly repair PPI defects at a pre-pulse intensity of 90dB in SI-treated Bdnf-e 6-/-mice (fig. 7F). At 71dB and 81dB AbB901,901 has a trend of effect, although statistical significance is not achieved (fig. 7G to 7H). From these data, abB a 901 has an increased likelihood of being used as a therapeutic agent for the clinical treatment of some schizophrenic patients, especially patients with a genetic variant of Bdnf (such as Bdnf val/met polymorphism) or a decrease in blood Bdnf levels.

Considering that postnatal stress or chronic administration of CORT elicits PPI defects in Bdnf-e 6-/-mice, researchers believe that blocking CORT signaling can also attenuate the SCZ internal phenotype in the GxE model. To test this point, the present inventors used mifepristone (RU-486) to investigate whether this drug could rescue PPI defects in hypoxia or PIR treated Bdnf-e 6-/-mice. Mifepristone, which is an 11 beta-dimethyl-amino-phenyl derivative of norethindrone, has a high affinity for the glucocorticoid receptor and has been shown to be an active glucocorticoid receptor inhibitor. It was found that RU-486 significantly increased the PPI ratio of PIR-treated Bdnf-e 6-/-mice at all pre-pulse intensities (71 dB, 81dB, 90 dB) (fig. 8A-8D, P <0.01, P < 0.001). RU-486 was also able to reduce PPI deficiency at partial pre-pulse intensity (P90 dB) and no reduction at other intensities (P71 dB, P81 dB) in postnatal hypoxic Bdnf-E6-/-mice (fig. 8E to 8H). These data indicate that CORT antagonist treatment can rescue PPI defects in Bdnf-e 6-/-mice experiencing postnatal stress. Furthermore, RU-486 treatment had no effect on PPI in adult isolated fed (AIR) -treated Bdnf-e 6-/-mice (fig. 14), indicating a significant impact of developmental stage stress. Interestingly, RU-486 induced a small but significant increase in PPI ratio in PIR-treated WT mice (fig. 15).

Finally, to determine whether RU-486 treatment could function in a non-genetic model, the present inventors administered MK801 intraperitoneally to adult WT mice (fig. 16A), a widely used pharmacological model of schizophrenia. The inventors pretreated adult WT animals with saline or RU-486 for one week and then induced SCZ with MK801 (0.2 mg/kg). RU-486 treatment increased PPI at 90dB pre-pulse intensity (fig. 16D).

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The invention is not limited to the specific embodiments provided in this specification. While the invention has been described with reference to the above description, the descriptions and illustrations of the embodiments herein should not be construed in a limiting sense. Many changes, modifications and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention will also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and their equivalents and methods and structures within the scope of these claims are hereby contemplated.

Claims

1. A medicament for the prevention, alleviation and/or treatment of schizophrenia comprising a tropomyosin-receptor-kinase B (TrkB) agonist and a glucocorticoid (e.g. cortisol, corticosterone or CORT) inhibitor.

2. The pharmaceutical product of claim 1, wherein the TrkB agonist comprises a TrkB agonistic antibody or antigen binding fragment thereof.

3. A pharmaceutical product according to any one of claims 1 to 2 wherein the TrkB agonist is capable of specifically binding to human TrkB.

4. A pharmaceutical product according to any one of claims 2 to 3, wherein the antigen binding fragment comprises a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv and/or dAb.

5. The pharmaceutical product of any one of claims 1-4, wherein the TrkB agonist comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3, and the LCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91.

6. The pharmaceutical product of claim 5, wherein the LCDR2 comprises an amino acid sequence of any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92.

7. The pharmaceutical product of any one of claims 5 to 6, wherein the LCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93.

8. The pharmaceutical product according to any one of claims 5 to 7, wherein the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98.

9. The pharmaceutical product of any one of claims 2-7, wherein the TrkB agonistic antibody comprises a light chain constant region, and the light chain constant region is a human igκ constant region or a human igλ constant region.

10. The pharmaceutical product of any one of claims 1 to 9, wherein the TrkB agonist comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94.

11. The pharmaceutical product of claim 10, wherein the HCDR2 comprises an amino acid sequence of any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95.

12. The pharmaceutical product according to any one of claims 10 to 11, wherein the HCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96.

13. The pharmaceutical product according to any one of claims 10 to 12, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97.

14. The pharmaceutical product of any one of claims 2-13, wherein the TrkB agonist antibody comprises a heavy chain constant region, and the heavy chain constant region is a human IgG constant region.

15. The pharmaceutical product according to any one of claims 1 to 14, wherein the glucocorticoid inhibitor is capable of reducing the amount of the glucocorticoid.

16. The pharmaceutical product according to any one of claims 1 to 15, wherein the glucocorticoid inhibitor is capable of inhibiting the activity of the glucocorticoid.

17. The pharmaceutical product according to any one of claims 1 to 16, wherein the glucocorticoid inhibitor comprises mifepristone (RU-486) or a functional derivative thereof.

18. A method for preventing, alleviating and/or treating schizophrenia in a subject in need thereof, the method comprising administering to the subject a TrkB agonist and/or glucocorticoid (e.g., cortisol, corticosterone or CORT) inhibitor, and the subject has a reduced level and/or activity of BDNF (e.g., BDNF-e 6) expression and an increased level and/or activity of glucocorticoid (e.g., cortisol, corticosterone or CORT).

19. The method of claim 18, wherein the subject has a defect in promoter VI of the Bdnf gene.

20. The method of any one of claims 18 to 19, wherein the subject has experienced post-natal stress.

21. The method of claim 20, wherein the post-natal stress comprises post-natal hypoxia and/or social isolation.

22. The method of any one of claims 18 to 21, wherein the subject has reduced expression levels and/or activity of BDNF (e.g. BDNF-e 6) in the hippocampus, prefrontal cortex and/or hypothalamus.

23. A method according to any one of claims 18 to 22, wherein the TrkB agonist comprises a TrkB agonist antibody or antigen binding fragment thereof.

24. A method according to any one of claims 18 to 23, wherein the TrkB agonist is capable of specifically binding to human TrkB.

25. The method of any one of claims 23 to 24, wherein the antigen binding fragment comprises a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv, and/or dAb.

26. The method of any one of claims 18 to 25, wherein the TrkB agonist comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3, and the LCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 9, 15, 21, 29, 37, 45, 53, 61, 69, 81 and 91.

27. The method of claim 26, wherein the LCDR2 comprises an amino acid sequence of any one of SEQ ID NOs 10, 16, 22, 20, 28, 46, 54, 62, 70, 82 and 92.

28. The method of any one of claims 26 to 27, wherein the LCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 11, 17, 23, 31, 39, 47, 55, 63, 71, 83 and 93.

29. The method of any one of claims 26 to 28, wherein the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 27, 35, 43, 51, 59, 67, 75, 88 and 98.

30. The method of any one of claims 23-29, wherein the TrkB agonistic antibody comprises a light chain constant region, and the light chain constant region is a human igκ constant region or a human igλ constant region.

31. The method of any one of claims 18 to 30, wherein the TrkB agonist comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence of any one of SEQ ID NOs 12, 18, 24, 32, 40, 48, 64, 72, 84 and 94.

32. The method of claim 31, wherein the HCDR2 comprises an amino acid sequence of any one of SEQ ID NOs 13, 19, 25, 33, 41, 49, 65, 73, 85 and 95.

33. The method of any one of claims 31 to 32, wherein the HCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 14, 20, 26, 34, 42, 50, 66, 74, 86 and 96.

34. The method of any one of claims 31 to 33, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 28, 36, 44, 52, 60, 68, 76, 87 and 97.

35. The method of any one of claims 23 to 34, wherein the TrkB agonist antibody comprises a heavy chain constant region, and the heavy chain constant region is a human IgG constant region.

36. A method according to any one of claims 18 to 35, wherein the glucocorticoid (e.g. cortisol, corticosterone or CORT) inhibitor is capable of reducing the amount of the glucocorticoid.

37. The method of any one of claims 18 to 36, wherein the glucocorticoid inhibitor is capable of inhibiting the activity of the glucocorticoid.

38. The method of any one of claims 18 to 37, wherein the glucocorticoid inhibitor comprises mifepristone (RU-486) or a derivative thereof.

39. A method for determining whether a subject has or is at risk of developing schizophrenia, comprising determining the level and/or activity of BDNF (e.g., BDNF-e 6) expression in the subject, and determining the level and/or activity of a glucocorticoid (e.g., cortisol, corticosterone, or CORT) in the subject.

40. A method according to claim 39, wherein said expression level and/or activity of BDNF (e.g. BDNF-e 6) is determined from a blood sample of said subject.

41. The method of any one of claims 39-40, wherein the level and/or activity of the glucocorticoid is determined from a blood sample of the subject.

42. A method according to any one of claims 39 to 41, further comprising selecting a subject having a reduced expression level and/or activity of BDNF (e.g. BDNF-e 6) and an elevated level and/or activity of said glucocorticoid, the selected subject being determined to be predisposed to or at risk of developing schizophrenia.

43. The method of any one of claims 39-42, further comprising determining whether the subject has a defect in promoter VI of the Bdnf gene.

44. The method of any one of claims 39 to 43, further comprising determining whether the subject has experienced post-natal stress.

45. The method of claim 44, wherein the post-natal stress comprises post-natal hypoxia and/or social isolation.

46. A method according to any one of claims 39 to 45, comprising determining the expression level and/or activity of BDNF (e.g. BDNF-e 6) in the hippocampus, prefrontal cortex and/or hypothalamus of the subject.

47. A method according to any one of claims 42 to 46, further comprising administering to the selected subject a TrkB agonist and/or a glucocorticoid (e.g. cortisol, corticosterone or CORT) inhibitor.

48. A system for determining whether a subject has or is at risk of developing schizophrenia, the system comprising: a first module for determining whether the subject has a reduced level and/or activity of BDNF (e.g., BDNF-e 6) expression, and a second module for determining whether the subject has an elevated level and/or activity of a glucocorticoid (e.g., cortisol, corticosterone, or CORT).

49. A system according to claim 48, wherein said BDNF (e.g. BDNF-e 6) is from the blood of said subject.

50. A system according to any one of claims 48 to 49, wherein the glucocorticoid (e.g. cortisol, corticosterone or CORT) is from the blood of the subject.