Optimized sigma-1 agonist method of responder selection and treatment

Abstract

To provide predictive biomarkers and methods for developing personalized protocols for treating patients in need of Sigma-1 receptor therapy.SOLUTION: The present disclosure provides genetic polymorphisms associated with an altered response of a subject to Sigma-1 receptor therapy. Also described is use of the polymorphisms to personalize treatment for subjects in need of Sigma-1 receptor therapy such as treatment of neurodevelopmental and neurodegenerative diseases and conditions.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 673,369, filed May 18, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates generally to methods for determining whether a subject is responsive or non-responsive to Sigma-1 receptor therapy. [Background technology]

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and other cognitive functions, resulting in interference in daily living activities. Currently, there is a lack of pharmacological treatments that have been shown to alter disease progression. Sigma-1 receptor (SIGMAR1), a regulator of calcium homeostasis and mitochondrial function, is a novel target associated with conditions variously known as neurodegenerative and neurodevelopmental disorders or indications, including AD. SIGMAR1 activation has been shown to reduce key pathophysiological processes in AD, including tau hyperphosphorylation, neuroprotection, neurodegeneration, and oxidative stress. SIGMAR1 activation also results in increased autophagic flux in vivo in human cells. However, the genetic heterogeneity of the AD patient population and the lack of objective efficacy measures or predictive biomarkers of response to treatment contribute to significant limitations in treatment efficacy. This heterogeneity is background noise that can obscure treatment success. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a need for predictive biomarkers and methods for developing personalized protocols for treating patients in need of Sigma-1 receptor therapy. [Means for solving the problem]

[0005] One aspect of the present disclosure encompasses a method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy. The method includes obtaining or having obtained the results of testing a biological sample from the subject to determine the presence of at least one polymorphism selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_1543V, and combinations thereof in the subject. The method further includes excluding the subject as non-responsive to Sigma-1 receptor agonist therapy if the polymorphism is present in the subject.

[0006] Another aspect of the present disclosure encompasses a method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy. The method includes detecting or having detected at least one polymorphism present at a locus in a biological sample obtained from the subject by sequencing the locus containing the polymorphism. The method further includes excluding the subject as non-responsive to Sigma-1 receptor agonist therapy if the polymorphism is present in the subject. The at least one polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_l543V, and combinations thereof.

[0007] Yet another aspect of the present disclosure encompasses a method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy.The method includes obtaining or obtaining the results of testing a biological sample from a subject, determining the expression level of RNA encoded by at least one gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, DPYD, and combinations thereof, and comparing the level of RNA in the test sample with the level of RNA in the subject that is responsive to Sigma-1 receptor therapy.The level of RNA in the test sample that is substantially similar to the level of RNA in the subject that is responsive to Sigma-1 receptor therapy identifies the subject that is responsive to Sigma-1 receptor therapy.

[0008] Another aspect of the present disclosure encompasses a method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy.The method includes determining or has determined the expression level of the RNA encoded by at least one gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, DPYD, and combinations thereof.The method also includes excluding the subject as non-responsive to Sigma-1 receptor agonist therapy if the level of RNA in the test sample is substantially different from the level of RNA in the subject that is responsive to Sigma-1 receptor therapy.

[0009] One aspect of the present disclosure encompasses a method for treating a subject in need of Sigma-1 receptor agonist therapy. The method includes obtaining or having obtained a result of testing a biological sample from the subject to detect the presence of at least one polymorphism selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_1543V, and combinations thereof in the subject. The method also includes administering a therapeutically effective amount of a Sigma-1 receptor agonist to the subject if the polymorphism is not present in the subject.

[0010] Another aspect of the present disclosure encompasses a method for treating a subject in need of Sigma-1 receptor agonist therapy.The method includes obtaining or having obtained the results of testing a biological sample from a subject, determining the expression level of RNA encoded by at least one gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, DPYD, and combinations thereof.The method further includes comparing the level of RNA in the test sample with the level of RNA in a subject that is responsive to Sigma-1 receptor therapy, and administering a therapeutically effective amount of a Sigma-1 receptor agonist to the subject when the level of RNA in the test sample is substantially similar to the level of RNA in a subject that is responsive to Sigma-1 receptor therapy.

[0011] Yet another aspect of the present disclosure encompasses a method for detecting the presence of a polymorphism associated with response to Sigma-1 receptor therapy in a subject. The method includes obtaining or having obtained a biological sample from the subject, and detecting the polymorphism in the biological sample by evaluating the sequence of a locus that contains the polymorphism. The at least one polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_l543V, and combinations thereof in the subject.

[0012] Color Shape Reference The application file will contain at least one photograph executed in color. A copy of this patent application publication with a color photograph will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0013] [Figure 1A] An exemplary overview of the study design, data availability, and analysis methods is shown in Figure 1. (a) A sequential clinical trial with two parts: Part A lasting 5 weeks and Part B lasting 52 weeks (57-week study), as well as a 208-week extension study planned immediately after the initial study for a cumulative duration of 265 weeks. [Figure 1B] (b) A summary of the integrated data sources. [Figure 1C] (c) A description of the number of patient descriptors incorporated in the two analytical steps applied in the study to model the two groups of ANAVEX2-73 concentrations: 1) an undependent FCA rule-based analysis of response at week 57; 2) a longitudinal validation using mixed-effects modeling of response over 148 weeks incorporating markers found at week 57. [Figure 2A]Figure 2 is a schematic representation of the dosing and plasma concentrations of ANAVEX2-73 and ANAVEX19-144. (a) Dose-administration crossover design including eight possible dosing schemes, with a single dose regimen given at each period (intravenously (IV) or orally (orally)), starting with the two possible doses randomly assigned IV or oral dosing. [Figure 2B] (b) Pharmacokinetic parameter, area under the curve from 0 to infinity (AUC0-inf), of ANAVEX2-73 after the first dose (first 24 hours of the study) in Part A of the 57-week study at different randomized doses. [Figure 2C] (c) Box plot of mean plasma concentrations of ANAVEX2-73. [Diagram 3] 3D crystal structure of SIGMAR1 visualized using VistaPDB 1.0 software. [Figure 4] Plots showing MMRM-LME models of MMSE and ADCS-ADL change from baseline over 148 weeks. (a) MMRM-LME adjusted slopes for the high intensity (green) cohort vs. the low and mid intensity patient cohort (magenta) over time (weeks) for adjusted delta MMSE. The mean adjusted values ​​for population-level residuals are plotted at each time point (dotted line). (b) MMRM-LME adjusted slopes for the high intensity (green) cohort vs. the low and mid intensity patient cohort (magenta) over time (weeks) for adjusted delta MMSE. The mean adjusted values ​​for population-level residuals are plotted at each time point (dotted line). [Diagram 5] Plot of unadjusted values ​​of delta ADCS-ADL over 148 weeks. The plot presents the (unadjusted) mean trajectory of delta ADCS-ADL scores through the intermediate 148 weeks for patients in the 208-week study according to biomarker status (absent or present) and for the subgroup of patients given standard treatment. Subgroups of patients in the 208-week study are represented in the plot (blue, green, orange, pink, purple, and turquoise) according to identified biomarkers and baseline reference characteristics. [Figure 6A]1A-B are plots showing treatment response showing longitudinal delta MMSE and delta ADCS-ADL over 148 weeks of the combination study. Patients shown in (a) and (b) have SIGMAR1 wild type, high mean plasma concentration levels of ANAVEX2-73 in Part B, and baseline MMSE ≧20. [Figure 6B] 1A-B are plots showing treatment response showing longitudinal delta MMSE and delta ADCS-ADL over 148 weeks of the combination study. Patients shown in (a) and (b) have SIGMAR1 wild type, high mean plasma concentration levels of ANAVEX2-73 in Part B, and baseline MMSE ≧20. [Figure 7A] 1 is a plot showing that the presence of a SIGMAR1 Pro2 mutation is associated with poor response to sigma-1 agonists as measured by both MMSE (a) and ADCS-ADL delta (b). [Figure 7B] Plots showing that the presence of SIGMAR1 Pro2 mutations is associated with poor response to sigma-1 agonists as measured by both MMSE (a) and ADCS-ADL delta (b). [Figure 8] (a) A plot showing that frameshift COMT Leu146 mutations are strongly associated with higher ADCS-ADL delta from baseline (BL). (b) A plot showing that absence of KANSL1 Pro304 / 946 / 1010 mutations are strongly associated with higher ADCS-ADL delta from BL. (c) A plot showing that absence of SIGMAR1 Pro2 mutations are strongly associated with higher ADCS-ADL delta from BL. (d) A plot showing that absence of frameshift COMT Leu146 mutations are strongly associated with higher MMSE delta from BL. (e) A plot showing that absence of KANSL1 Pro304 / 946 / 1010 mutations are strongly associated with higher MMSE delta from BL. (f) A plot showing that absence of SIGMAR1 Pro2 mutations are strongly associated with higher MMSE delta from BL. [Figure 9]Plot showing that high SIGMAR1 RNA expression is strongly associated with high ADCS-ADL slope values ​​from BL (Improvement). Analysis includes data from 100% of all patients (N=21). TPM means transcripts per kilobase per million. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure is based in part on the discovery of a method for determining whether a subject is responsive or non-responsive to Sigma-1 receptor therapy. The method may include detecting the presence of a polymorphism associated with an altered response to Sigma-1 receptor therapy. The method may also include determining the level of expression of an RNA encoded by a gene associated with an altered response to Sigma-1 receptor therapy. The method may be used to optimize treatment for subjects in need of Sigma-1 receptor therapy. Based on the subject's predicted responsiveness to therapy, a personalized treatment may be provided to each subject. For example, subjects who are responsive may be selected for treatment using a Sigma-1 receptor agonist, while non-responsive subjects may be provided with an alternative, more appropriate treatment regimen early in the disease development process and / or even before treatment is initiated. This is in contrast to current methods in which subjects are determined to be responsive or non-responsive after an extended period of treatment. The polymorphisms, methods for identifying the polymorphisms, and methods for using the polymorphisms are described below.

[0015] I. Polymorphisms Associated with Subject Response to Sigma-1 Receptor Therapy One aspect of the present invention encompasses genetic polymorphisms that predict the reduced therapeutic response to treatment using Sigma-1 receptor therapy.Polymorphisms of interest can be at any locus in the genome of a subject, provided that the polymorphism is associated with the subject's positive response to Sigma-1 receptor therapy.For example, polymorphisms can be at coding or non-coding regions of the genome of a subject, including mitochondrial DNA.Polymorphisms can result from insertions, deletions, or single nucleotide polymorphisms (SNPs) in genes or loci.

[0016] In some embodiments, the polymorphism associated with response to Sigma-1 receptor therapy is in a gene selected from SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, GAMT, IL10, MTUS1, PPARG, and combinations thereof. In one embodiment, the polymorphism is in a gene selected from SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, and combinations thereof. In another embodiment, the polymorphism is in a gene selected from SIGMAR1, COMT, KANSL1, and combinations thereof.

[0017] In some embodiments, the polymorphism associated with response to Sigma-1 receptor therapy is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_1543V, or a combination thereof. Table A details these polymorphisms.

[0018] [Table 1]

[0019] In one embodiment, the polymorphism associated with response to Sigma-1 receptor therapy is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs (rs113895332 / rs61143203), KANSL1_P1010L / P304L / P946L, and combinations thereof.In another embodiment, the polymorphism associated with response to Sigma-1 receptor therapy is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs (rs113895332 or rs61143203), KANSL1_P1010L / P304L / P946L, and combinations thereof.In yet another embodiment, the polymorphism associated with response to Sigma-1 receptor therapy is SIGMAR1_Q2P. In one embodiment, the polymorphism associated with response to Sigma-1 receptor therapy is COMT_L146fs (rs113895332 or rs61143203). In another embodiment, the polymorphism associated with response to Sigma-1 receptor therapy is KANSL1_P1010L / P304L / P946L.

[0020] II. Methods of Determining a Subject's Responsiveness to Sigma-1 Receptor Therapy Another aspect of the present invention provides a method for determining whether a subject is responsive or non-responsive to Sigma-1 receptor therapy.The method can include detecting the presence of a polymorphism associated with changes in response to Sigma-1 receptor therapy.The method can also include determining the level of expression of RNA encoded by at least one gene associated with changes in response to Sigma-1 receptor therapy.

[0021] In one embodiment, the method comprises detecting the presence of at least one polymorphism associated with altered response to Sigma-1 receptor therapy.The presence of at least one polymorphism identifies subjects who are responsive to Sigma-1 receptor therapy.The polymorphism and the gene that comprises the polymorphism are as described in Section I.

[0022] The presence of one or more polymorphisms can be detected. For example, the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymorphisms can be detected. In some embodiments, the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 polymorphisms can be detected. In other embodiments, the presence of 1, 2, 3, 4 or 5 polymorphisms can be detected. In still other embodiments, the presence of 1, 2 or 3 polymorphisms can be detected.

[0023] The presence or absence of a polymorphism can be detected by evaluating a nucleic acid sequence in a subject. Alternatively, the presence or absence of a polymorphism can be detected by evaluating expression of a nucleic acid or protein from a gene or locus that contains the polymorphism.

[0024] In some embodiments, the presence or absence of polymorphism can be detected by evaluating nucleic acid sequence for the presence of polymorphism at the locus containing polymorphism.The nucleic acid sequence can be DNA or RNA sequence expressed from the locus containing polymorphism.Methods suitable for detecting polymorphism are well known in the art.For example, methods include shotgun sequencing, bridge PCR, high-throughput methods, allele-specific real-time PCR, 5'-nuclease assay, template-directed dye terminator incorporation, molecular beacon allele-specific oligonucleotide assay, assay using invasive cleavage with flap nuclease, allele-specific hybridization (ASH), array-based hybridization, allele-specific ligation, primer extension, single base extension (SBE) assay, sequencing, pyrophosphate sequencing, real-time pyrophosphate sequencing, sequence length polymorphism analysis, restriction fragment polymorphism (RFLP), RFLP-PCR, single-strand conformation polymorphism (SSCP), PCR-SSCP, fragment sizing capillary electrophoresis, heteroduplex analysis, and mass array system. Analysis of the amplified sequence can be performed using various techniques such as microchips, fluorescence polarization assays, and matrix-assisted laser desorption / ionization (MALDI) mass spectrometry.High-throughput sequencing methods include massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, tunneling current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidic Sanger sequencing, microscope-based techniques, RNAP sequencing, and in vitro virus high-throughput sequencing.

[0025] In another embodiment, the method comprises determining the expression level of the RNA encoded by at least one gene associated with change in response to Sigma-1 receptor therapy.According to the method, the level of RNA substantially similar to the level of RNA in the subject that is responsive to Sigma-1 receptor therapy identifies the subject that is responsive to Sigma-1 receptor therapy.The gene associated with change in response to Sigma-1 receptor therapy is selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, DPYD, and combinations thereof.

[0026] For example, the level of expression of RNA encoded by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes can be detected. In some embodiments, the level of expression of RNA encoded by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes can be detected. In other embodiments, the level of expression of RNA encoded by 1, 2, 3, 4, or 5 genes can be detected. In still other embodiments, the level of expression of RNA encoded by 1, 2, or 3 genes can be detected.

[0027] Measuring the level of RNA expression can be achieved by various methods, including Northern blot, quantitative real-time PCR (qRT-PCR), nucleic acid microarray, Luminex microsphere, and nuclease protection assay.The method of determining the substantially different level of RNA expression is known in the art, and includes distribution analysis.In some embodiments, the substantially different level of RNA expression is determined by normalizing the RNA expression value as transcripts per kilobase per million (TPM).

[0028] In other embodiments, determining whether a subject is responsive or non-responsive to Sigma-1 receptor therapy can be detected by measuring the level of protein expressed from the gene containing polymorphism.Methods for measuring protein expression include various methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, Western blot, and protein immunostaining.

[0029] The determination of whether a subject is responsive or non-responsive to Sigma-1 receptor therapy can be detected in vivo or in vitro in a subject. Typically, the responsiveness of a subject to Sigma-1 receptor therapy can be obtained by analyzing nucleic acid or protein in vitro in a biological sample obtained from a subject. Nucleic acid or protein can be isolated from the biological sample using methods generally known in the art. For details, see Ausubel et al., 2003, or Sambrook & Russell, 2001. To isolate nucleic acid from a biological sample, commercially available nucleic acid and protein extraction kits or commercially available extraction reagents can be used.

[0030] Non-limiting examples of suitable biological samples include fluid samples, biopsy samples, skin samples, and hair samples. Fluid samples can include blood, serum, saliva, tears, and lymphatic fluid. In some embodiments, the biological sample is blood. Methods for collecting biological samples from subjects are well known in the art.

[0031] In some embodiments, the method of determining whether a subject is responsive or non-responsive to Sigma-1 receptor therapy comprises obtaining the results of testing a biological sample from the subject, which determines the presence of at least one polymorphism. In other embodiments, the method of determining the responsiveness of a subject to Sigma-1 receptor therapy comprises detecting whether at least one polymorphism is present at a locus in a biological sample obtained from the subject by sequencing the locus containing the polymorphism. In yet other embodiments, the method of determining whether a subject is responsive or non-responsive to Sigma-1 receptor therapy comprises obtaining the results of testing a biological sample from the subject, which determines the level of at least one RNA encoded by a gene associated with an altered response to Sigma-1 receptor therapy. In other embodiments, the method of determining the responsiveness of a subject to Sigma-1 receptor therapy comprises detecting whether at least one polymorphism is present at a locus in a biological sample obtained from the subject by determining the level of expression of at least one RNA encoded by a gene associated with an altered response to Sigma-1 receptor therapy.

[0032] III.How to use The method can be used to optimize treatment for subjects who need Sigma-1 receptor agonist therapy.For example, the method can be used to select subjects who are responsive to Sigma-1 receptor therapy before starting the treatment protocol of the subject.Conversely, the method can be used to exclude subjects before treatment if they are found to be unresponsive to Sigma-1 receptor therapy.The polymorphism can be used to determine whether a subject develops toxicity before treatment when treated with Sigma-1 receptor agonist.In addition, the method can be used to determine whether or when treatment with Sigma-1 receptor agonist should be discontinued in a subject.In addition, the method can be used in evaluating the response of a subject to substance withdrawal treatment.

[0033] The method includes detecting the presence of a polymorphism in a subject and determining whether the subject is a candidate for Sigma-1 receptor therapy. The method may also include determining a level of expression of RNA from one or more genes associated with an altered response to Sigma-1 receptor therapy. The polymorphism, the genes associated with an altered response to Sigma-1 receptor therapy, the method of detecting the polymorphism, and the method of determining the level of RNA expression may be as described in Section I, and the method of detecting the polymorphism may be as described in Section II.

[0034] As used herein, the term "responsiveness" can be used to describe any subject that experiences a positive outcome when treated with a Sigma-1 receptor agonist. The method of determining the responsiveness of a subject to Sigma-1 receptor therapy can vary and can be determined experimentally depending on the condition or disease, subject, treatment protocol, among other variables. Responsiveness can be determined at any time that is deemed appropriate by those skilled in the art for the disease or condition. For example, treatment outcome can be determined at about 57 weeks, about 148 weeks, or about 208 weeks after the start of treatment.

[0035] When the treatment is used for Alzheimer's disease (AD), a positive outcome may be as measured by delta or slope MMSE (Mini-Mental State Examination) or ADCS-ADL (Alzheimer's Disease Cooperative Study-Activities of Daily Living). Responsive subjects are characterized by having a positive or absent MMSE and / or ADCS-ASL slope, while non-responsive subjects are characterized by having a negative MMSE and / or ADCS-ASL slope. Responsive subjects are also characterized by having a high MMSE and / or ADCS-ASL delta, while non-responsive subjects are characterized by having a low MMSE and / or ADCS-ASL delta. A low MMSE delta may range from about -9 to about -6, about -6 to about -2, or about -9 to about -2, while a high MMSE delta may range from about -6 to -2, or about -2 to about 6. Methods for determining delta or slope MMSE or ADCS-ADL may be as described in the examples herein.

[0036] In some embodiments, the method is used to select subjects who are responsive to Sigma-1 receptor agonist therapy. The method includes obtaining the results of testing a biological sample from a subject to determine the presence of at least one polymorphism, and if the polymorphism is present in the subject, excluding the subject as non-responsive to Sigma-1 receptor agonist therapy. Alternatively, if the polymorphism is not present in the subject, the subject can be included as responsive to therapy. In one embodiment, the polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_l543V, and combinations thereof. In another embodiment, the polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, COMT_L146fs, KANSL1_P1010L / P304L / P946L, and combinations thereof. The presence of the polymorphism can be obtained by evaluating the nucleic acid sequence.

[0037] In another embodiment, the method is used to select subjects who are responsive to Sigma-1 receptor agonist therapy. The method includes detecting whether at least one polymorphism is present in a locus in a biological sample obtained from a subject by sequencing the locus containing the polymorphism, and excluding the subject as non-responsive to Sigma-1 receptor agonist therapy if the polymorphism is present in the subject. Alternatively, the subject can be included as responsive to therapy if the polymorphism is not present in the subject. In one embodiment, the polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_l543V, and combinations thereof. In another embodiment, the polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, and combinations thereof.The presence of the polymorphism can be detected by evaluating the nucleic acid sequence.

[0038] In another embodiment, the method is used to select subjects that are responsive to Sigma-1 receptor agonist therapy.The method includes obtaining the results of testing a biological sample from a subject, determining the expression level of the RNA encoded by at least one gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, and combinations thereof, and excluding the subject as non-responsive to Sigma-1 receptor agonist therapy if the level of RNA is substantially different from the level of RNA in subjects that are responsive to Sigma-1 receptor therapy.Alternatively, if the level of RNA is substantially similar to the level of RNA in subjects that are responsive to Sigma-1 receptor therapy, the subject can be included as responsive to therapy.In one embodiment, the expression level of the RNA encoded by the gene selected from the group consisting of SIGMAR1, COMT, KANSL1, and combinations thereof is determined.

[0039] In some embodiments, the method is used to select subjects that are responsive to Sigma-1 receptor agonist therapy. The method comprises detecting RNA expression in a biological sample test from a subject, determining the level of expression of RNA encoded by at least one gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, and combinations thereof. The method comprises excluding the subject as non-responsive to Sigma-1 receptor agonist therapy if the level of RNA is substantially different from the level of RNA in subjects that are responsive to Sigma-1 receptor therapy. Alternatively, the method comprises identifying the subject as responsive to Sigma-1 receptor agonist therapy if the level of RNA is substantially similar to the level of RNA in subjects that are responsive to Sigma-1 receptor therapy. In one embodiment, the level of expression of RNA encoded by a gene selected from the group consisting of SIGMAR1, COMT, KANSL1, and combinations thereof is determined.

[0040] In some embodiments, the method is used to treat a subject in need of Sigma-1 receptor agonist therapy. The method includes obtaining the results of a test of a biological sample from the subject, which determines the presence of at least one polymorphism. If the polymorphism is not present in the subject, a therapeutically effective amount of a Sigma-1 receptor agonist is administered to the subject. Alternatively, if the polymorphism is present in the subject, a therapeutically effective amount of a substitute for the Sigma-1 receptor agonist is administered to the subject. In one embodiment, the polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_l543V, and combinations thereof. In another embodiment, the polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, COMT_L146fs, KANSL1_P1010L / P304L / P946L, and combinations thereof. The presence of the polymorphism can be obtained by evaluating the nucleic acid sequence.

[0041] In another embodiment, the method is used to treat a subject who needs Sigma-1 receptor agonist therapy. The method includes obtaining the results of a test of a biological sample from a subject, determining the level of expression of RNA encoded by a gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, and combinations thereof. If the level of RNA in the test sample is substantially similar to the level of RNA obtained from a subject who is responsive to Sigma-1 receptor therapy, a therapeutically effective amount of a Sigma-1 receptor agonist is administered to the subject. Alternatively, if the level of RNA in the test sample is substantially different from the level of RNA obtained from a subject who is responsive to Sigma-1 receptor therapy, a therapeutically effective amount of a substitute of a Sigma-1 receptor agonist is administered to the subject. In another embodiment, the level of expression of RNA encoded by a gene selected from the group consisting of SIGMAR1, COMT, KANSL1, and combinations thereof is determined.

[0042] It will be appreciated that a subject may have more than one polymorphism.For example, a subject may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymorphisms.In some embodiments, a subject has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 polymorphisms.In other embodiments, a subject has 1, 2, 3, 4 or 5 polymorphisms.In still other embodiments, a subject has 1, 2 or 3 polymorphisms.

[0043] IV. TREATMENT OF A SUBJECT IN NEED OF A SIGMA-1 RECEPTOR AGONIST Sig-1R expression or activity is associated with neurodegeneration, and activation of Sig-1R is associated with neuroprotection in human subjects and different in vitro and in vivo models.Therefore, one aspect of the present disclosure includes the treatment of any Sigma-1 receptor-related disease or condition that primarily affects the neuronal system in subjects.Such conditions include those variously known as neurodevelopmental and neurodegenerative diseases and conditions, and can be used for neuroprotection. Non-limiting examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, prion diseases, Huntington's disease, motor neuron diseases (MND) such as amyotrophic lateral sclerosis, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), dementia, autism spectrum disorder, cerebral palsy, Rett syndrome, Angelman syndrome, Williams syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), multiple sclerosis, childhood disintegrative disorder, fragile X, infantile spasms and Smith-Magenis syndrome, schizophrenia, post-traumatic stress disorder (PTSD), and any neuronal injury such as injury resulting from stroke, traumatic brain injury, and spinal cord injury.

[0044] SIGMAR1 is also useful as a target for cancer therapy. As such, an embodiment of the present disclosure encompasses the treatment of any Sigma-1 receptor-associated cancer or tumor. As will be recognized by those skilled in the art, cancer as used throughout the present disclosure can be one or more tumors or cancers. A tumor can be malignant or benign, a cancer can be primary or metastatic, and a tumor or cancer can be early or late stage. Non-limiting examples of tumors or cancers that can be treated include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendicitis cancer, astrocytoma (pediatric cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, epidermoidoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway, and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoma, baculoblastoma ... Kitt's lymphoma, Carcinoid tumor (childhood, gastrointestinal cancer), Cancer of unknown primary, Central nervous system lymphoma (primary), Cerebellar astrocytoma, Cerebral astrocytoma / malignant glioma, Cervical cancer, Childhood cancer, Chronic lymphocytic leukemia, Chronic myeloid leukemia, Chronic myeloproliferative disorder, Colon cancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Renal pyeloma, Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, Extracranial germ cell tumor (childhood), Extragonadal germ cell tumor, Extrahepatic bile duct Cancer, Eye Cancer (Intraocular Melanoma, Retinoblastoma), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumors, Germ Cell Tumors (Pediatric Extracranial, Extragonadal, Ovarian), Gestational Trophoblastic Tumors, Gliomas (Adult, Pediatric Brainstem, Pediatric Cerebral Astrocytoma, Pediatric Visual Pathway and Hypothalamus), Gastric Carcinoid, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin's Lymphoma, Hypopharyngeal Cancer, Hypothalamic and Visual Pathway Gliomas (Pediatric), Intraocular Melanoma, Islet Cell Carcinoma, Kaposi's Sarcoma, Kidney Cancer (Renal Cell Carcinoma) alveolar carcinoma), laryngeal cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell), lip and oral cavity cancer, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma (AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia (Waldenstrom), malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma (adult malignant, childhood),Occult primary metastatic squamous cell carcinoma of the neck, oral cavity cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myeloid leukemia (chronic), myeloid leukemia (acute adult, acute childhood), multiple myeloma, myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal carcinoma, osteosarcoma / Malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumor (childhood), pituitary adenoma, plasma cell tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer, sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sezary syndrome, skin cancer (non-melanoma, melanoma), skin cancer (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, occult primary (metastatic) squamous cell carcinoma of the cervix, gastric cancer, supratentorial primitive neuroectodermal tumor (childhood), T-cell lymphoma (skin), testicular Cancer, pharyngeal cancer, goiter (childhood), goiter and thyroid cancer, thyroid cancer, thyroid cancer (childhood), transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor (gestational), unknown primary site (adult, child), transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine cancer (endometrium), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (childhood), vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor (childhood).

[0045] The method includes administering a therapeutically effective amount of a substitute Sigma-1 receptor agonist when the subject in need of Sigma-1 receptor agonist therapy is determined to be non-responsive to therapy.Non-limiting examples of substitute Sigma-1 receptor agonists include galantamine, memantine, and rivastigmine.

[0046] The method includes administering a therapeutically effective amount of a Sigma-1 receptor agonist if the subject in need of Sigma-1 receptor agonist therapy is determined to be responsive to the therapy. The Sigma-1 receptor agonist can be any therapeutic or active agent capable of treating the subject in need of Sigma-1 receptor agonist therapy. For example, the receptor agonist can be a biological compound such as an antibody or a peptide, or a small molecule active agent. Non-limiting examples of Sigma-1 receptor agonists include entacapone, nebicapone, nitecapone, opicapone, tolcapone, tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73, AV2-73, or A2-73), 1-(2,2-diphenyltetrahydrofuran-3-yl)-N-methylmethanamine hydrochloride (ANAVEX19-144, or A19-144), aminotetrahydrofuran derivative tetrahydro- Examples include N,N-dimethyl-5,5-diphenyl-3-furanmethanamine hydrochloride (ANAVEX 1-41), 1-(3-4(((1R,3S,5S)-adamantan-1-yl)(phenyl)methyl)propyl)-4-methylpiperazine (ANAVEX™ 1066 or "AV1066"), ANAVEX 3-71 (also known as AF710B), PRE-084, donepezil, fluvoxamine, amitriptyline, L-687,384, and combinations thereof. The Sigma-1 receptor agonist can also be an antibody.

[0047] In one embodiment, the Sigma-1 receptor agonist is A2-73. When the agonist is A2-73, the therapeutically effective amount of A2-73 can be in the range of about 0.5 mg to about 20 mg, about 1 mg to about 60 mg, about 30 mg to about 50 mg, or about 3 mg to about 5 mg. The therapeutically effective amount of A2-73 can be in the range of about 0.5 mg / day to about 100 mg / day, about 1 to about 60 mg / day, about 20 to about 50 mg / day, about 20 to about 30 mg / day, or about 15 to about 25 mg / day. Administering an anti-neurodegenerative effective amount of A2-73 can provide a blood level of A2-73 of about 10 ng / ml, about 12 ng / ml.

[0048] A2-73 may be administered to a subject once a day, or more than once a day. Additionally, A2-73 may be administered every 2, 3, 4, 5, 6, 7, 14, or 30 days. A2-73 may be administered for a period ranging from about 1 day to about 1 year, about 1 day to about 1 week, about 3 days to about 1 month, about 2 weeks to about 6 months, or about 2 months to about 4 months. A2-73 may also be administered for a period of about 1 day, about 7 days, about 30 days, about 60 days, about 120 days, or about 180 days or more. In some embodiments, A2-73 is administered for about 57 weeks, about 148 weeks, about 208 weeks, indefinitely, or until resolution of the condition being treated.

[0049] Other methods of administering A2-73 can be found, for example, in U.S. Patent No. 9,750,746, U.S. Patent Publication No. 2017 / 0360798, U.S. Patent Publication No. 2019 / 0022052, U.S. Patent Publication No. 2018 / 0360796, U.S. Patent Publication No. 2018 / 0169059, U.S. Patent Publication No. 2018 / 0177756, U.S. Patent Publication No. 2018 / 0169060, and U.S. Patent Publication No. 2019 / 0117615, the disclosures of which are incorporated herein in their entireties.

[0050] V. Pharmaceutical Formulations One aspect of the present disclosure includes a pharmaceutical formulation for the diagnosis and delivery of a Sigma-1 receptor agonist. The pharmaceutical formulation comprises a therapeutically effective amount of the agonist and any pharma- ceutical acceptable salts thereof.

[0051] Pharmaceutically acceptable salts of the compounds comprising Formula (II) or (IIa) include, without limitation, acetate, aspartate, benzoate, bitartrate, citrate, formate, gluconate, glucuronate, glutamate, fumarate, hydrochloride, hydrobromide, hydroiodide, hypophosphite, isobutyrate, isocitrate, lactate, malate, maleate, meconate, methyl bromide, methanesulfonate, monohydrate, mucate, nitrate, oxalate, phenylpropionate, phosphate, phthalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tannate, tartrate, terephthalate, valerate, and the like.

[0052] When the Sigma-1 receptor agonist is A2-73, the formulation may contain about 1 mg to about 50 g, about 0.1 to about 5 g, about 0.5 g to about 3 g, about 1 mg to about 55 mg, about 40 mg to about 60 mg, about 80 mg to about 120 mg, about 180 mg to about 220 mg, about 0.1 g to about 5 g, or about 0.5 g to about 3 g of A2-73. Formulations including A2-73 can be found, for example, in U.S. Patent No. 9,750,746, U.S. Patent Publication No. 2017 / 0360798, U.S. Patent Publication No. 2019 / 0022052, U.S. Patent Publication No. 2018 / 0360796, U.S. Patent Publication No. 2018 / 0169059, U.S. Patent Publication No. 2018 / 0177756, U.S. Patent Publication No. 2018 / 0169060, and U.S. Patent Publication No. 2019 / 0117615, the disclosures of which are incorporated herein in their entireties.

[0053] Sigma-1 receptor agonists can be formulated and administered to subjects by several different means.For example, compositions can generally be administered parenterally, intraperitoneally, intravascularly, transdermally, subcutaneously, or intrapulmonary in dosage unit formulations that contain conventional non-toxic pharmacologic adjuvants, carriers, excipients, and vehicles as required.The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrathecal, or intrasternal injection or infusion techniques.The formulation of pharmaceutical compositions is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY (1980).

[0054] Pharmaceutical preparations include one or more pharma- ceutically acceptable excipients.Non-limiting examples of excipients include chemical enhancers, wetting agents, pressure-sensitive adhesives, antioxidants, solubilizers, thickeners, plasticizers, adjuvants, carriers, excipients, vehicles, coatings, and any combination thereof.One or more excipients can be selected for oral, transdermal, parenteral, intraperitoneal, intravascular, subcutaneous, by inhalation spray, rectal, or pulmonary administration.

[0055] Sigma-1 receptor agonists can generally be formulated to improve patient compliance and prevent subjects from removing drug delivery devices. For example, Sigma-1 receptor agonists can be formulated to improve patient compliance and prevent removal of drug delivery devices by providing a formulation for extended delivery. The extended delivery can be for a period ranging from more than one day to several months. This can be particularly relevant for patients with impaired cognitive and / or motor control abilities. The extended delivery for a period can range from about 1 day to about 1 year, about 1 day to about 1 week, about 3 days to about 1 month, about 2 weeks to about 6 months, or about 2 months to about 4 months.

[0056] Extended release formulations can be used for substantially continuous delivery of the drug at a preselected rate. For example, for crystalline A2-73, the drug can be delivered at a rate of about 1 mg to about 100 mg / day, about 40 to about 60 gm / day, or about 10 to about 30 gm / day. The appropriate amount of crystalline A2-73 can be readily determined by one of skill in the art based, for example, on the intended duration of the drug by the extended release formulation, the delivery mechanism, the particular formulation, and the relative potency of the drug, among other factors.

[0057] i. Binder Non-limiting examples of binders suitable for the formulation of various embodiments include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 -C 18 These include fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, polypeptides, oligopeptides, and combinations thereof. A polypeptide can be any sequence of amino acids ranging from about 100 to about 300,000 daltons.

[0058] The binder may be introduced into the mixture to be granulated in a solid form, including but not limited to, crystals, particles, powder, or any other finely divided solid form known in the art. Alternatively, the binder may be dissolved or suspended in a solvent and sprayed onto the mixture in the granulation device as a binder fluid during granulation.

[0059] ii. Diluent Non-limiting examples of diluents (also referred to as "fillers" or "thinners") include carbohydrates, inorganic compounds, and biocompatible polymers such as polyvinylpyrrolidone (PVP). Other non-limiting examples of diluents include dibasic calcium sulfate, tribasic calcium sulfate, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, sugars such as sucrose, dextrose, lactose, microcrystalline cellulose, fructose, xylitol, and sorbitol, polyhydric alcohols, starches, pre-fabricated direct compression diluents, and mixtures of any of the foregoing.

[0060] iii. Disintegrants Disintegrants can be effervescent or non-effervescent.Non-limiting examples of non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, carob, karaya, pecithin and tragacanth.Suitable effervescent disintegrants include, but are not limited to, sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0061] iv. Preservatives Non-limiting examples of preservatives include, but are not limited to, ascorbic acid and its salts, ascorbyl palmitate, ascorbyl stearate, anoxomer, N-acetylcysteine, benzyl isothiocyanate, m-aminobenzoic acid, o-aminobenzoic acid, p-aminobenzoic acid (PABA), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid, canthaxanthin, alpha-carotene, beta-carotene, beta-carotene, beta-apocarotenic acid, carnosol, Carvacrol, catechin, cetyl gallate, chlorogenic acid, citric acid and its salts, clove extract, coffee bean extract, p-coumaric acid, 3,4-dihydroxybenzoic acid, N,N'-diphenyl-p-phenylenediamine (DPPD), dilauryl thiodipropionate, distearyl thiodipropionate, 2,6-di-tert-butylphenol, dodecyl gallate, edetic acid, ellagic acid, erythorbic acid, sodium erythorbate, esculetin, esculin, 6-ethoxy-1,2-dihydro-2,2,4-Trimethylquinoline, ethyl gallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA), eucalyptus extract, eugenol, ferulic acid, flavonoids (e.g., catechin, epicatechin, epicatechin gallate, epigallocatechin (EGC), epigallocatechin gallate (EGCG), polyphenol epigallocatechin-3-gallate), flavones (e.g., apigenin, chrysin, luteolin), flavonols (e.g., dathisitin, myricetin, denferro), flavanones, fraxetin, fumaric acid, gallic acid, gentian extract, gluconic acid, glycine, gumyu solanacearum (gum guaiacum), hesperetin, alpha-hydroxybenzylphosphinic acid, hydroxycinnamic acid, hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid, hydroxytrilosol, hydroxyurea, rice bran extract, lactic acid and its salts, lecithin, lecithin citrate, R-alpha-lipoic acid, lutein, lycopene, malic acid, maltol, 5-methoxytryptamine, methyl gallate, monoglyceride citrate, monoisopropyl citrate, morin, beta-naphthoflavone, nordihydroguaretic acid (NDGA), octyl gallate, oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine, phosphoric acid, phosphates, phytic acid, phytyl bichromel, pimento extract, gallic acid Propyl, polyphosphate, quercetin, trans-resveratrol, rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin, sinapic acid, succinic acid, stearyl citrate, syringic acid, tartaric acid, thymol, tocopherols (i.e., alpha-, beta-, gamma-, and delta-tocopherol), tocotrienols (i.e., alpha-, beta-, gamma-, and delta-tocotrienol), tyrosol, vanillic acid, 2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., Ionox 100), 2,4-(tris-3',5'-bi-tert-butyl-4'-hydroxybenzyl)-mesitylene (i.e., Ionox 330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxybutyrophenone, tryptamine, tyramine, uric acid, vitamin K and derivatives, vitamin Q10, wheat germ oil, zeaxanthin, or combinations thereof.

[0062] v. Flavor modifiers Suitable flavor modifiers include flavors, flavorings, sweeteners, etc. Flavors include, but are not limited to, synthetic flavor oils, flavor aromatics, and / or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.Other non-limiting examples of flavors include cinnamon oil, wintergreen oil, peppermint oil, clover oil, hay oil, anise oil, eucalyptus, vanilla, lemon oil, orange oil, citrus oils such as grape and grapefruit oil, fruit essential oils including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.

[0063] Flavoring agents include, but are not limited to, cellulose hydroxypropyl ethers (HPC), low substituted hydroxypropyl ethers (L-HPC), such as Klucel®, Nisswo HPC, and PrimaFlo HP22; cellulose hydroxypropyl methyl ethers (HPMC), such as Seppifilm-LC, Pharmacoat®, Metolose SR, Opadry YS, PrimaFlo, MP3295A, Benecel MP824, and Benecel MP843; methylcellulose polymers, such as Methocel® and Metolose®; ethylcellulose (EC), such as E461, Ethocel®, Aqualon®-EC, Surelease, and mixtures thereof; polyvinyl alcohols (PVA), such as Opadry AMB; hydroxyethylcelluloses, such as Natrosol®; carboxymethylcelluloses and salts of carboxymethylcellulose (CMC), such as Aualon®-CMC; Polyvinyl alcohol and polyethyl glycol copolymers such as IR®, monoglycerides (Myverol), triglycerides (KLX), polyethylene glycol, modified food starches, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit® EPO, Eudragit® RD100, and Eudragit® E100, Sepifilm such as mixtures of cellulose acetate phthalate, HPMC and stearic acid, cyclodextrins, and mixtures of these materials. In other embodiments, additional flavoring agents contemplated are those described in U.S. Patent Nos. 4,851,226, 5,075,114, and 5,876,759, each of which is incorporated herein by reference in its entirety.

[0064] Non-limiting examples of sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier), saccharin and its various salts such as sodium ene, dipeptide sweeteners such as aspartame, dihydrochalcone compounds, glycyrrhizin, Stevia rebaudiana (stevioside), chloro derivatives of sucrose such as sucralose, sugar alcohols such as sorbitol, mannitol, sciritol, hydrogenated starch hydrolysates, and the synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly the potassium salt (acesulfame-K), as well as the sodium and calcium salts thereof.

[0065] vi. Lubricants and Glidants Lubricant compositions may be utilized to lubricate the components forming the pharmaceutical composition. As glidants, lubricants facilitate removal of solid dosage forms during the manufacturing process. Non-limiting examples of lubricants and glidants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. Pharmaceutical compositions will generally contain about 0.01% to about 10% by weight of lubricant. In some embodiments, pharmaceutical compositions will contain about 0.1% to about 5% by weight of lubricant. In further embodiments, pharmaceutical compositions will contain about 0.5% to about 2% by weight of lubricant.

[0066] vii. Dispersants Dispersing agents may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isomorphous silicates, and microcrystalline cellulose as high hydrophilic lipophilic balance (HLB) emulsifier surfactants.

[0067] viii. Coloring agent Depending on the embodiment of the present disclosure, it may be desirable to include a coloring agent. Suitable color additives include, but are not limited to, Food, Drug, and Cosmetic Colors (FD&C), Drug and Cosmetic Colors (D&C), or External Drug and Cosmetic Colors (Ext. D&C). These colors or dyes, along with their corresponding lakes, and certain natural and derived colorants, may be suitable for use in various embodiments of the present disclosure.

[0068] ix. pH modifiers Non-limiting examples of pH modifiers include citric acid, acetic acid, tartaric acid, malic acid, fumaric acid, lactic acid, phosphoric acid, sorbic acid, benzoic acid, sodium carbonate, and sodium bicarbonate.

[0069] x. Chelating agents Chelators may be included as excipients to immobilize oxidative groups, including but not limited to metal ions, to inhibit oxidative degradation of the morphinan by these oxidative groups. Non-limiting examples of chelators include lysine, methionine, glycine, gluconate, polysaccharides, glutamate, aspartate, and disodium ethylenediaminetetraacetate (Na2EDTA).

[0070] xi. Antibacterial agents Antimicrobial agents may be included as excipients to minimize degradation of the compounds according to the present disclosure by microbial agents, including but not limited to bacteria and fungi. Non-limiting examples of antimicrobial agents include parabens, chlorobutanol, phenol, calcium propionate, sodium nitrate, sodium nitrite, Na2EDTA, and sulfites, including but not limited to sulfur dioxide, sodium bisulfite, and potassium bisulfite.

[0071] xii. Release control polymers The release-controlling polymer may be included in various embodiments of the solid dosage pharmaceutical composition incorporating the compound according to the present disclosure.In one embodiment, the release-controlling polymer may be used as tablet coating.In other embodiments, including but not limited to bilayer tablets, the release-controlling polymer may be mixed with granules and other excipients before forming tablets by known processes, including but not limited to compression in tablet molds.Suitable release-controlling polymers include but are not limited to hydrophilic polymers and hydrophobic polymers.

[0072] Suitable hydrophilic release controlling polymers include, but are not limited to, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose ethers, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, nitrocellulose, crosslinked starch, agar, casein, chitin, collagen, gelatin, maltose, mannitol, maltodextrin, pectin, pullulan, sorbitol, xylitol, polysaccharides, ammonia alginate, sodium alginate, calcium alginate, potassium alginate, propylene glycol alginate, sodium carmellose alginate, calcium carmellose, carrageenan, fucoidan, furcellaran, gum arabic, carrageen gum, gum ghatti, guar gum, karaya gum, locust bean gum, oleaginous ... Curragum, tragacanth gum, scleroglucan gum, xanthan gum, hypnea, laminaran, acrylic polymers, acrylate polymers, carboxyvinyl polymers, copolymers of maleic anhydride and styrene, copolymers of maleic anhydride and ethylene, copolymers of propylene maleic anhydride or copolymers of isobutylene maleic anhydride), crosslinked polyvinyl alcohol and poly N-vinyl-2-pyrrolidone, diesters of polyglucan, polyacrylamides, polyacrylic acids, polyamides, polyethylene glycols, polyethylene oxides, poly(hydroxyalkyl methacrylates), polyvinyl acetates, polyvinyl alcohols, polyvinyl chloride, polystyrene, polyvinylpyrrolidone, anionic and cationic hydrogels, and combinations thereof.

[0073] xiii. Coating A solid dosage containing a compound according to the present disclosure may include a coating, such a coating may control the release of the compound, function as a moisture barrier or buffer, or modify the pH. As used herein, a "control releasing coat" or "controlled release coat" is defined to mean a functional coating that may include, for example, at least one pH-independent polymer, pH-dependent polymer (e.g., enteric or reverse enteric type polymer), soluble polymer, insoluble polymer, lipid, lipid material, or a combination thereof. A coating may modify the release rate of a compound according to the present disclosure when applied onto a dosage form, slower (e.g., when applied to a normal release matrix dosage form), slower (e.g., when applied to a controlled release matrix dosage form), or when applied to an uncoated dosage form. For example, a controlled release coating may be designed such that when the controlled release coating is applied to a dosage form, the dosage form in combination with the controlled release coating may exhibit a release of a compound according to the present disclosure, such as "modified release", "controlled release", "sustained release", "extended release", "delayed release", "extended release", or a combination thereof. A "controlled release coating" may optionally include additional materials that may alter the function of the controlled release coating.

[0074] As used herein, the term "moisture barrier" refers to one that inhibits or retards the absorption of moisture. Compounds according to the present disclosure may be hygroscopic and therefore may be susceptible to degradation over time under high humidity conditions. The proportion of components of the moisture barrier and the amount of moisture barrier that is optionally applied on the controlled release coating or on the core are typically such that the moisture barrier does not fall within the USP definition and requirements of enteric coating. Preferably, the moisture barrier may comprise an enteric and / or acrylic polymer, preferably an acrylic polymer, optionally a plasticizer, and a permeation enhancer. The permeation enhancer is a hydrophilic material that allows water to enter without physically destroying the coating. The moisture barrier may additionally further comprise other conventional inert excipients, which may improve the processing of the extended release formulation.

[0075] Coating and matrix materials which may be used in accordance with the present invention are those known in the art for use in controlled release formulations, such as synthetic polymers of the polyvinyl type, e.g., polyvinyl chloride, polyvinyl acetate and copolymers thereof, polyvinyl alcohol, and polyvinylpyrrolidone; synthetic polymers of the polyethylene type, e.g., polyethylene and polystyrene; acrylic acid polymers; biopolymers or modified biopolymers, e.g., cellulose polymers, shellac, and gelatin; fats, oils, higher fatty acids, and higher alcohols (i.e., acids and alcohols containing alkyl chains of at least 10 carbon atoms), e.g., aluminum monostearate, cetyl alcohol, hydrogenated tallow, hydrogenated castor oil, 12-hydroxystearyl alcohol, glyceryl mono- or dipalmitate; glyceryl mono-, di-, or tristearate; myristyl alcohol, stearic acid, stearyl alcohol, and polyethylene glycol; waxes; sugars and sugar alcohols.

[0076] The pH buffering properties of the coating are determined by the groups of compounds commonly used in antacid preparations, such as magnesium oxide, hydroxide, or carbonate; aluminum or calcium hydroxide, carbonate, or silicate; complex aluminum / magnesium compounds, such as Al2O3·6MgO·CO2·12H2O, (Mg6Al2(OH) 16 CO3·4H2O), MgO·Al2O3·2SiO2.nH2O, aluminum bicarbonate coprecipitate, or similar compounds; or other pharma- ceutically acceptable pH buffering compounds such as sodium, potassium, calcium, magnesium, and aluminum salts of phosphate, carbonate, citric acid, or other suitable weak, inorganic, or organic acids; or suitable organic bases, including basic amino acids; and salts or combinations thereof.

[0077] The pH-dependent coating serves to release the drug in the desired region of the gastrointestinal (GI) tract, for example, the stomach or small intestine. If a pH-independent coating is desired, the coating is designed to achieve optimal release regardless of the pH changes in the environmental fluid, for example, the GI tract. When the coating is formulated to release the compound according to the present disclosure in the intestine (particularly the upper small intestine), the coating is often called an "enteric coating". The pH dependent coating may include, but is not limited to, acrylic acid polymers and copolymers, for example, polymers formed from acrylic acid, methacrylic acid, methyl acrylate, methyl acrylate, ammoniomethyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate (e.g., Eudragit™); cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate (CAP), cellulose acetate trimellitate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose succinate, and hydroxypropyl methyl cellulose succinate and sodium carboxymethyl cellulose; vinyl polymers and copolymers such as shellac (purified lac), polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate (PVAP), vinyl acetate chlorine acid copolymer, and ethylene vinyl acetate copolymer; zein, and salts and combinations thereof.

[0078] VI. Dosage form One embodiment of the present disclosure includes a dosage form of a Sigma-1 receptor agonist. When the agonist is A2-73, the dosage form may contain about 1 mg to about 50 g, about 1 mg to about 500 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 50 to about 400 mg, about 75 to about 150 mg, about 150 to about 200 mg, about 40 mg to about 60 mg, about 80 mg to about 120 mg, or about 180 mg to about 220 mg of A2-73. For example, the dosage form may contain 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 mg or more of A2-73. In some embodiments, the dosage form may contain from about 1 mg to about 500 mg, or from about 1 mg to about 100 mg of A2-73.

[0079] Dosage forms include those formulated for extended or sustained release and those formulated for immediate release. For example, immediate release dosage forms may contain crystalline forms of A2-73 as free base or A2-73 salt as disclosed herein. For example, fast disintegrating oral dosage forms may contain A2-73 salt, such as HCl salt. Alternatively, dosage forms may contain crystalline forms of A2-73 as free base or A2-73 salt formulated for inhalation drug delivery, either as dry powder or aerosol spray.

[0080] The dosage form also includes those formulated for topical administration. For example, the dosage form can be formulated as one or more of gel, ointment, emulsion, microemulsion, solution, suspension, paste, gel, foam, spray, lotion, or cream. In one embodiment, the topical administration dosage form is a transdermal patch. When the dosage form is formulated as a transdermal patch, the transdermal patch can contain about 40 mg to about 60 mg, about 80 mg to about 120 mg, or about 180 mg to about 220 mg of A2-73 free base in crystalline form.

[0081] Alternatively, dosage form can be formulated for oral administration.The dosage form formulated for oral administration can be the tablet, capsule and chewable capsule, powder, granule, tea, drops, or liquid medicine or syrup that can be swallowed, chewed, or dissolved in water or under the tongue.In some embodiments, dosage form is an enteric coated oral formulation.

[0082] When the dosage form is an enteric coated oral formulation, the formulation may contain from about 0.1 mg to about 60 mg of A2-73 free base, preferably from about 1 mg to about 50 mg of A2-73 free base.

[0083] The enteric coated oral formulation may also contain a crystalline form of A2-73 salt. The A2-73 salt may be a fumarate, sulfate, mesylate, dihydrogen phosphate, edisylate, benzoate, hydrochloride, and oxalate. In one embodiment, the A2-73 salt is a fumarate. When the A2-73 salt is a fumarate, the enteric coated oral formulation may contain about 0.1 to about 100 mg of A2-73 fumarate, preferably about 1 mg to about 55 mg of A2-73 fumarate.

[0084] Dosage forms also include those formulated for subcutaneous and / or intramuscular injection. For example, intramuscular dosage forms may contain A2-73 in free base form, dissolved in an oil matrix for intramuscular injection, or alternatively, prepared as a suspension of free base for intramuscular injection. Dosage forms formulated for subcutaneous or intramuscular injection may contain A2-73 in salt or free base form disclosed herein, prepared as microspheres using methods known in the art. Alternatively, A2-73 in free base or salt form may be coated with a thin layer coating, such as a coating of zinc oxide, for example, using atomic layer deposition (ALD) techniques, and used in formulations for subcutaneous or intramuscular injection. Alternatively, A2-73 free base may be dissolved in a biodegradable polymer matrix and then implanted subcutaneously (or used in a transdermal patch, as described in more detail below).

[0085] VII. Kit Further aspects of the present disclosure provide kits that include one or more reagents for use with the methods of the present disclosure. The kits may include cell growth media, selection media, nucleic acid sequencing and purification reagents, protein purification reagents, buffers, and the like. The kits provided herein generally include instructions for carrying out the methods detailed below. The instructions included in the kits may be affixed to packaging materials or included as a package insert. The instructions are typically written or printed materials, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" may include the address of an internet site that provides the instructions.

[0086] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.The following references provide those skilled in the art with many of the general definitions of the terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994), The Cambridge Dictionary of Science and Technology (Walker ed.,1988), The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991), and Hale&Marham, The Harper Collins Dictionary of Biology (1991).As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0087] When introducing an element of the disclosure or a preferred embodiment(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the element. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0088] Since various modifications can be made in the above cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below should be interpreted in an illustrative and not a limiting sense.

[0089] The term "comprising" means "including, but not necessarily limited to," and specifically indicates open-ended inclusion or membership in such listed combinations, groups, series, etc. As used herein, the terms "comprising" and "including" are inclusive and / or open-ended and do not exclude additional, unrecited elements or method steps. The term "consisting essentially of" is more restrictive than "comprising," but less restrictive than "consisting of." Specifically, the term "consisting essentially of" limits membership to specified materials or steps, and to materials or steps that do not materially affect the essential characteristics of the claimed invention.

[0090] As used herein, "expression" includes, but is not limited to, one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce a mature mRNA; mRNA stability; translation of mature mRNA into a protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, as necessary for proper expression and function.

[0091] As used herein, the term "gene" refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0092] As used herein, the term "genotype" means the unphased 5' to 3' sequence of nucleotide pairs found at one or more polymorphic sites at loci on homologous chromosome pairs in an individual. As used herein, genotype includes complete genotypes and / or subgenotypes.

[0093] As used herein, the terms "locus" or "genetic locus" are used interchangeably and refer to a location on a chromosome or DNA molecule that corresponds to a gene or a physical or phenotypic characteristic.

[0094] As used herein, the term "mutant" refers to any inherited variation from wild type that is the result of mutations, such as single nucleotide polymorphisms ("SNPs") and insertions / deletions. The term "mutant" is used interchangeably with the terms "marker," "biomarker," and "target" throughout this specification.

[0095] As used herein, the term "medical condition" includes, but is not limited to, any condition or disease that manifests itself as one or more physical and / or psychological symptoms for which treatment is desirable, including previously and newly identified diseases and other disorders.

[0096] As used herein, the term "nucleotide pair" means the nucleotides found at a polymorphic site on the two copies of a chromosome from an individual.

[0097] As used herein, the terms "polymorphism," "polymorphic variant," and "polymorphic site" are used interchangeably and refer to a position within a genetic locus at which at least two alternative sequences are found. Polymorphic variants of a gene can result in abnormal expression or production of an abnormal form of a protein, which may cause or be associated with disease.

[0098] As used herein, the term "polynucleotide" refers to any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules that contain DNA and RNA, which may be single-stranded or, more typically, may be double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions that contain RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains one or more modified bases, and DNA or RNA that has a backbone that is modified for stability or for other reasons.

[0099] As used herein, the term "polypeptide" refers to any polypeptide comprising two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptides refer to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences that are modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as in a voluminous research literature.

[0100] As used herein, the term "single nucleotide polymorphism (SNP)" refers to nucleotide variability at a single position in the genome where two alternative bases occur with significant frequency (i.e., >1%) in the human population. SNPs can occur within genes or within intergenic regions of the genome. SNP probes according to the present invention are oligonucleotides complementary to the SNP and its flanking nucleic acid sequence(s).

[0101] As used herein, the term "subject" means that the subject is preferably a mammal such as a human, but can also be an animal, such as domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., cynomolgus monkeys, rats, mice, guinea pigs, etc.).

[0102] As used herein, administration of an agent or drug to a subject or patient includes self-administration and administration by another. It is also understood that the various treatment or prevention modes of medical conditions as described are intended to mean "substantially" which includes complete treatment or prevention, but also includes less than complete treatment or prevention, and in which some biologically or medically relevant result is achieved. EXAMPLES

[0103] The publications discussed above are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0104] The following examples are included to demonstrate the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in implementing the present disclosure. However, those skilled in the art should understand in light of the present disclosure that many changes can be made in the present disclosure without departing from the spirit and scope of the present disclosure and still obtain the same or similar results, and therefore all matters described are to be interpreted as illustrative and not limiting.

[0105] Example 1. ANAVEX 2-73 Study Design, Safety, and Tolerability, and Patient Characteristics. ANAVEX2-73, an agonist of SIGMAR1, has been shown by PET scans to directly bind to its target and modulate cholinergic muscarinic receptors in mice. The clinical potential of ANAVEX2-73 in AD was evaluated first in mice, followed by a phase 1 study in healthy volunteers and a 57-week phase 2a AD study. This study, the 57-week study (Figure 1a), enrolled 32 patients with mild-to-moderate AD and met its primary endpoints of safety and tolerability (Table 1), leading to a 208-week extension study, referred to as the 208-week study (Figure 1a). Patients enrolled in the two studies were characterized at baseline and at each visit, including collection of data on clinical descriptors, physical examination, pharmacokinetics, and MMSE and ADCS-ADL as efficacy measures (Figure 1b and Table 3).

[0106] [Table 2]

[0107] [Table 3]

[0108] Example 2. Genomic biomarker identification. Genomic characterization of patient tumors is routinely performed in oncology trials to identify and rank genomic biomarkers of disease and treatment response. Genetic polymorphisms are associated with treatment response mutations, modulating treatment response in patient populations. In this study, whole exome sequencing (WES) and gene expression analysis (RNAseq) were performed at weeks 109-135 for the 21 patients remaining in the extension study (Figure 1b). Genomic data integrated with longitudinal clinical and other patient characteristics, including efficacy outcome measures and derived progression measures, will lead to phenotypic and genotypic precision medicine analyses (Figure 1b,c).

[0109] Small cohorts, where the patient observation scale is smaller than the number of attributes, constitute an analytical challenge. In this study, phenotypic and genotypic biomarkers were identified and ranked using a Galois lattice-based, non-objective formal concept analysis (FCA) method implemented in KEM software (v.3.6.2). In this analysis, no associations between biomarkers and outcomes are assumed, allowing for unconstrained data-driven hypothesis generation of all relationships.

[0110] A total of 3,145,630 relationships between all available features were explored. Stringent filtering excluded rules containing variables coding for the absence of DNA variants and intermediate RNA expression groups. Numerical filtering of the rules included the following thresholds: support (n>3), confidence (>50%), lift (≥1.2), and p-value (Fisher exact test or Mann-Whitney-Wilcoxon <0.05). The filtering step resulted in a subset of 1,019 association rules relating clinical, genomic, and transcriptomic patient characteristics to response at multiple time points. The second filtering step focused on relationships only with response at week 57, raising support ≥5, and lift ≥1.5, and excluded RNA expression, CYP variants, and dose / concentration from part A. This further reduced the number of rules to 15 correlations related to outcomes measured by delta (by binarized discretization, see Supplementary Table 4) or slope of MMSE or ADCS-ADL at week 57. The 15 rules are presented in order of highest confidence and highest lift. Stringent filtering focused on relationships of response at week 57 (end of part B) and reduced the number of correlations to only 15 related to outcomes measured by delta or slope MMSE or ADCS-ADL (Table 4). Within the 15 relationships identified, mean blood concentrations of ANAVEX2-73 above 4 ng / mL in part B increased the odds of improvement in MMSE outcome at week 57 by 1.88-fold (Table 4). Higher concentrations improved delta ADCS-ADL scores (p=0.03) (Figure 2c). A lower MMSE baseline score (<20) increased the relative odds (lift) of a worse MMSE outcome by 1.5-fold, and a higher MMSE baseline score (>20) increased the relative odds of MMSE improvement by 1.62-fold, both at week 57.

[0111] [Table 4]

[0112] Further focusing on the relationships showing 100% confidence and true for both delta MMSE and delta ADCS-ADL, two DNA variants, SIGMAR1_Q2P and COMT_L146fs, were identified. Table 5 shows a summary of the integrated data sources. A total of 2,527 features from 1,152 descriptors were used for each subject, including 837 genomic sequences with amino acid changes from a total of 27,155 annotated genes, and 185 RNA expression profiles. Patient descriptors are shown in grey and outcomes in pink. A description of the number of patient descriptors incorporated in the two analytical steps applied in the study to model the two groups of ANAVEX2-73 concentrations: 1) an undependent FCA rule-based analysis of response at week 57, and 2) a longitudinal validation using mixed-effects modeling of response over 148 weeks incorporating markers found at week 57.

[0113] [Table 5]

[0114] The 3D structural impact of SIGMAR1 Q2P was also investigated. The 3D crystal structure of SIGMAR1 was visualized using VistaPDB 1.0 software (Figure 3). Each monomer displays an N-terminal transmembrane alpha-helix. The N-terminus of the protein is thought to face the cytosol and interact with other ligands. The identified SIGMAR1 variant (glutamine (Gln) at position 2 changed to proline - SIGMAR1_p.Q2P) is located at the N-terminus of the protein and may therefore have structural and functional consequences. The change from Gln (Q) to proline (P) may affect the length of the side chain (extended with Gln) and thus the ability of the amino acid to form an alpha-helix. Proline is considered a "helix breaker" and has been attributed in some cases to unwinding the N-terminus of the a-helix. Retrospective exclusion of patients with SIGMAR1_Q2P or COMT_L146fs variants and low baseline MMSE scores improved MMSE and ADCS-ADL scores at week 57. Daily treatment with ANAVEX2-73 for the entire phase 2a cohort for 57 weeks had effect sizes of 0.57 and 0.18 for delta MMSE and delta ADCS-ADL, respectively, when compared with standard care (Table 6). When patients with SIGMAR1_Q2P and COMT_L146fs variants and low baseline MMSE scores (9 of 21 patients) were excluded, the effect sizes increased to 1.05 and 0.93 for MMSE and ADCS-ADL, respectively (Table 6), both considered "large" according to Cohen's d guidelines.

[0115] [Table 6]

[0116] Example 3. Verification of results. As an internal validation of the week 57 outcomes, predictors of response at week 57 were tested on the interim week 148 longitudinal data in the 208-week extension study using regression analysis. This analysis included 21 patients with genomic data and 287 observations for 14 time points (Table 7). ANAVEX2-73 concentrations in part B, identified as predictors of outcome at week 57, were used to define two arms for the MMRM-LME model. In addition to the parameters identified at week 57 (ANAVEX2-73 concentration levels, baseline MMSE score, SIGMAR1_Q2P and COMT_L146fs variants), age, sex, APOE ε4 status, and donepezil concomitant use were included in the MMRM-LME models for delta MMSE and delta ADCS-ADL. This analysis showed that the higher ANAVEX2-73 concentration arm had a favorable difference of 78% and 88% in adjusted MMSE and adjusted ADCS-ADL, respectively, when compared to the low / medium arm over 148 weeks (p-values ​​<0.0008 and <0.0001, respectively). In addition to time, APOE ε4 status (p<0.0001) and ANAVEX2-73 mean concentration were significant predictors (Figure 4a, 4b, and Table 8 and Table 6A). Furthermore, SIGMAR1_Q2P, COMT_L146fs, and APOE4 ε4 status interactions with time were significant variables. Unadjusted measures supported these results, with patients with positive biomarkers of response having better delta ADCS-ADL over 148 weeks than patients without biomarkers or the reference population receiving standard treatment (Figure 5 and Table 9). Two patients showed exceptional treatment response during the longitudinal study (148-week period). Both subjects had SIGMAR1 wild type, high mean plasma concentrations of ANAVEX2-73, and baseline MMSE > 20 (Figure 6). Reported clinical benefits included improved mood and attention, as well as increased independent activity.

[0117] JPEG2025020267000007.jpg121136

[0118] [Table 7]

[0119] [Table 8]

[0120] [Table 9]

[0121] Considerations of Examples 1 to 3 This study is, to our knowledge, the first reported genome-wide search for biomarkers associated with drug response in AD. The approach we used differed from standard classification or "signature" searches. We used FCA, based on the KEM framework, as a method for identifying and ranking predictors of response by association rules consistent with clinical trial data. As shown in this study, FCA machine learning methodology has been shown to provide a robust methodology for identifying and ranking predictors of response by association rules consistent with clinical trial data. 9 Search very large combinatorial spaces, typically 2-3×10 6 This allowed us to extract a smaller set of rules and finally focus on a few dozen significant relationships. In contrast to "black box" numerical methods, this approach allows the identification of patients that validate each identified rule (as shown in Tables 4 and 5) and introduces an objective and explicit filtering strategy to select the most relevant biomarkers.

[0122] The negative results generated around the "amyloid hypothesis" prompted the AD community to hypothesize that Aβ plaques could appear at advanced stages of the disease, which may be beyond the scope of existing anti-BACE or anti-tau interventions. However, it has also been difficult to characterize the disease at an early stage, as reliable genomic markers that could be prognostic for the development of MCI caused by AD have not yet been identified. APOE status alone does not have sufficient predictive power, as only about 25% of the population has the APOE4 genotype. Studies have identified a gene variant, TOMM40, that could be used for early detection of mild cognitive impairment (MCI). This hypothesis was tested in the TOMORROW study. However, the TOMORROW study, which included 3,494 patients (out of 24,136 screened), failed to reach its primary objective of biomarker qualification. In this context, the search for alternative druggable targets for AD is becoming a priority and SIGMAR1, targeted by ANAVEX2-73, may be a key drug target for maintaining cellular homeostasis, which could potentially slow or halt neurodegeneration and even enable compensatory responses.

[0123] This paper presents a data-driven unbiased analysis from two clinical trials that allowed the identification of parameters associated with improved outcomes. By searching and selecting biomarkers that are consistently associated with clinical response across multiple time points and multiple endpoints, reliable identification and ranking of patient selection markers is possible. Notably, the approach described in this study identifies ANAVEX2-73 mean concentration, baseline MMSE, and a specific variant of the SIGMAR1 gene (rs1800866) in addition to COMT variants (rs113895332 / rs61143203) as the top 20 drivers of clinical response among a subset of >3 million retained rules, without any a priori hypotheses. Low baseline MMSE is thought to be associated with advanced AD stages beyond the reach of many therapeutic approaches. SIGMAR1 is an in vivo confirmed target of ANAVEX2-73. COMT is a gene previously described as involved in memory and other neurobehavioral functions.

[0124] The MMRM-LME analysis at the interim 148-week point showed significant differences in the responses between the two concentration arms related to adjusted mean delta ADCS-ADL (88%) and adjusted mean delta MMSE (78%). Note that these differences are significant and rather large. These values ​​compare favorably with recent results obtained in other trials, for example with data presented at CTAD2018 for BAN2401 after 72 weeks: about 18% of the change could be attributed to APOE4 status imbalance in the respective arms, 47% in the adjusted mean change from baseline in ADAS-Cog and 30% in the adjusted mean change in ADCOMS54.

[0125] Analysis of 39,974 DNA variants (Table 10) showed that SIGMAR1_Q2P and COMT_L146fs were retained in the top correlations. Due to limited data availability, standard of care measures of delta MMSE and delta ADCS-ADL obtained from the literature to calculate effect sizes corresponded to 52 weeks rather than 57 weeks, the actual time point used in this study. Finally, RNA analysis was limited due to collection time (109–135 weeks) and no baseline transcriptome measures were available for comparison. However, exploratory analysis found that high SIGMAR1 expression was associated with better outcomes (Table 11).

[0126] [Table 10-1]

[0127] [Table 10-2]

[0128] [Table 10-3]

[0129] [Table 10-4]

[0130] [Table 10-5]

[0131] [Table 11]

[0132] The methodology described in this study opens the possibility of using data-driven unbiased biomarker identification early in the drug development process. The systematic approach of "white box" and formal concept analysis may be an ideal combination for the analysis of early data, where machine learning platforms may be essential to explain each finding and identify and rank patient-selected biomarkers for treatment response. These data analysis methods may be the basis for precision medicine drug discovery and development.

[0133] Methods of Examples 1 to 3 (a) Test Design The present clinical trial integrates and analyzes data and samples from two consecutive clinical trials: a 57-week study and a 208-week study. The 57-week study was a multicenter phase 2a clinical trial of the drug ANAVEX2-73 that recruited a sample of 32 subjects with mild-to-moderate AD. It consisted of two parts. Part A was a simple randomized, open-label, two-period, crossover, adaptive study lasting up to 5 weeks. Each period included one specific route of administration and two possible dose levels: in one period, an oral dose (30 mg or 50 mg) was administered, and in the other period, an intravenous (IV) form (3 mg or 5 mg) was administered, resulting in a total of eight possible dose administration schemes in part A (details are available in Figure 2a). The two periods were separated by a 12-day washout period during which no dose was given.

[0134] Part B, immediately following Part A, was an open-label extension of the oral daily dose for an additional 52 weeks. The primary endpoint of the 57-week study was to establish the safety and tolerability of the drug. Secondary endpoints aimed to establish the relationship between the dosing regimen and the pharmacokinetic-exploratory efficacy outcomes of cognition and function. Cognition was assessed by the Mini-Mental State Examination (MMSE) and functional capacity by the Alzheimer's Disease Cooperative Study-Activities of Daily Living Scale (ADCS-ADL). Additional cognitive measures included the Cogstate Brief Battery (CBB), as well as functional scales of electroencephalography and event-related potentials (EEG and ERP). Part B continued the adapted oral dosing scheme. Table 12 summarizes the mean oral dose administration during the 57-week study.

[0135] [Table 12]

[0136] Due to the favorable safety and tolerability profile of the 57-week study (Tables 1 and 2), and upon request from patients and caregivers, a 208-week open-label extension study (208-week study) was conducted immediately following completion of the 57-week study part B. The 208-week study enrolled 24 subjects who completed part B and were also administered the adapted daily oral dosing scheme. ANAVEX2-73-003 includes ongoing evaluation of safety and efficacy as measured by MMSE and ADCS-ADL. At week 148 (midpoint of analysis), data on subjects remaining in the study (n=21) were analyzed for efficacy outcomes. This report was prepared using data available at week 148 while ANAVEX2-73-003 was still ongoing.

[0137] (b) Target A total of 32 subjects with mild to moderate AD, aged 50-85 years, were enrolled across three sites in Australia between December 2014 and September 2015. Their diagnoses were consistent with the Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) and National Institute of Neurological and Communicative Disorders and Stroke / Alzheimer's Disease and Related Disorders Association criteria and included MRI and / or CT brain scan evaluation. The demographics and baseline characteristics of the 32 participants are summarized in Table 3 along with the group of participating subjects with genomic data collected after protocol amendment and patient consent (N=21). Table 1 shows patient disposition. Seven patients discontinued during the 57-week study, six of which withdrew consent and one due to an adverse event (AE). A total of three patients discontinued during the 208-week study by the cumulative interim of 148 weeks in the ongoing extension study.

[0138] The following assessment scores and criteria were recorded at baseline and each visit: MMSE, ADCS-ADL, Hamilton Rating Scale for Depression (HAM-D) assessment, Rosen-Modified Hachinski Ischemic Score (RmHis), CBB, and EEG / ERP. The CBB subtests consisted of six different subscores: Detection (DET, processing speed), Discrimination (IDN, attention / vigilance), One-Card Learning (OCL, visual recognition memory), One-Back (OBK, working memory), International Shopping List (ISLT, verbal learning), and International Shopping List Delayed Recall (ISRL, verbal memory).

[0139] (c) Study endpoints A. Safety Endpoints Safety was assessed by evaluation of the incidence and severity of adverse events (Table 2), vital signs, physical examinations, clinical laboratory parameters (hematology, serum chemistry, and urinalysis), and electrocardiograms (data not shown).

[0140] B. Efficacy Outcomes Exploratory clinical efficacy in this study was defined as the change from baseline in each patient's cognitive (MMSE) and functional ability (ADCS-ADL) scores. The MMSE57 is a widely used and validated instrument in clinical AD research, including cognitive outcome measures in drug trials. The scale ranges from 0 to 30, with higher scores indicating greater cognitive function. The ADCS-ADL58 is administered in a structured caregiver interview format to assess performance in activities of daily living in patients with AD. The scale ranges from 0 to 78, with higher scores indicating greater function. The ADCS-ADL has been shown to have good test-retest reliability59. The MMSE and ADCS-ADL were assessed during prespecified clinical site visits across 14 different time points during the 57-week study (weeks 0 and 5 in part A, and weeks 17, 31, 41, 53, and 57 in part B), as well as the 208-week study (weeks 70, 83, 96, 109, 122, 135, and 148).

[0141] The corresponding delta scores were calculated using the difference between the values ​​at each visit and at baseline for each individual patient, focusing on week 57. In addition, to reduce the effect of noise in the MMSE and ADCS-ADL scores, slopes were estimated using linear regression across all available time points for each individual patient. The derived scores describe the progression of exploratory efficacy data from baseline at several specific time points (delta) and over a set period of time (slope). The inclusion of subscores and measures derived from the same test score did not affect the analytical methods applied in this study, since each relationship was treated and described independently.

[0142] Table 13 summarizes the delta scores, relative to baseline, for MMSE and ADCS-ADL at 13 time points in the 57-week study (weeks 5, 12, 26, 36, 48, and 57) and ANAVEX2-73-003 (weeks 70, 83, 96, 109, 122, 135, and 148). Summary statistics for the slopes for MMSE and ADCS-ADL are shown in Table 13.

[0143] [Table 13]

[0144] C. Pharmacokinetics Pharmacokinetic analyses were performed only during Part A. Blood samples were collected at 12 and 11 time points, respectively, after the first oral or IV dose during the first and second periods of Part A. Pharmacokinetic parameters included in the analysis were maximum plasma drug concentration (C max ), time to maximum plasma concentration during the dosing interval (t max ), the area under the curve from time of administration to the last measurable concentration (AUC 0-t ), the mean elimination half-life (t1 / 2), and the total body clearance for extravascular administration divided by the fraction of the absorbed dose (CL / F). Figure 2b shows the AUC 0-inf Shows.

[0145] Steady-state plasma levels of ANAVEX2-73 and its metabolite ANAVEX19-144 were assessed in Part B through blood sample collection at pre-specified time points (weeks 12, 26, 36, and 48). The mean concentration values ​​in ng / mL for each patient were then averaged. Table 14 shows the mean plasma concentrations of ANAVEX2-73 and its metabolite ANAVEX19-144 during the 57-week study.

[0146] [Table 14]

[0147] The relationship between dose and plasma concentration of ANAVEX 2-73 was determined in a Phase 1 study 22 The dose-dependent increase in plasma concentrations of ANAVEX 2-73 and metabolites was also observed in the present study, as illustrated in Figure 2b.

[0148] D. DNA and RNA extraction and sequencing Blood samples were collected from all 21 still participating subjects at visit dates after protocol amendment and approval during the ANAVEX2-73-003 extension study, as well as weeks 103–135, depending on patient consent. These samples allowed DNA (whole exome) and RNA next-generation sequencing (NGS) data analysis. 408,551 DNA variants were identified within the cohort. Of these, 39,974 high or moderate impact variants driving amino acid modifications in proteins were retained, and RNA expression values ​​were normalized as transcripts per kilobase per million (TPM).

[0149] E. Focused Gene Subsets 243 genes were selected from 27,155 mapped sequences obtained from the NGS analysis introduced above (Supplementary Figure 1a, b, c). 102 genes were selected based on their involvement in neurodegenerative diseases (Supplementary Figure 1a). 20 genes were selected as part of the functional interactome of SIGMAR1 based on a confidence score of 0.150 (January 2018) obtained from the STRING database (Supplementary Figure 1c and d). In addition, 113 genes from the cytochrome P450 gene family and 10 genes from the methyltransferase gene family were also added (Supplementary Figure 1b).

[0150] [ka]

[0151] F. Data Analysis Data analysis using association rule-based FCA was performed to identify markers of response to treatment at week 57. In a subsequent step, a confirmatory analysis of the identified markers of response at week 148 was performed using linear mixed effects. Figure 1b shows the different types of data used in the analysis, and Figure 1c shows the two different types of analyses for the identification and validation of markers of drug response.

[0152] (d) Association Rules and the KEM® Platform KEM® (Knowledge Extraction and Management) v3.6.2 software was used to systematically extract relationships between all collected variables. Based on Galois Lattice Theory, or Formal Concept Analysis (FCA), KEM® identifies groups of objects with shared properties and generates corresponding association rules. Association rules have been developed to detect patterns and relationships in large heterogeneous databases. This methodology was introduced by Agrawal and Sirikant and has been successfully used in different domains including market basket analysis, drug discovery, and genomic feature analysis of complex diseases. The method has also been successfully used in drug efficacy and patient stratification studies. Association rules may reflect the existence of a causal relationship between two variables. However, the existence of a statistically significant correlation is neither necessary nor sufficient to indicate the existence of such a causal relationship. Therefore, the use of quality measures is described below.

[0153] An association rule is an association such as X → Y, where X is the antecedent (or combination of antecedents, also called the left side) and Y is the consequent (or combination of consequents, also called the right side) of the rule, allowing us to infer X as an explanation for Y. Given a dataset of N patients, X , n Y , and n XY are the number of patients who satisfy the antecedent(s) X, the consequent(s) Y, and both parts of the rule, respectively. For any given rule, xy It is possible to specifically identify a human patient.

[0154] Six main quality measures are used to describe and rank association rules: support, confidence, lift, two P-values ​​which are measures of significance, and Cohen's d which is a measure of effect size.

[0155] i. Support, Confidence, and Lift Support is defined as the number of subjects in which the correlation was observed: Support degree (X→Y)=n XY

[0156] Confidence is defined as the percentage of characterized patients that validate the rule:

[0157]

number

[0158] Lift (relative probability) is the ratio of observed support to the expected support if X and Y were independent. It measures the performance of a rule to identify subgroups from a larger population.

[0159]

number

[0160] ii. Significance tests P values ​​from two statistical tests were calculated for each association rule: a right-sided Fisher's exact test for categorical data to account for rule relationships and suitable for small sample sizes; a two-sided Mann-Whitney U test to compare continuous outcome measures by descriptor group. Statistical significance was determined at the 0.05 level for both tests.

[0161] iii. Effect size Cohen's d index is calculated for each correlation to quantify the effect size, from which the number of patients to be included in future studies to determine significant relationships can be calculated. Cohen's d indicates the standardized difference between two population means.

[0162] In this study, outcomes were defined by delta and slope values ​​of MMSE and ADCS-ADL. Control group values ​​for calculating effect sizes were obtained by combining available study results in the standard of care AD literature: control group mean delta MMSE at week 52 was defined as -3.7 with a standard deviation of 3.5, and mean delta ADCS-ADL at week 52 was -6.7 with a standard deviation of 8.0.

[0163] Continuous variables such as age were discretized using distributions (see summary of thresholds in Table 13). For example, baseline MMSE was discretized into two groups, "low" (defined as baseline MMSE<20) and "high" (defined as baseline MMSE≧20). Note that the value "20" is not a predefined limit. The MMSE baseline score threshold "<20" corresponded to the median observed value of all included patients.

[0164] (e) Linear mixed-effects model The application of mixed-effects models for repeated measures (MMRM) to model continuous primary endpoints in longitudinal clinical trials, and specifically to analyze outcomes in AD clinical trials, has become common.

[0165] Longitudinal associations of biomarkers identified at week 57 were assessed at week 148 using MMRM by allocating subjects to one of two arms (high vs. low / intermediate) based on ANAVEX2-73 mean blood concentrations in part B. Given the small cohort size, time was treated as a continuous variable in the analysis to avoid over-parameterization and improve robustness. Therefore, a linear component of a mixed effects model (LME) was applied to model time. MMRM-LME, implemented in R statistical software, was applied to model delta MMSE and ADCS-ADL scores with ANAVEX2-73 concentration levels over time, and other identified descriptors of response.

[0166] Example 4. DNA and RNA extraction and analysis A total of 27,155 annotated genomic locations corresponding to genes were identified in AD patient sequences, including 616 pathways. An initial panel of 243 genes was defined. The panel consisted of 102 genes involved in neurodegenerative diseases, 20 genes related to the SIGMAR1 functional interactome, as well as 12 methyltransferase genes and 111 CYP genes related to drug metabolism (Table 15).

[0167] [Table 15-1]

[0168] [Table 15-2]

[0169] [Table 15-3]

[0170] [Table 15-4]

[0171] [Table 15-5]

[0172] Distribution analysis was performed using TPM (transcripts per kilobase per million) values ​​for each RNA gene expression, defining three equally sized bins: low, medium, and high.

[0173] This is calculated as follows:

[0174]

number

[0175] Note X i : the read count of the mRNA being considered, l i : The length of the mRNA considered.

[0176] For COMT and COMT TPM (COMTt), levels below 10.1 are considered low, 10.1>COMTt>=15.2 are considered medium, and COMTt>15.2 are considered high.

[0177] For KANSL1, KANSL1 TPM (KANSL1t), levels below 16.9 are considered low, 16.9>KANSL1t>=27.5 are considered medium, and KANSL1t>27.5 are considered high.

[0178] For SIGMAR1, SIGMAR1 TPM (SIGMAR1t), levels below 12.7 are considered low, 12.7>SIGMAR1t>=19 are considered medium, and SIGMAR1t>19 are considered high.

[0179] Focusing on an initial panel of 243 genes, 185 RNA gene expression at the protein level and 837 DNA alterations were shown to be different for at least one subject out of a total set of 21 subjects. Subjects were further characterized using an additional set of 212 non-genomic features consisting of medical history, concomitant medications, and other baseline characteristics. A total of 40 endpoints were defined as follows:

[0180] >Mini-Mental State Examination ("MMSE") and Alzheimer's Disease Cooperative Study-Activities of Daily Living ("ADCS-ADL") endpoints were extracted from data collected in ANAVEX2-73-002.

[0181] Delta MMSE and ADCS-ADL were calculated using the differences between values ​​collected at the 6 time points and at baseline.

[0182] Similarly, multiple values ​​were combined to calculate the slope. The slope is calculated using linear regression across all available time points for each individual patient. The slope calculation has the advantage of smoothing the variability across observed values ​​of these scores.

[0183] A distribution analysis was performed on the calculated deltas for both MMSE and ADCS-ADL (Figures 7A and 1B). Patients were classified into categories based on their third tile.

[0184] - Delta ADCS-ADL: high (≥-1) and low (≤-2);

[0185] -Delta MMSE: high (≥-1) and low (≤-2).

[0186] The mid and low bins were combined to define a new low bin. The high bin corresponds to the highest tertile value. The slopes between baseline and week 57 for both MMSE and ADCS-ADL were considered as dichotomous endpoints. Responders were defined as having a positive or no slope. Non-responders were defined as displaying a negative slope.

[0187] The study contained a total of 2,481 features for each patient. Available data was analyzed using KEM® v3.6.2 (Knowledge Extraction and Management, available from Ariana Pharmaceuticals, SA). 3,040,276 relationships were generated and analyzed. Relationships were analyzed using set metrics to assess their strength.

[0188] - Support (n) shall mean the number of subjects in which a correlation is observed.

[0189] -Lift (relative probability) shall mean the frequency of the correlation divided by the frequency of the condition. This parameter measures the enrichment provided by a condition.

[0190] - Confidence means the probability of correlation if the condition is true

[0191] -Cohen's d is an effect size used to show the standardized difference between two measures. From the association rules, we identified several genetic features associated with improved or worsened outcome (delta MMSE or delta ADCSADL). For each feature, we defined group 1 by patients with the desired feature (if it was associated with improved outcome) or patients without the feature (if it was associated with worsened outcome). M 群1 and S.D. 群1 are the mean and standard deviation of outcomes in group 1, respectively. M 群2 and S.D. 群2 are mean and standard deviation reference values ​​for outcomes known in the art for a group of patients with mild Alzheimer's disease.

[0192]

number

[0193] [Table 16]

[0194] -P-value (Fisher's exact test, larger one-tailed) assesses the statistical significance of differences in 2x2 contingency tables.

[0195]

number

[0196] - Mann-Whitney-Wilcoxon (MWWilc.) is a non-parametric test of the null hypothesis that the distributions in both populations are equal. The MWWilc test ranks the values ​​of the combinations in the two populations and compares the sum of those ranks between the two populations. We compare the distribution of outcomes between patients with the feature and those without the feature.

[0197] -MAF (Minor Allele Frequency): refers to the frequency with which a mutation occurs in the general population from the 1000 Genomes database.

[0198] Filtering of relationships was performed in two steps: semantic filtering (1) and numerical filtering (2). In the semantic filtering step, we excluded parameters coding for the absence of DNA mutations, medium RNA expression bins, and slope MMSE and slope ADCS-ADL. In the numerical filtering step, we retained only relationships that fulfilled metrics based on support (n>3), confidence (>50%), relative probability (lift>=1.2), and p-value (Fisher or Mann-Whitney-Wilcoxon<0.05).

[0199] Filtering steps (1) and (2) result in a subset of 509 association rules that relate baseline clinical and genomic patient characteristics and RNA expression with response at multiple time points.

[0200] Focusing solely on response at week 57, this set was further reduced to nine, including 42 DNA mutation-related correlates, 27 RNA expression, and other parameters at baseline.

[0201] The 42 association rules relating the presence of DNA mutations to response correspond to 22 CYP proteins and 20 non-CYP proteins from the gene panel defined in Table 15. The 20 non-CYP associations correspond to 12 non-redundant SNPs associated with worse response at week 57 (Table 18) and 2 associated with better response at week 57 (Table 17).

[0202] Table 17 data was extracted from a systematic analysis of genomic data (185 gene panel and 837 variants). It identifies two SNP variants significantly associated with improved outcome measured by delta MMSE or delta ADCS-ADL at week 57: these are HLA-DRB1_p.LysThr134LysAsp and DPYD_p.l543V (re1801159). Rules: n>3 patients, confidence level>0.5, lift>=1.2, p-value (Fisher test or MWWilc.)<0.05.

[0203] [Table 17]

[0204] [Table 18]

[0205] The presence of altered COMT (stopped after L146), or DHCR7 T220 (DHCR7_p.M220T), or HLA-DRB1 T244 or T66 or S89 (HLA-DRB1_p.A244T or S66Y or Y89S), or KANSL1 Pro304 / 946 / 1010 (KANSL1_p.P1010L / P304L / P946L), or MS4A6E T59 (MS4A6E.p.M59T), or RIN3 R 215 (RIN3_p.H215R), or SIGMAR1 Pro2 (SIGMAR1_p.Q2P) mutations is associated with lower ADCS-ADL or MMSE delta at week 57 versus baseline.

[0206] Table 19 presents data on three mutations (SIGMAR1 Q2P, COMT_p.L146fs, KANSL1_p.P1010L / P304L / P946L) associated with both MMSE and ADCS-ADL delta at week 57. Systematic analysis of genomic data (185 gene panel and 837 mutations) identifies a significant strong association between SIGMAR1 Pro2 mutations and absence of response, measured using both MMSE and ADCS-ADL.

[0207] [Table 19]

[0208] Table 20 is a representation of one association rule showing that the presence of a Pro2 mutation in SIGMA1R implies a lower delta in MMSE with details on the rule metrics, extracted from the data shown in Table 18.

[0209] [Table 20]

[0210] The SIGMAR1 Gln(glutamine)2 to Pro(proline)2 mutation (SIGMAR1_p.Q2P) may have structural implications for the 3D conformation of the 3D structure of the SIGMAR1 protein, which may then change its function and / or interaction. The crystal structure of SIGMAR1 shows a trimer. Each monomer shows an N-terminal transmembrane alpha helix. The N-terminus of the protein is thought to face the cytosol and interact with other ligands. The Pro2 modification may change the conformation of the N-terminus of the helix by extending it, consistent with some modification of function.

[0211] The change from Gln (Q) to Pro (P) affects the length of the side chain (extended with Gln) and can affect the ability of the amino acid to form alpha helices. Proline is considered a "helix breaker" and has been attributed in some cases to unwinding a-helices.

[0212] KEM® (version 3.6.2) relationships relating the presence of SIGMAR1 Pro2 mutations to cognitive and functional endpoints are shown in Table 19. Cognitive and functional measures are both clinical endpoints in the regulatory approval of therapeutics in mild and mild-moderate Alzheimer's disease, with a primary endpoint of being significantly better than placebo.

[0213] Given that the 57-week Phase 2a ANAVEX2-73-002 trial did not include a placebo arm, the KEM® analysis measured two endpoints consisting of cognition (MMSE) and function (ADCS-ADL) in two ways: the delta of each patient's own MMSE and ADCS-ADL scores (delta from week 57 to baseline).

[0214] Importantly, increases in MMSE (positive delta) and ADCS-ADL (positive delta) over 57 weeks in mild-moderate Alzheimer's disease are rare positive events, as Alzheimer's disease is understood to be a progressive degenerative disease. Significantly, 100% of all patients who did not improve MMSE (delta, n=9) and ADCS-ADL (delta, n=8) did not have SIGMAR1 mutations. Additionally, 100% of patients with SIGMAR1 Pro2 (n=5) mutations did not improve MMSE or ADCS-ADL delta. Genetic data available for all (100%) patients (n=21).

[0215] Similar results were obtained for the COMT variant COMT_p.L146fs (rs113895332 / rs61143203). 100% of all patients (n=6) who showed improvement in MMSE (both delta and slope) and ADCS-ADL (both delta and slope) did not have the COMT_p.L146fs mutation. Furthermore, 100% of patients with the COMT_p.L146fs (n=5) mutation did not improve MMSE or ADCS-ADL slope. The COMT_p.L146fs mutation is also associated with lower delta MMSE and ADCS-ADL. Genetic data available for all (100%) patients (n=21).

[0216] As such, establishing the presence / absence of these mutations can be useful in therapeutic decision-making regarding SIGMAR1 agonist therapy.The absence of these mutations is diagnostic for enhanced SIGMAR1 agonist therapy, such as A2-73 therapy.Establishing the presence of either or both of these mutations is diagnostic for using other therapeutic agents as first-line treatment for neurodegenerative disorders.

[0217] [Table 21]

[0218] SIGMAR1 Pro2 mutations result in significant differences in numerical delta values ​​of ADCS-ADL and MMSE delta (Fig. 8c and f). The MWWilc test evaluates whether there is a significant difference in the median values ​​of continuous variables in the two populations (i.e., here two populations: SIGMAR1 Pro2 mutations vs. SIGMAR1 Gln2 mutations). Absence of SIGMAR1 Pro2 mutations is strongly associated with high ADCS-ADL delta (improvement) from BL. Similar results were found for COMT Leu146 mutations (Fig. 8a and d) and KANSL1 Pro304 / 946 / 1010 mutations (Fig. 8b and e).

[0219] As described above, 27 RNA expression was associated with improved or worsened response at week 57 versus baseline. Further selection of only non-CYP proteins and high levels of RNA expression (excluding low and moderate expression levels) shows that high expression levels of DHCR7, GAMT, IL10, KANSL1, MTUS1, PPARG, and SIGMAR1 are associated with improvement in ADCS-ADL or MMSE delta at week 57 versus baseline (Table 22).

[0220] [Table 22]

[0221] Focusing on the intersection between DNA and RNA analysis, and the subset of genes identified through DNA analysis that influence outcome as measured by both MMSE and ADCS-ADL, Table 23 shows that higher RNA expression of three genetic markers (SIGMAR1, COMT, KANSL1) associated with response as measured by MMSE or ADCS-ADL.

[0222] [Table 23]

[0223] In particular, high SIGMAR1 RNA expression is associated with high ANAVEX® 2-73 response (improvement), as shown in Table 24 and FIG.

[0224] [Table 24]

[0225] Both DNA and RNA analyses provide converging evidence for the role of SIGMAR1, KANSL1, and COMT as predictors of SIGMAR1 therapy outcome at 57 weeks, as measured by MMSE and ADCS-ADL variants.

[0226] Table 25 shows a systematic analysis of the CYP genomic data (from Table 15, 111 gene panel) identifying 11 SNPs significantly associated with outcome measured by delta MMSE or delta ADCS-ADL at week 57. Correlating with improvement in MMSE and / or ADCS-ADL scores is inclusion of variants CYP4V2, CYP27B1, CYP26C1. Correlating with exclusion based on decline in MMSE and / or ADCS-ADL scores is variants CYP39A1, CYP2D6, CYP2C8.

[0227] [Table 25]

[0228] Table 26 shows RNA expression of CYP4F2, CYP26A1, and CYP2D6 in relation to response as measured by MMSE or ADCS-ADL.

[0229] [Table 26]

[0230] Example 5. Sigma-1 agonist responder / non-responder determination Twelve subjects diagnosed with Alzheimer's disease, ages 57-71, are tested for the Q2P polymorphism in the SIGMAR1 gene. Two subjects are determined to have the amino acid proline at position 2 of the SIGMAR1 protein. The remaining 10 subjects are further determined to have glutamine at that position on the receptor protein. The group with the amino acid proline at position 2 of the SIGMAR1 protein is determined to be unsuitable for Sigma-1 agonist therapy. The group with the amino acid glutamine at position 2 of the SIGMAR1 protein is determined to be suitable for Sigma-1 agonist therapy.

[0231] Example 6. Sigma-1 agonist responder determination and therapy A 74 year old male presents with diagnosed Alzheimer's disease. His SIGMAR1 gene is analyzed to the extent that it is determined that he has a glutamine at position 2 of the SIGMAR1 gene. This is wild type. He is treated with A2-73 at an oral dose of 30 mg daily for 6 months. His disease appears to have stabilized and not progressed over that time period.

[0232] Example 7. Sigma-1 agonist responder determination and therapy A 74 year old male presents with diagnosed Alzheimer's disease. A cheek swab is taken and a genotype assay is performed to determine the presence of the SIGMAR1 2P genotype. Such analysis determines that the subject has a glutamine at position 2 of the SigmaR1 gene receptor (protein). He is treated with ANAVEX2-73 at an oral dose of 30 mg daily for 6 months. His disease appears to have stabilized and not progressed over that time period.

[0233] Example 8. Sigma-1 agonist responder determination and therapy A 66 year old female presents with diagnosed dementia. Her SIGMAR1 gene is analyzed to the extent that she is determined to have a proline at position 2 of the SIGMAR1 gene. She is denied treatment with Sigma-1 agonists. She is referred for other therapeutic treatment options.

[0234] Example 9. Sigma-1 agonist responder determination and therapy A 60 year old female presents with diagnosed dementia. Her SIGMAR1 gene is analyzed to the extent that she is determined to have a proline at position 2 of the SIGMAR1 gene. She is treated with ANAVEX2-73 at an oral dose of 80 mg daily combined with oral rivastigmine at 1.5 mg twice daily for six months. Her disease has remained stable over that time and appears not to have progressed.

[0235] Example 10. Sigma-1 agonist responder determination and therapy A 64 year old male presents with diagnosed Alzheimer's disease. His COMT gene is analyzed to the extent that he is determined to not have the COMT dbsnp ID rs113895332 / rs61143203 mutation. He is treated with A2-73 at an oral dose of 30 mg daily for 6 months. His disease appears to have stabilized and not progressed over that time period.

[0236] Example 11. Sigma-1 agonist responder determination and therapy A 71 year old female presents with diagnosed Alzheimer's disease. Her SIGMAR1 gene is analyzed to the extent that she is determined to not have SIGMAR1 rs1800866. She is treated with A2-73 at an oral dose of 30 mg daily for 6 months. Her disease appears to have stabilized and not progressed over that time period.

[0237] Example 12. Sigma-1 agonist responder determination and therapy An 81 year old male presents with diagnosed dementia. He is determined to have the KANSL1_p.P1010L / P304L / P946L mutation. He is declined treatment with Sigma-1 agonists and referred for other therapeutic treatment options.

[0238] Example 13. Sigma-1 agonist responder determination and therapy A 78 year old male presents with diagnosed dementia. He has a glutamine at position 2 of the SIGMAR1 gene. He is determined to have the KANSL1_p.P1010L / P304L / P946L mutation. He is treated with ANAVEX2-73 at an oral dose of 70 mg daily combined with oral memantine 7 mg daily for six months. His disease has remained stable over that time and appears not to have progressed. Exemplary aspects of the invention are described below. <1> 1. A method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy, the method comprising: a. obtaining or having obtained results of a test of a biological sample from said subject that determines the presence in said subject of at least one polymorphism selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_1543V, and combinations thereof; b. if the polymorphism is present in said subject, then ruling said subject out as non-responsive to Sigma-1 receptor agonist therapy. <2> 1. A method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy, the method comprising: a. detecting or having detected whether at least one polymorphism is present at a locus in a biological sample obtained from said subject by sequencing the locus containing said polymorphism; b. if the polymorphism is present in said subject, ruling said subject out as non-responsive to Sigma-1 receptor agonist therapy; The method of claim 1, wherein the at least one polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_l543V, and combinations thereof. <3> 1. A method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy, the method comprising: a. obtaining or having obtained results of a test of a biological sample from said subject, determining a level of expression of RNA encoded by at least a gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, DPYD, and combinations thereof, and combinations thereof; b. comparing the level of said RNA in said test sample to the level of said RNA in a subject who is responsive to Sigma-1 receptor therapy; A method wherein a level of said RNA in said test sample substantially similar to a level of said RNA in said subject responsive to Sigma-1 receptor therapy identifies a subject responsive to Sigma-1 receptor therapy. <4> 1. A method for selecting a subject that is responsive to Sigma-1 receptor agonist therapy, the method comprising: determining or having determined the level of expression of at least one RNA encoded by a gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, DPYD, and combinations thereof; b. Excluding the subject as non-responsive to Sigma-1 receptor agonist therapy if the level of the RNA in the test sample is substantially different from the level of the RNA in a subject who is responsive to Sigma-1 receptor therapy. <5> 1. A method of treating a subject in need of Sigma-1 receptor agonist therapy, the method comprising: a. obtaining or having obtained results of a test of a biological sample from said subject that detects the presence in said subject of at least one polymorphism selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S66Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_1543V, and combinations thereof; b. if the polymorphism is not present in said subject, administering to said subject a therapeutically effective amount of a Sigma-1 receptor agonist. <6> 1. A method of treating a subject in need of Sigma-1 receptor agonist therapy, the method comprising: a. obtaining or having obtained results of a test of a biological sample from said subject, determining a level of expression of RNA encoded by at least a gene selected from the group consisting of SIGMAR1, COMT, KANSL1, DHCR7, HLA-DRB1, MS4A6E, RIN3, DPYD, and combinations thereof; b. comparing the level of said RNA in said biological sample to the level of said RNA in a subject responsive to Sigma-1 receptor therapy; c. administering a therapeutically effective amount of a Sigma-1 receptor agonist to the subject if the level of the RNA in the biological sample is substantially similar to the level of the RNA in a subject responsive to Sigma-1 receptor therapy. <7> 1. A method for detecting the presence of a polymorphism associated with response to Sigma-1 receptor therapy in a subject, the method comprising: a. obtaining or having obtained a biological sample from said subject; b. detecting a pre-existing condition in the biological sample by assessing the sequence of the locus containing the polymorphism; and detecting the polymorphism, The method of claim 1, wherein the at least one polymorphism is selected from the group consisting of SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, DHCR7_M220T, HLA-DRB1_A244T, HLA-DRB1_S6Y, HLA-DRB1_Y89S, MS4A6E_M59T, RIN3_H215R, DPYD_l543V, and combinations thereof in the subject. <8> The at least one polymorphism is selected from SIGMAR1_Q2P, COMT_L146fs, KANSL1_P1010L / P304L / P946L, and combinations thereof; <1> , <3> , and <4> 2. The method according to claim 1 , <9> The polymorphism is SIGMAR1_Q2P; <1> , <2> , <5> 7>. A method according to any one of claims 1 to 6. <10> the agonist is selected from ANAVEX2-73, ANAVEX1-41, AV1066, ANAVEX3-71, PRE-084, donepezil, fluvoxamine, amitriptyline, L-687,384, SA-4503, dextromethorphan, dimethyltryptamine, (+)-pentazocine, and combinations thereof; <1> , <2> , <5> , and <7> 2. The method according to claim 1 , <11> The agonist is A2-73. <10> The method described above. <12> The therapeutically effective amount of A2-73 is in the range of about 0.5 to about 100 mg. <11> The method described above. <13> When administered orally, the therapeutically effective amount of A2-73 is in the range of about 20 to about 60 mg. <12> The method described above. <14> When administered intravenously, the therapeutically effective amount of A2-73 is about <1> ~<20mg range <12> The method described above. <15> The therapeutically effective amount of A2-73 is in the range of about 40 mg to about 60 mg per day. <12> The method described above. <16> The therapeutically effective amount provides a blood concentration of A2-73 of about 4 ng / mL. <12> The method described above. <17> and further comprising excluding said subject from Sigma-1 receptor agonist therapy if at least one polymorphism is present in said subject. <1> ~ <4> 2. The method according to claim 1 , <18> and administering to said subject an alternative to Sigma-1 receptor agonist therapy if at least one polymorphism is present in said subject. <6> 2. The method according to claim 1 , <19> The alternative to the Sigma-1 receptor agonist is galantamine, memantine, rivastigmine, or a combination thereof; <18> The method described above. <20> The subject is treated for a condition selected from the group consisting of neuroprotective, neurodegenerative, and neurodevelopmental conditions. <1> ~ <7> 2. The method according to claim 1 , <21> the condition is Alzheimer's disease or dementia; <20> The method described above.

Claims

[Claim 1] The invention described herein.

Images