WO2017031276A1 - Méthodes de fourniture d'une thérapie neuroprotectrice en lien avec l'administration d'un ligand du récepteur bêta des oestrogènes - Google Patents

Méthodes de fourniture d'une thérapie neuroprotectrice en lien avec l'administration d'un ligand du récepteur bêta des oestrogènes Download PDF

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WO2017031276A1
WO2017031276A1 PCT/US2016/047485 US2016047485W WO2017031276A1 WO 2017031276 A1 WO2017031276 A1 WO 2017031276A1 US 2016047485 W US2016047485 W US 2016047485W WO 2017031276 A1 WO2017031276 A1 WO 2017031276A1
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carboximidamide
hydroxy
dihydroxy
indole
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Rhonda R. Voskuhl
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University of California Berkeley
University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • MS Multiple sclerosis
  • brain and spinal cord central nervous system
  • MS affects more than 1 million people worldwide and is the most common neurological disease among young adults, particularly women. The exact cause of MS is still unknown.
  • MS is an autoimmune disease in which myelin sheaths surrounding neuronal axons are destroyed. This condition can cause weakness, impaired vision, loss of balance, and poor muscle coordination.
  • Relapsing-remitting MS is characterized by unpredictable relapses followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Deficits that occur during attacks may either resolve or leave sequelae, the latter in about 40% of attacks and being more common the longer a person has had the disease. This describes the initial course of 80% of individuals with MS. When deficits always resolve between attacks, this is sometimes referred to as benign MS, although people will still build up some degree of disability in the long term. On the other hand, the term malignant multiple sclerosis is used to describe people with MS having reached a significant level of disability in a short period of time.
  • the relapsing- remitting subtype usually begins with a clinically isolated syndrome (CIS).
  • CIS a person has an attack suggestive of demyelination but does not fulfill the criteria for multiple sclerosis; 30 to 70% of persons experiencing CIS go on to develop MS. Secondary-progressive MS occurs in around 65% of those with initial relapsing-remitting MS, who eventually have progressive neurologic decline between acute attacks without any definite periods of remission. Occasional relapses and minor remissions may appear. The median length of time between disease onset and conversion from relapsing-remitting to secondary progressive MS is 19 years.
  • Primary-progressive MS occurs in approximately 10-20% of individuals, with no remission after the initial symptoms. It is characterized by progression of disability from onset, with no, or only occasional and minor, remissions and improvements.
  • the usual age of onset for the primary progressive subtype is later than of the relapsing-remitting subtype, but similar to the age that secondary-progressive MS usually begins in relapsing-remitting MS, around 40 years of age.
  • the following agents are approved by the U.S. Food and Drug Administration (FDA) to reduce disease activity and disease progression for many people with relapsing forms of MS, including relapsing-remitting MS, as well as secondary-progressive and progressive-relapsing MS in those people who continue to have relapses: dimethyl fumarate (Tecfidera®), fingolimod (Gilenya®), glatiramer acetate (Copaxone®), interferon beta- la (Avonex® and Rebif®), interferon beta- lb (Betaseron® and Extavia®), peginterferon beta- la (Plegridy®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), alemtuzumab (Lemtrada®), and teriflunomide (Aubagio®).
  • FDA U.S. Food and Drug Administration
  • the invention relates to a method for treating multiple sclerosis in a subject who does not present with active lesions (e.g., gadolinium-enhancing lesions), comprising administering an estrogen receptor ⁇ ligand ("ERP ligand") to the subject without conjointly administering a second immunotherapeutic agent for treating multiple sclerosis.
  • active lesions e.g., gadolinium-enhancing lesions
  • the invention relates to a method for treating multiple sclerosis in a subject receiving treatment with an immunotherapeutic agent who does not present with active lesions (e.g., gadolinium-enhancing lesions), comprising administering an ERP ligand to the subject and discontinuing treatment with the immunotherapeutic agent.
  • an immunotherapeutic agent who does not present with active lesions (e.g., gadolinium-enhancing lesions)
  • administering an ERP ligand to the subject and discontinuing treatment with the immunotherapeutic agent.
  • the invention relates to a method for treating multiple sclerosis in a subject, comprising determining whether the brain of the subject presents with active lesions (e.g., gadolinium- enhancing lesions), administering an ERP ligand to the subject, and
  • active lesions e.g., gadolinium- enhancing lesions
  • Figure 1 Study overview.
  • Figure 1 includes two panels, identified as panels (A) and (B).
  • Panel A shows the disposition of subjects enrolled in a clinical trial of estriol for treating multiple sclerosis.
  • Panel B shows the study design.
  • "Taper” indicates a period of reduction of either estriol or placebo over the course of 4 weeks at end of study, after month 24 clinic visit. Specifically, the dose of estriol was reduced by half (from 8 mg to 4 mg) for 2 weeks, then reduced by half again (from 4 mg to 2 mg) for 2 weeks, then discontinued,
  • "x” indicates the administration of a progestin (0.7 mg norethindrone) orally each day for 2 weeks every three months, beginning at study month 6.
  • "o” indicates the administration of a placebo for the progestin orally each day for 2 weeks every three months, beginning at study month 6.
  • Figure 2 Estriol levels and relapsing disease activity in Estriol + glatiramer acetate (GA) as compared to Placebo + GA treatment groups.
  • Figure 2 includes three panels, identified as panels (A), (B), and (C).
  • Panel (A) shows that serum estriol concentrations are significantly increased at each time point after baseline (month 0) in the Estriol + GA group (-*-), while remaining below the assay detection limit in the Placebo + GA group (— ).
  • Estriol levels are expressed as mean ⁇ SE in ng/mL.
  • Figure 3 Disabilities and Brain Volumes.
  • Figure 3 includes nine panels, identified as panels (A), (B), (C), (D), (E), (F), (G), (H), and (I).
  • EDSS Expanded Disability Status Scale
  • MFIS Modified Fatigue Impact Scale
  • Figure 4 Trends for MS Quality of Life and Depression.
  • Figure 4 includes three panels, identified as panels (A), (B), and (C).
  • Panel (B) shows MSQOL composite scores for Mental outcomes had trends similar to MSQOL Physical.
  • BDI Beck Depression Inventory
  • Figure 5 Change in PASAT: Subgroups by Baseline Performance.
  • Figure 5 shows the percent change in PASAT scores at 12 months from baseline for all subjects (All, left bars), those with disability scores of less than 55 at baseline ( ⁇ 55, middle bars), and those with scores from 55 to the maximum of 60 at baseline (>55, right bars).
  • a perfect PASAT score is 60, and scores lower than 55 depict disability.
  • the estriol group displayed a significant benefit as assessed by PASAT scores, and the subgroup of estriol subjects with PASAT scores less than 55 at baseline displayed a significant benefit.
  • the data is expressed as mean % change ⁇ SE.
  • Figure 6 Voxel-wise Gray Matter Atrophy.
  • Figure 6 shows maximum intensity projections of voxel- wise gray matter atrophy superimposed onto 3 orthogonal planes through the brain.
  • significant localized gray matter loss was observed in the Placebo + GA group as compared to baseline (top) and in the Estriol + GA group as compared to baseline (middle), each shown in gray against a black background in the 3 planes.
  • Regions of significantly more gray matter loss in the Placebo + GA group as compared to the Estriol + GA group on between group comparisons are shown in white in the 3 planes (bottom).
  • Gray matter loss is also visualized as projected onto a surface rendering of the mean template (lower right corner of each panel). All results are corrected for multiple comparisons by controlling the FDR at P ⁇ 0.05.
  • FIG. 9 Rotarod times for female C57BL/6 mice that received high dose AC-186 treatments (30 mg/kg) in sesame oil versus control animals (Veh). No differences in AC-186 treatment using the sesame oil vehicle.
  • FIG. 10A EAE scores (Fig. 10A) and rotarod times (Fig. 10B) for a positive control DPN in the vehicle of 10% ethanol / miglyol solution.
  • Figure 11 EAE scores for male C57BL/6 mice that received high dose AC-186 treatments (30 mg/kg) in either sesame oil or miglyol or the vehicle alone. Miglyol carrier with AC-186 shows benefit while sesame oil carrier with AC-186 does not.
  • FIG. 1 Rotarod times for NOD males under various treatment conditions.
  • FIG. 1 EAE scores for C57BL/6 females under various treatment conditions.
  • FIG. 20 AC-186 post-treatment during EAE: effects on axonal densities, beta- APP, and myelin in spinal cord.
  • A top row
  • EAE mice were sacrificed at EAE day 60.
  • Axons and myelin were stained with NF200 and MBP, respectively (A, top three rows).
  • Representative lOxconfocal images of spinal cord sections were stained for axonal damage using beta-APP (A, bottom row).
  • the AC-186 lOmg/kg treatment group showed a trend of less beta-APP staining compared with the Vehicle treated EAE group, but the difference with this dose did not reach significance.
  • MBP staining showed that both AC- 186 treatment groups had a trend for somewhat higher MBP staining intensity as compared to vehicle but this did not reach statistical significance.
  • Four mice in each treatment group were examined for each treatment group, p- values were determined by one-way ANOVA.
  • FIG. 21 AC-186 post-treatment during EAE: effects on macrophages and T cells in spinal cord.
  • A top row
  • EAE mice were sacrificed at EAE day 60.
  • Immune cells in the CNS were stained with pan-leucocyte marker CD45. The tissues were counterstained by DAPI.
  • A middle row
  • A, bottom row Representative lOxconfocal images of spinal cord sections stained with CD3.
  • Vehicle treated EAE (Veh) as compared to matched healthy controls (Cont) showed significantly increased CD45 staining (left), increased Iba-1 globoid cells (middle), and increased CD3 cells (right).
  • Four mice in each treatment group or three mice for Veh and AC-186 1 Omg/kg groups in CD45 staining were examined for each treatment group, p-values were determined by one-way ANOVA.
  • FIG 22 EAE scores and rotarod times for female C57BL/6 mice who received 30 mg/kg AC-186 in miglyol for mice that were subject to MRI and neuropathologic analyses of cerebrum and cerebellum.
  • FIG. 23 Whole Brain, Cortical and Cerebellar Atrophy in EAE is Ameliorated by AC- 186 Treatment.
  • A A graph of the mean whole brain volume in healthy controls (data points with least decay in each graph), AC-186-treated mice with EAE (data points with steepest decay in each graph) and vehicle-treated mice with EAE (data points with intermediate decay in each graph) at dO, d30 and d60.
  • AC-186-treated EAE mice exhibit less brain atrophy than vehicle-treated EAE mice as early as d30.
  • B A graph of the mean cerebral cortex volume in healthy controls, AC-186-treated mice with EAE and vehicle-treated mice with EAE at dO, d30 and d60.
  • AC-186-treated EAE mice exhibit less atrophy in the cerebral cortex than vehicle- treated EAE mice by d60.
  • C A graph of the mean cerebellar volume in healthy controls, AC- 186-treated mice with EAE and vehicle-treated mice with EAE at dO, d30 and d60. AC- 186- treated EAE mice exhibit less cerebellar atrophy than vehicle-treated EAE mice by d60.
  • FIG. 24 AC-186 treatment protects against loss of cerebral and cerebellar neurons and synapses in gray matter.
  • FIG 25 AC-186 treatment during EAE: effects on axonal loss, axonal damage, and myelin in spinal cord.
  • EAE mice were sacrificed at the end of the experiments depicted in Figures 22 (clinical data) and 23 (MRI data). Quantification is shown of axonal densities by NF200 staining (left), beta-APP expression for axonal damage (middle), and myelin staining intensity by MBP (right).
  • the vehicle treated (Veh) group as compared to matched healthy controls (Cont) showed significantly reduced axon numbers (left), increased beta-APP (middle) and reduced myelin (right).
  • the AC-186 30mg/kg (AC186) treated EAE group showed significantly more axon numbers compared with the Vehicle (Veh) treated EAE group (p ⁇ 0.05, Veh vs AC-186 30mg/kg).
  • the beta-APP staining showed that the AC-186 30 mg/kg (AC186) treated group had significantly less expression of beta-APP compared with the Vehicle (Veh) treated EAE group (p ⁇ 0.04, Veh vs AC-186 30mg/kg).
  • MBP staining showed that the AC-186 treatment group had higher MBP staining intensity as compared to vehicle (p ⁇ 0.004, Veh vs AC- 186 30mg/kg).
  • Three to five mice were examined for each treatment group, p-values were determined by one-way ANOVA.
  • FIG 26 AC-186 treatment during EAE: effects on macrophages and T cells in spinal cord.
  • EAE mice were sacrificed at end of the experiments depicted in Figure 22 (clinical data) and 23 (MRI data). Quantification of CD45 immunoreactivity to quantify all immune cells (left), Iba-1 globoid to quantify macrophage like cells (middle), and CD3 to quantify T lymphocytes (right) was done. Vehicle treated EAE (Veh) as compared to matched healthy controls (Cont) showed significantly increased CD45 staining (left), increased Iba-1 globoid cells (middle), and increased CD3 cells (right).
  • Three to five mice were examined for each treatment group, p-values were determined by one-way ANOVA.
  • FIG. 27 AC-186 treatment during EAE: effects on cerebellar white matter.
  • EAE mice were sacrificed at end of the experiments depicted in Figures 22 (clinical data) and 23 (MRI data). Quantification is shown of myelin staining intensity by MBP (left) and axonal densities by NF200 staining (right).
  • the vehicle treated (Veh) group as compared to matched healthy controls (Cont) showed significantly reduced myelin (left) and reduced axon numbers (right).
  • MBP staining showed that the AC- 186 treated group had higher MBP staining intensity as compared to vehicle (p ⁇ 0.0176, Veh vs AC-186 30mg/kg) (left).
  • the AC-186 30mg/kg treated EAE group showed significantly more axon numbers compared with the Vehicle treated EAE group (p ⁇ 0.01, Veh vs AC-186 30mg/kg) (right). Three to five mice were examined for each treatment group, p-values were determined by one-way ANOVA.
  • FIG 28 AC-186 treatment during EAE: effects on cerebral white matter.
  • EAE mice were sacrificed at end of the experiments depicted in Figure 22 (clinical data) and 23 (MRI data). Quantification is shown of myelin staining intensity by MBP (left) and axonal densities by NF200 staining (right) in the splenium of the corpus callosum of the cerebrum.
  • Figure 29 consists of four panels, labeled panels A, B, C, and D.
  • Panel A shows that 30 mg/kg AC-186 administered every other day significantly improved EAE clinical severity scores and rotarod performance in female mice (p ⁇ 0.0001).
  • Panel B shows that 30 mg/kg AC-186 administered every other day significantly improved EAE clinical severity scores and rotarod performance in male mice (p ⁇ 0.0001).
  • Panel C shows that 30 mg/kg AC-186 administered every other day significantly reduced spinal cord white matter axonal loss, as evidenced by NF200 staining (p ⁇ 0.05), axonal damage, as evidenced by beta-APP staining (p ⁇ 0.05), and demyelination, as evidenced by MBP staining (p ⁇ 0.005) relative to vehicle-treated controls.
  • Panel D shows that 30 mg/kg AC-186 administered every other day significantly reduced inflammation, as evidenced by CD45 staining (p ⁇ 0.05), and macrophage/activated microglia, as evidenced by Iba-1 globoid staining (p ⁇ 0.05), but not T lymphocyte counts relative to vehicle- treated controls.
  • Some aspects of the invention are based on the finding that the estrogen receptor ⁇ ligand estriol protects gray matter (e.g., cortical gray matter) in subjects with relapsing-remitting multiple sclerosis who do not present with active lesions (see, e.g., Figure 3, Panel I).
  • gray matter e.g., cortical gray matter
  • ⁇ ligand therapies are efficacious for treating multiple disease states, not only relapsing- remitting multiple sclerosis, but also secondary progressive multiple sclerosis and primary progressive multiple sclerosis.
  • ⁇ ligand therapies are likely generally efficacious for treating various other forms of neurodegenerative disease that are not primarily autoimmune response driven.
  • the invention relates to a method for treating a neurodegenerative disease, such as multiple sclerosis, in a subject who does not present with active lesions ⁇ e.g., gadolinium-enhancing lesions), comprising administering an ⁇ ligand to the subject without conjointly administering a second immunotherapeutic agent.
  • a neurodegenerative disease such as multiple sclerosis
  • active lesions e.g., gadolinium-enhancing lesions
  • the invention relates to a method for treating a neurodegenerative disease, such as multiple sclerosis, in a subject receiving treatment with an immunotherapeutic agent who does not present with active lesions (e.g., gadolinium-enhancing lesions), comprising administering an ⁇ ligand to the subject and discontinuing treatment with the active lesions (e.g., gadolinium-enhancing lesions), comprising administering an ⁇ ligand to the subject and discontinuing treatment with the
  • the invention relates to a method for treating a neurodegenerative disease, such as multiple sclerosis, in a subject, comprising determining whether the brain of the subject presents with active lesions (e.g., gadolinium-enhancing lesions), administering an ⁇ ligand to the subject, and discontinuing any second immunotherapeutic agent that the subject is receiving if the subject lacks active lesions.
  • active lesions e.g., gadolinium-enhancing lesions
  • the invention relates to a method for treating a neurodegenerative disease, such as multiple sclerosis, in a subject, comprising administering an ⁇ ligand to the subject, determining whether the brain of the subject presents with active lesions, and conjointly administering to the subject a second immunotherapeutic agent if the brain of the subject presents with active lesions.
  • a neurodegenerative disease such as multiple sclerosis
  • active lesion refers to inflammation in the central nervous system associated with immune cells, such as T lymphocytes.
  • T lymphocytes may cross the blood-brain barrier in a relapsing-remitting multiple sclerosis patient.
  • T cells may mount an autoimmune response against myelin.
  • other blood cells may cross the blood-brain barrier, such as white blood cells, e.g., other lymphocytes and monocytes.
  • An active lesion may be detected, for example, by gadolinium- enhanced magnetic resonance imaging (MRI).
  • MRI gadolinium- enhanced magnetic resonance imaging
  • Gadolinium-based contrast agents generally do not cross the blood-brain barrier, and thus a gadolinium-enhanced MRI scan may be used to detect an active lesion, termed a "gadolinium-enhancing lesion” or simply an "enhancing lesion”. Additionally, active lesions may be diagnosed by other methods. For example, an active lesion may be detected by MRI without a gadolinium- based contrast agent, e.g., by identifying a new or enlarging T2 hyperintensity in the brain, relative to a previous MRI, indicating that the subject had a recent active lesion. Active lesions may also be identified by diagnosing a relapse in a multiple sclerosis patient ⁇ e.g., by significant worsening on a walking, balance, or visual acuity test that has an acute onset, such as over 1-7 days).
  • central nervous system is used herein as defined in the art and includes the brain and spinal cord.
  • brain is used synonymously with “central nervous system” and thus, unless otherwise apparent from context, the term “brain” includes both the brain as defined in the art and the spinal cord.
  • a subject presents with active lesions if the subject has been recently diagnosed (e.g., within the last two months, preferably within the last month) with at least one active lesion, e.g., by gadolinium-enhanced MRI.
  • a subject does not present with active lesions if the subject has not been recently diagnosed with any active lesions. For example, a subject does not present with active lesions if a gadolinium-enhanced MRI scan of the brain of the subject suggests that no gadolinium crossed the blood-brain barrier of the subject, e.g., relative to a control scan or control region of the brain. Additionally, a patient diagnosed with secondary progressive multiple sclerosis or primary progressive multiple sclerosis presents with few or no active lesions. Similarly, a subject with relapsing-remitting multiple sclerosis who is in clinical remission may or may not present with active lesions on an MRI scan.
  • Whether the brain of a subject presents with active lesions may be determined directly, e.g., by diagnosing the subject, or indirectly, by obtaining a prior diagnosis. For example, a physician may determine whether the brain of a subject presents with active lesions by identifying a breach in the blood-brain barrier of the subject, e.g., by gadolinium-enhanced MRI or by assaying the cerebrospinal fluid of the subject for a biomarker. Similarly, a physician may determine whether the brain of a subject presents with active lesions by diagnosing the subject with a relapse of multiple sclerosis, e.g., by identifying a new onset walking or other difficulty.
  • whether the brain of a subject presents with active lesions may be determined indirectly, e.g., by obtaining a diagnosis from a physician, technician, nurse, medical chart, MRI scan, or from the subject.
  • a doctor or other prescriber may administer an ERP ligand to a subject after determining whether the brain of a subject presents with active lesions by reviewing the medical records of the subject and/or discussing the medical history of the subject with the subject or another caretaker of the subject.
  • An active lesion may be, for example, an infratentorial lesion, juxtacortical lesion, periventricular lesion, or spinal cord lesion.
  • determining whether the brain of a subject presents with an active lesion comprises evaluating the cerebrospinal fluid of the subject, e.g. for immunoglobulin abnormalities (assessing the IgG Index in cerebrospinal fluid).
  • the subject may display evidence of cognitive decline.
  • the evidence of cognitive decline may be worsening performance on the Paced Auditory Serial Addition Test ("PASAT").
  • PASAT Paced Auditory Serial Addition Test
  • the subject may display evidence of brain atrophy.
  • the evidence of brain atrophy may be determined by MRI.
  • the brain atrophy may be cortical gray matter atrophy.
  • the brain atrophy may be a decrease in whole brain volume.
  • the subject may present with a cognitive disability.
  • the evidence of cognitive disability may be determined by performance on the Paced Auditory Serial Addition Test ("PASAT”) or on a Symbol Digit Modalities Test (SDMT).
  • PASAT Paced Auditory Serial Addition Test
  • SDMT Symbol Digit Modalities Test
  • the method includes the steps of administering an ERP ligand to a subject.
  • the ERP ligand may be administered on a continuous basis, e.g., daily.
  • the term "ERP ligand” as used herein refers to an estrogen receptor ⁇ agonist, including steroidal and non-steroidal agents that bind to and/or cause a change in activity or binding of the estrogen receptor ⁇ .
  • One agent useful in this invention is the ERP ligand known as AC- 186 (Compound I), or compounds substantially similar in structure and function thereto (such as those compounds disclosed in U.S. Patent Application Publication No's 2009/0131510 and 2014/0275284, and PCT Patent Application Publication No. WO 2014/125121, hereby incorporated by reference, especially for the molecules disclosed therein).
  • the ERP ligand is a compound selected from the compounds disclosed in U.S. Patent Application Publication No's 2012/0128435, 2012/0202853, 2012/0202861, 2013/0131061, or 2014/0323518 and PCT Patent Application Publication No's WO
  • the ERP ligand is KBRVl or KBRV2 (Karo Bio, Huddinge, Sweden).
  • the ERP ligand may be any one of the following compounds:
  • the ERP ligand is Compound II, III, or IV.
  • the ERP ligand may be 5-(3,5-dimethylisoxazol-4-yl)-N'-hydroxy-l-(4-hydroxyphenyl)- 3 -methyl- lH-pyrazole-4-carboximidamide; 5-(3,5-dimethylisoxazol-4-yl)-l-(4-hydroxyphenyl)- 3 -methyl- lH-pyrazole-4-carboxamide; 5-(3,5-dimethylisoxazol-4-yl)-l-(4-hydroxyphenyl)-3- methyl-lH-pyrazole-4-carbaldehyde oxime; 5-(3,5-dimethylisoxazol-4-yl)-l -(4-hydroxyphenyl)
  • the ERP ligand may be 2-(3,5-dimethylisoxazol-4-yl)-3-(4-hydroxyphenyl)-lH-indole-l carbonitrile; 2-(3,5-dimethylisoxazol-4-yl)-N'-hydroxy-3-(4-hydroxyphenyl)-lH-indole-l- carboximidamide; 2-(3,5-dimethylisoxazol-4-yl)-3-(4-hydroxyphenyl)-lH-indole-l- carboxamide; 2-(3,5-dimethylisoxazol-4-yl)-3-(4-hydroxyphenyl)-N,N-dimethyl-lH-indole-l- sulfonamide; 2-(3,5-dimethylisoxazol-4-yl)-3-(4-hydroxyphenyl)-lH-indole-l-carbaldehyde oxime; 4-(2-(3,5-dimethyl
  • the ERP ligand may be 2-(3,5-dimethylisoxazol-4-yl)-N'-hydroxy-3-(4-hydroxyphenyl)- 2,4,5,6- tetrahydrocyclopenta[c]pyrrole-l -carboximidamide; 2- (3,5-dimethylisoxazol-4-yl)-3-(2 fluoro-4-hydroxyphenyl)-N'-hydroxy-5,6- dihydrocyclopenta[b]pyrrole- 1 (4H)- carboximidamide; 3-(2,6-difluoro-4-hydroxyphenyl)-2-(3,5-dimethylisoxazol-4-yl)-N'-hydroxy- 5,6- dihydrocyclopenta[b]pyrrole- 1 (4H)-carboximidamide; 3- (2,5-difluoro-4-hydroxyphenyl)- 2-(3,5-dimethylisoxazol-4-yl)-N'-hydroxy-5,6-di
  • the ERP ligand may be N',4'-dihydroxy-2-(3-methylthiophen-2-yl)-5-propyl-[l,l'- biphenyl] -3 -carboximidamide; 2-(3,5-dimethylisoxazol-4-yl)-N',4'-dihydroxy-5-methyl-[l,l'- biphenyl] -3 -carboximidamide; 2-(3,5-dimethylisoxazol-4-yl)-N',4'-dihydroxy-5-propyl-[l,l'- biphenyl] -3 -carboximidamide; 2-(3,5-dimethylisoxazol-4-yl)-4'-hydroxy-5-propyl-[l,l '- biphenyl]-3-carboxamide; 2-(3,5-dimethylisoxazol-4-yl)-4'-hydroxy-5-propyl-[l,l '- biphenyl]-3-car
  • the ERP ligand may be 2-Bromo-l-(4-hydroxy-phenyl)-lH-indole-3-carbonitrile; l-(4- Hydroxy-phenyl)-2-thiophen-3-yl-lH-indole-3-carbonitrile; 2-(3-Cyano-furan-2-yl)-l-(4- hydroxy-phenyl)-lH-indole-3-carbonitrile; 1 -(4-Hydroxy-phenyl)-2-pyrrol-l -yl-lH-indole-3- carbonitrile; 2-Dimethylamino-l -(4-hydroxy-phenyl)-lH-indole-3-carbonitrile; 1 -(4-Hydroxy- phenyl)-2-isopropyl-lH-indole-3-carbonitrile; 2-Acetyl-l-(4-hydroxy-phenyl)-lH-indole-3
  • the ERP ligand is a compound selected from the ERP ligands disclosed in U.S. Patent No. 8,334,280, hereby incorporated by reference.
  • the ERP ligand is 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-l,3- benzoxazol-5-ol (ERB-041 ; Wyeth).
  • the ERP ligand may be a substituted benzoxazole, such as any of the compounds disclosed in U.S. Patent No. 6,794,403 or U.S. Patent Application
  • the ERP ligand may be 2-(5-hydroxy-l,3-benzoxazol-2-yl) benzene- 1,4-diol; 3-(5- hydroxy-l,3-benzoxazol-2-yl)benzene-l,2-diol; 2-(3-fluoro-4-hydroxyphenyl)-l,3-benzoxazol-5- ol; 2-(3-chloro-4-hydroxyphenyl)-l,3-benzoxazol-5-ol; 2-(2-chloro-4-hydroxyphenyl)-l,3- benzoxazol-5-ol; 2-(3-fluoro-4-hydroxyphenyl)-l ,3-benzoxazol-6-ol; 2-(3-tert-butyl-4- hy droxypheny 1)- 1 ,3 -benzoxazol-6-ol; 2-(6-hy droxy- 1 ,3 -benzoxazol-2-y
  • the ERP ligand may be 2-(3-fluoro-4-hydroxyphenyl)-l,3-benzoxazol-5-ol, 2-(3-chloro- 4-hydroxyphenyl)-l ,3-benzoxazol-5-ol, 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-l ,3-benzoxazol-5- ol, 2-(2-chloro-4-hydroxyphenyl)-l ,3-benzoxazol-5-ol, 2-(3-fluoro-4-hydroxyphenyl)-l ,3- benzoxazol-6-ol, 2-(3-tert-butyl-4-hydroxyphenyl)-l,3-benzoxazol-6-ol, 2-(3-chloro-4- hydroxyphenyl)-l ,3-benzoxazol-6-ol, 6-chloro-2-(3-fluoro-4-hydroxyphenyl)-l ,3-benzoxazol
  • the ERP ligand is a compound selected from the ERP ligands disclosed in U.S. Patent Application Publication No. 2007/0021495 or 2013/0274344, each of which is hereby incorporated by reference.
  • the ERP ligand is a halogen-substituted phenyl-2H-indazole, such as indazole chloride (see, e.g., Moore, S. M. et al. Proc. Nat'l Acad. Sci. USA 111(5): 18061-66 (2014), herby incorporated by reference).
  • the ERP ligand is not estriol.
  • the ERP ligand may be a non- steroidal compound. In some embodiments, the ERP ligand is not a steroid hormone.
  • Some ERP ligand receptors such as AC- 186, KBRVl, and KBRV2, do not require the co-administration of a progestogen.
  • the method does not comprise the co-administration of a progestogen, such as norethindrone, e.g., either periodically or continuously.
  • a progestogen such as norethindrone
  • the method does not comprise the co-administration of a gestagen or progestin.
  • a patient may not be taking any one of chlormadinone acetate, cyproterone acetate, desogestrel, dienogest, 5a-dihydroprogesterone, drospirenone (Yasmin®), ethinodiol acetate, ethynodiol diacetate, etonogestrel (Nexplanon®), gestodene, 17-hydroxyprogesterone, levonorgestrel (Alesse®), medroxyprogesterone acetate (17a-hydroxy-6a-methylprogesterone acetate; Provera®), megestrol, megestrol acetate (17a-acetoxy-6-dehydro-6- methylprogesterone), nestorone, nomegestrol acetate, norethindrone, norethindrone acetate (also known as norethisterone acetate), norethynodrel (Enovid®
  • the ERP ligand may be a compound having the structure of formula I, and it may be administered at a dose sufficient to achieve a mean blood concentration of the compound between 1 ng/ml and 1000 ng/ml.
  • the compound may be administered at a dose sufficient to achieve a mean blood concentration of the compound between 1 ng/ml and 1000 ng/ml or about 1-100 mg/kg/day.
  • the compound may be administered at a dose sufficient to achieve a mean blood concentration of the compound between 10 ng/ml and 50 ng/ml, between 25 ng/ml and 75 ng/ml, between 50 ng/ml and 100 ng/ml, between 75 ng/ml and 125 ng/ml, between 100 ng/ml and 150 ng/ml, between 125 ng/ml and 175 ng/ml, between 100 ng/ml and 200 ng/ml, between 150 ng/ml and 250 ng/ml, between 200 ng/ml and 300 ng/ml, between 250 ng/ml and 350 ng/ml, between 300 ng/ml and 400 ng/ml, between 350 ng/ml and 450 ng/ml, between 400 ng/ml and 500 ng/ml, between 450 ng/ml and 650 ng/ml, between 550 ng/ml and 750 ng/ml
  • the compound may be administered at a dose sufficient to achieve a mean blood concentration of the compound between 100 ng/ml and 200 ng/ml. In other embodiments, the compound may be administered at a dose sufficient to achieve a mean blood concentration of the compound between 10 ng/ml and 20 ng/ml. In some embodiments, the compound is administered at a dose sufficient to achieve a mean blood concentration of the compound between 10 ng/ml and 500 ng/ml.
  • the ERP ligand may be a compound having the structure of formula I, and it may be administered at a dose between 10 mg and 10 g per day, such as between 80 mg and 8000 mg per day, or between 200 mg and 2000 mg per day.
  • the ERP ligand may be a compound having the structure of formula I, and it may be administered at a dose of about 5 ⁇ g/kg per day to about 100 mg/kg/day, such as about 50 ⁇ g/kg per day to about 50 mg/kg/day or about 500 ⁇ g/kg per day to about 5 mg/kg/day.
  • the ERP ligand may be a compound having the structure of formula I, and it may be administered at a dose between 1 mg/kg and 100 mg/kg per day, such as between 4 mg/kg and 80 mg/kg per day, or between 10 mg/kg and 50 mg/kg per day.
  • the ERP ligand may be KBRVl or KBRV2, and it may be administered at a dose between 1 mg and 10 g per day, such as between 5 mg and 1000 mg per day, or between 10 mg and 500 mg per day.
  • the ERP ligand may be KBRVl or KBRV2, and it may be administered at a dose between 0.1 mg/kg and 100 mg/kg per day, such as between 0.5 mg/kg and 100 mg/kg per day, or between 1 mg/kg and 10 mg/kg per day.
  • an “effective amount,” as used herein, refers to an amount that is sufficient to achieve a desired biological effect.
  • a “therapeutically effective amount,” as used herein refers to an amount that is sufficient to achieve a desired therapeutic effect.
  • a therapeutically effective amount can refer to an amount that is sufficient to improve at least one sign or symptom of multiple sclerosis.
  • the dosage of the ERP ligand may be selected for an individual patient depending upon the route of administration, severity of disease, age and weight of the patient, other medications the patient is taking and other factors normally considered by the attending physician, when determining the individual regimen and dosage level as the most appropriate for a particular patient. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected. In vitro or in vivo assays can be employed to help identify optimal dosage ranges.
  • the therapeutically effective dose of the ERP ligand included in the dosage form is selected at least by considering the type of ERP ligand selected and the mode of administration.
  • the dosage form may include the ERP ligand in combination with other inert ingredients, including adjuvants and pharmaceutically acceptable carriers for the facilitation of dosage to the patient as known to those skilled in the pharmaceutical arts.
  • the dosage form may be any form suitable to cause the ERP ligand to enter into the tissues of the patient.
  • Pharmaceutically acceptable carriers can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
  • Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical excipients can include, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutically acceptable excipients are sterile when administered to a subject.
  • Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the dosage form of the ERP ligand is an oral preparation (liquid, tablet, capsule, caplet, or the like), which, when consumed results in elevated serum ERP ligand levels.
  • the oral preparation may comprise conventional carriers including diluents, binders, time-release agents, lubricants, and disintegrants.
  • the dosage form of the ERP ligand is a sublingual preparation, which results in elevated serum ERP ligand levels when consumed.
  • the dosage form of the ERP ligand may be provided in a topical preparation (lotion, cream, ointment, patch, or the like) for transdermal application.
  • the dosage form may be provided as a suppository or the like for transvaginal or transrectal application.
  • the dosage form may also allow for preparations to be applied subcutaneously, intravenously, intramuscularly, or via the respiratory system.
  • the ERP ligand is preferably administered to the subject on a continuous basis, e.g., for at least one treatment period.
  • a continuous basis is daily, i.e. , on consecutive days.
  • the ERP ligand may be administered to the subject for multiple treatment periods ⁇ e.g., multiple consecutive treatment periods), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, or 24 treatment periods.
  • a “treatment period” refers to a period of time during which a subject is receiving an ERP ligand, on a continuous or daily basis, for the purpose of treating a
  • each treatment period is at least 28 consecutive days, at least 56 consecutive days, at least 84 consecutive days, at least 112 consecutive days, at least 140 consecutive days, or at least 168 consecutive days.
  • each treatment period may be 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106
  • each treatment period is at least 4 consecutive weeks, at least 8 consecutive weeks, at least 12 consecutive weeks, at least 16 consecutive weeks, at least 20 consecutive weeks, or at least 24 consecutive weeks.
  • each treatment period may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 consecutive weeks.
  • each treatment period is at least one month, at least two consecutive months, at least three consecutive months, at least four consecutive months, at least five consecutive months, or at least six consecutive months.
  • each treatment period may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive months.
  • a given dose of each formulation can comprise one or more pills, tablets, capsules, or the like (i.e., unit doses).
  • an 800 mg dose of an ERP ligand can be administered as four 200 mg capsules.
  • a given dose of each formulation may comprise one or more tablets or lozenges (i.e., unit doses) or a volume of liquid (e.g., one or more drops) or a volume of spray (e.g., one or more spray pumps).
  • a given dose of any agent involves administration of more than a single unit dose, e.g., four 200 mg capsules of an ERP ligand
  • the individual unit doses can be administered at essentially the same time, or they can be administered at different times on a given day, provided the entire daily dose is administered within a single day.
  • four 200 mg capsules of an ERP ligand can be taken together essentially once a day, or they may be taken two at a time twice a day, or they may be taken one at a time four times a day.
  • Additional schedules are contemplated by the invention, again provided the entire daily dose is administered within a single day. While it may be preferable that the subject follow the same schedule from one day to the next, such is not required, once again provided the entire daily dose is administered within a single day.
  • subject refers to a living mammal and may be interchangeably used with the term "patient.”
  • the subject is a human.
  • a human subject is female, such as a woman.
  • the subject is a
  • the subject is a
  • the subject is a perimenopausal woman. In certain embodiments, the subject is a postmenopausal woman.
  • the subject may have multiple sclerosis.
  • the multiple sclerosis is relapsing-remitting multiple sclerosis.
  • the multiple sclerosis is secondary-progressive multiple sclerosis.
  • the multiple sclerosis is primary-progressive multiple sclerosis.
  • the multiple sclerosis is progressive-relapsing multiple sclerosis.
  • the subject has a mild form of any one of the foregoing subtypes of MS.
  • the subject has a moderate form of any one of the foregoing subtypes of MS.
  • the subject has an aggressive form of any one of the foregoing subtypes of MS.
  • the multiple sclerosis is, more accurately, so-called clinically isolated syndrome (CIS).
  • CIS clinically isolated syndrome
  • An ERP ligand can be used, in accordance with the invention, to prevent or delay the onset of relapsing-remitting MS in subjects having CIS.
  • the subject has radiologically isolated syndrome.
  • neurodegenerative diseases such as, by way of illustration, Alzheimer's disease, Parkinson's disease, stroke, amyotrophic lateral sclerosis, cerebellar ataxia, frontotemporal dementia, prion disease, Huntington's Disease, cerebral ischemia, idiopathic Morbus Parkinson, Parkinson syndrome, Morbus Alzheimers, cerebral dementia syndrome, infection- induced neurodegeneration disorders (e.g., AIDS-encephalopathy, Creutzfeld- Jakob disease, encephalopathies induced by rubiola and herpes viruses and borrelioses), metabolic- toxic neurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-, hypo- or
  • the neurodegenerative disease is multiple sclerosis.
  • the patient is a woman.
  • the patient is a premenopausal or perimenopausal woman.
  • the patient is a postmenopausal woman.
  • the various methods disclosed herein can be methods for improving walking, vision, balance, cognition, or other symptoms in a subject, such as a subject with multiple sclerosis, and/or methods for improving multiple sclerosis functional composite (MSFC), EDSS, or MSSS scores in a subject, such as a subject with multiple sclerosis.
  • MSFC multiple sclerosis functional composite
  • EDSS EDSS
  • MSSS scores in a subject, such as a subject with multiple sclerosis.
  • the methods of treatment disclosed herein include methods for stabilizing or improving disability in a patient, whereby the patient's disability score (as measured by either of these tests or another suitable test) after six months, one year, or two years of therapy is at least about 10%, at least about 25%, at least about 40%, at least about 50%, or even at least about 60% higher relative to a control patient not receiving the ERP ligand therapy (but otherwise receiving the same treatment as the ERP ligand-treated patient).
  • the patient's disability score (as measured by either of these tests or another suitable test) after six months, one year, or two years of therapy is within about 2% or within about 5% of an earlier assessment, or at least about 2%, at least about 5%, at least about at least about 10%, at least about 25%, at least about 40%, at least about 50%, or even at least about 60% higher than the earlier assessment.
  • the progression of a walking disability can be tested using a walking test, e.g., assessing the subject's performance on a 25-foot walk test at different points in time, such as at 0 months (baseline), 6 months, 1 year, and 2 years.
  • the walking test may be a distance test (e.g., a 25 foot walk test) or a timed test (e.g., a 6 minute walk test), for example, or another multiple sclerosis walk scale may be employed.
  • the subject if there is documented worsening in walking (takes more seconds) by 20 percent as compared to baseline (optionally if this worsening is confirmed on a subsequent walk test (e.g., 3 months later)), then the subject is deemed to have progressive worsening in walking.
  • the subject demonstrating the progressive walking disability commences treatment with ERP ligand.
  • the walking test may be repeated (e.g., at 1 year and/or 2 years from the start of ERP ligand treatment) to assess whether the ERP ligand treatment slowed or halted any further worsening in walking performance, e.g., as measured by the walking test.
  • Improvements in cognition outcomes associated with MS therapy can be assessed using the PAS AT (e.g., PAS AT 2 or PAS AT 3) or SDMT test, or alternatively the MS-COG test (see Erlanger et al., JNeuro Sci 340: 123-129 (2014)).
  • PAS AT e.g., PAS AT 2 or PAS AT 3
  • SDMT test e.g., SDMT test
  • MS-COG test See Erlanger et al., JNeuro Sci 340: 123-129 (2014).
  • the methods of treatment disclosed herein include methods for stabilizing or improving cognition in a patient, whereby the patient's cognition outcome after one year of therapy is at least about 2%, at least about 5%, at least about 10%, at least about 25%, at least about 40%, at least about 50%, or even at least about 60% higher relative to a control patient not receiving the ERP ligand therapy (but otherwise receiving the same treatment as the ERP ligand- treated patient), e.g., as measured by any of the preceding tests.
  • the patient's cognition outcome after six months, one year, or two years of therapy may be within about 2% or within about 5% of an earlier assessment, or at least about 2%, at least about 5%, at least about 10%, at least about 25%, at least about 40%, at least about 50%, or even at least about 60% higher than the earlier assessment, e.g., as measured by any of the preceding tests at different times.
  • Methods of treatment disclosed herein include methods for stabilizing and/or improving fatigue and/or depression in a patient.
  • the fatigue and/or depression of the patient after one year of therapy may be reduced by at least about 2%, at least about 5%, at least about 10%, at least about 25%, at least about 40%, at least about 50%, or even at least about 60% relative to a control patient not receiving the ERP ligand therapy (but otherwise receiving the same treatment as the ERP ligand-treated patient), e.g., as measured by a Modified Fatigue Impact Scale (MFIS), Beck Depression Inventory, MS Quality of Life score, or Patient-Reported Outcomes
  • MFIS Modified Fatigue Impact Scale
  • Beck Depression Inventory Beck Depression Inventory
  • MS Quality of Life score MS Quality of Life score
  • a subject who scores below 50 on PAS AT may be deemed to have cognitive disability.
  • the subject demonstrating the cognitive disability may commence treatment with ERP ligand.
  • the cognitive test may be repeated (e.g., at about six months from the start of ERP ligand treatment) to assess whether the ERP ligand treatment slowed or halted any further worsening in cognitive performance, e.g., as measured by the PAS AT test.
  • the patient's score may increase by at least 3 points over the course of six to twelve months of ERP ligand therapy.
  • any of these methods further includes the step of administering to the subject an immunotherapeutic agent, wherein the
  • immunotherapeutic agent is not an ERP ligand. That is, in certain embodiments the subject is administered, in addition to the ERP ligand, a second agent useful in the treatment of MS.
  • agents useful in the treatment of MS are, in general, immunotherapeutic agents. At least in connection with MS, such agents are sometimes referred to as disease-modifying therapies or disease-modifying therapeutics (DMTs).
  • ERP ligands may be used in patients who are at risk of developing a gynecological problem or cancer in which an estrogen receptor a agonist is contraindicated.
  • the patient may present with a history of breast cancer, ovarian cancer, and/or uterine cancer.
  • the subject may have a family history of breast cancer, ovarian cancer, and/or uterine cancer ⁇ e.g., a parent, sister, grandparent, aunt, or cousin of the subject may have had breast cancer, ovarian cancer, or uterine cancer).
  • the subject may be at risk of developing breast cancer, ovarian cancer, and/or uterine cancer.
  • immunotherapeutic agent refers to a compound, other than an ERP ligand as defined herein, with an objectively measurable effect on at least one aspect of the immune system or an immune response.
  • the immunotherapeutic agent is immunosuppressive, i.e., it exerts an objectively measurable inhibitory effect on at least one aspect of the immune system or an immune response.
  • the immunotherapeutic agent is immunosuppressive, i.e., it exerts an objectively measurable inhibitory effect on at least one aspect of the immune system or an immune response.
  • the immunotherapeutic agent is anti-inflammatory.
  • the immunotherapeutic agent is a small molecule (molecular weight less than or equal to about 1.5 kDa) pharmaceutical compound or composition.
  • the immunotherapeutic agent is a biological compound or composition, e.g., an antibody, peptide, nucleic acid, etc.
  • the immunotherapeutic agent is selected from dimethyl fumarate
  • Tecfidera®; BG-12 fingolimod (Gilenya®), glatiramer acetate (Copaxone®, for example "longer-lasting" 40 mg/ml or 20 mg/ml versions), interferon beta- la (Avonex® or Rebif®), interferon beta- lb (Betaseron® or Extavia®), peginterferon beta- la (Plegridy®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), alemtuzumab (Lemtrada®), and teriflunomide (Aubagio®), mycophenolate mofetil, paclitaxel, cyclosporine, corticosteroids (e.g., prednisone, methylprednisolone), azathioprine, cyclophosphamide, methotrexate, cladribine, 4- aminopyridine, and tizanidine.
  • the immunotherapeutic agent is selected from dimethyl fumarate (Tecfidera®; BG-12), fingolimod (Gilenya®), glatiramer acetate (Copaxone®), interferon beta- la (Avonex® or Rebif®), interferon beta- lb (Betaseron® or Extavia®), peginterferon beta- la (Plegridy®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), alemtuzumab (Lemtrada®), and teriflunomide (Aubagio®).
  • the immunotherapeutic agent is not mitoxantrone (Novantrone®).
  • the immunotherapeutic agent is not glatiramer acetate (Copaxone®).
  • the immunotherapeutic agent is dimethyl fumarate (Tecfidera®; BG-12). In certain embodiments, the immunotherapeutic agent is fingolimod (Gilenya®). In certain embodiments, the immunotherapeutic agent is glatiramer acetate (Copaxone®). In certain embodiments, the immunotherapeutic agent is interferon beta- la (Avonex® or Rebif®). In certain embodiments, the immunotherapeutic agent is interferon beta- lb (Betaseron® or Extavia®). In certain embodiments, the immunotherapeutic agent is peginterferon beta- la (Plegridy®). In certain embodiments, the immunotherapeutic agent is mitoxantrone
  • the immunotherapeutic agent is natalizumab
  • the immunotherapeutic agent is alemtuzumab
  • the immunotherapeutic agent is teriflunomide
  • the subject is receiving treatment with an immunotherapeutic agent.
  • the method may comprise discontinuing treatment with the immunotherapeutic agent, e.g., if the central nervous system (i.e., brain) of the subject does not present with active lesions, e.g., gadolinium-enhancing lesions.
  • the method may comprise discontinuing treatment with the immunotherapeutic agent if no active lesions (e.g., gadolinium-enhancing lesions) have been detected in the central nervous system (i.e., brain) of the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, or 52 weeks.
  • active lesions e.g., gadolinium-enhancing lesions
  • the method may comprise discontinuing treatment with the immunotherapeutic agent if no active lesions (e.g., gadolinium-enhancing lesions) have been detected in the central nervous system (i.e., brain) of the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months.
  • the method may comprise discontinuing treatment with the immunotherapeutic agent if no active lesions (e.g., gadolinium-enhancing lesions) have been detected in the central nervous system (i.e. , brain) of the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the method comprises administering an ERP ligand to the subject without conjointly administering an immunotherapeutic agent to the subject.
  • the method may comprise administering an ERP ligand to the subject without conjointly administering an immunotherapeutic agent to the subject, for example, if no active lesions (e.g., gadolinium- enhancing lesions) have been detected in the central nervous system (i.e., brain) of the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, or 52 weeks.
  • active lesions e.g., gadolinium- enhancing lesions
  • the method may comprise administering an ERP ligand to the subject without conjointly administering an immunotherapeutic agent to the subject, for example, if no active lesions (e.g., gadolinium-enhancing lesions) have been detected in the central nervous system (i.e., brain) of the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months.
  • the method may comprise administering an ERP ligand to the subject without conjointly administering an immunotherapeutic agent to the subject, for example, if no active lesions (e.g., gadolinium-enhancing lesions) have been detected in the central nervous system (i.e., brain) of the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • This example describes a randomized, double-blind, placebo-controlled human clinical trial for the treatment of multiple sclerosis using glatiramer acetate (GA) and estriol.
  • GA glatiramer acetate
  • Eligible patients were females, an age of 18-50 years, a diagnosis of relapsing remitting multiple sclerosis as defined according to the McDonald criteria (Polman C. et al, Neurology 64:987 (200)), a baseline score of 0 to 4.5 on the Expanded Disability Status Scale (EDSS, which ranges from 0 to 10, with higher scores indicating more severe disability), and disease activity as evidenced by at least two documented relapses in the previous 24 months before screening or as evidenced by at least one documented relapse within 24 months before screening with a history of at least one gadolinium-enhancing lesion on a brain or cord magnetic resonance imaging (MRI) scan performed at least 3 months before or 3 months after the clinical relapse.
  • MRI magnetic resonance imaging
  • adrenocorticotropic hormone (ACTH)
  • corticosteroids corticosteroids
  • intravenous immunoglobulins or other listed MS treatments within 2 months before screening, those who were pregnant, breastfeeding, or trying to get pregnant, those not willing to discontinue other hormonal treatments, those who underwent surgical or natural menopause for longer than 1 or 3 years, respectively, with no hormone replacement therapy, and those who had ever been treated with a major
  • Gadolinium-enhancing lesions number 1.0 ⁇ 2.3 0.9 ⁇ 2.0 Active lesions on brain MRI - no. (%)
  • Patients may have received more than one prior multiple sclerosis medication. Patients may have received other non-approved therapies for multiple sclerosis before enrollment in the study. The percentage of patients receiving medication for multiple sclerosis before study entry was balanced across treatment groups.
  • H Scores on the Expanded Disability Status Scale ranged from 0 to 10, with higher scores indicating a greater degree of disability.
  • One patient in the Estriol + GA group did not have a confirmed relapse within 24 months prior to randomization, with enrollment based on disease activity evidenced by MRI enhancing lesions.
  • Standardized neurologic assessments including an EDSS assessment, were performed at months 0, 3, 6, 12, 18 and 24, and at the time of a suspected relapse (as an additional unscheduled visit).
  • EDSS assessments were performed by physicians who were trained either by in-person training or online (www.Neurostatus.net). MRI scans were obtained at screening and at months 0, 3, 6, 12 and 24. Subjects were seen or contacted every 3 months for compliance assessments and for dispensing medications.
  • the primary efficacy end point was the annualized relapse rate.
  • a relapse was defined as the appearance of new neurological symptoms or the worsening of pre-existing symptoms, lasting at least 48 hours in a subject who had been neurologically stable or improving in the previous 30 days, accompanied by an objective change in a neurological examination ⁇ i.e., a worsening of 0.5 or more points on the EDSS or a worsening by 1.0 or more points on the pyramidal, cerebellar, brainstem or visual functional system scores, not due to fatigue alone and not associated with fever or infection).
  • the treating physician made the decision concerning whether the relapse criteria had been met, incorporating whether a change in EDSS had been documented by the examining physician. Both treating and examining physicians were unaware of study group assignments.
  • the standard treatment for relapse was a 3-5 day course of glucocorticoids at the discretion of the treating neurologist.
  • Secondary efficacy end points included the proportion of subjects with a relapse over all 24 months, the proportion of subjects with positive MRI scans for gadolinium enhancing lesions, a change in PASAT cognitive testing, a sustained improvement in PASAT cognitive testing (as defined by an increase of at least 3 points sustained over at least 6 months), a change in EDSS scores from baseline, disability progression (as defined by an increase in EDSS of at least 1.0 point in subjects with a baseline score of 1.0 or higher, or by an increase of at least a 1.5 points in subjects with a baseline score of 0, each sustained for at least 6 months).
  • Tertiary end points included gray matter atrophy on MRI, and changes in results from baseline on questionnaires including the Modified Fatigue Impact Scale, Beck Depression Inventory, and MS Quality of Life.
  • Safety and Adverse Events Safety assessments including clinical, blood laboratory safety testing and assessments of estriol levels, occurred at months 0, 3, 6, 12, 18, and 24.
  • blood tests included complete blood count (CBC) with differential and platelets; chemistry panel including sodium, potassium, creatinine, BUN, glucose, total protein, albumin, bilirubin (total), alkaline phosphatase, AST (SGOT), and ALT (SGPT), and lipid profile (HDL, LDL and triglycerides, cholesterol.
  • CBC complete blood count
  • chemistry panel including sodium, potassium, creatinine, BUN, glucose, total protein, albumin, bilirubin (total), alkaline phosphatase, AST (SGOT), and ALT (SGPT), and lipid profile (HDL, LDL and triglycerides, cholesterol.
  • Gynecologic exams were done at month 0, 6, 18 and at month 24 exit, with uterine ultrasounds at months 6, 18 and at month 24 exit. Mammograms were done in screening and at month 24 exit. Adverse event analysis was based on the percentage of patients who discontinued the study and the percentage of patients who discontinued the study possibly due to adverse events.
  • the sample size was determined based on the primary end point of annualized relapse rate. A total sample of 150 eligible patients would provide approximately 80% power at a two- sided significance level of 0.10 for this phase II clinical trial to detect the difference in the annualized relapse rate of 0.76 versus 1.18 for Estriol plus GA group and the Placebo plus GA group in 2 years.
  • Intention-to-treat analyses were carried out for all end points.
  • a negative binomial regression model was used to compare both 12 months and 24 months annualized relapse rates between Estriol + GA versus Placebo + GA groups adjusted for covariates.
  • a sequential testing procedure was applied to control the overall type I error.
  • a hierarchical statistical approach was used whereby results in the first 12 months of treatment would be assessed, and, if and only if, significance were met, results in the entire 24 months of treatment would be assessed.
  • the earlier timepoint was compared first since GA requires time to reach full efficacy, potentially providing a greater window to detect efficacy 12 months after initiation of GA and study drug treatment. Consistent with a phase 2 study using a clinical outcome, a p-value ⁇ 0.10 was considered statistically significant.
  • proportional hazards model was used to compare the time to relapse free probabilities between two groups adjusting for covariates.
  • the fixed effects include treatment groups (Estriol + GA vs Placebo + GA), baseline lesion number, age, and baseline EDSS score.
  • the random effect of subject is included in the model to account for within subject correlation.
  • Mixed effects negative binomial regression model and linear mixed effects model were used to compare enhancing lesion volume (log-transformed) between treatment groups at all follow-ups, and mixed effects logistic model was used to compare the number of subjects positive for gadolinium enhancing lesions.
  • Linear mixed effects model was carried out to compare the percent change in whole gray matter and cortical gray matter between treatment groups. For the exploratory endpoints of EDSS, PASAT, fatigue, depression, quality of life and brain volume measures, linear mixed effects model was used to compare treatment groups at 12 and 24 months.
  • Mixed effects models were used to assess the association among outcomes and estriol levels at all follow-ups and using subjects in both treatment groups.
  • Mixed effects logistic regression model was used to evaluate the association between the number of enhancing lesions and the occurrence of relapse at all follow-up intervals.
  • Linear mixed effects model was carried out to evaluate the association between PASAT change and percent brain volume change, as well as between PASAT change and estriol levels.
  • the pattern mixture model provides the analysis with the possibility of non-random dropout.
  • the missing data were sequentially imputed by the follow up time and the imputation model assumed that the treatment effect for patients after drop out is the same as taking placebo.
  • the sample size of 150 patients was used to provide approximately 80% power detect a one third reduction in relapse rates in Estriol + GA compared to Placebo + GA at a two-sided significance level of 0.10 for this phase 2 clinical trial to detect a difference in annualized relapse rates with an estimated rate of 0.75 versus 1.18 for Estriol + GA versus Placebo + GA, respectively, in 2 years.
  • Serum total estriol levels are expressed as means ⁇ SE in units of ng/rriL. Free estriol levels were also measured and followed a similar pattern of change within individuals as total levels, with absolute free levels a fraction of the magnitude of the absolute total levels as expected.
  • the primary outcome measure for efficacy was annualized relapse rate including all subjects on an intent-to-treat basis.
  • Plus-minus values are means ⁇ SD.
  • CI denotes confidence interval, E+GA for Estriol+GA , and P+GA for Placebo+GA.
  • Relapse rate ratio was estimated using negative binomial regression with adjustment for age, baseline EDSS ( ⁇ 2 vs. >2), number of relapse 12 months prior study entry (0-1 vs. >1), MS duration ( ⁇ 1 vs. >1 year), prior GA treatment (never vs. past/current), and prior interferon treatment (yes vs. no). % Values were calculated using the Kaplan-Meier product-limit method. Progression defined as EDSS increase of at least 1.0 point in subjects with baseline score of 1.0 or higher or increase of at least 1.5 points with baseline score of 0, each sustained for at least 6 months. Hazard ratio was estimated using Cox proportional hazard regression. For relapse, age, baseline EDSS ( ⁇ 2 vs.
  • EDSS Expanded Disability Status Scale
  • P+GA indicates Placebo+GA.
  • J Values were calculated using the Kaplan-Meier product-limit method. Progression defined as EDSS increase of at least 1.0 point in subjects with baseline score of 1.0 or higher or increase of at least 1.5 points with baseline score of 0, each sustained for at least 6 months.
  • MSFC Functional Composite
  • PASAT Auditory Serial Addition Test
  • Mass SIX iss 0.10 ⁇ 0.35 0.00.;: 0.42 s «er « - .4 f»*ile*ts *
  • VBM voxel-based morphometry
  • Patk « &h etOtaacteg ksloas at h &m
  • linear mixed effects model for the difference of the two groups means, adjusted for baseline volume and enhancing lesions present or absent.
  • Plus-minus values are means ⁇ SD.
  • CI denotes confidence interval
  • E+GA indicates Estriol+GA
  • P+GA indicates Placebo+GA.
  • Estriol plus GA was found to be safe and well tolerated with regard to adverse events including gynecological outcomes (Table 8). Regarding adverse events, irregular menses occurred more with Estriol + GA (P ⁇ 0.001), while vaginal infections occurred more with Placebo + GA (P ⁇ 0.05), with no increase in discontinuations due to either.
  • Migraine headache related eye pain 1 [ i, i%] 0
  • Pace maker implantation 1 [ i, i%] 0
  • the main analysis seeks to compare the relapse event rate between treatment groups based on the negative binomial regression.
  • recurrent events analysis was performed based on Andersen Gill model to compare the relapse hazard rate between treatment groups. Both analyses showed similar results, and significant and meaningful reduction in relapse rates was found in the Estriol plus GA group as compared to the Placebo plus GA group.
  • Misskg data msl sis for some se ondar d oixtis - Com anag the difference ofmeaas betweea the two stsdy roups at Mosfhs i 2 and 24
  • PASAT3 score 12 1,5 (-0,1. 4.3), FTM 0.06 1.6 (0.0, 3,2), P » 0,05
  • ⁇ Values were calculated based on liaear mixed effect model adjusted ibr baseliae volume and mhancing lesions (present vs. absent).
  • MRI scans were performed at 0, 3, 6, 12 and 24 months using a standardized protocol implemented at each site that consisted of the following: Tl -weighted 3D volume, pre and post contrast: TR2200, TE3.4, Tl 900, 176 slices, 1mm 3 .
  • Dual-echo fast spin echo TR10000, TE12/95, 50 slices, 1x1x3mm.
  • MRI data in Dicom format were fully anonymized prior to transfer and then uploaded to the central MRI reading center database.
  • each site Prior to study onset, each site provided a dummy scan utilizing the standardized sequences for review by the central MRI reading center to verify scan quality and fidelity. Quality control was maintained at each site using standard procedures for clinical scanners (daily phantoms, stability testing). Quarterly phantoms were collected from 12 of the 15 sites, most using the standard American College of Radiology (ACR) phatom. One site upgraded from a Siemens 1.5T to a 3.0T in November 2013, resulting in the acquisition of one month 24 scan on the new scanner. One site upgraded from a Phillips Achieva 3.0T to a Pillips Intera 3.0T after the first subject completed month 24. All subsequent studies were performed on the Intera.
  • ACR American College of Radiology
  • T2 lesion areas were determined using a semi-automated intensity based segmentation procedure by a trained, experienced researcher verified by a single investigator (NLS).
  • MRI brain whole gray matter, whole white matter and cortical gray matter volumes were determined using a pairwise Jacobian integration (PJI) method.
  • Pre-processing for structural Tl- weighted images included 1) N3 non- uniformity correction, 2) histogram-based intensity normalization, 3) linear standard space registration using ICBM 2009c nonlinear symmetric template, 4) patch-based brain extraction, and 5) lesion-inpainting.
  • Inputs to PJI were a pair of baseline and follow-up pre-processed structural Tl -weighed images.
  • the PJI consisted of 1) linear skull-constrained symmetric registration, 2) halfway transformation and resampling, 3) nonlinear symmetric registration using ANTS, and 4) voxel wise Jacobian determinant calculation on the warp field.
  • Whole gray matter and whole white matter tissue masks were generated by SPM8 Segment function. Additional nonlocal means denoising was applied.
  • For whole brain tissue masks, the whole gray matter and whole white matter masks were combined.
  • cortical gray matter mask a standard cortical mask was nonlinearly transformed and merged with gray matter mask.
  • the standard template was the ICBM (ICBM 2009c nonlinear symmetric version), and the nonlinear registration was performed by ANTS.
  • the Jacobian determinants were averaged within the masks for percent volume change in cortical gray matter, whole gray matter, whole white matter, and whole brain.
  • VBM Voxel-based morphometry
  • Brain images were preprocessed utilizing SPM8 and the VBM8 toolbox.
  • White matter lesions were in-painted to minimize their impact based on manual delineations that were used for the analysis of new T2 lesions.
  • these manually delineated lesion masks were coregistered to the Tl -weighted images, corrected if necessary, and used for lesion in-painting as described by Chard et al. (J. Magn. Reson. Imaging 34:223 (2010)).
  • the lesion in-painted images were subsequently realigned for each subject using halfway-registrations and corrected for bias-field inhomogeneities.
  • the realigned, bias corrected images were then tissue-classified into gray matter, white matter, and cerebrospinal fluid and registered to MNI space through linear and non-linearly transformations (see http://dbm.neuro.uni-jena.de/vbm8/VBM8-
  • tissue classification was based on maximum a posteriori segmentations, accounted for partial volume effects, and was refined by applying a spatially adaptive non-local means denoising filter as well as a hidden Markov random field model.
  • the estrogen receptor ⁇ ligand AC-186 was dissolved in either Miglyol 812N liquid oil (Sasol North America) or sesame oil (Sigma Aldrich) as following concentration; 1.5 mg/mL for 3 mg/kg group, 5 mg/mL for 10 mg/kg group, and 15 mg/mL for 30 mg/kg group.
  • the estrogen receptor ⁇ ligand diary lpropionitrile (DPN, Tocris Biosciences) was dissolved in 10% molecular-grade ethanol and diluted with 90% of either Miglyol 812N liquid oil or sesame oil. EAE and Treatments.
  • Rotarod Testing Motor behavior was tested up to two times per week for each mouse using a rotarod apparatus (Med Associates Inc., St. Albans, VT). Briefly, animals were placed on a rotating horizontal cylinder for a maximum of 200 seconds. The amount of time the mouse remained walking on the cylinder, without falling, was recorded. Each mouse was tested on a speed of 3-30 rpm and given three trials for any given day. The three trials were averaged to report a single value for an individual mouse, and then averages were calculated for all animals within a given treatment group.
  • mice were exposed to a lethal dose of isoflurane and perfused transcardially with ice-cold 1 * PBS for 8-15 min, followed by 10% formalin for 10-15 min.
  • Spinal cords and brains were dissected and submerged in 10% formalin overnight at 4°C, followed by 30% sucrose in PBS for 24 h.
  • Tissues were embedded in 75% gelatin/15% sucrose solution for cryostat sectioning then post-fixed overnight in 10% formalin and cryoprotected in 30% sucrose. The embedded tissues were stored in - 80 °C after flash frozen in dry ice.
  • tissue sections Prior to histological staining, 40-mm thick free-floating sections were thoroughly washed with IX PBS to remove residual sodium azide.
  • tissue sections were processed with an additional 1 h incubation with 5% glacial acetic acid in 100-proof ethanol at room temperature (RT). After washing tissue sections were permeabilized with 0.3% TritonX-100 and 2% normal goat serum in IX PBS for 30 min at RT and blocked with 10% normal goat serum in IX PBS for 1 hr. Tissues were then incubated with primary antibodies overnight in 4 °C.
  • Rat anti- MBP (Millipore) at 1 : 1000 dilutions
  • Rabbit anti-NF200 (Sigma Aldrich) at 1 :750 dilutions
  • Rabbit anti-beta- APP (Life Technologies) at 1 :200 dilutions
  • Mouse anti-NeuN at 1 : 1000 dilutions (Millipore)
  • Rabbit anti-PSD95 and Rabbit anti-Synapsinl at 1 :500 dilutions (Millipore)
  • Rat anti-CD45 at 1 : 1500 dilutions (Millipore).
  • Tissue sections were thoroughly washed with lx PBS to remove residual sodium azide and treated with 3% hydrogen peroxide for 30 min at RT and then simultaneously blocked with 10% NGS and permeabilized with 0.3% Triton X-100 in lx PBS for 1 h at room temperature. Tissues were then incubated with primary antibodies overnight in 4 °C. The following primary antibodies were used: Rat anti-CD3 at 1 :2000 dilutions (BD Pharmigen), anti-Calbindin D28K at 1 : 1000 dilutions (Millipore), and Rabbit anti-Ibal at 1 : 10000 dilutions (Wako Chemicals), were added for 2 hour at RT, and then placed in 4°C overnight.
  • Tissue sections then followed with secondary Ab labeling at 1 : 1000 dilutions (Vector labs) for 1 h at room temperature and then with Avidin-Biotin Conjugation solution (Vector Labs) for 1 hour at RT. Tissue sections were treated with DAB peroxidase substrate (Vector labs) according to manufacturer instructions. IgG-control experiments were performed for all primary Ab, and only non-immunoreactive tissues under these conditions were analyzed.
  • Cerebellar Purkinje (Calbindin + ) cells were manually counted using a brightfield lOx microscope over the entire sagittal cerebellum. PSD-95 and Synapsinl density was measured and reported as a percentage of the sampled area.
  • CD45 + , CD3 + , and Ibal + cells in spinal cord cross-sections were manually quantified under either of a confocal lOx microscope for CD45 + cells or a brightfield lOx microscope for CD3 + and Ibal + cells.
  • MRI Acquisition Mice were anesthetized with isofluorane and their heads secured with bite and ear bars. Respiration rate was monitored and the mice were maintained at 37° C using a circulating water pump.
  • In vivo magnetic resonance imaging was performed using a 200 mm horizontal bore 7.0 T Bruker imaging spectrometer with a micro-imaging gradient insert with a maximum gradient strength of 100 G/cm and 30 mm birdcage RF coil (Bruker Instruments, Billerica, MA). An actively decoupled quadrature surface coil array was used for signal reception and a 72-mm birdcage coil was used for transmission. Images were acquired and reconstructed using ParaVision 5.1 software.
  • RARE relaxation enhancement
  • MRI Analysis Images were skull-stripped using the Brain Surface Extractor (BSE) and residual non-brain signal was removed by a single operator manually editing the masks using BrainSuite 11a and bias-field inhomogeneities removed using the N3 correction. After inhomogeneity correction, a minimum deformation atlas (MDA) was produced. Images were spatially and intensity normalized to the MDA using a rigid-body transformation and an intensity rescaling cost function in Alignlinear (AIR). This process permits the comparison of images in a standard space correcting for both gross positional and intensity differences, yet preserving anatomically significant local changes. Following creation of this atlas, cerebral cortices and cerebella were manually labeled on the atlas.
  • BSE Brain Surface Extractor
  • AIR intensity rescaling cost function in Alignlinear
  • the labels were then warped onto the individual spatially normalized images to produce standardized estimates of gray matter volumes in individual subjects. All automated image processing was performed using the LONI Pipeline Processing Environment on an 8-processor core Mac Pro computer (Apple, Cupertino, CA).
  • Cerebral cortex and cerebellum labels were based on the Mouse Atlas Project 2003 mouse brain atlas. For clarity and consistency, the cerebral cortex label was bounded ventrally by the plane inferior to the most anterior point of the corpus callosum at midline. Importantly, this label contained the somatosensory regions (primary and secondary) and the motor cortex (primary and secondary). Additional anatomical information was obtained from the Franklin and Paxinos mouse brain atlas (Franklin and Paxinos, 2008).
  • ERp ligands protect cerebellar function
  • Three doses of the AC- 186 compound were tested in C57BL/6 female mice: low (3mg/kg, every other day), medium (10 mg/kg, every other day), and high (30 mg/kg, every other day), each injected subcutaneously in a sesame oil vehicle.
  • the ERP ligand diarylpropionitrile (DPN) was tested as a positive control because DPN can partially ameliorate EAE and because the treatment effects in EAE experiments can vary based on the level of disease severity within an experiment. Direct comparisons between treatments were only made between treatment groups within a single EAE experiment and not between treatment groups of different EAE experiments.
  • FIG. 7 shows graphs in which the vehicle consisted of sesame oil for AC- 186 (all doses) and vehicle, while the DPN vehicle was 10% ethanol in sesame oil.
  • the difference in improvement between the 30 mg/kg AC-186 group and all other groups increased with time.
  • Rotarod performance is likely more aligned with coordination and cerebellar function than standard EAE scores, which reflect principally walking and spinal cord pathology. No significant effect was observed for mice receiving 30 mg/kg AC-186 in sesame oil relative to those receiving vehicle only; however, very late in disease, at the time when ERP ligands are known to start working, the performance curves trended toward divergence, with AC-186 treated mice trending toward improved performance. Further, the rotarod test is insensitive in detecting differences when the vehicle group performs well, and the vehicle group performed well in this case, staying on the rotarod for approximately 150 seconds. Additionally, during the final two time-points, on days 48 and 50, the AC-186 group performed perfectly during the 200 second test, and thus, a significant effect may have been masked by the experimental design.
  • DPN protects against EAE better when administered in a Miglyol vehicle rather than in a sesame oil vehicle, and thus, the effect of the choice of vehicle on the efficacy of AC-186 was assessed. Accordingly, the AC-186 compound was assessed in C57BL/6 males using sesame oil and miglyol as vehicles. Male mice receiving AC-186 administered at 30 mg/kg in miglyol performed significantly better than mice receiving miglyol alone as assessed by both EAE score (figure 11) and rotarod performance (figure 12). In contrast, male mice receiving AC-186 administered at 30 mg/kg in sesame oil did not perform significantly better than mice receiving sesame oil alone as assessed by EAE score (figure 11) and rotarod performance (figure 12).
  • AC-186 solution dissolved more rapidly in miglyol than sesame oil, and miglyol could dissolve AC-186 by merely pipetting for 30 seconds.
  • AC-186 required mixing/nutating for 5 minutes to dissolve the compound in sesame oil.
  • Sesame oil has been shown previously to have some nonspecific immunostimulatory effects, and thus is not most commonly used as a vehicle in EAE. As shown in figure 13, the different vehicle type does not affect EAE differentially when given without an ERP ligand. Rather, the different vehicle type likely affects the ability of a given ERP ligand to protect in EAE, with Miglyol enabling better EAE protection than sesame oil when either DPN or AC-186 are administered.
  • AC-186 was assessed in female NOD mice using a MOG-induced EAE model.
  • the efficacy of AC-186 was tested in miglyol and sesame oil vehicles.
  • AC-186 improved EAE scores relative to vehicle only for mice receiving 10 mg/kg or 30 mg/kg in miglyol (p ⁇ 0.001), and the 30mg/kg group trended toward increased efficacy relative to the 10 mg/kg group (figure 14).
  • the positive control DPN which had not previously been tested in the NOD EAE model, also significantly ameliorated EAE, appearing similar to the disease reduction observed with the 30 mg/kg dose of AC-186.
  • AC-186 was assessed in male NOD mice using a MOG-induced EAE model.
  • the efficacy of AC-186 was tested in miglyol vehicle only.
  • AC-186 improved EAE scores relative to vehicle only for mice receiving 30 mg/kg in miglyol (p ⁇ 0.0001) (figure 16).
  • Mice treated with AC-186 displayed no significant improvement in performance in the rotarod experiment relative to mice treated with vehicle only (figure 17).
  • NF200 indicated axonal integrity with decreases indicating axonal loss
  • beta-APP also indicated axonal integrity with increases indicating axonal damage
  • MBP staining indicated myelin integrity with decreases indicating demyelination during EAE.
  • AC-186 30 mg/kg/every other day treatment in EAE significantly preserved axon numbers (NF200) and reduced axonal damage (beta-APP), with a trend for sparing myelin (MBP).
  • mice treated with AC-186 at 30 mg/kg underwent in vivo, longitudinal MRI scanning at day 0, 30, and 60 after EAE induction.
  • Whole brain, cerebral cortex and cerebellar volumes were determined at each EAE time point in female C57B1/6 mice that were treated with either AC-186 (AC-186) or vehicle (EAE), as well as in age-and sex-matched healthy control mice (NOR).
  • mice underwent in vivo, longitudinal MRI scanning at day 0, 30, and 60 after EAE induction.
  • Whole brain, cerebral cortex and cerebellar volumes were determined at each EAE time point in female C57B1/6 mice that were treated with either AC-186 (AC-186, 30mg/kg/every other day) or vehicle (EAE), as well as in age-and sex-matched healthy control mice (NOR).
  • AC-186 AC-186, 30mg/kg/every other day
  • EAE vehicle-and sex-matched healthy control mice
  • Cerebral and cerebellar neuropathology revealed that AC-186 treatment prevented neuronal cell (NeuN) and synaptic (PSD-95) loss in cerebral cortex gray matter ( Figure 24 top panel) and Purkinje neuronal cell (Calbindin) and synaptic (PSD-95) loss in the cerebellar cortex gray matter ( Figure 24 bottom panel).
  • AC-186 displayed a protective effect at a dose of 30 mg/kg/every other day for cerebellar cells (Calbindin+ Purkinje cells) and synapses (PSD-95 and Synapsin 1). Further, AC-186 displayed a protective effect at a dose of AC-186 30 mg/kg/every other day for cerebral cells (NeuN+ neurons) and synapses (PSD-95) (figure 30). No effect was observed for Synapsin 1 in cerebral gray matter, in contrast to cerebellar gray matter.

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Abstract

L'invention concerne des méthodes pour traiter une maladie neurodégénérative, telle que la sclérose en plaques, chez un sujet qui ne présente pas de lésions actives (par ex.,, des lésions rendues plus visibles par le gadolinium), comprenant l'administration d'un ligand du récepteur β des œstrogènes au sujet sans l'administration conjointe d'un second agent immunothérapeutique. Par exemple, n'importe quel agent immunothérapeutique reçu par le sujet peut être interrompu, par ex., lorsqu'il est déterminé que le cerveau du sujet ne présente pas de lésions actives. La méthode peut consister à déterminer si le cerveau du sujet présente des lésions actives, par ex., par IRM renforcée par du gadolinium.
PCT/US2016/047485 2015-08-19 2016-08-18 Méthodes de fourniture d'une thérapie neuroprotectrice en lien avec l'administration d'un ligand du récepteur bêta des oestrogènes Ceased WO2017031276A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019203752A3 (fr) * 2017-12-14 2020-01-16 Sanovel Ilac Sanayi Ve Ticaret Anonim Sirketi Combinaisons comprenant un agent de relaxation des muscles squelettiques et un agent de traitement de la sclérose en plaques
WO2020122839A3 (fr) * 2018-12-12 2021-02-18 Sanovel Ilac Sanayi Ve Ticaret Anonim Sirketi Association comprenant un agent contre la sclérose en plaques et au moins un agent relaxant musculaire

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WO2013017619A1 (fr) * 2011-08-01 2013-02-07 Acadia Pharmaceuticals Inc. Dérivés de cyclohexane à substitution diphényle, utiles en tant que modulateurs des récepteurs bêta des œstrogènes

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WO2013017619A1 (fr) * 2011-08-01 2013-02-07 Acadia Pharmaceuticals Inc. Dérivés de cyclohexane à substitution diphényle, utiles en tant que modulateurs des récepteurs bêta des œstrogènes

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WISDOM ,AMY J. ET AL.: "Estrogen receptor-beta ligand treatment after disease onset is neuroprotective in the multiple sclerosis model", J NEUROSCI RES., vol. 91, no. 7, 30 April 2013 (2013-04-30), pages 901 - 908, XP055363530 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019203752A3 (fr) * 2017-12-14 2020-01-16 Sanovel Ilac Sanayi Ve Ticaret Anonim Sirketi Combinaisons comprenant un agent de relaxation des muscles squelettiques et un agent de traitement de la sclérose en plaques
WO2020122839A3 (fr) * 2018-12-12 2021-02-18 Sanovel Ilac Sanayi Ve Ticaret Anonim Sirketi Association comprenant un agent contre la sclérose en plaques et au moins un agent relaxant musculaire

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