[go: up one dir, main page]

WO2025213007A1 - S-hydroxychloroquine, optionally with tauroursodeoxycholic acid and/or 3,3'-diindolylmethane, for treating and/or preventing neurological disorders - Google Patents

S-hydroxychloroquine, optionally with tauroursodeoxycholic acid and/or 3,3'-diindolylmethane, for treating and/or preventing neurological disorders

Info

Publication number
WO2025213007A1
WO2025213007A1 PCT/US2025/023133 US2025023133W WO2025213007A1 WO 2025213007 A1 WO2025213007 A1 WO 2025213007A1 US 2025023133 W US2025023133 W US 2025023133W WO 2025213007 A1 WO2025213007 A1 WO 2025213007A1
Authority
WO
WIPO (PCT)
Prior art keywords
hcq
active agent
concentration
tudca
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023133
Other languages
French (fr)
Inventor
Madhav Thambisetty
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
Original Assignee
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Publication of WO2025213007A1 publication Critical patent/WO2025213007A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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

  • This disclosure concerns use of S-hydroxychloroquine, optionally in combination with tauroursodeoxycholic acid and/or 3,3'-diindolylmethane, for treating and/or preventing neurological disorders.
  • an active agent comprising S-hydroxychloroquine (S-HCQ) for treatment or prevention of neurological disorders.
  • the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of HCQ.
  • the active agent may further comprise tauroursodeoxycholic acid (TUDCA), 3,3'-diindolylmethane (DIM, arundine), or a combination thereof.
  • the compounds at least partially normalize an abnormal pathological characteristic of Alzheimer’s disease (AD) and/or AD risk.
  • a method includes administering to a subject an amount of the active agent effective to at least partially normalize an aberrant level of one or more indicators, wherein the indicators comprise extracellular amyloid beta ( A[>) concentration, plasma neurofilament light chain protein (NfL) concentration, neuroinflammation, tau phosphorylation, total tau, plasma glial fibrillary acidic protein (GFAP) concentration, hippocampal synaptic plasticity, or any combination thereof.
  • A[>) concentration plasma neurofilament light chain protein (NfL) concentration, neuroinflammation, tau phosphorylation, total tau, plasma glial fibrillary acidic protein (GFAP) concentration, hippocampal synaptic plasticity, or any combination thereof.
  • normalizing the aberrant level of the one or more indicators reduces extracellular A(3 concentration, reduces plasma NfL concentration, reduces neuroinflammation, reduces tau phosphorylation, reduces plasma GFAP concentration, increases hippocampal synaptic plasticity, or any combination thereof.
  • the active agent at least partially normalizes aberrant levels of at least two or three of the indicators.
  • the subject is administered S- HCQ.
  • the subject is administered a combination of S-HCQ and TUDCA.
  • the subject is administered a combination of S-HCQ and DIM.
  • the subject is administered a combination of S-HCQ, TUDCA, and DIM.
  • the method may further include receiving data comprising an initial level of at least one of the indicators prior to administering the active agent to the subject.
  • the method may further include receiving data comprising a post-administration level of at least one of the indicators following administration of the active agent to the subject, and selecting an adjusted amount of the active agent for administration to the subject based at least in part on the post-administration level.
  • selecting an adjusted amount of the active agent comprises selecting an adjusted amount of at least one component of the active agent.
  • the subject is diagnosed as having a neurological disorder prior to administering the active agent.
  • the neurological disorder is Alzheimer’s disease (AD) and the method further includes identifying the subject as being at risk of developing AD by (i) identifying the subject as being an APOE e4 carrier, or (ii) identifying the subject as having an elevated level of the one or more indicators relative to a normal level of the one or more indicators, or (iii) both (i) and (ii).
  • the active agent is administered to the subject prophylactically in the absence of any cognitive, behavioral, mood, or psychological signs or symptoms of a neurological disorder.
  • FIGS. 17A-17H show plasma NIL measured in wild-type (WT) (FIG. 17A) and 5xFAD mice (FIG. 17B) treated with saline over 10 weeks, as well as 5xFAD mice treated with racemic HCQ (FIG. 17C), HCQ nano (FIG. 17D), saline (FIG. 17E) R-HCQ (FIG. 17F), S-HCQ (FIG. 17G), TUDCA (FIG. 17H), and amiloride (FIG. 171).
  • FIGS. 33A and 33B are bar graphs showing impact of S-HCQ on A 1-42 phagocytosis.
  • BV-2 cells were treated with S-HCQ and A01-42 for 3 h; thereafter supernatant and cell lysates were harvested and analyzed for: the changes in A(3 level in the supernatant as pg/ml (FIG. 33A) as well as the content of A01-42 in cell lysates as pg/pg total protein (FIG. 33B).
  • VC vehicle control 0.1% DMSO, S-HCQ - 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6).
  • Statistical Analysis One-way ANOVA followed by Dunnell’s multiple comparison test versus VC: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • FIGS. 34 A and 34B are bar graphs showing impact of R-HCQ on A01-42 phagocytosis.
  • BV-2 cells were treated with R-HCQ and AP 1 -42 for 3 h; thereafter supernatant and cell lysates were harvested and analyzed for: the changes in AP level in the supernatant as pg/ml (FIG. 34A) as well as the content of Api-42 in cell lysates as pg/pg total protein (FIG. 34B).
  • VC vehicle control 0.1% DMSO, R-HCQ - 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6).
  • Statistical Analysis One-way ANOVA followed by Dunnett’s multiple comparison test versus VC: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • S-HCQ concentrations - 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6); RI DAPT.
  • Statistical Analysis One-way ANOVA followed by Dunnett’s multiple comparison test versus vehicle control (VC): *p ⁇ 0.05; **p ⁇ 0.01;***p ⁇ 0.001.
  • Statistical Analysis One-way ANOVA followed by Dunnett’s multiple comparison test versus vehicle control (VC): *p ⁇ 0.05; **p ⁇ 0.01;***p ⁇ 0.001.
  • FIGS. 37A and 37B are graphs showing that racemic HCQ free base has limited effectiveness in rescuing synaptic plasticity in APP/PS1 mice at 50 pM (FIG. 37 A) and is partially effective at 25 pM (FIG. 37B).
  • FIGS. 38A and 38B are graphs showing that both 25 pM R-HCQ (FIG. 38A) and 25 pM S-HCQ (FIG. 38B) rescue late long-term potentiation (L-LTP) in APP/PS1 mice.
  • FIGS. 39 A and 39B are graphs showing that both 50 pM R-HCQ (FIG. 39 A) and 50 pM S-HCQ (FIG. 39B) rescue L-LTP in APP/PS1 mice.
  • FIGS. 40A and 40B are graphs showing the effects of 25 pM HCQ enantiomers (FIG. 38 A) and 50 pM HCQ enantiomers (FIG. 38B) on L-LTP rescue in APP/PS1 mice.
  • FIGS. 41A and 41B are graphs showing that application of 10 pM DIM rescues L-LTP in APP/PS1 slices (FIG. 41 A) and a 3-hour pre-incubation with 10 pM DIM no longer results in rescue of L-LTP in APP/PS1 slices (FIG 4 IB).
  • FIG. 42 is a graph showing that a mixture of 50 pM HCQ sulfate and 10 pM DIM had a deleterious effect on synaptic plasticity in APP/PS1 slices.
  • FIG. 43 is a graph showing that 100 pM TUDCA had a deleterious effect on synaptic plasticity in APP/PSl slices.
  • FIGS. 44A-44D show quantification of A [140 and A [142 in DEA soluble and FA soluble fraction of the cortex: A [140 in FA soluble fraction (FIG. 44A); A(342 in FA soluble fraction (FIG. 44B); A [140 in DEA soluble fraction (FIG. 44C); A 42 in DEA soluble fraction (FIG. 44D).
  • mice t.g., saline
  • G - t.g., vehicle a - transgenic mice (t.g.), saline; C - t.g., TUDCA 500 mg/kg + HCQ 100 mg/kg; D - t.g., DIM 50 mg/kg; E - t.g., DIM 100 mg/kg; F - t.g., DIM 200 mg/kg; G - t.g., vehicle.
  • FIGS. 45A and 45B show effect of HCQ sulfate/DIM on Ap 1 -42 phagocytosis in BV-2 cells.
  • FIG. 45A shows the A 1 -42 level in the supernatant
  • FIG. 45B shows the Api -42 level in the cell lysates.
  • VC vehicle control 0.1% DMSO
  • cl 25 pM HCQ/10 pM DIM
  • c2 25 pM HCQ/3 pM DIM
  • c3 10 pM HCQ/10 pM DIM.
  • Statistical Analysis One-way ANOVA followed by Bonferroni’s multiple comparison test versus VC: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • VC vehicle control HzO;
  • cl 25 pM HCQ/10 pM DIM,
  • Statistical Analysis One-way ANOVA followed by Dunnett’s multiple comparison test versus vehicle control (VC): *p ⁇ 0.05;
  • Reference item (RI) dexamethasone.
  • Statistical Analysis One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • Reference item (RI) dexamethasone.
  • Statistical Analysis Oneway ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p ⁇ 0.05; **p ⁇ 0.01;
  • Reference item (RI) dexamethasone.
  • Statistical Analysis One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • Reference item (RI) dexamethasone.
  • Statistical Analysis One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • FIG. 62B shows change in escape latency w.r.t Saline treated 5xFAD (left) expressed as posterior mean (filled black circles) and 95% credible interval (black error bars), and 2 x log Bayes Factor (BF) quantifying evidence (right) for each of three alternative hypotheses Hl: slight improvement, partial rescue, full rescue. 2 x log BF > 2, 6, 10 is considered as moderate, strong and very strong evidence for Hl relative to HO (no change in escape latency).
  • This disclosure concerns use of an active agent comprising an enantiomeric excess of the S- enantiomer of hydroxychloroquine (S-HCQ) for treatment or prevention of neurological disorders.
  • the active agent may further comprise tauroursodeoxycholic acid (TUDCA), 3,3'-diindolylmethane (DIM, arundine), or a combination thereof.
  • aberrant refers to deviating from an accepted standard value.
  • the term “aberrant level” refers to a level that deviates from a range considered to represent normal levels of the biomarker.
  • Active agent A drug, medicament, pharmaceutical, therapeutic agent, nutraceutical, or other compound that may be administered to a subject to effect a change, such as treatment, amelioration, or prevention of a disease or disorder or at least one symptom associated therewith.
  • the active agent may be a “small molecule,” generally having a molecular weight of about 2000 daltons or less.
  • the active agent may also be a “biological active agent.”
  • Biological active agents include proteins, antibodies, antibody fragments, peptides, oligonucleotides, vaccines, and various derivatives of such materials.
  • Administration To provide or give a subject an agent, such as one or more compounds provided herein, by any effective route.
  • Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraosseous, intracerebroventricular, intrathecal, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • injection such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraosseous, intracerebroventricular, intrathecal, and intratumoral
  • sublingual rectal
  • transdermal intranasal, vaginal and inhalation routes.
  • Effective amount An amount sufficient to provide a beneficial, or therapeutic, effect to a subject or a given percentage of subjects, such as an amount effective to elicit a desired biological or medical response in a tissue, system, subject or patient; to treat a specified disorder or disease; to ameliorate or eradicate one or more of its symptoms; and/or to prevent the occurrence of the disease or disorder.
  • the amount of a compound which constitutes an “effective amount” may vary depending on the compound, the desired result, the disease state and its severity, the age of the patient to be treated, and the like.
  • Enantiomeric excess A measurement of purity for chiral substances, reflecting the degree to which one enantiomer is present in greater amount than the other enantiomer. Enantiomeric excess (ee) is calculated as the difference between the two enantiomers as the percentage of a mixture. A racemic mixture with 50% of each enantiomer has an ee of zero. A pure enantiomer has an ee of 100%. If a mixture has, for example, 70% of one enantiomer and 30% of the other, the ee is 40% (70% - 30%).
  • Normalizing the aberrant level of the one or more indicators may reduce extracellular A concentration, reduce tau phosphorylation, reduce plasma NfL concentration, reduce ILl-beta concentration, decrease neuroinflammation, reduce plasma GFAP concentration, increase hippocampal synaptic plasticity, or any combination thereof.
  • the active agent is effective in at least partially normalizing aberrant levels of at least two of the indicators.
  • the active agent may at least partially normalize levels of both A(3 concentration and plasma NfL concentration.
  • the active agent at least partially normalizes aberrant levels of at least three indicators.
  • compositions disclosed herein include those formed from pharmaceutically acceptable salts and/or solvates of the disclosed active agents.
  • Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids.
  • Particular disclosed active agents may possess at least one basic group that can form acid-base salts with acids. Examples of basic groups include, but are not limited to, amino and imino groups. Examples of inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid.
  • Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids).
  • Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations.
  • biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.
  • compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use.
  • Sterile solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • methods of preparation include vacuum drying and freeze-drying which yields a powder of the active agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the actual dosage of the active agent will vary according to factors such as the disease indication and particular status of the subject (for example, the subject’ s age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the active agent for eliciting the desired response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. An effective amount is also one in which any toxic or detrimental side effects of the active agent is outweighed in clinical terms by therapeutically beneficial effects.
  • a non-limiting range for an effective amount of an active agent within the methods and formulations of the disclosure is about 0.01 mg/kg body weight to about 20 mg/kg body weight, such as about 0.05 mg/kg to about 10 mg/kg body weight, about 0.2 mg/kg to about 10 mg/kg body weight, or about 1 mg/kg to about 10 mg/kg.
  • Dosage can be varied by the attending clinician as previously described, such as based on a determined level of the one or more indicators associated with AD. Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, intraosseous, or intranasal delivery versus intravenous or subcutaneous or intramuscular delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.
  • an S-HCQ dosage for an adult human may be from 50 mg to 1000 mg, such as from 200 mg to 500 mg, or from 0.5 mg/kg body weight to 20 mg/kg body weight, such as from 2 mg/kg body weight to 10 mg/kg body weight, daily in one dose or two or more divided doses.
  • a TUDCA dosage for an adult human may be from 100 mg to 2000 mg, such as from 500 mg to 1000 mg, or from 1 mg/kg body weight to 40 mg/kg body weight daily, such as from 5 mg/kg body weight to 20 mg/kg body weight, daily in one dose or two or more divided doses.
  • an DIM dosage for an adult human may be from 25 mg to 200 mg, such as from 100 mg to 150 mg, or from 0.25 mg/kg body weight to 4 mg/kg body weight, such as from 1 mg/kg body weight to 3 mg/kg body weight, daily in one dose or two or more divided doses.
  • the active agent can be administered to the subject by the oral route or in a single bolus delivery, via continuous delivery (for example, continuous intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol).
  • the effective dosage of the active agent can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms associated with AD and/or at least partially normalize a level of one or more indicators associated with AD.
  • Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of AD symptoms or at least partially normalize the level of the one or more indicators in the subject.
  • Suitable models in this regard include, for example, murine, rat, avian, dog, sheep, porcine, feline, non-human primate, and other accepted animal model subjects known in the art.
  • effective dosages can be determined using in vitro models. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer an effective amount of the active agent.
  • the subject is administered the composition for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, at least 5 years, at least 10 years, or indefinitely for the remainder of the subject’s life.
  • the subject may further be administered additional therapeutic agents.
  • the subject may be administered one or more additional therapeutic agents used for treating a neurological disorder or for treating one or more signs or symptoms associated with a neurological disorder.
  • the additional therapeutic agent may be a therapeutic agent used for treating AD, Parkinson’ s disease, Huntington’ s disease, vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, or any combination thereof.
  • Preparation and dosing schedules for the additional agent may be used according to manufacturer's instructions or as determined empirically by the skilled practitioner.
  • the combination therapy may provide synergy and prove synergistic, that is, the effect achieved when the active agent and therapeutic agent used together is greater than the sum of the effects that results from using the active agent and therapeutic agent separately.
  • a synergistic effect may be attained when the active agent and additional therapeutic agent are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation, a synergistic effect may be attained when the active agent and therapeutic agent are administered or delivered sequentially, for example by different injections in separate syringes.
  • kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects.
  • Kits for diagnostic use are also provided.
  • these kits include a container or formulation that contains one or more of the active agents described herein.
  • this component is formulated in a pharmaceutical preparation for delivery to a subject.
  • the active agent is optionally contained in a bulk dispensing container or unit or multi-unit dosage form.
  • Optional dispensing means can be provided, for example a pulmonary or intranasal spray applicator.
  • Packaging materials optionally include a label or instruction indicating for what treatment purposes and/or in what manner the pharmaceutical agent packaged therewith can be used.
  • DIVIO pre-aggregated A 1-42 (Bachem 4061966, final concentration 10 pM, 48h at 4°C) was added to the cells in the presence or absence of Hydroxychloroquine sulfate (TargetMol, T0951) at 25 pM, 2.5 pM or 0.25 pM concentrations.
  • TargetMol Hydroxychloroquine sulfate
  • Covariates A large number of covariates were identified, which were measured in the 365- day baseline period preceding the cohort entry date. The following set of variables were included: 1) demographic factors such as age, gender, race, socioeconomic status proxies, 2) risk factors for ADRD identified in previous studies such as diabetes, stroke, and depression, 3) lifestyle factors such as smoking as well as use of preventive services, including screening mammography and vaccinations, to account for healthy-user effects 13 ; measures for use of various healthcare services before cohort entry including number of distinct prescriptions filled, number of emergency department visits, hospitalizations, and number of physician office visits to account for patients’ general health and contact with the healthcare system to minimize the possibility of differential surveillance; frailty indicators based on composite scoring scheme to address potential confounding by frailty, 4) comorbid conditions and comedications including prior use of pain medications such as steroids and opioids.
  • FIG. 5 summarizes cumulative incidence of ADRD among HCQ initiators compared to MTX initiators; results from all four analyses indicate that after approximately 2 years of treatment, individuals on HCQ had lower cumulative incidence of ADRD compared to MTX.
  • FIG. 6 summarizes the crude and PS-matched comparative risk of ADRD in HCQ compared to MTX groups; results indicated that risk of ADRD was consistently lower among HCQ initiators.
  • HCQ initiators had an 8% lower risk of ADRD compared to MTX initiators (HR, 95% CI 0.92, 0.83-1.00).
  • C3aRl C3 and C3a receptor
  • HCQ impacts the three principal pathogenic molecular pathways in AD, i.e., neuroinflammation, A clearance and tau phosphorylation
  • L-LTP impaired late long-term potentiation
  • L-LTP is a protein-synthesis dependent form of synaptic plasticity important in hippocampal memory formation (Binhibit et al., FEES J 2021, doi: 10.1111/febs.16065).
  • Previous studies have observed impaired L-LTP in APP/PS 1 mice at 3-4 months of age, prior to the accumulation of Ap plaques (Li et al., PNAS USA 2017, 114(21):5527-5532). This observation allowed testing of pharmacological interventions at the earliest stages of AD, prior to the accumulation of Ap pathology.
  • the results show that HCQ restores L-LTP in the hippocampus of APP/PS 1 mice.
  • HCQ and other 4-aminoquinolones have been shown to preferentially accumulate in acidic cellular compartments such as lysosomes at millimolar levels (Browning, Hydroxychloroquine and Chloroquine Retinopathy 2014, 35-63). Furthermore, HCQ has a well-established safety profile with serious side effects of retinopathy and cardiac toxicity being relatively rare (Nirk et al., EMBO Mol Med 2020, 12:el2476).
  • HCQ was confirmed to exhibit anti-inflammatory effects in a model of neuroinflammation using microglial cells from the brains of a transgenic AD mouse model.
  • the results showed that, similar to observations in BV2 microglial cells, HCQ lowered the release of several cytokines, including IL6, ILl-b, IL-10, and IL-12p70.
  • the results are shown in FIG. 8.
  • MACS microglia isolation and cultivation Adult microglia were isolated from 9 months old 5xFAD mice via magnetic cell sorting (MACS). The mice were terminally anesthetized by i.p. injection of Pentobarbital (600 mg/kg, dosing 10 pL/g body weight) and brains were transcardially perfused with DPBS. Brains were removed, the brainstem discarded and the remaining brain minced for cell dissociation. Cell dissociation was performed using Miltenyi Adult Brain Dissociation Kit (Miltenyi, 130-107-677).
  • MACS cell separation was performed using CD1 lb (Microglia) MicroBeads, mouse (Miltenyi, 130-093-634) and MS columns on OctoMACSTM cell separator (Miltenyi). Isolated microglia were seeded onto 0.01% PLL coated plates at a density of 10.000 cells per well in 384 well plate in DMEM containing 10% FBS,1% penicillin/streptomycin, and 2 mM L- glutamine.
  • LPS-induced neuroinflammation on MACS isolated 5xFAD microglia On DIV7 HCQ at 0,.25 pM, 2.5 pM and 25, u.M was applied 1 hour before LPS stimulation (Sigma-Aldrich; L6529;
  • TUDCA 100 pM (Cl), 10 pM (C2), or 1.0 pM (C3)
  • MSD MSD® 96-well MULTI-SPOT® 6E10 Abeta Triplex Assay
  • Aft Clearance 20,000 BV2 cells per well (uncoated 96-well plates were plated out. After changing cells to treatment medium, drug compounds were administered 1 hour before A stimulation (final concentration in well 200 ng/mL; dilutions in medium). Cells treated with vehicle and cells treated with A[3 alone served as controls. After 3 hours of A [3 stimulation, cell supernatants were collected for the A[3 measurement and cells were carefully washed twice with PBS and thereafter lysed in 35 pL of cell lysis buffer (50 mM tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% SDS) supplemented with protease inhibitors.
  • cell lysis buffer 50 mM tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% SDS
  • PLS-HCQ is more brain penetrant than HCQ.
  • the efficacy of PLS-HCQ will be compared with HCQ for amelioration of AD pathology and ability to slow cognitive decline in a transgenic mouse model of AD.
  • HCQ sulfate 10 g (Sigma PHR1682; pharmaceutical secondary standard), was dissolved in 300 mL H2O (18.2 ) and gently sonicated at room temperature to ensure complete dissolution of the salt.
  • 20 mL (8.3 equiv, 190 mmol) of diethylamine (Sigma 471216; > 99.5%) was added, which precipitated the free -base HCQ as a dense and white suspension.
  • the suspension was poured into a separatory funnel and leftover material was transferred by rinsing the flask with dichloromethane (Sigma 320269; > 99.5%).
  • Amiloride preparation Dissolved in water; treatment dosage 12 mg/kg; i.p. injection 10 pL/g b.w.
  • R-HCQ preparation The compound was dissolved in DMSO. This stock solution was stored at room temperature protected from light. The dosing solution was made from this stock solution once a week. For the dosing solution, the test item was dissolved in the vehicle (10% DMSO, 10% Cremophor® (BASF Corp.) water) and stored at 4°C and protected from light. Treatment dosage 100 mg/kg; i.p. injection 10 pL/g b.w.
  • S-HCQ preparation The compound was dissolved in DMSO. This stock solution was stored at room temperature protected from light. The dosing solution was made from this stock solution once a week. For the dosing solution, the test item was dissolved in the vehicle (10% DMSO, 10% Cremophor® (BASF Corp.) water) and stored at 4°C and protected from light. Treatment dosage 100 mg/kg; i.p. injection 10 pL/g b.w.
  • Animal management Animals were housed in individual ventilated cages on standardized rodent bedding supplied by Rettenmaier. Each cage contained a maximum of five mice. The temperature in the keeping room was maintained between 20 to 24 °C and the relative humidity was maintained between 45 to 65 %. Animals were housed under a constant light-cycle (12 hours light/dark). Dried, pelleted standard rodent chow (Altromin) as well as normal tap water was available to the animals ad libitum. Only animals in apparently good health condition were included to the study. Randomization of group allocation was done per cage. If possible, animals were assigned to different starting groups (cohorts) comprising animals of all treatment groups. The number of animals in a starting group was limited to ensure same age and uniform handling. Age at treatment start 7.5 months ⁇ 0.5 months.
  • mice A total of 160 mice were used for the study. 112 transgenic 5xFAD mice were randomly allocated to 7 treatment groups A - G, each consisting of 16 animals. 48 age matched wild type littermates were randomly allocated to 3 treatment groups H, I and J each consisting of 16 animals. All animals were treated with test compound or vehicle for the whole study period by i.p. injection (Table 1). Group A was dosed daily for the whole treatment period. Group G and I were dosed twice per week. All other groups were dosed daily in the first 4 weeks, three times per week in weeks 5 to 8, and once weekly until the end of the study.
  • the 5xFAD mouse mimics the most crucial phenotypic symptoms of amyloidogenic neurodegeneration, neuroinflammation as well as learning and memory deficits and is a suitable model for Alzheimer’ s disease to study effects of drugs on biochemical, histological and behavioral hallmarks.
  • MWM test was performed on 4 days with 4 trials per day and a probe trial on day 5.
  • a computerized video tracking system was used to quantify escape latency and distance travelled. The mice were tested in a randomized order.
  • MWM Morris Water Maze
  • the platform was removed from the pool and the number of crossings over the former target position as well as the abidance in the target quadrant was recorded.
  • escape latency the time [sec] to find the hidden platform
  • pathway the length of the trajectory [meters] to reach the target
  • target zone crossings the abidance in the target quadrant in the PT
  • mice were euthanized by IP injection of 600 mg/kg pentobarbital. After confirmation of deep anesthesia, CSF was obtained by dissection of the muscles and exposure of the foramen magnum. Upon exposure, a Pasteur pipette was inserted in an approximate depth of 0.3 - 1 mm into the cisterna magna. CSF was collected by suction and capillary action until flow fully ceases in 0.2 ml polypropylene PCR tubes. The tubes were spun briefly and immediately frozen in an upright position on dry ice. CSF quality was documented as score from 0 (no visible blood contamination) to 3 (severe blood contamination).
  • Terminal blood was collected by heart puncture in EDTA coated tubes. Blood plasma was collected by centrifugation (3000 x g for 10 minutes at room temperature) and plasma aliquots were transferred to 1.5 mL tubes, frozen on dry ice and stored at -80 °C.
  • Amyloid-0-positive plaques, microglia, and tau phosphorylation were evaluated using immunofluorescence labeling on a uniform systematic random set of five sections per mouse (one section each from levels 2, 4, 6, 8, 10; total of 270 sections).
  • Microglia activation, phosphorylated tau at serine 202/threonine 205 (pSer202/Thr205 Tau, pTau), and amyloid 0-positive plaques (fibrils) were evaluated using quadruple immunofluorescent labeling with the following protocol.
  • Sections were washed 3 x 5 minutes each in DPBS. 6. Sections were incubated with primary antibodies in M.O.M. diluent (Vector Laboratories) over night at 4°C in a damp chamber - rabbit anti- amyloid-0 fibrils (LOC) polyclonal antibody (Merck Millipore, AB2287), 1:1000; guinea pig monoclonal [Gp311H9] anti-Ibal (Synaptic Systems, 234 308), 1:3000; mouse anti-pSer202/Thr205 Tau monoclonal [AT8] antibody (Thermo Fisher, MN1020), 1:300. 7. Sections were washed 3 x 5 minutes each in DPBS. 8.
  • S-HCQ is a promising therapeutic candidate for AD as well as other neurological disorders such as ALS and Parkinson's disease (PD) and multiple sclerosis (MS) that are associated with an increase in plasma NfL levels.
  • PD Parkinson's disease
  • MS multiple sclerosis
  • the studies in the 5x-FAD AD mouse model also tested a novel HCQ nano-formulation that uses a selective polylysine succinylated (PLS) drug delivery platform, which can pass through the BBB by scavenger receptor Al (SR-A1)- mediated transcytosis.
  • PLS polylysine succinylated
  • SR-A1 scavenger receptor Al
  • DAPT y-Secretase Inhibitor IX, Lot 3218570
  • Calbiochem Cat. No. 565770, > 95%) and stored at -20 °C.
  • the working solution as prepared fresh in 0.1% DMSO from stored DMSO aliquots.
  • the treatment dosage was 400 nM.
  • BV-2 Cells The murine microglial cell line BV-2 was cultivated in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% FCS, 1% penicillin/streptomycin and 2 mM L-glutamine (culture medium). For the A(3 clearance assay, 20 000 BV-2 cells per well (uncoated 96 well plates) were plated out. After 24 hours, medium was changed to treatment medium (DMEM, 5% fetal calf scrum (FCS), 2 mM L-glutaminc) and cells were maintained in treatment medium for the remaining culture period.
  • DMEM Modified Eagle medium
  • FCS fetal calf scrum
  • FCS fetal calf scrum
  • test items were administered 1 hour before A
  • cell supernatants were collected for the Ap measurement and cells were carefully washed twice with PBS and thereafter lysed in 35 pl cell lysis buffer (50 mM Tris- HC1, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS) supplemented with protease inhibitors.
  • murine microglial cell line BV-2 was treated with test items and Api -42 for 3 h. Thereafter supernatant and cell lysates were harvested and analyzed for remaining Api-42 in the supernatant as well as possibly increased intracellular Api-42.
  • Transgenic 5xFAD mice were randomly allocated to treatment groups. The expression of the 5xFAD transgene is driven by the neuron specific Thyl promoter. The five mutations cause an early onset of the cognitive decline and increasing Af! 1-40 and 1-42 levels in the brain and cerebrospinal fluids, over age. Histological analysis revealed plaque load and beta sheet formation accompanied with neuroinflammation. Thus, the 5xFAD mouse mimics the most crucial phenotypic symptoms of amyloidogenic neurodegeneration, neuroinflammation as well as learning and memory deficits and is a suitable model for Alzheimer’s disease to study effects of drugs on biochemical, histological and behavioral hallmarks. All animals were treated with test compound or vehicle for the whole study period by i.p.
  • the frozen cortex samples from animals of each group were homogenized in lysis buffer (THB; 250 mM Sucrose, 1 mM EDTA, 1 mM EGTA, 20 mM Tris pH 7.4) including lx protease inhibitor (Calbiochem).
  • THB lysis buffer
  • the tissue was homogenized with a beadmill (UPHO, Geneye) at 55Hz for 50sec. Aliquots were stored at 80°C until further use.
  • UPHO UPHO, Geneye
  • 1 aliquot of THB homogenate was mixed with 1 part diethylamine (DEA) solution (0.4% DEA, lOOmM NaCl). The mixture was centrifuged for 120 min at 20,000xg, 4°C.
  • DEA diethylamine
  • the third aliquot of homogenate was substituted with Triton® X-100 (Dow Chemical Company, Midland, MI) so that the final concentration as 1 % in the homogenate. After vortexing and 10 min incubation on ice, the homogenates were cleared from cell debris by centrifugation at 20,800 x g at 4°C for 10 minutes in a tabletop centrifuge and the supernatants were collected for the measurement of cytokines and stored at -80°C until further use as Triton® fraction.
  • Triton® X-100 Low Chemical Company, Midland, MI
  • Afi 1-40 and Afi 1-42 levels in DEA and FA fraction' were measured in duplicates in the fractions described above (DEA and FA fractions) using Ap 1-42 with MSD® Human (6E10) V-plex kit (K151LBE-2, Mesoscale Discovery) and A 1-4O with MSD® Human (6E10) V-plex kit (K150SKE-2, Mesoscale Discovery) according to the instructions of the manufacturer. Plates were read on Quickplex SQ 120 sector imager (Mesoscale Discovery). A[> levels in study samples were evaluated in comparison to calibration curves provided in the kit and are expressed as pg per mg brain wet weight.
  • TUDCA+HCQ treatment resultsed in statistically significant lowering of A [3 1 -40 and A[3 1 -42 levels in the FA fraction of the cortex compared to saline-treated transgenic mice (group A) (FIGS. 44A-44B).
  • the murine microglial cell line BV-2 was cultivated in DMEM medium supplemented with 10% FCS, 1% penicillin/streptomycin and 2 mM L-glutamine (culture medium).
  • A0 clearance assay 20,000 BV-2 cells per well (uncoated 96 well plates) were plated out. After 24 hours, medium was changed to treatment medium (DMEM, 5% FCS, 2 mM L-glutamine) and cells were maintained in treatment medium for the remaining culture period. After changing cells to treatment medium, the test items were administered 1 hour before A[> stimulation (Bachem 4061966; final concentration in well: 200 ng/mL (dilutions in medium)). Cells treated with vehicle, cells treated with A(3 alone, as well as wells with A(3 but no cells served as controls. All wells were handled the same way.
  • DMEM 5% FCS, 2 mM L-glutamine
  • Cytokine measurement Levels of 10 cytokines (IFN-y, IL-i , IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-a) were measured in supernatants (1:2 diluted) of BV-2 cells collected 24 h after LPS stimulation. Cytokines were measured by an immunosorbent assay (V-PLEX Proinflammatory Panel 1 Mouse Kit, K15048D, Mesoscale) according to the instructions of the manufacturer and evaluated in comparison to calibration curves provided in the kit. Results are given as pg per mL.
  • IL-6 levels were significantly increased at the lowest concentration FIG. 47C).
  • KC/GRO was significantly increased at all concentrations (FIG. 47D).
  • the MTT Assay indicated higher viability of samples treated with low and middle dose of HCQ/DIM, which may be associated with the increased cytokine secretion (FIG. 47 F).
  • cell supernatants were collected for GFAP measurement and cells were harvested in 30 pL/well RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1% Nonidet P40, 2%SDS, Protease Inhibitor Cocktail I (Calbiochem), Phosphatase Inhibitor Cocktail III (Sigma)), protease inhibitors.
  • HCQ sulfate, S-HCQ, or R-HCQ and LPS were treated with racemic HCQ sulfate, S-HCQ, or R-HCQ and LPS for 24 h. Thereafter supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/mL as well as the content of GFAP in cell lysates as pg/mL lysate.
  • HCQ sulfate significantly increased the concentration of GFAP in the lysate of primary astrocytes at the third treatment concentration (FIGS. 48A and 48B). No effects of the test items were observed in the supernatant or for any other concentration in the lysate.
  • S-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate of primary astrocytes (FIGS. 49A, 49B).
  • R-HCQ free base also had no significant impact on GFAP levels in the supernatant or lysate of primary astrocytes (FIGS. 50A, 50B).
  • TUDCA/HCQ sulfate racemic
  • TUDCA/S-HCQ free base TUDCA/R-HCQ free base
  • TUDCA alone and LPS for 24 h.
  • supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/ml as well as the content of GFAP in cell lysates as pg/ml lysate.
  • TUDCA/HCQ sulfate significantly increased GFAP levels in the supernatant of primary astrocytes, for the third concentration and in the cell lysate no significant effects were observed (FIGS. 51 A and 5 IB).
  • TUDCA/S-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate (FIGS. 52A and 52B).
  • TUDCA/R-HCQ free base also had no significant impact on GFAP levels in the supernatant or lysate (FIGS. 53A and 53B).
  • TUDCA alone had no significant impact on GFAP levels in the supernatant or lysate (FIGS. 54A and 54B).
  • HCQ sulfate (racemic), S-HCQ free base, or R- HCQ free base and LPS for 24 hours. Thereafter supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/mL as well as the content of GFAP in cell lysates as ng/pg lysate.
  • HCQ sulfate significantly increased the concentration of GFAP in the supernatant of brain slices at the highest tested concentration; no effects were observed in the supernatant for any other concentration or in the lysate (FIGS. 55A and 55B).
  • S-HCQ free base significantly reduced secreted GFAP in the supernatant at the lowest tested concentration, while no impact on GFAP levels in the lysate of brain slices was detected (FIGS. 56A and 56B).
  • R-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 57 A and 57B).
  • Organotypic brain slices also were treated with TUDCA/HCQ sulfate (racemic), TUDCA/S- HCQ free base, TUDCA/R-HCQ free base, or TUDCA alone and LPS for 24 h. Thereafter supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/ml as well as the content of GFAP in cell lysates as ng/ pg lysate.
  • TUDCA/HCQ sulfate had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 58A and 58B).
  • TUDCA/S-HCQ free base significantly reduced secreted GFAP in the supernatant at the highest tested concentration, while no significant impact on GFAP levels in the lysate of brain slices was detected (FIGS. 59A and 59B).
  • TUDCA/R-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 60A and 60B).
  • TUDCA alone had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 61 A and 6 IB).
  • mice in this study are split into four groups including two treatment groups: 1) 5x Familial Alzheimer’s disease (FAD) transgenic (TG) mice that receive the TUDCA + (S)-HCQ test compounds; 2) wild-type (WT) mice that receive the TUDCA + (S)-HCQ test compounds; and two vehicle control groups: 3) 5xFAD TG mice that receive saline; 4) WT mice that receive saline.
  • Animals are dosed daily for the entire 15-week trial and serial, in-vivo blood samples are collected for biomarker analyses. All animals are tested twice using the Y-maze and once using the Morris Water Maze (MWM) to determine the effect of treatment/ vehicle control on cognitive deficits.
  • MMM Morris Water Maze
  • the 5xFAD mouse model has five mutations including three in the amyloid precursor protein (APP695) gene [APP K670N/M671L (Swedish), I716V (Florida), V717I (London)] and two mutations in the presenilin 1 gene [PSI M146L, L286V].
  • the 5xFAD mouse mimics phenotypic symptoms of Alzheimer’s disease including amyloidogenic neurodegeneration, neuroinflammation,
  • AD Alzheimer’s disease
  • mice receive intraperitoneal (IP) injections of 500 mg/kg Tauroursodeoxycholic acid (TUDCA) on Monday and Friday throughout the 15 week trial duration. Mice receive IP injections of 100 mg/kg of (S)-hydroxychloroquine ((S)-HCQ) on Tuesday through Thursday and Saturday and Sunday during weeks 1-4, Tues through Thursday on weeks 5-8, and Wednesday only on week 9 through the end of the trial.
  • IP Intrperitoneal
  • TDCA Tauroursodeoxycholic acid
  • in-vivo blood samples are collected by mandibular sampling from each animal at 4 time points: baseline (prior to the start of treatment) and after 4, 8, and 12 weeks of treatment.
  • K2EDTA potassium ethylenediaminetetraacetic acid
  • Samples are centrifuged (3000 x g for 10 minutes at room temperature) and plasma aliquots are transferred to 1.5 mL tubes, frozen on dry ice and stored at -80 °C.
  • the cortex and hippocampus from a selection of animals per group are prepared. Briefly, brain tissue samples are homogenized by adding 9 or 19 volumes (to cortex and hippocampus samples respectively) of tissue homogenization buffer (THB; 250 mM Sucrose, 1 mM EDTA, 1 mM EGTA, 20 mM Tris pH 7.4) including lx protease inhibitor (Calbiochem). The tissue is homogenized with a beadmill (UPHO, Geneye) at 55Hz for 50 seconds. Three aliquots are stored at -80°C until further use.
  • TLB tissue homogenization buffer
  • UPHO UPHO, Geneye
  • the third aliquot of homogenate is substituted with Triton X-100 so that the final concentration is 1% in the homogenate. After vortexing and 10 minutes incubation on ice, the homogenates are cleared from cell debris by centrifugation at 20,800 x g at 4 °C for 10 minutes in a tabletop centrifuge and the supernatants are collected for the measurement of cytokines and stored at -80 °C until further use as the Triton fraction.
  • the Morris Water Maze is the primary, cognitive outcome of the trial.
  • the MWM is a tool frequently used in drug trials to test the effect of drug exposure on spatial learning and memory deficits in animals modelling neurologic diseases including AD. All animals are assessed in the MWM on week 14. The assessment has two consecutive parts: the training probe (days 1-4) and the test probe (on day 5).
  • Neurofilament light chain (NfL) are measured at all 4 in-vivo blood timepoints with NF- light® (Neurofilament-light) ELISA 10-7001 CE from UmanDiagnostics. Samples are diluted (1:3) in assay buffer and analyzed according to manufacturer protocol. After dilution, 100 pl of sample are added to the pre-coated wells and incubated for 1.5 hours at room temperature (RT) with gentle agitation (800 rpm). Wells are washed three times with assay wash buffer and 100 pl of the tracer antibody are added. After 45 minutes incubation (RT, 800 rpm) wells are washed again three times.
  • RT room temperature
  • 800 rpm gentle agitation
  • TUDCA or HCQ separately had no effect in TG mice on any cytokine level except for TUDCA on IL-1 P level, which had similar magnitude and statistical significance to the combined TUDCA + HCQ treatment (FIG. 69, right panel).
  • TUDCA + HCQ treated TG mice were the compared to saline treated TG in terms of the measured Nfl trajectory.
  • FIG. 71 bottom compares raw, untransformed, trajectories without baseline correction and shows that Nil level is significantly lower in TUDCA + HCQ treated TG than in saline treated TG 8 weeks after treatment onset, suggesting that TUDCA + HCQ treatment protects against pathological increase in Nfl level in TG.
  • FIG. 72 presents results from an alternative approach based on log-transformed, baseline- corrected Nfl level. This approach, too, showed a decrease in Nil level at week 8 in TUDCA + HCQ treated TG relative to saline treated TG, but this decrease (FIG. 72 bottom left) was not statistically significant (FIG. 72 bottom right).

Landscapes

  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Psychology (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

A subject is administered an amount of an active agent effective to treat and/or prevent a neurological disorder, wherein the active agent comprises S-hydroxychloroquine with an enantiomeric excess of at least 20% of the S-enantiomer of hydroxychloroquine. The active agent may further comprise tauroursodeoxycholic acid and/or 3,3'-diindolylmethane. The subject may be diagnosed with a neurological disorder or may be identified as being at risk of developing a neurological disorder. Administering the active agent may at least partially normalize an aberrant level of an indicator characteristic of the neurological disorder.

Description

S-HYDROXYCHLOROQUINE, OPTIONALLY WITH TAUROURSODEOXYCHOLIC ACID AND/OR 3,3'-DIINDOLYLMETHANE, FOR TREATING AND/OR PREVENTING NEUROLOGICAL DISORDERS
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of the earlier filing date of U.S. provisional patent application No. 63/574,481, filed April 4, 2024, which is incorporated herein by reference in its entirety.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Project No. Z01 AG000436-03 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD
[0003] This disclosure concerns use of S-hydroxychloroquine, optionally in combination with tauroursodeoxycholic acid and/or 3,3'-diindolylmethane, for treating and/or preventing neurological disorders.
SUMMARY
[0004] This disclosure concerns use of an active agent comprising S-hydroxychloroquine (S-HCQ) for treatment or prevention of neurological disorders. In some aspects, the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of HCQ. The active agent may further comprise tauroursodeoxycholic acid (TUDCA), 3,3'-diindolylmethane (DIM, arundine), or a combination thereof. In some embodiments, the compounds at least partially normalize an abnormal pathological characteristic of Alzheimer’s disease (AD) and/or AD risk.
[0005] In some embodiments, a method includes administering to a subject an amount of the active agent effective to at least partially normalize an aberrant level of one or more indicators, wherein the indicators comprise extracellular amyloid beta ( A[>) concentration, plasma neurofilament light chain protein (NfL) concentration, neuroinflammation, tau phosphorylation, total tau, plasma glial fibrillary acidic protein (GFAP) concentration, hippocampal synaptic plasticity, or any combination thereof. In some embodiments, normalizing the aberrant level of the one or more indicators reduces extracellular A(3 concentration, reduces plasma NfL concentration, reduces neuroinflammation, reduces tau phosphorylation, reduces plasma GFAP concentration, increases hippocampal synaptic plasticity, or any combination thereof. In certain implementations, the active agent at least partially normalizes aberrant levels of at least two or three of the indicators. In one aspect, the subject is administered S- HCQ. In an independent aspect, the subject is administered a combination of S-HCQ and TUDCA. In another independent aspect, the subject is administered a combination of S-HCQ and DIM. In still another independent aspect, the subject is administered a combination of S-HCQ, TUDCA, and DIM. [0006] In any of the foregoing or following embodiments, the method may further include receiving data comprising an initial level of at least one of the indicators prior to administering the active agent to the subject. In any of the foregoing or following embodiments, the method may further include receiving data comprising a post-administration level of at least one of the indicators following administration of the active agent to the subject, and selecting an adjusted amount of the active agent for administration to the subject based at least in part on the post-administration level. When the active agent comprises S-HCQ in combination with TUDCA, DIM, or a combination thereof, selecting an adjusted amount of the active agent comprises selecting an adjusted amount of at least one component of the active agent.
[0007] In some embodiments, the subject is diagnosed as having a neurological disorder prior to administering the active agent. In some implementations, the neurological disorder is Alzheimer’s disease (AD) and the method further includes identifying the subject as being at risk of developing AD by (i) identifying the subject as being an APOE e4 carrier, or (ii) identifying the subject as having an elevated level of the one or more indicators relative to a normal level of the one or more indicators, or (iii) both (i) and (ii). In certain embodiments, the active agent is administered to the subject prophylactically in the absence of any cognitive, behavioral, mood, or psychological signs or symptoms of a neurological disorder.
[0008] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0010] FIGS. 1 A-1C show that HCQ rescues molecular phenotypes relevant to AD. FIG. 1 A shows greater A^ 1.42 clearance in microglia. FIG. IB shows reduction in tau phosphorylation in neuroblastoma cells overexpressing human mutant tau. FIG. 1C shows lowering of bacterial lipopolysaccharide induced neuroinflammation in microglia. Data are presented as bar graphs with group mean and error bars representing standard deviation (SD). Individual values arc shown as dots (n=6 per group). Values were compared by one-way ANOVA followed by Dunnetts multiple comparison test versus vehicle control (VC): *p<0.05; **p<0.01; ***p<0.001. HCQ: hydroxychloroquine; AU: arbitrary units; VC: vehicle control (0.1% DMSO); LPS: lipopolysaccharide, RI: reference item; dexamethasone lOpM. [0011] FIGS. 2A-2G show that rescue of AD phenotypes by HCQ is associated with STAT3 inactivation. FIGS. 2A and 2B show increase in A[31.42 clearance in microglia. FIGS. 2C and 2D show reduction in tau phosphorylation. FIGS. 2E-2G show lowering of bacterial lipopolysaccharide- induced neuroinflammation. Group differences were evaluated using two-sample t-tests: *p<0.05; **p<0.01; VC: vehicle control; HCQ: hydroxychloroquine; VC: vehicle control (0.1% DMSO); p- STAT3: phosphorylated STAT3; Tyr705: tyrosine 705; Ser727: serine 727.
[0012] FIGS. 3A-3H show that HCQ rescues late-long-term potentiation (LTP) in hippocampal CAI synapses of APP/PS 1 mice. FIG. 3A is a schematic representation of a hippocampal slice with electrodes located in the CAI region. ‘Rec’ represents the recording electrode positioned in the CAI region flanked by two stimulating electrodes represented as S 1 and S2 in the stratum radiatum to stimulate two independent pathways to a single neuronal population in Schaffer collateral pathway (sc). FIG. 3B shows induction of late-LTP by STET in synaptic input SI in WT mice resulted in a potentiation that remained stable for 180 min (filled circles, n=7). FIG. 3C shows induction of late- LTP by STET in synaptic input SI in APP/PS 1 mice resulted only in early-LTP in SI (filled circles, n=6). FIG. 3D shows treatment of hippocampal slices with 25 pM HCQ resulted in partial restoration of late-LTP in S 1 in APP/PS 1 mice (filled circles, n=8) that was however significantly lower than WT late-LTP (D vs B). FIG. 3E shows that treatment of hippocampal slices with 50 pM HCQ resulted in restoration of late-LTP in SI in APP/PS 1 mice (filled circles, n=7) that was similar to WT late-LTP (E vs B). FIG. 3F shows that treatment of hippocampal slices from wild type mice with 50 pM HCQ does not alter L-LTP compared to untreated wild type hippocampal slices, indicating that HCQ affects synaptic plasticity only in the APP/PS 1 transgenic mice. In FIGS. 3B-3F control input S2 remained stable throughout the recording (open circles). In FIG. 3G, a comparison of input-output curves showed no significant change between WT and APP/PS 1 before and after HCQ application. In FIG. 3H, a comparison of Paired Pulse Ratio (PPR) also revealed no significant change in Paired Pulse Facilitation (PPF) ratio between WT and APP/PS 1 mice before and after HCQ application (n=12). Error bars in all the graphs indicate +SEM. Analog traces represent typical field excitatory postsynaptic potentials (fEPSPs) of inputs SI and S2, recorded 15 min before (dotted line), 30 min after (dashed line), and 180min (solid line) after tetanization in SI and the corresponding time points in S2. Three solid arrows represent the time of induction of late-LTP by STET. Solid rectangular bar in FIGS. 3D-3F represents the time of application of HCQ. Scale bars: vertical, 2 mV; horizontal, 3 ms.
[0013] FIGS. 4A-4B show that HCQ reduces levels of hippocampal p-STAT3 in APP/PS 1 mice. FIG. 4A is a western blot analysis of hippocampal p-STAT3 (Tyr705) and total STAT3 levels between WT, APP/PS 1, WT + 50pM HCQ and APP/PS 1 + 50 pM HCQ mice (N = 3 for each group). Levels of p-STAT3 (Tyr705) were significantly higher in APP/PS 1 mice compared to WT mice and were similar between WT and WT + 50, u M HCQ mice. FIG. 4B shows p-STAT3/total STAT3 ratio (N = 3 for each group). The values of the individual groups were calculated in relation to the control group, while tubulin serves as a loading control. Asterisk indicates significant differences between groups (two-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001). Error bars indicate ± SEM.
[0014] FIG. 5 shows analyses of cumulative incidence of Alzheimer’s and related dementia (ADRD) in rheumatoid arthritis patients treated with methotrexate or HCQ; Medicare data 2007-2017. The four analyses were designed to address various uncertainties associated with claims-based analyses of ADRD risk including: Analysis 1: ‘As-treated’ follow-up approach; Analysis 2: ‘As-started’ followup approach incorporating a 6-month induction period; Analysis 3: Incorporating a 6-month ‘symptom to diagnosis’ period’ and Analysis 4: Alternate outcome definition. HCQ: hydroxychloroquine; MTX: methotrexate.
[0015] FIG. 6 shows the comparative risk of ADRD in rheumatoid arthritis patients treated with HCQ vs. MTX; Medicare data 2007-2017. The four analyses are those described in FIG. 5. HCQ: hydroxychloroquine; MTX: methotrexate; PS: propensity score.
[0016] FIGS. 7A and 7B are fluorescence images (FIG. 7 A) and a bar graph (FIG. 7B) showing that HCQ pretreatment enhances microglial uptake of AP1.42 preferentially into acidic cellular compartments such as lysosomes.
[0017] FIG. 8 shows that HCQ lowers release of several cytokines in microglial cells from the 5xFAD transgenic AD mouse model.
[0018] FIG. 9 shows levels of A01-38, A 1-4O, and A 1-42 in supernatant after 24 hours treatment of human APP overexpressing H4-hAPP cells with tauroursodeoxycholic Acid (TUDCA) at TUDCA concentrations of 100 pM (Cl), 10 pM (C2), and 1.0 pM (C3).
[0019] FIG. 10 shows levels of A 1-38, A01-4O, and A01-42 in supernatant after 24 hours treatment of human APP ovcrcxprcssing H4-hAPP cells with HCQ and TUDCA at concentrations of 25 pM HCQ/0.1 pM TUDCA (Cl), 2.5 pM HCQ/100 pM TUDCA (C2), and 2.5 pM HCQ/10 pM TUDCA (C3).
[0020] FIG. 11 shows aggregated performance of TUDCA and DIM in 9 experiments where performance is characterized by the posterior probability of a protective, neutral, and adverse effect, effect (hypothesis Hl, HO, H2, respectively). Prior probabilities are marked by vertical lines as a reference. The probabilities were calculated by averaging the probabilities across all assays in any given experiment. Scores greater than zero indicate a protective effect probability, with higher scores indicating sponger evidence for a protective effect.
[0021] FIGS. 12A-12D are bar graphs showing quantification of A [140 and A [142 in DEA (diethylamine) soluble and FA (formic acid) soluble fraction of the cortex: A [140 in FA soluble fraction (FIG. 12A), A [142 in FA soluble fraction (FIG. 12B), A [140 in DEA soluble fraction (FIG. 12C), and A042 in DEA soluble fraction (FIG. 12D). Data are given as pg per mg tissue and represented as group means +SEM (n=6 per group), Statistics: one-way ANOVA + Dunnett’s multiple comparisons test. All data were analyzed using group D as reference group for pairwise comparisons. *p<0,05; **p<0,01; ***P<0,001. Groups: A = tg (transgenic), amiloride, B - tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA.
[0022] FIGS. 13A-13D are bar graphs showing quantification of A[>40 and A 042 in DEA soluble and FA soluble fraction of the hippocampus: A04O in FA soluble fraction (FIG. 13A), A042 in FA soluble fraction (FIG. 13B), A04O in DEA soluble fraction (FIG. 13C), and A042 in DEA soluble fraction (FIG. 13D). Data are given as pg per mg tissue and represented as group means +SEM (n=6 per group), Statistics: one-way ANOVA + Dunnett’s multiple comparisons test. All data were analyzed using group D as reference group for pairwise comparisons. *p<0,05; **p<0,01;
***P<0,001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA.
[0023] FIGS. 14A and 14B are bar graphs showing quantification of IL-6 in the cortex (FIG. 14A) and hippocampus (FIG. 14B). Data are given as pg per g protein and represented as group means +SEM (n=6 per group), Statistics: one-way ANOVA + Dunnett’s multiple comparisons test for cortex and Kruskal- Wallis test + Dunn’s multiple comparison test for hippocampus. All data were analyzed using group D as reference group for pairwise comparisons. *p<0,05; **p<0,01; ***P<0,001.
Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0024] FIGS. 15A and 15B are bar graphs showing quantification of IL-1 in the cortex (FIG. 15A) and hippocampus (FIG. 15B). Data are given as pg per g protein and represented as group means +SEM (n=6 per group), Statistics: one-way ANOVA + Dunnett’s multiple comparisons test for cortex and Kruskal- Wallis test + Dunn’s multiple comparison test for hippocampus. All data were analyzed using group D as reference group for pairwise comparisons. *p<0,05; **p<0,01; ***P<0,001.
Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0025] FIGS. 16A-16D are bar graphs showing quantification of NfL in plasma at different timepoints baseline (FIG. 16A), after 4 weeks (FIG. 16B), after 8 weeks (FIG. 16C), and after 12 weeks (FIG. 16D). Data are given as pg per ml plasma and represented as group means +SEM (n=6 per group), Statistics: Data were analyzed by one-way ANOVA and Dunnett’s post hoc test. Group D was defined as reference group for pairwise comparisons. *p<0,05; **p<0,01; ***P<0,001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0026] FIGS. 17A-17H show plasma NIL measured in wild-type (WT) (FIG. 17A) and 5xFAD mice (FIG. 17B) treated with saline over 10 weeks, as well as 5xFAD mice treated with racemic HCQ (FIG. 17C), HCQ nano (FIG. 17D), saline (FIG. 17E) R-HCQ (FIG. 17F), S-HCQ (FIG. 17G), TUDCA (FIG. 17H), and amiloride (FIG. 171).
[0027] FIG. 18 is a graph showing the estimated effects of the drugs of FIGS. 17C-17H.
[0028] FIG. 19 is a bar graph showing quantification of GFAP in terminal plasma samples. Data are given as pg per ml plasma and represented as group means +SEM (n=5-6 per group), Statistics: Data were analyzed by one-way ANOVA and Dunnett’s post hoc test. Group D was defined as reference group for pairwise comparisons. *p<0,05; **p<0,01; ***P<0,001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0029] FIG. 20 includes images showing immunofluorescent labeling of ThioS + LOC + Ibal + pTau on sagittal sections of transgenic animals treated with amiloride (group A) or racemic HCQ (group B); nuclei are labeled with DAPI. Single channel magnifications show labeling in layer 5 of the cortex and in the subiculum of the hippocampal formation; images were taken at the positions indicated by the rectangles in the overview images. Examples of pTau-positive puncta are indicated by arrows.
[0030] FIG. 21 includes images showing immunofluorescent labeling of ThioS + LOC + Ibal + pTau on sagittal sections in transgenic animals treated with HCQ nano (group C) or saline vehicle (group D); nuclei are labeled with DAPI. Single channel magnifications show labeling in layer 5 of the cortex and in the subiculum of the hippocampal formation; images were taken at the positions indicated by the rectangles in the overview images. Examples of pTau-positive puncta are indicated by arrows.
[0031] FIG. 22 includes images showing immunofluorescent labeling of ThioS + LOC + Ibal + pTau on sagittal sections in transgenic animals treated with R-HCQ (group E) or S-HCQ (group F); nuclei are labeled with DAPI. Single channel magnifications show labeling in layer 5 of the cortex and in the subiculum of the hippocampal formation; images were taken at the positions indicated by the rectangles in the overview images. Examples of pTau-positive puncta are indicated by arrows.
[0032] FIG. 23 includes images showing immunofluorescent labeling of ThioS + LOC + Ibal + pTau on sagittal sections in transgenic animals treated with TUDCA (group G) or wt animals treated with saline vehicle (group H); nuclei are labeled with DAPI. Single channel magnifications show labeling in layer 5 of the cortex and in the subiculum of the hippocampal formation; images were taken at the positions indicated by the rectangles in the overview images. Examples of pTau-positive puncta are indicated by arrows. Note absence of plaques and low pTau signal in wt (group H). [0033] FIG. 24 includes images showing immunofluorescent labeling of ThioS + LOC + Ibal + pTau on sagittal sections in wt animals treated with TUDCA (group I) or 10% DMSO/10% Cremophor/water vehicle (group J); nuclei are labeled with DAPI. Single channel magnifications show labeling in layer 5 of the cortex and in the subiculum of the hippocampal formation; images were taken at the positions indicated by the rectangles in the overview images. Examples of pTau- positive puncta are indicated by arrows. Note absence of plaques and low pTau signal in wt mice.
[0034] FIG. 25 includes bar graphs showing quantification of ThioS histofluorescence in the cortex. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (object intensity, object density) or by Kruskal -Wallis test and Dunn’s post hoc test (immunoreactive area, object size). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM. * p < 0.05, *** p < 0.001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0035] FIG. 26 includes bar graphs showing quantification of ThioS histofluorescence in the hippocampus. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (object intensity, object density) or by Kruskal- Wallis test and Dunn’s post hoc test (immunoreactive area, object size). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM. * p < 0.05, *** p < 0.001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0036] FIG. 27 includes bar graphs showing quantification of LOC immunofluorescence in the cortex. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (immunoreactive area, object density) or by Kruskal- Wallis test and Dunn’s post hoc test (object intensity, object size). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM.
* p < 0.05, *** p < 0.001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G — tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0037] FIG. 28 includes bar graphs showing quantification of LOC immunofluorescence in the hippocampus. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (immunoreactive area, object density) or by Kruskal- Wallis test and Dunn’s post hoc test (object intensity, object size). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0038] FIG. 29 includes bar graphs showing quantification of Ibal immunofluorescence in the cortex. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (object intensity, object size, object density) or by Kruskal-Wallis test and Dunn’s post hoc test (immunoreactive area). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM. * p < 0.05, *** p < 0.001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0039] FIG. 30 includes bar graphs showing quantification of Ibal immunofluorescence in the hippocampus. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (object intensity, object size, object density) or by Kruskal- Wallis test and Dunn’s post hoc test (immunoreactive area). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM. * p < 0.05, *** p < 0.001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0040] FIG. 31 includes bar graphs showing quantification of pTau immunofluorescence in the cortex. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (object intensity) or by Kruskal- Wallis test and Dunn’s post hoc test (immunoreactive area, object size, object density). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM. * p < 0.05, ** p < 0.01. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0041] FIG. 32 includes bar graphs showing quantification of pTau immunofluorescence in the hippocampus. Graphs present the means of signal on 5 brain sections per mouse (n = 6 per group). Data were analyzed by one-way ANOVA and Dunnett’s post hoc test (object intensity) or by Kruskal- Wallis test and Dunn’s post hoc test (immunoreactive area, object size, object density). Group D was defined as reference group for pairwise comparisons. Bar graphs represent group means + SEM. * p < 0.05, *** p < 0.001. Groups: A = tg (transgenic), amiloride, B = tg, HCQ, C = tg, HCQ nano, D = tg, vehicle (saline), E = tg, R-HCQ, F = tg, S-HCQ, G = tg, TUDCA, H = wt (wild type) vehicle (saline), I = wt TUDCA, J = wt, vehicle (10% DMSO, 10% Cremophor, water).
[0042] FIGS. 33A and 33B are bar graphs showing impact of S-HCQ on A 1-42 phagocytosis. BV-2 cells were treated with S-HCQ and A01-42 for 3 h; thereafter supernatant and cell lysates were harvested and analyzed for: the changes in A(3 level in the supernatant as pg/ml (FIG. 33A) as well as the content of A01-42 in cell lysates as pg/pg total protein (FIG. 33B). Data are presented as bar graph with group mean+SEM (n=6 per group). VC= vehicle control 0.1% DMSO, S-HCQ - 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6). Statistical Analysis: One-way ANOVA followed by Dunnell’s multiple comparison test versus VC: *p<0.05; **p<0.01; ***p<0.001.
[0043] FIGS. 34 A and 34B are bar graphs showing impact of R-HCQ on A01-42 phagocytosis. BV-2 cells were treated with R-HCQ and AP 1 -42 for 3 h; thereafter supernatant and cell lysates were harvested and analyzed for: the changes in AP level in the supernatant as pg/ml (FIG. 34A) as well as the content of Api-42 in cell lysates as pg/pg total protein (FIG. 34B). Data are presented as bar graph with group mean+SEM (n=6 per group). VC= vehicle control 0.1% DMSO, R-HCQ - 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6). Statistical Analysis: One-way ANOVA followed by Dunnett’s multiple comparison test versus VC: *p<0.05; **p<0.01; ***p <0.001.
[0044] FIGS. 35A-35C are bar graphs showing levels of A i-38 (FIG. 35 A), A i-40 (FIG. 35B) and A -42 (FIG. 35C) in supernatant after 24 h treatment of human APP overexpressing H4-hAPP cells with S-HCQ. Levels were assessed with MSD V-plex are given as pg/ml. Data are presented as bar graphs with group mean+SEM (n=6 per group). VC = vehicle control H2O; S-HCQ concentrations - 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6); RI = DAPT. Statistical Analysis: One-way ANOVA followed by Dunnett’s multiple comparison test versus vehicle control (VC): *p<0.05; **p<0.01;***p<0.001.
[0045] FIGS. 36A-36C are bar graphs showing levels of A 1-38 (FIG. 36A), A 1-4O (FIG. 36B) and Af)-42 (FIG. 36C) in supernatant after 24 h treatment of human APP overexpressing H4-hAPP cells with R-HCQ. Levels were assessed with MSD V-plex are given as pg/ml. Data are presented as bar graphs with group mean+SEM (n=6 per group). VC = vehicle control H2O; R-HCQ concentrations - 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6); RI = DAPT. Statistical Analysis: One-way ANOVA followed by Dunnett’s multiple comparison test versus vehicle control (VC): *p<0.05; **p<0.01;***p<0.001.
[0046] FIGS. 37A and 37B are graphs showing that racemic HCQ free base has limited effectiveness in rescuing synaptic plasticity in APP/PS1 mice at 50 pM (FIG. 37 A) and is partially effective at 25 pM (FIG. 37B).
[0047] FIGS. 38A and 38B are graphs showing that both 25 pM R-HCQ (FIG. 38A) and 25 pM S-HCQ (FIG. 38B) rescue late long-term potentiation (L-LTP) in APP/PS1 mice.
[0048] FIGS. 39 A and 39B are graphs showing that both 50 pM R-HCQ (FIG. 39 A) and 50 pM S-HCQ (FIG. 39B) rescue L-LTP in APP/PS1 mice. [0049] FIGS. 40A and 40B are graphs showing the effects of 25 pM HCQ enantiomers (FIG. 38 A) and 50 pM HCQ enantiomers (FIG. 38B) on L-LTP rescue in APP/PS1 mice.
[0050] FIGS. 41A and 41B are graphs showing that application of 10 pM DIM rescues L-LTP in APP/PS1 slices (FIG. 41 A) and a 3-hour pre-incubation with 10 pM DIM no longer results in rescue of L-LTP in APP/PS1 slices (FIG 4 IB).
[0051] FIG. 42 is a graph showing that a mixture of 50 pM HCQ sulfate and 10 pM DIM had a deleterious effect on synaptic plasticity in APP/PS1 slices.
[0052] FIG. 43 is a graph showing that 100 pM TUDCA had a deleterious effect on synaptic plasticity in APP/PSl slices.
[0053] FIGS. 44A-44D show quantification of A [140 and A [142 in DEA soluble and FA soluble fraction of the cortex: A [140 in FA soluble fraction (FIG. 44A); A(342 in FA soluble fraction (FIG. 44B); A [140 in DEA soluble fraction (FIG. 44C); A 42 in DEA soluble fraction (FIG. 44D). Data are given as pg per mg tissue and represented as group means +SEM (n=6 per group), Statistics: one-way ANOVA + Dunnett’s multiple comparisons test. All data were analyzed using group A as reference group for pairwise comparisons. *p<0,05; **p<0,01; ***P<0,001. Groups: A - transgenic mice (t.g.), saline; C - t.g., TUDCA 500 mg/kg + HCQ 100 mg/kg; D - t.g., DIM 50 mg/kg; E - t.g., DIM 100 mg/kg; F - t.g., DIM 200 mg/kg; G - t.g., vehicle.
[0054] FIGS. 45A and 45B show effect of HCQ sulfate/DIM on Ap 1 -42 phagocytosis in BV-2 cells. FIG. 45A shows the A 1 -42 level in the supernatant, and FIG. 45B shows the Api -42 level in the cell lysates. Data are presented as bar graph with group mean+SEM (n=6 per group). VC= vehicle control 0.1% DMSO, cl = 25 pM HCQ/10 pM DIM, c2 = 25 pM HCQ/3 pM DIM, c3 = 10 pM HCQ/10 pM DIM. Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus VC: *p<0.05; **p<0.01; ***p<0.001.
[0055] FIGS. 46A-46D show effects of HCQ sulfate/DIM on A01-38 (FIG. 46A), A01-4O
(FIG. 46B), A01-42 (FIG. 46C), and MTT (FIG. 46D) in supernatant of H4h-APP cells after 24 hours. Viability assessed with MTT assay given as % of the vehicle control. Data are presented as bar graph with group mean+SEM (n=6 per group). VC = vehicle control HzO; cl = 25 pM HCQ/10 pM DIM, c2 = 25 pM HCQ/3 pM DIM, c3 = 10 pM HCQ/10 pM DIM. Statistical Analysis: One-way ANOVA followed by Dunnett’s multiple comparison test versus vehicle control (VC): *p<0.05;
**p<0.01;***p<0.001.
[0056] FIGS. 47A-47F show levels of secreted cytokines as well as cell viability after 24 h treatment in LPS-stimulated BV2 microglial cells: TNF-a (FIG. 47 A), IL- 10 (FIG. 47B) IL-6 (FIG. 47C), KC/GRO (FIG. 47D), IL- 10 assessed with MSD V-plex (FIG. 47E) given as pg/mL, cell viability assessed with MTT assay given as percent of LPS control (FIG. 47F). Data are presented as bar graph with group mean+SEM (n=6 per group). LPS= stimulated vehicle control, VC = unstimulated vehicle control H2O; cl = 25 pM HCQ/10 pM DIM, c2 = 25 pM HCQ/3 pM DIM, c3 = 10 pM HCQ/10 pM DIM. Statistical Analysis: One-way ANOVA followed by Dunnett’ s multiple comparison test versus stimulated vehicle control (LPS): *p<0.05; **p<0.01;***p<0.001.
[0057] FIGS. 48 A and 48B show effects of HCQ sulfate (racemic) on extracellular (FIG. 48 A) and intracellular (FIG. 48B) GFAP in astrocytes treated with HCQ sulfate at three concentrations: cl = 25 pM, c2 = 2.5 pM, c3 = 0.25 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=6 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0058] FIGS. 49A and 49B show effects of S-HCQ free base on extracellular (FIG. 49A) and intracellular (FIG. 49B) GFAP in astrocytes treated with S-HCQ free base at three concentrations: cl = 25 pM, c2 = 2.5 pM, c3 - 0.25 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=6 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0059] FIGS. 50A and 50B show effects of R-HCQ free base on extracellular (FIG. 50A) and intracellular (FIG. 50B) GFAP in astrocytes treated with R-HCQ free base at three concentrations: cl = 25 pM, c2 = 2.5 pM, c3 = 0.25 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=6 per group).
Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0060] FIGS. 51 A and 5 IB show effects of TUDCA/HCQ sulfate (racemic) on extracellular (FIG. 51 A) and intracellular (FIG. 51B) GFAP in astrocytes treated with TUDCA/HCQ sulfate at three concentrations: cl = 100 pM/25pM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=6 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0061 ] FIGS. 52 A and 52B show effects of TUDCA/S-HCQ free base on extracellular (FIG. 52A) and intracellular (FIG. 52B) GFAP in astrocytes treated with TUDCA/S-HCQ free base at three concentrations: cl = 100 pM/25pM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=6 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0062] FIGS. 53A and 53B show effects of TUDCA/R-HCQ free base on extracellular (FIG. 53A) and intracellular (FIG. 53B) GFAP in astrocytes treated with TUDCA/R-HCQ free base at three concentrations: cl = 100 pM/25pM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=6 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0063] FIGS. 54A and 54B show effects of TUDCA on extracellular (FIG. 54A) and intracellular (FIG. 54B) GFAP in astrocytes treated with TUDCA at three concentrations: cl = 100 pM, c2 = 10 pM, c3 = 1 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=6 per group). Statistical Analysis: Oneway ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01;
***p<0.001.
[0064] FIGS. 55A and 55B show effects of HCQ sulfate (racemic) on extracellular (FIG. 55A) and intracellular (FIG. 55B) GFAP in organotypic brain slices treated with HCQ sulfate at three concentrations: cl = 25 pM, c2 = 2.5 pM, c3 - 0.25 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=5-8 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0065] FIGS. 56A and 56B show effects of S-HCQ free base on extracellular (FIG. 56A) and intracellular (FIG. 56B) GFAP in organotypic brain slices treated with S-HCQ free base at three concentrations: cl = 25 pM, c2 = 2.5 pM, c3 = 0.25 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=5-8 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0066] FIGS. 57A and 57B show effects of R-HCQ free base on extracellular (FIG. 57 A) and intracellular (FIG. 57B) GFAP in organotypic brain slices treated with R-HCQ free base at three concentrations: cl = 25 M, c2 = 2.5 pM, c3 = 0.25 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=5-8 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0067] FIGS. 58A and 58B show effects TUDCA/HCQ sulfate (racemic) on extracellular (FIG. 58A) and intracellular (FIG. 58B) GFAP in organotypic brain slices treated with TUDCA/HCQ sulfate (racemic) at three concentrations: cl = 100 pM/25pM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=5-8 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0068] FIGS. 59A and 59B show effects TUDCA/S-HCQ free base on extracellular (FIG. 59A) and intracellular (FIG. 59B) GFAP in organotypic brain slices treated with TUDCA/S-HCQ free base at three concentrations: cl = 100 pM/25pM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=5-8 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0069] FIGS. 60A and 60B show effects TUDCA/R-HCQ free base on extracellular (FIG. 60A) and intracellular (FIG. 60B) GFAP in organotypic brain slices treated with TUDCA/R-HCQ free base at three concentrations: cl = 100 pM/25|iM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=5-8 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0070] FIGS. 61 A and 61B show effects TUDCA on extracellular (FIG. 61 A) and intracellular (FIG. 61B) GFAP in organotypic brain slices treated with TUDCA at three concentrations: cl = 100 pM, c2 = 10 pM, c3 - 1 pM. Reference item (RI) = dexamethasone. LC= Lesion control LPS + 0.1% DMSO. Data are presented as bar graphs with group mean+SEM (n=5-8 per group). Statistical Analysis: One-way ANOVA followed by Bonferroni’s multiple comparison test versus LC: *p<0.05; **p<0.01; ***p<0.001.
[0071] FIG. 62 A shows the effect of TUDCA + HCQ on the escape latency as a function of training days, with observed data for male and female mouse subjects. Thick light line: Bayesian fitted curve (posterior mean). Thin black lines: curves sampled from the posterior distribution.
[0072] FIG. 62B shows change in escape latency w.r.t Saline treated 5xFAD (left) expressed as posterior mean (filled black circles) and 95% credible interval (black error bars), and 2 x log Bayes Factor (BF) quantifying evidence (right) for each of three alternative hypotheses Hl: slight improvement, partial rescue, full rescue. 2 x log BF > 2, 6, 10 is considered as moderate, strong and very strong evidence for Hl relative to HO (no change in escape latency).
[0073] FIG. 63 shows that there is no change of escape latency in WT mice by combined TUDCA + HCQ treatment.
[0074] FIG. 64 demonstrates that combined TUDCA + HCQ treatment improved spatial memory of 5xFAD TG mice during the test probe of MWM experiments. The upper and lower row of panels show two different sets of experiments. The left column of panels provides the maximum-likelihood (ML) estimate of the fraction of time spent in each quadrant of the MWM. The middle and right columns present spatial memory performance in terms of Information gain and Likelihood ratio test, respectively. In both cases, performance is evaluated relative to both a trained control (Saline TG) and an untrained control (modeled by a uniform distribution). Note that the combined TUDCA + HCQ treatment leads to the greatest increases in the fraction of time spent in the target quadrant at the expense of the opposite quadrant. This translates to the most information gained during MWM training and the smallest p-values for rejecting Ho in likelihood-ratio tests. Also note that while all TG and WT treatment groups outperform the untrained control statistically significantly (a=0.01 middle vertical dashed line, right column), only TUDCA + HCQ treatment outperforms the trained control (ct=0.05 right vertical dashed line, right column).
[0075] FIG. 65 shows the levels of the A/J-40 and 42 alloforms in the cortex and hippocampus region of 5xFAD and WT mice measured in the FA and DEA fraction.
[0076] FIG. 66 provides levels of the A/J-40 and 42 alloforms in the cortex and hippocampus region of 5xFAD mice measured in the FA and DEA fraction, and demonstrates that combined TUDCA + HCQ treatment decreased A/? levels for all alloforms, brain regions and fractions.
[0077] FIG. 67 provides levels of the A/J-40 and 42 alloforms in the cortex and hippocampus region of 5xFAD mice measured in the FA and DEA fraction, and demonstrates that, when applied separately, TUDCA leads to a relatively small decrease in A/3 level, while HCQ has no effect.
[0078] FIG. 68A is a table providing multiple regression analysis of A/? level in 5xFAD TG mice treated with TUDCA + HCQ combined. The linear model included terms for treatment, A/? alloform, brain region, and fraction of brain homogenate, as well as pairwise interactions of those terms.
[0079] FIG. 68B is a table providing multiple regression analysis of A (3 level in 5xFAD TG mice treated with TUDCA or HCQ separately. The linear model included terms for treatment, A/3 alloform, brain region, and fraction of brain homogenate, as well as pairwise interactions of those terms.
[0080] FIG. 69 shows the effect of combined or separate application of TUCA and HCQ on cytokine levels in 5xFAD TG mice. Shown are estimates (circles) and their standard error (error bars). The TUDCA and/or HCQ drug effect is compared to the effect of WT genotype. * p < 0.05, ** p < 0.01, *** p<0.001
[0081] FIG. 70 shows the effect of TUDCA, HCQ and combined TUDCA + HCQ treatment on Nfl level in 5xFAD TG mice, and demonstrates a dramatic increase of Nfl level in 5xFAD TG compared to WT mice.
[0082] FIG. 71 provides t-tests comparing Saline and drug treatment in TG mice in terms of untransformed Nfl level at given time points without baseline correction for the results in FIG. 70.
[0083] FIG. 72 provides the same comparisons as FIG. 70 but in terms of log-transformed, baseline- corrected Nfl level at week 8.
DETAILED DESCRIPTION
[0084] In some instances, compared to a racemic mixture, a single enantiomer of a chiral active agent may possess an improved therapeutic index through increased potency, increased selectivity, decreased side effects, faster onset of action, reduced drug-drug interactions, and/or a reduced effective dosage. Commercially available hydroxychloroquine is available as a racemic mixture of R- and S-cnantiomcrs. Data from rats suggest that racemic HCQ is preferentially metabolized to R-HCQ which accumulates at greater concentrations (1.6-1.7x) in the brain compared to S-HCQ (Wei et al., Chirality 1996, 7:598-604). However, the inventors have surprisingly discovered that S-HCQ is the more desirable enantiomer as a therapeutic for treating and/or preventing neurological disorders. In some aspects, the inventors have discovered that an enantiomeric excess of S-HCQ provides unexpectedly superior results compared to the racemic free base mixture or R-HCQ alone.
[0085] This disclosure concerns use of an active agent comprising an enantiomeric excess of the S- enantiomer of hydroxychloroquine (S-HCQ) for treatment or prevention of neurological disorders. The active agent may further comprise tauroursodeoxycholic acid (TUDCA), 3,3'-diindolylmethane (DIM, arundine), or a combination thereof.
S-HCQ R-HCQ
[0086] In some embodiments, the active agent at least partially normalizes an abnormal pathological characteristic of a neurological disorder and/or neurological disorder risk. The neurological disorder may be a neurodegenerative disease or other neurological disorder not primarily characterized by neurodegeneration. Exemplary neurological disorders include, but are not limited to, Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’ s disease (HD), vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, exhaustion disorder (ED, a stress-induced disorder characterized by extreme fatigue, cognitive impairment, and stress intolerance), post-acute cognitive impairment and fatigue (i.e., cognitive impairment and fatigue following a coronavirus disease 2019 (COVID-19) infection), or neuropsychiatric manifestations of systemic lupus erythematosus (SLE). In certain aspects, the neurological disorder is AD.
I. Definitions and Abbreviations
[0087] The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. [0088] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.
[0089] The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, percentages, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited.
[0090] Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (ed.), Hawley's Condensed Chemical Dictionary, published by John Wiley & Sons, Inc., 2016 (ISBN 978- 1-118-13515-0). Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.
[0091] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms arc provided:
[0092] Aberrant: As used herein, the term “aberrant” refers to deviating from an accepted standard value. As used herein with respect to a biomarker, the term “aberrant level” refers to a level that deviates from a range considered to represent normal levels of the biomarker.
[0093] Active agent: A drug, medicament, pharmaceutical, therapeutic agent, nutraceutical, or other compound that may be administered to a subject to effect a change, such as treatment, amelioration, or prevention of a disease or disorder or at least one symptom associated therewith. The active agent may be a “small molecule,” generally having a molecular weight of about 2000 daltons or less. The active agent may also be a “biological active agent.” Biological active agents include proteins, antibodies, antibody fragments, peptides, oligonucleotides, vaccines, and various derivatives of such materials. [0094] Administration: To provide or give a subject an agent, such as one or more compounds provided herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraosseous, intracerebroventricular, intrathecal, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
[0095] Effective amount: An amount sufficient to provide a beneficial, or therapeutic, effect to a subject or a given percentage of subjects, such as an amount effective to elicit a desired biological or medical response in a tissue, system, subject or patient; to treat a specified disorder or disease; to ameliorate or eradicate one or more of its symptoms; and/or to prevent the occurrence of the disease or disorder. The amount of a compound which constitutes an “effective amount” may vary depending on the compound, the desired result, the disease state and its severity, the age of the patient to be treated, and the like.
[0096] Enantiomeric excess: A measurement of purity for chiral substances, reflecting the degree to which one enantiomer is present in greater amount than the other enantiomer. Enantiomeric excess (ee) is calculated as the difference between the two enantiomers as the percentage of a mixture. A racemic mixture with 50% of each enantiomer has an ee of zero. A pure enantiomer has an ee of 100%. If a mixture has, for example, 70% of one enantiomer and 30% of the other, the ee is 40% (70% - 30%).
[0097] Enantiomers: Optically active isomers containing one or more asymmetric carbons that are non-superimposable mirror images of one another.
[0098] Indicator: As used herein, the term “indicator” refers to a measurable component indicative of a pathology, such as a component indicative of Alzheimer’s disease pathology.
[0099] Normalize: As used herein, the term “normalize” means to move a value (e.g., a level of a biomarker) closer to a “normal” or accepted standard value of the biomarker.
[0100] Pharmaceutically acceptable: A substance that can be taken into a subject without significant adverse toxicological effects on the subject. The term "pharmaceutically acceptable form" means any pharmaceutically acceptable derivative or variation, such as stereoisomers, stereoisomer mixtures, enantiomers, solvates, hydrates, isomorphs, polymorphs, pseudomorphs, neutral forms, salt forms, and prodrug agents.
[0101] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions and additional pharmaceutical agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some examples, the pharmaceutically acceptable carrier may be sterile to be suitable for administration to a subject (for example, by parenteral, intramuscular, or subcutaneous injection). In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In some examples, the pharmaceutically acceptable carrier is a non-naturally occurring or synthetic carrier. The carrier also can be formulated in a unit-dosage form that carries a preselected therapeutic dosage of the active agent, for example in a pill, vial, bottle, or syringe.
[0102] Subject: An animal (human or non-human) subjected to a treatment, observation or experiment. In some embodiments, the subject is a human having, or at risk of developing, Alzheimer’s disease.
[0103] Therapeutic time window: The length of time during which an effective, or therapeutic dose, of a compound remains therapeutically effective in vivo.
[0104] Treating or treatment: With respect to disease, either term includes (1) preventing the disease, e.g., causing the clinical symptoms of the disease not to develop in an animal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, e.g., arresting the development of the disease or its clinical symptoms, or (3) relieving the disease, e.g., causing regression of the disease or its clinical symptoms.
II. Treating or Preventing Neurological disorders with S-HCQ, Optionally in Combination with TUDCA and/or DIM
[0105] A neurological disorder is a disorder in which cells of the central nervous system stop working or die. As used herein, the term “neurological disorder” refers to diseases which are characterized by aberrant levels of one or more of the following indicators: extracellular amyloid beta (Af) concentration, tau phosphorylation, interleukin- 1 beta (ILl-beta) concentration, neuroinflammation, plasma neurofilament light chain protein (NIL) concentration, plasma glial fibrillary acidic protein (GFAP) concentration, and hippocampal synaptic plasticity. Impaired hippocampal synaptic plasticity is a well-established neurobiological surrogate of learning and memory. The neurological disorder may be characterized by increased extracellular Af> concentration, increased tau phosphorylation, increased neuroinflammation, increased plasma NfL concentration, increased plasma GFAP concentration, and/or impaired hippocampal synaptic plasticity. Exemplary neurological disorders that may be treated or prevented with S-HCQ, optionally in combination with TUDCA and/or DIM, include but are not limited to Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’ s disease (HD), vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, exhaustion disorder (ED), post-acute cognitive impairment and fatigue, or neuropsychiatric manifestations of systemic lupus erythematosus (SLE). In some aspects, the neurological disorder is AD, PD, HD, vascular dementia, ED, post-acute cognitive impairment and fatigue, or neuropsychiatric manifestations of SLE.
[0106] AD is characterized by well-known signs and symptoms, including cognitive impairment (e.g., mental decline, difficulty thinking and/or understanding, delusion, disorientation, forgetfulness, mental confusion, difficulty concentrating, inability to create new memories, inability to simple math, inability to recognize common objects), behavioral signs and symptoms (e.g., aggression, agitation, difficulty with self-care, irritability, meaningless repetition of own words, personality changes, restlessness, lack of restraint, wandering and getting lost), mood signs and symptoms (e.g., anger, apathy, general discontent, loneliness, mood swings), psychological signs and symptoms (e.g., depression, hallucination, paranoia), and other signs and symptoms (e.g., inability to combine muscle movements, jumbled speech, loss of appetite).
[0107] AD and the risk of developing AD also are characterized by certain biomarkers. For example, the risk of developing AD can be predicted by certain genetic markers. The e4 allele of the apolipoprotein-E (APOE) gene is the most robust genetic risk factor for sporadic or late-onset AD. Heterozygous carriers of the e4 allele are at 3-4 times greater risk of AD, while homozygous individuals are at 10 times greater risk relative to non-carriers (Farrer et al., JAMA 1997, 278: 1349- 1356; Neu et al., JAMA Neurol 2017, 74: 1178-1189; Genin et al., Mol Psychiatry 2011, 16:903-907). APOE e4 carriers accumulate AD neuropathology early in adulthood. A neuroimaging meta-analysis showed that 15% of non-demented APOE e4 homozygous individuals showed evidence of cerebral amyloid accumulation at 40 years of age (Jansen et al., JAMA 2015, 313: 1924-1938). Additionally, a study of regional cerebral glucose metabolism demonstrated that young, cognitively normal APOE e4 carriers between 20-39 years showed regional patterns of hypometabolism similar to those observed in patients with AD (Reiman et al., PNAS USA 2004, 101 :284-289). This suggests that APOE e4 contributes to AD risk over the course of several decades prior to disease onset. Other biomarkers characteristic of AD include, but are not limited to, increased levels of extracellular Af> concentration, tau phosphorylation, neuroinflammation, increased plasma NfL concentration, increased plasma GFAP concentration, or any combination thereof. For example, increased GFAP levels, a marker of neuronal injury, are observed a decade prior to AD onset suggesting that astrocyte activation is a plausible drug target in AD (and other neurode generative diseases as well as MS). STAT3 is a well- established regulator of GFAP expression. This further suggests that HCQ- mediated STAT3 inactivation not only underlies the rescue of AD phenotypes, but also is linked to GFAP activity (Hagemann et a/. , Cells, 2023, 12(7):978). [0108] In addition to the above biomarkers, a number of proteins exhibit altered expression in both AD and also in subjects at risk of developing AD but without significant AD pathology and/or symptoms. Based on the altered expression pattern of these proteins, the inventor, along with others, has established an AD proteomic signature in subjects with AD as well as an incipient AD proteomic signature in young APOE e4 carriers. The proteomic signatures are indicative of very early biological perturbations occurring during the long preclinical phase of AD and may present novel therapeutic molecular targets for disease modification. In some embodiments, administering an active agent targeting one or more of the proteins in the proteomic signature may at least partially normalize levels of one or more indicators of AD. In some embodiments, administering the active agent may modify development and/or progression of AD. Additional details of the proteomic signature are disclosed in WO 2023/059867 Al, which is incorporated by reference herein.
[0109] In some embodiments, a method includes administering to a subject an amount of an active agent effective to at least partially normalize an aberrant level of one or more indicators wherein the indicators comprise, or consist of, extracellular A[> concentration, tau phosphorylation, total tau, plasma NfL concentration, neuroinflammation, plasma GFAP concentration, hippocampal synaptic plasticity, or any combination thereof. In some aspects, the indicators may comprise ratios of certain indicators in cerebrospinal fluid, such as A[> I -42/A[> I -40, and phosphorylated tau/A(31-42. These indicators are associated with pathology of some neurological disorders. The indicators may be measured by any suitable means. In some embodiments, the indicators are measured by a positron emission tomography (PET) scan, e.g., brain amyloid deposition. A[i concentration, phosphorylated tau, and total tau also can be measured by cerebrospinal fluid and/or blood assays. NfL and GFAP concentration may be measured by blood tests. Hippocampal synaptic plasticity may be measured with neuroimaging methods, such as functional magnetic resonance imaging (fMRI) to determine functional connectivity and positron emission tomography (PET) to measure synaptic vesicle protein 2 A (SV2A).
[01 10] The active agent comprises an enantiomeric excess (ee) of the S-enantiomer of HCQ (S- HCQ). In some aspects, the ee of the S-HCQ is at least 20%, i.e., an HCQ composition comprising at least 60% of the S-enantiomer of the R-enantiomer of HCQ. In some implementations, the ee is at least 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the S-HCQ. In certain implementations, the ee is 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 70% to 100%, 80% to 100%, 90% to 100% of the S-HCQ, or even 95% to 100% of the S-HCQ. In any of the foregoing or following aspects, the active agent may further comprise TUDCA, DIM, or a combination thereof.
[0111] Normalizing the aberrant level of the one or more indicators may reduce extracellular A concentration, reduce tau phosphorylation, reduce plasma NfL concentration, reduce ILl-beta concentration, decrease neuroinflammation, reduce plasma GFAP concentration, increase hippocampal synaptic plasticity, or any combination thereof. In some embodiments, the active agent is effective in at least partially normalizing aberrant levels of at least two of the indicators. For example, the active agent may at least partially normalize levels of both A(3 concentration and plasma NfL concentration. In certain embodiments, the active agent at least partially normalizes aberrant levels of at least three indicators. For instance, the active agent may at least partially normalize levels of A(3 concentration, plasma NfL concentration, and IL 1 -beta concentration/neuroinflammation. Or, the active agent may at least partially normalize levels of A concentration, plasma NfL concentration, and hippocampal synaptic plasticity. Advantageously, an active agent that targets multiple pathologies (i.e., by at least partially normalizing levels of multiple indicators) may reduce or eliminate the need to treat the subject with multiple active agents or interventions, thereby facilitating treatment, facilitating subject compliance with the treatment regimen, and/or reducing likelihood of adverse side effects.
[0112] In one implementation, reducing neuroinflammation comprises reducing a concentration of one or more interleukins and/or inflammatory cytokines, such as interleukin-6 (IL-6), interleukin- 1 P (IL-1 P), interleukin IL-12pl70 (IL-12pl70), interleukin- 10 (IL-10), tumor necrosis factor alpha (TNF-a), or any combination thereof. In an independent implementation, reducing extracellular Ap concentration comprises reducing Ap secretion, increasing Ap clearance, or both.
[0113] The active agents disclosed herein are S-HCQ, a combination of S-HCQ and TUDCA, a combination of S-HCQ and DIM, or a combination of S-HCQ, TUDCA, and DIM. In any of the foregoing or following embodiments, the subject may not have previously received the active agent, e.g., for another indication.
[0114] In some aspects, the active agent is a combination of S-HCQ with TUDCA and/or DIM. Administering a combination can include administering (i) a single pharmaceutical composition comprising S-HCQ with TUDCA and/or DIM, or (ii) administering two or more pharmaceutical compositions, each pharmaceutical composition comprising at least one active agent, e.g., one pharmaceutical composition comprising S-HCQ and a second pharmaceutical composition comprising TUDCA and/or DIM. When administering two or more pharmaceutical compositions, the pharmaceutical compositions may be administered simultaneously or sequentially in any order. When administered sequentially, the pharmaceutical compositions preferably are administered such that therapeutic time windows of each of the active agents at least partially overlap. The pharmaceutical compositions may be administered by the same or different routes.
[0115] S-HCQ advantageously at least partially normalizes the levels of extracellular A concentration, tau phosphorylation, plasma NfL concentration, and neuroinflammation. S-HCQ also may at least partially normalize plasma GFAP concentration. S-HCQ increases A[3 clearance in microglial cells [0116] TUDCA, a bile acid, reduces Af> levels by reducing A |3 secretion. In some embodiments, TUDCA is administered in combination with S-HCQ or in combination with S-HCQ and DIM.
[0117] In some implementations, TUDCA is administered in combination with S-HCQ. The combination provides synergy with S-HCQ at least partially normalizing the levels of extracellular A(3 concentration by increasing A [5 clearance and/or decreasing A[i secretion and TUDCA reducing A[i secretion. The S-HCQ also provides additional benefits, e.g., increasing hippocampal synaptic plasticity as previously described. In one aspect, S-HCQ and TUDCA are administered in a single pharmaceutical composition comprising both S-HCQ and TUDCA. For example, S-HCQ and TUDCA may be combined into an oral dosage form, such as a suspension, tablet, or capsule comprising both S-HCQ and TUDCA. In an independent aspect, S-HCQ and TUDCA are administered simultaneously or sequentially in separate pharmaceutical compositions, one pharmaceutical composition comprising HCQ and one pharmaceutical composition comprising TUDCA. When administering two or more pharmaceutical compositions sequentially, the pharmaceutical compositions may be administered in any order. When administered sequentially, the pharmaceutical compositions preferably are administered such that therapeutic time windows of each of the active agents at least partially overlap. The pharmaceutical compositions may be administered by the same or different routes. For example, S-HCQ and TUDCA may be administered individually as oral dosage forms, such as suspensions, tablets, or capsules. In any of the foregoing or following aspects, S-HCQ may be administered as in the form of nanoparticles (nano-S-HCQ). In some aspects, nano-S-HCQ is in the form of poly(L-lysine succinylated)-S-HCQ (PLS-S-HCQ).
[0118] DIM is the dimeric product of the natural product indole-3-carbinole. DIM reduces A(3 levels and neuroinflammation, such as LPS-induced neuroinflammation. DIM has been shown to inhibit oxidative stress-induced apoptosis in hippocampal neuronal cells (Lee et al., Antioxidants 2019, 9( 1 ):3) and protect hippocampal cell cultures from ischemia-induced apoptosis and autophagy (Rzemieniec et al., Apoptosis: An International Journal on Programmed Cell Death, 2019, 24:435- 452). DIM has been shown to target the RGS4 gene (regulator of G-protein signaling 4) and to bind to the aryl hydrocarbon receptor, product of the AHR gene (Rzemieniec et al., Apoptosis: An International Journal on Programmed Cell Death, 2019, 24:435-452).
[0119] In some implementations, DIM is administered in combination with S-HCQ. The combination provides synergy with both S-HCQ and DIM at least partially normalizing the levels of extracellular A(3 concentration by increasing Af> clearance. Both S-HCQ and DIM also reduce neuroinflammation. In one aspect, S-HCQ and DIM are administered in a single pharmaceutical composition comprising both S-HCQ and DIM. For example, S-HCQ and DIM may be combined into an oral dosage form, such as a suspension, tablet, or capsule comprising both S-HCQ and DIM. In an independent aspect, S-HCQ and DIM are administered simultaneously or sequentially in separate pharmaceutical compositions, one pharmaceutical composition comprising S-HCQ and one pharmaceutical composition comprising DIM. When administering two or more pharmaceutical compositions sequentially, the pharmaceutical compositions may be administered in any order. When administered sequentially, the pharmaceutical compositions preferably are administered such that therapeutic time windows of each of the active agents at least partially overlap. The pharmaceutical compositions may be administered by the same or different routes. For example, S-HCQ and DIM may be administered individually as oral dosage forms, such as suspensions, tablets, or capsules. In any of the foregoing or following aspects, S-HCQ may be administered as in the form of nanoparticles (nano-S-HCQ). In some aspects, nano-S-HCQ is in the form of polylL-lysine succinylated)-S-HCQ (PLS-S-HCQ).
[0120] In some aspects, S-HCQ, TUDCA, and DIM are administered in combination. In one aspect, S-HCQ, TUDCA and DIM are administered in a single pharmaceutical composition comprising all three active agents. For example, S-HCQ, TUDCA, and DIM may be combined into an oral dosage form, such as a suspension, tablet, or capsule comprising all three active agents. In an independent aspect, S-HCQ, TUDCA, and DIM are administered simultaneously or sequentially in separate pharmaceutical compositions, one pharmaceutical composition comprising S-HCQ, one pharmaceutical composition comprising TUDCA, and one pharmaceutical composition comprising DIM. For example, S-HCQ, TUDCA, and DIM may be administered individually as oral dosage forms, such as suspensions, tablets, or capsules. Alternatively, S-HCQ, TUDCA, and DIM are administered simultaneously or sequentially in separate pharmaceutical compositions, where one pharmaceutical composition comprises two of the active agents, and another pharmaceutical composition comprises the third active agent. When administering two or more pharmaceutical compositions sequentially, the pharmaceutical compositions may be administered in any order. When administered sequentially, the pharmaceutical compositions preferably are administered such that therapeutic time windows of each of the active agents at least partially overlap. The pharmaceutical compositions may be administered by the same or different routes. In any of the foregoing or following aspects, S-HCQ may be administered as in the form of nanoparticles (nano-S-HCQ). In some aspects, nano-S-HCQ is in the form of poly(L-lysine succinylated)-S-HCQ (PLS-S-HCQ).
[0121] In any of the foregoing or following embodiments, the subject may be diagnosed as having a neurological disorder prior to administering the active agent(s). In some embodiments, the subject may have an aberrant level of one or more indicators of neurological pathology, such as increased levels of extracellular A concentration, increased plasma NfL concentration, increased neuroinflammation, increased plasma GFAP concentration, and/or impaired hippocampal synaptic plasticity.
[0122] In any of the foregoing or following embodiments, the subject may be at risk of developing a neurological disorder, and the method may further comprise identifying the subject as being at risk of developing a neurological disorder prior to administering the active agent(s) prophylactically. In any of the foregoing or following embodiments, the subject may further be identified as not having previously received the active agent(s).
[0123] In some embodiments, the subject is identified as having a neurological disorder based on an aberrant level of one of more indicators, such as increased extracellular A(3 concentration, presence of neuroinflammation and/or increased neuroinflammation, increased plasma NfL concentration, increased plasma GFAP concentration, and/or impaired hippocampal synaptic plasticity. The subject may be identified as being at risk of developing a neurological disorder by identifying the subject as having an elevated level of the one or more indicators (e.g., in the brain, cerebrospinal fluid, and/or blood) relative to a normal level of the one or more indicators in the absence of any physical, cognitive, behavioral, mood, or psychological signs or symptoms of a neurological disorder.
[0124] Less commonly, the subject may be identified as having a neurological disorder or being at risk of developing a neurological disorder by measuring initial levels of one or more of the indicators, administering the active agent, and subsequently measuring a post-administration level of the indicator/ s). In certain implementations, the subject is administered the active agent for an effective period of time prior to measuring the post-administration level. The effective period of time may be, for example, two weeks, one month, two months, three months, six months, or longer. A postadministration level of the indicator(s) that is lower than the initial level may be indicative of the subject having a neurological disorder or being at risk of developing a neurological disorder.
[0125] As previously discussed, exemplary neurological disorders that may be treated and/or prevented with the disclosed active agents include, but are not limited to, AD, PD, HD, vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, and cognitive impairment no dementia. In some aspects, the neurological disorder is AD, PD, HD, vascular dementia, ED, post-acute cognitive impairment and fatigue, or ncuropsychiatric manifestations of SLE. In certain aspects, the neurological disorder is AD.
[0126] In some embodiments, the subject is identified as being at risk of a neurological disorder based on an aberrant level of one of more indicators, such as extracellular A(5 concentration, plasma NfL concentration, neuroinflammation, plasma GFAP concentration, and/or hippocampal synaptic plasticity, or any combination thereof. In some implementations, the subject is identified as having AD or being at risk of developing AD by identifying the subject as being an APOE s4 carrier. The subject may be identified as being at risk of developing AD by identifying the subject as being an APOE e4 carrier and/or as having an elevated level of the one or more indicators relative to a normal level of the one or more indicators in the absence of any cognitive, behavioral, mood, or psychological signs or symptoms of AD. Alternatively, the subject may be identified as having a neurological disorder or being at risk of developing a neurological disorder by measuring initial levels of one or more of the indicators, administering the active agent, and subsequently measuring a postadministration level of the indicator(s) as described above. A post-administration level of the indicator(s) that is lower than the initial level may be indicative of the subject having a neurological disorder or being at risk of developing a neurological disorder. In some implementations, the subject at risk of developing a neurological disorder is administered an active agent comprising S-HCQ. In certain implementations, the active agent further comprises TUDCA, DIM, or a combination thereof. [0127] In any of the foregoing or following embodiments, it may be beneficial to monitor levels of the one or more indicators to assess severity and/or progression of the pathology, to assess treatment efficacy, or both. In some embodiments, the method further includes receiving data comprising an initial level of the one or more indicators prior to administering the active agent to the subject. For example, imaging evaluation, such as PET scans, may determine levels of A plaques in the brain (e.g., as evidenced by extent of A[> deposition), extent of neurofibrillary tangles, and/or severity neuroinflammation. Extracellular A concentration, levels of phosphorylated and total taumay also be determined by laboratory evaluation, such as cerebrospinal fluid and/or blood-based assays. Plasma or cerebrospinal fluid levels of NIL and/or GFAP concentration may also be measured by blood-based assays. In humans, hippocampal synaptic dysfunction may be assessed by methods such as fMRI and quantitative electroencephalography (qEEG). However, fEPSPs can only be measured in animal models. In certain implementations, the method may further include receiving data comprising a post-administration level of the one or more indicators following administration of the active agent to the subject, and selecting an adjusted amount of the active agent for administration to the subject based at least in part on the post-administration level of the one or more indicators. When the active agent comprises S-HCQ in combination with TUDCA and/or DIM, selecting an adjusted amount of the active agent comprises selecting an adjusted amount of at least one component of the active agent. For example, when the active agent comprises S-HCQ and TUDCA, the amount of S-HCQ, TUDCA, or both may be adjusted. Similarly, when the active agent comprises S-HCQ and DIM, the amount of S-HCQ, DIM, or both may be adjusted.
[0128] In any of the foregoing or following embodiments, the method may comprise administering the active agent to the subject prophylactically in the absence of any physical, cognitive, behavioral, mood, or psychological signs or symptoms of a neurological disorder. For example, the active agent may be administered prophylactically to a subject identified as being at risk of developing a neurological disorder.
III. Pharmaceutical Compositions and Dosing
[0129] Another aspect of the disclosure includes pharmaceutical compositions prepared for administration to a subject and which include an effective amount of one or more of the active agents disclosed herein. The therapeutically effective amount of a disclosed active agent will depend on the route of administration and the physical characteristics of the subject being treated. Specific factors that can be taken into account include disease severity and stage, weight, diet and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed active agents is understood by those of skill in the art.
[0130] Pharmaceutical compositions for administration to a subject can include at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. Remington ’s Pharmaceutical Sciences, by E. W. Mar“n, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the active agents herein disclosed.
[0131] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In some embodiments, the formulation may comprise a plurality of nanoparticles, the nanoparticles comprising the active agent.
[0132] Pharmaceutical compositions disclosed herein include those formed from pharmaceutically acceptable salts and/or solvates of the disclosed active agents. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Particular disclosed active agents may possess at least one basic group that can form acid-base salts with acids. Examples of basic groups include, but are not limited to, amino and imino groups. Examples of inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid. Basic groups also can form salts with organic carboxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, and, in addition, with amino acids, for example with a-amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2-hydroxy methanesulfonic acid, ethane- 1,2-disulfonic acid, benzenedisulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2- sulfonic acid, 2- or 3- phosphoglycerate, glucose-6-phosphate or A'-cyclohcxy Isul famic acid (with formation of the cyclamates) or with other acidic organic compounds, such as ascorbic acid. In particular, suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. [0133] Certain active agents may include at least one acidic group that can form an acid-base salt with an inorganic or organic base. Examples of salts formed from inorganic bases include salts of the presently disclosed active agents with alkali metals such as potassium and sodium, alkaline earth metals, including calcium and magnesium and the like. Similarly, salts of acidic compounds with an organic base, such as an amine (as used herein terms that refer to amines should be understood to include their conjugate acids unless the context clearly indicates that the free amine is intended) are contemplated, including salts formed with basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, pyridines, guanidines and amidines. In addition, quaternary ammonium counterions also can be used.
[0134] Particular examples of suitable amine bases (and their corresponding ammonium ions) for use in the present active agents include, without limitation, pyridine, A'.A'-dinicthylaminopyridinc, diazabicyclononane, diazabicycloundecene, A nethyl-A'-ethy larni ne, diethylamine, triethylamine, diisopropylethylamine, mono-, bis- or tris- (2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, tris(hydroxymethyl)methylamine, A',A,-dimethyl-A'-('2- hydroxyethyl)amine, tri-(2- hydroxyethyl)amine and A'-methy l-D-glucaminc. For additional examples of "pharmacologically acceptable salts," see Berge et al., J. Pharm. Sci. 66:1 (1977).
[0135] The pharmaceutical compositions can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the compositions can be administered by non- mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes.
[0136] To formulate the pharmaceutical compositions, the active agent can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the active agent. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (for example, benzyl alcohol), isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween 80 or Miglyol 812), solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund’s adjuvant, MPL™ (3-0- deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL- 12 (Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, can be included in the compositions. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7.
[0137] The active agent can be dispersed in a base or vehicle, which can include a hydrophilic compound having a capacity to disperse the active agent, and any desired additives. The base can be selected from a wide range of suitable compounds, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl (meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or vehicle, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The vehicle can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to a mucosal surface.
[0138] The active agent can be combined with the base or vehicle according to a variety of methods, and release of the active agent can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the active agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, for example, Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time. For example, S-HCQ may be administered in nanoparticulate form, such as in S-HCQ-loaded liposomes, polymer-based nanoparticles or micelles, lipid-based nanoparticles, nanoemulsions, and metallic nanoparticle conjugates (Stevens et al., Molecules 2021, 26(1): 175). Exemplary polymers include, but are not limited to, methoxy PEG-b-poly(L-lactic acid), poly(lactic-co-glycolic acid) (PLGA), poly(N-isopropylacrylamide-co-acrylic acid), and poly(L- lysine). In some aspects, S-HCQ is administered in nanoparticulate form as poly(L-lysine succinylated)-S-HCQ (PLS-S-HCQ).
[0139] The compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
[0140] Pharmaceutical compositions for administering the active agent can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the active agent can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
[0141] In certain embodiments, the active agent can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearatc hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body. [0142] Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include polyglycolic acids and polylactic acids, poly(DL-lactic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(E-caprolactone), poly(E-caprolactone-CO-lactic acid), poly(s- caprolactone-CO-glycolic acid), poly([3-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels, such as polyihydroxyethyl methacrylate), polyamides, poly(amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), polylester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release microcapsules (U.S. Patent Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Patent Nos. 4,677,191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Patent No. 4,675,189).
[0143] The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active agent plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
[0144] The effective amount of the active agent will depend upon the severity of the disease and the general state of the subject's health. An effective amount is that which provides either subjective relief of one or more signs or symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer. In one embodiment, an effective amount is the amount necessary to at least partially normalize a level of one or more indicators associated with AD. The effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated.
[0145] The actual dosage of the active agent will vary according to factors such as the disease indication and particular status of the subject (for example, the subject’ s age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the active agent for eliciting the desired response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. An effective amount is also one in which any toxic or detrimental side effects of the active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for an effective amount of an active agent within the methods and formulations of the disclosure is about 0.01 mg/kg body weight to about 20 mg/kg body weight, such as about 0.05 mg/kg to about 10 mg/kg body weight, about 0.2 mg/kg to about 10 mg/kg body weight, or about 1 mg/kg to about 10 mg/kg.
[0146] Dosage can be varied by the attending clinician as previously described, such as based on a determined level of the one or more indicators associated with AD. Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, intraosseous, or intranasal delivery versus intravenous or subcutaneous or intramuscular delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.
[0147] In some aspects, an S-HCQ dosage for an adult human may be from 50 mg to 1000 mg, such as from 200 mg to 500 mg, or from 0.5 mg/kg body weight to 20 mg/kg body weight, such as from 2 mg/kg body weight to 10 mg/kg body weight, daily in one dose or two or more divided doses. In some aspects, a TUDCA dosage for an adult human may be from 100 mg to 2000 mg, such as from 500 mg to 1000 mg, or from 1 mg/kg body weight to 40 mg/kg body weight daily, such as from 5 mg/kg body weight to 20 mg/kg body weight, daily in one dose or two or more divided doses. In some aspects, an DIM dosage for an adult human may be from 25 mg to 200 mg, such as from 100 mg to 150 mg, or from 0.25 mg/kg body weight to 4 mg/kg body weight, such as from 1 mg/kg body weight to 3 mg/kg body weight, daily in one dose or two or more divided doses.
[0148] For prophylactic and therapeutic purposes, the active agent can be administered to the subject by the oral route or in a single bolus delivery, via continuous delivery (for example, continuous intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The effective dosage of the active agent can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms associated with AD and/or at least partially normalize a level of one or more indicators associated with AD.
Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of AD symptoms or at least partially normalize the level of the one or more indicators in the subject. Suitable models in this regard include, for example, murine, rat, avian, dog, sheep, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer an effective amount of the active agent.
[0149] Treatment can involve daily or multi-daily doses of active agent(s) over a period of a few days to months, or even years. Thus, the dosage regimen will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner. In particular examples, the subject is administered a therapeutic composition that includes one or more of the disclosed active agents on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years. In one example, the subject is administered the composition for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, at least 5 years, at least 10 years, or indefinitely for the remainder of the subject’s life.
[0150] In some embodiments, the subject may further be administered additional therapeutic agents. For example, the subject may be administered one or more additional therapeutic agents used for treating a neurological disorder or for treating one or more signs or symptoms associated with a neurological disorder. For example, the additional therapeutic agent may be a therapeutic agent used for treating AD, Parkinson’ s disease, Huntington’ s disease, vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, or any combination thereof. Preparation and dosing schedules for the additional agent may be used according to manufacturer's instructions or as determined empirically by the skilled practitioner. The combination therapy may provide synergy and prove synergistic, that is, the effect achieved when the active agent and therapeutic agent used together is greater than the sum of the effects that results from using the active agent and therapeutic agent separately. A synergistic effect may be attained when the active agent and additional therapeutic agent are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation, a synergistic effect may be attained when the active agent and therapeutic agent are administered or delivered sequentially, for example by different injections in separate syringes. In general, during alternation, an effective dosage of the active agent and of the therapeutic agent is administered sequentially, i.c. serially, whereas in combination therapy, effective dosages of the active agent and therapeutic agent are administered together. [0151] The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Kits for diagnostic use are also provided. In one embodiment, these kits include a container or formulation that contains one or more of the active agents described herein. In one example, this component is formulated in a pharmaceutical preparation for delivery to a subject. The active agent is optionally contained in a bulk dispensing container or unit or multi-unit dosage form. Optional dispensing means can be provided, for example a pulmonary or intranasal spray applicator.
Packaging materials optionally include a label or instruction indicating for what treatment purposes and/or in what manner the pharmaceutical agent packaged therewith can be used.
V. Examples
[0152] Example 1
[0153] Effect ofHCQ on AD-Related Phenotypes in Cell Cultures
[0154] The ability of HCQ to rescue molecular phenotypes relevant to AD including A 1-42 clearance, A[3 secretion, A toxicity, tau phosphorylation, lipopolysaccharide (LPS)-induced neuroinflammation, cell death due to trophic factor withdrawal and neurite outgrowth was investigated.
[0155] Methods
[0156] A i i-42 clearance: For Abeta (AP1.42) clearance assay, 20,000 BV-2 cells per well (uncoated 96 well plates) were plated out. After changing cells to treatment medium, HCQ (0.25pM, 2.5 pM and 25 pM) was applied 1 hour before AP1.42 stimulation (Bachem 4061966; final concentration in well: 200 ng/mL (dilutions in medium)). Cells treated with vehicle (0.1% DMSO) and cells treated with A M2 alone served as controls. After 3 h of APj.42 stimulation, cell supernatants were collected for the APM2 measurement and cells were carefully washed twice with PBS and thereafter lysed in 35 pL cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS) supplemented with protease inhibitors. Supernatants and cell lysates were analyzed for human AP1.42 with MSD® V- PLEX Human Ap42 Peptide (6E10) Kit (K151LBE, Mesoscale Discovery). The immune assay was carried out according to the manufacturer’s manual and plates were read on the MESO QuickPlex SQ 120.
[0157] Ajffi.42 clearance-Protonex assay: For Abeta (AP1.42) clearance Protonex assay, 5000 BV-2 cells per well (uncoated 96 well plates) were plated out. After changing cells to treatment medium, HCQ (25 pM) was applied 1 hour before stimulation with Protonex™ Green 500, SE (21216, AAT Bioquest) labelled AP1-42 (Bachem 4061966; final concentration in well: 200 ng/mL (dilutions in medium)). Cells treated with vehicle and cells treated with AP1.42 alone served as controls. After 3 h of A [31 -12 stimulation, cells were imaged on Cytation 5 multimode reader (Biotek) and green fluorescence was measured.
[0158] A/> secretion: Human APP overexpressing H4-hAPP cells were cultivated in Opti-MEM supplemented with 10% FCS, 1% penicillin/streptomycin 200 pg/mL Hygromycin B and 2.5 pg/mL Blasticidin S. H4-hAPP cells were seeded into 96 well plates (2 x 104 cells per well). On the next day, cells in 96 well plates were treated HCQ (0.25 M, 2.5 pM and 25 pM) or the reference item (DAPT 400 nM), a y-secretase inhibitor vehicle. 24 h later, supernatants were collected for further A0 measurements by MSD® (V-PLEX A(3 Peptide Panel 1 (6E10) Kit, K15200E, Mesoscale Discovery).
[0159] Aft toxicity: Primary hippocampal neurons were prepared from E18.5 timed pregnant C57BL/6JRccHsd mice as previously described. Cells were seeded in poly-D-lysine pre-coated 96- well plates at a density of 4x104 cells/well and cultivated until DIVIO (Neurobasal, 2% B-27, 0.5 mM glutamine, 25 pM glutamate, 1% Penicillin-Streptomycin). On DIVIO pre-aggregated A 1-42 (Bachem 4061966, final concentration 10 pM, 48h at 4°C) was added to the cells in the presence or absence of Hydroxychloroquine sulfate (TargetMol, T0951) at 25 pM, 2.5 pM or 0.25 pM concentrations. On DIV 16 cells were subject to MTT assay to determine cell viability.
[0160] Tau phosphorylation: SH-SY5Y-hTau441(V337M/R406W) cells were maintained in culture medium (DMEM medium, 10% FCS, 1% NEAA, 1% L-Glutamine, 100 pg/mL Gentamycin, 300 pg/mL Geneticin G-418) and differentiated with 10 pM retinoic acid (RA) for 5 days changing medium every 2 to 3 days. Prior to the treatment, cells were seeded onto 24-well plates at a cell density of 2 x 105 cells per well (DIV1). HCQ (25 pM) was applied on DIV2. After 24 h of incubation (DIV3), cells on 24-well plates were harvested in 60 pL RIPA-Buffer [50 mM Tris pH 7.4, 1% Nonidet P40, 0.25% Na-deoxy-cholate, 150 mM NaCl, 1 mM EDTA supplemented with freshly added 1 pM NaF, 0.2 mM Na-ortho-vanadate, 80 pM Glycerophosphate, protease (Calbiochem) and phosphatase (Sigma) inhibitor cocktail]. Protein concentration was determined by BCA assay (Pierce, ThermoFisher) and samples were adjusted to a uniform total protein concentration. Total Tau and phosphorylated Tau were determined by immunosorbent assay from Mcsoscalc Discovery (Phospho(Thr231)/Total Tau Kit K15121D, Mesoscale Discovery).
[0161] Lipopolysaccharide (LPS)-induced neuroinflammation: The murine microglial cell line BV-2 was cultivated in DMEM medium supplemented with 10% FCS, 1% penicillin/streptomycin and 2 mM L-glutamine (culture medium). For LPS stimulation assay, 5000 BV-2 cells per well (uncoated 96 well plates) were plated out and the medium was changed to treatment medium (DMEM, 5% FCS, 2 mM L-glutamine). After changing cells to treatment medium, HCQ (0.25 pM, 2.5 pM and 25 pM) was applied 1 hour before LPS stimulation (Sigma-Aldrich; L6529; 1 mg/ml stock in ddH O. final concentration in well: 100 ng/mL (dilutions in medium)). Cells treated with vehicle, cells treated with LPS alone, as well as cells treated with LPS plus reference item (dexamethasone 10 pM, Sigma D4902) served as controls. After 24 h of stimulation, cell supernatants were collected for the cytokine measurement (V-PLEX Proinflammatory Panel 1 Mouse Kit, K15048D, Mesoscale) and cells were subjected to MTT assay.
[0162] Cell death due to trophic factor withdrawal: Primary cortical neurons from E18 C57B1/6 mice were prepared as previously described. On the day of preparation (DIV1), cortical neurons were seeded on poly-D-lysine pre-coated 96-well plates at a density of 3 x 104 cells per well. Every 4-6 days, a half medium exchange using full medium (Neurobasal, 2% B-27, 0.5 mM glutamine, 1% Penicillin-Streptomycin) was carried out. On DIV8, a full medium exchange to B-27 free medium (Neurobasal, 0.5 mM glutamine, 1% Penicillin-Streptomycin) was performed and HCQ (0.25pM.
2.5 pM and 25 pM) was applied thereafter. The experiment was carried out with n=6 technical replicates per condition, vehicle treated cells served as control. After 28 h on B-27 free medium, cells were subject to YOPRO/PI and MTT as well as LDH assay.
[0163] YO-PRO™-1 (Invitrogen; Y3603) assay was carried out to detect apoptotic cells in combination with Propidium iodide (PI; P4864 Sigma Aldrich) staining for necrotic cells. Part of the supernatant of the cultivated cells was sucked off, so that 90 pL remained per well. 50 pM YO-PRO 1 solution was prepared out of the 1 mM YO-PRO 1 stock solution in DMSO. The stock solution was diluted in a ratio of 1 :20 in PBS and Propidium iodide (PI) was added to the same stock to a final concentration of 1 pg/mL. 10 pL of this 50 pM YO-PRO 1/lpg/mL PI solution in PBS was added to the remaining 90 pL to result in a final concentration of 5 pM YO-PRO 1 in the well. Incubation for 15 min in the incubator at 37°C was performed (light protected). Supernatant was aspirated completely and discarded. 140 pL PBS was added to well. Plate was measured at the multimodereader (Cytation 5, BioTek).
[0164] MTT solution was added to each well in a final concentration of 0.5 mg/mL. After 2 h, the MTT containing medium was aspirated. Cells were lysed in 3% SDS and the formazan crystals were dissolved in isopropanol/HCl. Optical density was measured with a Cytation 5 (Biotek) multimode reader at wavelength 570 nm. Values were calculated as percent of control values (vehicle control or lesion control).
[0165] The Lactate dehydrogenase (LDH) toxicity assay was carried out on the supernatants collected after treatment using the Cytotoxicity Detection Kit (Roche Diagnostics, Cat. No: 11 644 793 001). 70 pL of cell culture supernatant was transferred to clear 96-well plates. 70 pL freshly prepared reaction mixture was added to each well and the mixture was incubated for 1 h at room temperature protected from light. Absorbance was measured at 492 nm and 620 nm as reference wavelength with a Cytation 5 (Biotek) multimode reader. Values of culture medium were subtracted as background control. Values were calculated as percent of control values (vehicle control or lesion control).
[0166] Neurite outgrowth and neurogenesis: Primary hippocampal neurons were prepared from E18.5 timed pregnant C57BL/6JRccHsd mice as previously described. Cells were seeded in poly-D- lysine pre-coated 96- well plates at a density of 2.6x104 cells/well in (Neurobasal, 2% B-27, 0.5 mM glutamine, 25 pM glutamate, 1% Penicillin-Streptomycin). Directly on DIV1 HCQ (TargetMol, T0951) at 25 pM, 2.5 pM or 0.25 pM concentrations or vehicle control was applied. On DIV2 10 pM Bromodeoxyuridine (BrdU; B5002 Sigma Aldrich) was added and cells were fixed after additional 24h. Cells were permeabilized with 0.1% Triton-X and incubated with primary Beta Tubulin Isotype III (T8660, Sigma Aldrich) and BrdU antibodies (MAS250c, AHrlan-Sera Lab) overnight at 4°C. Afterwards cells were washed two times with PBS and incubated with fluorescently labeled secondary antibodies and DAPI for 1.5 hour at RT in the darkness. Cells were rinsed three times with PBS imaged with the Cytation 5 Multimode reader (BioTek) at lOx magnification (6 images per well). BrdU positive cells were counted as marker for neurogenesis and Beta Tubulin Isotype III signal was used for macro-based quantification of neurite outgrowth.
[0167] Statistical analyses: Statistical analysis was performed in GraphPad Prism 9.1.2. Group differences were evaluated for each test item separately by One-way ANOVA followed by Dunnett’s multiple comparison test versus vehicle or lesion control.
[0168] Western blot analyses: Samples for western blot analyses were prepared from 3 assays which provided evidence of HCQ-associated rescue (i.e., Abw2 clearance, tau phosphorylation, and LPS- induced neuroinflammation). Samples were prepared identically as described above in section: Effect of HCQ on AD-related phenotypes in cell culture, and scaled up to 24-well format to allow for adequate sample volume. After HCQ treatment, cells were washed once with PBS and harvested in 50 pL 50 M HEPES + 2% SDS lysis buffer. The extracts were then boiled at 95° C for 5 mins. Analytic methods were identical to those described above in Example 4.
[0169] Statistical analyses: Statistical analysis was performed using STATA 16.1. Group differences were evaluated using two-sample t-tests (parametric) to calculate differences in p-STAT3 levels between HCQ treated and control samples. We additionally used the Wilcoxon rank-sum test (non-parametric) to confirm that results were robust to distributional assumptions. Significant differences were indicated as p < 0.05.
[0170] Results
[0171] To test whether HCQ rescues molecular outcomes relevant to AD in cell culture -based phenotypic assays, its effects on AP1.42 clearance, secretion, and toxicity, tau phosphorylation, lipopolysaccharide (LPS)-induced neuroinflammation, cell death due to trophic factor withdrawal and neurite outgrowth and neurogenesis were evaluated.
[0172] In murine BV-2 microglial cells, HCQ (25 pM) increased A|3M2 clearance as shown by reduced levels of A|3M2 in the supernatant and a lowering of the AP1.42 supernatant:lysate ratio (i.e. phagocytized A 1.42 in cells) without adverse effects on cell viability (FIG. 1A). HCQ (25 pM) reduced levels of total tau and phosphorylated tau (ptau231) in SH-SY5Y cells overexpressing human mutant tau-hTau441(V337M/R406W) (FIG. IB). In the murine BV-2 microglial cell line following stimulation with LPS, HCQ (2.5 pM, 25 pM) reduced levels of secreted pro-neuroinflammatory cytokines in a dose dependent manner. A reduction in TNF-alpha secretion was observed at the highest HCQ concentration (25pM) and a dose dependent (2.5 pM<25 pM) reduction in IL-6, IL- lb, IL-12p70 and IL-10 occurred without any adverse effects on cell viability (FIG. 1C).
[0173] In human APP over-expressing H4 neuroglioma cells, HCQ did not alter levels of secreted and had no effects on toxicity due to exogenous Af> in primary hippocampal neurons. No effects of HCQ on cell death were observed in response to trophic factor withdrawal in primary cortical neurons, or neurite outgrowth and neurogenesis in primary hippocampal neurons.
[0174] To test whether HCQ-related amelioration of molecular abnormalities relevant to AD was associated with inactivation of STAT3, levels of total and phosphorylated STAT3 were examined to determine whether they were reduced by HCQ treatment in the phenotypic assays of A [3 , 1 clearance, tau phosphorylation, and LPS-induced neuroinflammation. Cell homogenates from each of these assays were harvested and tested to determine whether levels of total STAT3 and phosphorylated STAT3 (Tyr705 and Ser727 epitopes) differed between HCQ-treated cells and the relevant vehicular control (VC) condition. As shown in FIGS. 2A-2B, western blot results, and the accompanying graphs, showed that in the A [U clearance assay, the highest concentration of HCQ (25pM) lowered p-STAT3 (Tyr705) relative to vehicular control (VC). In the tau phosphorylation assay (FIGS. 2C- 2D), HCQ lowered p-STAT3 (Tyr705) levels relative to VC at 25pM and 2.5pM concentrations, and p-STAT3 (Ser727) levels at 25pM, 2.5pM, and 0.25 pM concentrations. In the LPS-induced neuroinflammation assay (FIGS. 2E-2G), p-STAT3 (Tyr705) was significantly lower than the VC at the highest HCQ concentration (25pM).
[0175] Example 2
[0176] HCQ Rescue of Impaired Long-Term Potentiation in the Hippocampus
[0177] Methods
[0178] Animals: All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore. We used a transgenic mouse model of AD, which expresses a mutated chimeric mouse/human APP and the exon-9-deleted variant of human PSI, both linked to familial AD, under the control of a prion promoter element (APPSwe/PSldE9), which we denote as APP/PS1 (Borchelt et al., Neuron 1997, 19(4):939-45). A total of 35 hippocampal slices (21 APP/PS1 and 14 WT) were prepared from 9 APP/PS1 mice and 7 WT mice. Animals were housed under 12h light/12h dark conditions with food and water available ad libitum. [0179] Hippocampal slice preparation: Animals were anaesthetized briefly using CO2 and were decapitated. Brains were quickly removed in 4 °C artificial cerebrospinal fluid (aCSF), a modified Krebs-Ringer solution containing the following (in mM): 124 NaCl, 3.7 KC1, 1.2 KH2PO4, 1 MgSO4-7H2O. 2.5 CaC12-2H2O, 24.6 NaHCO3, and 10 D-glucose. The pH of aCSF was between 7.3 and 7.4 when bubbled with 95% oxygen and 5% carbon dioxide (carbogen). Both right and left hippocampi were dissected out in cold (2-4 °C) aCSF, which was being continuously bubbled with carbogen. Transverse hippocampal slices of 400 pm thickness were prepared from the right and left hippocampus using a manual tissue chopper (Stoelting, Wood Dale, Illinois), and transferred onto a nylon net placed in an interface chamber (Scientific Systems Design, Ontario, Canada) and incubated at 32 °C at an aCSF flow rate of 1 ml/min and carbogen consumption of 16 1/h. The entire process of animal dissection, hippocampal slice preparation and placement of slices on the chamber was done within approximately five minutes to ensure that hippocampal slices were in good condition for electrophysiology studies. The slices were incubated for at least 3 h before starting the experiments.
[0180] Field potential recordings: In all the electrophysiology recordings, two-pathway experiments were performed. Two monopolar lacquer-coated stainless-steel electrodes (5MQ; AM Systems, Sequim) were positioned at an adequate distance within the stratum radiatum of the CAI region for stimulating two independent synaptic inputs SI and S2 of one neuronal population, thus evoking field excitatory postsynaptic potentials (fEPSP) from Schaffer collateral/commissural-CAl synapses (FIG. 3A). One electrode (5MQ; AM Systems) represented as ‘rec’ was placed in the CAI apical dendritic layer for recording fEPSP. After the pre-incubation period, a synaptic input-output curve (afferent stimulation vs. fEPSP slope) was generated. Test stimulation intensity was adjusted to elicit fEPSP slope of 40% of the maximal slope response for both synaptic inputs SI and S2. The signals were amplified by a differential amplifier, digitized using a CED 1401 analog-to-digital converter (Cambridge Electronic Design, Cambridge, UK) and monitored online with custom-made software. To induce late long-term potentiation (L-LTP), a “strong” tetanization (STET) protocol consisting of three trains of 100 pulses at 100 Hz (single burst, stimulus duration of 0.2 ms per polarity), with an inter-train interval of 10 min, was used. In all experiments, a stable baseline was recorded for at least 30 min using four 0.2-Hz biphasic constant-current pulses (0.1 ms per polarity) at each time point. Four 0.2-Hz biphasic, constant current pulses (spaced at 5 s) given every live minutes were used for post-induction recordings also and the average slope values from the four sweeps was considered as one repeat while used for plotting. Initial slopes of fEPSPs were expressed as percentages of baseline averages. A series of pulses ranging from 0, 10, 20, 30, 40, 50, 70, 100 microamperes were applied to generate an input output curve. Graphs are plot as stimulus intensity versus fEPSP slope. Paired pulse ratio (PPR) was evoked using an interstimulus interval of 50 ms at 40 % of maximum stimulus intensities. PPR was expressed as the ratio of the fEPSP slope of second stimulus to the first stimulus. [0181] Pharmacology: Hydroxychloroquine sulphate (HCQ) (Selleckchem, catalog, No-S4430) was stored at -20°C as 50 mM stock in deionized water. Before application, the stock solution was diluted to a final concentration of 25 pM or 50 pM in aCSF and bath-applied for a total of 60 min, 30 min before and 30 min after the STET or unless otherwise specified.
[0182] Statistical analysis: All data are represented as mean ± SEM. The fEPSP slope value expressed as percentages of average baseline values per time point was subjected to statistical analysis using Graph Pad Prism (Graph Pad, San Diego, CA, USA). Wilcoxon signed rank test (Wilcox test) was used to compare fEPSP values within one group and Mann-Whitney U test (U test) was used when data were compared between groups. Statistical comparisons for input-output (VO) curve and paired pulse facilitation (PPF) experiments were performed using two-way ANOVA test. P<0.05 was considered as the cutoff for statistically significant differences.
[0183] Results
[0184] To test whether HCQ may rescue impaired hippocampal synaptic plasticity in AD, its effects on late-long term potentiation (L-LTP), a form of synaptic plasticity that has been shown to be impaired before the accumulation of AD pathology in the APP/PS1 transgenic mouse model of AD, were studied. FIG. 3A illustrates the location of electrodes in hippocampal slice preparations from APP/PS1 transgenic and WT mice (age 12 weeks). As a control, using hippocampal slices from WT mice, L-LTP was first recorded by applying strong tetanization (STET; SI electrode) the, following the recording of a stable baseline for 30 min (FIG. 3B). This resulted in a long-lasting and stable L- LTP during a recorded time period of 180 min (FIG. 3B: filled circles). The control input remained stable throughout the recording time period. (FIG. 3B: open circles). Statistically significant differences were observed from 1 min until 180 min when compared with its own baseline and with control input S2 (1 min Wilcox, P=0.01, 1 min U-test, P=0.0006; 60 min Wilcox, P=0.01, 60 min U- test, P=0.0006; 120 min Wilcox, P=0.01, 120 min U-test, P=0.0006; 180 min Wilcox, P=0.01, 180 min U-tcst, P=0.0006).
[0185] Next, the same experimental paradigm was used to study L-LTP in hippocampal slices from APP/PS 1 mice. As indicated in FIG. 3C, applying STET to S 1 resulted in only short-lasting form of LTP (early-LTP) (FIG. 3C: filled circles) while the control input S2 remained stable (FIG. 3C: open circles). Significant difference was observed only until 120 min (175 min Wilcox, P=0.15, 120 min U-test, P=0.09).
[0186] The effect of HCQ (25 pM applied before and after induction of STET) was then tested on L- LTP in hippocampal slices from APP/PS 1 mice. As shown in FIG. 3D, bath application of HCQ induced late-LTP which was significantly different from 1 min up to 180 min when compared to its own baseline and S2 (1 min Wilcox, P=0.007, 1 min U-tcst, P=0.0002; 60 min Wilcox, P=0.007, 60 min U-test, P=0.0002; 120 min Wilcox, P=0.007, 120 min U-test, P=0.0003; 180 min Wilcox, P=0.01, 180 min U-test, P=0.01). At this concentration, HCQ (25 pM) induced L-LTP in APP/PS1 hippocampus that was similar in magnitude to wild type up to 60 minutes (i.e. no statistically significant differences between L-LTP in Tg versus WT; 1 min Wilcox, P-X, 1 min U-test, P=X; 60 min Wilcox, P-X, 60 min U-test, P-X) (FIG. 3D vs FIG. 3B). From 70 minutes through 180 minutes, L-LTP in APP/PS1 hippocampus after HCQ application (25 pM) was significantly lower than in WT (70 min, U-test, P=0.009; 180 min, U-test, P=0.01) (FIG. 3D vs FIG. 3B), suggesting that HCQ at 25 pM partially rescued impaired L-LTP in APP/PS 1 mice.
[0187] Finally, a higher concentration of HCQ (50 pM applied before and after induction of STET) was tested on L-LTP in hippocampal slices from APP/PS 1 mice. As indicated in FIG. 3E, HCQ induced L-LTP (S l)(filled circles) that was significantly different when compared to its own baseline and S2 (open circles) up to 180 min (1 min Wilcox, P=0.01, 1 min U-test, P=0.0006; 60 min Wilcox, P=0.01, 60 min U-test, P=0.0006; 120 min Wilcox, P=0.01, 120 min U-test, P=0.0006; 180 min Wilcox, P=0.01, 180 min U-test, P=0.004). At this higher concentration, HCQ-induced L-LTP in APP/PS 1 hippocampus was similar in magnitude to WT L-LTP throughout the recording duration of 180 minutes ((i.e. no statistically significant differences between L-LTP in Tg versus WT;1 min U- test, P=0.62; 60 min U-test, P=0.07; 120 min U-test, P=0.62; 180 min U-test, P=>0.99) (FIG. 3E vs FIG. 3B), suggesting that HCQ at 50 pM fully rescued impaired L-LTP.
[0188] Comparison of input-output (I/O) curves between WT and APP/PS 1 before and after HCQ application did not show any significant differences (Two-way ANOVA, P= 0.99) (FIG. 3G). Comparison of paired pulse ratio (PPR) in WT and APP/PS 1 before and after HCQ did not show a significant difference, indicating that HCQ does not affect basal synaptic transmission in either WT or APP/PS 1 mice (FIG. 3H) (ANOVA, P= 0.09).
[0189] To test if the rescue of impaired L-LTP by HCQ may be through inactivation of STAT3, levels of total STAT3 and phosphorylated STAT3 (p-STAT3; Tyr705) were examined to determine whether they were reduced by HCQ treatment. Specifically, differences in STAT3 and pSTAT3 levels were tested in hippocampal homogenate samples across four groups: WT, APP/PS1, WT + 50pM HCQ, and APP/PS 1 + 50pM HCQ mice (5-months old). All hippocampal slices were incubated in the interphase chamber and HCQ was applied 30 minutes before and after STET. In each group, hippocampal slices were collected one hour after STET. Levels of total STAT3 and p-STAT3 (Tyr705) were significantly higher in APP/PS 1 mice compared to WT mice and were similar between WT and WT + 50pM HCQ mice (FIGS. 4A and 4B). Levels of p-STAT3 (Tyr705) were significantly lower in APP/PS 1 + 50pM HCQ mice compared to untreated APP/PS 1 mice. In addition, levels of total STAT3 were significantly higher in both APP/PS 1 and APP/PS 1 + 50pM HCQ mice compared to WT and WT + 50pM HCQ mice. However, no significant difference in the level of total STAT3 was found between APP/PS 1 and APP/PS 1 + 50pM HCQ mice. This suggests that the rescue of impaired L-LTP by HCQ in APP/PS 1 mice may be partially mediated through inactivation of STAT3 by regulating the expression of plasticity-related proteins which are important for the maintenance of synaptic plasticity.
[0190] Example s
[0191] HCQ Lowers Risk of AD and Related Dementias
[0192] Methods
[0193] The full study protocol for patient-level analysis in Medicare claims was pre-registered on clinicaltrials.gov prior to data analysis (NCT04691505) and contains detailed information on implementation including all codes that were used to identify study variables to allow future replication. The following sections summarize key methodologic details.
[0194] Data source: Medicare Fee-For-Service claims data from 2007 through 2017 were used. Medicare Part A (hospitalizations), B (medical services), and D (prescription medications) claims are available for research purposes through the Centers for Medicare and Medicaid Services. A signed data use agreement with the CMS was available and the Brigham and Women’s Hospital’s Institutional Review Board approved this study.
[0195] Study cohort: A new user, active comparator, observational cohort study design comparing methotrexate to hydroxychloroquine was employed. The patients were required to have continuous enrollment in Medicare parts A, B, and D during the baseline period of 365 days before initiation date of methotrexate or hydroxychloroquine, which was defined as the cohort entry date. Patients were required to have >1 diagnosis codes of rheumatoid arthritis during the baseline period but no prior use of any disease modifying antirheumatic treatments. Patients with existing diagnosis of Alzheimer’s disease and related dementia (ADRD) any time prior to and including cohort entry date were excluded to focus on incident events. Patients with nursing home admission in 365 days prior to and including cohort entry date also were excluded as medication records for short nursing home stays are unavailable in Medicare claims.
[0196] Outcome measurement: The endpoint of ADRD was identified based on diagnosis codes, recorded on 1 inpatient claim or 2 outpatient claims, of Alzheimer’ s disease, vascular dementia, senile, presenile, or unspecified dementia, or dementia in other diseases classified elsewhere. When validated against a structured in-home dementia assessment, Medicare claims- based dementia identification is reported to have a positive predictive value (PPV) in the range of 65% to 78%.
[°197J Alternative analytic approaches: To accommodate various uncertainties involved in pharmacoepidemiologic investigations focused on ADRD risk, the following alternative analyses were employed. Analysis 1- ‘As-treated’ follow-up approach: In this approach, the follow-up started on the day following the cohort entry date and continued until treatment discontinuation or switch (to comparator treatment), insurance disenrollment, death, or administrative endpoint (December 2017). A 90-day ‘grace period’ after the end of the expected days-supply of the most recently filled prescription was considered to define the treatment discontinuation date to accommodate for suboptimal adherence during treatment periods. Analysis 2- ‘As-started’ follow-up approach incorporating a 6-month ‘induction’ period: In this approach, a 6-month induction period was incorporated after the cohort entry date before beginning the follow-up for ADRD and followed patients for a maximum of 3 years regardless of subsequent treatment changes or discontinuation, similar to an intent-to-treat approach in randomized controlled trials. This follow-up approach addresses concerns related to informative censoring if patients discontinue or if physicians deprescribe the treatments under consideration because of memory problems associated with ADRD, but the diagnosis is not recorded in the electronic healthcare records until after the end of follow-up due to treatment-related censoring. Analysis 3- incorporating a 6-month ‘symptoms to diagnosis’ period: In this approach, an outcome date was assigned that was 6 months before the first recorded ADRD date and excluded last 6 months of follow-up for those who are censored without an event to account for the possibility that ADRD symptoms likely appear some time before a formal diagnosis is recorded in insurance records, which leads to misclassification of ADRD onset. Analysis 4- Alternate outcome definition: In this approach, the outcome was defined using a combination of diagnosis code and >1 prescription claim for a symptomatic treatment [donepezil, galantamine, rivastigmine, and memantine] occurring within 6 months of each other with outcome date assigned to second event in the sequence. Use of medication records to identify dementia has been reported to result in >95% PPV in a previous validation study.
[0198] Covariates: A large number of covariates were identified, which were measured in the 365- day baseline period preceding the cohort entry date. The following set of variables were included: 1) demographic factors such as age, gender, race, socioeconomic status proxies, 2) risk factors for ADRD identified in previous studies such as diabetes, stroke, and depression, 3) lifestyle factors such as smoking as well as use of preventive services, including screening mammography and vaccinations, to account for healthy-user effects13; measures for use of various healthcare services before cohort entry including number of distinct prescriptions filled, number of emergency department visits, hospitalizations, and number of physician office visits to account for patients’ general health and contact with the healthcare system to minimize the possibility of differential surveillance; frailty indicators based on composite scoring scheme to address potential confounding by frailty, 4) comorbid conditions and comedications including prior use of pain medications such as steroids and opioids.
[0199] Statistical analyses: A propensity- score (PS)-based approach was used to account for measured confounding in this study. The PS were calculated as the predicted probability of initiating the exposure of interest versus the reference drug conditional on baseline covariates using multivariable logistic regression. On average, patients with similar PSs have similar distribution of potential confounders used to estimate the PS. Therefore, analyses conditioned on the PS provide effect estimates that are free from measured confounding. For all our analyses, initiators of each exposure of interest were matched with initiators of the reference exposure based on their PS. Pair matching was conducted using a nearest-neighbor algorithm, which seeks to minimize the distance between propensity scores in each pair of treated and reference patients, and a caliper of 0.025 on the natural scale of the PS was used to ensure similarity between the matched patients. Multiple diagnostics for PS analysis were evaluated including PS distributional overlap before and after matching to ensure comparability of these groups and balance in each individual covariate between two treatment groups using standardized differences.
[0200] In the PS matched sample, incidence rates along with 95% confidence intervals for the outcome were estimated for the treatment and reference groups. The competing risk of death could be of concern for the current set of analyses if mortality is frequent among patients included in the cohort and if differences in the risk of mortality between treatment and reference groups are substantial. Therefore, we calculated cumulative incidence using cumulative incidence functions that account for competing risk by death and provided cause-specific hazard ratios from Cox proportional hazards regression model. Pre-specified subgroup analyses were conducted based on age, sex, and baseline cardiovascular disease.
[0201] Results
[0202] To test whether exposure to HCQ lowers AD risk in older individuals, insurance claims data from the Medicare program were used to evaluate the comparative risk of incident AD among patients with rheumatoid arthritis who initiated HCQ versus methotrexate (MTX) (clinicaltrials.gov protocol NCT04691505). A new-user active comparator design with propensity-score (PS) based adjustment for confounding, was used to estimate treatment effects in four alternative analyses designed to address various uncertainties associated with claims-based analyses of dementia risk including exposed person-time misclassification, reverse causation, informative censoring, and misclassification of outcome onset as described previously ( (Zhao et al., Cell Death Differ2019, 26:1600-1614).
[0203] Of the 881,432 patients filling at least one prescription for the drugs of interest (HCQ or MTX) during the study period, 109,124 patients who met all inclusion criteria (54,562 hydroxychloroquine initiators 1 : 1 PS matched to 54,562 methotrexate initiators) were included. Average age of included patients was 74 years, 76% were females, and 84% were white; exposure groups were comparable on all measured characteristics after PS matching.
[0204] FIG. 5 summarizes cumulative incidence of ADRD among HCQ initiators compared to MTX initiators; results from all four analyses indicate that after approximately 2 years of treatment, individuals on HCQ had lower cumulative incidence of ADRD compared to MTX. FIG. 6 summarizes the crude and PS-matched comparative risk of ADRD in HCQ compared to MTX groups; results indicated that risk of ADRD was consistently lower among HCQ initiators. In Analysis 1 where patient follow-up time was censored at discontinuation of the initial treatment (“as-treated” approach), HCQ initiators had an 8% lower risk of ADRD compared to MTX initiators (HR, 95% CI 0.92, 0.83-1.00). In Analysis 2 where a 6-month induction period was incorporated to eliminate reverse causality and continued follow-up for 3 years regardless of treatment change or discontinuation to minimize the impact of informative censoring in an ‘as started’ follow-up scheme, HCQ was significantly associated with a 13% lower risk (HR, 95% CI 0.87, 0.81-0.93). In Analysis 3 where a 6-month ‘symptom to claims’ period was accommodated to address misclassification of outcome onset, HCQ was significantly associated with a 16% lower risk (HR, 95% CI 0.84 [0.76- 0.93). Finally, in Analysis 4, which required symptomatic treatment with cholinesterase inhibitors or Memantine along with diagnosis codes to overcome outcome misclassification due to low sensitivity of the approach relying only on diagnosis codes, effect estimates were largely consistent with other approaches (HR, 95% CI 0.87, 0.75-1.01). No evidence of heterogeneity in treatment effects was observed across subgroups of age, gender, and baseline cardiovascular disease history.
[0205] Discussion of Examples 1-3
[0206] The results suggest that HCQ impacts key features of microglial function relevant to AD pathophysiology that may mediate its therapeutic effects (Lee et al., J Neural Transm (Vienna), 2010, 117(8):949-60; Grubman et al., Nature Comm 2021, 12, article 3015). These include countering neuroinflammation by lowering release of pro-inflammatory cytokines and blocking their downstream effects by inactivating signaling through the cytokine transducer STAT3 (FIG. 1C, 2C). Impaired phagocytosis and poor clearance of extracellular Ap by activated microglia is another important pathogenic mechanism in AD. HCQ also appears to enhance the physiological role of microglia in clearing extracellular A[31 As shown in FIG. 2A, pretreatment of BV2 microglial cells with 25 pM HCQ significantly enhanced A 1.42 clearance as indicated by lower levels in the supernatant and lower ratio of supernatant:lysate AP1.42 levels. To further assess potential mechanisms underlying this finding, the pH-sensitive fluorescent label, Protonex Green, was used to label AP1 2 and demonstrated that HCQ pretreatment enhances microglial uptake of AP1-42 preferentially into acidic cellular compartments such as lysosomes (FIGS. 7A, 7B). While HCQ-associated attenuation of neuroinflammation was associated with lower levels of total STAT3, the enhancement of AP1.42 clearance was associated with reduction in p-STAT3 levels in microglia (FIGS. 7A, 7B). Together, these results indicate that HCQ restores critical microglial functions known to be perturbed in AD.
[0207] The observation that HCQ reduces tau phosphorylation in neuroblastoma cells overexpressing human mutant tau implicates yet another potential therapeutic mechanism of action in AD. While the precise mechanisms mediating this effect have not been investigated, the finding that lowering of p- tau levels by HCQ is associated with an accompanying reduction in p-STAT3 is consistent with prior studies suggesting that pro-inflammatory cytokine signaling through STAT3 may lead to tau hyperphosphorylation through cyclin-dependent kinase 5 (cdk5). IL-6 induces Alzheimer-type phosphorylation of tau protein by deregulating the cdk5/p35 pathway. Another mechanism that implicates STAT3 in tau phosphorylation is by complement-mediated signaling through C3 and C3a receptor (C3aRl). Complement C3aRl inactivation attenuates tau pathology and reverses an immune network dysregulated in tauopathy models and AD. Given that HCQ impacts the three principal pathogenic molecular pathways in AD, i.e., neuroinflammation, A clearance and tau phosphorylation, the next goal was to examine whether HCQ may restore impaired synaptic plasticity which is believed to trigger cognitive impairment in AD. This was evaluated by studying the effects of HCQ on abnormal hippocampal synaptic plasticity characterized by impaired late long-term potentiation (L-LTP) in the APP/PS 1 transgenic AD mouse model. L-LTP is a protein-synthesis dependent form of synaptic plasticity important in hippocampal memory formation (Bin Ibrahim et al., FEES J 2021, doi: 10.1111/febs.16065). Previous studies have observed impaired L-LTP in APP/PS 1 mice at 3-4 months of age, prior to the accumulation of Ap plaques (Li et al., PNAS USA 2017, 114(21):5527-5532). This observation allowed testing of pharmacological interventions at the earliest stages of AD, prior to the accumulation of Ap pathology. The results show that HCQ restores L-LTP in the hippocampus of APP/PS 1 mice. At a dose of 25pM HCQ, restoration of L-LTP was observed that was previously absent in untreated APP/PS 1 hippocampal slices, although the magnitude of L-LTP at this dose of HCQ was still lower than that observed in wild type mice. However, at a concentration of 50pM HCQ, L-LTP in APP/PS 1 mice was identical to that in wild type mice, suggesting a complete recovery of impaired hippocampal synaptic plasticity. The western blot analyses suggest that this rescue of L-LTP at the higher concentration of HCQ is associated with inactivation of STAT3 as reflected in lower p-STAT3 levels. The findings are consistent with recent studies showing that pharmacological inactivation of STAT3 rescues impaired learning and memory in the 5xFAD and APP/PS 1 mouse models of AD. (Choi M. et al., Inhibition of STAT3 phosphorylation attenuates impairments in learning and memory in 5XFAD mice, an animal model of Alzheimer's disease, J. Pharmacol. Sci, 2020, vol. 143(4): 290-299; Reichenbach, N. et al., Inhibition of STAT3-mediated astrogliosis ameliorates pathology in an Alzheimer’s disease model, EMBO Mol. Med. 2019, vol. 1 l(2):e9665.) A potential mechanism proposed to explain these findings is through correction of astrocytic and neuronal hyperactivity, attenuation of neuroinflammation, restoration of A[3 clearance by microglia and a reduction in dystrophic neurites. Previous findings that STAT3 signaling may mediate A -induced neuronal cell death in AD is also a potential mechanism underlying the rescue of hippocampal function by HCQ-induced STAT3 inactivation. (Wan, J. et al., Tyk2/STAT3 signaling mediates amyloid-induced neuronal cell death, J. Neurosci., 2010, vol.
30(20):6873-81.) [0208] Building on the cell culture-based phenotypic assays and in vitro studies of synaptic function in APP/PS1 mice that showed promising therapeutic effects of HCQ, an evaluation of whether exposure to HCQ lowers AD risk in humans was performed. A large real-world clinical dataset using Medicare-based claims data was leveraged and a new-user, active comparator observational cohort study was implemented to compare the effect of methotrexate to hydroxychloroquine on incident AD risk in older individuals diagnosed with rheumatoid arthritis. A rigorous study design was implemented that addressed several common pitfalls in pharmacoepidemiological studies that may lead to spurious results in analyses of claims-based data. The findings indicate that exposure to HCQ in older individuals prior to AD diagnosis is associated with 8-16% lowering of incident AD relative to the active comparator, methotrexate. These results suggest that the observations from cell culturebased and transgenic AD model-based experiments indicating therapeutic effects of HCQ in AD may be translated into humans to modify the trajectory of AD. The current state of knowledge of AD progression and potential disease-modifying treatments converges around the consensus that such interventions must be deployed well before symptoms begin and precisely targeted at individuals likely to benefit the most (see, e.g., Cummings et al., Alzheimer’s Research and Therapy 2016, 8:39).
[0209] Several features make HCQ an attractive candidate for repurposing in AD including its permeability across the blood brain barrier and effective partitioning into the brain. Doses of HCQ of 6.0-6.5 mg/kg/day based on an ideal body weight and typically used in RA patients, yield serum concentrations of 1.4 to 1.5 micromolar (Mackenzie, Am J Med 1983, 75(1 A):5- 10). Previous studies have shown that brain tissue concentrations of HCQ are several fold higher than in plasma (Kp brain/plasma 21.99 + 0.11 in macaques, 4-31 in albino rats) (Browning, Hydroxychloroquine and Chloroquine Retinopathy 2014, 35-63; Belkhir et al., Eur J Drug Metab Pharmacokinet 2020, 45(6):703- 13). These studies indicate that at doses of HCQ reflecting those used in RA patients, there is a substantial penetration of HCQ into the brain and accumulation at levels within the range of the doses used in our in vitro studies. Moreover, HCQ and other 4-aminoquinolones have been shown to preferentially accumulate in acidic cellular compartments such as lysosomes at millimolar levels (Browning, Hydroxychloroquine and Chloroquine Retinopathy 2014, 35-63). Furthermore, HCQ has a well-established safety profile with serious side effects of retinopathy and cardiac toxicity being relatively rare (Nirk et al., EMBO Mol Med 2020, 12:el2476).
[0210] In summary, it was established that the commonly used RA drug, HCQ inactivates STAT3 and targets multiple pathogenic mechanisms in AD including neuroinflammation, Af> clearance, tau phosphorylation and synaptic dysfunction. HCQ also lowers AD risk in older individuals. The results provide compelling evidence that this safe and inexpensive drug is a promising disease-modifying treatment for AD. [0211] Example 4
[0212] HCQ Reduces Neuroinflammation in AD Mouse Model
[0213] HCQ was confirmed to exhibit anti-inflammatory effects in a model of neuroinflammation using microglial cells from the brains of a transgenic AD mouse model. The results showed that, similar to observations in BV2 microglial cells, HCQ lowered the release of several cytokines, including IL6, ILl-b, IL-10, and IL-12p70. The results are shown in FIG. 8.
[0214] Methods
[0215] MACS microglia isolation and cultivation: Adult microglia were isolated from 9 months old 5xFAD mice via magnetic cell sorting (MACS). The mice were terminally anesthetized by i.p. injection of Pentobarbital (600 mg/kg, dosing 10 pL/g body weight) and brains were transcardially perfused with DPBS. Brains were removed, the brainstem discarded and the remaining brain minced for cell dissociation. Cell dissociation was performed using Miltenyi Adult Brain Dissociation Kit (Miltenyi, 130-107-677). MACS cell separation was performed using CD1 lb (Microglia) MicroBeads, mouse (Miltenyi, 130-093-634) and MS columns on OctoMACS™ cell separator (Miltenyi). Isolated microglia were seeded onto 0.01% PLL coated plates at a density of 10.000 cells per well in 384 well plate in DMEM containing 10% FBS,1% penicillin/streptomycin, and 2 mM L- glutamine.
[0216] LPS-induced neuroinflammation on MACS isolated 5xFAD microglia: On DIV7 HCQ at 0,.25 pM, 2.5 pM and 25, u.M was applied 1 hour before LPS stimulation (Sigma-Aldrich; L6529;
1 mg/ml stock in ddH2O, final concentration in well: 50 ng/mL (dilutions in medium)). Cells treated with vehicle, cells treated with LPS alone, as well as cells treated with LPS plus reference item (dexamethasone 10 pM, Sigma D4902) served as controls. After 24 h of stimulation, cell supernatants were collected for the cytokine measurement (V-PLEX Proinflammatory Panel 1 Mouse Kit, K15048D, Mesoscale).
[0217] Example 5
[0218] Treatment with Tauroursodeoxycholic Acid (TUDCA ) or HCQ+TUDCA
[0219] TUDCA was evaluated alone and in combination with HCQ. Prior work has shown that bile acid metabolism may be a plausible drug target in AD and related dementias (Varma et al., PLOS Medicine, May 27, 2021, 18(5):e 1003615). The cell culture-based results in FIG. 9 show that TUDCA lowers the secretion of Api-38, A 1-4O, and Api-42, whereas HCQ increased microglial clearance of exogenous Api-42 (FIG. 1A). FIG. 10 shows the effect of HCQ in combination with TUDCA on the secretion of Api-38, Api-40, and Api-42.
[0220] Methods [0221] Cultivation and treatment of H4-hAPP cells: H4-hAPP cells were thawed and cultivated in Opti-MEM supplemented with 10% FCS, 1% penicillin/streptomycin 200 pg/ml Hygromycin B and 2.5 pg/ml Blasticidin S (=culture medium). H4-hAPP cells were seeded into 96 well plates (2 x 104 cells per well). On the next day, cells in 96 well plates were treated with TUDCA (100 pM (Cl), 10 pM (C2), or 1.0 pM (C3)), R.I. (DAPT 400 nM) or vehicle. Alternatively, cells in 96 well plates were treated with 25 pM HCQ/0.1 pM TUDCA (Cl), 2.5 pM HCQ/100 pM TUDCA (C2), and 2.5 pM HCQ/10 pM TUDCA (C3). 24 h later, supernatants were collected for further A0 measurements by MSD. The experiment was performed in n=6 technical replicates for all groups.
[0222] A[138, 40, 42 measurement: Supernatants were diluted 1 : 10 and analyzed for human A 38, 40, and 42 with MSD® 96-well MULTI-SPOT® 6E10 Abeta Triplex Assay (Mesoscale Discovery, Rockville, MD). The immune assay was carried out according to the manual and plates are read on the MESO QuickPlex SQ 120 multiplexing instrument. Analyte levels were evaluated according to adequate A[> peptide standards (MSD) as pg per mL.
[0223] Statistics: Basic statistical analysis was performed. Data are presented as mean ± standard error of mean (SD) and group differences are evaluated for each Test items separately by one-way ANOVA in GraphPad Prism 9. In FIGS. 9 and 10, data are presented as bar graphs with group mean+SD (n=6 per group). VC= vehicle control 0.1% DMSO. In FIG. 20, C1-C3 are 100 pM, 10 pM, and 1.0 pM TUDCA, respectively. In FIG. 21, C1-C3 are 25 pM HCQ/0.1 pM TUDCA, 2.5 pM HCQ/100 pM TUDCA, and 2.5 pM HCQ/10 pM TUDCA, respectively. One-way ANOVA followed by Dunnetts multiple comparison test versus VC: *p<0.05; **p<0.01;***p<0.001.
[0224] Example 6
[0225] TUDCA and DIM Evaluation in AD-Related Cell-Based Assays
[0226] TUDCA and DIM were investigated to determine whether they could rescue molecular phenotypes relevant to AD, including tau phosphorylation, Ap 1-42 clearance, A(3 secretion, A toxicity, lipopolysaccharide (LPS)-induced neuroinflammation, cell death due to trophic factor withdrawal, and neurite outgrowth and neurogenesis (Varma, PloS one 2020,
15(32271844):e02314446; Desai el al., Brain Communications 2022, 4(5): 10). TUDCA and DIM were selected based on brain penetration/permeability across the blood brain barrier, close network proximity of the drug target genes to established AD risk genes through short path lengths connected by single mediator genes, and prior evidence for roles in molecular mechanisms relevant to neurodegeneration. In all assays, cells treated with drugs were compared to either a vehicle control (VC) or lesion control. [0227] Methods
[0228] Cell-based assays: For each combination of drugs and assay, the posterior probability P(H,|data) of protective, neutral, and adverse drug effect (formal hypotheses Hi, Ho, H2, respectively) were evaluated. Dose-response relationships (x-y data) were modeled with a Bayesian nonlinear regression model:
. = 171
[0229] The parameter yo, i was interpreted as expected bioactivity at zero and saturating drug concentration, and fold change was defined as FCy = yjyo. A gamma prior density was chosen for the fold change as:
FCy ~ T a = 5, /? = 5) and Bayesian estimation of the posterior density of the fold change was performed using default settings of the Markov chain Monte Carlo samples implemented by the PyMC python library. For efficient sample, each bioactivity data set was standardized such that 10 units corresponded to one standard deviation. Diagnostic criteria (posterior sample of fitted curves, effective sample sizes, values of the f statistic, and Markov chain standard errors) were inspected for the fit and three poorly fitted data sets out of a total of 91 data sets were excluded.
[0230] Aft Clearance: 20,000 BV2 cells per well (uncoated 96-well plates were plated out. After changing cells to treatment medium, drug compounds were administered 1 hour before A stimulation (final concentration in well 200 ng/mL; dilutions in medium). Cells treated with vehicle and cells treated with A[3 alone served as controls. After 3 hours of A [3 stimulation, cell supernatants were collected for the A[3 measurement and cells were carefully washed twice with PBS and thereafter lysed in 35 pL of cell lysis buffer (50 mM tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% SDS) supplemented with protease inhibitors. Supernatants and cell lysates were analyzed for human A 42 with the MSD V-PLEX Human A[342 Peptide (6E10) Kit (K151LBE, Mesoscale Discovery). The immune assay was carried out according to the manual, and plates were read on MESO QuickPlex SQ 120 (Meso Scale Diagnostics, Rockville, MD).
[0231] Afi Secretion: H4-hAPP cells were cultivated in Opti-MEM supplemented with 10% FCS, 1% pcnicillin/strcptomycin, hygromycin B (200 pg/ ml), and blasticidin S (2.5 pg/ml). H4-hAPP cells were seeded into 96-well plates (2 x 104 cells per well). On the next day, cells in 96-well plates were treated with compounds, reference item [400 nM N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S- phenylglycine t-butyl ester (DAPT)], or vehicle. Twenty-four hours later, supernatants were collected for further A measurements by MSD [V-PLEX A Peptide Panel 1 (6E10) Kit, K15200E, Mesoscale Discovery].
[0232] Tau Phosphorylation: SH-SY5Y-hTau441(V337M/R406W) cells were maintained in culture medium [DMEM medium, 10% FCS, 1% nonessential amino acids (NEAA), 1% 1-glutamine, gentamycin (100 pg/ml), and geneticin G-418 (300 pg/rnl)] and differentiated with 10 pM retinoic acid for 5 days changing medium every 2 to 3 days. Before the treatment, cells were seeded onto 24- well plates at a cell density of 2 x 105 cells per well on day one of in vitro culture (DIV1). Drug compounds were applied on DIV2. After 24 hours of incubation (DIV3), cells on 24-well plates were harvested in 60 pL of RIPA buffer [50 mM tris (pH 7.4), 1 % NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA supplemented with freshly added 1 pM NaF, 0.2 mM Na-orthovanadate, 80 pM glycerophosphate, protease (Calbiochem, Millipore Sigma, Burlington, MA), and phosphatase (Sigma- Aldrich, St. Louis, MO) inhibitor cocktail]. Protein concentration was determined by BCA assay (Pierce, Thermo Fisher Scientific, Waltham, MA), and samples were adjusted to a uniform total protein concentration. Total tau and phosphorylated tau were determined by immunosorbent assay from Mesoscale Discovery [Phospho(Thr231)/Total Tau Kit K15121D, Mesoscale Discovery, Meso Scale Diagnostics].
[0233] LPS-induced Neuroinflammation'. The murine microglial cell line BV2 was cultivated in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, and 2 mM 1-glutamine (culture medium). For LPS stimulation assay, 5000 BV2 cells per well (uncoated 96-well plates) were plated out and the medium was changed to treatment medium (DMEM, 5% FCS, and 2 mM 1-glutamine). After changing cells to treatment medium, drug compounds were administered 1 hour before LPS stimulation [Sigma- Aldrich; L6529; 1 mg/mL stock in ddH2O; final concentration in well, 100 ng/mL (dilutions in medium)]. Cells treated with vehicle, cells treated with LPS alone, and cells treated with LPS plus reference item (dexamethasone, 10 pM; Sigma-Aldrich, D4902) served as controls. After 24 hours of stimulation, cell supernatants were collected for the cytokine measurement (V-PLEX Proinflammatory Panel 1 Mouse Kit, K15048D, Mesoscale) and cells were subjected to 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
[0234] Neurite Outgrowth and Neurogenesis: Primary hippocampal neurons were prepared from El 8.5 timed pregnant C57BL/6JRccHsd mice as previously described. Cells were seeded in poly-D- lysine pre-coated 96-well plates at a density of 2.6 x 104 cells/well in medium (Neurobasal, 2% B-27, 0.5 mM glutamine, 25 pM glutamate, 1% Penicillin-Streptomycin). Directly on DIV1, the drug of interest or VC was applied. On DIV2, 10 pM bromodeoxyuridine (BrdU; B5002 Sigma Aldrich) was added and cells were fixed after an additional 24h. Cells were permeabilized with 0.1% Triton-x and incubated with primary Beta Tubulin Isotype III (T8660, Sigma Aldrich) and BrdU antibodies (MAS25°C, AHrlan-Sera Lab) overnight at 4 °C. Afterwards, cells were washed two times with PBS and incubated with fluorescently labelled secondary antibodies and DAPI for 1.5 h at room temperature (RT) in the dark. Cells were rinsed three times with PBS and imaged with the Cytation 5 Multimode reader (BioTek, Winooski, VT) at 10 x magnification (six images per well). BrdU- positive cells were counted as a marker of neurogenesis and Beta Tubulin Isotype III signal was used for macro-based quantification of neurite outgrowth.
[0235] Trophic Factor Withdrawal: Primary cortical neurons from embryonic day 18 (E18) C57B1/6 mice were prepared as previously described. On the day of preparation (DIV1), cortical neurons were seeded on poly-d-lysine pre-coated 96-well plates at a density of 3 x 104 cells per well. Every 4 to 6 days, a half medium exchange using full medium (Neurobasal, 2% B-27, 0.5 mM glutamine, and 1 % penicillin-streptomycin) was carried out. On DIV8, a full medium exchange to B-27 free medium (Neurobasal, 0.5 mM glutamine, and 1 % penicillin-streptomycin) was performed and drug compounds were applied thereafter. The experiment was carried out with six technical replicates per condition, and vehicle-treated cells served as control. After 28 hours on B-27-free medium, cells were subjected to YO-PRO/propidium iodide (PI) and MTT as well as lactate dehydrogenase (LDH) assay.
[0236] Results:
[0237] TUDCA and DIM were evaluated experimentally in 33 AD-related cell-based assays from 9 experiments. Both TUDCA and DIM were found to be protective with high probability in several assays. However, each drug was also found to have an adverse effect in other assays. A high-level summary of the results is presented in FIG. 11. Both TUDCA and DIM exhibited strongly protective effects in A[> clearance (BV2 cells) and LPS induced neuroinflammation (iPSCs). Moreover, TUDCA also proved to be protective in A[> release (H4 cells), whereas DIM was protective in LPS induced neuroinflammation (BV2 cells).
[0238] Discussion
[0239] Network pharmacology analyses were combined with cell-based experimental validation to discover novel drug repurposing opportunities in AD. Using cell culture-based phenotypic assays, both DIM and the TUDCA were found to exhibit protective effects given several molecular perturbations relevant to AD pathogenesis.
[0240] Example 7
[0241] Poly(L-lysine succinylated)-hydroxychloroquine (PLS-HCQ)
[0242] PLS-HCQ is more brain penetrant than HCQ. The efficacy of PLS-HCQ will be compared with HCQ for amelioration of AD pathology and ability to slow cognitive decline in a transgenic mouse model of AD. [0243] Example 8
[0244] S-HCQ with TUDCA or DIM
[0245] Methods:
[0246] Separation of S-HCQ and R-HCQ: HCQ sulfate, 10 g (Sigma PHR1682; pharmaceutical secondary standard), was dissolved in 300 mL H2O (18.2 ) and gently sonicated at room temperature to ensure complete dissolution of the salt. To this solution, 20 mL (8.3 equiv, 190 mmol) of diethylamine (Sigma 471216; > 99.5%) was added, which precipitated the free -base HCQ as a dense and white suspension. The suspension was poured into a separatory funnel and leftover material was transferred by rinsing the flask with dichloromethane (Sigma 320269; > 99.5%). Free-base HCQ was extracted with dichloromethane (5 x 100 mL washes). The organic phase was pooled, dried over M SO4 (EDM Millipore MX0075; anhydrous), and concentrated in vacuo to afford ~ 8g free-base HCQ, which was chirally separated without further manipulation.
[0247] HCQ preparation’. Treatment dosage 100 mg/kg, vehicle saline, intraperitoneal (i.p.) injection 5 pL/g b.w.
[0248] HCQ nano (PLS -HCQ) preparation’. Add 2.5 mL of saline to each vial of lyophilized material on each dose day. Vortex briefly to mix. Prepare fresh vial daily. Solution will be pale yellow in color. Protect from light. Discard remaining material after animals are successfully dosed each day. Vehicle 100 mg/kg, i.p. injection 10 pL/g b.w.
[0249] Amiloride preparation: Dissolved in water; treatment dosage 12 mg/kg; i.p. injection 10 pL/g b.w.
[0250] TUDCA preparation: Dissolved in saline; treatment dosage 500 mg/kg; i.p. injection 10 pL/g b.w.
[0251] R-HCQ preparation: The compound was dissolved in DMSO. This stock solution was stored at room temperature protected from light. The dosing solution was made from this stock solution once a week. For the dosing solution, the test item was dissolved in the vehicle (10% DMSO, 10% Cremophor® (BASF Corp.) water) and stored at 4°C and protected from light. Treatment dosage 100 mg/kg; i.p. injection 10 pL/g b.w.
[0252] S-HCQ preparation: The compound was dissolved in DMSO. This stock solution was stored at room temperature protected from light. The dosing solution was made from this stock solution once a week. For the dosing solution, the test item was dissolved in the vehicle (10% DMSO, 10% Cremophor® (BASF Corp.) water) and stored at 4°C and protected from light. Treatment dosage 100 mg/kg; i.p. injection 10 pL/g b.w.
[0253] Animal management: Animals were housed in individual ventilated cages on standardized rodent bedding supplied by Rettenmaier. Each cage contained a maximum of five mice. The temperature in the keeping room was maintained between 20 to 24 °C and the relative humidity was maintained between 45 to 65 %. Animals were housed under a constant light-cycle (12 hours light/dark). Dried, pelleted standard rodent chow (Altromin) as well as normal tap water was available to the animals ad libitum. Only animals in apparently good health condition were included to the study. Randomization of group allocation was done per cage. If possible, animals were assigned to different starting groups (cohorts) comprising animals of all treatment groups. The number of animals in a starting group was limited to ensure same age and uniform handling. Age at treatment start 7.5 months ± 0.5 months.
[0254] Study Design: A total of 160 mice were used for the study. 112 transgenic 5xFAD mice were randomly allocated to 7 treatment groups A - G, each consisting of 16 animals. 48 age matched wild type littermates were randomly allocated to 3 treatment groups H, I and J each consisting of 16 animals. All animals were treated with test compound or vehicle for the whole study period by i.p. injection (Table 1). Group A was dosed daily for the whole treatment period. Group G and I were dosed twice per week. All other groups were dosed daily in the first 4 weeks, three times per week in weeks 5 to 8, and once weekly until the end of the study. 5xFAD (Familiar Alzheimer Disease) mice bear five mutations, three in the amyloid precursor protein (APP695) gene [APP K670N/M671L (Swedish), 1716V (Florida), V717I (London)] as well as two mutations in the presenilin 1 gene [PSI M146L, L286V] (Oakley ct al., 2006). The expression of the 5xFAD transgcnc is driven by the neuron specific Thyl promoter. The five mutations cause an early onset of the cognitive decline and increasing A(3 1-40 and 1-42 levels in the brain and cerebrospinal fluids, over age. Histological analysis revealed plaque load and beta sheet formation accompanied with neuroinflammation. Thus, the 5xFAD mouse mimics the most crucial phenotypic symptoms of amyloidogenic neurodegeneration, neuroinflammation as well as learning and memory deficits and is a suitable model for Alzheimer’ s disease to study effects of drugs on biochemical, histological and behavioral hallmarks.
Table 1 - Treatment Groups tg = transgenic; wt = wild type
[0255] In-vivo blood samples were collected by mandibular sampling from each animal, at baseline (before treatment start), and after 4, 8 and 12 weeks of treatment. Maximum allowed blood volume was sampled into K2EDTA (potassium ethylenediaminetetraacetic acid) tubes. The tube was inverted thoroughly to facilitate homogeneous distribution of the EDTA and prevent clotting. Blood plasma was isolated by centrifugation (3000 x g for 10 minutes at room temperature (22 °C)) and plasma aliquots were transferred to 1.5 mL tubes, frozen on dry ice and stored at -80°C.
[0256] All animals were tested twice in the Y-maze and once in Morris Water Maze (MWM) to assess the test item's effects on the cognitive deficits of the mice. MWM test was performed on 4 days with 4 trials per day and a probe trial on day 5. A computerized video tracking system was used to quantify escape latency and distance travelled. The mice were tested in a randomized order.
[0257] Spatial working learning capacities of all animals were tested in the Y-Maze. The Y-Maze apparatus consists of three identical white arms (length x width x height = 38 x 6.5 x 13 cm). The light was set to 10 Lux. Each mouse was placed individually in the center of the maze and allowed to move freely through the maze during an 8 minute test session. To avoid odor traces the maze was cleaned with 70% isopropanol between different animals. The process was video recorded and analyzed using Noldus Ethovision software. The sequence of arm entries as well as the number of total arm visits were recorded automatically. An alternation was defined as entering each of the three arms consecutively. The maximum number of alternations was the total number of arms entered minus two. The percentage of alternations was calculated as actual alternations per maximum alternations.
[0258] Spatial learning capacities of all animals were tested in the Morris Water Maze (MWM). The MWM was performed using the following pattern: four trials on each of four consecutive days were performed. In all trials, the platform was located in the northeast (NE) quadrant of the pool. Mice started from predefined positions (southeast (SE), southwest (SW), northwest (NW)). A single trial lasted for a maximum of 60 seconds. In case the mouse did not find the hidden, diaphanous platform within this time, the experimenter guided the mouse to the target. Mice were allowed to rest on the platform for 10-15 sec to orientate in the surrounding. On day five mice were tested in the probe trial (PT). During the PT, the platform was removed from the pool and the number of crossings over the former target position as well as the abidance in the target quadrant was recorded. For the quantification of escape latency (the time [sec] to find the hidden platform), of pathway (the length of the trajectory [meters] to reach the target), of target zone crossings and of the abidance in the target quadrant in the PT, a computerized video tracking system (Noldus Observer) was used.
[0259] At the end of the treatment phase, all mice were euthanized by IP injection of 600 mg/kg pentobarbital. After confirmation of deep anesthesia, CSF was obtained by dissection of the muscles and exposure of the foramen magnum. Upon exposure, a Pasteur pipette was inserted in an approximate depth of 0.3 - 1 mm into the cisterna magna. CSF was collected by suction and capillary action until flow fully ceases in 0.2 ml polypropylene PCR tubes. The tubes were spun briefly and immediately frozen in an upright position on dry ice. CSF quality was documented as score from 0 (no visible blood contamination) to 3 (severe blood contamination).
[0260] Terminal blood was collected by heart puncture in EDTA coated tubes. Blood plasma was collected by centrifugation (3000 x g for 10 minutes at room temperature) and plasma aliquots were transferred to 1.5 mL tubes, frozen on dry ice and stored at -80 °C.
[0261] Brains were dissected after transcardial perfusion with 0.9% saline and hemisected at midline. The left hemi brains were further dissected in hippocampus, cortex, and rest brain, all parts were weighed and snap frozen on dry ice for biochemical analysis. The right hemi brains were fixed by immersion in freshly prepared 4% paraformaldehyde in PB (pH 7.4) for 2 hours at room temperature. The samples were then transferred to 15% sucrose in PBS and stored at 4°C until sunk. Hemispheres were then embedded in OCT and frozen in cryomolds with ice-cold isopentane on dry ice, and stored at -80°C.
[0262] Six right hemibrains per group (total of 54 hemibrains) were cryo-sectioned (60 sections per brain collected). Five consecutive cryosections were sagittally cut at 10 pm thickness on a Leica CM1950 or Thermo Scientific NX70 cryotome. The next 25 sections per level were discarded. This collection scheme was repeated for 12 levels and was somewhat modified if required by unusual brain size. In total 12 x 5 = 60 sections were collected. Collection of sections started at a level ~0.2 mm lateral from midline and extended through the hemisphere in order to ensure systematic random sampling through the target regions. Sections were stored at -20°C.
[0263] Amyloid-0-positive plaques, microglia, and tau phosphorylation were evaluated using immunofluorescence labeling on a uniform systematic random set of five sections per mouse (one section each from levels 2, 4, 6, 8, 10; total of 270 sections). Microglia activation, phosphorylated tau at serine 202/threonine 205 (pSer202/Thr205 Tau, pTau), and amyloid 0-positive plaques (fibrils) were evaluated using quadruple immunofluorescent labeling with the following protocol.
[0264] ThioS + LOC + Ibal + pTau + DA Ph All steps were executed in Dulbecco’s phosphate buffered saline pH 7.2-7.8 (DPBS) at room temperature unless noted otherwise. 1. Cryosections were air-dried for 45 minutes and washed in DPBS for 10 minutes. 2. Sections were incubated in 0.5 % Thioflavin S in ddH2O for 7 minutes, then differentiated in 80 % ethanol for 2 x 3 minutes. 3. Sections were washed 3 x 5 minutes each in DPBS. 4. Unspecific binding sites were blocked with M.O.M. blocking reagent in 0.1% Triton X-100 in DPBS for 60 minutes in a damp chamber. 5. Sections were washed 3 x 5 minutes each in DPBS. 6. Sections were incubated with primary antibodies in M.O.M. diluent (Vector Laboratories) over night at 4°C in a damp chamber - rabbit anti- amyloid-0 fibrils (LOC) polyclonal antibody (Merck Millipore, AB2287), 1:1000; guinea pig monoclonal [Gp311H9] anti-Ibal (Synaptic Systems, 234 308), 1:3000; mouse anti-pSer202/Thr205 Tau monoclonal [AT8] antibody (Thermo Fisher, MN1020), 1:300. 7. Sections were washed 3 x 5 minutes each in DPBS. 8. Sections were incubated with secondary antibodies in M.O.M. diluent (Vector Laboratories) for 60 minutes in a damp chamber (light protected) - donkey anti-rabbit IgG H+L Alexa Fluor 750 conjugated (Abeam, abl75728), 1:500; donkey anti-guinea pig IgG H+L Cy3 Conjugated, (Jackson ImmunoResearch, 706-165- 148), 1:500; donkey anti-mouse IgG H+L DyLight 650-conjugated (Thermo Scientific, SA5-10169), 1:500. 9. Sections were washed 3 x 5 minutes each in DPBS (light protected). 10. Sections were incubated with DAPI working solution for 15 minutes (light protected). 11. Sections were washed 2 x 5 minutes in DPBS (light protected). 12. Sections were washed 5 minutes in ddH2O (light protected). 13. Sections were automatically covered with Mowiol and coverslips (light protected) using a Leica CV5O3O coverslipper.
[0265] Mosaic images of the stained sections were recorded on a Zeiss automatic microscope AxioScan Z1 with high aperture lenses, equipped with a Zeiss Axiocam 506 mono and a Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 3.3 software.
[0266] The target areas (isocortex and hippocampus) were identified by drawing an area of interest (ROI) on the images. A second ROI excludes wrinkles, air bubbles, or any other artifacts interfering with the measurement and defines the area for quantitative image analysis. Afterwards, signal of IBA1, LOC, Tau phosphorylation, and ThioS was quantitatively evaluated within the identified areas. Background correction was used if necessary and immunoreactive objects were detected by adequate thresholding and morphological filtering (size, shape). Typical readouts were size and intensity of objects, number of objects per mm2 (numerical object density), and the percentage of the ROI area that was covered by immunopositive objects. Once the parameters of the targeted objects were defined in a test run, the quantitative image analysis was run automatically so that the results were operator-independent and fully reproducible.
[0267] Biochemistry
[0268] The frozen hippocampus and cortex samples from 6 animals of each group (total of 108 samples) were homogenized in lysis buffer (THB; 250 mM Sucrose, 1 mM EDTA, 1 mM EGTA, 20 mM Tris pH 7.4) including lx protease inhibitor (Calbiochem). The tissue was homogenized with a beadmill (UPHO, Geneye) at 55Hz for 50sec. Aliquots were stored at -80°C until further use. For extraction of non-plaque associated proteins, 1 aliquot of THB homogenate was mixed with 1 part diethylamine (DEA) solution (0.4% DEA, lOOmM NaCl). The mixture was centrifuged for 120 min at 20,000xg, 4°C. The supernatant was neutralized with 1/10 of the volume 0.5 M Tris-HCl, pH 6.8 and vortexed briefly. Aliquots were stored at -80°C as DEA fraction (soluble fraction). For extraction of deposited proteins, a second aliquot of THB homogenate was mixed with 2.2 parts cold formic acid (FA), sonicated for 30 sec on ice and centrifuged for 120 min at 20,000xg, 4°C. The supernatant was mixed with 19 parts FA Neutralization Solution (IM Tris, 0.5 M Na2HPO4, 0.05% NaN3). Aliquots were stored at -80°C as FA fraction (insoluble fraction). For measurement of inflammation markers, the third aliquot of homogenate was substituted with Triton® X-100 (Dow Chemical Company, Midland, MI) so that the final concentration as 1 % in the homogenate. After vortexing and 10 min incubation on ice, the homogenates were cleared from cell debris by centrifugation at 20,800 x g at 4°C for 10 minutes in a tabletop centrifuge and the supernatants were collected for the measurement of cytokines and stored at -80°C until further use as Triton® fraction.
[0269] A ? 1-40 andAf 1-42 levels in DEA and FA fraction'. A0 1-40 and A0 1-42 were measured in duplicates in the fractions described above (DEA and FA fractions; 72 samples per assay) using A01- 42 with MSD® Human (6E10) V-plex kit (K151LBE-2, Mesoscale Discovery) and A01-4O with MSD® Human (6E10) V-plex kit (K150SKE-2, Mesoscale Discovery) according to the instructions of the manufacturer. Plates were read on Quickplex SQ 120 sector imager (Mesoscale Discovery). A [J levels in study samples were evaluated in comparison to calibration curves provided in the kit and are expressed as pg or ng per mg brain wet weight.
[0270] Inflammation markers'. The levels of ten different cytokines (IFN-y, IL- 10, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-a) were measured in the 36 aliquots of the brain homogenates (Triton® fraction) using an immunosorbent assay (V-PLEX Custom Mouse Cytokine, Mesoscale Discovery cat.nr. K15048D)) and cytokine levels were statistically evaluated. [0271] NfL levels'. The NF-light® (Neurofilament-light) ELISA 10-7001 CE from UmanDiagnostics was used for analysis. The following samples were analyzed: terminal CSF, terminal blood plasma as well as all 4 timepoints from the in vivo bleedings from 6 animals per groups (3 groups; 18 animals, 108 samples in total). Samples were diluted (1:3 for plasma, 1:35 for CSF) in assay buffer and analyzed according to the manufacturers protocol. CSF and in vivo samples are analyzed in single replicates only, due to limited sample volume. In brief, after dilution, 100 pl of sample are added to the pre-coated wells and incubated for 1.5 hours at RT with gentle agitation (800 rpm). Wells were washed three times with assay wash buffer and 100 pL of the tracer antibody were added. After 45 min incubation (RT, 800 rpm) wells were again washed three times. Thereafter 100 pL of conjugate were added and incubated for 30 min (RT, 800 rpm). After 3 x washing 100 pl of TMB substrate were added to each well and incubated for 15 min at RT. 50 pL stop reagent were added and after short gentle agitation, the plate was read at 450 nm (reference 620- 650 nm) on the Cytation® 5 multimode reader (Biotek, Agilent Technologies, Inc., Santa Clara, CA). Data were evaluated in comparison to calibration curves provided in the kit and are expressed as pg/ml plasma.
[0272] GFAP levels'. Plasma samples of n=6 animals per group (10 groups, total n=60) were analyzed for GFAP level using Mouse GFAP Elisa Kit (ab233621). Samples were applied at 1:50 dilution and assessed according to the instructions of the manufacturer and evaluated in comparison to calibration curves provided in the kit. Plates are read on the Cytation® 5 multimode reader and values were expressed as pg/mL plasma.
[0273] Statistics: All raw data was analyzed in GraphPad Prism™ 10 (GraphPad Software Inc., USA). Graphs were displayed with group means and standard error of the mean (SEM). Normality distribution was analyzed by Kolmogorov-Smirnov tests. If more than 2 groups were compared with each other, significance was calculated by One-way or Two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Significance was defined as * p < 0.5, ** p < 0.01 and *** p < 0.001.
[0274] Results
[0275] Body weight: Body weights were recorded on a weekly basis throughout the in-vivo phase of the study, starting on the first day of vehicle or test compound treatment. As expected, 5xFAD mice displayed a lower body weight compared to nTg control littermates. Nevertheless, body weights in all treatment groups increased over the course of the study, indicating that the animals tolerated i.p. treatment with either vehicle or test item well.
[0276] Morris water maze: The Morris Water Maze (MWM) is a widely used behavioral test to study spatial learning and memory in rodents. Typically, an animal learns to escape from the water by locating a hidden platform with help of visual cues. Lesions in distinct brain regions like hippocampus, striatum, basal forebrain, cerebellum and cerebral cortex are shown to impair MWM accomplishment. Spatial learning capacities of all animals were tested in the Morris Water Maze (MWM) in treatment week 14, at an age of -9 months. As part of the test, escape latency, swim distance and thigmotaxis (indicative of strategy learning) were assessed during training days 1-4. Motor performance and emotional status (velocity and floating, respectively) were calculated. Furthermore, target zone crossings and abidance in the target quadrant were assessed on the probe trial (day 5). As expected, the 5xFAD animals exhibited pronounced learning deficits during the MWM test, as indicated by significantly increased escape latency and swim distance compared to non- transgenic littermates. The 5xFAD animals of group D (Tg, vehicle) displayed significantly increased escape latency on day 2 and 3, which was paralleled by significantly higher swim distance on day 2 and 3. Swim speed, thigmotaxis and floating behavior were not significantly changed between treatment groups, suggesting comparable motor performance and emotional status. However, the 5xFAD animals of groups H and I displayed a noticeable trend towards reduced swim velocity. During the probe trial, no significant group differences in target zone crossings could be detected. All animals except Amiloride, HCQ nano and R-HCQ treated 5xFAD mice (groups A, C and E) showed a significantly higher abidance in the target quadrant (compared to the by-chance to stay, which is 25%). This indicates that these animals have a learning behavior. That indicates a strategic learning performance for these groups. Overall abidance in either quadrant of the water maze was not significantly changed between groups. The learning curves of the vehicle or any test compound treated 5xFAD animals showed no statistically significant differences, indicating that strategic learning performance was unaffected by any treatment.
[0277] Y maze The Y-maze test is routinely employed to assess spatial working memory in rodent models. The test is based on the natural behavior of rodents to explore new environments. Rodents typically prefer to investigate new environments rather than familiar ones, so animals will explore a new arm of a maze before returning to the one that was previously visited. Many parts of the brain including the hippocampus, septum, basal forebrain, and prefrontal cortex are involved in this task. Spontaneous alternation in the Y-Maze was tested in all animals in treatment week 12 and 13 at an age of ~9 months. No significant differences were detected between groups.
[0278] Histology: Immunofluorescence signal was quantified in the cerebral cortex and hippocampal formation for the following markers: Amyloid-beta was stained with ThioS. Highly significant differences between genotypes were evident by statistical analysis. In contrast, no significant effects of treatment were detected. Fibrillary Amyloid was labeled with rabbit polyclonal LOC antibody. Significant differences between genotypes were evident by statistical analysis. In contrast, no significant effects of treatment were detected. Ibal was labeled with guinea pig monoclonal antibody. Significant differences between genotypes were evident by statistical analysis. The size of microglia was significantly enlarged in R-HCQ-trcatcd mice compared to vehicle controls, and this effect occurred in both cortex and hippocampus. pTau was labeled with mouse monoclonal antibody. Significant differences between genotypes were evident by statistical analysis. In contrast, no significant effects of treatment were detected.
[0279] Biochemistry.
[0280] Human A0 1-40 and A0 1-42 levels were measured in FA and DEA soluble fraction of cortex and hippocampus of 5xFAD mice after receiving different treatments. No significant changes in A[> 1- 40 and A[> 1-42 levels were observed after any of the treatments in any of the fractions of cortex and hippocampus of transgenic animals compared to vehicle treated transgenic control group D (FIGS. 12A-12D, 13A-13D).
[0281] Different cytokines were measured in the cortex of hippocampus of 5xFAD mice and WT littermates after receiving different treatments. The levels of IFN y. IE- 10, II- 12p70, IL-2, IL-4 and IL-5 in cortex as well as levels of IL-4 in the hippocampus were below or close to LLOD. KC/GRO, TNF a and IL- 1 levels were significantly lower in cortex and hippocampus of all wild type animals than the 5xFAD vehicle control, reflecting the known genotype differences. As expected, saline treated wild type mice showed significantly lower levels of IL-6 in both cortex and hippocampus, compared to vehicle treated 5xFAD.
[0282] R-HCQ treatment led to significant decrease of TNF-a and IL- 10 in the hippocampus compared to 5xFAD vehicle control. While IL-6 was significantly increased in the cortex of S-HCQ treated 5xFAD mice (FIGS. 14A, 14B), compared to vehicle treated transgenic control, significantly decreased IL-1 0 in cortex and hippocampus were detected (FIGS. 15A, 15B).
[0283] Plasma NIL levels are increasingly accepted as a surrogate biomarker of neurodegeneration. NIL was quantified in the plasma of 5xFAD and wild type littermates at baseline (before treatment start), and after 4, 8 and 12 weeks of treatment (FIGS. 16A-16D). At all timepoints measured wild type animals showed significantly lower levels of NfL in their plasma. After 4 weeks of treatment S- HCQ treatment led to significant decrease of NfL in treated 5xFAD mice (FIG. 16B). After 8 weeks mice receiving S-HCQ and TUDCA showed significantly lower levels of NfL (FIG. 16C). Together, these results suggest that S-HCQ and TUDCA may attenuate neurodegeneration in a transgenic mouse model of AD. No effects were observed on plasma NfL levels after treatment with either racemic HCQ or R-HCQ.
[0284] Analyses were performed to test whether candidate AD drugs rescue the peak in plasma NfL levels observed in drug-treated Tg 5xFAD versus saline treated animals. These analyses accounted for the individual variability in NfL levels between animals. NfL was measured in blood plasma drawn at 0, 4, 8, and 12 weeks after injection. Both wild type (FIG. 17A) and 5xFAD mice (FIG. 17B), injected with saline, showed a pronounced NfL peak between 4 and 8 weeks, relative to the baseline NfL level at week 0 (FIG. 17A). FIGS. 17C-17H show the effects on NfL concentration in 5xFAD mice treated with racemic HCQ (FIG. 17C), HCQ nano (FIG. 17D), saline (FIG. 17E), R-HCQ (FIG. 17F), S-HCQ (FIG. 17G), TUDCA (FIG. 17H), and amiloride (FIG. 171); HCQ doses were 100 mg/kg and the TUDCA dose was 500 mg/kg. To gain quantitative support for this finding, the log-transformed baseline-to-peak fold-change of NfL on the indicator variable of drug treatment was regressed and the corresponding regression coefficient beta J was estimated (FIG. 18). HCQ nano and S-HCQ both fully prevented the peak in plasma NfL levels compared to saline-treated transgenic mice (p < 0.001 and p < 0.01), respectively. TUDCA and HCQ partially lowered the peak plasma NfL levels compared to saline-treated transgenic mice (p < 0.05).
[0285] GFAP levels were assessed in terminal plasma samples from animals of all groups (FIG. 19). In terminal plasma, no significant treatment effects were detected between transgenic groups, however a strong trend towards reduced plasma GFAP level in group E (R-HCQ) compared to transgenic vehicle group D was observed (p=0.087). Also no significant genotype effect was found when comparing TG vehicle group D with wildtype groups G, I or J.
[0286] Histology. The brains were processed as described for histological analysis. Measurements in repeated sections led to comparable background throughout all sections from all animals of all groups. No differences in tissue quality were detected during histological procedures. Examples of fluorescent labeling of ThioS + LOC + Ibal + pTau are provided in FIGS. 20-24.
[0287] ThioS'. Histofluorescence of Amyloid-beta was detected with ThioS staining, and the signal was quantified in the cerebral cortex (FIG. 25), and hippocampal formation (FIG. 26). As expected, highly significant differences between genotypes were detected by statistical analysis. In contrast, no significant effects of treatment occurred by statistical analysis with one-way ANOVA.
[0288] LOC: Immunofluorescence of fibrillary amyloid was detected with rabbit polyclonal LOC antibody, and the signal was quantified in the cerebral cortex (FIG. 27), and hippocampal formation (FIG. 28). As expected, highly significant differences between genotypes were detected by statistical analysis. In contrast, no significant effects of treatment occurred by statistical analysis with one-way ANOVA.
[0289] Ibal : Immunofluorescence of Ibal was detected with guinea pig monoclonal antibody, and the signal was quantified in the cerebral cortex (FIG. 29), and hippocampal formation (FIG. 30). As expected, highly significant differences between genotypes were detected by statistical analysis. The size of microglia was significantly enlarged in R-HCQ-treated mice compared to vehicle controls, and this effect occurred in both cortex and hippocampus, as detected by statistical analysis with one-way ANOVA.
[0290] pTau Immunofluorescence of pSer202/pThr205-Tau (pTau) was detected with mouse monoclonal AT8 antibody, and the signal was quantified in the cerebral cortex (FIG. 31), and hippocampal formation (FIG. 32). As expected, highly significant differences between genotypes were detected by statistical analysis. In contrast, no significant effects of treatment occurred by statistical analysis with one-way ANOVA. [0291] Discussion
[0292] Prescription HCQ sulfate is a racemic mixture of two enantiomers, S-HCQ and R-HCQ. Prior studies have found that after administration of racemic HCQ, there is a preferential accumulation of R-HCQ relative to S-HCQ (1.6-1 ,7x) in the brain. The results of the A[1 studies in cell culture and NfL studies in 5xFAD mice provide strong evidence that chiral switching to derive S-HCQ generates a novel HCQ derivative that exerts substantially superior efficiency over racemic HCQ and R-HCQ in ameliorating molecular abnormalities (A 42 clearance and A(3 release) relevant to AD. It also ameliorates increase in plasma NfL levels- an established biomarker of neurodegeneration in a transgenic mouse model of AD. This effect is not observed with treatment by either racemic HCQ or R-HCQ. These findings suggest that S-HCQ is a promising therapeutic candidate for AD as well as other neurological disorders such as ALS and Parkinson's disease (PD) and multiple sclerosis (MS) that are associated with an increase in plasma NfL levels. S-HCQ offers a novel and substantial enhancement in efficacy over both racemic HCQ and R-HCQ. The variability of HCQ levels in patients as well as the finding that it is the less efficacious R-enantiomer that preferentially accumulates in the brain following administration of racemic HCQ, offers a compelling therapeutic strategy to utilize S-HCQ as a superior therapeutic in neurological disorders as well as neuroinflammatory conditions such as MS. The results showing a lowering of plasma NfL levels by the bile acid TUDCA also offer the potential of combination treatments with S-HCQ and TUDCA in neurodegenerative and neuroinflammatory diseases. The studies in the 5x-FAD AD mouse model also tested a novel HCQ nano-formulation that uses a selective polylysine succinylated (PLS) drug delivery platform, which can pass through the BBB by scavenger receptor Al (SR-A1)- mediated transcytosis. This HCQ-PLS nano-formulation also reduces the peak plasma NfL levels in 5xFAD transgenic mice relative to saline-treated animals.
[0293] Example 9
[0294] Af> Clearance and Secretion
[0295] Methods'.
[0296] Compound preparation'. lOOOx stock solutions were prepared in DMSO and further diluted in culture medium so that a maximum final concentration of 0.1% DMSO was present in the wells. Items that did not need DMSO to solubilize were also adjusted to 0.1% DMSO to have the same conditions in all wells. DMSO stocks were aliquoted and stored at -20°C. Working dilutions were always prepared freshly on the day of experiment. Test item concentrations were 25 pM (cl), 2.5 pM (c2), 0.25 pM (c3), 0.1 pM (c4), 0.05 pM (c5), and 0.01 pM (c6). [0297] DAPT, y-Secretase Inhibitor IX, Lot 3218570, was obtained from Calbiochem (Cat. No. 565770, > 95%) and stored at -20 °C. The working solution as prepared fresh in 0.1% DMSO from stored DMSO aliquots. The treatment dosage was 400 nM.
[0298] A Clearance:
[0299] Culture and Treatment of BV-2 Cells: The murine microglial cell line BV-2 was cultivated in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% FCS, 1% penicillin/streptomycin and 2 mM L-glutamine (culture medium). For the A(3 clearance assay, 20 000 BV-2 cells per well (uncoated 96 well plates) were plated out. After 24 hours, medium was changed to treatment medium (DMEM, 5% fetal calf scrum (FCS), 2 mM L-glutaminc) and cells were maintained in treatment medium for the remaining culture period. After changing cells to treatment medium, the test items were administered 1 hour before A|3 stimulation (Bachem 4061966; final concentration in well: 200 ng/mL (dilutions in medium)). Cells treated with vehicle, cells treated with Ap alone, as well as wells with A but no cells served as controls. All wells were handled the same way. After 3 h of Ap stimulation, cell supernatants were collected for the Ap measurement and cells were carefully washed twice with PBS and thereafter lysed in 35 pl cell lysis buffer (50 mM Tris- HC1, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS) supplemented with protease inhibitors. Cells were incubated for 15 min at RT on a plate shaker, samples were frozen at -80°C until further use. The experiment was performed in n=6 technical replicates for all groups.
[0300] A 42 Measurement: Supernatants were diluted 1:250, cell lysates 1 :2.5 and analyzed for human Ap42 with MSD® V-PLEX Human Ap42 Peptide (6E10) Kit (K151LBE, Mesoscale Discovery). The immune assay was carried out according to the manual and plates were read on the MESO QuickPlex SQ 120. Analyte levels were evaluated according to adequate Ap peptide standards (MSD) as pg per mL. Protein concentration in the cell lysates was determined by BCA assay for potential normalization of Ap42 levels.
[0301] Results:
[0302] To assess the impact of the R-HCQ and S-HCQ on microglial Api-42 phagocytosis, murine microglial cell line BV-2 was treated with test items and Api -42 for 3 h. Thereafter supernatant and cell lysates were harvested and analyzed for remaining Api-42 in the supernatant as well as possibly increased intracellular Api-42.
[0303] S-HCQ (TI 16/17) treatment at the highest concentration did not lead to a significant reduction of Api-42 in the supernatant, while all other concentrations did (FIG. 33A). However, no significant increase of intracellular Api-42 could be detected (FIG. 33B).
[0304] The two highest and three lowest concentrations of R-HCQ (TI.18/19) led to significant reduction of Api-42 in the supernatant (Figure 34A); however no significant increase of intracellular Abetal-42 could be detected (FIG. 34B). [0305] The results show that S-HCQ is more effective at clearing A [142 in microglia compared to HCQ and R-HCQ.
[0306] Ap Secretion:
[0307] Cultivation and treatment of H4-hAPP cells: H4-hAPP cells were thawed and cultivated in Opti-MEM supplemented with 10% FCS, 1% penicillin/streptomycin 200 pg/ml Hygromycin B and 2.5 pg/ml Blasticidin S (^culture medium). H4-hAPP cells were seeded into 96 well plates (2 x 104 cells per well). On the next day, cells in 96 well plates were treated with T.I., R.I. (DAPT 400 nM) or vehicle. 24 h later, supernatants were collected for further A measurements. The experiment was performed in n=6 technical replicates for all groups.
[0308] A 38, 40, 42 measurement: Supernatants were diluted 1:10 and analyzed for human A038, 40, and 42 with MSD® 96-well MULTI-SPOT® 6E10 Abeta Triplex Assay (Mesoscale Discovery). The immune assay was carried out according to the manual and plates were read on the MESO QuickPlex® SQ 120 instrument (Mesoscale Discovery). Analyte levels were evaluated according to adequate A0 peptide standards (MSD) as pg per mL.
[0309] Results:
[0310] Neuroblastoma cells H4 overexpressing hAPP were treated with R-HCQ and S-HCQ at three concentrations and A[> secretion into the supernatant was assessed 24 h after treatment start. Ap 1-38, 1-40 and 1-42 were assessed using MSD assay (FIGS. 35A-35C, 36A-36C). All values were well within the detection range of the assay, whereas A[> I -40 was the most prominent A species, as expected. The reference item DAPT (y-secretase inhibitor) significantly reduced all tested species similarly on all plates.
[0311] The lowest concentration of S-HCQ significantly reduced levels of all analyzed Ap species (FIGS 35A-35C, c6). However, medium concentration was found to increase A01-38 and Ap 1 -42 (FIGS. 35A, 35C, c3). In contrast, no significant change in A0 secretion due to R-HCQ could be detected (FIGS. 36A-36C).
[0312] Example 10
[0313] Effect of HCQ Enantiomers, DIM, and TUDCA on Synaptic Plasticity in APP/PS1 Mice
[0314] To investigate the effect of HCQ racemic free base on APP/PS 1 hippocampal synaptic plasticity, electrophysiological experiments were performed utilizing the mixture at both 25 itM and 50 pM concentrations. Hippocampal slices from 4-5 month old APP/PS 1 mice were perfused with 50 pM HCQ racemic free base (FIG. 37A) or 25 pM HCQ racemic free base (FIG. 37B) for 1 hour (-30min to +30min) after 30 min stable baseline recordings were obtained. Thirty minutes after the introduction of racemic HCQ, late long-term potentiation (L-LTP) was induced in synaptic input 1 (SI) via a strong tetanizing (STET - 8% sucrose, 5% Triton® X-100, 50 mM EDTA, 50 mM Tris HO - G-Biosciences, St. Louis, MO) protocol, consisting of 3 tetanizing trains (arrows). 25 .M racemic HCQ resulted in significant potentiation (155.545 + 12.309 %) that sustained till the end of the experiment (118.003 ± 7.753 %), with SI slope values being significantly different from unstimulated S2 pathway throughout the experiment (FIG. 37B. However, 50 pM racemic HCQ resulted in slight potentiation (128.809 ± 8.116 %) that was no longer statistically relevant by the end of the experiment (119.117 + 8.167 %). The SI slope values were mostly significantly different up until the 160th minute. The independent S2 pathways in both conditions were stable throughout the experiments.
* represents p < 0.05, t-test at each time point between SI and S2.
[0315] Effects of the HCQ racemic free base differed from that of the previously tested HCQ sulfate. 25 pM HCQ racemic resulted in partial rescue of L-LTP in APP/PS 1 mice, similar the HCQ sulfate. However, 50 pM HCQ racemic had a diminished initial potentiation which did not last, as compared to 50 pM HCQ sulfate, which resulted in a complete rescue of L-LTP. Concentration-dependent effects may have manifested in the 50 pM trials. A preliminary (n=l) test using age-matched WT mice supported this hypothesis with the observation that L-LTP was partially impaired in these mice with 50 pM HCQ racemic free base.
[0316] To investigate potential differences in R-HCQ and S-HCQ in rescuing impaired L-LTP in APP/PS 1 mice at a threshold concentration, field electrophysiological experiments were performed utilizing the two enantiomers at 25 pM or 50 pM. Hippocampal slices from 4-5 month old APP/PS 1 mice were perfused with 25 pM R-HCQ (FIG. 38A), 25 pM S-HCQ (FIG. 38B), 50 pM R-HCQ (FIG. 39A), or 50 pM S-HCQ (FIG. 39B) for 1 hour (-30min to +30min) after 30 min stable baseline recordings were obtained. Thirty minutes after the introduction of HCQ, L-LTP was induced in synaptic input 1 (SI) via a strong tetanizing (STET) protocol, consisting of 3 tetanizing trains (arrows).
[0317] 25 pM R-HCQ resulted in strong potentiation (166.451 12.702 %) that sustained to the end of the experiment (130.644 12.132 %) (FIG. 38A). Similarly, 25 pM S-HCQ resulted in significant potentiation (152.676 + 7.545 %) that lasted for at least 3 hours (131.360 ± 12.007 %) (FIG. 38B). Independent synaptic pathways S2, designated as control pathways, were stable in both groups throughout the experiments. As seen in FIGS. 38A and 38B, 25 pM R-HCQ and 25 pM S-HCQ had similar magnitudes of potentiation throughout the evaluation.
[0318] 50 pM R-HCQ resulted in strong potentiation (158.239 + 15.477 %) that sustained to the end of the experiment (131.428 + 7.469 %) (FIG. 39A). Similarly, 50 pM S-HCQ resulted in significant potentiation (140.492 ± 7.120 %) that last for at least 3 hours (137.275 + 11.517%) (FIG. 39B). Independent synaptic pathways S2, designated as control pathways, were stable in both groups throughout the experiments. As seen in FIGS. 39A and 39B, 50 pM R-HCQ and 50 pM S-HCQ had similar magnitudes of potentiation throughout the evaluation. [0319] FIGS. 40A and 40B show the effects of 25 pM HCQ enantiomers (FIG. 40A) and 50 pM HCQ enantiomers (FIG. 40B) on L-LTP. Statistical comparisons between concentrations of either enantiomer are not significant.
[0320] To investigate efficacy of DIM in rescuing impaired L-LTP in APP/PS 1 mice at a threshold concentration, field electrophysiological experiments were performed utilizing 10 pM DIM. DIM had an acute rescue effect on L-LTP in APP/PS 1 slices. FIG. 41 A shows that the application of 10 pM DIM rescued L-LTP in APP/PS 1 slices. FIG. 41B shows that a 3-hour pre-incubation with 10 pM DIM no longer resulted in rescue of L-LTP in APP/PS 1 slices.
[0321] Application of a mixture of 40 pM HCQ sulfate and 10 pM DIM blocked expression of carly- LTP upon delivery of STET in APP/PS 1 slices (FIG. 42). TUDCA was also found to have a deleterious effect on expression of LTP. STET no longer induced LTP in APP/PS 1 mice when applied under the influence of 100 pM TUDCA (FIG. 43).
[0322] Example 11
[0323] Correlation in Whole Blood Levels of HCQ with Plasma Biomarker Concentrations
[0324] The relationships between whole blood HCQ levels in patients with a lupus cohort and plasma biomarkers of AD will be evaluated. Plasma concentrations of A 42 and A [340. phosphorylated tau (p-tau), neurofilament-light chain; NfL and glial fibrillary acidic protein; GFAP in serially collected plasma samples in this cohort will be assayed using established immunoassay methods. A secondary aim is to assay whole blood levels of stereoisomers of HCQ. Female patients aged 55-80 years will be included in the study. Immunoassays for A/?40, A ? 42, GFAP, and NfL will be performed. Phosphorylated tau (p-tau 181 and p-tau231) will be measured. Whole blood assays of HCQ enantiomers will be performed by a supercritical chromatography (SFC) method using a ColumnTek® EnantioCel® C2-5 pm column (available through Fisher Scientific, Waltham, MA).
[0325] Analyses will be performed both cross-sectionally (at individual time points where concordant data are available for whole blood HCQ levels and plasma AD biomarker concentrations) and longitudinally (change in whole blood HCQ levels with change in plasma AD biomarker concentrations). Covariates included will be lupus disease severity, duration, and age. Additional analyses will be performed both cross-sectionally (at individual time points where concordant data are available for whole blood R-HCQ and S-HCQ levels and plasma AD biomarker concentrations) and longitudinally (change in whole blood R-HCQ and S-HCQ levels with change in plasma AD biomarker concentrations). Covariates included will be lupus disease severity, duration, and age. [0326] Example 12
[0327] Effects of HCQ, DIM, and TUDCA on A[>l-4() and 1-42 in 5xFAD mice
[0328] Transgenic 5xFAD mice were randomly allocated to treatment groups. The expression of the 5xFAD transgene is driven by the neuron specific Thyl promoter. The five mutations cause an early onset of the cognitive decline and increasing Af! 1-40 and 1-42 levels in the brain and cerebrospinal fluids, over age. Histological analysis revealed plaque load and beta sheet formation accompanied with neuroinflammation. Thus, the 5xFAD mouse mimics the most crucial phenotypic symptoms of amyloidogenic neurodegeneration, neuroinflammation as well as learning and memory deficits and is a suitable model for Alzheimer’s disease to study effects of drugs on biochemical, histological and behavioral hallmarks. All animals were treated with test compound or vehicle for the whole study period by i.p. injection and then euthanized. Male mice administered TUDCA + HCQ were dosed with TUDCA on M/F and HCQ all other days for weeks 1-4, then TUDCA on M/F and HCQ on T/W/Th for weeks 5-8, and then TUDCA on M/F and HCQ on W for weeks 9-14. Animals or mixed sex in other groups were administered the pertinent compounds daily for the 14- week period.
[0329] The frozen cortex samples from animals of each group were homogenized in lysis buffer (THB; 250 mM Sucrose, 1 mM EDTA, 1 mM EGTA, 20 mM Tris pH 7.4) including lx protease inhibitor (Calbiochem). The tissue was homogenized with a beadmill (UPHO, Geneye) at 55Hz for 50sec. Aliquots were stored at 80°C until further use. For extraction of non-plaque associated proteins, 1 aliquot of THB homogenate was mixed with 1 part diethylamine (DEA) solution (0.4% DEA, lOOmM NaCl). The mixture was centrifuged for 120 min at 20,000xg, 4°C. The supernatant was neutralized with 1/10 of the volume 0.5 M Tris-HCl, pH 6.8 and vortexed briefly. Aliquots were stored at -80°C as DEA fraction (soluble fraction). For extraction of deposited proteins, a second aliquot of THB homogenate was mixed with 2.2 parts cold formic acid (FA), sonicated for 30 sec on ice and centrifuged for 120 min at 20,000xg, 4°C. The supernatant was mixed with 19 parts FA Neutralization Solution (IM Tris, 0.5 M Na2HPO4, 0.05% NaN3). Aliquots were stored at 80°C as FA fraction (insoluble fraction). For measurement of inflammation markers, the third aliquot of homogenate was substituted with Triton® X-100 (Dow Chemical Company, Midland, MI) so that the final concentration as 1 % in the homogenate. After vortexing and 10 min incubation on ice, the homogenates were cleared from cell debris by centrifugation at 20,800 x g at 4°C for 10 minutes in a tabletop centrifuge and the supernatants were collected for the measurement of cytokines and stored at -80°C until further use as Triton® fraction.
[0330] Afi 1-40 and Afi 1-42 levels in DEA and FA fraction'. A[3 1-40 and A(3 1 -42 were measured in duplicates in the fractions described above (DEA and FA fractions) using Ap 1-42 with MSD® Human (6E10) V-plex kit (K151LBE-2, Mesoscale Discovery) and A 1-4O with MSD® Human (6E10) V-plex kit (K150SKE-2, Mesoscale Discovery) according to the instructions of the manufacturer. Plates were read on Quickplex SQ 120 sector imager (Mesoscale Discovery). A[> levels in study samples were evaluated in comparison to calibration curves provided in the kit and are expressed as pg per mg brain wet weight.
[0331] Human A 1-40 and A[3 1-42 levels were measured in the FA and DEA soluble fractions of the cortex of 5xFAD mice after receiving different treatments (FIGS. 44A-44D). Group A - transgenic mice, saline; Group C - tg mice, TUDCA 500 mg/kg + HCQ 100 mg/kg; Group D - tg mice, DIM 50 mg/kg; Group E - tg mice, DIM 100 mg/kg; Group F - tg mice, DIM 200 mg/kg; Group G - tc mice, vehicle (10% DMSO, 10% pEG300, 10% Tween® 80 surfactant (Sigma-Aldrich, St. Louis, MO), 70% H2O). TUDCA+HCQ treatment (group C) resulted in statistically significant lowering of A [3 1 -40 and A[3 1 -42 levels in the FA fraction of the cortex compared to saline-treated transgenic mice (group A) (FIGS. 44A-44B).
[0332] Example 13
[0333] Effect of HCQ and DIM on A/3 clearance and LPS-induced neuroinflammation in cells
[0334] Methods
[0335] Compound preparation: lOOOx stock solutions were prepared in DMSO and further diluted in culture medium so that a max final concentration of 0.1% DMSO was present in the wells. Items that did not need DMSO to solubilize were also adjusted to 0.1% DMSO to have the same conditions in all wells. DMSO stocks were aliquoted and stored at -20°C. Working dilutions were always prepared freshly on the day of experiment.
[0336] Culture and treatment of BV-2 cells'. The murine microglial cell line BV-2 was cultivated in DMEM medium supplemented with 10% FCS, 1% penicillin/streptomycin and 2 mM L-glutamine (culture medium).
[0337] For A0 clearance assay, 20,000 BV-2 cells per well (uncoated 96 well plates) were plated out. After 24 hours, medium was changed to treatment medium (DMEM, 5% FCS, 2 mM L-glutamine) and cells were maintained in treatment medium for the remaining culture period. After changing cells to treatment medium, the test items were administered 1 hour before A[> stimulation (Bachem 4061966; final concentration in well: 200 ng/mL (dilutions in medium)). Cells treated with vehicle, cells treated with A(3 alone, as well as wells with A(3 but no cells served as controls. All wells were handled the same way. After 3 h of A(3 stimulation, cell supernatants were collected for the A[> measurement and cells were carefully washed twice with PBS and thereafter lysed in 35 pl cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS) supplemented with protease inhibitors. Cells were incubated for 15 min at RT on a plate shaker, samples were frozen at -80°C until further use. The experiment was performed in n=6 technical replicates for all groups.
[0338] For LPS stimulation assay, 5000 BV-2 cells per well (uncoated 96 well plates) were plated out. After 48 hours, medium was changed to treatment medium (DMEM, 5% FCS, 2 mM L- glutamine) and cells were maintained in treatment medium for the remaining culture period. After changing cells to treatment medium, the HCQ/DIM concentrations were administered 1 hour before LPS stimulation (Sigma- Aldrich; L6529; 1 mg/ml stock in ddHzO, final concentration in well: 100 ng/mL (dilutions in medium)). Cells treated with vehicle, cells treated with LPS alone, as well as cells treated with LPS plus reference item (dexamethasone, 10 pM) served as controls. All wells were handled the same way. After 24 h of stimulation, cell supernatants were collected for the cytokine measurement and cells were subjected to MTT assay. The experiment was performed in n=6 technical replicates for all groups.
[0339] Culture and treatment of H4-hAPP cells'. H4-hAPP cells were thawed and cultivated in Opti- MEM supplemented with 10% FCS, 1% penicillin/streptomycin 200 iig/rnl Hygromycin B and 2.5 pg/ml Blasticidin S (=culture medium). H4-hAPP cells were seeded into 96 well plates (2 x 104 cells per well). On the next day, cells in 96 well plates were treated with T.I., R.I. (DAPT 400 nM) or vehicle. 24 h later, supernatants were collected for further A(i measurements by MSD. The experiment was performed in n=6 technical replicates for all groups.
[0340] Af>42 measurement in BV-2 cells'. Supernatants were diluted 1:250, cell lysates 1:2.5 and analyzed for human A 42 with MSD® V-PLEX Human A 42 Peptide (6E10) Kit (K151LBE, Mesoscale Discovery). The immune assay was carried out according to the manual and plates were read on the MESO QuickPlex SQ 120. Analyte levels were evaluated according to adequate A[3 peptide standards (MSD) as pg per mL. Protein concentration in the cell lysates was determined by BCA assay for potential normalization of A[342 levels.
[0341] Results'. HCQ sulfate/DIM - cl = 25 pM HCQ/10 pM DIM, c2 = 25 pM HCQ/3 pM DIM, c3 = 10 pM HCQ/10 pM DIM. Treatment at none of the concentrations led to a significant reduction of Ap 1-42 in the supernatant (FIG. 45A). However, significant increase of intracellular Ap 1 -42 could be detected in the lysate of cells treated with the second highest concentration (FIG. 45B).
[0342] Af38, 40, 42 measurement in H4-hAPP cells'. Supernatants were diluted 1: 10 and analyzed for human A 38, 40, and 42 with MSD® 96-well MULTI-SPOT® 6E10 Abeta Triplex Assay (Mesoscale Discovery). The immune assay was carried out according to the manual and plates were read on the MESO QuickPlex SQ 120. Analyte levels were evaluated according to adequate A[3 peptide standards (MSD) as pg per mL.
[0343] Results'. Neuroblastoma cells H4 overexpressing hAPP were treated with HCQ sulfate and DIM at 3 concentrations: cl = 25 pM HCQ/10 pM DIM, c2 = 25 pM HCQ/3 pM DIM, c3 = 10 pM HCQ/10 pM DIM. A(3 secretion into the supernatant was assessed 24 h after treatment start. A i-38, 1-40 and 1-42 were assessed using MSD assay. All values were well within the detection range of the assay, whereas A i-40 was the most prominent A species, as expected. The reference item DAPT (gamma-secretase inhibitor) significantly reduced all tested species similarly on all plates. None of the concentrations of HCQ/DIM had any significant effect on Ap levels, the reference Item significantly reduced all A|3 species. No toxicity of the test item could be seen in the MTT assay (FIGS. 46A-46D).
[0344] Cytokine measurement: Levels of 10 cytokines (IFN-y, IL-i , IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-a) were measured in supernatants (1:2 diluted) of BV-2 cells collected 24 h after LPS stimulation. Cytokines were measured by an immunosorbent assay (V-PLEX Proinflammatory Panel 1 Mouse Kit, K15048D, Mesoscale) according to the instructions of the manufacturer and evaluated in comparison to calibration curves provided in the kit. Results are given as pg per mL.
[0345] Results: Microglial BV2 cells were stimulated with LPS and cytokine secretion as well as cell viability were assessed (FIGS. 47A-47F). As expected, a significant increase of pro- inflammatory cytokines due to LPS stimulation was observed, which could be reversed with RI dexamethasone treatment on all plates. Treatment with HCQ sulfate/DIM was performed at 3 concentrations: cl = 25 pM HCQ/10 pM DIM, c2 = 25 pM HCQ/3 pM DIM, c3 = 10 pM HCQ/10 pM DIM. Treatment led to significant increase in TNF-a at all concentrations (FIG. 47 A). IL-6 levels were significantly increased at the lowest concentration FIG. 47C). KC/GRO was significantly increased at all concentrations (FIG. 47D). The MTT Assay indicated higher viability of samples treated with low and middle dose of HCQ/DIM, which may be associated with the increased cytokine secretion (FIG. 47 F).
[0346] Example 14
[0347] Effect of HCQ and TUDCA on GFAP in Astrocytes and Brain Slices
[0348] Methods
[0349] Compound preparation: lOOOx stock solutions were prepared in DMSO and further diluted in culture medium so that a maximum final concentration of 0.1% DMSO was present in the wells. Items that did not need DMSO to solubilize were also adjusted to 0.1% DMSO to have the same conditions in all wells. DMSO stocks were aliquoted and stored at -20°C. Working dilutions were always prepared freshly on the day of experiment.
[0350] Culture and treatment of primary mouse astrocytes: The murine primary astrocytes (generated and stored frozen at QPS America Inc., Independence, Ohio) were thawed according to SOP NMET050 and cultivated in DMEM medium supplemented with 10% FCS, 1% penicillin/streptomycin and 2 mM L-glutamine (culture medium). After thawing, astrocytes were seeded onto PDL (poly-D-lysine)-coated plates at a cell density of 2 xlO4 cells/well (96-well plate). 24 hours after seeding, medium was changed to treatment medium (DMEM, 5% FCS, 2 mM L- glutamine) and cells were maintained in treatment medium for the remaining culture period. [0351] After changing cells to treatment medium, the test items (HCQ sulfate, S-HCQ free base, R- HCQ free base, TUDCA/HCQ, TUDCA/S-HCQ, TUDCA/R-HCQ, and TUDCA) and reference item (dexamethasone) were administered 1 hour before LPS stimulation (Sigma-Aldrich; L6529; 1 mg/mL stock in ddH2O, final concentration in well: 1000 ng/mL (dilutions in medium)). Cells treated with vehicle and cells treated with LPS alone served as controls. All wells are handled the same way. After 24 h of stimulation, cell supernatants were collected for GFAP measurement and cells were harvested in 30 pL/well RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1% Nonidet P40, 2%SDS, Protease Inhibitor Cocktail I (Calbiochem), Phosphatase Inhibitor Cocktail III (Sigma)), protease inhibitors. The experiment is performed in n=6 technical replicates for all groups.
[0352] Culture and treatment of organotypic brain slices’. P9/P10 mouse pups (C57BL/6JRccHsd) were decapitated, skin and skull gently removed and brains immersed in slicing medium (Opti-MEM 1, 20 uM glucose). Brains were hemisected and hippocampi were isolated. Hippocampi were placed on the cutting disc of a McIlwain Tissue Chopper. 300 pm thick organotypic brain slices were chopped transversely. A number of 4 slices per hippocampus were placed onto porous (0.4 pm) transparent membrane inserts (12 mm in diameter for 24 well plate) and incubated for 1 h on ice in Hanks’ balanced salt solution (HBSS) containing 10 mM glucose. Afterwards inserts were transferred to fresh 24- well plates containing 300 pL culture medium (50% MEM/Earlc’ s balanced salt solution (EBSS), 25% horse serum, 25%CMF-HBSS, 25 mM glucose, 0.5% pen/strep). Slices were maintained at 37°C and 5% CO2. The full medium was changed on days 3-5. In total, 36 wells were prepared. On day 8, culture medium was replaced with serum reduced medium = treatment medium (MEM/EBSS, 5% horse serum, 25 mM glucose) and slices were maintained in this medium for the remaining treatment period.
[0353] Test item (HCQ sulfate, S-HCQ free base, R-HCQ free base, TUDCA/HCQ, TUDCA/S- HCQ, TUDCA/R-HCQ, TUDCA), reference item (dexamethasone), or vehicle was administered to the well 1 h before LPS (100 ng/ml) stimulation by addition of 15 pL 22x stock. LPS stimulation (Sigma-Aldrich; L6529; 1 mg/mL stock in ddH2O, final concentration in well: 100 ng/ml) is applied 1 h after addition of test or reference item by addition of another 15 pL 22x stock LPS solution to achieve a final concentration of 100 ng/well. Slices treated with vehicle (no LPS), LPS alone, or reference item served as controls. All control and compound treated wells were handled the same way. For each condition, 5 replicates were made. All 6 replicates of one condition were from 6 different animals and mixed left and right hippocampi. Cell media were collected, stored at -80°C for the measurement of cytokines 24h after LPS stimulation. Furthermore, slices were lysed in RIPA buffer after 24 h LPS treatment and lysates were also stored at -80°C until GFAP analysis. 24 h LPS stimulated brain slices were lysed with 100 pL/wcll RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1% Nonidet P40, 2%SDS, Protease Inhibitor Cocktail I (Calbiochem), Phosphatase Inhibitor Cocktail III (Sigma)). Total protein content of the sample was assessed by using BCA assay (Pierce, 23225). Cell lysates were stored at -80°C for further analysis.
[0354] Astrocyte results:
[0355] Primary astrocytes were treated with racemic HCQ sulfate, S-HCQ, or R-HCQ and LPS for 24 h. Thereafter supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/mL as well as the content of GFAP in cell lysates as pg/mL lysate. HCQ sulfate, S-HCQ, and R-HCQ were evaluated at three concentrations: cl = 25 pM, c2 = 2.5 pM, c3 = 0.25 pM.
[0356] HCQ sulfate significantly increased the concentration of GFAP in the lysate of primary astrocytes at the third treatment concentration (FIGS. 48A and 48B). No effects of the test items were observed in the supernatant or for any other concentration in the lysate.
[0357] S-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate of primary astrocytes (FIGS. 49A, 49B). R-HCQ free base also had no significant impact on GFAP levels in the supernatant or lysate of primary astrocytes (FIGS. 50A, 50B).
[0358] Primary astrocytes also were treated with TUDCA/HCQ sulfate (racemic), TUDCA/S-HCQ free base, TUDCA/R-HCQ free base, or TUDCA alone and LPS for 24 h. Thereafter supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/ml as well as the content of GFAP in cell lysates as pg/ml lysate. The TUDCA/HCQ combinations were evaluated at three concentrations: cl = 100 pM/25pM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. TUDCA was evaluated at three concentrations: cl = 100 pM, c2 = 10 pM, c3 = 1 pM.
[0359] TUDCA/HCQ sulfate significantly increased GFAP levels in the supernatant of primary astrocytes, for the third concentration and in the cell lysate no significant effects were observed (FIGS. 51 A and 5 IB). TUDCA/S-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate (FIGS. 52A and 52B). TUDCA/R-HCQ free base also had no significant impact on GFAP levels in the supernatant or lysate (FIGS. 53A and 53B). Additionally, TUDCA alone had no significant impact on GFAP levels in the supernatant or lysate (FIGS. 54A and 54B).
[0360] Organotypic brain slice results:
[0361] Organotypic brain slices were treated with HCQ sulfate (racemic), S-HCQ free base, or R- HCQ free base and LPS for 24 hours. Thereafter supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/mL as well as the content of GFAP in cell lysates as ng/pg lysate. HCQ sulfate, S-HCQ, and R-HCQ were evaluated at three concentrations: cl = 25 pM, c2 = 2.5 pM, c3 = 0.25 pM.
[0362] HCQ sulfate (racemic) significantly increased the concentration of GFAP in the supernatant of brain slices at the highest tested concentration; no effects were observed in the supernatant for any other concentration or in the lysate (FIGS. 55A and 55B). S-HCQ free base significantly reduced secreted GFAP in the supernatant at the lowest tested concentration, while no impact on GFAP levels in the lysate of brain slices was detected (FIGS. 56A and 56B). R-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 57 A and 57B).
[0363] Organotypic brain slices also were treated with TUDCA/HCQ sulfate (racemic), TUDCA/S- HCQ free base, TUDCA/R-HCQ free base, or TUDCA alone and LPS for 24 h. Thereafter supernatant and cell lysates were harvested and analyzed for the changes in GFAP level in the supernatant as pg/ml as well as the content of GFAP in cell lysates as ng/ pg lysate. The TUDCA/HCQ combinations were evaluated at three concentrations: cl = 100 pM/25pM, c2 = 10 pM/25 pM, c3 = 100 pM/2.5 pM. TUDCA was evaluated at three concentrations: cl = 100 pM, c2 = 10 pM, c3 = 1 pM.
[0364] TUDCA/HCQ sulfate (racemic) had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 58A and 58B). TUDCA/S-HCQ free base significantly reduced secreted GFAP in the supernatant at the highest tested concentration, while no significant impact on GFAP levels in the lysate of brain slices was detected (FIGS. 59A and 59B). TUDCA/R-HCQ free base had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 60A and 60B). Additionally, TUDCA alone had no significant impact on GFAP levels in the supernatant or lysate of brain slices (FIGS. 61 A and 6 IB).
[0365] Example 15
[0366] Effect of(S)-HCQ and TUDCA on cognitive and molecular pathology in an Alzheimer’s mouse model
[0367] Methods
[0368] Trial Design
[0369] The mice in this study are split into four groups including two treatment groups: 1) 5x Familial Alzheimer’s disease (FAD) transgenic (TG) mice that receive the TUDCA + (S)-HCQ test compounds; 2) wild-type (WT) mice that receive the TUDCA + (S)-HCQ test compounds; and two vehicle control groups: 3) 5xFAD TG mice that receive saline; 4) WT mice that receive saline. Animals are dosed daily for the entire 15-week trial and serial, in-vivo blood samples are collected for biomarker analyses. All animals are tested twice using the Y-maze and once using the Morris Water Maze (MWM) to determine the effect of treatment/ vehicle control on cognitive deficits. At the end of the 15-week trial, all animals are euthanized; terminal blood is collected and brains are dissected and frozen for histological and biochemical analyses.
[0370] Animals
[0371] The 5xFAD mouse model has five mutations including three in the amyloid precursor protein (APP695) gene [APP K670N/M671L (Swedish), I716V (Florida), V717I (London)] and two mutations in the presenilin 1 gene [PSI M146L, L286V]. The 5xFAD mouse mimics phenotypic symptoms of Alzheimer’s disease including amyloidogenic neurodegeneration, neuroinflammation,
- T - and learning and memory deficits. This is a suitable model to study the effects of potential treatment drugs on biochemical, histological, and behavioral hallmarks of Alzheimer’s disease (AD).
[0372] Drug treatment dosage and schedule
[0373] Mice receive intraperitoneal (IP) injections of 500 mg/kg Tauroursodeoxycholic acid (TUDCA) on Monday and Friday throughout the 15 week trial duration. Mice receive IP injections of 100 mg/kg of (S)-hydroxychloroquine ((S)-HCQ) on Tuesday through Thursday and Saturday and Sunday during weeks 1-4, Tues through Thursday on weeks 5-8, and Wednesday only on week 9 through the end of the trial.
[0374] In-vivo blood sampling
[0375] To measure biomarker progression over the course of the trial, in-vivo blood samples are collected by mandibular sampling from each animal at 4 time points: baseline (prior to the start of treatment) and after 4, 8, and 12 weeks of treatment. K2EDTA (potassium ethylenediaminetetraacetic acid) tubes are inverted thoroughly to ensure homogenous distribution of EDTA and to prevent clotting. Samples are centrifuged (3000 x g for 10 minutes at room temperature) and plasma aliquots are transferred to 1.5 mL tubes, frozen on dry ice and stored at -80 °C.
[0376] Brain tissue sampling and preparation
[0377] To measure A I-40, A/J-42 and inflammatory marker differences between between treatment and control groups, after the end of the trial, animals are transcardially perfused with 0.9% saline. A 23-guage needle connected to a 0.9% saline is inserted into the left ventricle. The thoracic aorta is clamped with hemostatic forceps to block the flow of blood to the abdomen but allowing blood to flow to the brain. The right atrium is opened with scissors. A constant pressure of 100-120 mm Hg was maintained on the perfusion solution (manometer-controlled air compressor). Perfusion is continued until the skull surface turns pale and only perfusion solution is exiting the right atrium. [0378] The animal’s skull is then opened and the brain carefully removed. The left hemi-brain is dissected on a cooled surface into the hippocampus, cortex and rest of the brain. Brain sections are then weighed, snap frozen on dry ice and stored at -80°C.
[0379] The cortex and hippocampus from a selection of animals per group are prepared. Briefly, brain tissue samples are homogenized by adding 9 or 19 volumes (to cortex and hippocampus samples respectively) of tissue homogenization buffer (THB; 250 mM Sucrose, 1 mM EDTA, 1 mM EGTA, 20 mM Tris pH 7.4) including lx protease inhibitor (Calbiochem). The tissue is homogenized with a beadmill (UPHO, Geneye) at 55Hz for 50 seconds. Three aliquots are stored at -80°C until further use.
[0380] For measurement of A/7-40, A//-42 levels, the DEA fraction (soluble fraction), non-plaque associated proteins are extracted as follows: 1 aliquot of THB homogenate is mixed with 1 part diethylamine (DEA) solution (0.4% DEA, lOOmM NaCl). The mixture is centrifuged for 120 minutes at 20,000xg, 4 °C. The supernatant is neutralized with 1/10 of the volume 0.5 M Tris-HCl, pH 6.8 and vortexed briefly. Aliquots are stored at -80 °C. For the FA fraction (insoluble fraction), plaque associated proteins are extracted as follows: a second aliquot of THB homogenate is mixed with 2.2 parts cold formic acid (FA), sonicated for 30 seconds on ice and centrifuged for 120 minutes at 20,000xg, 4 °C. The supernatant is mixed with 19 parts FA Neutralization Solution (IM Tris, 0.5 M NaiHPO i, 0.05% NaNi). Aliquots are stored at -80 °C.
[0381] For measurement of inflammation markers, the third aliquot of homogenate is substituted with Triton X-100 so that the final concentration is 1% in the homogenate. After vortexing and 10 minutes incubation on ice, the homogenates are cleared from cell debris by centrifugation at 20,800 x g at 4 °C for 10 minutes in a tabletop centrifuge and the supernatants are collected for the measurement of cytokines and stored at -80 °C until further use as the Triton fraction.
[0382] Cognitive outcome - Morris Water Maze
[0383] The Morris Water Maze (MWM) is the primary, cognitive outcome of the trial. The MWM is a tool frequently used in drug trials to test the effect of drug exposure on spatial learning and memory deficits in animals modelling neurologic diseases including AD. All animals are assessed in the MWM on week 14. The assessment has two consecutive parts: the training probe (days 1-4) and the test probe (on day 5).
[0384] MWM training probe
[0385] On all four days of the MWM training probe, the platform is placed in the northeast quadrant of the pool and the animal start position (southeast, southwest, northwest) is predefined and varied across days. Each subject is allowed one and only one swim-trial a day in order to “escape” the MWM by finding the platform. Each swim-trial ends at 60 seconds or at the escape latency (the time needed to find the platform), whichever time is shorter. Thus, the escape latency data are censored. Note that the escape latency is expected to decrease with not only cognitive ability but also time as the subject learns and memorizes the location of the platform increasingly better with time during the training probe.
[0386] In addition to escape latency, the following are also recorded and included in modeling the treatment effect: drug treatment, training day, the sex and subject ID, cohort, swimming velocity, time spent in thigmotaxis, and time spent floating.
[0387] MWM test probe
[0388] On day 5, during the test probe, the platform is removed from the pool and the time spent in each quadrant is recorded. Performance is evaluated based on the notion that the better a mouse remembers the location of the platform, the higher fraction of time the mouse spends in the target quadrant (containing the platform’s former location) and the lower fraction of time in the opposite quadrant. For each mouse group the mean fraction of time for each quadrant is estimated using the Dirichlet distribution.
[0389] The estimated fractions are then used to quantify both the size of an effect and its statistical significance. Effect size is quantified using the information gain of a test condition relative to a reference condition (e.g Saline WT relative to Saline TG). Information gain is defined as Hret - Htest, where Hx is the information entropy of the Dirichlet distribution estimated for the particular condition x. The statistical significance of an effect is obtained from the likelihood ratio test comparing the likelihood of the test condition to that of the reference condition, p-values are calculated based on the standard assumption that the likelihood ratio test statistic is Chi2-distributed.
[0390] Biomarker outcomes
[0391] Affl-40, A/T 1-42
[0392] A[> 1-40 and Ap 1-42 are measured in duplicates in the soluble and insoluble fractions described above. Ap 1-40 is measured with MSD® Human (6E10) V-plex kit (K151LBE-2, Mesoscale Discovery) and A 1-42 is measured with MSD® Human (6E10) V-plex kit (K150SKE-2, Mesoscale Discovery) following instructions provided by the manufacturer. Plates are read on Quickplex SQ120 sector imager (Mesoscale Discovery). Ap 1-40 and 1-42 levels in the brain tissue samples are evaluated in comparison to calibration curves provided in the kit and are typically expressed as pg per mg tissue wet weight.
[0393] Inflammation markers
[0394] Levels of ten cytokines (IFN-y, IL-1 P, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL- 10, IL-12p70, TNF-a) are measured in the brain homogenate aliquots using an immunosorbent assay (V-PLEX Custom Mouse Cytokine, Mesoscale Discovery cat. nr. K15048D)).
[0395] Nfl measurements
[0396] Neurofilament light chain (NfL) are measured at all 4 in-vivo blood timepoints with NF- light® (Neurofilament-light) ELISA 10-7001 CE from UmanDiagnostics. Samples are diluted (1:3) in assay buffer and analyzed according to manufacturer protocol. After dilution, 100 pl of sample are added to the pre-coated wells and incubated for 1.5 hours at room temperature (RT) with gentle agitation (800 rpm). Wells are washed three times with assay wash buffer and 100 pl of the tracer antibody are added. After 45 minutes incubation (RT, 800 rpm) wells are washed again three times. [0397] Then, 100 pl of conjugate is added and incubated for 30 minutes (RT, 800 rpm). After 3 x washing 100 pl of TMB substrate are added to each well and incubated for 15 minutes at RT. 50 pl stop reagent is added and after a short gentle agitation, the plate is read at 450 nm (reference 620-650 nni) on the Cytation 5 multimode reader (Biotek). Data are evaluated in comparison to calibration curves provided in the kit and are expressed as pg/ml plasma.
[0398] Example 16
[0399] Effect of (S)-HCQ and TUDCA on cognitive and molecular pathology in an Alzheimer's mouse model
[0400] Methods
[0401] Trial Design [0402] The trial was run according to the protocol described in Example 15, but using racemic hydroxychloroquine (HCQ) in place of the S-enantiomer ((S)-HCQ) that is referred to in Example 15.
[0403] Results
[0404] Effect of TUDCA + HCQ treatment on impaired spatial learning in 5xFAD mice
[0405] The study was performed to determine whether combined treatment with HCQ and TUDCA resulted in improved learning in transgenic 5xFAD mice relative to saline-treated littermates.
Impaired spatial learning in this AD model has been well characterized previously and was reflected in prolonged escape latency in the Morris water maze (MWM) task. This metric assesses the time required for the animal to find a submerged and hidden platform in a circular water pool across a given number of learning trials. Previously reported impairment in spatial learning in transgenic 5xFAD mice relative to wild type littermates was first confirmed by showing that escape latency in saline-treated 5xFAD transgenic mice was 1.5-2 fold higher relative to saline-treated WT mice (data not shown). A striking improvement in escape latency was observed in HCQ+TUDCA treated transgenic mice relative to saline-treated transgenic littermates. Escape latencies in transgenic 5xFAD mice treated with HCQ and TUDCA were similar to those of wild type mice.
[0406] The hypothetical cognitive benefits of TUDCA, HCQ and combined TUDCA + HCQ treatment were tested in Morris Water Maze (MWM) experiments (Methods). Strong, statistically significant effects were observed in these experiments, which are presented in two parts below: (1) MWM training probe and (2) test probe.
[0407] Full rescue of 5xFAD’s spatial memory loss in the MWM training probe
[0408] During the MWM training probe saline treated 5xFAD TG mice displayed 1.5-2 fold increase in escape latency relative to saline treated WT mice (FIG. 62A, left and right) indicating severe defects in spatial learning and memory. Strikingly, however, TUDCA + HCQ treated TG mice escaped the maze at latencies comparable to WT (FIG. 62A middle) suggesting that the treatment fully rescued learning deficits in the 5xFAD phenotype. This was further investigated quantitatively using Bayesian estimation of escape latencies (FIG. 62B left) and Bayesian hypothesis testing (FIG. 62B right).
[0409] Consistent with the findings above, 25.3% and 44.2% mean posterior reduction of escape latencies was estimated in saline treated WT mice relative to saline treated TG littermates in two mouse litters (FIG. 62B left top and bottom). Bayesian hypothesis testing was then performed with three alternative hypotheses from the strongest to the weakest: (1) full rescue defined as the estimated mean reduction in saline treated WT mice given a litter; (2) partial rescue as half of that reduction, and (3) slight improvement as any reduction of escape latency, no matter how small (FIG. 62B right). The Bayes factor (BF) was evaluated for each of these hypotheses for each condition relative to saline treated TG. As expected, 2 x log BF > 10 was found for the saline WT condition for all three hypotheses, including full rescue, which means very strong statistical support. [0410] Turning to the main question of interest, very strong statistical support was found for the full rescue of learning deficits in TG mice by TUDCA + HCQ (FIG. 62B right bottom). Evidently, the support for partial rescue and for slight improvement in TG mice by TUDCA + HCQ was even larger (FIG. 62B right bottom). The results were qualitatively similar in a subset of data restricted to male mouse subjects. Next, these major findings of the study were extended in two ways:
[0411] First, the effect of TUDCA and HCQ in TG mice was tested separately (FIG. 62B top left). In contrast to the combined TUDCA + HCQ treatment, essentially no change was found in escape latency compared to saline treated TG (FIG. 62B top left). This translated to nonexistent or negligible statistical support for both the weakest alternative hypothesis (slight improvement), or for partial or full rescue (FIG. 62B top left). This suggest that the full rescue by the combined TUDCA + HCQ treatment is by and large a synergistic effect of the two drugs.
[0412] Second, WT mice were treated with TUDCA + HCQ and compared to saline treated WT. Similarly, in contrast to the same combined drug treatment in TG mice, no change was identified in escape latency in WT mice (FIG. 63).
[0413] Improved spatial memory in the MWM test probe
[0414] In the MWM test probe, spatial memory of TG and WT mice was additionally evaluated under various treatments in terms of their abidance to the target quadrant and their avoidance of the opposite quadrant of the maze (FIG. 64).
[0415] First, it was confirmed that the mice indeed learned information about the position of the hidden platform in the target quadrant during the training probe: substantial information gain was identified relative to the expected fully random performance of untrained animals (modeled by the uniform distribution of time spent in each quadrant), as shown by the open circles in FIG. 64, middle. Uikelihood ratio test showed that the information gain was significantly greater than zero for all conditions (p<102 FIG. 64 left).
[0416] Next, we asked whether TUDCA + HCQ treatment in TG mice enhanced the information gain during training, relative to saline treated TG. Indeed, a substantial, 3.14 unit increase was identified in information gain by TUDCA + HCQ (FIG. 64 middle bottom), which was statistically significant (likelihood ratio test, p<5xl0’2, FIG. 64 left bottom). This further indicatessuggested that TUDCA + HCQ treatment enhances spatial memory in TG 5xFAD mice.
[0417] A similar result was observed in a subset of data restricted to male mice, although information gain increased to a lesser extent (2.37 vs 3.14) and, consistent with that and the smaller sample size, the increase was not statistically significant (data not shown). It was also found that TUDCA + HCQ treatment increased information gain by 1.05 units even in the WT background, although that was not significantly different from zero (likelihood ratio test).
[0418] Surprisingly, however, the information gain was not significantly different from zero in saline treated WT (FIG. 64 right). Moreover, separate TUDCA or HCQ treatment of TG mice also failed to enhance spatial information gain (FIG. 64 left bottom). [0419] TUDCA + HCQ improves the 5xFAD molecular pathology
[0420] The second major focus of the study was to test the hypothesis that TUDCA and HCQ ameliorate molecular pathologies that 5xFAD mice exhibit. Thus, the level of amyloid-/? (A/?), phosphorylated tau (pTau), a panel of 10 cytokines, neurofilament-L (NfL) and glial acidic fibrillar protein (GFAP) was characterized in TG and WT brains and plasma. No effect of drug treatment was found on pTau or GFAP (data not shown). However, the data on A/? some cytokines and Nil were consistent with the protective effect of TUDCA + HCQ treatment in TG mice as described below. [0421] Reduction of pathological amyloid-/? {A/?) levels
[0422] Concentrations of A/7-40 and A/J-42) were measured in two brain regions (cortex, hippocampus) in two fractions of brain tissue homogenates (soluble; Diethylamine;: DEA, insoluble; formic acid FA). These factors were combined with genotype (WT, TG) and treatment condition (saline, TUDCA + HCQ, TUDCA, HCQ) in the statistical analyses).
[0423] Exploratory analysis showed that among all factors, genotype had the largest effect as expected: A ? level was substantially higher in TG mice relative to WT (FIG. 65) confirming the known severe amyloid pathology of the 5xFAD strain. Further exploration of TG brain samples showed that A/? level depended not only on drug treatment and other factors specified above but also on combinations of these (FIG. 66).
[0424] Does TUDCA + HCQ reduce A/3?
[0425] To disentangle the observed complex dependencies in TG brains, Af level was regressed on all factors with their pairwise interaction terms (Table 1). Although TUDCA + HCQ treatment decreased Af level in TG brains under all conditions, the degree of decrease depended greatly on all other factors. Our regression analysis of interaction terms involving drug treatment revealed (FIGS. 68A and 68B) that the most pronounced decrease by TUDCA + HCQ was observed in A/J-40 levels in the cortex and in the insoluble (FA) fraction yielding p=l .5 x 10-5 (cf. FIG. 66 upper left). Similar results were observed in the hippocampus of TG mice TUDCA + HCQ (p=l .1 x 10-2, FIGS. 68A and 68B). We obtained similar findings from a subset of data restricted to male TG mice (FIGS. 68A and 68B).
[0426] Separate experiments revealed that TUDCA alone also decreased A/? level in TG brains, while HCQ alone had no significant effect (FIG. 67). However, TUDCA was less protective alone than with HCQ under all conditions studied (compare FIG. 66 and FIG. 67). For instance, A/7-40 level in the FA fraction of TG cortices was changed -0.64 units by TUDCA + HCQ (p=1.5 x 10'5), while only -0.33 units by TUDCA alone (p=1.8 x 10’2).
[0427] Taken together, these findings support the hypothesis that TUDCA + HCQ treatment significantly lowers amyloid pathology in 5xFAD mice. Moreover, TUDCA and HCQ act synergistically on lowering brain A/? levels as they do on rescue of spatial learning and memory. [0428] Reduction of pathological interleukin Ifi (IL-lfi) levels
[0429] A panel of 10 cytokines in the cortex and hippocampus region was measured to test the hypothesis that TUDCA + HCQ may ameliorate pathological neuroinflammation associated with the 5xFAD TG phenotype.
[0430] Although brain region had an impact on cytokine levels, it showed no significant statistical interactions with drug treatment or genotype (data not shown). Therefore, each cytokine’s level was modeled without interaction terms, while still correcting for the effect of brain region. This analysis revealed that three of the ten cytokines (TNF-a, KC/GRO, and IL-P) had lower levels in saline treated WT than TG at significance level (a=0.001), which confirms the expected chronic inflammation in the 5xFAD TG brain (FIG. 69, left and right panel, black symbols).
[0431] IL-ip levels showed statistically significant (a=0.01) reduction by combined TUDCA + HCQ treatment in TG mice (FIG. 69, left panel, orange symbols). This drug effect is underscored by the finding that also IL- 1 P displayed the largest difference between WT and TG (FIG. 69, left and right panel, black symbols).
[0432] TUDCA or HCQ separately had no effect in TG mice on any cytokine level except for TUDCA on IL-1 P level, which had similar magnitude and statistical significance to the combined TUDCA + HCQ treatment (FIG. 69, right panel).
[0433] These results, in particular the reduction of IL- 1 P, by combined TUDCA + HCQ and separate TUDCA treatment, were corroborated in the males-only dataset (data not shown).
[0434] Effects on pathological Neurofilament-L (Nfl) levels
[0435] The longitudinal trajectory of Nfl levels in the plasma of the TG and WT mice were measured just before treatment (week 0) as well as 4, 8 and 12 weeks after treatment. We previously found that plasma Nfl levels initially increase to a peak around 9 months of age, after which Nfl level declines. This earlier finding was confirmed along with the significantly increased Nfl level in TG mice (FIG. 70).
[0436] TUDCA + HCQ treated TG mice were the compared to saline treated TG in terms of the measured Nfl trajectory.
[0437] FIG. 71 bottom compares raw, untransformed, trajectories without baseline correction and shows that Nil level is significantly lower in TUDCA + HCQ treated TG than in saline treated TG 8 weeks after treatment onset, suggesting that TUDCA + HCQ treatment protects against pathological increase in Nfl level in TG.
[0438] FIG. 72 presents results from an alternative approach based on log-transformed, baseline- corrected Nfl level. This approach, too, showed a decrease in Nil level at week 8 in TUDCA + HCQ treated TG relative to saline treated TG, but this decrease (FIG. 72 bottom left) was not statistically significant (FIG. 72 bottom right).
[0439] The effect of TUDCA and HCQ treatment was characterized separately using both statistical approaches. This showed that both drugs decrease Nfl level at week 8 but the decrease appeared smaller than that for the combined TUDCA + HCQ treatment, especially in the case of HCQ (FIG. 71 top and FIG. 72 top).
[0440] In view of the many possible aspects to which the principles of the disclosure may be applied, it should be recognized that the illustrated aspects are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as the disclosure all that comes within the scope and spirit of these claims.

Claims

We claim:
1. A method, comprising: administering to a subject an effective amount of an active agent comprising S- hydroxychloroquine (S-HCQ) to at least partially normalize an aberrant level of one or more indicators, wherein the indicators comprise extracellular amyloid beta (A ) concentration, plasma neurofilament light chain protein (NfL) concentration, neuroinflammation, tau phosphorylation, total tau, plasma glial fibrillary acidic protein (GFAP) concentration, hippocampal synaptic plasticity, or any combination thereof, and wherein the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of hydroxychloroquine.
2. The method of claim 1, wherein the active agent further comprises tauroursodeoxycholic acid (TUDCA), 3,3'-diindolylmethane (DIM), or a combination thereof.
3. The method of claim 1 or claim 2, wherein normalizing the aberrant level of the one or more indicators reduces extracellular Ap concentration, reduces plasma NfL concentration, reduces neuroinflammation, reduces tau phosphorylation, reduces plasma GFAP concentration, increases hippocampal synaptic plasticity, or any combination thereof.
4. The method of claim 3, wherein reducing extracellular Ap concentration comprises reducing Ap secretion, increasing Ap clearance, or both.
5. The method of claim 3, wherein reducing neuroinflammation comprises reducing a concentration of interleukin-6, interleukin- ip, interleukin- 12p70, interleukin- 10, tumor necrosis factor alpha, or any combination thereof.
6. The method of any one of claims 2-5, wherein the active agent comprises: an enantiomeric excess of at least 20% of S-HCQ in combination with TUDCA; or an enantiomeric excess of at least 20% of S-HCQ in combination with DIM; or an enantiomeric excess of at least 20% of S-HCQ in combination with TUDCA and DIM.
7. The method of any one of claims 1-6, wherein the active agent at least partially normalizes an aberrant level of extracellular Ap concentration.
8. The method of claim 7, wherein the active agent comprises a combination of an enantiomeric excess of at least 20% of S-HCQ and TUDCA, and at least partially normalizing the aberrant level of extracellular A0 concentration comprises reducing A secretion and increasing A clearance.
9. The method of any one of claims 1-8, wherein the active agent at least partially normalizes the level of at least two of the one or more indicators.
10. The method of claim 9, wherein the active agent reduces at least two of extracellular A concentration, plasma NfL concentration, and neuroinflammation.
11. The method of any one of claims 1-10, further comprising receiving data comprising an initial level of at least one of the indicators prior to administering the active agent to the subject.
12. The method of any one of claims 1-11, further comprising: receiving data comprising a post- administration level of at least one of the indicators following administration of the active agent to the subject; and selecting an adjusted amount of the active agent for administration to the subject based at least in part on the post-administration level.
13. The method of claim 12, wherein the active agent comprises S-HCQ in combination with TUDCA, DIM, or a combination thereof, and selecting an adjusted amount of the active agent comprises selecting an adjusted amount of at least one component of the active agent.
14. The method of any one of claims 1-13, wherein the subject is diagnosed as having a neurological disorder prior to administering the active agent.
15. The method of claim 14, wherein the neurological disorder is Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, exhaustion disorder (ED), post-acute cognitive impairment and fatigue, or neuropsychiatric manifestations of systemic lupus erythematosus (SLE).
16. The method of claim 15, wherein the neurological disorder is AD.
17. The method of claim 15 or claim 16, wherein the subject is diagnosed as having AD prior to administering the active agent.
18. The method of any one of claims 1-13, further comprising administering the active agent to the subject prophylactically in the absence of any physical, cognitive, behavioral, mood, or psychological signs or symptoms of a neurological disorder.
19. The method of any one of claims 1-13 or 18, further comprising, prior to administering to the subject the active agent, identifying the subject as being at risk of developing a neurological disorder by:
(i) identifying the subject as being an APOE e4 carrier; or
(ii) identifying the subject as having an abnormal level of the one or more indicators relative to a normal level of the one or more indicators; or
(iii) both (i) and (ii).
20. The method of any one of claims 1-19, wherein the enantiomeric excess of S-HCQ is from 30% to 100%.
21. A method for inhibiting progression of a neurological disorder, comprising administering to a subject diagnosed as having a neurological disorder an amount of an active agent effective to at least partially normalize an aberrant level of one or more indicators wherein the indicators comprise extracellular amyloid beta (AP) concentration, plasma NfL concentration, neuroinflammation, tau phosphorylation, total tau, plasma GFAP concentration, hippocampal synaptic plasticity, or any combination thereof, and wherein the active agent comprises an enantiomeric excess of at least 20% of the S -enantiomer of HCQ.
22. A method for inhibiting or preventing development of a neurological disorder, comprising: identifying a subject as being at risk of developing a neurological disorder; and administering to the subject at risk of developing a neurological disorder an amount of an active agent effective to at least partially normalize an aberrant level of one or more indicators, wherein the indicators comprise extracellular amyloid beta (A ) concentration, plasma NfL concentration, neuroinflammation, tau phosphorylation, total tau, plasma GFAP concentration, hippocampal synaptic plasticity, or any combination thereof, and wherein the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of HCQ.
23. The method of claim 21 or claim 22, wherein the active agent further comprises
TUDCA, DIM, or a combination thereof.
24. The method of any one of claims 21-23, wherein the neurological disorder is AD, PD, HD, vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, ED, post-acute cognitive impairment and fatigue, or neuropsychiatric manifestations of SLE.
25. The method of claim 24, wherein the neurological disorder is AD.
26. The method of claim 24 or claim 25, wherein the subject is identified as being at risk of developing AD by identifying the subject as:
(i) being an APOE e4 carrier; or
(ii) having an elevated level of the one or more indicators relative to a normal level of the one or more indicators in the absence of any cognitive, behavioral, mood, or psychological signs or symptoms of AD; or
(iii) both (i) and (ii).
27. The method of any one of claims 21-26, wherein the enantiomeric excess of S-HCQ is from 30% to 100%.
28. An active agent for use in a method of at least partially normalizing an aberrant level of one or more indicators, wherein the indicators comprise extracellular A concentration, plasma NfL concentration, neuroinflammation, tau phosphorylation, total tau, plasma GFAP concentration, hippocampal synaptic plasticity, or any combination thereof, the method comprising administering to a subject an amount of the active agent effective to at least partially normalize the aberrant level of the one or more indicators, wherein the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of HCQ.
29. An active agent for use in a method of treating a neurological disorder, the method comprising administering to a subject diagnosed as having a neurological disorder, an amount of an active agent effective to at least partially normalize an aberrant level of one or more indicators of neurological disorder pathology, wherein the indicators comprise extracellular A concentration, plasma NfL concentration, neuroinflammation, tau phosphorylation, total tau, plasma GFAP concentration, hippocampal synaptic plasticity, or any combination thereof, and wherein the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of HCQ.
30. The active agent for use in the method of claim 28 or claim 29, wherein the active agent further comprises TUDCA, DIM, or a combination thereof.
31. The active agent for use in the method of any one of claims 30-32, wherein the neurological disorder is AD, PD, HD, vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, ED, post-acute cognitive impairment and fatigue, or neuropsychiatric manifestations of SLE.
32. The active agent for use in the method of claim 31, wherein the neurological disorder is AD.
33. The active agent for use in the method of any one of claims 28-32, wherein the enantiomeric excess of S-HCQ is from 30% to 100%.
34. Use of an active agent for at least partially normalizing an aberrant level of one or more indicators of neurological disorder pathology in a subject diagnosed as having a neurological disorder, wherein the active agent comprises an enantiomeric excess of at least 20% of the S- enantiomer of HCQ, and wherein the indicators comprise extracellular A concentration, plasma NfL concentration, neuroinflammation, tau phosphorylation, total tau, plasma GFAP concentration, hippocampal synaptic plasticity, or any combination thereof.
35. Use of an active agent for the treatment of a neurological disorder, wherein the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of HCQ.
36. The use of the active agent of claim 34 or claim 35, wherein the active agent further comprises TUDCA, DIM, or a combination thereof.
37. The use of the active agent of any one of claims 34-36, wherein the neurological disorder is AD, PD, HD, vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Down syndrome, Lewy body dementia, human immunodeficiency virus dementia, cerebral amyloid angiopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, cognitive impairment no dementia, ED, post-acute cognitive impairment and fatigue, or neuropsychiatric manifestations of SLE.
38. The use of the active agent of claim 37, wherein the neurological disorder is AD.
39. The use of the active agent of any one of claims 34-38, wherein the enantiomeric excess of S-HCQ is from 30% to 100%.
40. An active agent for use in a method of inhibiting or preventing development of AD, the method comprising administering to a subject identified as being at risk of AD an amount of an active agent effective to at least partially normalize an aberrant level of one or more indicators of AD pathology, wherein the indicators comprise extracellular A concentration, plasma NfL concentration, neuroinflammation, tau phosphorylation, total tau, plasma GFAP concentration, hippocampal synaptic plasticity, or any combination thereof, and wherein the active agent comprises an enantiomeric excess of at least 20% of the S -enantiomer of HCQ.
41. The active agent for use in the method of claim 40, wherein the active agent further comprises TUDCA, DIM, or a combination thereof.
42. The active agent for use in the method of claim 40 or claim 41 , wherein the subject is identified as being at risk of developing AD on the basis of:
(i) being an APOE e4 carrier; or
(ii) having an elevated level of the one or more indicators relative to a normal level of the one or more indicators in the absence of any cognitive, behavioral, mood, or psychological signs or symptoms of AD; or
(iii) both (i) and (ii).
43. The active agent for use in the method of any one of claims 40-42, wherein the enantiomeric excess of S-HCQ is from 30% to 100%.
44. Use of an active agent for inhibiting or preventing development of AD in a subject identified as being at risk of developing AD, wherein the active agent comprises an enantiomeric excess of at least 20% of the S-enantiomer of HCQ.
45. The use of the active agent of claim 44, wherein the active agent further comprises TUDCA, DIM, or a combination thereof.
46. The use of the active agent of claim 44 or claim 45, wherein the enantiomeric excess of S-HCQ is from 30% to 100%.
PCT/US2025/023133 2024-04-04 2025-04-04 S-hydroxychloroquine, optionally with tauroursodeoxycholic acid and/or 3,3'-diindolylmethane, for treating and/or preventing neurological disorders Pending WO2025213007A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463574481P 2024-04-04 2024-04-04
US63/574,481 2024-04-04

Publications (1)

Publication Number Publication Date
WO2025213007A1 true WO2025213007A1 (en) 2025-10-09

Family

ID=95583501

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/023133 Pending WO2025213007A1 (en) 2024-04-04 2025-04-04 S-hydroxychloroquine, optionally with tauroursodeoxycholic acid and/or 3,3'-diindolylmethane, for treating and/or preventing neurological disorders

Country Status (1)

Country Link
WO (1) WO2025213007A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4652441A (en) 1983-11-04 1987-03-24 Takeda Chemical Industries, Ltd. Prolonged release microcapsule and its production
US4675189A (en) 1980-11-18 1987-06-23 Syntex (U.S.A.) Inc. Microencapsulation of water soluble active polypeptides
US4677191A (en) 1984-07-06 1987-06-30 Wada Pure Chemical Ind., Ltd. Copolymer and method for producing the same
US4728721A (en) 1985-05-07 1988-03-01 Takeda Chemical Industries, Ltd. Polymer, production and use thereof
WO2004004660A2 (en) * 2002-07-09 2004-01-15 Alpha Research Group, L.L.C. Compositions and methods for the treatment of parkinson’s disease and tardive dyskinesias
WO2023059867A1 (en) 2021-10-08 2023-04-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compounds for treating or preventing alzheimer's disease

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4675189A (en) 1980-11-18 1987-06-23 Syntex (U.S.A.) Inc. Microencapsulation of water soluble active polypeptides
US4652441A (en) 1983-11-04 1987-03-24 Takeda Chemical Industries, Ltd. Prolonged release microcapsule and its production
US4917893A (en) 1983-11-04 1990-04-17 Takeda Chemical Industries, Ltd. Prolonged release microcapsules
US4677191A (en) 1984-07-06 1987-06-30 Wada Pure Chemical Ind., Ltd. Copolymer and method for producing the same
US4728721A (en) 1985-05-07 1988-03-01 Takeda Chemical Industries, Ltd. Polymer, production and use thereof
WO2004004660A2 (en) * 2002-07-09 2004-01-15 Alpha Research Group, L.L.C. Compositions and methods for the treatment of parkinson’s disease and tardive dyskinesias
WO2023059867A1 (en) 2021-10-08 2023-04-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compounds for treating or preventing alzheimer's disease

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Molecular Biology and Biotechnology: a Comprehensive Desk Reference", 1995, WILEY, JOHN & SONS, INC.
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT, WILLIAMS, & WILKINS
"Sustained and Controlled Release Drug Delivery Systems", 1978, MARCEL DEKKER, INC.
"The Encyclopedia of Molecular Biology", 1994, BLACKWELL PUBLISHERS
BELKHIR ET AL., EUR J DRUG METAB PHARMACOKINET, vol. 45, no. 6, 2020, pages 703 - 13
BERGE ET AL., J. PHARM. SCI., vol. 66, 1977, pages 1
BIN IBRAHIM ET AL., FEBS J, 2021
BORCHELT ET AL., NEURON, vol. 19, no. 4, 1997, pages 939 - 45
BROWNING, HYDROXYCHLOROQUINE AND CHLOROQUINE RETINOPATHY, vol. 35-63, 2014
CHOI M. ET AL.: "Inhibition of STAT3 phosphorylation attenuates impairments in learning and memory in 5XFAD mice, an animal model of Alzheimer's disease", J. PHARMACOL. SCI, vol. 143, no. 4, 2020, pages 290 - 299
CUMMINGS ET AL., ALZHEIMER'S RESEARCH AND THERAPY, vol. 8, 2016, pages 39
DESAI ET AL., BRAIN COMMUNICATIONS, vol. 4, no. 5, 2022, pages 10
FARRER ET AL., JAMA, vol. 278, 1997, pages 1349 - 1356
GENIN ET AL., MOL PSYCHIATRY, vol. 16, 2011, pages 903 - 907
GRUBMAN ET AL., NATURE COMM, vol. 12, 2021
HAGEMANN ET AL., CELLS, vol. 12, no. 7, 2023, pages 978
JANSEN ET AL., JAMA, vol. 313, 2015, pages 1924 - 1938
LEE ET AL., 1 NEURAL TRANSM, vol. 117, no. 8, 2010, pages 949 - 60
LEE ET AL., ANTIOXIDANTS, vol. 9, no. 1, 2019, pages 3
LEWIN: "Genes VII", 2000, OXFORD UNIVERSITY PRESS
LI ET AL., PNAS, vol. 114, no. 21, 2017, pages 5527 - 5532
LI GUANGUAN ET AL: "Enantiomers of Chloroquine and Hydroxychloroquine Exhibit Different Activities Against SARS-CoV-2 in vitro , Evidencing S -Hydroxychloroquine as a Potentially Superior Drug for COVID-19", BIORXIV, 22 August 2020 (2020-08-22), BIORXIV, XP093292160, DOI: 10.1101/2020.05.26.114033 *
MACKENZIE, AM J MED, vol. 75, no. 1, 1983, pages 5 - 10
MICHAEL ET AL., J. PHARMACY PHARMACOL., vol. 43, 1991, pages 1 - 5
NEU ET AL., JAMA NEUROL, vol. 74, 2017, pages 1178 - 1189
NIRK ET AL., EMBO MOL MED, vol. 12, 2020, pages 12476
RAMAKRISHNA KAKARLA ET AL: "Current understanding and future directions of cruciferous vegetables and their phytochemicals to combat neurological diseases", PHYSIOTHERAPY RESEARCH, JOHN WILEY & SONS LTD. CHICHESTER, GB, vol. 38, no. 3, 12 January 2024 (2024-01-12), pages 1381 - 1399, XP072601766, ISSN: 0951-418X, DOI: 10.1002/PTR.8122 *
REICHENBACH, N. ET AL.: "Inhibition of STAT3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model", EMBO MOL. MED., vol. 11, no. 2, 2019, pages 9665, XP093076950, DOI: 10.15252/emmm.201809665
REIMAN ET AL., PNAS, vol. 101, 2004, pages 284 - 289
RZEMIENIEC ET AL., APOPTOSIS: AN INTERNATIONAL JOURNAL ON PROGRAMMED CELL DEATH, vol. 24, 2019, pages 435 - 452
STEVENS ET AL., MOLECULES, vol. 26, no. 1, 2021, pages 175
VARMA ET AL., PLOS MEDICINE, vol. 18, no. 5, 27 May 2021 (2021-05-27), pages 1003615
VARMA, PLOS ONE, vol. 15, no. 32271844, 2020, pages 02314446
WAN, J. ET AL.: "Tyk2/STAT3 signaling mediates amyloid-induced neuronal cell death", J. NEUROSCI., vol. 30, no. 20, 2010, pages 6873 - 81
WEI ET AL., CHIRALITY, vol. 7, 1996, pages 598 - 604
ZHAO ET AL., CELL DEATH DIFFER, vol. 26, 2019, pages 1600 - 1614

Similar Documents

Publication Publication Date Title
Haghparast et al. Apelin-13 ameliorates cognitive impairments in 6-hydroxydopamine-induced substantia nigra lesion in rats
US20200339591A1 (en) Compositions and methods of treatment for neurological disorders comprising a dementia
US8143221B2 (en) Use of ADNF polypeptides for treating peripheral neurotoxicity
US20200325148A1 (en) Compositions and methods of treatment for neurological disorders comprising motor neuron diseases
US20090048288A1 (en) Method of treating stress-mediated depression
JP7642735B2 (en) Compositions Comprising Anti-Abeta Protofibril Antibodies and Beta-Secretase BACE1 Inhibitors for Treating Alzheimer&#39;s Disease - Patent application
CN108289930A (en) Methods and compositions for treating neurodegenerative and neuroinflammatory disorders
JP2018514531A (en) NMDA antagonists for the treatment of cardiac disorders associated with the development of aggressive and / or impulsive behavior
Leung et al. Microglia activation mediates fibrillar amyloid-β toxicity in the aged primate cortex
US20240269128A1 (en) Compounds for treating or preventing alzheimer&#39;s disease
US20100247688A1 (en) Pirenzepine and derivatives thereof as anti-amyloid agents
WO2020165802A1 (en) Compositions and methods relating to use of agonists of alpha5-containing gabaa receptors
WO2025213007A1 (en) S-hydroxychloroquine, optionally with tauroursodeoxycholic acid and/or 3,3&#39;-diindolylmethane, for treating and/or preventing neurological disorders
AU2022277299A1 (en) Methods and compositions for treating neurological conditions
JP7277458B2 (en) Methods and compositions for treating neurological disorders
KR20250070087A (en) Methods for reducing neurodegeneration associated with neurodegenerative diseases
EP3429671A1 (en) COMPOSITION AND METHODS FOR STIMULATING CLEARANCE OF AMYLOID-ß PROTEIN
Mali et al. Screening of nootropics: An overview on preclinical evaluation techniques
EP3122363B1 (en) Galantamine carbamates for the treatment of autism
US20240285680A1 (en) Method of treating alzheimer&#39;s disease
JP5714572B2 (en) Method of modulating KCNQ potassium channel activity for the treatment of psychiatric disorders and symptoms
CN108635358A (en) Aspirin eugenol ester is preparing the application in preventing or treating Alzheimer disease drugs
Verhaeg et al. Investigating the effects of prednisolone on behavior in mouse models of Duchenne muscular dystrophy
CA2944017C (en) Treatment of rett syndrome
EP2776131B1 (en) Composition for treating or preventing neurodegenerative disorders

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25722689

Country of ref document: EP

Kind code of ref document: A1