3TC tablets for treating alzheimer's disease and related tauopathies

Abstract

Embodiments are directed to methods for treating neurodegenerative disease by administering anti-viral compounds to subjects having or at risk of developing neurodegenerative diseases such as Alzheimer's disease.

Description

3TC TABLETS FOR TREATING ALZHEIMER’ S DISEASE AND RELATED TAUOPATHIESRELATED APPLICATION S

[0001] This Application is an International Application claiming priority to US Provisional Applications 63 / 557,522 filed 2 / 24 / 2024; 63 / 574,838 filed 4 / 4 / 2024; and 63 / 741,963 filed 1 / 5 / 2025 each of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant number NS 112391 awarded bv the National Institutes of Health. The government has certain rights in the inventionFIELD OF THE INVENTION

[0003] Embodiments are directed generally to the field of neuroscience and medicine. Embodiments are directed in particular to methods for treating neurodegenerative diseases.BACKGROUND

[0004] Transposon biology. Transposable elements, known colloquially as “jumping genes,” constitute approximately 45% of the human genome1. Transposable elements are a diverse superfamily of genomic DNA species that have the ability to either copy themselves and insert the DNA copy into a new genomic location (retrotransposons) or excise themselves from the genome and insert in a new genomic location (transposons). Retrotransposons outnumber DNA transposons 13 to 1 in the human genome2. Over the course of human evolution, most retrotransposons and all DNA transposons have lost the ability to mobilize due to truncation and mutation. Some human retrotransposons, however, retain mobilization potential, including specific long and short interspersed nuclear element (LINE and SINE, respectively) subfamilies3. Retrotransposon activation has been identified as a disease mechanism in several human disorders, including cancer4, Aicardi Goutieres Syndrome5, amyotrophic lateral sclerosis (ALS)6, frontotemporal dementia7, Alzheimer’s disease and related tauopathies8 10.

[0005] Retrotransposons are structurally akin to retroviruses. Retrotransposition occurs through a copy-and-paste mechanism involving transcription of retrotransposon DNA to RNA, reverse transcription of retrotransposon RNA into a new DNA copy, and reinsertion of the DNAcopy into the genome3. Retrotransposition is carried out by proteins, including a reverse transcriptase, encoded by retrotransposons.

[0006] Evidence of retrotransposon activation in Alzheimer 's disease and related tauopathy. Alzheimer’s disease is a progressive neurodegenerative disorder that affects over 5.8 million Americans and has no curative treatment11. Recent evidence suggests that Alzheimer’ s disease pathophysiology begins decades prior to symptoms12. A poor understanding of mediators that drive disease onset and progression during the long prodromal, asymptomatic stages presents a large barrier for developing effective therapeutic strategies. We have reported that pan-neuronal transgenic expression of human tau in Drosophila activates retrotransposons by disrupting two arms of retrotransposon control: 1) heterochromatin- and 2) piRNA-mediated silencing8. Consistent with the increase in DNA copy number of select retrotransposons in brains of tau transgenic Drosophila9, we demonstrated active retrotransposition as a consequence of human tau in neurons of the adult Drosophila brain. We found that genetic manipulation of retrotransposon regulatory machinery modifies tau-induced neurodegeneration, supporting a causal link between retrotransposon dysregulation and neurodegeneration8.

[0007] RNA-seq analysis of brain lysates from post-mortem human controls, Alzheimer’s disease and progressive supranuclear palsy, a “primary” tauopathy, revealed elevated levels of specific retrotransposon transcripts, including LINE-1, human endogenous retrovirus (HERV), and SVA, and decreased levels of Alu family members8. Consistent with our work, Shulman and colleagues reported a significant association between decreased cognitive performance in the year prior to death and elevation of specific HERV subfamilies in human Alzheimer’s disease brain, as well as an association between tau tangle burden and increased transcript levels of select LINE-1 and HERV elements9. We now have additional data suggesting that retrotransposon activation occurs early in human Alzheimer’s disease, which we describe below. Together with reports of heterochromatin relaxation13,14and piRNA dysregulation15,16in post-mortem human Alzheimer’s disease brain, these studies suggest that tau-induced heterochromatin decondensation, piRNA dysregulation and consequent transposable element activation is a novel, conserved, pharmacologically targetable driver of neurodegeneration in Alzheimer’s disease and related tauopathies.

[0008] Use of antiviral reverse transcriptase inhibitors in human diseases associated with retrotransposon activation. Based on the similarities between exogenous retroviruses andretrotransposons, numerous studies have investigated the therapeutic efficacy of antiviral medications, including NRTIs, to prevent rctrotransposition8 17 l 9. The Lighthouse trial (NCT02868580), for example, an open label, multi-center study to investigate the safety and tolerability of Triumeq in patients with ALS, has recently completed phase 2a. Triumeq consists of two NRTIs, abacavir and 3TC, and an integrase inhibitor, dolutegravir, which blocks episomal DNA integration into genomic DNA. 35 out of 40 patients completed the 24-week treatment period. Non-serious adverse events that occurred in three or more participants included nausea and rash. While five deaths were expected based on modeling20, only one death was observed five months after conclusion of the trial. DNA copy number of retrotransposon DNA was significantly reduced by the end of the trial, and the ALS functional rating scale demonstrated a declining trend21. The trial is slated for phase 3, in which overall survival and disease progression will be measured in a larger cohort. An additional trial (NCT02437110) for a retroviral combination therapy (darunavir, ritonavir, dolutegravir, and tenofovir alafenamide,) in ALS is currently in phase 1. A recently-completed 12 month clinical trial (NCT02363452) in children with Aicardi- Goutieres Syndrome, in which the body mounts a viral response due to activation of retrotransposons, reports that patients administered 3TC, zidovudine, and abacavir have a significantly reduced blood interferon score after treatment22.

[0009] 3TC was approved by the FDA for the treatment of HIV-1 and hepatitis B virus in the late 1990’s, and continues to be prescribed as both a mono- and combination therapy for retroviral infection. Combination therapies are warranted in HIV, as the virus demonstrates a high mutation rate that can overcome an individual NRTI23. It is not clear, however, that retrotransposons have a mutational capacity that requires a combinatorial approach. We aim to reduce the risk of negative health consequences associated with polypharmacy in older patients by using 3TC as a monotherapy at a dose that is cost-effective and currently recommended for treatment of HIV+ patients in the clinic24. Of relevance to older patients, who are often subject to polypharmacy, 3TC has few clinically-relevant pharmacological interactions due to its low metabolic clearance, minimal binding to plasma protein, and no detectable effects on liver function25. While 3TC is considered to be generally safe and is widely prescribed, no study to date has specifically investigated the use of 3TC in older individuals with cognitive impairment. Outcomes of this 24- week open label phase 2a clinical trial include i) target engagement, ii) CNS penetration, iii)efficacy based on cognitive scores and biomarkers of neurodegeneration, and iv) safety and tolerability of 3TC in patients with early stage Alzheimer’s disease.SUMMARY

[0010] Embodiments are directed to methods of using an anti-retrovial, e.g., lamivudine / 3TC for the treatment of AD and / or other tauopathies. In particular aspects the anti-retrovial is administered as a high dose relative to the dose used for treating retroviruses, e.g., HIV. High dose administration has been shown in a clinical trial to improve cognitive function of a subject. A therapeutically effective dose of the current invention achieves an in-vivo (brain) tissue concentration.

[0011] Certain embodiments are directed to methods of treating a tauopathy or neurodegenerative disease comprising administering a high dose, therapeutic amount of a reverse transcriptase inhibitor (e.g., lamivudine) to a subject having or at risk of developing a tauopathy or neurodegenerative disease. The dose can be about 250, 300, 350, 400, 450, 500, 550, 600, 65, 700, 750, 800, 850, 900, 950, to 1000 mg or more. The tauopathy or neurodegenerative disease can be Alzheimer’s disease. In certain aspects the reverse transcriptase inhibitor (e.g., lamivudine) is administered at a dose of 250 mg to 500 mg daily. In certain aspects the reverse transcriptase inhibitor (e.g., lamivudine) is administered at a dose greater than 300 mg daily. The method can include administering the reverse transcriptase inhibitor (e.g., lamivudine) chronically, or for 10 to 30 days, or 2 to 5 weeks, or 1 to 6 months, 1 to 20 years. The therapeutic composition can be administrated by oral administration. A tablet, capsule, pill, powder, sustained release formulation, solution, or suspension containing the reverse transcriptase inhibitor (e.g., lamivudine) can be administered orally.

[0012] “Reverse transcriptase inhibitor (RTI)” refers to a class of antiretroviral drugs that work by inhibiting the activity of reverse transcriptase, an enzyme that retroviruses use to convert their RNA into DNA, a crucial step in their replication process. By blocking this enzyme, RTIs prevent the virus from multiplying within the host’s cells. RTIs are divided into two main categories: Nucleoside / Nucleotide Reverse Transcriptase Inhibitors (NRTIs) or Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). NRTIs mimic the natural building blocks of DNA (nucleosides or nucleotides). When the reverse transcriptase enzyme incorporates them into the viral DNAchain, the chain cannot be completed, halting viral replication. Lamivudine is an example of an NRTI. NNRTIs bind directly to the reverse transcriptase enzyme at a site away from the active site (allosteric site), altering its shape and preventing it from functioning properly. NRTIs include but are limited to Lamivudine (3TC), Zidovudine (AZT), Tenofovir (TDF or PMPA), Emtricitabine (FTC), Abacavir (ABC), Didanosine (ddl), and Stavudine (d4T). NNRTIs include but are not limited to Efavirenz (EFV), Nevirapine (NVP), Rilpivirine (RPV), Etravirine (ETR), and Doravirine (DOR).

[0013] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of a pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; stabilization of symptoms, pathology, or condition, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and / or psychiatric or cognitive evaluations.

[0014] “Effective amount” and “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a drug as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, the biological or therapeutic results disclosed herein. In certain aspects a dose of drug is administered that results in a drug concentration in the cerebrospinal fluid of at least 150, 200, 250, 300, 350 ng / ml or more of a reverse transcriptase inhibitor, e.g., lamivudine (3TC).

[0015] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0016] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0017] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0018] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0019] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.

[0020] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0021] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0022] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0025] FIG. 1. Quantification of biomarkers associated with neurodegeneration in CSF and plasma. Quanterix was used to detect the indicated proteins in CSF (A-D) and plasma (E-H) of participants at baseline and after 24 weeks of 3TC treatment. CSF, n=9; Plasma, n=12. Normality testing was based on the POST-PRE difference for each target; P values are based on two-sided paired sample t-test (normal distribution) or Wilcoxon matched-pairs signed rank test (non-normal distribution).

[0026] FIG. 2A-2B. Levels of 3TC in plasma and CSF based on HPLC / MS / MS. Plasma, n=12; CSF, n=9.

[0027] FIG. 3. Reverse transcriptase activity in plasma. Reverse transcriptase activity was quantified using the EnzCheck Reverse Transcriptase Activity assay. N=12.

[0028] FIG. 4. Difference among participants in PACC-5 z-score from baseline to posttreatment. denotes self- or provider-reported COVID-19 infection.

[0029] FIG. 5. Neuroinflammatory biomarkers in CSF. The MSD platform was used to detect the indicated proteins in CSF of participants at baseline (PRE) and after 24 weeks of 3TC treatment (POST). Normality testing was based on the POST-PRE difference for each target. N=9. Significant P value is noted; all other P values are provided in Table 5. P values are based on two- sided paired sample t-test (normal distribution) or Wilcoxon matched-pairs signed rank test (nonnormal distribution).

[0030] FIG. 6. Neuroinflammatory biomarkers in plasma. The MSD platform was used to detect the indicated proteins in plasma of participants at baseline (PRE) and after 24 weeks of 3TC treatment (POST). Normality testing was based on the POST-PRE difference for each target. N=12. Significant P value is noted; all other P values are provided in Table 5. P values are based on two-sided paired sample t-test (normal distribution) or Wilcoxon matched-pairs signed rank test (non-normal distribution).

[0031] FIG. 7. 3TC levels in CSF and cognitive function. Participant with the highest levels of 3TC in their cerebrospinal fluid (and thus brain) had an improvement in cognitive function over the course of the six-month trial.DESCRIPTION

[0032] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment, and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0033] Described herein is a study to evaluate central nervous system (CNS) penetration, target engagement, efficacy of lamivudine based on fluid-based biomarkers of neurodegeneration and neuropsychological assessment, and safety in older adults with early Alzheimer’s disease as an initial proof-of-concept.Lamivudine (3TC)

[0034] Traditionally, therapeutic development in Alzheimer’s disease has been focused on targeting amyloid beta, pathological forms of tau protein, or strategies to boost neuronal function. Rather than targeting the initiating proteins that have been present in brains of affected individuals for decades prior to symptom onset, we will target a mechanism that is downstream of pathogenic forms of tau that is well-connected to neuronal death.

[0035] Evidence of transposable element activation in brains of tau transgenic mice. Based on our previous work showing that transposable element mobilization is significantly increased in an age-dependent manner in tau transgenic Drosophila and that endogenous retroviruses are aberrantly expressed in late-stage Alzheimer’s disease and progressive supranuclear palsy8, we determined the time course of retrotransposon activation in a mouse model of tauopathy. We analyzed RNA-sequencing data from non-transgenic controls versus INPL326human tau transgenic mice that harbor the disease-associated tau P301L mutation. We find that elevated retrotransposon expression occurs prior to neurofibrillary tangle formation and neuronal loss in this model, suggesting that tau drives aberrant transposable element expression early in disease, considerably before symptom onset.

[0036] Staging of transposable element activation in human Alzheimer ’s disease. Increased retrotransposon DNA copy number suggests that a given retrotransposon is activated and mobilization-competent. We have quantified retrotransposon DNA copy number in Braak III and V / VI postmortem human Alzheimer’s disease brain relative to control (tissue provided by Dr. Dennis Dickson). Since some retrotransposons reverse transcribe in the cytoplasm and then importthe new DNA copy into the nucleus, we isolated DNA from nuclei in order to reduce contribution from episomal cytoplasmic DNA. To quantify DNA copy number, we created a custom NanoString codeset to detect 52 human transposable elements. DNA copy was normalized to ten single-copy, invariant internal control genes, and is presented relative to control brains. We find that 49 out of 52 retrotransposons have significantly increased DNA copy number at Braak III. Interestingly, Braak V / VI brains do not feature significant elevation of retrotransposon DNA copy number with this relatively small sample size. As our experiments in Drosophila indicate that retrotransposon activation is toxic, we speculate that cells that undergo retrotransposon activation die, and that cells surviving into a Braak V / VI brain are cells in which transposable elements have not been activated. These data, along with data from JNPL3 tau transgenic mice, which we find exhibit a similar early increase and late decrease in retrotransposon expression at the transcript level, serve as rationale for targeting early-stage Alzheimer’s disease in our clinical trial.

[0037] 3TC mechanism of action and pharmacokinetics. 3TC 5 ’-triphosphate, the active analog of 3TC, competitively inhibits viral reverse transcriptase, resulting in proviral DNA chain extension termination. 3TC is one of two NRTIs used in the most well -tolerated and common antiretroviral therapy, Trium eq, and has mild brain penetration in pediatric patients25. Importantly, we8and others19,27,28have shown that 3TC effectively inhibits activation of retrotransposons, which, like retroviruses, encode a reverse transcriptase.

[0038] 3TC is absorbed rapidly after oral administration, with an absolute bioavailability of 82% in adults. 3TC 5 ’-triphosphate peaks in serum within 0.5-1.5 h, with an elimination half-life of 5-7 hours. 2-4 hours following treatment, the concentration of 3TC in cerebrospinal fluid (CSF) is 4-8% of serum concentrations in adults25. In collaboration with Dr. Marty Javors, we have successfully detected 3TC in brains of older mice and in CSF of humans who have taken 3TC. For mouse studies, we administered a five day, 50mg / kg dose of 3TC in drinking water, corresponding to the current recommended dosage for HIV adjusted to body area (0.026 and 0.30 m2 / kg of body weight in humans vs. mice, respectively18). The quantity of 3TC dissolved into drinking water was determined based on a daily liquid consumption of 5 mb per mouse. Daily liquid intake is not affected by 3TC treatment, and stability of 3TC in drinking water is 90-103% of the initial value over one week18. Using HPLC / MS / MS chromatography29, we measured 3TC levels in plasma and brains of 24-25 month old 3TC-treated mice, and calculated the brain to plasma ratio. Similarly, we have used HPLC / MS / MS chromatography to quantify 3TC levels inplasma and CSF of humans who are actively taking 3TC. With this very limited human sample size, 3TC CNS penetration does not significantly differ between younger individuals and older individuals (young=average age of 28 years old, old=average age of 65 years old). In mice and in younger and older human individuals, we find that the CNS harbors approximately 20% of the levels of circulating 3TC.

[0039] The study will consist of a screening / baseline period of 30 days pre-medication, a 24- week open label treatment period, and a post-intervention visit one month following treatment. During the treatment period, daily 300 mg 3TC will be taken orally with or without food. The selected dosage is what is currently prescribed for HIV-1 patients. 3TC is widely prescribed and well-tolerated. While 3TC has fewer side effects than many other antiretroviral treatments, side effects can include cause vomiting, weakness, diarrhea, headache, and nausea.

[0040] Drug Properties. 3TC is a first-generation, synthetic nucleoside analogue reverse transcriptase inhibitor approved in the US in 1995 for the treatment of HIV and Hepatitis B virus. The CAS number for 3TC is 134678-17-4. The molecular formula is C8H11N3O3S, which corresponds to a formula weight of 229.26 g / mol. 3TC is a white to off-white crystalline solid with a melting point of 160-162°C. 3TC is soluble in water (70 mg / mL) at 20°C. 3TC tablets contain the following inactive ingredients: black iron oxide, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, polysorbate 80, sodium starch glycolate and titanium dioxide.

[0041] Mechanism of Action. The nucleoside analog 3TC inhibits the activity of reverse transcriptase to prevent retroviral (or retrotransposon27) replication. 3TC is phosphorylated intracellularly to its active metabolite, 3TC 5 ’-triphosphate, which is incorporated into newly synthesized DNA by reverse transcriptase, causing DNA chain termination. Antiviral activity is detected at concentrations without toxicity in humans and is detected at low nanomolar concentrations (EC50 of 0.003-15 mM) in cell culture25.

[0042] HIV inhibitors comprise nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (Pls), entry inhibitors, fusion inhibitors, gp41 inhibitors, gpl20 inhibitors, integrase inhibitors, co-receptors inhibitors (e.g. CCRS, CXCR4, . . . ), budding / maturation inhibitors, etc.

[0043] HIV nucleoside reverse transcriptase inhibitors include those compounds whose mechanism of action comprises an inhibition of the viral reverse transcriptase enzyme. As example, and with no limitation to existing and future new compounds, HIV nucleoside reverse transcriptase inhibitors include zidovudine (AZT), lamivudine (3TC), stavudine (d4T), zalcitabine (ddC), didanosine (ddl), abacavir (ABC)

[0044] HIV non-nucleoside reverse transcriptase inhibitors include those compounds whose mechanism of action comprises an inhibition of the viral reverse transcriptase enzyme. As example, and with no limitation to existing and future new compounds, HIV non-nucleoside reverse transcriptase inhibitors include nevirapine, delavirdine, efavirenz, TMC120, TMC125, capravirine, calanolide, UC781, SJ-1366, benzophenones, PETT compounds, TSAO compounds.

[0045] Nucleotide reverse transcriptase inhibitors include those compounds whose mechanism of action comprises an inhibition of the viral or transposon reverse transcriptase enzyme. Examples of nucleoside reverse transcriptase inhibitors include but are not limited to zidovudine, didanosine, stavudine, zalcitabine, and lamivudine. As an example, and with no limitation to existing and future new compounds, non-nucleotide reverse transcriptase inhibitors include nevirapine, delaviradine, adefovir (PMEA), tenofovir (PMPA), and other functional similar compounds.I. Pharmaceutical Compositions and Administration Thereof

[0046] In light of the current specification, the determination of an appropriate treatment regimen is within the skill of the art. For administration, the components described herein will be formulated in a unit dosage form (solution, suspension, emulsion, etc.) in association with a pharmaceutically acceptable carrier. Such vehicles are usually nontoxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. The vehicle may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0047] The therapeutic compositions described herein, as well as their biological equivalents, can be administered independently or in combination by any suitable route. Examples of parenteral administration include intravenous, intraarterial, intramuscular, intraperitoneal, and the like. The routes of administration described herein are merely an example and in no way limiting.

[0048] The dose of the therapeutic compositions administered to an animal, particularly in a human, in accordance with embodiments of the invention, should be sufficient to result in a desired response in the subject over a reasonable time frame. It is known that the dosage of therapeutic compositions depends upon a variety of factors, including the strength of the particular therapeutic composition employed, the age, species, condition or disease state, and the body weight of the animal. Further disclosed herein, in certain embodiments, is a pharmaceutical composition comprising reverse transcriptase inhibitor or a pharmaceutically acceptable salt, or prodrug thereof. In some embodiments, the pharmaceutical composition is in a form suitable for oral administration. In further or additional embodiments, the pharmaceutical composition is in the form of a tablet, capsule, pill, powder, sustained release formulation, solution, or suspension; for parenteral injection as a sterile solution, suspension or emulsion. In further or additional embodiments, the pharmaceutical composition is in unit dosage forms suitable for single administration of precise dosages. In further or additional embodiments, the amount of a compound disclosed herein is in the range of about 0.001 to about 2000 mg / kg body weight / day. In further or additional embodiments, the amount of a compound disclosed herein is in the range of about 5 to about 500 mg / kg / day. In further or additional embodiments, the amount of a compound disclosed herein is about 300, 400, 500, 600, 700, 800, 900, to about 1000 mg / day. In further or additional embodiments, dosage levels above the upper limit of the aforesaid ranges are required.

[0049] In certain embodiments a compound(s) disclosed herein is administered in a single dose, once daily. In further or additional embodiments, a compound disclosed herein is administered in multiple doses, more than once per day. In further or additional embodiments, a compound disclosed herein is administered twice daily. In further or additional embodiments, a compound disclosed herein is administered three times per day. In further or additional embodiments, a compound disclosed herein is administered four times per day. In further or additional embodiments, a compound disclosed herein is administered more than four times per day. In some embodiments, the pharmaceutical composition is for administration to a mammal. In further or additional embodiments, the mammal is human. In further or additional embodiments, the pharmaceutical composition further comprises a pharmaceutical carrier, excipient and / or adjuvant. In further or additional embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable salt of a compound disclosed herein.

[0050] Disclosed herein, in certain embodiments, is a method for inhibiting a reverse transcriptase enzyme in the central nervous system of a subject. Certain aspects are directed to a method for inhibiting a reverse transcriptase enzyme in the brain of a subject. In some embodiments, the method comprises contacting said reverse transcriptase enzyme with an amount of a composition comprising a compound disclosed herein or a pharmaceutically acceptable salt, or prodrug thereof, sufficient to inhibit a reverse transcriptase enzyme. In further or additional embodiments, the contacting occurs within a cell. In further or additional embodiments, the cell is a mammalian cell. In further or additional embodiments, the mammalian cell is a human cell. In further or additional embodiments, the reverse transcriptase enzyme is inhibited with a composition comprising a pharmaceutically acceptable salt of a compound disclosed herein.

[0051] Disclosed herein, in certain embodiments, is a method of treating a neurodegenerative disease or tauopathy in an individual suffering from said disease comprising administering to said individual an effective amount of a composition comprising a compound disclosed herein or a pharmaceutically acceptable salt, or prodrug thereof.

[0052] Disclosed herein, in certain embodiments, is a method of treatment of neurodegenerative disease in an individual diagnosed with or at risk of developing a neurodegenerative disease, e.g., Alzhemier’s disease, comprising administering to said individual an effective amount of a composition comprising a compound disclosed herein or a pharmaceutically acceptable salt, or prodrug thereof. In some embodiments, the composition comprising a compound disclosed herein is administered orally, intraduodenally, parenterally (including intravenous, subcutaneous, intramuscular, intravascular or by infusion), topically or rectally. In some embodiments, the pharmaceutical composition is in a form suitable for oral administration. In further or additional embodiments, the pharmaceutical composition is in the form of a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. Certain aspects are directed to nasal delivery of a drug involving administering through the nasal passages or inhalation of a drug, where it can be absorbed into the bloodstream. The nasal mucosa is highly vascular, allowing for quick absorption of drugs into the systemic circulation, which can lead to a rapid onset of action. Drugs administered nasally can avoid the liver's metabolism, potentially increasing bioavailability for drugs that are significantly metabolized when taken orally. Compared to injections, nasaldelivery is less invasive, which can improve patient compliance. The most common form for drug delivery is a nasal spray. A spray pump can be used to deliver a fine mist of the drug solution or suspension into the nasal cavity. Nasal drops are a less commonly used due to less precise dosing, but can be used for hydration or in certain treatments where the drug needs to stay in contact with the nasal mucosa for longer. Nasal powders can be used for drugs that are not stable in liquid form. They might be administered via a device that can precisely deliver the powder. Nasal gels can provide a longer contact time with the nasal mucosa, which can be beneficial for drugs requiring prolonged local action or for moisture retention in dry nasal conditions. The drug can be formulated to ensure stability, effective particle size for absorption, and compatibility with the nasal environment. In further or additional embodiments, the pharmaceutical composition is in unit dosage forms suitable for single administration of precise dosages. In further or additional embodiments, the pharmaceutical composition further comprises a pharmaceutical carrier, excipient and / or adjuvant.

[0053] A nasal spray formulation can include an active pharmaceutical ingredient (API) in a vehicle / solvent and one or more of preservatives, pH buffers, viscosity enhancers, humectants, surfactants / emulsifiers, and / or osmotic agents. An API) is the drug that will be delivered through the nasal mucosa. The solubility, stability, and concentration of the API are crucial for the formulation. A vehicle / solvent is typically water or saline because of biocompatibility. However, for drugs that are not soluble in water, might use co-solvents like ethanol or propylene glycol. Preservatives are used to prevent microbial growth, preservatives like benzalkonium chloride, chlorobutanol, or parabens might be included. The choice depends on compatibility with the drug and nasal mucous membranes. pH Buffers are used to maintain the stability of the drug and ensure comfort for the nasal mucosa, buffers like citrate or phosphate might be used to keep the pH within a range of 4.5 to 6.5. Viscosity enhancers include ingredients like hydroxypropyl methylcellulose (HPMC) or microcrystalline cellulose can be added to increase the viscosity, which can help in better drug distribution and retention in the nasal cavity. Humectants include glycerin or sorbitol that can be used to maintain moisture, reducing irritation from the spray. Surfactants / emulsifiers can be used if the drug is not completely soluble, surfactants like polysorbate 80 could be employed to help emulsify the solution. Osmotic agents include sodium chloride to adjust the osmolarity of nasal fluids, preventing discomfort upon administration.

[0054] Formulation Process can include solubilization - dissolving or suspending the API in the solvent. This step might involve heating, stirring, or sonication depending on the drug's properties. Adding buffers, preservatives, and adjust pH and osmolarity. Adjusting viscosity by adding viscosity enhancers if needed. Filtration of the solution through a 0.22 pm or smaller pore size fdter to ensure sterility. Filling the solution into the nasal spray container (which can include a metering valve to deliver a consistent dose). Ensuring the product is sterile, which might include terminal sterilization if the drug and formulation components can withstand it.

[0055] Moreover, dose and dosage regimen, will depend mainly on the type of biological damage to the host, the type of subject, the history of the subject, and the type of therapeutic composition being administered. The size of the dose will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of a particular therapeutic composition and the desired physiological effect. It is also known that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

[0056] Therefore, the amount of the therapeutic composition must be effective to achieve an enhanced therapeutic index. If multiple doses are employed, the frequency of administration will depend, for example, on the type of subject. One skilled in the art can ascertain upon routine experimentation the appropriate route and frequency of administration in a given subject that are most effective in any particular case.

[0057] The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers, or diluents, are well known and readily available. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert with respect to the therapeutic composition and one that has no detrimental side effects or toxicity under the conditions of use.

[0058] The choice of excipient will be determined, in part, by the particular therapeutic composition, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical composition used in the embodiments of the invention. For example, the non-limiting formulations can be injectable formulations such as, but not limited to, those for intravenous, subcutaneous, intramuscular, intraperitoneal injection, and the like, and oral formulations such as, but not limited to, liquid solutions, including suspensions and emulsions, capsules, sachets, tablets, lozenges, and the like. Non-limiting formulations suitable for parenteral administration include aqueous and non-aqueousisotonic sterile injection solutions, including non-active ingredients such as antioxidants, buffers, bacteriostats, solubilizers, thickening agents, stabilizers, preservatives, surfactants, and the like. The solutions can include oils, fatty acids, including detergents and the like, as well as other known and common ingredients in such compositions, without limitation.

[0059] In certain aspects a subject will be provided a drug and drug schedule, subjects can be given a two-month supply of drug, e.g., 3TC, which consists of a bottle of 60 tablets. Two-month refills can be provided at appropriate times.IL Examples

[0060] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1A PILOT STUDY TO INVESTIGATE THE SAFETY AND FEASIBILITY OF ANTIRETROVIRAL THERAPY FOR ALZHEIMER’S DISEASE (ART -AD)

[0061] Studies in Drosophila, mice, cultured cells, and human brain indicate that retrotransposons are activated in settings of tauopathy, including Alzheimer’s disease, and causally drive neurodegeneration. Retrotransposons are viral-like DNA sequences that constitute approximately 41% of the human genome. The anti-retroviral medication 3TC (lamivudine), a nucleoside analog reverse transcriptase inhibitor, limits retrotransposon activation and suppresses neurodegeneration in tau transgenic Drosophila, two mouse models of tauopathy, and in brain assembloids derived from patients with sporadic Alzheimer’s disease. A 24-week phase 2 openlabel clinical trial of 300 mg daily oral 3TC (NCT04552795) was performed in 12 participants aged 52-83 years with a diagnosis of mild cognitive impairment due to suspected Alzheimer’s disease. Primary outcomes included blood brain penetration, safety and target engagement. Secondary outcomes included changes in cognition and fluid-based biomarkers of neurodegeneration and neuroinflammation. All participants completed the six-month trial; oneadverse event was reported. 3TC was detected in blood and cerebrospinal fluid (CSF) of all participants, suggestive of strong adherence to study drug and effective brain penetration. Reverse transcriptase assays did not support evidence of target engagement. While cognitive measures remained stable throughout the study, significant changes in several fluid biomarkers were detected. Glial fibrillary acidic protein (GFAP) (P=0.03) and Fltl ( / 1.05) were significantly reduced in CSF over the treatment period; Ab42 / 40 ( =0.009) and IL-15 (P=0.006) were significantly elevated in plasma. NfL, a fluid-based biomarker of neurodegeneration, exhibited a trending decrease in CSF (P=0.116) and increase in plasma (P=0.0954) over the treatment period. While this study is based on a small sample size and lacks a placebo control, the significant decrease of some neurodegeneration- and neuroinflammation-related biomarkers in CSF, significantly elevated levels of plasma Ab42 / 40, and a trending decrease of CSF NfL after six months of 3TC exposure suggest a beneficial effect on subjects with mild cognitive impairment due to suspected Alzheimer’s disease. Safety, tolerability, and central nervous system (CNS) penetration assessments further support clinical evaluation of 3TC in a larger placebo-controlled, multi-dose phase 2 clinical trial in participants with early Alzheimer’s disease.A. RESULTS

[0062] Study design, participant characteristics and drug adherence. Primary outcomes of this 24-week open-label phase 2a trial were to evaluate safety and tolerability, CNS penetration, and target engagement of the nucleoside analog reverse transcriptase inhibitor 3TC in subjects with mild cognitive impairment due to suspected Alzheimer’s disease. Secondary outcomes explored cognitive performance and changes in CSF and plasma biomarkers related to neurodegeneration and neuroinflammation from baseline to post-treatment. 22 candidates were pre-screened to identify 12 participants aged 50-99 years with a Clinical Dementia Rating (CDR) of 0.5 and Mini -Mental State Examination (MMSE) score between 24 and 30. Eligibility criteria are included in Table IB. Participants were enrolled between March 18, 2021 and September 15, 2022. Baseline participant characteristics are summarized in Table 1A; subject-specific demographic data are included in Table 2B. No significant differences were detected in years of education among subjects.Table 1A. Study participant characteristicsTable IB. Eligibility criteria

[0063] After the initial screening visit (visit -1), enrolled subjects initiated 300 mg oral 3TC daily at visit one (Table 2A). Visit one also included a physical exam, blood draw, and comprehensive neuropsychological exam. A lumbar puncture was performed to collect CSF prior to treatment initiation. Participants returned to the clinic at weeks 8, 16, and 24 of treatment to complete medication checks, physical examinations, brief cognitive screening, and blood draw. At week 24 of treatment, participants completed a post-treatment comprehensive neuropsychological exam, blood draw, and lumbar puncture. One month after the final dose of medication, participants returned to the clinic for a final safety assessment and disenrollment. All study participants completed the trial. CSF was not collected from three of 12 participants due to procedure failure, procedure-related pain, or patient-reported illness. Two participants (ART-AD-11 and ART-AD- 22) reported a COVID- 19 infection during the treatment period.

[0064] 3TC is absorbed rapidly after oral administration, reaching peak serum concentrations within 1.5-3 hours (Else etal., 2012, doi: 10.1128 / AAC.05599-11). Previous analysis of 3TC brain penetration reports that the concentration of 3TC in CSF is 4-8% of serum concentrations 2-4 hours following treatment. Blood draws and lumbar punctures for 3TC analyses were performed within 24 hours of drug administration. 3TC was detected in plasma and CSF in all participants at all time points (FIG. 2A, 2B), indicative of brain penetration and in line with full medication adherence asreported by all participants at each visit. Plasma and CSF 3TC levels varied widely among participants and study visits, likely due to the short half-life of 3TC.Table 2A. Study visit schedule.Table 2B. Subject-specific demographic data.

[0065] Safety and treatment-emergent adverse events. 3TC is widely used in antiretroviral therapy regimens due to its potent and long-lasting antiviral efficacy and low toxicity. Adverse events defined as unfavorable medical occurrences related to participation in the research were reviewed at visits 2-5 and at the follow-up visit one month following the last administration of 3TC. One adverse event of gastrointestinal bleeding was recorded; participant ART-AD-02, on daily aspirin, presented to the emergency room with complaints of abdominal pain and was found to have an ulcer. The remaining participants reported no treatment-emergent adverse events. Serial laboratory studies did not reveal signs of liver toxicity (e.g., elevated liver enzymes, bilirubin,hypoglycemia, anemia) or cytopenias, which are previously-reported 3TC-related adverse events (Lebensztejn and Kaczmarski, Am J Gastroenterol 97, 2687-88, 2002; Ormseth et al., American Journal of Gastroenterology 96, 1619-22, 2001 ; Kakubu et al. , OxfMed Case Reports 2023 , 106- 109, 2023). Given that weight loss has been reported in previous studies of 3TC-containing antiretroviral regimens (Bosch et al., Clinical Infectious Diseases 76, 1492-97, 2022), we compared standing weights at baseline and after 24 weeks of treatment. On average, subjects lost 4.03 lbs, ranging from a 19 lb. loss (ART-AD-02) to 8.8 lb. gain (ART-AD-22) in weight, after daily 3TC for 24 weeks. No deaths occurred within the treatment period or one month after treatment.

[0066] 3TC target engagement. Some retrotransposons encode a viral-like reverse transcriptase that acts to convert retrotransposon RNA into new DNA copies. Reverse transcriptase activity was quantified in plasma collected from participants at baseline and after 24 weeks of 3TC treatment using a modified version of the EnzCheck Reverse Transcriptase Assay, a fluorescent dye-based assay that quantifies the ability of a given sample to generate RNA:DNA heteroduplexes from a poly(A) template (Chang et al., J Virol Methods 65, 45-54, 1997). Levels of reverse transcriptase activity at baseline and following 24 weeks of 3TC treatment were highly variable among participants (FIG. 3); no evidence of target engagement was detected based on this assay.

[0067] Effects of 3TC on cognition and functional status. Following 24 weeks of treatment, we measured cognitive change from baseline using a comprehensive neuropsychological battery (Table 3). There was no significant change in cognition among participants from baseline to follow-up assessment. In addition to exploring post-treatment changes in cognition across the test battery, secondary aims assessed changes in the Preclinical Alzheimer Cognitive Composite (P ACC-5) score. Although typically relegated to individuals with prodromal and asymptomatic disease, the PACC-5 was included given its sensitivity to Alzheimer’s disease-specific cognitive change (Papp et al, Alzheimer ’s and Dementia: Translational Research and Clinical Interventions, 2017). Similarly, no statistically significant changes were observed. The trending significance based on MMSE were due primarily to a single outlier. Our oldest participant (ART- AD-11) was recovering from COVID-19 during follow-up assessment; the observed cognitive changes are likely a byproduct of the effects of the illness as opposed to a cognitive change due to 3TC. Additional information detailing cognitive performance for each participant is included in Table 4B and FIG. 4.Table 3. Neuropsychological battery.Table 4A. Cognitive assessments. IQR=interquartile range. P value is based on Wilcoxon signed rank test with continuity correction. n=12,Table 4B. Subject-specific cognitive data. Subjects are ordered based on PACC-5 POST-PRE z- scores.

[0068] Effects of 3TC on neurodegeneration-related biomarkers. Next the effects of 24- week 3TC treatment on biomarkers that have been previously linked to neurodegeneration were analyzed (Chatterjee et al., Alzheimer ’s and Dementia 19, 1117-34, 2023). Commercial SIMOA (Single Molecule Array) assays were performed on an HD-X platform (Quanterix) to quantify NfL, GFAP, Ab42, Ab40, and pTaul81. In CSF, an insignificant trending reduction in NfL from baseline to post-treatment was detected (FIG. 1A) and significant reduction of GFAP with treatment (FIG. IB). The Ab42 / 40 ratio was unchanged (FIG. 1C), as was pTaul81 (FIG. ID). In plasma, an insignificant trending increase in NfL was detected (FIG. IE). While plasma GFAP is unchanged with treatment (FIG. IF), significant elevation of Ab42 / 40 was detected (FIG. 1G), and trending increase in pTaul81 in plasma after 24 weeks of 3TC (FIG. 1H).

[0069] Effects of 3TC on biomarkers of neuroinflammation. The Mesoscale Discoveries (MSD) V-PLEX Neuroinflammation Panel 1 was used to analyze CSF and plasma levels of 37 biomarkers that are associated with a neuroinflammatory response. Biomarkers that were eithernot detected in more than three participants or those in which the % coefficient of variation (%CV) exceeded 15 were removed from the analysis. This left 21 high-confidence targets for analysis in CSF and 30 high-confidence targets for analysis in plasma (Table 5, FIG. 5, FIG. 6). In CSF, we detect a significant reduction in Fltl from baseline to post-treatment ( =0.05). In plasma, we detect a significant increase in IL-15 from baseline to post-treatment (P=0.006). The remaining neuroinflammatory biomarkers were not significantly changed over the course of treatment.Table 5. CSF and plasma-based inflammatory biomarker analysis. A value of “NA” is included for targets in which three or more participants had undetectable values or %CV values exceeding 15. POST-PRE values are in fg / ml. CSF, N=9; Plasma, N=12. Normality testing was based on the POST-PRE difference for each target; P values are based on two-sided paired sample t-test (normal distribution) or Wilcoxon matched-pairs signed rank test (non-normal distribution). IQR=Interquartile Range.B. METHODS

[0070] Study design and intervention. ART -AD is an investigator-initiated, single center, open-label phase 2 clinical trial. Enrolled subjects were stable on current medications for at least eight weeks prior to initiating 300 mg oral 3TC (McKesson Medical Supplies, product #1121405) daily at the initial visit. This dose was maintained throughout the entire 24-week study with excellent medication adherence as assessed by pill count. All subjects received the same dose and frequency. Subjects presented to the clinic at regular visits according to the protocol outlined in Table 2. This study was conducted according to US and international standards of Good Clinical Practice (FDA Title 21 part 312) applicable government regulations. The study protocol was approved by the University of Texas Health San Antonio Institutional Review Board. The trial was registered at ClinicalTrials.gov NCT04552795.

[0071] Fluid collection and processing. Fasting peripheral venous blood was collected into BD Paxgene® DNA tubes, BD EDTA coated tubes, and BD serum separator tubes (SST) for DNA, hematology, and chemistry analyses, respectively. Peripheral blood mononuclear cells (PBMC) were isolated from plasma using BD CPT tubes that contain Ficoll-Hypaque. Plasma and PBMCs were aliquoted into polypropylene tubes and stored at -80 °C. Complete blood count with differential and platelets, coagulation, metabolic, and lipid panels, and hemoglobin Ale were analyzed by LabCorp.

[0072] Lumbar punctures were performed at the Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases after overnight fasting. CSF was collected into a polypropylene tubevia gravity drip using a spinal needle. Resulting CSF was centrifuged to remove cellular debris, then aliquoted into polypropylene tubes and stored at -80 °C.

[0073] Neuropsychological assessment. All participants completed comprehensive neuropsychological assessment (see Table 3) with ACS or a Master’s-level psychometrist. These evaluations were completed at baseline (prior to the initiation of 3TC), and then again following 24-weeks of continued treatment. The PACC-5 score was calculated as a mean normative z-score across five measures, including MMSE, Logical Memory Delayed Recall, Digit-Symbol Coding Test, Category Fluency, and Free and Cued Selective Reminding Test.

[0074] HPLC / MS / MS chromatography. 3TC ((-)-L-2',3'-dideoxy-3 '-thiacytidine) and internal standard (3-guanidinopropionic acid (3GPA)) were obtained from Millipore Sigma. All other HPLC-grade reagents were purchased from Thermo Fisher Scientific. All solutions were prepared with Milli-Q water. A 3TC super stock was prepared in methanol at a concentration of 1 mg / ml and stored in aliquots at -80 °C. The working stock solution of 3TC was prepared each day from the super stock at a concentration of 100 pg / ml to spike the calibrators.

[0075] Reference calibrators were prepared by spiking control plasma to concentrations of 0, 5, 10, 25, 50, 100, 500, 1,000 ng / ml of 3TC, and control CSF to concentrations of 0, 2, 10, 50, 100, 500, 1,000, 5,000 ng / ml of 3TC. An identical concentration of internal standard, 10 pl (100 pg / ml 3-GPA), was spiked into each of the calibrators and blinded samples. 100 pl of mobile phase B (0.1% formic acid in acetonitrile) was added to each tube, vortexed thoroughly, and spun for 5 minutes at 17,000xg. 10 pl of the supernatant was injected into the HPLC with MS detection.

[0076] The LC / MS / MS system consisted of a Shimadzu SIL 20A HT autosampler, two LC- 20AD pumps, and an AB Sciex API 4000 tandem mass spectrometer with turbo ion spray. The LC analytical column was an ACE Excel C18-PFP (75 x 3.0 mm, 3 micron) purchased from Mac- Mod Analytical and was maintained at 24 °C during the chromatographic runs using a Shimadzu CT-20A column oven. Mobile phase A containing 0.1% formic acid dissolved in water was run at 0.255 ml / min. Mobile phase B consisted of 0.1% formic acid dissolved in 100% HPLC grade acetonitrile and run at 0.045 ml / min. The total flow rate of the mobile phase was 0.3 ml / min. 3TC and the internal standard, 3-GPA, were eluted isocratically with a 5 minute run. 3TC eluted at 3.69 min, while 3-GPA eluted at 3.25 min. Drug transitions were detected in positive mode at m / z 230 a 112 for 3TC and m / z 132 a 72 for 3-GPA. The ratio of 3TC peak area to the internal standardpeak area for each blinded sample was compared against a linear regression of the ratios obtained by the calibration peak areas to quantify 3TC.

[0077] Reverse transcriptase activity. Quantification of reverse transcriptase activity was performed using the EnzCheck reverse transcriptase assay kit (Invitrogen, E22064). Frozen plasma samples were thawed on ice and centrifuged at 2,000 rpm for 10 minutes to remove debris. Protein within the resulting supernatant was concentrated using 50K molecular weight cut off concentrators (Thermo Fisher Scientific, 88539). Reverse transcriptase reactions were run with diluted samples in freshly prepared enzyme dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT, pH 8) to microplate wells with and without the reaction mixture containing template / primer solution in polymerization buffer. The reaction was incubated for six hours at room temperature, after which 200 mM EDTA was added to stop the reaction. The RNA-DNA heteroduplexes formed in the reaction were detected via PicoGreen; fluorescence was measured using a microplate reader (Promega GloMax Discover). To correct for nonspecific binding, the fluorescence level of the sample (plasma sample with reaction mixture) was subtracted from the background (plasma sample without reaction mixture). The corrected data was used to calculate reverse transcriptase activity based on the reverse transcriptase standard curve.

[0078] Fluid biomarker analysis. Plasma and CSF samples were analyzed in the Fluid Biomarkers Laboratory at the Brown University Center for Alzheimer’s Disease Research. Samples and controls were run in duplicate and calibrators were run in triplicate. The measurements were performed blinded to diagnosis and clinical data in one round of experiments using one batch of reagents. Biomarkers related to neurodegeneration and neuroinflammation were quantified from the same CSF or plasma sample for each subject. The mean inter-assay and intraassay CV values were below 10% for all markers. Samples with CV values greater than 15% were removed from the analysis. Targets lacking data for more than three participants (due to lack of detection or CV > 15%) were not included in the analysis.

[0079] Measurement of Ab40, Ab42, NfL, GFAP and pTaul81. Plasma and CSF levels of the analytes of interest were determined by commercial SIMOA (Single Molecule Array) assays on an HD-X platform (Quanterix). Levels of A 42, A04O, GFAP, and NfL were quantified using the Neurology 4-plex E kit (103670; Quanterix) in CSF (400-fold dilution) and plasma (4-fold dilution). Levels of pTaul81 were quantified using the pTaul81 Advantage kit version 2.1 (104111; Quanterix) in CSF (10-fold dilution) and plasma (4-fold dilution).

[0080] Samples were thawed briefly on wet ice. Immediately after thawing, CSF samples used for A[34O, A 42, NIL and GFAP assays were diluted 400x in N4PE CSF Sample Diluent. All calibrators, controls, and samples were then incubated at room temperature for one hour. Samples and internal controls were centrifuged at 10,000 x g for five minutes. All plasma samples were diluted 4x onboard using the provided sample diluent. CSF samples were diluted at the bench and run neat. A four-parameter logistic curve fit, l / y2 weighted was used for GFAP, NfL and pTaul81 while a 5-paramter logistic curve fit, l / y2 weighted was used for A04O and A042. Two control samples of known concentration (high-control and low-control) provided in each kit as well as two internal controls of pooled CSF and plasma were included as quality control.

[0081] Measurement of inflammatory markers. Inflammatory proteins were measured according to manufacturer’s instructions using the V-PLEX Neuroinflammation Panel 1 (K15210D; Meso Scale Diagnostics) on the Meso QuickPlex SQ 120 (MSD). Plasma and CSF samples were diluted 2-fold for the V-plex Pro-inflammatory panel 1 (fFN-y, IL-ip, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13 and TNF-a), Cytokine Panel 1 (1L-Ia IL-5, IL-7, IL-12 / 23p40, IL-15, IL-16, IL-17A, TNF-P and VEGFA) and Angiogenesis panel 1 (VEGF-C, VEGF-D, Tie-2, Flt-1, PIGF and bFGF). Plasma and CSF samples were diluted 4-fold for the Chemokine Panel 1 (Eotaxin, MIP-10, Eotaxin-3, TARC, IP- 10, MIP-la, MCP-1, MDC and MCP-4). CSF samples were diluted 5-fold and plasma samples were diluted 1000-fold for the Vascular Injury Panel 1 (SAA, CRP, VCAM-1 and ICAM-1). Levels of MCP-4 (CSF), MIP-la (plasma), TARC (CSF), IL-17a (CSF), IL-10 (plasma), IL-5 (plasma), IL-17a (plasma) fell below the lower limit of detection (LLOD) during the initial screen. The assay was repeated for those markers only using a 2-fold dilution with an overnight incubation at 4°C (Janelidze et al., Neurology 91, e867-e877, 2018).

[0082] Samples were thawed briefly on wet ice and centrifuged at 2,000 x g for three minutes. After washing of the precoated plates with Phosphate Buffered Saline plus 0.05% Tween-20, 50 ml of calibrator control and diluted sample was added to each well. Plates were sealed and incubated for two hours at room temperature or overnight at 4 °C with shaking at 700 rpm. After washing, 25 pL of detection antibody solution was added to each well and the plate was incubated for two hours at room temperature with shaking at 700 rpm for all assays except Vascular Injury Panel 1, which was incubated for one hour. The plates were washed again followed by the addition of 150 pl Read Buffer T to each well. Plates were read immediately with the exception ofChemokine Panel 1 , which was incubated for ten minutes at room temperature prior to reading. A four-parameter logistic curve fit, l / y2 weighted was used for all assays. Control samples of known concentration were included as quality control.

[0083] Statistical analyses. Cognitive assessments. Given the small sample size, nonparametric (Wilcoxon matched-pairs signed rank test) tests were used to examine cognitive change following 24-weeks of 3TC. SPSS was used for statistical analyses of cognitive data. Fluid biomarker analyses. For each target, the range of POST-PRE values among all participants was tested for normality using the D’Agostino and Pearson test. A paired two-sided t-test was used to calculate P values for targets in which POST-PRE values passed the normality test (alpha = 0.05), while P values for targets that failed the normality test were calculated using the Wilcoxon matched-pairs signed rank test. A P value of less than or equal to 0.05 was considered significant. Multiple comparison testing was not performed due to the small sample size. GraphPad Prism was used for statistical analyses and figure preparation.C. Discussion

[0084] Activation of retrotransposons has been reported in post-mortem human tissue and fluids from patients with Alzheimer’s disease and related tauopathies (Sun etal., Nat Neurosci 21, 1038-48, 2018; Guo et al., CellReports 23, 2874-80, 2018; Macciardi et al., Geroscience 44, 1525-50, 2022), as well as in Drosophila (Sun et al., Nat Neurosci 21, 1038-48, 2018; Ochoa et al., Sci Adv 9, eabq5423, 2023; Guo el al., Cell Rep 5, 2874-80, 2018), mouse (Ramirez el al., Prog Neurobiol 208, 102181, 2022; Valles-Saiz et al., Int J Mol Sci 24, 2023; Wahl et al., Aging Cell el3798, 2023), and cell culture-based models (Valles-Saiz et al. , Int J Mol Sci 24, 2023; Sun et al., bioRxiv 2023.05.24.542155, 2023). Mechanistically, data suggest that retrotransposon activation is a consequence of the effects of pathogenic forms of tau on chromatin architecture and small RNA-mediated silencing of retrotransposon transcripts (Sun et al., Nat Neurosci 21, 1038— 48, 2018). Studies in Drosophila (Sun et al., Nat Neurosci 21, 1038-48, 2018) and mouse models (Valles-Saiz et al., Int J Mol Sci 24, 2023; Wahl et al., Aging Cell el3798, 2023 doi: 10.1111 / acel.13798) of tauopathy indicate that tau-induced retrotransposon activation causally drives neurodegeneration and is pharmacologically targetable using the nucleoside analog reverse transcriptase inhibitor 3TC. The example described herein provides results of the first clinical trialtesting the effects of 3TC in participants with mild cognitive impairment due to suspected Alzheimer’s disease.

[0085] The inventors find that 24-week treatment with 300 mg daily oral 3TC (the standard dosing regimen for HIV and hepatitis B) is safe and well -tolerated, and that 3TC effectively penetrates the blood brain barrier in this population. 3TC is absorbed rapidly after oral administration, with an absolute bioavailability of 82% in adults (Johnson et al., Clinical Pharmacokinetics Preprint 1999). While 3TC was detected in plasma and CSF of all participants, indicating 100% adherence to the study drug, 3TC levels were variable among participants and between study visits. The variability of 3TC detected in fluid among participants likely reflects the short half-life of 3TC, as drug levels in plasma and CSF depend on the amount of time that has passed between drug administration and time of fluid collection. The inventors contemplate controlled timing between dosing and fluid sampling to decrease variability among subjects and timepoints. The active anabolite of 3TC, 3TC 5 ’-triphosphate (3TC-TP), has a relatively longer half-life of approximately 15 hours within cells (Else et al., Pharmacokinetics of Lamivudine and Lamivudine-Triphosphate after Administration of 300 Milligrams and 150 Milligrams Once Daily to Healthy Volunteers: Results of the ENCORE 2 Study. 2012). While analysis of 3TC-TP would provide a quantitative assessment of active drug in each participant, this approach requires measurements of drug within isolated cells, which is a challenge for quantification in CSF.

[0086] The effects of 3TC on reverse transcriptase activity in plasma was determined as a measure of potential target engagement. The high degree of variability in reverse transcriptase activity among participants indicates that 300 mg of daily oral 3TC did not sufficiently block reverse transcriptase activity in plasma. While these data fail to show evidence of target engagement, the approach has several caveats. First, as the EnzCheck assay was performed on cell-free plasma, it is possible that levels of secreted reverse transcriptase are insufficient for assessing target engagement and / or do not reflect the level of reverse transcriptase activity within cells. Second, any type of double stranded nucleic acid, whether it is produced via reverse transcription or another mechanism, will be detected by the assay. The Lighthouse trial (NCT02868580), an open label, multi-center study to investigate the safety and tolerability of Triumeq (3TC, abacavir and dolutegravir) in subjects with amyotrophic lateral sclerosis (ALS), analyzed DNA copy number of human endogenous retrovirus K (HERV-K) elements as a measure of target engagement (Gold et al., Safety and tolerability of Triumeq in amyotrophic lateralsclerosis: the Lighthouse trial. Amyotroph Lateral Scler Frontotemporal Degener 2019). While analysis of retrotransposon DNA copy number could potentially be leveraged to assess 3TC target engagement in future studies of 3TC in Alzheimer’s disease and related tauopathies, the normalization of retrotransposon DNA copies, which are highly abundant and repetitive within the human genome, to a single-copy control gene using quantitative or digital PCR-based methods is a technical caveat of this approach.

[0087] While the study was not powered to assess efficacy, significant changes were detected in biomarkers related to neurodegeneration and neuroinflammation. In longitudinal studies, low levels of plasma Ab42 / 40 and high levels of plasma pTaul 81, GFAP, and NfL are associated with subsequent cognitive decline (Chatteij ee et al., Alzheimer ’s and Dementia 19, 1117-34, 2023). In the current analysis, significant changes in fluid levels of Ab42 / 40 and GFAP were found, and trending changes in NfL after six months of 3TC treatment. Plasma Ab42 / 40 is inversely correlated with amyloid burden in the brain such that individuals with positive amyloid PET have significantly lower levels of Ab42 / 40 (Schindler et al., High-precision plasma (3-amyloid 42 / 40 predicts current and future brain amyloidosis 2019; Rembach etal., Alzheimer ’s and Dementia 10, 53-61, 2014). A lower Ab42 / 40 ratio is also associated with amnestic mild cognitive impairment, and individuals with lower baseline levels of Ab42 / 40 have increased risk of progression to dementia (Verberk et al., Ann Neurol 84, 648-58 2018; Perez-Grijalba et al., J Prev Alzheimers Dis 6, 34-41, 2019). Significant elevation of Ab42 / 40 in plasma was found after six months of 3TC treatment compared to baseline, suggestive of a potentially protective effect of 3TC. In line with these data, significant reduction of GFAP in CSF was detected after six months of 3TC treatment. GFAP is a marker of reactive astrogliosis that is elevated in preclinical, prodromal, and Alzheimer’s disease dementia in plasma and CSF (Chatterjee etal., Alzheimer ’s and Dementia 19, 1117-34, 2023; Verberk etal., Ann Neurol 84, 648-58 2018; Fukuyama etal., Eur Neurol 46, 35- 38 2001; Jesse et al., Journal of Alzheimer 's Disease 17, 541-51, 2009; Oeckl et al., Journal of Alzheimer ’s Disease 67, 481-88, 2019). The trending decrease in CSF levels of NfL, a marker of axonal damage that is elevated in CSF (Zetterberg etal., JAMA Neurol 73, 60-672016) and plasma (Mattsson et al., JAMA Neurol 74, 557, 2017) of individuals with mild cognitive impairment, Alzheimer’s dementia, and other neurodegenerative disorders (Simren et al., Current Opinion in Neurobiology 2020), further suggests a benefit due to 3TC treatment. Among neuroinflammatory biomarkers, significant reduction of CSF Fit- 1 was detected, also known as vascular endothelialgrowth factor receptor 1 , and significant elevation of the proinfl am m at ory cytokine IL-15 in plasma. Longitudinal biomarker analyses indicate that Fit- 1 and IL-15 are elevated within CSF at preclinical, prodromal and dementia stages of Alzheimer’s disease, and are associated with cortical thinning and subsequent cognitive decline (Janelidze et al., Neurology 91, e867-e877, 2018). While analyses of plasma levels of IL-15 in individuals with Alzheimer’s disease are limited, a smaller study of 20 subjects with Alzheimer’s disease compared to 15 controls reports that IL-15 is slightly but significantly reduced in plasma of individuals with Alzheimer’s disease (Rentzos et al., Journal of Neuropsychiatry and Clinical Neurosciences 19, 318-25, 2007); another study of 52 subjects with Alzheimer’s disease compared to 18 controls found no difference in IL-15 in CSF or plasma (Johansson et al., Reduced cerebrospinal fluid concentration of interleukin- 12 / 23 subunit p40 in patients with cognitive impairment, 2017). Taken together, the fluid biomarker analysis points toward compelling potential benefits of 3TC in individuals with mild cognitive impairment due to suspected Alzheimer’s disease.

[0088] Based on the similarities between exogenous retroviruses and retrotransposons, numerous studies have investigated the therapeutic efficacy of antiviral medications, including nucleoside analog reverse transcriptase inhibitors, to suppress retrotransposon activation. The Lighthouse trial, for example, which tested the effects of Triumeq in participants with ALS, has completed phase 2a. Triumeq consists of two reverse transcriptase inhibitors, abacavir and 3TC, and an integrase inhibitor, dolutegravir, which blocks episomal DNA integration into genomic DNA. 35 out of 40 subjects completed the 24-week study. Non-serious adverse events that occurred in three or more participants included nausea and rash. While five deaths were expected based on modeling, only one death was observed five months after conclusion of the trial. The ALS functional rating scale demonstrated a declining trend. The trial is currently recruiting for phase 3, in which overall survival and disease progression will be measured in a larger cohort. An additional trial (NCT02437110) for a retroviral combination therapy (darunavir, ritonavir, dolutegravir, and tenofovir alafenamide,) in ALS is currently in phase 1. A 12-month trial (NCT02363452) in children with Aicardi-Goutieres Syndrome, in which the body mounts a viral response due to activation of retrotransposons, reports that participants administered 3TC, zidovudine, and abacavir have a significantly reduced blood interferon score after treatment (Rice et al., New England Journal of Medicine 2018). Ongoing trials leveraging reverse transcriptase inhibitors in tauopathies include LINE-AD, a phase 1 study in which emtricitabine is being testedin participants with mild cognitive impairment or Alzheimer’s disease (NCT04500847), and a phase 2a trial testing the effects of TPN-101 in individuals with progressive supranuclear palsy (NCT0499 768).

[0089] In summary, findings are presented from the first clinical trial testing the effects of reverse transcriptase inhibition in participants with mild cognitive impairment due to suspected Alzheimer’s disease. It was found that 3TC is safe and well -tolerated among participants, and gains access to the CNS. While no evidence of target engagement was obtained, the 3TC treatment has beneficial effects on biomarkers associated with neurodegeneration and neuroinflammation, strongly supporting the initiation of a larger, placebo-controlled phase II study of 3TC in older adults with Alzheimer’s disease and, potentially, primary tauopathies.

Claims

CLAIMS1. A method of treating a tauopathy or neurodegenerative disease comprising administering a high dose, therapeutic amount of lamivudine to a subject having or at risk of developing a tauopathy, wherein the dose is about 350 mg to 1000 mg.

2. The method of claim 1, wherein the tauopathy is Alzheimer’s disease.

3. The method of claim 1, wherein lamivudine is administered at a dose of lOOmg to 500mg daily.

4. The method of claim 1, wherein lamivudine is administered at a dose greater than 300 mg daily.

5. The method of claim 1, wherein lamivudine is administered for 10 to 30 days or 2 to 5 weeks or 1 to 6 months.

6. The method of claim 1, wherein administration is oral or nasal administration.

7. The method of claim 6, wherein a tablet, capsule, pill, powder, sustained release formulation, solution, or suspension is orally administered.

8. The method of claim 1, wherein the dose administered results in a concentration of at 150 ng / ml of reverse transcriptase inhibitor in the cerebrospinal fluid (CSF).

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