WO2023105283A1 - Nucleoside reverse transcriptase inhibitors for use in down syndrome and alzheimer`s disease therapy - Google Patents

Abstract

The present invention relates to nucleoside reverse transcriptase inhibitors (NRTI) or their pharmaceutically acceptable salts for use in (a) the treatment of Down syndrome or the consequences thereof or (b) the prevention, inhibition of progression or treatment of Alzheimer`s disease or the consequences thereof. Pharmaceutical compositions comprising such NRTI and combinations of NRTI are provided. Kits including NRTI, combinations of NRTI, and pharmaceutical compositions comprising such NRTI and combinations of NRTI are also described.

Description

NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS FOR USE IN DOWN SYNDROME

AND ALZHEIMER'S DISEASE THERAPY

Field of the Invention

The present invention relates to nucleoside reverse transcriptase inhibitors (NRTI) or their pharmaceutically acceptable salts for use in (a) the treatment of Down syndrome or the consequences thereof or (b) the prevention, inhibition of progression or treatment of Alzheimer' s disease or the consequences thereof. Pharmaceutical compositions comprising such NRTI and combinations of NRTI are provided. Kits including NRTI, combinations of NRTI, and pharmaceutical composition comprising such NRTI and combinations of NRTI are also described.

Background of the Invention

Down syndrome (DS) is the most commonly known genetic disorder associated with moderate to severe intellectual disability due to a total or partial trisomy of the autosomal chromosome 21 (HSA21) and a genetic form of Alzheimer’s disease (AD) (Dierssen M, et al., Nat Rev Neurosci 2012; 13(12):844-858). Overexpression of HSA21 gene products such as DYRK1A, SOD1 or SIOOB has been proposed to contribute to the neurological and neurob ehavi oral DS phenotypes (Altafaj X, et al, Hum Mol Genet 2001; 10(18): 1915-1923, Gulesserian T, et aL, J Investig Med 2001; 49(l):41-46, Griffin W, et al., Proc Natl Acad Sci USA 1989; 86(19):7611-7615). However, treatments targeted to candidate genes or mechanisms have only been partially successful (de la Torre R, et al., Lancet Neurol 2016; 15(8): 801 -810, Fernandez F, et al., Lancet Neurol 2016; 15(8):776-777). This is possibly explained because besides the HSA21 gene-dosage effects, triplication of HSA21 also leads to a genome wide transcriptional deregulation (De Toma I, et al., PLoS Comput Biol 2021; 17(9):el009317).

Recently, single-nucleus long-read RNA sequencing has revealed thousands of unannotated RNA transcripts containing intra-exonic junctions, involving a myriad of genes, including the amyloid precursor protein (APP) (Palmer C, et al. , Proc Natl Acad Sci USA 2021 ; 118(47)). Notably, increased brain transcription and increased copy numbers of APP have been linked to APP somatic gene recombination associated with sporadic AD and with DS, and could contribute to their cognitive deficits (Kaeser G, et al., Front Genet 2020; 11 :390). Retrotransposable elements like the long interspersed nuclear element 1 (LINE1 or LI - 6kb) are thought to participate in this process in mammals (Muotri A, etal., Nature 2005; 435(7044): 903-910). Increased retrotransposition is observed in cell senescence and with ageing and has been implicated in several neurodegenerative diseases including AD, frontotemporal dementia, prion disease, but also in developmental disorders such as Rett’s syndrome, autism or fragile X syndrome (Gorbunova V, et al., Nature 2021; 596(7870):43-53, Li W, et al, PLoS One 2012; 7(9):e44099).

Nucleoside reverse transcriptase inhibitors (NRTIs) have been used to suppress retrotransposition, and thus NRTIs could theoretically improve these pathologies. Preclinical studies have shown that the reverse transcriptase inhibitor lamivudine [(-)-L-2',3'-dideoxy-3'- thiacytidine (3TC)] improves cognition in senescence-accelerated prone 8 (SAMP8) mice, a model for studying human ageing and age-related diseases (Li M, et al., J Cell Mol Med 2021; 25(17)8490-8503. Although a contribution of retrotransposition to developmental disorders has also been suggested, NRTIs have not been explored in DS. Thus, the possible therapeutic effects of lamivudine on DS, AD and/or the consequences thereof remains to be further explored.

Summary of the Invention

In one aspect the present invention is directed to nucleoside reverse transcriptase inhibitors (NRTI) or their pharmaceutically acceptable salts for use in (a) the treatment of Down syndrome or the consequences thereof or (b) the prevention, inhibition of progression or treatment of Alzheimer' s disease or the consequences thereof. In some embodiments, the NRTI comprises abacavir, didanosine, dideoxycytidine, emtricitabine, entecavir, lamivudine, stavudine, tenofovir, zidovudine, their pharmaceutically acceptable salts or a combination thereof. In some embodiments the NRTI is lamiduvine or its pharmaceutically acceptable salts. In some embodiments, the consequences thereof comprise cognitive deficit.

In another aspect, the present invention refers to a pharmaceutical composition comprising at least one NRTI or its pharmaceutically acceptable salts, a combination of NRTI their pharmaceutically acceptable salts or according to the invention for use in (a) the treatment of Down syndrome or (b) the prevention, inhibition of progression or treatment of Alzheimer's disease or the consequences thereof. In some embodiments, the NRTI or the combination thereof may be in the form of pharmaceutically acceptable salts, as generally described below. In some embodiments, the consequences thereof comprise cognitive deficit.

In a further aspect, the present invention refers to Down syndrome and Alzheimer' s disease therapies based on the use of the NRTI, combinations of NRTI, and pharmaceutical compositions of the invention. In some embodiments, the present invention relates to a method for treating Down syndrome or the consequences thereof (e.g., cognitive deficit) which comprises administering a therapeutically effective amount of (a) at least one NRTI or a combination thereof (b) a pharmaceutical composition according to the present invention to a subject in need thereof. In some embodiments, the present invention is directed to a method for preventing, inhibiting the progression or treating Alzheimer’s disease or the consequences thereof (e.g., cognitive deficit) which comprises administering a therapeutically effective amount of (a) at least one NRTI or a combination thereof or (b) a pharmaceutical composition according to the present invention to a subject in need thereof.

In an additional aspect, the present invention refers to kits comprising, according to the invention, (a) at least one NRTI or a pharmaceutically acceptable salt thereof, (b) a combination of NRTI or their pharmaceutically acceptable salts, (c) a pharmaceutical composition comprising at least one NRTI, a pharmaceutically acceptable salt thereof or a combination of NRTI or their pharmaceutically acceptable salts thereof. The kits of the invention are used for (i) treating Down syndrome or the consequences or (ii) preventing, inhibiting the progression or treating Alzheimer’s disease or the consequences thereof in a subject in need thereof.

Brief Description of the Drawings

Fig- 1- Locomotor activity and anxiety-like behavior in TS and respective WT in basal conditions and after 1 or 4 months of treatment with lamivudine. A) Total activity and B) circadian activity for 24 hours measured as ambulatory (X axis) beam breaks. In B) the dark square under the X-axis represents the dark phase of the cycle (WT n = 20, TS n = 15). Anxietylike behavior was assessed in elevated plus maze. C) Total distance travelled, D) number of entries, and E) percentage of time spent in each zone of the elevated plus maze (WT n = 20, TS n = 12). Data are represented as mean ± SEM. Genotype effect * p < 0.05, ** p < 0.01, *** p < 0.005, Treatment effect & p <0.05, && p <0.01; &&& p < 0.005. Fig. 2. Cognitive assessment in novel object recognition test in TS and respective WT in basal conditions and after 1 or 4 months of treatment with lamivudine. A) Total time of exploration during the familiarization phase and B) discrimination index 24 hours after familiarization (treated: Wild type (WT-T) n = 17, Ts65Dn (TS-T) n = 13; non-treated: Wild type (WT-NT) n = 9, Ts65Dn (TS-NT) n = 9). Boxplots extend from the 25th to 75th percentiles and the median is represented as a line in the box. The whiskers correspond to the maximum and minimum value excluding outliers. * p < 0.05, ** p < 0.01, *** p < 0.005.

Detailed Description of the Invention

1. Definitions of general terms and expressions

The term “AIDS”, as used herein, refers to the symptomatic phase of HIV infection, and includes both acquired immune deficiency syndrome (commonly known as AIDS) and “ARC,” or AIDS-Related Complex (Adler M, etal., Brit. Med. J. 1987; 294: 1145-1147). The immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and cancers resulting from immune deficiency.

The term “Alzheimer' s disease” or “AD”, as used herein, refers to a progressive neurologic disorder that causes the brain to shrink (i.e., atrophy) and brain cells to die. Alzheimer's disease is the most common cause of dementia (i.e., a continuous decline in thinking, behavioral and social skills that affects a person's ability to function independently). AD consequences include, but are not limited to, (a) cognitive deficit (e.g., persistent memory lapses, impaired thinking, and reasoning), (b) reduced ability for making judgements and taking decisions, (c) decreased capacity for conducting routinary activities (e.g., bathing, cooking, driving), and (d) changes in personality and behavior (e.g., mood swings, depression, aggressiveness).

The term “comprising” or “comprises”, as used herein, discloses also “consisting of’ according to the generally accepted patent practice.

The term “Down syndrome” or “DS”, as used herein, refers to a genetic disorder caused when abnormal cell division results in an extra full or partial copy of chromosome 21. This extra genetic material causes the developmental changes and physical features of Down syndrome. DS consequences include, but are not limited to, moderate cognitive deficit (e.g., impaired thinking and reasoning, delayed language development, reduced short- and long-term memory).

The term “enteral administration”, and the related term “enterally” as used herein refer to any administration of a NRTI (e.g., lamivudine) or combination thereof according to the present invention or a pharmaceutical composition comprising said NRTI or combination thereof via the gastrointestinal tract. Enteral administration includes, but is not limited to, the oral, sublingual, and rectal routes of administration.

The term “HIV”, as used herein, include HIV-1 and HIV-2, SHIV and SIV. “HIV-1” means the human immunodeficiency virus type-1. HIV-1 includes, but is not limited to, extracellular virus particles and the forms of HIV-1 associated with HIV-1 infected cells. The HIV-1 virus may represent any of the known major subtypes (Classes A, B, C, D E, F, G and H) or outlying subtype (Group O) including laboratory strains and primary isolates. “HIV-2” means the human immunodeficiency virus type-2. HIV-2 includes, but is not limited to, extracellular virus particles and the forms of HIV-2 associated with HIV-2 infected cells. The term “SIV” refers to simian immunodeficiency virus which is an HIV-like virus that infects monkeys, chimpanzees, and other nonhuman primates. SIV includes, but is not limited to, extracellular virus particles and the forms of SIV associated with SIV infected cells.

The term “kit”, as used herein, refers to a product containing the different reagents necessary for carrying out the uses and methods of the invention which is packed so as to allow their transport and storage. Materials suitable for packing the components of the kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper or envelopes.

The term “lamivudine”, as used herein, refers to 4-amino-l-[(2R,5S)-2- (hydroxymethyl)-l,3-oxathiolan-5-yl]pyrimidin-2-one, a compound of chemical formula C8H11N3O3S and CAS [134678-17-4], Lamivudine is a nucleoside analogue and reverse transcriptase inhibitor used for the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections. In HIV, lamivudine inhibits HIV-1 reverse transcriptase (RT) via DNA chain termination after incorporation of the nucleoside analogue into viral DNA. In HBV, the incorporation of lamivudine into viral DNA by HBV polymerase results in DNA chain termination. Lamivudine is a weak inhibitor of mammalian DNA polymerases alpha and beta, and mitochondrial DNA polymerase (NCI04). Lamivudine is combined with other active agents, such as zidovudine, as part of the HIV antiretroviral therapy (ART) protocol.

The term “NRTI” or “nucleoside reverse transcriptase inhibitor”, as used herein, refers to active inhibitors of reverse transcriptase found in retroviruses such as the human immunodeficiency virus (HIV). The different nucleoside reverse transcriptase inhibitors may be activated differently but they have the same mechanism of action. NRTIs are activated generally by phosphorylation to the triphosphate form by cellular enzymes. It then competes with cellular triphosphates, which are substrates for proviral DNA by viral reverse transcriptase. NRTIs are used in the treatment of HIV infection and acquired immune deficiency syndrome (AIDS). Examples ofNRTI, or combinations comprising NRTI, include, but are not limited to, abacavir sulfate (Ziagen®), abacavir and lamivudine (Epzicom®), abacavir, zidovudine, and lamivudine (Trizivir®), delay ed-release didanosine (Videx EC®), didanosine (Videx®), dideoxycytidine (Hivid®), emtricitabine (Emtriva®), entecavir (Baraclude®), lamivudine (Epivir®), lamivudine and zidovudine (Combivir®), stavudine (Zerit®), tenofovir disoproxil fumarate (Viread®), tenofovir disoproxil fumarate and emtricitabine (Truvada®), tenofovir alafenamide and emtricitabine (Descovy®), and zidovudine (Retrovir®).

The term “parenteral administration” and the related term “parenterally”, as used herein, refer to the administration of a NRTI or a combination thereof according to the present invention or a pharmaceutical composition according to the present invention characterized by the physical breaching of a tissue of a subject and the administration of the NRTI, combination or pharmaceutical composition through said breach in the tissue. Parenteral administration includes, but is not limited to, the administration of a NRTI (e.g., lamivudine) or a combination thereof according to the present invention or a pharmaceutical composition comprising said NRTI or combination, by the application of the NRTI, combination or pharmaceutical composition through, for instance, a surgical incision or through a tissue-penetrating non- surgical wound. In particular, parenteral administration includes, but is not limited to, the epidural, intraarterial, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal injection, intravascular, intravenous, intravenous infusion, spinal, subcutaneous, and subcutaneous depot routes of administration.

The expression “pharmaceutically acceptable carrier”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible with the NRTI of the invention.

The terms “prevent,” “preventing” and “prevention”, as used herein, refer to inhibiting the inception or decreasing the occurrence of a disease in a subject. The prevention may be complete (e.g., the total absence of pathological cells in a subject). The prevention may also be partial, such as, for example, lowering the occurrence of pathological cells in a subject. Prevention also refers to a reduced susceptibility to a clinical condition. Within the context of the present invention, the terms “prevent,” “preventing” and “prevention”, refer specifically to averting or reducing the probability of that a subject develops Alzheimer's disease or any of the consequences related to Alzheimer' s disease (e.g., cognitive deficit).

The term “subject”, as used herein, refers to an individual or animal, such as a human, a nonhuman primate (e.g., chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats, and guinea pigs. The term does not denote a particular age or sex. The term “subject” encompasses an embryo and a fetus. In some embodiments, the subject is a human.

The term “therapeutically effective amount”, as used herein, refers to the dose or amount of the NRTI or combination thereof according to the present invention or the pharmaceutical compositions of the present invention that produce a therapeutic response or desired effect in a subject.

The term “topical”, and the related term “topically” as used herein refer to any administration of a NRTI (e.g., lamivudine) or combination thereof according to the present invention or a pharmaceutical composition comprising said NRTI or combination by applying the NRTI, combination or pharmaceutical composition to a particular place on or in the body, such as the skin or a mucous membrane. Topical administration includes, but is not limited to, the aural, cutaneous, nasal, transdermal, urethral, vaginal, and urethral routes of administration.

The term “treat” or “treatment”, as used herein, refers to the administration of at least one NRTI, a combination of NRTI or a pharmaceutical composition according to the present invention for controlling the progression of a disease after its clinical signs have appeared. Control of the disease progression is understood to mean the beneficial or desired clinical results that include, but are not limited to, reduction of the symptoms, reduction of the duration of the disease, stabilization of pathological states (specifically to avoid additional deterioration), delaying the progression of the disease, improving the pathological state and remission (both partial and total). The control of progression of the disease also involves an extension of survival compared with the expected survival if treatment was not applied. Within the context of the present invention, the terms “treat” and “treatment” refer specifically to stopping or slowing the consequences (e.g., cognitive deficit) of (a) Down syndrome or (b) Alzheimer' s disease in a subject afflicted with such syndrome or disease. “Treatment” can also mean prolonging survival of a subject afflicted with Down syndrome or Alzheimer' s disease as compared to the expected survival of the subject if the subject does not receive any of the NRTI or pharmaceutical compositions according to the present invention.

2. NRTI

In one aspect, the present invention refers to nucleoside reverse transcriptase inhibitors (NRTI) or their pharmaceutically acceptable salts for use in (a) the treatment of Down syndrome or the consequences thereof or (b) the prevention, inhibition of progression or treatment of Alzheimer' s disease or the consequences thereof. In some embodiments, the NRTI comprises abacavir, didanosine, dideoxycytidine, emtricitabine, entecavir, lamivudine, stavudine, tenofovir, zidovudine, their pharmaceutically acceptable salts or a combination thereof. In some embodiments the NRTI is lamiduvine or its pharmaceutically acceptable salts. In some embodiments, the consequences thereof comprise cognitive deficit.

3. Pharmaceutical compositions

In another aspect, the present invention refers to a pharmaceutical composition comprising at least one NRTI or a combination of NRTI according to the invention for use in (a) the treatment of Down syndrome or (b) the prevention, inhibition of progression or treatment of Alzheimer' s disease or the consequences thereof. In some embodiments, the NRTI or the combination thereof may be in the form of pharmaceutically acceptable salts, as generally described below. In some embodiments, the consequences thereof comprise cognitive deficit.

Pharmaceutically acceptable salts of the NRTI of the present invention include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. See Stahl P, et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, Weinheim, DE, 2008).

The NRTI or combinations thereof described herein may be administered in the form of a pharmaceutical composition containing prodrugs of said NRTI or their combinations. A prodrug can include a covalently bonded carrier which releases the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include, for example, compounds wherein a hydroxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol functional groups in the compounds. Methods of structuring a compound as prodrugs are known in the art. See Testa B, et al., Hydrolysis in Drug and Prodrug Metabolism (John Wiley, Hoboken, NJ, USA, 2006). Typical prodrugs form the active metabolite by transformation of the prodrug by hydrolytic enzymes, the hydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxides or the cleavage of esters of inorganic acids.

Pharmaceutical compositions for use according to the present invention typically comprise an effective amount of at least on NRTI or a combination thereof and at least one pharmaceutical acceptable carrier. The preparations may be prepared in a manner known in the art, which usually involves mixing the at least one NRTI according to the invention with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary, under aseptic conditions. See US6372778, US6369086, US6369087, US6372733 and Remington: The Science and Practice of Pharmacy, 21st Ed. (Pharmaceutical Press, Philadelphia, PA, USA, 2011).

Generally, for pharmaceutical use, the compounds may be formulated as a pharmaceutical preparation comprising at least one compound and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.

The pharmaceutical preparations of the present invention are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use.

The NRTI, combinations thereof and pharmaceutical compositions of the invention can be administered by a variety of routes including the topical, enteral or parenteral routes, depending mainly on the specific preparation used. A “therapeutically effective amount” of the NRTI or combination thereof would generally be administered to the subject in need thereof. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. See US6372778, US6369086, US6369087, US6372733, and Remington, 2011, supra.

Depending upon the manner of introduction, the NRTI or combinations thereof of the invention may be formulated in a variety of ways. Formulations containing one or more NRTI can be prepared in various pharmaceutical forms including, but not limited to, granules, tablets, capsules, suppositories, powders, controlled release formulations, suspensions, emulsions, creams, gels, ointments, salves, lotions or aerosols. In some embodiments, these formulations are employed in solid dosage forms suitable for simple, and preferably oral, administration of precise dosages. Solid dosage forms for oral administration include, but are not limited to, tablets, soft or hard gelatin or non-gelatin capsules, and caplets. However, liquid dosage forms, such as solutions, syrups, suspension, and shakes can also be utilized. In some embodiments, the formulation is administered topically. Suitable topical formulations include, but are not limited to, lotions, ointments, creams, and gels. In some embodiments, the topical formulation is a gel. In another embodiment, the formulation is administered intranasally.

Formulations containing one or more of the NRTI of the present invention may be prepared using a pharmaceutically acceptable carrier composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. As generally used herein “carrier” includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, pH modifying agents, preservatives, antioxidants, solubility enhancers, and coating compositions.

Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Delayed release, extended release, and/or pulsatile release dosage formulations may be prepared as described in standard references. See Liberman A, et al.. Eds, Pharmaceutical Dosage Form Tablets (Marcel Dekker, Inc., New York City, NY, USA 1989), and Ansel H, et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 10^th Edition, (Lippincott Williams & Wilkins, Baltimore, MD, 2014), and Remington, 2011, supra. Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, methacrylic resins, and polysaccharides.

Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants.

Diluents, also referred to as “fillers”, are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and Veegum®, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil. Disintegrants are used to facilitate dosage form disintegration or breakup after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross-linked polymers, such as crosslinked polyvinylpyrrolidone (PVP).

Surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include, but are not limited to, sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2- ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl- -alanine, sodium N-lauryl-|3- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the tablets, beads, granules or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents or preservatives.

The concentration of the compound(s) to carrier and/or other substances may vary from about 0.5 to about 100 wt % (weight percent). For oral use, the pharmaceutical formulation will generally contain from about 5 to about 100% by weight of the active material. For other uses, the pharmaceutical formulation will generally have from about 0.5 to about 50 wt % of the active material.

The compositions described herein can be formulation for modified or controlled release. Examples of controlled release dosage forms include extended-release dosage forms, delayed release dosage forms, pulsatile release dosage forms, and combinations thereof. The extended-release formulations are generally prepared as diffusion or osmotic systems, according to procedures known in the art. See Remington, 2011, supra. A diffusion system typically consists of two types of devices, a reservoir and a matrix. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses such as hydroxypropyl-cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and Carbopol® 934, polyethylene oxides and mixtures thereof. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil or mixtures thereof.

In some embodiments, the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers.

The devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units. Examples of multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads or granules. An immediate release portion can be added to the extended-release system by means of either applying an immediate release layer on top of the extended-release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

Extended-release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation or dry granulation. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin, and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.

Extended-release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In the congealing method, the drug is mixed with a wax material and either spray-congealed or congealed and screened and processed.

Delayed release formulations are created by coating a solid dosage form with a polymer film, which is insoluble in the acidic environment of the stomach, and soluble in the neutral environment of the small intestine.

The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a “coated core” dosage form or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. In some embodiments, coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and/or enzymatically degradable polymers, and may be conventional “enteric” polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available such as Eudragit® (Rohm Pharma, Westerstadt, DE), including Eudragit® L30D-55 and L100-55 (soluble at pH 5.5 and above), Eudragit® L-100 (soluble at pH 6.0 and above), Eudragit® S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and Eudragit® NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.

A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition.

Alternatively, each dosage unit in the capsule may comprise a plurality of drugcontaining beads, granules or particles. As is known in the art, drug-containing “beads” refer to beads made with drug and one or more excipients or polymers. Drug-containing beads can be produced by applying drug to an inert support, e.g., inert sugar beads coated with drug or by creating a “core” comprising both drug and one or more excipients. As is also known, drugcontaining “granules” and “particles” comprise drug particles that may or may not include one or more additional excipients or polymers. In contrast to drug-containing beads, granules and particles do not contain an inert support. Granules generally comprise drug particles and require further processing.

Generally, particles are smaller than granules, and are not further processed. Although beads, granules and particles may be formulated to provide immediate release, beads and granules are generally employed to provide delayed release.

In some embodiments, the compound is formulated for topical administration. Suitable topical dosage forms include lotions, creams, ointments, and gels. A “gel” is a semisolid system containing a dispersion of the active agent (e.g., Nox inhibitor), in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Methods for preparing lotions, creams, ointments, and gels are well known in the art.

The NRTI, combinations and pharmaceutical compositions of the invention can be administered adjunctively with other active compounds. These compounds include but are not limited to analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxiolytics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics, and anti-narcoleptics. “Adjunctive administration”, as used herein, means the compounds can be administered in the same dosage form or in separate dosage forms with one or more other active agents.

The pharmaceutical compositions of the invention may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic aqueous solution. Examples of suitable diluents are normal isotonic saline solution, 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulations are especially suitable for parenteral administration but may also be used for oral administration. Excipients, such as polyvinylpyrrolidinone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate, may also be added.

Alternatively, these compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols or water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing, and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion or an aqueous or nonaqueous suspension. Such a liquid formulation may be administered directly or filled into a soft gelatin capsule.

The pharmaceutical compositions of the application may be in the form of a sterile injectable preparation. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents as described before.

4. Methods of treatment and prevention

In a further aspect, the present invention refers to Down syndrome and Alzheimer' s disease therapies based on the use of the NRTI, combinations of NRTI, and pharmaceutical compositions of the invention. In some embodiments, the present invention relates to a method for treating Down syndrome or the consequences thereof (e.g., cognitive deficit) which comprises administering a therapeutically effective amount of (a) at least one NRTI or a combination thereof (b) a pharmaceutical composition according to the present invention to a subject in need thereof. In some embodiments, the present invention is directed to a method for preventing, inhibiting the progression or treating Alzheimer’s disease or the consequences thereof (e.g., cognitive deficit) which comprises administering a therapeutically effective amount of (a) at least one NRTI or a combination thereof or (b) a pharmaceutical composition according to the present invention to a subject in need thereof. Alternatively, the present invention relates also to the use of NRTI in the manufacture of a medicament for (a) treating Down syndrome or the consequences thereof or (b) preventing, inhibiting the progression or treating Alzheimer’s disease or the consequences thereof. In some embodiments, the subject is human. In some embodiments, the subject is also infected with HIV and/or is afflicted with AIDS. In some embodiments, the (a) at least one NRTI or a combination thereof or (b) the pharmaceutical composition according to the present invention is administered to the subject via a topical, enteral or parenteral route of administration. The NRTI, combination of NRTI, and pharmaceutical compositions of the invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route or mode of administration will vary depending upon the desired results. The active agents of the invention can be prepared with carriers that will protect the agent against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems, as described above. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are generally known to in the art. See Robinson J, et al., Eds., Sustained and Controlled Release Drug Delivery Systems (Marcel Dekker, Inc., New York, NY, USA, 1978).

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased, as indicated by the exigencies of the therapeutic situation.

It is especially advantageous to formulate enteral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is dictated by, and directly dependent on, the unique characteristics of the active agent and the particular therapeutic effect to be achieved.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art. The amount of active agent which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active agent which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, this amount will range from about 0.001% to about 90% of active agent, preferably from about 0.005% to about 70% and, most preferably, from about 0.01% to about 30%.

Actual dosage levels ofNRTI, combinations of NRTI and pharmaceutical compositions according to the present invention may be varied for attaining the desired therapeutic response in a subject. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular agent of the invention employed, its amount, the route of administration, the time of administration, the rate of excretion or expression of the particular active agent employed, the duration of the treatment, other drugs, compounds or materials used in combination with the particular pharmaceutical compositions employed, the age, sex, weight, condition, general health, and prior medical history of the subject being treated and other similar factors known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the active agent(s) required. For example, the physician or veterinarian could start doses of the active agents of the invention employed in the pharmaceutical composition at levels lower than that required for achieving the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the NRTI, combinations of NRTI and pharmaceutical compositions according to the present invention which is the lowest dose to be therapeutically effective. Such therapeutically effective dose will generally depend upon the factors described above. If desired, the therapeutically effective daily dose the NRTI, combinations of NRTI and pharmaceutical compositions according to the present invention may be administered as two, three, four, five, six or more sub-doses applied separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for an active agent of the invention to be administered alone, it is preferable to administer said agent as a pharmaceutical composition.

5. Kits

In an additional aspect, the present invention refers to kits comprising, according to the invention, (a) at least one NRTI or a pharmaceutically acceptable salt thereof, (b) a combination of NRTI or their pharmaceutically acceptable salts, (c) a pharmaceutical composition comprising at least one NRTI, a pharmaceutically acceptable salt thereof or a combination thereof. The kits of the invention are used for (i) treating Down syndrome or the consequences or (ii) preventing, inhibiting the progression or treating Alzheimer’s disease or the consequences thereof in a subject in need thereof. The components of the kits of the invention may be optionally packed in suitable containers and be labeled for preventing, inhibiting the progression, and treating Down syndrome, Alzheimer’s disease or the consequences thereof (e.g., cognitive deficit). The components of the kits may be stored in unit or multi-dose containers as a solid or aqueous, preferably sterile, solution. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kits may further comprise more containers comprising a pharmaceutically acceptable carrier. They may further include other materials desirable from a commercial and user standpoint, including, but not limited to, buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable host cells or other active agents. The kits can contain instructions customarily included in commercial packages of diagnostic and therapeutic products that contain information, for example, about the indications, usage, dosage, manufacture, administration, contraindications or warnings concerning the use of such diagnostic and therapeutic products.

All publications mentioned herein are incorporated in their entirety by reference. Having now generally described the invention, the same will be more readily understood through reference to the following general procedures, which are provided by way of illustration and are not intended to be limiting of the present invention, unless specified.

General Procedures

1. Materials and methods

Trisomic (Ts65Dn, TS) male mice were obtained by breeding B6EiC4Sn.BLiA- Ts(1716)65Dn/DnJ females with C57BL/6 x 6J01aHsd (B6C3Fl/01aHsd) hybrid males. Mice were genotyped, and ~25% of the offspring presented with trisomy. Wild-type (WT) littermates were used as controls. After weaning, male mice were group housed with 3-4 animals per cage on a conventional 12:12 light cycle in controlled environmental conditions of humidity (50-70%) and temperature (21 ± 1°C). Water and standard rodent chow were available ad lib. Experiments were conducted using 3-4 month-old male mice. TS and WT mice were assigned using a simple randomization to either control conditions or lamivudine 3 mg/kg (Epivir, 10 mg/mL, oral solution) in the drinking water. 15 TS and 20 WT mice were treated with lamivudine and 14 TS and 18 WT mice were used as control group and received water. The treatment lasted 4 months. Behavioral testing was performed before the treatment and 1 and 4 months after starting the treatment. Behavioral experiments were conducted during the light phase of the light/dark cycle between 10 am and 2 pm and were performed by trained observers blind to genotype. The novel object recognition test and plus maze were videotaped using a computer-assisted data acquisition system (SMART, Panlab Harvard Apparatus, ES). Less stressful tests were performed first, and each behavioral test was separated by at least 24 h. The order of the tests was as follows: (1) locomotor activity, (2) novel object recognition, and (3) elevated plus maze. All procedures were conducted according local, national, and European regulations relating to the use of animals for scientific purposes.

Spontaneous locomotor activity in the home cage was measured using an automated infrared light-beam monitoring system (ActiMot2, TSE system, DE) and the ambulatory beam breaks (X axis) were counted for 24 h. Total general activity was analyzed. Mice were isolated for assessing locomotor activity during 24 hours and immediately regrouped after the test.

The apparatus consisted of a rectangular open-field arena (50 cm long x 50 cm wide x 50 cm high) made of black Plexiglas. Animals’ behavior was monitored using System Motor Activity Record and Tracking software (SMART, Panlab Harvard Apparatus, ES). On the first day, mice were habituated to the arena for 5 min. On the second day, mice were presented with two identical objects, for 10 min. Subjects failing to complete a minimum of 20 s of exploration during the familiarization session were excluded for posterior analysis. 24 h after (test session), mice were presented with one familiar object and one novel one, for 5 min. The discrimination index was calculated as ((time exploring the novel object - time exploring the familiar object)/total time of exploration) * 100 [48], Exploratory behavior was defined as the animal directing its nose towards the object at a distance of < 2 cm and manually registered by the experimenter. Sitting on or resting against the object was not considered as exploration. All the objects used were plastic made and induced similar exploration levels. The arena and objects were deeply cleaned between animals to avoid olfactory cues.

The elevated plus maze paradigm was used to study anxiety-related behavior. The apparatus consists of a cross-shaped platform (four arms faced two to two with a length of 40 cm and 8 cm width) elevated 40 cm from the floor. Two opposing arms are protected by black walls and the other two are left unprotected. Because of the distance to the ground, the open arm is an aversive environment for the mouse, so it tends to remain in the closed arms. The proportion of time spent in open versus closed arms during 5 min is considered a measure of anxiety. Distance travelled was analyzed as a measure of the activity in the apparatus.

Two-way analysis of variance (ANOVA) repeated measures was used for testing genotype and treatment differences in the locomotor activity and elevated plus maze test, and for the analysis of the novel object recognition test. Tukey or Bonferroni post hoc tests were used as a correction between pairwise comparisons. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (version 19.0) and GraphPad Prism (version 8.01).

2. Results

2.1 Lamivudine ameliorated hyperactivity in TS mice

The locomotor activity of WT and TS mice in baseline conditions and the effect of lamivudine in both genotypes one and four months after starting the treatment were analyzed. Two-way ANOVA repeated measures showed a significant genotype effect (F(l,22) = 15.76 p < 0.01). In baseline conditions, TS mice showed significantly increased total locomotor activity as compared to WT mice (Bonferroni post-hoc, p < 0.005, Figure 1A), as shown by an increased distance travelled during 24 hours.

After one month of treatment with lamivudine, total locomotor activity was reduced in TS mice with respect to baseline levels, although not completely rescued to WT levels (Bonferroni post-hoc, p < 0.05, Figure 1A), and the same tendency was observed after four months of treatment, although no statistically significant differences were observed between both genotypes (Bonferroni post-hoc, N.S., Figure 1A). Two-way repeated measures ANOVA revealed a significant effect of lamivudine (treatment effect (F2, 60) = 8.07, p < 0.005). However, the distance travelled was significantly reduced after one month of treatment only in TS mice (Tukey post-hoc, TS basal vs TS-1M; p < 0.05).

These results indicate an amelioration of hyperactivity in Ts65Dn after treatment with lamivudine. When analyzing the circadian pattern of activity (Figure IB), it was observed that the treatment-associated changes of locomotor activity are mainly detected during the active (dark) phase of the circadian cycle. The increase in locomotor activity in TS mice during the dark phase are significant in basal conditions (One way ANOVA repeated measures, F(l, 33) = 13.53, p < 0.005), and are significantly reduced with the treatment (1 month of treatment: F(l, 33) = 9.09, p < 0.01 and 4 month of treatment F(l, 33) = 5.88, p < 0.05).

2.2 Lamivudine increased anxiety-like behavior in WT and TS mice

The anxiety-like behavior in the elevated plus maze paradigm was also assessed to understand whether TS mice would be more sensitive to this side effect of lamivudine (Figure 1C-E). Locomotor activity (distance travelled) was first analyzed (Figure 1C). No genotypedependent differences were detected, but both genotypes travelled a reduced distance in the maze after treatment (Two-way ANOVA repeated measures, treatment effect F(2, 51) = 34.67, p < 0.005. Tukey's post-hoc test, WT BASAL v WT1M p < 0.005, WTBASAL vs WT4M p < 0.005, TSBASAL vs TS1M p < 0.05, TS BASAL vs TS4M p < 0.005, Figure 1C). Possibly related to this, although no significant differences between genotypes were detected in the number of entries in open or closed arms, upon treatment we detected a reduction of entries in both open (Two-way ANOVA repeated measures, treatment effect, F(l, 38) = 15.10, p <0.005, Figure ID) and closed arms (Two-way ANOVA repeated measures, treatment effect, F(2, 52) = 12.55, p <0.005). The reduction in the number of entries was maintained along the treatment at 1 and 4 months both in open (Tukey’s post-hoc test, WT-BASAL vs WT-1M p< 0.005; WTBASAL vs WT-4M p < 0.005; TS-1M vs TS-4M p<0.05) and in closed arms (Tukey’s post- hoc test, WT-BASAL vs WT-1M p< 0.01; WT-BASAL vs WT-4M p < 0.05; TS-BASAL vs TS-4M p<0.05, TS-1M vs TS-4M p<0.05). As both the entries in open and closed arm were reduced, this global reduction in activity as measured also by the distance travelled may reflect the habituation to the apparatus after repeated exposures.

Anxiety-like behavior is expressed as an increased time spent in the close arms with respect to open arms. No differences between genotypes in the number of entries nor in the time spent in neither open nor closed were observed, indicating similar anxiety-related behavior in TS and WT mice. However, a treatment effect in the time spent in closed arms (Two-way ANOVA repeated measures, treatment effect, F(1.5, 46) = 15.18, p < 0.005, Figure IE) was detected, that was slightly increased in both genotypes and in the center (Two-way ANOVA repeated measures, treatment effect, F(1.8, 55) = 11.82, p < 0.005), in which permanence was slightly reduced. Upon lamivudine treatment, WT mice spent significantly more time in closed arms (Tukey’s post-hoc test, WT-BASAL vs WT-1M p < 0.005; WT-BASAL vs WT-4M p < 0.005) and less time in open arms (non-significant) and in the center of the maze (Tukey’s post- hoc test, WT-BASAL v WT-1M p < 0.005; WT-BASAL vs WT-4M p < 0.005) as compared to basal conditions, suggesting anxiety-like effects. In the case of the TS, although the tendency is similar, no significant differences were observed.

2.3 Lamivudine rescues recognition memory deficits in TS mice

Recognition memory was evaluated using the novel object recognition paradigm that has been shown to be a robust and reproducible test for studying the Ts65Dn strain (Sierra C, et al., Front Behav Neurosci 2021; 15:772734). An independent group of WT and TS receiving water in basal conditions and after 1 or 4 months to discard possible carry-over effects of the test, and track the possible age-related differences in recognition memory, was used for this assay. During the familiarization test, no differences in the time of exploration were observed among the groups at baseline, 1 month or 4 months (Figure 2A). In basal conditions, TS mice showed recognition memory impairment, as shown by the significantly reduced discrimination index (Two-way ANOVA, F(3,47) = 1.28 p < 0.005, Tukey’s post-hoc test: WT-NT v TS-NT p < 0.01 WT-T vs TS-T p < 0.05). One month after starting non-treated mice continued showing cognitive impairment (Two-way ANOVA, F(3,47)= 5.27 p < 0.005, Tukey’s post-hoc test: WT-NT vs TS-NT p < 0.05), while TS treated with lamivudine, showed a complete rescue of the cognitive deficit reaching WT performance levels (Tukey’s post-hoc test: WT-T vs TS-T p =0.98, Figure 2B). The cognitive rescue in TS mice was maintained after 4 months of treatment (Two-way ANOVA, F(3,47)= 6.28 p < 0.005, Tukey’s post-hoc test: WT-T vs TS-T p = 0.82, Figure 2B), whereas non-treated Ts65Dn mice showed an even worse discrimination index (Tukey’s post-hoc test: WT-NT v TS-NT p < 0.005, Figure 2B), probably due to age- associated cognitive decline.

References

Altafaj X, et al.. Neurodevel opmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing DyrklA (minibrain), a murine model of Down's syndrome. Hum Mol Genet, 2001. 10(18): p. 1915-23.

De Cecco M, et al., LI drives IFN in senescent cells and promotes age-associated inflammation. Nature, 2019. 566(7742): p. 73-78. de la Torre R, et al., Safety and efficacy of cognitive training plus epigallocatechin-3 -gallate in young adults with Down's syndrome (TESDAD): a double-blind, randomised, placebo- controlled, phase 2 trial. Lancet Neurol, 2016. 15(8): p. 801-810.

De Toma I, et a , Meta-analysis of transcriptomic data reveals clusters of consistently deregulated gene and disease ontologies in Down syndrome. PLoS Comput Biol, 2021. 17(9): p. el009317.

Dierssen M, Down syndrome: the brain in trisomic mode. Nat Rev Neurosci, 2012. 13(12): p. 844-58.

Escorihuela R, et al., Impaired short- and long-term memory in Ts65Dn mice, a model for Down syndrome. Neurosci Lett, 1998. 247(2-3): p. 171-4.

Fernandez F, et al., Pharmacotherapy in Down's syndrome: which way forward? Lancet Neurol, 2016. 15(8): p. 776-777.

Fructuoso M, et al., Increased levels of inflammatory plasma markers and obesity risk in a mouse model of Down syndrome. Free Radic Biol Med, 2018. 114: p. 122-130.

Gardner E, et al., Contribution of retrotransposition to developmental disorders. Nat Commun, 2019. 10(1): p. 4630.

Gensous N, et al., Down syndrome, accelerated aging and immunosenescence. Semin Immunopathol, 2020. 42(5): p. 635-645.

Gorbunova V, et al., The role of retrotransposable elements in ageing and age-associated diseases. Nature, 2021. 596(7870): p. 43-53.

Griffin W, et al., Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc Natl Acad Sci U S A, 1989. 86(19): p. 7611-5.

Gulesserian T, et al., Superoxide dismutase SOD1, encoded on chromosome 21, but not SOD2 is overexpressed in brains of patients with Down syndrome. J Investig Med, 2001. 49(1): p. 41-6. Kaeser G, et al., Mosaic Somatic Gene Recombination as a Potentially Unifying Hypothesis for Alzheimer's Disease. Front Genet, 2020. 11 : p. 390.

Lee M, et al., Somatic APP gene recombination in Alzheimer's disease and normal neurons. Nature, 2018. 563(7733): p. 639-645.

Li M, et al., Lamivudine improves cognitive decline in SAMP8 mice: Integrating in vivo pharmacological evaluation and network pharmacology. J Cell Mol Med, 2021. 25(17): p. 8490-8503.

Li W, et al., Transposable elements in TDP-43 -mediated neurodegenerative disorders. PLoS One, 2012. 7(9): p. e44099.

Ling K, et al., Functional transcriptome analysis of the postnatal brain of the TslCje mouse model for Down syndrome reveals global disruption of interferon-related molecular networks. BMC Genomics, 2014. 15: p. 624.

Manti S, et al., Inflammatory biomarkers and intellectual disability in patients with Down syndrome. J Intellect Disabil Res, 2018. 62(5): p. 382-390.

Muotri A, et al., Somatic mosaicism in neuronal precursor cells mediated by LI retrotransposition. Nature, 2005. 435(7044): p. 903-10.

Palmer C, etal., Altered cell and RNA isoform diversity in aging Down syndrome brains. Proc Natl Acad Sci U S A, 2021. 118(47).

Patterson D, et al., Down syndrome as a model of DNA polymerase beta haploinsufficiency and accelerated aging. Meeh Ageing Dev, 2012. 133(4): p. 133-7.

Rodgers S, et al., Transgenic APP expression during postnatal development causes persistent locomotor hyperactivity in the adult. Mol Neurodegener, 2012. 7: p. 28.

Siangphoe U, et al., Associations of antiretroviral therapy and comorbidities with neurocognitive outcomes in HIV- 1 -infected patients. AIDS, 2020. 34(6): p. 893-902.

Sierra C, et al., Social Factors Influence Behavior in the Novel Object Recognition Task in a Mouse Model of Down Syndrome. Front Behav Neurosci, 2021. 15: p. 772734.

Simon M, et al., LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation. Cell Metab, 2019. 29(4): p. 871-885 e5.

Sullivan K, et al., Trisomy 21 consistently activates the interferon response. Elife, 2016. 5.

Claims

27 Claims

1. NRTI for use in the treatment of Down syndrome or the consequences thereof.

2. NRTI for use in the prevention, inhibition of progression or treatment of Alzheimer’s disease or the consequences thereof.

3. A pharmaceutical composition comprising at least one NRTI or a combination thereof for use in the treatment of Down syndrome or the consequences thereof.

4. A pharmaceutical composition comprising at least one NRTI or a combination thereof for use in the prevention, inhibition of progression or treatment of Alzheimer’s disease or the consequences thereof.

5. NRTI and pharmaceutical compositions for use according to any one of the preceding claims wherein the NRTI comprises abacavir, didanosine, dideoxycytidine, emtricitabine, entecavir, lamivudine, stavudine, tenofovir, zidovudine, their pharmaceutically acceptable salts or a combination thereof.

6. A method for treating Down syndrome or the consequences thereof which comprises administering a therapeutically effective amount of (a) at least one NRTI or a combination thereof (b) a pharmaceutical composition according to any one of the preceding claims to a subject in need thereof.

7. A method for preventing, inhibiting the progression or treating Alzheimer’s disease or the consequences thereof which comprises administering a therapeutically effective amount of (a) at least one NRTI or a combination thereof or (b) a pharmaceutical composition according to any one of the preceding claims to a subject in need thereof.

8. NRTI and pharmaceutical compositions for use according to claims 1-5 and the methods according to claims 6-7 wherein the consequences of the Down syndrome or Alzheimer’s disease is cognitive deficit.

9. The methods according to claims 6-7 wherein the subject is human.

10. The method according to claim 7 wherein the subject is infected with HIV or suffers AIDS.

11. The methods according to any one of the preceding claims wherein the (a) at least one NRTI or a combination thereof or (b) the pharmaceutical composition is administered to the subject via a topical, enteral or parenteral route of administration.

12. A kit comprising the NRTI and pharmaceutical compositions for use according to claims 1-5 or a combination thereof and instructional materials for their use.