Oral gemfibrozil for the treatment of CLN3-related juvenile neuronal ceroid lipofuscinosis

Abstract

The present disclosure relates generally to improved pharmaceutical compositions useful for the treatment of juvenile neuronal ceroid lipofuscinosis (JNCL) or juvenile Batten disease, which are fatal inherited neurodegenerative disorders of children caused by dysfunction of the Cln3 gene. More specifically, the present disclosure relates to pharmaceutical compositions comprising oral gemfibrozil for the treatment of juvenile neuronal ceroid lipofuscinosis (JNCL) or juvenile Batten disease.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Application No. 63 / 439,723, filed January 18, 2023, the entire contents of which are incorporated herein by reference.

[0002] [Government License Rights Clause] This invention was made with government support under awards AG050431 and AT010980 from the National Institutes of Health (NIH) and 1K6BX004982 from the U.S. Department of Veterans Affairs. The government has certain rights in this invention.

[0003] [Sequence table] The Sequence Listing, prepared in accordance with WIPO Standard ST.26, was prepared on January 17, 2024, has a file size of approximately 7,000 bytes, and is hereby submitted as part of this disclosure.

[0004] [Field of the Invention] The present disclosure relates generally to methods and pharmaceutical compositions useful for treating juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, a fatal genetic neurodegenerative disorder of childhood caused by mutations in molecular pathways or direct mutations in the Cln3 gene. More specifically, the present disclosure relates to therapeutic methods and pharmaceutical compositions comprising oral gemfibrozil for the treatment of JNCL. [Background technology]

[0005] Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is a fatal, autosomal recessive, childhood-onset neurodegenerative disorder caused by mutations in the Cln3 gene. With an estimated incidence of 1 in 12,500 live births, JNCL is considered a rare neurological disorder, typically presenting between the ages of 4 and 10 years and presenting with progressive vision loss, seizures, blindness, motor and cognitive decline, mental and intellectual decline, epilepsy, and ultimately premature death in the teens or twenties. Despite intensive research, no effective treatment for JNCL exists.

[0006] Gemfibrozil is an FDA-approved lipid-lowering drug prescribed for its ability to lower plasma triglyceride levels and reduce the risk of hyperlipidemia in patients. Commonly known as "Lopid," gemfibrozil was successfully marketed in 1976.

[0007] With this background in mind, the following disclosure describes the potential therapeutic use of gemfibrozil in patients with JNCL. Summary of the Invention

[0008] Batten disease appears to be caused, at least in part, by two mutant copies of the Cln3 gene. This disclosure utilizes the findings that gemfibrozil treatment reduces microglial and astroglial activation, alleviates neuroinflammation, restores TFEB (a master regulator of lysosomal biogenesis) levels, and reduces the accumulation of storage materials, including mitochondrial ATP synthase subunit c (SCMAS), in the somatosensory barrel field (SBF) cortex of a JNCL mouse model. Without being bound by theory, it is believed that the molecular dysfunction caused by mutations in at least one copy of the Cln3 gene can be rescued or alleviated by the introduction of gemfibrozil, which stimulates the recruitment of PPARα to the Tfeb gene promoter in vivo in the SBF cortex. Additionally, gemfibrozil introduction reduced microglial and astroglial activation, which reduced neuroinflammation; increased TFEB levels, which are low in JNCL patients; and reduced SCMAS accumulation in the SBF. Furthermore, oral treatment in a JNCL mouse model showed improvement in locomotor function. Taken together, this data suggests that gemfibrozil could be used as a continuous treatment for patients with diagnosed or suspected JNCL.

[0009] JNCL is characterized by glial activation and the accumulation of autofluorescent storage material containing mitochondrial ATP synthase subunit c (SCMAS), ultimately leading to neuronal loss. To date, no effective treatment is available for JNCL. This study highlights the therapeutic potential of gemfibrozil, a prototypic activator of peroxisome proliferator-activated receptor α (PPARα) and a Food and Drug Administration (FDA)-approved lipid-lowering drug, in animal models of JNCL. Oral gemfibrozil treatment significantly reduced Cln3 activity. Δex7 / 8In the somatosensory barrel cortex (SBF) cortex of Cln3ΔJNCL mice, gemfibrozil treatment reduced microglial and astroglial activation, attenuated neuroinflammation, restored TFEB (a master regulator of lysosomal biogenesis) levels, and reduced accumulation of the storage compound SCMAS. Accordingly, gemfibrozil treatment also improved locomotor activity in Cln3ΔJNCL mice. Investigating the mechanism, we found that PPARα was significantly lost in the SBF cortex of Cln3ΔJNCL mice, which increased after gemfibrozil treatment. Oral gemfibrozil also stimulated PPARα recruitment to the Tfeb gene promoter in vivo in the SBF cortex of Cln3ΔJNCL mice, indicating that gemfibrozil treatment increased Tfeb transcription in the CNS via PPARα. Furthermore, in Cln3ΔJNCL mice (Cln3ΔJNCL), which lack PPARα, gemfibrozil treatment significantly improved locomotor activity in Cln3ΔJNCL mice. ΔPPARα ) exacerbated the pathology, and gemfibrozil reduced SCMAS accumulation and glial cell activation, resulting in Cln3ΔJNCL ΔPPARα However, the locomotor performance of the mice remained unimproved. These results suggest that PPARα activation may be beneficial for JNCL and that gemfibrozil could be used to treat this incurable disease.

[0010] This study demonstrated that gemfibrozil inhibits Cln3 Δex7 / 8 To investigate whether orally administered gemfibrozil could halt and / or slow the disease course of JNCL in Cln3ΔJNCL (Cln3ΔJNCL) mice, we found that orally administered gemfibrozil significantly suppressed glial cell activation, increased TFEB levels, and reduced SCMAS accumulation, thereby activating Cln3ΔJNCL through PPARα. ΔPPARα Here, we provide evidence that oral administration of the lipid-lowering drug gemfibrozil improved locomotor activity in Cln3 mice. Δex7 / 8We demonstrate that PPARα-deficient Cln3ΔJNCL (Cln3ΔJNCL) mice exhibit reduced glial inflammation, normalized and / or upregulated TFEB, reduced the accumulation of autofluorescent storage material in the SBF cortex, and improved locomotor activity. ΔPPARα ) and gemfibrozil was shown to worsen the condition in Cln3ΔJNCL ΔPPARα The failure to reduce SCMAS accumulation, suppress glial cell activation, and improve locomotor performance in mice highlights the critical role of PPARα in this process.

[0011] In various embodiments, the present disclosure provides methods for treating juvenile neuronal ceroid lipofuscinosis (JNCL) in a patient in need thereof.

[0012] In another embodiment, a method for treating or correcting dysfunction of the lysosomal membrane glycoprotein Cln3 is provided.

[0013] In either embodiment, the method comprises administering to a patient in need thereof an effective amount of an oral pharmaceutical composition comprising gemfibrozil. Gemfibrozil can be identified by its CAS number 25812-30-0.

[0014] In any of the disclosed embodiments, the pharmaceutical composition may be administered to the patient any number of times per day, including once per day, twice per day, and three times per day.

[0015] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages of the present disclosure will be described hereinafter, which form the subject of the claims of this application. Those skilled in the art will appreciate that the conception and specific implementations disclosed may be readily utilized as a basis for modifying or designing other implementations for carrying out the same purposes of the present disclosure. It will also be appreciated by those skilled in the art that such equivalent implementations do not depart from the spirit and scope of the present disclosure as set forth in the appended claims. [Brief explanation of the drawings]

[0016] [Figures 1A-1D] Figure 1A shows a graph depicting the amount of GFAP compared to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 1B shows a graph depicting the amount of Iba1 compared to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 1C shows a graph depicting the number of GFAP-positive cells. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 1D shows a graph showing the number of Iba-1 positive cells, where the first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. [Figures 2A-2D]Figure 2A shows a graph depicting the amount of iNOS compared to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 2B shows a graph depicting the amount of Pro IL-1β compared to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 2C shows a graph depicting the amount of mature IL-1β compared to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 2D shows a graph showing the amount of TNF-α compared to the WT control, with the first bar closest to the y-axis representing the WT, the second bar representing the JNCL mouse model, the third bar representing the 4 mg / kg / day mouse model, and the fourth bar representing the 8 mg / kg / day mouse model. [Figures 3A-3D]Figure 3A shows a graph depicting the MFI of neuronal TFEB. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 3B shows a graph depicting the MFI of NeuN. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 3C shows a graph depicting the amount of TFEB compared to the WT control. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. Figure 3D shows a graph showing the MFI amounts of SCMAS, where the first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the 4 mg / kg / day mouse model, and the fourth bar represents the 8 mg / kg / day mouse model. [Figures 4A-4D] Figure 4A shows a graph depicting TPP1 levels relative to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the 8 mg / kg / day mouse model. Figure 4B shows a graph depicting p62 levels relative to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the 8 mg / kg / day mouse model. Figure 4C shows a graph depicting MFI levels of neuronal TPP1. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the 8 mg / kg / day mouse model. Figure 4D shows a graph depicting MFI levels of neuronal p62. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the 8 mg / kg / day mouse model. [Figures 5A-5F]Figure 5A shows a graph depicting velocity (cm / sec), with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 4 mg / kg / day mouse model, and the fourth bar representing the 8 mg / kg / day mouse model. Figure 5B shows a graph depicting cumulative duration in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 4 mg / kg / day mouse model, and the fourth bar representing the 8 mg / kg / day mouse model. Figure 5C shows a graph depicting distance traveled in centimeters, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 4 mg / kg / day mouse model, and the fourth bar representing the 8 mg / kg / day mouse model. Figure 5D shows a graph of center point movement in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 4 mg / kg / day mouse model, and the fourth bar representing the 8 mg / kg / day mouse model. Figure 5E shows a graph of pole latency in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 4 mg / kg / day mouse model, and the fourth bar representing the 8 mg / kg / day mouse model. Figure 5F shows a graph of rotarod latency in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 4 mg / kg / day mouse model, and the fourth bar representing the 8 mg / kg / day mouse model. [Figures 6A-6D]Figure 6A shows a graph showing the amount of PPARα compared to the WT control. The first bar closest to the y-axis represents the WT, the second bar represents the JNCL mouse model, and the third bar represents the 8 mg / kg / day mouse model. Figure 6B shows a graph showing the MFI amount of PPARα. The first bar closest to the y-axis represents the WT, the second bar represents the JNCL mouse model, and the third bar represents the 8 mg / kg / day mouse model. Figure 6C shows a schematic diagram of the mouse Tfeb gene promoter containing the PPRE. Figure 6D shows a graph showing quantitative PCR of PPARα, CBP, RNA Pol, IgG, and Input (control). The first bar closest to the y-axis represents the WT, the second bar represents the JNCL mouse model, and the third bar represents the 8 mg / kg / day mouse model. [Figures 7A-7D] Figure 7A shows a graph depicting Iba1 levels relative to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the JNCL and PPARα null models. Figure 7B shows a graph depicting GFAP levels relative to WT controls. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the JNCL and PPARα null models. Figure 7C shows a graph depicting MFI levels of SCMAS. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the JNCL and PPARα null models. Figure 7D shows a graph depicting pole T turns in seconds. The first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, and the third bar represents the JNCL and PPARα null models. [Figure 7E-7J]Figure 7E shows a graph showing pole latency in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, and the third bar representing the JNCL and PPARα null models. Figure 7F shows a graph showing the rotarod test in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, and the third bar representing the JNCL and PPARα null models. Figure 7G shows a graph showing speed in centimeters per second, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, and the third bar representing the JNCL and PPARα null models. Figure 7H shows a graph showing cumulative duration in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, and the third bar representing the JNCL and PPARα null models. Figure 7I shows a graph depicting distance traveled in centimeters, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, and the third bar representing the JNCL and PPARα null models. Figure 7J shows a graph depicting migration, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, and the third bar representing the JNCL and PPARα null models. [Figure 8A-8B] Figure 8A shows a graph depicting the MFI of TFEB, where the first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the JNCL model administered at 8 mg / kg / day, the fourth bar represents the JNCL and PPARα null model, and the fifth bar represents the JNCL and PPARα null model administered at 8 mg / kg / day. Figure 8B shows a graph depicting the MFI of SCMAS, where the first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the JNCL model administered at 8 mg / kg / day, the fourth bar represents the JNCL and PPARα null model, and the fifth bar represents the JNCL and PPARα null model administered at 8 mg / kg / day. [Figures 9A-9C]Figure 9A shows a graph depicting the number of microglia per square millimeter, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 8 mg / kg / day JNCL model, the fourth bar representing the JNCL and PPARα null model, and the fifth bar representing the 8 mg / kg / day JNCL and PPARα null model. Figure 9B shows a graph depicting the number of astrocytes per square millimeter, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL mouse model, the third bar representing the 8 mg / kg / day JNCL model, the fourth bar representing the JNCL and PPARα null model, and the fifth bar representing the 8 mg / kg / day JNCL and PPARα null model. Figure 9C shows a graph showing the number of iNOS(+) cells per square millimeter, where the first bar closest to the y-axis represents WT, the second bar represents the JNCL mouse model, the third bar represents the JNCL model administered at 8 mg / kg / day, the fourth bar represents the JNCL and PPARα null model, and the fifth bar represents the JNCL and PPARα null model administered at 8 mg / kg / day. [Figures 10A-10C] Figure 10A shows a graph depicting velocity measured in centimeters per second, with the first bar closest to the y-axis representing WT, the second bar representing JNCL and PPARα null model, the third bar representing JNCL and PPARα null model at 8 mg / kg / day, and the fourth bar representing vehicle-treated JNCL and PPARα null model. Figure 10B shows a graph depicting cumulative duration in seconds, with the first bar closest to the y-axis representing WT, the second bar representing JNCL and PPARα null model, the third bar representing JNCL and PPARα null model at 8 mg / kg / day, and the fourth bar representing vehicle-treated JNCL and PPARα null model. Figure 10C shows a graph of distance traveled in centimeters, with the first bar closest to the y-axis representing WT, the second bar representing JNCL and PPARα null model, the third bar representing JNCL and PPARα null model administered at 8 mg / kg / day, and the fourth bar representing JNCL and PPARα null model administered with vehicle. [Figures 10D-10G] Figure 10D shows a graph depicting center point shift, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL and PPARα null model, the third bar representing the JNCL and PPARα null model at 8 mg / kg / day, and the fourth bar representing the JNCL and PPARα null model at vehicle administration. Figure 10E shows a graph depicting rotarod latency in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL and PPARα null model, the third bar representing the JNCL and PPARα null model at 8 mg / kg / day, and the fourth bar representing the JNCL and PPARα null model at vehicle administration. Figure 10F shows a graph of pole T-turns in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL and PPARα null model, the third bar representing the JNCL and PPARα null model at 8 mg / kg / day, and the fourth bar representing the JNCL and PPARα null model at vehicle administration. Figure 10G shows a graph of pole latency in seconds, with the first bar closest to the y-axis representing WT, the second bar representing the JNCL and PPARα null model, the third bar representing the JNCL and PPARα null model at 8 mg / kg / day, and the fourth bar representing the JNCL and PPARα null model at vehicle administration. DETAILED DESCRIPTION OF THE INVENTION

[0017] Throughout this disclosure, various quantities, such as amounts, sizes, dimensions, ratios, etc., are presented in range format. It should be understood that the description of quantities in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges within that range as well as all individual numerical values ​​within that range, unless the context clearly dictates otherwise. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values ​​within that range, e.g., 1.1, 2, 2.3, 4.6, 2, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, which are also encompassed within the disclosure, except for any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly indicates otherwise.

[0018] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiments. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It is further understood that, as used in this specification, the terms "includes," "comprises," "including," and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The term "and / or," as used herein, includes any and all combinations of one or more of the associated listed items. Additionally, items in a list of the form "at least one of A, B, and C" can be understood to mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, an item listed in the format "at least one of A, B, or C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).

[0019] Unless otherwise specified or apparent from the context, as used herein, the term "about" in reference to a numerical value or range of numerical values ​​is understood to mean the stated number plus or minus 10% of that number, or, for values ​​recited for a range, 10% below the lower recited value and 10% above the upper recited value.

[0020] The present disclosure provides compositions and methods for treating juvenile neuronal ceroid lipofuscinosis (JNCL) in a patient in need thereof. In other embodiments, methods are provided for treating or correcting dysfunction of the lysosomal membrane glycoprotein Cln3. In any embodiment, the methods comprise administering to a patient in need thereof an effective amount of an oral pharmaceutical composition comprising gemfibrozil. The disclosed methods contemplate that the pharmaceutical composition comprising gemfibrozil may be administered to the patient any number of times daily, including once daily, twice daily, and three times daily.

[0021] As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid excipient, diluent, encapsulating material or formulation auxiliary of any kind.

[0022] As used herein, the term "treating" means alleviating or alleviating symptoms associated with a disease.

[0023] Some examples of materials which may serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, sucrose, and the like; starches such as corn starch, potato starch, and the like; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate; powdered tragacanth; malt; gelatin; talc; additives such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate, ethyl laurate, and the like; agar; buffers such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, phosphate buffer, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavorings and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the discretion of the formulator. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences" (Mack Pub. Co., New Jersey, 1991), the contents of which are expressly incorporated herein by reference.

[0024] Methods of formulation are well known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995)). Pharmaceutical compositions used in accordance with the present disclosure may be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, lyophilized powders, or other forms known in the art.

[0025] Solid dosage forms for oral administration include, by way of illustrative, non-limiting example, capsules, tablets, pills, powders, films, and granules. In solid dosage forms, the active compound may be mixed with at least one inert pharmaceutically acceptable excipient or carrier. Illustrative, non-limiting examples of excipients or carriers include sodium citrate or dicalcium phosphate, and / or a) one or more excipients or fillers (excipients or fillers may be one or more selected from, but not limited to, starch, lactose, sucrose, glucose, mannitol, and silicic acid), b) one or more binders (binders may be selected from, but not limited to, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia), c) one or more humectants (humectants may be, but are not limited to, glycerol), d) one or more disintegrants (disintegrants may be, but are not limited to, agar, calcium carbonate, potato, or tapioca starch, alginic acid, silicates, and sodium carbonate), e) one or more solution retarders (for example, but not limited to, paraffin), f) one or more absorption enhancers (for example, but not limited to, quaternary ammonium compounds), g) one or more wetting agents (for example, but not limited to, acetyl alcohol and glycerol monostearate), h) one or more absorbents (for example, but not limited to, kaolin and bentonite clay), and i) one or more lubricants (for example, but not limited to, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate). For example, in the case of capsules, tablets, and pills, dosage forms may also include buffering agents.

[0026] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0027] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells. Illustrative, non-limiting examples of coatings and shells include enteric coatings and other coatings / shells well known in the pharmaceutical formulation art. These may optionally contain opacifying agents and may be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include, but are not limited to, polymeric substances and waxes.

[0028] The active compound may be in microencapsulated form with one or more additives, as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells. The coatings or shells can be, but are not limited to, enteric coatings, release-controlling coatings, and other coatings commonly used in the pharmaceutical formulation arts. In solid dosage forms, the active compound may be mixed with at least one inert diluent. Inert diluents can include, but are not limited to, one or more of sucrose, lactose, or starch. Dosage forms can also contain additional substances other than inert diluents. Additional substances can include, but are not limited to, tableting lubricants and other tableting aids. Tableting lubricants and other tableting aids can include, but are not limited to, magnesium stearate and microcrystalline cellulose. For example, in the case of capsules, tablets, and pills, dosage forms can also include buffering agents. They can also contain opacifying agents. They can be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract. Release may be in a delayed manner.Examples of embedding compositions that can be used include, but are not limited to, polymeric substances and waxes.

[0029] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain one or more inert diluents. The inert diluents may be selected from those commonly used in the art. Illustrative, non-limiting examples of inert diluents include water or other solvents, solubilizers, emulsifiers (including, but not limited to, ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, and mixtures thereof). Oral compositions may contain one or more adjuvants. Illustrative, non-limiting examples of adjuvants include wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0030] The liquid dosage form may be in the form of a pharmaceutical suspension, which is understood to be a liquid dosage form containing finely divided insoluble material (suspended matter) dispersed more or less uniformly throughout a suspending medium (suspension vehicle) in which the drug exhibits minimal solubility.

[0031] In yet other embodiments, it is contemplated that the pharmaceutical composition may be part of a thin film dosage form. See Karki et al. (2016), "Thin films as an emerging platform for drug delivery," Asian J Pharmaceutical Sci. 11:559-574. Generally, a thin film is also referred to as a thin, flexible polymer layer, with or without a plasticizer. Thin films provide a means of targeting sensitive sites that may not be possible with tablet or liquid formulations. Thin films have shown the ability to improve drug onset, reduce dosing frequency, and increase drug efficacy.

[0032] The amount of active ingredient (wherein the active ingredient is oral gemfibrozil, which can be combined with optional carrier materials to create a single dosage form) can vary depending on the host treated and the particular method of administration. The specific dosage level for any particular patient can depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disorder or disease being treated. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of an ordinary clinician.

[0033] According to certain methods of treatment disclosed in the present application, the progression of various disorders is slowed or halted in a patient (the patient may be a human, a lower mammal, or a warm-blooded animal) by administering to the patient effective amounts of: i) oral gemfibrozil and / or gemfibrozil and vitamin A; or ii) cinnamic acid, in amounts and for periods of time necessary to achieve the desired result. An amount of a compound effective to slow or halt the progression of a disease or disorder can mean the amount of compound sufficient to treat the disease or disorder, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0034] The total daily dosage of the compounds and compositions of the present disclosure can be determined by the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particular patient can depend on various factors, including the disease or disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, diet; the time of administration, the route of administration, the excretion rate of the specific compound used; the duration of treatment; and drugs used in combination or simultaneously with the specific compound used.

[0035] An "effective amount" or dose of a compound of the present disclosure, such as i) gemfibrozil, administered to a warm-blooded animal (e.g., a human) can vary depending on the disease being treated, the method or form of drug administration, and the desire to minimize known side effects. For certain neurodegenerative diseases, such as juvenile Batten disease, an effective amount can be from about 0.01 mg / kg / day to about 5.0 g / kg / day, or any amount or subrange therein. In a preferred embodiment, the dosage ranges from about 0.01 g / kg / day to about 200 mg / kg / day.

[0036] Administration can be once daily, twice daily, or three or more times daily. Additionally, in some embodiments, patients can receive the active ingredient by multiple administration methods, including a combination of oral and buccal or sublingual administration. The present disclosure encompasses any combination of administration techniques described or contemplated herein.

[0037] In some embodiments, compositions comprising gemfibrozil are provided to children in the age range of about 6 months to about 10 years to reduce or prevent the formation of symptoms prior to the onset of JNCL. In some embodiments, the compositions are administered to children at least about 6 months, at least about 1 year, at least about 1.5 years, at least about 2 years, at least about 2.5 years, at least about 3 years, at least about 3.5 years, at least about 4 years, at least about 4.5 years, at least about 5 years, at least about 5.5 years, at least about 6 years, at least about 6.5 years, at least about 7 years, at least about 7.5 years, at least about 8 years, at least about 8.5 years, at least about 9 years, at least about 9.5 years, or at least about 10 years of age.

[0038] In some embodiments, the composition comprising gemfibrozil is administered continuously for the life of the human patient.

[0039] In some embodiments, the method of treatment further comprises administering a composition comprising gemfibrozil in combination with a second therapy, which can be a gene therapy, an oral medication, a dietary therapy, or the like.

[0040] In addition to the aspects and embodiments described and provided elsewhere in this disclosure, the following non-limiting list of embodiments is also contemplated.

[0041] 1. A method for treating a patient in need of treatment diagnosed with or suspected of having juvenile neuronal ceroid lipofuscinosis (JNCL), comprising administering to the patient a composition comprising a therapeutically effective amount of gemfibrozil or a pharmaceutically acceptable salt thereof, wherein administration alleviates symptoms associated with JNCL, thereby treating the patient. 2. The method of paragraph 1, wherein the patient is at least about 6 months of age. 3. The method of paragraph 1 or 2, wherein the patient is at least about 3 years of age. 4. The method of any one of paragraphs 1 to 3, wherein the patient is between about 6 months and about 10 years of age. 5. The method of any one of paragraphs 1 to 4, wherein the composition further comprises at least one pharmaceutically acceptable excipient or carrier. 6. The method of any one of paragraphs 1 to 5, wherein the composition is formulated for oral administration to the patient. 7. The method of any one of paragraphs 1 to 6, wherein the patient does not exhibit symptoms of JNCL prior to administration of the composition. 8. The method of any one of paragraphs 1 to 7, wherein the composition is administered at least once daily. 9. The method of any one of paragraphs 1 to 8, wherein the composition is administered at least twice daily. 10. The method according to any one of items 1 to 9, further comprising the concomitant administration of a second treatment. 11. The method of claim 10, wherein the second therapy is a gene therapy. 12. A composition comprising gemfibrozil, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating JNCL in a patient. 13. The composition of paragraph 12, formulated for oral administration to a patient. 14. The composition of paragraph 12 or 13, wherein the patient is between about 3 and about 10 years of age. 15. The composition of paragraphs 12 to 14, wherein the patient is at least about 6 months of age. 16. A composition comprising gemfibrozil or a pharmaceutically acceptable salt thereof for use in a method of therapy for treating JNCL in a patient. 17. The composition of paragraph 16, formulated for oral administration to a patient. 18. The composition of paragraph 16 or 17, wherein the patient is between about 3 and about 10 years of age. 19. The composition of any one of paragraphs 16 to 18, wherein the patient is at least about 6 months of age. 20. A method for treating or repairing defective lysosomal transmembrane glycoprotein Cln3 in a patient in need of treatment or repair, comprising administering to the patient in need of treatment or repair an effective amount of a pharmaceutical composition comprising gemfibrozil and at least one pharmaceutically acceptable excipient or carrier.

[0042] Materials and Methods Reagents: Various molecular biology-grade reagents were obtained from Sigma-Aldrich. Gemfibrozil and methylcellulose were purchased from Spectrum Chemicals. Primary antibodies, their suppliers, and concentrations used are listed in Table 1. Alexa Fluor antibodies used for immunostaining were purchased from Jackson ImmunoResearch Laboratories. IR dye-labeled reagents used for immunoblotting were received from Li-Cor Biosciences.

[0043] Animals and gemfibrozil treatment: Homozygous CLN3 (Cln3 Δex7 / 8 (Jackson Laboratory) mice were used as a JNCL model. These mice will be referred to as Cln3ΔJNCL in the remainder of this document. Cln3ΔJNCL mice were screened by genotyping, performed by PCR on DNA taken from tail biopsy samples using the following primers: Common: 5'-CACTTGGGAGATTGTGAATTTG-3' SEQ ID NO: 1 Mutant Reverse: 5'-GGTGCTCCCAGCCTCTAGGT-3' SEQ ID NO: 2 Wild-type reverse: 5'-GAGATAGGGTTTTGCTGTGC-3' SEQ ID NO: 3

[0044] Wild-type (WT) mice of the same background were used as controls. Cln3ΔJNCL mice were crossed with PPARα null mice to generate Cln3ΔJNCLΔPPARα bigenic mice. Animal care and experiments were performed in accordance with National Institutes of Health guidelines and approved by the Rush University Medical Center Animal Care and Use Committee. Three-month-old Cln3ΔJNCL and Cln3ΔJNCL mice were used. ΔPPARα Mice (male and female) were treated with different doses of gemfibrozil (4 and 8 mg / kg body weight / day) in 100 μl of 0.1% methylcellulose (MC) by gavage for 3 months, followed by monitoring of locomotor activity and biochemical parameters. A separate group of Cln3ΔJNCL mice also received MC alone as a vehicle.

[0045] TIFF2026504932000002.tif214170

[0046] WB, Western blot; IF, immunofluorescence; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule 1; ChIP, chromatin immunoprecipitation; IL-1β, interleukin-1β; TNFα, tumor necrosis factor α; PPARα, peroxisome proliferator-activated receptor α; TFEB, transcription factor EB.

[0047] [Immunohistochemistry (IHC)] For immunohistochemistry, mice were anesthetized and intracardially perfused with 1X PBS followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Brains were postfixed in PFA overnight at 4°C and then transferred to phosphate buffer containing 30% sucrose at 4°C. Somatosensory barrel cortex sections were prepared and stored continuously at -20°C until immunostaining. For this purpose, hemibrains incubated in 30% sucrose were thoroughly washed with PBS and cryosectioned using a sliding microtome (American Opticals 860). Prior to staining, 40 μM free-floating somatosensory barrel cortex sections were thoroughly washed with PBS. Sections were blocked for 1 hour using 2% BSA in PBSTT (PBS + Triton X-100 + Tween-20). Next, sections were incubated with primary antibodies in 1% PBSTT overnight at 4°C. The next day, sections were washed with PBSTT and incubated with 488- or 647-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 3 hours at room temperature. After washing with PBSTT, sections were mounted on glass slides (Patel et al., 2019; Raha et al., 2020). Samples were visualized under an Olympus BX41 fluorescence microscope equipped with a Hamamatsu ORCA-03G camera. For DAB staining, somatosensory-motor cortex sections were stained for GFAP and Iba1 using the Vectastain DAB protocol, mounted, and viewed under an Olympus brightfield microscope. Optical density measurements were obtained using ImageJ software (Raha et al., 2020; Paidi et al., 2021b) as previously described (Varghese et al., 2014). Counting analysis was performed using the Touch Count module ( Corbett et al., 2015 ) with Olympus MicrosuiteV software for imaging applications (Waltham, MA, USA).

[0048] [Immunoblotting] Western blotting was performed as previously described (Jana et al., 2012; Rangasamy et al., 2018; Chandra and Pahan, 2019). After 12 weeks of treatment, mice were perfused with PBS, and somatosensory motor neuron regions were harvested from half of the mouse brain and homogenized in RIPA buffer. The supernatants were collected and analyzed for protein concentration using the Bradford method (Bio-Rad). Protein samples were added to SDS sample buffer and boiled for 5 minutes. Denatured samples were electrophoresed on 10% or 12% Bis-Tris SDS polyacrylamide gels in a sequential buffer system and transferred to nitrocellulose membranes (Bio-Rad) using a Thermo-Pierce Fast Semi-Dry Blotter. The membranes were then washed with TBS + Tween 20 (TBST) for 15 minutes and blocked with BSA in TBST for 1 hour. The membranes were then incubated with primary antibodies at 4°C under shaking conditions, followed by washing with TBST for 1 hour. The membranes were then incubated with secondary antibodies for 1 hour at room temperature, washed for another 1 hour, and visualized under an Odyssey® infrared imaging system (Li-COR, Lincoln, NE, USA). Blots were converted to binary and analyzed using ImageJ (NIH), with β-actin normalized as a loading control.

[0049] [In situ chromatin immunoprecipitation (ChIP) assay] In situ ChIP was performed as described. Briefly, animals were perfused with PBS followed by 4% paraformaldehyde in PBS, and the somatosensory barrel cortex (SBF) was isolated for DNA isolation using the phenol-chloroform-isopropyl alcohol method. Cell lysates were incubated overnight at 4°C with 2 μg of anti-PPARα, anti-CBP, or anti-RNA polymerase II antibodies, followed by a 2-hour incubation with Protein G agarose (Santa Cruz Biotechnology). The beads were then washed with cold IP buffer, and a total of 100 μl of 10% Chelex (10 g / 100 ml H2O) was added to the washed Protein G beads and vortexed. The Chelex / Protein G bead suspension was boiled for 10 minutes and then allowed to cool to room temperature. Proteinase K (100 μg / ml) was then added, and the beads were incubated at 55°C for 30 minutes with shaking, followed by an additional 10-minute boiling period. The suspension was centrifuged, and the supernatant was collected. Semi-quantitative and real-time PCR were performed using this eluate. A PPRE-containing fragment of the mouse Tfeb promoter was amplified using the following primers: sense: 5'-GAA CAT TCC AGG TGG AGG CA-3' (SEQ ID NO: 5), antisense: 5'-CCC CCA ACA CAT GCT TCT CT-3' (SEQ ID NO: 6). For real-time PCR, data were normalized by input values, and fold changes relative to untreated controls were calculated.

[0050] [Behavior analysis] Open field test: This test was performed as previously described (Patel et al., 2018; Raha et al., 2020; Paidi et al., 2021b). Briefly, each mouse was allowed to freely explore the open field arena for 5 min. The test apparatus was a typical open field (i.e., a 40 cm x 40 cm square wooden floor arena with 30 cm wall height). A video camera (Basler Gen I Cam - Basler acA 1300-60) connected to a Noldus computer system was installed on top of the box. Each mouse was placed individually in the center of the arena, and locomotor activity and other parameters, such as speed, total distance traveled, and center time frequency, were monitored for 5 min using a live video tracking system (Noldus System). The center area was arbitrarily defined as a 20 x 20 cm square (half of the total area).

[0051] Rotarod test: The rotarod uses a motor-driven rotating rod to measure limb coordination and balance in mice. This measurement was performed using a rotarod apparatus (ENV-576M; Med-associates) using a previously described protocol (Chandra and Pahan, 2019; Patel et al., 2019; Raha et al., 2020). Briefly, mice were transported (in their home cages) and allowed to acclimate to the testing room for 1 h before the start of the test. Prior to acquisition, parameters of the rotarod system, which is equipped with an automatic fall detector, such as starting velocity and acceleration, were carefully checked. Each mouse was placed in the enclosed section of the rod, and the test began with a smoothly increasing speed from 4 rpm to 40 rpm over a 5-minute period. If the mouse did not fall off the rod, it was removed from the rod after 5 minutes.

[0052] Pole test: To examine motor coordination and spatial awareness, the vertical test was performed as previously described (Chandra et al., 2016; Raha et al., 2020). A vertical wooden pole (50 cm high, 1 cm diameter) with a rough surface was placed in the home cage. Mice were allowed to acclimate to the pole for three trials of 120 s each. Each trial was separated by a 60 s interval, and each mouse was tested three times during the behavioral test.

[0053] Storage Material Detection: Detection of mitochondrial ATP synthase subunit C (SCAMS) was performed by monitoring by immunofluorescence as previously described (Ghosh et al., 2017). See Table 1 for antibody dilution details. DAPI was used to monitor nuclei. SCAMS-associated fluorescence intensity was quantified using Olympus Microsuite V software. Briefly, captured images were opened in Infinity Image Viewer, and a contour line was drawn around the granules to obtain fluorescence intensity.

[0054] Densitometric analysis: Protein blots were analyzed using ImageJ (NIH, Bethesda, MD), and bands were normalized to their respective β-actin loading controls. Data represent the average fold change relative to controls in three independent experiments.

[0055] Statistical analysis: Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA). Behavioral parameters of mice were examined by independent one-way analysis of variance (ANOVA) using SPSS. Homogeneity of variance between test groups was examined using Levene's test. Post-hoc analysis was performed using Tukey's test. Other data were expressed as the mean ± SD of three independent experiments. Statistical differences between means were calculated by Student's t-test (two-tailed). A p-value of less than 0.05 (p<0.05) was considered statistically significant.

[0056] The following examples are intended to illustrate some embodiments of the present disclosure and are not intended to limit the scope of the disclosure or claims in any way. [Example]

[0057] Example 1: Oral administration of gemfibrozil attenuates glial cell activation in the somatosensory barrel field (SBF) cortex of Cln3ΔJNCL mice Because gemfibrozil inhibits glial activation and associated inflammation, we investigated whether gemfibrozil treatment could suppress astroglial activation in a JNCL animal model. To mimic the human mutation, we generated multiple JNCL mouse models affecting the orthologous mouse Cln3 gene. Among these, the Cln3Δex7 / 8 knockin mouse, which carries a 1.02 kb genomic deletion in Cln3, deletes exons 7 and 8 surrounding the intronic DNA, corresponding to the deletion most commonly found in JNCL patients. Therefore, we selected the Cln3Δex7 / 8 knockin (Cln3ΔJNCL) mouse, an accepted JNCL model, to study the efficacy of gemfibrozil. Figures 1A–1D show the effect of gemfibrozil on glial activation in the somatosensory barrel field (SBF) cortex of Cln3ΔJNCL mice. Briefly, the graphs show the results of treating 3-month-old Cln3ΔJNCL mice (n = 6 per group) with different doses of gemfibrozil (4 or 8 mg / kg body weight) by daily gavage for 3 months, followed by monitoring the protein levels of various pro-inflammatory molecules by Western blot (not shown). Western blot bands were scanned, and the values ​​presented (cytokine / actin) compared to WT controls are plotted in the bar graphs shown in Figure 1A (GFAP) and Figure 1B (Iba1). Cell counts were measured using DAB staining of SBF cortical sections for GFAP (not shown) and Iba1 (not shown). Cell count results from specific brain sections are plotted in bar graphs; Figure 1C shows the counts of GFAP-positive cells, and Figure 1D shows the counts of Iba-1-positive cells. Cell counts were measured in one section (two images per section) for each of six different mice (n = 6) per group. ** p<0.01; *** p<0.001.

[0058] As expected, increased protein levels of the astroglial marker GFAP were observed in the somatosensory barrel cortex (SBF) cortex of 6-month-old Cln3ΔJNCL mice compared with age-matched WT mice (Figure 1A). However, as shown in Figure 1A, oral administration of different doses of gemfibrozil (4 and 8 mg / kg body weight / day) significantly reduced GFAP protein levels. Immunohistochemical analysis of the SBF cortex of Cln3ΔJNCL mice also demonstrated morphological features of reactive astrogliosis with strong GFAP immunoreactivity (not shown). The number of activated astrocytes / mm was significantly higher in Cln3ΔJNCL mice compared with WT mice. 2 However, gemfibrozil treatment significantly inhibited astrogliosis in the SBF cortex of Cln3ΔJNCL mice (Figure 1C).

[0059] Similarly, gemfibrozil treatment had a significant effect on microgliosis in Cln3ΔJNCL mice. Reactive microglia are thought to be an important contributor to several neurodegenerative diseases, including JNCL, and several studies have provided evidence that early microglial activation can predict areas where neuronal loss occurs later in the disease process in JNCL mouse models. Western blot analysis showed that Iba1 protein levels were significantly increased in the SBF cortex of Cln3ΔJNCL mice compared with WT mice, and gemfibrozil treatment normalized Iba1 levels in Cln3ΔJNCL mice (Figure 1B). To confirm this finding, we also performed immunohistochemistry using an antibody against Iba1 and found that microglia that appeared normal in WT mice were reactive in Cln3ΔJNCL mice (Figure 1D). However, oral gemfibrozil treatment inhibited microglial activation and reduced the number of activated microglia in the SBF cortex of Cln3ΔJNCL mice ( Figure 1 D).

[0060] Oral gemfibrozil suppresses proinflammatory molecules in the SBF cortex of Cln3ΔJNCL mice. Several reports have shown increased expression of proinflammatory genes in the SBF cortex of Cln3ΔJNCL mice compared with WT mice. Figures 2A–2D show the effect of gemfibrozil on the levels of various proinflammatory molecules in the SBF cortex of Cln3ΔJNCL mice. Three-month-old Cln3ΔJNCL mice (n = 6 per group) were treated daily by gavage with different doses of gemfibrozil (4 and 8 mg / kg body weight) for 3 months, and then the protein levels of various proinflammatory molecules were monitored using Western blotting (not shown). Western blot bands were scanned, and values ​​(cytokines / actin) compared with those of WT controls are presented (Figure 2A, iNOS; Figure 2B, Pro-IL-1β; Figure 2C, IL-1β; Figure 2D, TNFα). Immunostaining of SBF cortical sections for GFAP (not shown) and Iba1 (not shown). Results represent analysis of one section from each of six different mice in each group. Results are the mean ± SEM of six mice in each group. * p<0.05; ** p<0.01; ***p<0.001. Because activated glial cells produce different proinflammatory molecules and gemfibrozil treatment inhibited glial cell activation, we investigated the status of different proinflammatory molecules in gemfibrozil-treated and untreated Cln3ΔJNCL mice. Western blot analysis of SBF cortical tissue showed that the protein levels of iNOS, pro-IL-1β, IL-1β, and TNFα were significantly elevated in Cln3ΔJNCL mice compared with wild-type mice (Figures 2A-2D). However, consistent with the inhibition of gliosis, gemfibrozil treatment reduced the levels of iNOS, pro-IL-1β, IL-1β, and TNFα in the SBF cortex of Cln3ΔJNCL mice (Figures 2A-2D). This effect was more pronounced with a high dose (8 mg / kg body weight / day) of gemfibrozil (Figures 2A-2D). Furthermore, the expression of the nitrosative stress marker inducible nitric oxide synthase (iNOS) was upregulated in GFAP-positive astrocytes and Iba1-positive microglia in the SBF cortex of Cln3ΔJNCL mice, which was strongly inhibited by gemfibrozil treatment (not shown).

[0061] Example 2: Oral gemfibrozil upregulates TFEB and reduces storage load in the SBF cortex of Cln3ΔJNCL mice Accumulation of autofluorescent storage material and glial activation are early neuropathological features of CLN3 Batten disease and are also observed in Cln3ΔJNCL mice. Several studies have demonstrated that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, plays a critical role in cell clearance in neurodegenerative storage diseases. Figures 3A–3D show the effect of gemfibrozil on the levels of TFEB and storage material in the SBF cortex of Cln3ΔJNCL mice. Three-month-old Cln3ΔJNCL mice (n = 6 per group) were treated daily by gavage with different doses of gemfibrozil (4 and 8 mg / kg body weight) for 3 months, followed by double-labeling of SBF cortical sections with antibodies against TFEB and NeuN (not shown). The mean fluorescence intensity (MFI) of TFEB (Figure 3A) and NeuN (Figure 3B) was quantified using NIH Image J software in two different sections (two images per section) from each of six different mice (n = 6) per group. SBF cortical homogenates were immunoblotted for TFEB. Actin was run as a loading control (not shown). Figure 3C shows the immunoblot bands scanned and values ​​(TFEB / actin) compared to the WT control. Results are the mean ± SEM for six mice per group. Immunofluorescence analysis of mitochondrial ATP synthase subunit c (SCMAS) revealed storage dye in SBF cortical sections. DAPI was used for nuclear visualization (not shown). The MFI of SCMAS was quantified using NIH Image J software in one section (two images per section) from each of six different mice (n = 6) per group (Figure 3D). ***p<0.001. Double-label immunofluorescence analysis of SBF cortical sections for TFEB and NeuN revealed that both TFEB (Figure 3A) and NeuN (Figure 3B) were significantly reduced in 6-month-old Cln3ΔJNCL mice compared with age-matched WT mice. Meanwhile, gemfibrozil treatment increased and / or normalized the levels of both TFEB (Figure 3A) and NeuN (Figure 3B) in the SBF cortex of Cln3ΔJNCL mice. Western blot analysis of TFEB in SBF cortical tissue also substantiates this finding (Figure 3C).

[0062] TFEB is a transcription factor whose target genes are ultimately involved in lysosomal biogenesis and autophagy. Therefore, to confirm TFEB activation, we monitored the status of tripeptidyl peptidase 1 (TPP1) and p62, molecules regulated by TFEB, in the SBF cortex of Cln3ΔJNCL mice. Figures 4A–4D show the effect of gemfibrozil on the autophagy pathway in the SBF cortex of Cln3ΔJNCL mice. Three-month-old Cln3ΔJNCL mice (n = 6 per group) were treated with gemfibrozil (8 mg / kg body weight) daily by gavage for 3 months, after which tripeptidyl peptidase 1 (TPP1) and p62 levels were monitored by Western blotting (not shown). Actin was run as a loading control. Western blot bands were scanned, and values ​​(TPP1 / actin, Figure 4A; p62 / actin, Figure 4B) are presented relative to WT controls. Results are the mean ± SEM of four mice per group. SBF cortical sections were double-labeled with antibodies against NeuN and TPP1 (not shown) and NeuN and p62 (not shown). The MFI of TPP1 (Figure 4C) and p62 (Figure 4D) was quantified in two different sections (two images per section) from each of six different mice (n = 6) per group using NIH Image J software. ***p<0.001; NS, not significant. As revealed by Western blot analysis, gemfibrozil treatment significantly increased the levels of both TPP1 (Figure 4A) and p62 (Figure 4B) in the SBF cortex of Cln3ΔJNCL mice. To further confirm this finding, we performed double-label immunofluorescence analysis, which also demonstrated a significant increase in both TPP1 (Figure 4C) and p62 (Figure 4D) in the SBF cortex of Cln3ΔJNCL mice upon gemfibrozil treatment. These results suggest that gemfibrozil treatment can stimulate the active form of TFEB and that gemfibrozil increases autophagy in the CNS of Cln3ΔJNCL mice.

[0063] Next, we investigated whether gemfibrozil treatment could reduce the levels of autofluorescent storage materials, including SCMAS, in the SBF cortex of Cln3ΔJNCL mice. As expected, we found that SCMAS accumulated significantly more in the SBF cortex of Cln3ΔJNCL mice than in WT mice (Figure 3D). However, consistent with the upregulation of TFEB, gemfibrozil treatment resulted in a significant reduction of SCMAS in the SBF cortex of Cln3ΔJNCL mice (Figure 3D).

[0064] Example 3: Oral gemfibrozil improves locomotor activity in Cln3ΔJNCL mice: Alleviating functional impairment is clearly a therapeutic goal for neuroprotection in JNCL patients. Similar to what is observed in JNCL patients, Cln3ΔJNCL mice also exhibit motor deficits. Therefore, we examined locomotor activity in gemfibrozil-treated and untreated 6-month-old Cln3ΔJNCL mice. Referring to the figures, Figures 5A–5F show the effect of gemfibrozil on locomotor activity in Cln3ΔJNCL mice. Three-month-old Cln3ΔJNCL mice (n = 6 per group) were treated daily by gavage with different doses of gemfibrozil (4 and 8 mg / kg body weight). After 3 months of treatment, open-field behavior was monitored using Noldus tracking software and visualized as heat maps (not shown); Figure 5A, speed; Figure 5B, cumulative distance; Figure 5C, distance traveled; Figure 5D, center point shift; Figure 5E, pole latency; Figure 5F, rotarod latency. Results are means ± SEM of 6 mice per group. ** p<0.01; *** p<0.001. As expected, Cln3ΔJNCL mice showed reduced horizontal activity (not shown), speed (Figure 5A), cumulative duration (Figure 5B), total distance traveled (Figure 5C), and center shift (Figure 5D) compared with age-matched WT mice. In the pole test, WT mice quickly descended the pole and touched the bottom of the pole without hesitation, whereas Cln3ΔJNCL mice exhibited abnormal behaviors, including looking up, falling off the pole, descending slowly, freezing on the pole, and hesitation to touch the bottom of the pole (Figure 5E). Similarly, as evidenced by rotarod latency (Figure 5F), Cln3ΔJNCL mice performed very poorly on the rotarod, confirming the impaired motor coordination and muscle experience of these animals. However, oral administration of gemfibrozil significantly improved open-field, pole, and rotarod activity in Cln3ΔJNCL mice (Figures 5A-5F), indicating that gemfibrozil improved the locomotor capacity of Cln3ΔJNCL mice. Therefore, we speculate that the reduction in Batten lesions in gemfibrozil-treated Cln3ΔJNCL mice is positively correlated with the improvement of motor function.

[0065] Example 4: Oral administration of gemfibrozil stimulates recruitment of PPARα to the TFEB gene promoter in the SBF cortex of Cln3ΔJNCL mice Next, we investigated the mechanism by which gemfibrozil upregulates TFEB and suppresses storage material accumulation in Cln3ΔJNCL mice. Because gemfibrozil is known to activate peroxisome proliferator-activated receptor α (PPARα), we investigated the role of PPARα. Figures 6A–6D show that oral administration of gemfibrozil increases PPARα levels and PPARα recruitment to the Tfeb promoter in the SBF cortex of Cln3ΔJNCL mice. Three-month-old Cln3ΔJNCL mice (n = 6 per group) were treated daily with gemfibrozil (8 mg / kg body weight) by gavage for 3 months, after which PPARα protein levels were monitored by Western blotting (not shown). Western blot bands were scanned, and values ​​(PPARα / actin) compared with those of WT controls are presented (Figure 6A). Immunostaining of SBF cortical sections for PPARα and NeuN (not shown). Using NIH Image J software, the mean fluorescence intensity (MFI) of PPARα was quantified in two different sections (two images per section) from each of six different mice (n = 6) per group (Figure 6B). A schematic diagram of the mouse Tfeb gene promoter containing PPRE (SEQ ID NO: 4) (Figure 6C). In situ ChIP of PPARα, CBP, and RNA polymerase was performed on SBF cortex, followed by semi-quantitative PCR (not shown) and quantitative PCR (Figure 6D) analysis. ***p<0.001. Western blot results show that PPARα protein levels in the SBF cortex of Cln3ΔJNCL mice were significantly reduced compared with those of wild-type mice (Figures 6A and 6B). Meanwhile, gemfibrozil treatment significantly elevated and / or restored PPARα levels in the CNS of Cln3ΔJNCL mice (Figures 6A and 6B). Double-label immunofluorescence for NeuN and PPARα in cortical sections from gemfibrozil-treated and untreated Cln3ΔJNCL mice also supports this finding (Figure 6D).

[0066] We demonstrated that PPARα activation upregulates Tfeb transcription and stimulates lysosomal biogenesis in brain cells. Therefore, using in situ ChIP, we investigated whether oral gemfibrozil treatment stimulated PPARα recruitment to the Tfeb promoter in vivo in the SBF cortex of Cln3ΔJNCL mice. Figure 6C shows the presence of a consensus PPRE (SEQ ID NO: 4) in the Tfeb promoter. By immunoprecipitating chromatin fragments with an antibody against PPARα, we were able to amplify a 200-bp fragment encompassing the PPRE in the Tfeb promoter in the SBF cortex of WT mice but not in Cln3ΔJNCL mice (Figure 6D), indicating reduced PPARα recruitment to the Tfeb promoter in the CNS of Cln3ΔJNCL mice. However, gemfibrozil treatment significantly restored / increased PPARα recruitment to the Tfeb gene promoter in the SBF cortex of Cln3ΔJNCL mice (Figure 6D). Similarly, we observed decreased utilization of CREB-binding protein (CBP), a key histone acetyltransferase, and RNA polymerase at the Tfeb promoter in the CNS of ΔCln3 mice, which was restored by gemfibrozil treatment (Figure 6D). These results are specific, as no amplification of products was observed in immunoprecipitates using control IgG. Taken together, these results demonstrate that oral gemfibrozil stimulates PPARα recruitment to the Tfeb promoter in vivo in the CNS of Cln3ΔJNCL mice.

[0067] Example 5: PPARα knockdown exacerbates JNCL pathology and worsens locomotor performance in Cln3ΔJNCL mice Figures 7A-7J show that PPARα deletion exacerbates the disease course in Cln3ΔJNCL mice. Genetic screening of Cln3ΔJNCL mice lacking PPARα (Cln3ΔJNCLΔPPARα) (not shown). Three-month-old Cln3ΔJNCL and Cln3ΔJNCLΔPPARα mice (n = 6 per group) were treated with gemfibrozil (8 mg / kg body weight) daily by gavage for 3 months, after which Iba-1 and GFAP protein levels were monitored by Western blotting (not shown). Bands from the Western blot were scanned, and values ​​(cytokine / actin) compared to WT controls are presented (Figure 7A; Iba1; Figure 7B; GFAP). Immunostaining of SBF cortical sections for SCMAS (not shown). Using NIH Image J software, the MFI of the SCMAS was quantified in two different sections (two images per section) from six different mice (n = 6) per group (Figure 7C). Mice were monitored for the pole test (Figure 7D, pole turn; Figure 7E, pole latency), the rotarod test (Figure 7F), and open-field behavior (Figure 7G, speed; Figure 7H, cumulative duration; Figure 7I, distance traveled; Figure 7J, locomotion). Results are the mean + SEM of six mice per group. * p<0.05; ** p<0.01; *** p<0.001. To further clarify the role of PPARα in JNCL pathology, we crossed Cln3ΔJNCL mice with PPARα- / - mice to generate Cln3ΔJNCLΔPPARα mice (not shown). Interestingly, deletion of PPARα in Cln3ΔJNCL mice increased glial activation in the SBF cortex, as evidenced by upregulation of Iba-1 and GFAP (Figures 7A and 7B). Immunofluorescence analysis also demonstrated increased accumulation of SCMAS in the SBF cortex of Cln3ΔJNCLΔPPARα mice compared with Cln3ΔJNCL mice (Figure 7C).

[0068] Next, we compared locomotor activity between Cln3ΔJNCL and Cln3ΔJNCLΔPPARα mice. As evidenced by the pole test, Cln3ΔJNCLΔPPARα mice took longer to complete T-turns (Figure 7D) and descend a vertical pole (Figure 7E) compared with Cln3ΔJNCL mice. Similarly, Cln3ΔJNCLΔPPARα mice exhibited poorer performance on the rotarod compared with Cln3ΔJNCL mice (Figure 7F). Consistently, Cln3ΔJNCL mice were more efficient in overall locomotor activity, including speed (Figure 7G), cumulative duration (Figure 7H), distance traveled (Figure 7I), and amount of movement (Figure 7J), than Cln3ΔJNCLΔPPARα mice. These results suggest that PPARα may play an important role in JNCL.

[0069] Example 6: Oral gemfibrozil upregulates TFEB via PPARα, reduces storage materials, and attenuates glial activation in the SBF cortex of Cln3ΔJNCL mice To confirm that gemfibrozil indeed requires PPARα to exert its neuroprotective effects in Cln3ΔJNCL mice, we monitored TFEB levels and the accumulation of the storage compound SCMAS in the SBF cortex of gemfibrozil-treated Cln3ΔJNCL and Cln3ΔJNCLΔPPARα mice. Referring to the figures, Figures 8A and 8B show the effects of gemfibrozil on the levels of TFEB and storage compounds in the SBF cortex of Cln3ΔJNCL and Cln3ΔJNCLΔPPARα mice. Three-month-old Cln3ΔJNCL and Cln3ΔJNCLΔPPARα mice (n = 6 per group) were treated with gemfibrozil (8 mg / kg body weight) daily by gavage for 3 months, after which SBF cortical sections were double-labeled for NeuN and TFEB (not shown). The MFI of TFEB was quantified in two different sections (two images per section) from six different mice (n = 6) per group using NIH Image J software (Figure 8A). SBF cortical sections were also immunostained with an antibody against SCMAS (not shown). DAPI was used to visualize nuclei. The MFI of SCMAS was quantified in two different sections (two images per section) from six different mice (n = 6) per group using NIH Image J software (Figure 8B). Results are the mean + SEM of six mice per group. *** p<0.001; NS, not significant. In contrast to the upregulation of TFEB and the reduction of storage reserves in Cln3ΔJNCL mice, gemfibrozil treatment failed to increase TFEB (Figure 8A) and reduce storage reserves (Figure 8B) in Cln3ΔJNCLΔPPARα mice. Neuronal loss was observed in Cln3ΔJNCL mice, and gemfibrozil protected NeuN-positive neurons in Cln3ΔJNCL mice (Figure 3B), but not in Cln3ΔJNCL. ΔPPARαNo such neuroprotection by gemfibrozil was observed in mice (not shown). These results suggest that gemfibrozil mediates PPARα in the upregulation of TFEB, depletion of storage materials, and neuroprotection in the CNS of Cln3ΔJNCL mice.

[0070] Next, we investigated whether gemfibrozil reduces glial activation in the CNS of Cln3ΔJNCL mice through PPARα expression. Figures 9A–9C show the effect of gemfibrozil on glial activation in the SBF cortex of Cln3ΔJNCL and Cln3ΔJNCLΔPPARα mice. Three-month-old Cln3ΔJNCL and Cln3ΔJNCLΔPPARα mice (n = 6 per group) were treated daily by gavage with gemfibrozil (8 mg / kg body weight). After 3 months of treatment, mice were perfused, and SBF cortical sections were double-labeled for Iba1 and iNOS (not shown) and GFAP and iNOS (not shown). DAPI was used to visualize nuclei. Iba1-positive (Figure 9A), GFAP-positive (Figure 9B), and iNOS-positive (Figure 9C) cells were counted in one section (two images per section) from six different mice (n = 6) in each group. Results represent the analysis of one section from six different mice in each group. *** p<0.001. Gemfibrozil at a dose of 8 mg / kg body weight / day significantly reduced microglial (Fig. 9A) and astroglial (Fig. 9B) activation and iNOS levels (Fig. 9C) in the SBF cortex of Cln3ΔJNCL mice. ΔPPARα In mice, gemfibrozil failed to inhibit glial cell activation and reduce iNOS expression, suggesting that oral gemfibrozil also fails to suppress glial inflammation in the absence of PPARα.

[0071] Example 7: Gemfibrozil improves locomotor activity in Cln3ΔJNCL mice via PPARα. As shown in Figures 5A-5F, gemfibrozil treatment restored locomotor activity in Cln3ΔJNCL mice, so we investigated whether such protection was also dependent on PPARα. ΔPPARα Mice were orally treated with gemfibrozil for 3 months, after which open-field activity, pole test, and rotarod performance were monitored. Figures 10A–10G show the effects of gemfibrozil on locomotor activity in Cln3ΔJNCLΔPPARα mice. Three-month-old Cln3ΔJNCLΔPPARα mice (n = 6 per group) were treated daily with gemfibrozil (8 mg / kg body weight) by gavage. After 3 months of treatment, locomotor activity was monitored by open-field behavior (Figure 10A, speed; Figure 10B, cumulative duration; Figure 10C, distance traveled; Figure 10D, center point shift), rotarod performance (Figure 10E), and pole test (Figure 10F, pole T-turn; Figure 10G, pole latency). Results are the mean ± SEM of six mice per group. *** p<0.001; NS: not significant. Consistent with the failure of gemfibrozil to reduce SCMAS accumulation and inhibit glial cell activation, gemfibrozil also inhibited Cln3ΔJNCL ΔPPARα Gemfibrozil failed to improve the overall open-field performance (not shown), speed (Figure 10A), cumulative duration (Figure 10B), distance traveled (Figure 10C), and center point shift (Figure 10D) of the mice. Thus, gemfibrozil significantly improved Cln3ΔJNCL mice's performance on the rotarod (Figure 10E) and pole descent (Figures 10F and 10G). ΔPPARα It also did not improve the performance of the mice, indicating that gemfibrozil enhances locomotor activity in Cln3ΔJNCL mice via PPARα.

[0072] [Consideration] Neuronal ceroid lipofuscinosis (NCL), or Batten disease, is a group of recessively inherited fatal lysosomal storage disorders that occur in infants and rarely in adults (Kim et al., 2017; Johnson et al., 2019). Each type of NCL is caused by a different genetic mutation, which determines the age of onset, symptoms, and rate of disease progression. However, all NCLs result in long-term disability and ultimately death. Apart from Brineura therapy for NCL, no effective treatment is available. Recently, various therapeutic approaches (e.g., enzyme replacement therapy, immunosuppressive therapy, cell therapy, and gene therapy) have been explored to slow or halt the progression of these devastating rare diseases. However, unlike CLN1 and CLN2, the transmembrane protein CLN3 is not secreted, limiting its uptake by nearby cells. Furthermore, because the disease process of NCL spreads to various parts of the brain, recombinant functional proteins or viral vectors may not be able to penetrate the densely packed cell populations and reach the appropriate target cell type during intracranial delivery. While gene therapy trials are underway, the development of neuroprotective therapies to slow disease progression, improve locomotor function, and increase survival rates in JNCL patients is crucial. Cln3Δex7 / 8 (Cln3ΔJNCL) mice are useful for determining novel therapeutic strategies for JNCL and testing the efficacy of new drugs. Here, we demonstrate for the first time that oral administration of gemfibrozil, an FDA-approved drug for human hyperlipidemia, reduces CNS storage accumulation, attenuates glial activation, and improves locomotor function in Cln3ΔJNCL mice. Due to increased proinflammatory molecules in the CNS of ΔCln3 mice, the observed motor deficits may be due to pathological behavior rather than structural changes in the CNS. Although gemfibrozil treatment reduces storage load and improves neuronal health, as monitored by increased NeuN, further studies are needed to confirm that gemfibrozil-induced motor performance improvement in Cln3ΔJNCL mice is not due to suppression of neuroinflammation. Nevertheless, these results suggest that oral gemfibrozil may be beneficial for patients with JNCL.

[0073] The accumulation of lipophilic, ceroid-like, autofluorescent storage material in both neuronal and non-neuronal cells is a major characteristic of NCL, including JNCL. The mechanisms leading to the clearance of storage material are only beginning to emerge. Autophagy is a cellular method for removing unnecessary or dysfunctional components through a lysosome-dependent degradation process to maintain cellular homeostasis. Because abnormal lysosomal function and autophagy are associated with multiple lysosomal storage and neurodegenerative diseases, upregulation of this lysosomal-autophagy pathway has emerged as an attractive therapeutic strategy. TFEB is known as a master regulator of lysosomal biogenesis, and several studies have shown that overexpression of TFEB can alleviate neurodegenerative pathologies through upregulation of the autophagy-lysosomal pathway. Recently, we employed a different approach to upregulate TFEB. We demonstrated that the Tfeb gene promoter harbors a consensus peroxisome proliferator response element (PPRE) and that activation of PPARα, but not PPARβ or PPARγ, leads to transcriptional upregulation of Tfeb. Because gemfibrozil is a known agonist of PPARα, we investigated the role of this molecule and found that gemfibrozil treatment reduced the deposition of autofluorescent storage material in the CNS, demonstrating neuroprotective effects via PPARα. Our conclusions are based on the following: First, PPARα levels were lower in the SBF cortex of 6-month-old Cln3ΔJNCL mice compared with age-matched WT mice, and were increased and / or normalized by oral gemfibrozil. Second, we found reduced PPARα enrollment to the Tfeb promoter in the SBF cortex of Cln3ΔJNCL mice compared with WT mice, suggesting that the JNCL disease process reduces PPARα recruitment to the Tfeb gene promoter. However, gemfibrozil treatment was able to stimulate PPARα recruitment to the Tfeb promoter in vivo in the CNS of Cln3ΔJNCL mice. Third, TFEB levels in the CNS of Cln3ΔJNCL mice, which were increased by gemfibrozil treatment, were also reduced.Fourth, gemfibrozil failed to reduce stores and improve locomotor performance in Cln3ΔJNCL mice lacking PPARα.

[0074] Similar to other neurodegenerative diseases, astrocyte and microglial activation are early pathological events in human JNCL and the Cln3ΔJNCL mouse model of JNCL. It has also been reported that early-onset microgliosis is closely associated with light-induced retinal degeneration in Cln3ΔJNCL mice and progressive loss of photoreceptor cells in the nclf mouse model of CLN6 disease. Here, we also found upregulation of GFAP and Iba-1, along with the expression of IL-1β, TNFα, and iNOS, in the SBF cortex of Cln3ΔJNCL mice. However, consistent with the inhibition of proinflammatory molecules in cultured astrocytes and microglia, oral gemfibrozil treatment suppressed glial activation and inflammation in Cln3ΔJNCL mice. However, gemfibrozil did not inhibit glial inflammation in Cln3ΔJNCL mice lacking PPARα, indicating an essential role for PPARα in this process. Suppressors of cytokine signaling (SOCS) proteins also play an important role in inhibiting cytokine signaling and inflammatory gene expression in various cell types, including glial cells. Similarly, IL-1R antagonist (IL-1Ra) inhibits proinflammatory cell signaling by binding to IL-1R, the receptor for IL-1α and IL-1β. We found that gemfibrozil can upregulate both SOCS3 and IL-1Ra in brain cells. Gemfibrozil treatment also increased the levels of SOCS3 and IL-1Ra in the striatum and cortex of Cln2(- / -) mice, demonstrating neuroprotective effects (Ghosh et al., 2017). Recently, we demonstrated that PPARα is involved in aspirin-induced transcription of both SOCS3 and IL-1Ra in astrocytes, suggesting that gemfibrozil, a prototypic agonist of PPARα, may involve PPARα in the upregulation of SOCS3 and IL-1Ra, thereby exerting anti-inflammatory properties.

[0075] Gemfibrozil has several advantages over other promising neuroprotective agents. For example, it is an oral drug and is fairly non-toxic. After oral administration, gemfibrozil can cross the blood-brain barrier. Gemfibrozil is neuroprotective and has been shown to inhibit the growth of Cln2, an animal model of LINCL. (- / -) It can extend the lifespan of mice. Gemfibrozil has been reported to be safe for lipid lowering in children. Although the primary site of CLN3 disease is the CNS, accumulation of lysosomal storage materials occurs in different parts of the body, and as a result, cardiac abnormalities are also observed in children as the disease progresses. In this context, gemfibrozil may be able to prevent cardiac disease in JNCL patients. Furthermore, gemfibrozil exhibits neurotrophic and promyelinating effects, supporting memory and learning.

[0076] In summary, this study demonstrates that oral administration of gemfibrozil, an FDA-approved human lipid-lowering drug, exhibits neuroprotective effects in an animal model of JNCL. Notably, gemfibrozil reduced storage reserves, attenuated glial inflammation, and improved locomotor activity in the JNCL mouse model via PPARα, suggesting that gemfibrozil may not exert neuroprotective effects in JNCL in the absence of PPARα. Although the in vivo condition of Cln3ΔJNCL mice does not fully resemble the in vivo scenario in JNCL patients, and much remains unknown about the status of PPARα in JNCL, our results suggest that PPARα activation by oral gemfibrozil may have therapeutic significance in JNCL.

[0077] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While this invention may be embodied in many different forms, there are described in detail herein certain preferred embodiments of the invention. The present disclosure exemplifies the principles of the invention and is not intended to limit the invention to the particular embodiments exemplified.

[0078] Furthermore, the present invention encompasses any and all possible combinations of some or all of the various embodiments described herein. It should also be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its intended advantages. Accordingly, it is intended that such changes and modifications be covered by the appended claims.

Claims

1. A method for treating a patient in need of treatment who has been diagnosed with or is suspected of having juvenile neuronal ceroid lipofuscinosis (JNCL), comprising administering to the patient a composition comprising a therapeutically effective amount of gemfibrozil or a pharmaceutically acceptable salt thereof, wherein administration alleviates symptoms associated with JNCL, thereby treating the patient.

2. 10. The method of claim 1, wherein the patient is at least about 6 months old.

3. 10. The method of claim 1, wherein the patient is at least about 3 years old.

4. 10. The method of claim 1, wherein the patient is between about 6 months and about 10 years of age.

5. The method of claim 1 , wherein the composition further comprises at least one pharmaceutically acceptable excipient or carrier.

6. 10. The method of claim 1, wherein the composition is formulated for oral administration to a patient.

7. 10. The method of claim 1, wherein the patient is free of symptoms of JNCL prior to administration of the composition.

8. 10. The method of claim 1, wherein the composition is administered at least once daily.

9. 10. The method of claim 1, wherein the composition is administered at least twice daily.

10. 10. The method of claim 1, further comprising concurrently administering a second therapy.

11. 11. The method of claim 10, wherein the second therapy is a gene therapy.

12. A composition comprising gemfibrozil, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of JNCL in a patient.

13. 13. The composition of claim 12, formulated for oral administration to a patient.

14. 13. The composition of claim 12, wherein the patient is between about 3 and about 10 years of age.

15. 13. The composition of claim 12, wherein the patient is at least about 6 months of age.

16. A composition comprising gemfibrozil, or a pharmaceutically acceptable salt thereof, for use in a therapy for treating JNCL in a patient.

17. 17. The composition of claim 16, formulated for oral administration to a patient.

18. 18. The composition of claim 17, wherein the patient is between about 3 and about 10 years of age.

19. 20. The composition of claim 18, wherein the patient is at least about 6 months old.

20. A method for treating or repairing defective lysosomal transmembrane glycoprotein Cln3 in a patient in need of treatment or repair, comprising administering to the patient in need of treatment or repair an effective amount of a pharmaceutical composition comprising gemfibrozil and at least one pharmaceutically acceptable excipient or carrier.

Images