Methods for treating diffuse midline glioma (DMG) or diffuse intrinsic pontine glioma (DIPG)

Abstract

Disclosed is a method of treating diffuse midline glioma (DMG), such as diffuse intrinsic pontine glioma (DIPG), with a compound that possesses inhibitory activity against histone acetyltransferase p300 (EP300) and CREB-binding protein (CBP).

Description

Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO METHODS FOR TREATING DIFFUSE MIDLINE GLIOMA (DMG) OR DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63 / 643,685, filed May 7, 2024, which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Diffuse midline gliomas (DMG) are primary central nervous system (CNS) tumors and is the second most common type of primary, high grade brain tumor in children. A subtype is known as diffuse intrinsic pontine glioma (DIPG), which is a fast-growing type of brain tumor that starts in the part of the brain stem called the pons. The brain stem is the part of the brain above the back of the neck that is connected to the spinal cord. The brain stem controls many vital functions such as breathing, heart rate, and blood pressure, and the nerves and muscles used in seeing, hearing, walking, talking, and eating. Categorized as a glioma, DMG starts in the brain stem's glial cells that support and protect the brain's nerve cells. In the United States, about 300 children are diagnosed with DMG each year. Rare in adults, DMG primarily affects children between the ages of 5 and 10 years but can occur in younger children and teens. See, National Cancer Institute website.

[0003] The primary genetic drivers in DMG are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3.3 and H3.1) wherein lysine 27 is mutated to methionine (H3K27M) (Schwartzentruber et al., Nature 482:226-231 (2012); Wu et al., Nat Genet 44:251- 253 (2012)). The H3K27M oncohistones do not contain direct enzymatic function, and have been considered undruggable.

[0004] H3K27-altered DMG, e.g., wherein the H3K27M mutation is present, accounts for 10 percent of all childhood central nervous system tumors. See, Dana Farber Cancer Institute website. Accordingly, therapies for DMG such as DIPG are urgently needed. SUMMARY

[0005] The present disclosure includes methods for treating diffuse midline glioma (DMG), such as H3K27-altered DMG, especially diffuse intrinsic pontine glioma (DIPG). 1 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO

[0006] A first aspect of the present disclosure is directed to a method of treating DMG wherein cells of the DMG express EP300 and optionally CBP (and / or the DMG has EP300 and optionally CBP activity), comprising administering a compound selected from: a

[0007] The following compounds are known amongst other lysine acetyltransferases (KAT) inhibitors and degraders: A-485 (Lasko et al., (2017) Nature; 550; 128-132), iP300w (Bosnakovski et al., (2019) Sci. Adv.; 5; eaaw7781), CPI-1612 (Wilson et al., (2020) A.C.S. Med. Chem. Lett.; 11; 1324-1329), B026 (Yang et al., (2020) J. Med. Chem.; 63; 1337-1360) and dCBP-1 (Vannam et al., (2021) Cell Chem. Biol.; 25; 503-514.e12). Vannam et al., (2021) describes other active compounds, such as GNE-781.

[0008] As illustrated in various figures, disclosed compounds are more active against DMG cell lines than other inhibitors or degraders of EP300 (also known as E1A Binding Protein P300 and histone acetyltransferase p300) or CBP (also known as CREB-binding protein or CEBBP), 2 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO such as GNE-049. Confocal microscopy confirmed the activity of the compounds correlated with decrease in the acetylation of lysine 27 on histone H3 (H3K27Ac), a reduction of levels of glioblastoma stem cell marker OLIG2, and resulted in cell cycle arrest and cell death. In contrast, the effect was caspase-independent with no effect observed on caspase activity or PARP cleavage. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 depicts the drug profiling of EP300 / CBP bromodomain (BRD) and KAT (lysine acetyltransferase) inhibitors / degraders as area under the curve (AUC) with blue depicting 100% viable cells and red depicting 100% toxicity of the compounds applied.

[0010] FIG. 2A and FIG. 2B are confocal microscopy images showing that the H3K27M glioma differential sensitivity to EP300 / CBP BRD and KAT inhibitors and degraders is directly related with their effect in reducing H3K27ac.

[0011] FIG. 3 is a series of confocal microscopy images showing that EP300 / CBP KAT inhibitors and degraders result in decreased OLIG2 levels and increased GFAP levels suggesting that EP300 / CBP inhibition induces glioma cells to transition from a stem-like OPC state to a more mature AC-like state.

[0012] FIG.4A-FIG.4D are a series of bar graphs showing the treatment of H3K27M glioma cells with EP300 / CBP KAT inhibitors and degraders for 3 days results in cell cycle arrest.

[0013] FIG.5A-FIG.5D are a series of bar graphs showing the treatment of H3K27M glioma cells with EP300 / CBP KAT inhibitors and degraders for 5 days leads to cell death in selected cell lines.

[0014] FIG.6A-FIG. 6E are a series of graphs showing caspase activity assays in H3K27M glioma cells upon treatment with EP300 / CBP KAT inhibitors and degraders reveals no increase in Caspase activity. Similar plots were obtained for BT245, DIPGXXV, DIPGXIIIP*.

[0015] FIG. 7A-FIG. 7D is a series of immunoblots showing the treatment of H3K27M glioma cells with EP300 / CBP KAT inhibitors and degraders for 3 / 5 days does not lead to increased PARP cleavage.

[0016] FIG. 8A and FIG. 8B show that dCBP1 reduces CBP and EP300 levels consistently across cell lines while, LS and QL were more CBP specific. H3K27M cells sensitivity to EP300 / CBP degraders was directly related with their ability to degrade EP300, suggesting that between the EP300 and CBP paralogs, H3K27M glioma cells depend on EP300 or on both EP300 and CBP. 3 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO DETAILED DESCRIPTION

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure.

[0018] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Therefore, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0019] Unless stated otherwise, the term “about” means within 10% (e.g., within 5%, 2%, or 1%) of the particular value modified by the term “about.”

[0020] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group that that minimum number of heteroatoms. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the disclosure.

[0021] Methods of the present disclosure utilize the following compounds: ,4 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO a.

[0022] Compounds of the disclosure may be in the form of a free acid or free base, or a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" in the context of a salt refers to a salt of the compound that does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the compound in salt form may be administered to a subject without causing undesirable biological effects (such as dizziness or gastric upset) or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The term "pharmaceutically acceptable salt" refers to a product obtained by reaction of the compound of the present disclosure with a suitable acid or a base. Examples of pharmaceutically acceptable salts of the compound of this disclosure include those derived from suitable inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Al, Zn and Mn salts. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate or p-toluenesulfonate salts and 5 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO the like. The compound of the disclosure can form pharmaceutically acceptable salts with various organic bases such as lysine, arginine, guanidine, diethanolamine or metformin.

[0023] Administration of the compounds of the disclosure may be facilitated by formulating them with a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising a formulation, and / or the patient being treated therewith. Suitable carriers may include, for example, liquids (both aqueous and non- aqueous alike, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), and gases, that function to carry or transport the compound from one organ, or portion of the body, to another organ, or portion of the body. A carrier is “acceptable” in the sense of being physiologically inert to and compatible with the other ingredients of the formulation and not injurious to the subject or patient. Depending on the type of formulation, the carrier may be a pharmaceutically acceptable excipient, a pharmaceutically acceptable diluent, a pharmaceutically acceptable vehicle, and / or a pharmaceutically acceptable solvent.

[0024] Broadly, the compounds and their pharmaceutically acceptable salts may be formulated into a given type of composition in accordance with conventional pharmaceutical practice such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (20thed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York), each of which is incorporated herein by reference in its entirety. The type of formulation depends on the mode of administration which may include enteral (e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), and intrasternal injection, or infusion techniques, intra-ocular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, interdermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal).

[0025] Representative types of compositions suitable for oral administration include tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral administration use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. They may further contain one or more of sweetening agents, flavoring agents, coloring agents and preserving agents. 6 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO

[0026] Tablets contain the compound in admixture with a non-toxic pharmaceutically acceptable excipient. Representative examples of excipients include inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate. The tablets may be uncoated, or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes.

[0027] Compositions intended for oral administration use may be formulated as hard gelatin capsules wherein the compound is in admixture with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the compound is in admixture with water or an oily medium, such as peanut oil, liquid paraffin, or olive oil.

[0028] Aqueous suspensions may contain the compound in admixture with an excipient. Representative examples of excipients include suspending agents, e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; and dispersing or wetting agents, e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as example polyoxy ethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyoxyethylene sorbitan monooleate.

[0029] Aqueous suspensions may also contain a preservative, such as for example, ethyl or n-propyl p-hydroxybenzoate, and / or a coloring agent such as sucrose or saccharin.

[0030] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, representative examples of which include arachis oil, olive oil, sesame oil, 7 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO coconut oil, and a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent such as, for example beeswax, hard paraffin or cetyl alcohol.

[0031] Sweetening agents, such as those set forth above, and flavoring agents may be added. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compound in admixture with a dispersing or wetting agent, a suspending agent and a preservative. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.

[0032] The pharmaceutical compositions may be formulated as an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents include naturally occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soybean lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, for example sorbitan mono-oleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol and sucrose. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolize to glucose or which only metabolize a very small amount to glucose.

[0033] Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

[0034] Representative types of compositions suitable for parenteral administration (e.g., subcutaneous, intravenous, intramuscular, intrasternal, or by infusion techniques) include sterile injectable aqueous and oleaginous suspensions. The suspensions may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents as described above. The injectable compositions may also be a sterile injectable solution or suspension in a non-toxic paternally acceptable diluent or solvent, for example as a solution in 1,3-butane diol.

[0035] Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. 8 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO

[0036] In other embodiments, the compositions may be formulated for oral, buccal, sublingual administration (examples of which include tablets, lozenges and gels), inhalation, topical, ophthalmic and rectal administration.

[0037] The amount of the compound administered that may be effective in producing the desired therapeutic response. Therefore, the amount of the compound or a pharmaceutically acceptable salt thereof, may for example, achieve remission of DMG, prevent or delay progression of DMG, alleviate one or more of the symptoms of DMG, or kill or inhibit the growth of DMG cells, such as DIPG cells.

[0038] The total daily dosage of the compounds and usage thereof may be decided in accordance with standard medical practice, e.g., by the attending physician using sound medical judgment. The specific dose for any particular subject may depend upon a variety of factors including the disease or disorder being treated and the severity thereof (e.g., its present status); the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the compound; and like factors well known in the medical arts (see, for example, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10thEdition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001), which is incorporated herein by reference in its entirety. The daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case. Typically, however, the dosage adopted for each route of administration when a compound is administered alone to humans is 0.0001 to 50 mg / kg, most commonly in the range of 0.001 to 10 mg / kg, body weight, for instance 0.01 to 1 mg / kg. Such a dosage may be given, for example, from 1 to 5 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 1 mg / kg body weight, preferably from 0.0001 to 0.1 mg / kg body weight. In some embodiments, the dosage range may be 1 to 20 mg, e.g., 2.5 to 10 mg and 5 to 10 mg.

[0039] A daily dosage can be administered as a single dosage or according to a divided dose schedule.

[0040] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone to or suffering from DMG or DIPG. In some embodiments, the subject is a mammal, e.g., a human or a non-human mammal. A subject “in need of” treatment according to the present disclosure may be “suffering from or suspected of suffering from” DMG or DIPG may have been positively diagnosed or otherwise presents with a sufficient number of risk factors or a sufficient number or combination of signs or symptoms such that a medical 9 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO professional could diagnose or suspect that the subject was suffering from DMG or DIPG. Thus, subjects suffering from, and suspected of suffering from, DMG or DIPG are not necessarily two distinct groups. In some embodiments, the subject is a pediatric patient. In some embodiments, the pediatric patient is from 5 to 9 years old. In some embodiments, the subject is a young adult.

[0041] These and other aspects of the present disclosure will be further appreciated upon consideration of the following Example, which is intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims. EXAMPLE

[0042] Example 1: Blood Brain Barrier Penetrance Compound Route Brain / Plasma Max Conc. Brain Dosage (ng / mL)e co pou s a o e e e a s e e o ce us g e ou e and concentrations as depicted.30 min post dosing, mice were sacrificed, and their blood and brains were collected. Concentrations of the EP300 / CBP inhibitors were determined by LC-MS.

[0044] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0045] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims. 10 AFSDOCS:302452409.1

Claims

Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO What is claimed is:

1. A method of treating a diffuse midline glioma (DMG), comprising administering, to a subject in need thereof, a compound selected from: a2. The method of claim 1, wherein the (A-485), or a pharmaceutically acceptable salt11 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO 3. The method of claim 1, wherein the (B026), or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein the (iP300w), or a pharmaceutically acceptable salt5. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt12 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO 6. The method of claim 1, wherein the compound is 1), or a7. A method of treating a diffuse midline glioma (DMG), comprising administering, to a subject in need thereof, a pharmaceutical composition comprising a compound selected from: ,13 AFSDOCS:302452409.1Date of Deposit: May 6, 2025 Attorney Docket No.046094-796001WO a8. The method of any one of claims 1-7, wherein the subject is a human.

9. The method of claim 8, wherein the human is a pediatric patient.

10. The method of claim 9, wherein the pediatric patient is from 5 to 9 years old.

11. The method of any one of claims 1-10, wherein the DMG is diffuse intrinsic pontine glioma (DIPG). 14 AFSDOCS:302452409.1

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