TATR: modeling a target and timed-release strategy for TRKB agonist drug delivery to cochlear bone

Abstract

A target and timed-release platform having formula (1): or a pharmaceutically acceptable salt thereof; wherein: BP is a bisphosphonate group for bone targeting and retention; LK is a cleavable linking group; and Dr is a drug cargo, wherein the drug cargo is released upon cleavage of the cleavable linking group.

Description

[0001] 2024-054-01 TATR: MODELING A TARGET AND TIMED-RELEASE STRATEGY FOR TRKB AGONIST DRUG DELIVERY TO COCHLEAR BONE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. provisional application Serial No. 63 / 544,161 filed October 14, 2023, the disclosure of which is hereby incorporated in its entirety by reference herein. TECHNICAL FIELD In at least one aspect, the present invention is related to platforms for treating hearing loss. BACKGROUND Hearing loss affects more than two-thirds of the elderly population.1Sensorineural hearing loss (SNHL) related to noise exposure or aging is associated with loss of inner ear sensory hair cells (HCs), cochlear spiral ganglion neurons (SGNs), and ribbon synapses between HCs and SGNs. This has stimulated intense interest in therapies to regenerate synaptic function. Neurotrophin- 3 (NT-3) and brain-derived neurotrophic factor (BDNF) represent the primary neurotrophins in the inner ear, where they promote SGN survival and enhance neurite outgrowth and synaptogenesis.3Selective and potent small molecule analogs of neurotrophins agonizing tropomyosin receptor kinase B (TrkB) or C (TrkC) are known to promote the survival of SGNs. For example, 7,8- DHF and 1Aa, small-molecule neurotrophin mimetics that respectively agonize TrkB and TrkC, can stimulate neurite outgrowth and synapse regeneration in vitro4,5However, local delivery of drugs to the inner ear and maintenance of an effective drug concentration in situ remains a challenge due to several factors, including clearance by inner ear fluids. Accordingly, there is a need for improved methods to treat hearing loss. SUMMARY 2024-054-01 In at least one aspect, a target and timed-release platform that is useful for treating hearing loss is provided. The target and timed-release platform is described by formula 1: BP LK Dr(1)or a pharmaceutically acceptable salt thereof, wherein: BP is a bisphosphonate for bone targeting and retention; LK is a cleavable linking group; and Dr is a drug cargo. Advantageously, the drug cargo is released upon cleavage of the cleavable linking group. The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein: FIGURES 1A and 1B. (A) SGM neurite outgrowth in vitro. Neurites were stained with neuronal market TuJ (red), and nuclei were labeled with DAPI (blue). The scale bar represents 100 µm. Images are representative of four independent experiments. (B) Quantification of neurite outgrowth in µm compared to DMSO control and DHF (n.s., not significant; **** represent p ≤ 0.0001).5FIGURE 2A and 2B. Examples of the TATR platform. 2024-054-01 FIGURES 3A and 3B. Fluorescence results for experiments in which 6-FAM-ZOL was imbedded in hydrogel material which was placed on RWM.7(A) 0.1X of systemic BP dose applied to RMW, observe weak fluorescent signal (B) 0.3X of systemic BP dose applied to RWM, a significant increase in signal. FIGURE 4. Schematic of the operation of the TATR platform. FIGURE 5A. Synthetic scheme for preparing the TATR platform. FIGURE 5B. Modulating groups (Mod) for the TATR platform. FIGURE 5C. Scheme for synthesizing BP-DBCO. FIGURE 6A. Flowchart depicting a method for determining TATR in vitro cleavage rates. FIGURE 6B. Plot of the ln of the TATR1 concentration as a function of time. FIGURE 6C. Plot of the ln of the TATR2 concentration as a function of time. FIGURE 7. Plot of the ln of the TATR2 concentration as a function of time for TATR2 pre-adsorbed onto HAP. FIGURE 8A. In Vivo Pharmacokinetic model. FIGURE 8B. Drug concentrations as a function of time. FIGURE 9. Table 1. Half-lives for in-solution cleavage of TATR1, TATR2, and TATR5. * represents data as mean ± SD (n = 3),†represents data at mean ± SD (n = 2), and represents data as single point (n = 1). FIGURE 10. Cleavage over time graph for TATR2 in HEPES (pH 7.4). FIGURE 11. Cleavage over time graph for TATR1 in HEPES (pH 7.4). 2024-054-01 FIGURE 12. Cleavage over time graph for TATR5 in HEPES (pH 7.4). FIGURE 13. Cleavage over time graph for TATR2 in 1X PBS (pH 7.4). FIGURE 14. Cleavage over time graph for TATR5 in 1X PBS (pH 7.4). FIGURE 15. Cleavage over time graph for TATR2 in Borate (pH 9.3). FIGURE 16. Table 2: Half-lives for on HAP cleavage of TATR1, TATR2 and TATR5.†represents data at mean ± SD (n = 2), and represents data as single point (n = 1). FIGURE 17. Cleavage over time graph for TATR1 in 1X PBS (pH 7.4) on HAP FIGURE 18. Cleavage over time graph for TATR2 in 1X PBS (pH 7.4) on HAP FIGURE 19. Cleavage over time graph for TATR2 in HEPES (pH 7.4) on HAP FIGURE 20. Cleavage over time graph for TATR2 in Borate (pH 9.3) on HAP FIGURE 21. Cleavage over time graph for TATR5 in 1X PBS (pH 7.4) on HAP DETAILED DESCRIPTION Reference will now be made in detail to presently preferred compositions, embodiments, and methods of the present invention, which constitute the best modes of practicing the invention presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting but merely as a representative basis for any aspect of the invention and / or as a representative basis for teaching one skilled in the art to variously employ the present invention. Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and / or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. 2024-054-01 Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: all R groups (e.g. Ri where i is an integer) include hydrogen, alkyl, lower alkyl, C1-6 alkyl, C6-10aryl, C6-10heteroaryl, -NO2, -NH2, -N(R’R”), -N(R’R”R”’)+L-, Cl, F, Br, -CF3, -CCl3, -CN, - SO3H, -PO3H2, -COOH, -CO2R’, -COR’, -CHO, -OH, -OR’, -O-M+, -SO3-M+, -PO3-M+, -COO-M+, - CF2H, -CF2R’, -CFH2, and -CFR’R” where R’, R” and R”’ are C1-10 alkyl or C6-18 aryl groups M is a metal atom (e.g., Na, K, Li, etc.) and L- is a counter anion (e.g., Cl-, Br-, tosylate, etc.); single letters (e.g., "n" or "o") are 1, 2, 3, 4, or 5; in the compounds disclosed herein including compounds described by formula or by name, a CH bond can be substituted with alkyl, lower alkyl, C1-6 alkyl, C6-10 aryl, C6- 10 heteroaryl, -NO2, -NH2, -N(R’R”), -N(R’R”R”’)+L-, Cl, F, Br, -CF3, -CCl3, -CN, -SO3H, -PO3H2, - COOH, -CO2R’, -COR’, -CHO, -OH, -OR’, -O-M+, -SO3-M+, -PO3-M+, -COO-M+, -CF2H, -CF2R’, - CFH2, and -CFR’R” where R’, R” and R”’ are C1-10alkyl or C6-18aryl groups M is a metal atom (e.g., Na, K, Li, etc.) and L- is a negative counterion; percent, "parts of," and ratio values are by weight; the term "polymer" includes "oligomer," "copolymer," "terpolymer," and the like; molecular weights provided for any polymers refers to weight average molecular weight unless otherwise indicated; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property. The term “alkyl” refers to C1-20inclusive, linear (i.e., “straight-chain”), branched, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl, or propyl, is attached to a linear alkyl chain. “Lower alkyl” 2024-054-01 refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way. It must also be noted that, as used in the specification and the appended claims, the singular form "a," "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components. The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps. The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. With respect to the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. 2024-054-01 The phrase “composed of” means “including” or “comprising.” Typically, this phrase is used to denote that an object is formed from a material. It should also be appreciated that integer ranges explicitly include all intervening integers. For example, the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 includes 1, 2, 3, 4. . . .97, 98, 99, 100. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper limit and the lower limit divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1, the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits. In the examples set forth herein, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In a refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In another refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 10 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. The compounds set forth herein may be used per se or as pharmaceutically acceptable derivatives. The latter term includes salts, esters, and other derivatives generally considered acceptable by pharmaceutical standards. Useful derivatives, for example, include salts of organic and inorganic acids such as sulfates, phosphates, hydrohalide salts, carboxylate salts, etc., as well as esters of carboxylic acid or hydroxyl substituents, ethers of hydroxyl substituents, amides of amino substituents, as well as carbamates, ureas, etc. Synthesis of these derivatives is conventional and well-known to those skilled in pharmaceutical chemistry. For example, compounds bearing hydroxyl groups may be converted to esters by customary techniques of organic chemistry, such as a reaction with an acyl halide, carboxylic acid anhydride, or by esterification with an acid while removing byproduct water. In some cases, derivation may be desired to facilitate compounding of the pharmaceutical into an 2024-054-01 acceptable form such as tablets, powder, aqueous dispersion, capsule, etc., or may be useful in assisting bioavailability of the drug following administration, for example, by rendering the compound more or less soluble. In many cases, such as, for example, esters, ureas, carbamates, ethers, etc., the derivative may function as a “prodrug,” which liberates the active form by biological transformation, i.e., by enzymatic hydrolysis of an ester functionality, as is well known to the pharmaceutical chemist. Throughout this application, where publications are referenced, the disclosures of these publications in their entirety are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. The term "chemical linker" refers to a chemical moiety or bond that covalently attaches two or more components, such as a bisphosphonate and a drug cargo in the target and timed-release (TATR) platform. The linker may be a cleavable linker, such as a pH-sensitive (acid-labile) linker, a labile carbamate group, a labile thiocarbamate group, a disulfide linker, or a peptide-based linker, which facilitates drug release upon cleavage. The linker may also be a non-cleavable linker, such as an alkyl, alkylamido, or amide linker, which maintains the attachment between the components without cleavage. Covalent bonding of the bisphosphonate, chemical linker, and drug cargo can occur through the formation of amide, ester, or imino bonds between acid, aldehyde, hydroxyl, amino, or carbamate groups. Linkage may be achieved by covalent attachment using a variety of suitable methods. For example, the bisphosphonate and drug cargo in the target and timed-release (TATR) platform may be linked using bifunctional reagents (linkers) capable of reacting with both the bisphosphonate and the drug cargo, forming a bridge between the two components. The terms “cleavable linker” or “cleavable linking group,” as used herein, refer to a divalent or monovalent moiety that is capable of being separated (e.g., detached, split, hydrolyzed, or broken) into distinct entities. In the context of the target and timed-release (TATR) platform, a cleavable linker is specifically cleavable in response to external stimuli such as changes in pH (e.g., physiological pH), nucleophilic or basic reagents, or other chemical triggers. A chemically cleavable linker refers to a linker capable of being split in response to chemical agents, such as hydroxide ions 2024-054-01 at physiological pH, weak acids, or reducing agents. Importantly, these cleavable linkers in the TATR platform are non-enzymatically cleavable, meaning cleavage occurs without enzyme catalysis. In embodiments, the cleavable linker in the TATR platform includes a labile carbamate group or a labile thiocarbamate group that can be cleaved through β-elimination by hydroxide ions at physiological pH, facilitating controlled drug release. The term “non-cleavable linker” or “non-cleavable linker section” refers to a chemical moiety that covalently connects two or more components, such as a targeting moiety and a drug cargo, through a stable bond that does not degrade or break under physiological conditions. As used herein, non-cleavable linkers, which may include amide bonds, are designed to resist cleavage due to the inherent stability of these bonds, which do not readily break down under mild conditions such as physiological pH (7.4) or in the presence of hydroxide ions (in concentrations at physiological conditions). Unlike cleavable linkers, which are designed to release their cargo via specific chemical reactions (e.g., β-elimination), non-cleavable linkers maintain a permanent connection between the components, ensuring long-term stability in the biological environment. Therefore, “non-cleavable linkers” or “non-cleavable linker sections” are more resistant to cleavage under physiological conditions than “cleavable linkers” or “cleavable linking groups.” Abbreviations: “7,8-DHF” means 7,8-dihydroxyflavone. "6-FAM" means 6-carboxyfluorescein. "7-AMC" means 7-amino-4-methylcoumarin. "BDNF" means brain-derived neurotrophic factor. "BP" means bisphosphonate. "DBCO" means dibenzylcyclooctyne. "DMSO" means dimethyl sulfoxide. 2024-054-01 "HAP" means hydroxyapatite. "HC" means hair cell. "HEPES" means 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. "M" means molarity. "RWM" means round window membrane. "SGN" means spiral ganglion neuron. "SNHL" means sensorineural hearing loss. "TATR" means target and timed-release. "Trk" means tropomyosin receptor kinase. In at least one aspect, a target and timed-release platform that can be useful for treating hearing loss is provided. The target and timed-release platform has formula 1: BP LK Dr(1)or a pharmaceutically acceptable salt thereof, wherein: BP is a bisphosphonate group for bone targeting and retention or a pharmaceutically acceptable salt thereof; LK is a cleavable linking group; and Dr is a drug cargo, and in particular, a drug cargo for treating hearing loss. Characteristically, the drug cargo is released upon cleavage of the cleavable linking group. Advantageously, cleavage of the linking group can occur at physiological pH. In a refinement, the bisphosphonate group is described as follows: 2024-054-01 PO3H2wherein R1is H, OH, halo, or alkyl. In another aspect, the cleavable linking group LK includes a labile carbamate group or labile thiocarbamate. The attachment of the drug cargo may utilize an available amine function to form the labile carbamate or thiocarbamate group. In a further refinement, LK includes a β-hydrogen. Advantageously, the β-hydrogen can be abstracted by hydroxide ions at physiological pH. Typically, physiological pH is from about 7.35 to 7.45, which corresponds to a hydroxyl ion range of about 2.24×10−7M to about 2.82×10−7M. In another aspect, the target and timed-release platform is described by formula 2, which includes a labile carbamate group or a labile thiocarbamate group: Mod or a X is O or S; R1 is H, OH, halo, or alkyl; 2024-054-01 mod is a chemical moiety that adjusts or modulates the cleavage rate of the cleavable linking group; LK1is a non-cleavable linking section between the bisphosphonate and the labile carbamate group or thiocarbamate or a labile thiocarbamate group; and DR is the drug cargo. It should be appreciated that in formula 2, the cleavable linking group is: Mod . In a refinement, mod includes a ^-hydrogen. In a includes a component selected from methylene units (–CH2–), alkyl chains (–(CH2)n– where n is an integer typically from 1 to 20 or more), thioether linkages (–S–CH2), ether linkages (–O–CH2–), ethylene glycol units (-CH2CH2O-), amide linkages (– C(=O)–NH–), aryl groups such as phenylene (–C₆H₄–), heteroaryl groups such as pyridine (–C₅H₄N– ), imidazole (–C₃H₃N₂–), thiophene (–C₄H₃S–), cycloalkyl groups such as cyclohexyl (–C6H11–), and heterocycloalkyl groups such as piperidine (–C₅H₁₁N–), morpholine (–C₄H₈ON–) or azocane (C7H15N). In some variations, drug cargo Dr includes a constitutive primary or secondary amine, or equivalent functionality, for incorporation into the labile carbamate group or a labile thiocarbamate group. In another aspect, the TATR platform, when adsorbed onto bone or bone-like tissue (e.g., tissue containing hydroxyapatite), provides a range of half-lives for drug release that are highly beneficial for therapeutic applications. This extended drug release is due to the strong affinity of bisphosphonates for bone, which unexpectedly modifies (e.g., extends) the time release, allowing the drug cargo to remain adsorbed for extended periods. Once adsorbed, the cleavable linker in the TATR platform facilitates controlled drug release over a broad range of timescales (half-lives from about 8 to 680 hours), depending on the specific linker and environmental conditions. This tunability ensures 2024-054-01 that the drug remains within therapeutic concentration levels over an extended duration, optimizing treatment efficacy while minimizing systemic exposure and the need for frequent administration. In another aspect, the target and timed-release platform is described by formula 3, which includes a labile carbamate group: EWGHb; or a wherein: X is O or S; R1 is H, OH, halo, or alkyl; Hb is the β-hydrogen; LK2is a non-cleavable linking section between the bisphosphonate and the labile carbamate group or a labile thiocarbamate group; EWG is an electron-withdrawing group; and Dr is the drug cargo, and in particular, a drug cargo for treating hearing loss. In a refinement, the non-cleavable linking section LK2includes a component selected from methylene units (–CH2–), alkyl chains (–(CH2)n– where n is an integer typically from 1 to 20 or more), thioether linkages (–S– CH2), ether linkages (–O–CH2–), ethylene glycol units (-CH2CH2O-), amide linkages (–C(=O)–NH– ), aryl groups such as phenylene (–C₆H₄–), heteroaryl groups such as pyridine (–C₅H₄N–), imidazole (–C₃H₃N₂–), thiophene (–C₄H₃S–), cycloalkyl groups such as cyclohexyl (–C6H11–), and heterocycloalkyl groups such as piperidine (–C₅H₁₁N–), morpholine (–C₄H₈ON–) or azocane (C7H15N). In some variations, drug cargo Dr includes a constitutive primary or secondary amine, or equivalent functionality, for incorporation into the labile carbamate group or a labile thiocarbamate group. 2024-054-01 In a variation, electron-withdrawing groups operate as modulating groups that can adjust the time-release properties (e.g., modulate the half-life). In a refinement, the electron- withdrawing group includes an electron-withdrawing moiety selected from the group consisting of F, Cl, Br, I, -NO2, C=O, -CN, -SO3R, -NO, -COOR, -PO3R2, -SO₂NH₂, -COCF₃ -SO₂CF₃ , -SO2R, and combinations thereof where R is H, alkyl, or OH. In a further refinement, the electron-withdrawing group is a phenyl substituted by one or more electron-withdrawing moieties. Specific examples of electron-withdrawing groups include Cl or ,where a wavy line indicates the point of bonding. In a refinement, the electron group is F, Cl, Br, I, -NO2, C=O, -CN, -SO3R, -NO, COOR, PO3R2, -SO₂NH₂, -COCF₃ - SO₂CF₃ , -SO2R, and combinations thereof where R is H, alkyl, or OH. In another aspect, the TATR platform may be used to deliver therapeutic agents Dr commonly used to treat sensorineural hearing loss and related conditions. In a refinement, Dr is a TrkB agonist. In another refinement, Dr is a TrkC agonist. Advantageously, Dr can be 1Aa with the NH group of the carbamate or thiocarbamate being contributed by 1Aa when present. Additional examples of drug cargo Dr include but are not limited to 7,8-DHF or a derivative thereof, dexamethasone, prednisolone, methylprednisolone, betamethasone, BDNF, neurotrophic factors (such as BDNF and NT-3), N-acetylcysteine (NAC), glucocorticoids, TrkB agonists (e.g., 7,8-dihydroxyflavone, LM22A- 4, TDP6, deoxygedunin, R13, amifostine, and antioxidants (e.g., vitamin E and vitamin C). These drugs are aimed at reducing inflammation, protecting neurons, and promoting the regeneration of 2024-054-01 sensory cells and neurons in the inner ear. Some drug cargos are described in Kempfle JS, Duro MV, Zhang A, Amador CD, Kuang R, Lu R, Kashemirov BA, Edge AS, McKenna CE, Jung DH. A Novel Small Molecule Neurotrophin-3 Analogue Promotes Inner Ear Neurite Outgrowth and Synaptogenesis In vitro. Front Cell Neurosci. 2021 Jul 15;15:666706. doi: 10.3389 / fncel.2021.666706. PMID: 34335184; PMCID: PMC8319950 and Zaccaro, M. C., Lee, H. B., Pattarawarapan, M., Xia, Z., Caron, A., L'Heureux, P. J., Bengio, Y., Burgess, K., & Saragovi, H. U. (2005). Selective small molecule peptidomimetic ligands of TrkC and TrkA receptors afford discrete or complete neurotrophic activities. Chemistry & Biology, 12(9), 1015–1028. https: / / doi.org / 10.1016 / j.chembiol.2005.06.015; the entire disclosures of which are hereby incorporated by reference. In another aspect, the drug cargo Dr includes a backbone selected from the group consisting of: R1H N R2

[0002] 2024-054-01 R1H N R2. It should be appreciated R1 and R2 are selected to define the following dipeptides in the backbone: IK (a), NN (b), GK (c), EK (d), IR (e), TG (f), KG (g), IN (h), KT (i), EN (j), or RG (k). The letters in parentheses are just labels for identifying the dipeptide. The dipeptides are provided as one-letter amino acid codes where I is isoleucine, K is lysine, N is asparagine, G is glycine, E is glutamic acid (glutamate), R is arginine, and T is threonine. For example, “1Aa” refers to the following structure: ONH2. 2024-054-01 The bifunctional linkers described herein provide diverse options for covalently attaching the bisphosphonate and drug cargo in the TATR platform, allowing for precise control over the stability and release of the drug cargo based on the specific linker chemistry employed. In another embodiment, a method for treating hearing loss with the target and timed- release platform set forth herein is provided. The method includes a step of administering a therapeutically effective amount of the target and timed-release platform. Advantageously, the target and timed-release platform can be introduced via a subject’s round window membrane. In a refinement, the target and timed-release platform are absorbed in the cochlea bone. In another aspect, the time release of the drug cargo adsorbed on bone or bone-like tissue is modified as compared to the time release in solution. In a refinement, the time release of the drug cargo adsorbed on bone or bone-like tissue is extended as compared to the time release in solution. In a further refinement, the time release of the drug cargo adsorbed on bone or bone-like tissue has a half-life from about 8 hours to about 680 hours. In another aspect, the dosage range for the TATR platform can be reduced (e.g., reduced by 50 to 90%) as compared to the normal dosage range for the drug cargo alone. Typically, the dosage range for the TATR platform can be from 1 mg / (kg of a patient’s weight per day) to 100 mg / (kg of a patient's weight) per day. For example, if a typical systemic dose for 7,8-dihydroxyflavone is 100–500 mg / day for humans, the TATR platform could reduce this dose by 50%–90%, resulting in an estimated dose of 10–50 mg / day. For LM22A-4, where the systemic dose is 3–10 mg / kg in animal studies, the TATR platform might lower it to 0.3–5 mg / kg. In the case of deoxygedunin, with systemic doses of 10–50 mg / kg, the TATR platform could reduce the required dose to approximately 1–25 mg / kg. Similarly, for R13, where typical doses range from 10–40 mg / kg, the TATR platform could lower the dosage to around 1–20 mg / kg. In another aspect, the TATR platform can be delivered to the round window membrane (RWM) via a transtympanic or intratympanic injection method. This process begins with the application of local anesthesia to numb the ear, followed by, in some cases, a small incision (myringotomy) in the eardrum to access the middle ear. The TATR platform, formulated in a solution 2024-054-01 or embedded in a hydrogel, is then injected near the RWM using a small needle or catheter. The gel or solution helps to keep the drug in place, ensuring contact with the membrane. The semi-permeable RWM allows the TATR platform to diffuse into the cochlea, where the bisphosphonate moiety binds to the cochlear bone. This binding enables the platform to provide localized and sustained drug delivery as the drug cargo is released over time through the degradation of a cleavable linker. This method allows for prolonged therapeutic effects while minimizing systemic exposure, making it particularly suitable for treating conditions such as sensorineural hearing loss. In another aspect, TATR platform can be delivered to the round window membrane via a transtympanic or intratympanic injection. The solution used for this delivery typically contains a solvent that is biocompatible and maintains physiological stability, such as saline (0.9% NaCl) or phosphate-buffered saline (PBS). These solvents help ensure that the TATR platform is stable, safe, and effective in the inner ear environment. PBS or other buffers, such as HEPES, can be included to maintain a physiological pH (7.4), which is crucial for the stability and function of the TATR platform. In many cases, the TATR platform may be embedded in a hydrogel or polymer matrix to allow for sustained release. Hydrogels such as polyethylene glycol (PEG) or hyaluronic acid provide a medium that helps retain the drug at the RWM and release it gradually over time. This controlled release ensures that the therapeutic agent is delivered effectively to the cochlear bone, where it can be absorbed and released in a sustained manner as the cleavable linker in the TATR platform degrades. Additional components like surfactants or stabilizers may be used to ensure the drug cargo remains soluble and stable in the solution. Polysorbate 80 (Tween 80) or Pluronic F-127 are commonly included to improve the solubility of hydrophobic drugs. These ingredients further enhance the delivery efficiency and ensure that the formulation is well-suited for the inner ear environment. Optionally, preservatives such as benzyl alcohol may be added to maintain sterility, preventing contamination during storage and administration. Overall, the formulation for delivering the TATR platform to the RWM includes a solvent, a hydrogel or polymer matrix, stabilizers, and potentially preservatives. This ensures that the drug cargo is delivered in a controlled and sustained manner to the cochlea, optimizing the therapeutic effect while minimizing systemic exposure. 2024-054-01 In another aspect, a pharmaceutical composition including the TATR platform is provided. The concentration of the TATR platform in solution depends on several factors, including the therapeutic dose of the drug cargo, the delivery mechanism, and the desired release profile. For localized delivery, such as to the cochlear bone via the round window membrane (RWM), the concentration is typically lower than systemic doses due to the targeted nature of the platform. As set forth above, the TATR platform allows for reduced dosages compared to systemic delivery, often lowering the required dose by 50% to 90%. For potent active ingredients, such as neurotrophic factors or TrkB agonists, the concentration of the TATR platform in solution could range between 1 mg / mL and 50 mg / mL, depending on the drug’s potency and the desired release rate. In formulations where the TATR platform is embedded in a hydrogel matrix for sustained release, the concentration of the drug is generally between 0.1 mg / mL and 10 mg / mL, ensuring gradual diffusion over time. The concentration is adjusted to balance effective delivery with the sustained release mechanism, ensuring consistent therapeutic action over the course of hours or days. Typical pharmaceutical formulations for targeted delivery often use concentrations in the range of 1 mg / mL to 10 mg / mL, but in some cases, higher concentrations are required depending on the potency of the drug cargo and the specific needs of the treatment. Thus, the concentration of the TATR platform in solution is typically between 1 mg / mL and 50 mg / mL, optimized for the drug’s activity and the specific release kinetics desired. The following examples illustrate the various embodiments of the present invention. Those skilled in the art will recognize many variations that are within the spirit of the present invention and the scope of the claims. Bisphosphonates strongly complex calcium ions in hydroxyapatite matrices, enabling them to tightly bind to the bone. This property offers the potential for bone-specific drug delivery, which has been demonstrated to provide more effective concentrations of antibiotics to infected bone tissue by attachment of the drug cargo to a bisphosphonate via a pH-sensitive linker that cleaves in situ. Bisphosphonate-conjugated fluorescent imaging probes triggered by cathepsin K secretion at bone resorption sites have also been recently implemented. In the case of SNHL, cochlear bone is an intriguing target for bisphosphonates, but an instigating drug release mechanism other than bone resorption is indicated, and it is further desirable 2024-054-01 to be able to adjust the rate of drug release from the bone-anchored conjugate at physiological pH by effecting synthetically accessible structural changes in the linker moiety (‘target and timed-release,’ TATR) (Fig. 1A). To this end, we have synthesized a small library of TATR platforms incorporating a standard fluorophore (AMC) as the drug cargo to facilitate the determination of release kinetics from their bisphosphonate-derived model conjugates. The conjugates were designed to exhibit half-lives ranging from 36 h to 450 h, with the drug release timing dependent on synthetic adjustment of the cleavable linker structure. Release kinetics were determined experimentally at pH 7.4 and the concentrations as a function of time with the simulation of in situ dilution were calculated for a bolus drug, the parent conjugate, and released drug using FluidSIM4. An example of the results is shown in Fig.1B. Under the selected conditions, the TATR-generated drug concentration was maintained within the therapeutic dose range of 5 - 0.5 ^M well over tenfold longer than the bolus drug concentration, demonstrating the potential of this new approach to bone-targeted drug delivery. Several methodologies are known for inner ear drug delivery. The indirect transtympanic route is suitable for small drug formulations. However, for the indirect transtympanic route more drug is needed, and sustained delivery options are required due to rapid drug clearance. The indirect transtympanic route can be safer for hearing preservation. The direct intracochlear route allows for the delivery of larger molecules such as proteins. However, the direct intracochlear route comes with a great risk of damaging hearing. Figures 3A and 3B provide fluorescence results for experiments in which 6-FAM-ZOL was embedded in hydrogel material which was placed on RWM.7(A) 0.1X of systemic BP dose applied to RMW, observe weak fluorescent signal (B) 0.3X of systemic BP dose applied to RWM, a significant increase in signal. Another route for inner ear drug delivery involves the development of a BP-based platform. Bisphosphonates (BPs) bind tightly to bone and remain adsorbed for several months.6BP- coupled fluorophores bind to cochlear bone and can be introduced via the round window membrane (RWM).7Therefore, linking a BP to a drug via a cleavage linker would allow for sustained delivery of the drug from the cochlear bone. In this regard, non-enzymatic cleavable linkers with tunable release rates were previously reported.8 2024-054-01 Referring to Figure 4, the assembly of TATR components is illustrated.5, 6Target and timed-release (TATR) platform 10 includes BP moiety 12 for bone targeting and retention and a labile carbamate linker 14 that upon cleavage (β-elimination) releases drug cargo 18. In a refinement, target and timed-release platform 10 includes an electron-withdrawing group (EWG) 20 that modulates the abstraction rate of β-hydrogen (in red). In the examples depicted, Fluorophore (7-AMC) is used as a drug model cargo for the evaluation of cleavage rates. Figures 5A, 5B, and 5C depict a general synthetic route for the target and timed-release platform. In this regard, click chemistry is used to attach a portion of the target and timed-release platform with bisphosphonate and a portion of the drug cargo. The mod functional group can be adjusted to produce each of the compounds provided in Figures 2A and 2B. Figures 6A, 6B, and 6C summarize experiments for determining TATR in vitro cleavage rates. In these experiments, TATR1 uses the following modulating group:

[0003] 2024-054-01 Figure 7 provides a plot of TATR in vitro cleavage rates (determined by a linear fit) for pre-adsorbed onto HAP. In this experiment, TATR2 was pre-adsorbed onto HAP. TATR2 was incubated in 0.1 M HEPES buffer (pH 7.4) at 37 °C. The buffer was analyzed by UV-Vis to determine the concentration of released 7-AMC. Surprisingly, the cleavage rate of TATR2 was significantly reduced when adsorbed onto HAP. Figure 8A provides a schematic of an in vivo pharmacokinetic model simulating drug concentration over time. Experimental half-lives in solution and simulated (FluidSim49) clearance rates are used to model predicted drug concentrations in cochlear fluid. Comparison of k1 to k2 shows that clearance >> drug release for bolus drug. Figure 8B shows the drug concentrations as a function of time. Suppression of k2 by BP-immobilization at cochlear bone is predicted to provide an effective concentration of drug in situ (red trace). 2024-054-01 Table 1. Experimental and Simulated Rate Constants (h-1) Drug k1k2k3 In conclusion, TATR platforms display cleavage rates consistent with previously reported conjugates. Surprisingly, adsorption of TATR platforms to HAP significantly reduced the cleavage rate. These results show that the cleavage rate must be adjusted by changing the structure of the TATR platform, appropriately as described herein. This is required for therapeutic effectiveness. Cleavage Studies Referring to Figures 9 to 21, analysis of the cleavage rates for the TATR1, TATR2, and TATR5 platforms, both in solution and on hydroxyapatite (HAP), reveals significant differences in their stability and behavior under various conditions. Referring to Figures 9 to 15, in solution, TATR1 cleaves in 0.1 M HEPES (pH 7.4) with a half-life of 44.53 hours. TATR2 exhibits faster cleavage in 1X PBS (pH 7.4) with a half-life of 7.58 hours, while its half-life increases to 17.81 hours in 0.1 M HEPES (pH 7.4). TATR5 demonstrates the shortest half-lives among the platforms, cleaving in 3.96 hours in 0.1 M HEPES and 2.46 hours in 1X PBS, indicating that it is the least stable in solution. Referring to Figures 16 to 21, on HAP, the cleavage rates slow down significantly, indicating greater stability when adsorbed onto a bone-like substrate. TATR1 has an extended half- life of 239.6 hours on HAP in 1X PBS. TATR2 shows a drastically extended half-life of 676 hours on HAP in 0.1 M HEPES, but in 1X PBS, its half-life is shorter at 47.63 hours. In 0.2 M borate (pH 9.3), TATR2 cleaves more quickly, with a half-life of 8.16 hours. TATR5 exhibits a relatively short half- life on HAP at 15.64 hours in 1X PBS. 2024-054-01 Overall, the data unexpectedly shows that the TATR platforms exhibit significantly slower cleavage on HAP compared to in solution when adsorbed onto a bone-like substrate. The assay buffer used plays a significant role in the cleavage rates, with TATR2 showing the most variability. TATR5 remains the least stable, while TATR1 demonstrates the longest half-life in both solution and on HAP, making these findings useful for optimization with respect to tissue targeting. Collectively, TATR1, TATR2, TATR3, TATR4, and TATR5 provide a range of half-lives, which enable the application of the TATR platform to either bone-like tissue (e.g., bone) or non-bone tissue targets. While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implementing embodiments may be combined to form further embodiments of the invention. References 1. Lin F, Thorpe R, Gordon-Salant S, and Ferrucci. Hearing loss prevalence and risk factors among older adults in the United States. J. Gerontol., Ser. A. 2011; 66(5): 582-590 2. Wu et al. Primary neural degeneration in the human cochlea: evidence for hidden hearing loss in the aging eat. Neuroscience. 2019; 407: 8 -20 3. Suzuki J, Corfas G, and Liberman M. C. Round-window delivery of neurotrophin 3 regenerates cochlear synapses after acoustic overexposure. Sci. Rep. 2016; 6: 1-11 4. Kempfle et al. Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss. Bioconjugate Chem. 2018; 29(4): 1240-1250 5. Kempfle et al. A Novel Small Molecule Neurotrophin-3 Analogue Promotes Inner Ear Neurite Outgrowth and Synaptogenesis In Vitro. Frontiers in Cellular Neuroscience. 2021; 15: 1-15 2024-054-01 6. McKenna C. E. et al. Chemistry of Bisphosphonates. Encyclopedia of Bone Biology, 2020; 3: 551-565 7. Kang W. S., et al. Non-Ototoxic Local Delivery of Bisphosphonate to the Mammalian Cochlea. Otol. Neurotol.2015; 36(6): 953-960 8. Santi V. D. et al. Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates. PNAS, 2012; 109(16): 6211- 6216 9. https: / / alecsalt.com / index.php / simulations / fluidsim4

Claims

2024-054-01 WHAT IS CLAIMED IS:

1. A target and timed-release platform having formula 1: (1) or a pharmaceutically acceptable salt thereof, wherein: BP is a bisphosphonate for bone targeting and retention; LK is a cleavable linking group; and Dr is a drug cargo, wherein the drug cargo is released upon cleavage of the cleavable linking group.

2. The target and timed-release platform of claim 1, wherein cleavage of the cleavable linking group occurs at physiological pH.

3. The target and timed-release platform of claim 1, wherein LK includes a labile carbamate group or a labile thiocarbamate group.

4. The target and timed-release platform of claim 3, wherein the target and timed-release platform is described by formula 2, which includes a labile carbamate group or a labile thiocarbamate group:or a pharmaceutically acceptable salt thereof,2024-054-01 X is O or S; R1 is H, OH, halo, or alkyl; mod is a chemical moiety that adjusts or modulates cleavage rate of the cleavable linking group; LK1 is a non-cleavable linking section between the bisphosphonate and the labile carbamate group or a labile thiocarbamate group; and Dr is the drug cargo.

5. The target and timed-release platform of claim 4, wherein Dr includes a constitutive primary or secondary amine, or equivalent functionality, for incorporation into the labile carbamate group or the labile thiocarbamate group.

6. The target and timed-release platform of claim 4, wherein mod includes a β-hydrogen.

7. The target and timed-release platform of claim 6, wherein the β-hydrogen can be abstracted by hydroxide ions at physiological pH.

8. The target and timed-release platform of claim 3 having formula 3, which includes a labile carbamate group or a labile thiocarbamate group:wherein: X is O or S; R1 is H, OH, halo, or alkyl; Hb is a β-hydrogen; LK2is a linking section between the bisphosphonate and the labile carbamate group;2024-054-01 EWG is an electron-withdrawing group; and Dr is the drug cargo.

9. The target and timed-release platform of claim 8, wherein Dr includes a constitutive primary or secondary amine, or equivalent functionality, for incorporation into the labile carbamate group or a labile thiocarbamate group.

10. The target and timed-release platform of claim 8, wherein the electron-withdrawing group operates as a modulating group that can adjust time-release properties.

11. The target and timed-release platform of claim 8, wherein the electron-withdrawing group includes an electron-withdrawing moiety selected from the group consisting of F, Cl, Br, I, - NO2, C=O, -CN, -SO3R, -NO, COOR, PO3R2, -SO2R, and combinations thereof where R is H, alkyl, or OH.

12. The target and timed-release platform of claim 8, wherein the electron-withdrawing group is a phenyl substituted by one or more electron-withdrawing moieties.

13. The target and timed-release platform of claim 8, wherein the electron-withdrawing group is selected from the group consisting of:2024-054-01 .The target and timed-release platform of claim 1 wherein Dr is a drug cargo for treating hearing loss.

15. The target and timed-release platform of claim 1 wherein Dr is a TrkB agonist.

16. The target and timed-release platform of claim 1, wherein Dr is a TrkC agonist.

17. The target and timed-release platform of claim 1, wherein Dr is 1Aa with an NH group of a carbamate or thiocarbamate is contributed by 1Aa.

18. A target and timed-release platform comprising a compound selected from the group consisting of: O ,2024-054-01 H2H22024-054-01 aof claims 1 to 18, the pharmaceutical composition being a solution or gel.

20. A method for treating hearing loss with a target and timed-release platform having formula 1: (1) or a pharmaceutically acceptable salt thereof, wherein: BP is a bisphosphonate for bone targeting and retention; LK is a cleavable linking group; and2024-054-01 Dr is a drug cargo, wherein the drug cargo is released upon cleavage of the cleavable linking group, the method comprising administering a therapeutically effective amount of the target and timed-release platform.

21. The method of claim 20, wherein the target and timed-release platform is introduced via a subject’s round window membrane.

22. The method of claim 20, wherein the target and timed-release platform is absorbed in cochlea bone.

23. The method of claim 20, wherein LK includes a labile carbamate group or a labile thiocarbamate group.

24. The method of claim 23, wherein the target and timed-release platform is described by formula 2, which includes a labile carbamate group or a labile thiocarbamate group:or a X is O or S; R1is H, OH, halo, or alkyl; mod is a chemical moiety that adjusts or modulates cleavage rate of the cleavable linking group; LK1is a non-cleavable linking section between the bisphosphonate and the labile carbamate group or the labile thiocarbamate group; and2024-054-01 Dr is the drug cargo.

25. The method of claim 24, wherein the Dr includes a constitutive primary or secondary amine, or equivalent functionality, for incorporation into the labile carbamate group or a labile thiocarbamate group.

26. The method of claim 24, wherein mod includes a β-hydrogen.

27. The method of claim 26, wherein the β-hydrogen can be abstracted by hydroxide ions at physiological pH.

28. The method of claim 23 having formula 3, which includes a labile carbamate group or a labile thiocarbamate group: wherein:X is O or S; R1 is H, OH, halo, or alkyl; Hbis a β-hydrogen; LK2is a linking section between the bisphosphonate and the labile carbamate group or the labile thiocarbamate group; EWG is an electron-withdrawing group; and DR is the drug cargo.

29. The method of claim 28, wherein the Dr includes a constitutive primary or secondary amine, or equivalent functionality, for incorporation into the labile carbamate group or the labile thiocarbamate group.2024-054-01 30. The method of claim 20, wherein Dr is a TrkB agonist.

31. The method of claim 20, wherein Dr is a TrkC agonist.

32. The method of claim 20, wherein Dr is 1Aa with an NH group of a carbamate or thiocarbamate contributed by 1Aa.

33. The method of claim 20, wherein time release of the drug cargo adsorbed on bone or bone-like tissue is modified as compared to the time release in solution.

34. The method of claim 33, wherein the time release of the drug cargo adsorbed on bone or bone-like tissue is extended as compared to the time release in solution.

35. The method of claim 34, wherein the time release of the drug cargo adsorbed on bone or bone-like tissue has a half-life from about 8 hours to about 680 hours.

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