Compositions and methods for treating V-type ATPase dysfunction

Abstract

A method for treating V-ATPase dysfunction can include identifying a dysfunction of at least a portion of the V-ATPase protein, where the dysfunction occurs in the transmembrane portion of the V-ATPase protein. Further, the method can include preparing a glycosylated precursor and preparing a P2Y12 inhibitor. Further, the method can include combining the glycosylated precursor and the P2Y12 inhibitor to obtain a novel cocktail, and delivering the novel cocktail using a prodrug delivery system, where the prodrug delivery system includes a prodrug.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is related to U.S. Provisional Patent Application No. 63 / 399,964, filed August 22, 2022, entitled "A COMPOSITION FOR THE TREATMENT OF AN ATP6AP2 DEFICIENCY," U.S. Provisional Patent Application No. 63 / 423,854, filed November 9, 2022, entitled "A METHOD, COMPOSITION, AND SYSTEM FOR TREATING V-ATPASE MALFUNCTION," and U.S. Provisional Patent Application No. 63 / 423,854, filed November 9, 2022, entitled "COMPOSITION FOR THE TREATMENT OF AN ATP6AP2 DEFICIENCY." This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 413,424, filed October 5, 2022, entitled "A METHOD FOR IMPROVING A ... FIELD OF THE INVENTION The present invention relates generally to the field of therapeutics. In particular, the present invention is directed to methods, compositions and systems for treating V-type ATPase dysfunction. [Background technology]

[0002] (background) V-ATPase (V-ATPase) is a protein complex that functions as a proton pump in the membranes of cells and organelles. Some individuals have dysfunctional V-ATPases due to mutations that cause subunits or other related proteins to be insufficiently produced or incorrectly folded, for example. Defects in V-ATPases are evident in various diseases and disorders, including, but not limited to, Parkinson's disease, distal renal acidosis, myopathy, lysosomal storage diseases, and congenital disorders of glycosylation. Defects in V-ATPases can occur at one or more positions in the V-ATPase protein. For example, ATP6AP2 is an assembly factor that mediates the formation of multiunit V-ATPases. ATP6AP2 can interact with and bind to ATP6AP1, chaperoning the connections between the subunits of the proton pump. Defects in ATP6AP2 can cause diseases associated with insufficient functional V-ATPase. It may be advantageous and beneficial to ensure that V-ATPase and ATP6AP2 function properly in an attempt to correct any possible defects and / or diseases. Summary of the Invention [Means for solving the problem]

[0003] (Summary of the Disclosure) In one embodiment, a method for treating one or more V-type ATPase dysfunctions includes identifying a dysfunction of at least a portion of a V-type ATPase protein in a subject, preparing a composition comprising a glycosylated precursor, a modulator, and a delivery vehicle, and administering the composition to the subject.

[0004] In another embodiment, the pharmaceutical composition may comprise a glycosylation precursor, a modulator, and a delivery vehicle.

[0005] These and other aspects and features of non-limiting embodiments of the present invention will become apparent to those skilled in the art upon reading the following description of specific non-limiting embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, the drawings show aspects of one or more embodiments of the invention. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentality shown in the drawings. [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 shows a diagram of a V-type ATPase protein according to an embodiment of the present invention. [Figure 2] FIG. 2 shows a block diagram of an exemplary composition for treating V-type ATPase dysfunction. [Figure 3] FIG. 3 shows a flow diagram of an exemplary method for treating V-type ATPase protein dysfunction according to an embodiment of the present invention. [Figure 4] FIG. 4 shows an illustrative example of the lysosomal process. [Figure 5] FIG. 5 shows an exemplary diagram of the pH environment within a cell. [Figure 6] 6A-6D show exemplary embodiments of prodrug structures. [Figure 7] FIG. 7 shows a diagram of an exemplary prodrug delivery system using ManNAc. [Figure 8] FIG. 8 shows the ATP6ap2 subunit and the V-type ATPase with the V1 and V2 regions labeled. [Figure 9] FIG. 9 shows a proton pump that creates an acidic environment across a membrane gradient. [Figure 10] FIG. 10 shows the addition of sialic acid to the synthesized protein. [Figure 11] FIG. 11 shows glycosylation precursors and the chemical reactions that convert them to sialic acid. [Figure 12] FIG. 12 shows the autophagy-lysosome pathway (ALP) involved in lysosomal fusion and waste degradation. [Figure 13] FIG. 13 shows the interplay of TFEB, AMPK, and V-type ATPases in relation to lysosomal function and biogenesis. [Figure 14] Figure 14 shows a glycosylation therapeutic pathway. The drawings are not necessarily to scale and may be represented by phantom lines, diagrammatic representations, and partial views. In certain cases, details that are not necessary for understanding the embodiments or that make other details difficult to perceive may be omitted. DETAILED DESCRIPTION OF THE INVENTION

[0007] (Detailed explanation) Broadly, embodiments of the present disclosure are directed to systems and methods for treating V-type ATPase dysfunction.

[0008] Aspects of the present disclosure can be used to treat patients suffering from mutations in V-type ATPases (e.g., when V-type ATPases cannot assemble properly), which cause the V-type ATPases to lose function or have reduced function. Reduced or lost V-type ATPase function can cause the pH of cells and / or organelles to differ from their physiologically appropriate levels. This can cause various problems, including insufficient protein glycosylation and impaired autophagy. Exemplary embodiments illustrating aspects of the present disclosure are described below in the context of several specific examples.

[0009] The term "subject" is used to refer to animals (including humans and non-human animals) to which the present apparatus and methods may be applied. The term "user" is used to refer to a person who applies a device to a human or non-human animal. The subject and user may, but need not, be the same person.

[0010] Referring now to Figure 1, an exemplary embodiment of a V-type ATPase is shown. V-type ATPases (also called vacuolar-type ATPases) are a type of ATPase that utilizes energy derived from ATP hydrolysis to transport protons across cell membranes. It should be noted that V-type ATPases are composed of several subunits. Mutations in one or more of these subunits, or mutations in non-coding regions that cause the subunits to be produced inefficiently, can cause the V-type ATPase to fail to assemble properly at normal levels and / or to function improperly. V-type ATPase subunits include ATP6AP1, ATP6V0E1, ATP6V0E2, ATP6V0D1, ATP6V0D2, ATP6V0B, ATP6V0C, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V1H, ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1E2, ATP6V1G1, ATP6V1G2, ATP6V1G3, and ATP6V1F. V-type ATPase subunits include the V0 subunit 104 and the V1 subunit 108. V0 may comprise the portion of the V-type ATPase 100 responsible for translocating protons from one side of the membrane to the other. V 1 (108) may comprise the portion of the V-ATPase 100 responsible for hydrolyzing ATP, as shown. 1 108 can receive ATP from other organelles of the cell. In particular, mitochondria can generate ATP for delivery to various organelles and proteins of the cell. Furthermore, ATP can be used as an energy source for proper functioning of the V-type ATPase 100. A properly functioning V-type ATPase 100 can facilitate H+ (proton) movement across the membrane. In some cases, moving protons across the membrane 112 can be active transport, which may require an energy source (e.g., ATP) to facilitate that active transport. Active transport can be used due to higher proton concentrations in the extracellular environment adjacent to the cell membrane 112. In some cases, the V-type ATPase 100 can function as an assembly factor and / or an ion channel stabilizing factor.

[0011] 1, ATPase H+ transport accessory protein 2 (ATP6AP2) is a protein associated with type V ATPases. In some embodiments, mutations in coding or non-coding regions associated with ATP6AP2 can cause type V ATPases to fail to assemble and / or function properly.

[0012]

[0003] Still referring to Figure 1, V-ATPases are enzymes that use energy derived from ATP to pump protons (i.e., H+ ions) across membranes upstream of a gradient, thereby creating the pH or acidity level and / or electrical charge required for organelle function. Inappropriate cellular acidity can be lethal or debilitating to an organism on a large scale. On a smaller scale, each organelle within an animal cell may have a specific pH to perform its own function. Thus, inappropriate or suboptimal pH in a cell or organelle can be harmful in various ways to an organ, cell, or other part of an organism. Although V-ATPases are located in organelle membranes and can regulate their pH, generating acidity that promotes various functions, they are commonly referred to as lysosomal accessory proteins because they are abundantly expressed in lysosomal membranes.

[0013] With further reference to FIG. 1 , the V-type ATPase 100 can comprise a transmembrane protein. A transmembrane protein extends from the interior portion of a cell or organelle to the exterior portion of the cell or organelle. It should be noted that a transmembrane protein can transport any substance across the membrane of a cell or organelle against a concentration gradient, transport large substances through the membrane of the cell or organelle, or any similar process. As a non-limiting example, a portion of the V-type ATPase 100 can be located in a hydrophobic region of the membrane 112. As shown in FIG. 1 , the V-type ATPase 100 can be located in a hydrophobic region of the membrane 112. 0 (104) may be disposed in the film 112. 0 (104) can extend from hydrophilic heads in the interior portion of a cell or organelle to hydrophilic heads in the exterior portion of the cell or organelle. 0 The (104) portion may include ATP6AP2, which is an assembly factor in V-ATPase 100.

[0014] With further reference to Figure 1, a properly functioning V-type ATPase 100 can be described as a proton pump. Proton pumps change the acidity of a cell's or organelle's environment by actively transporting protons across membranes. V-type ATPases can be found in the plasma membrane as well as in the membranes of various organelles (e.g., membranes of the endoplasmic reticulum, Golgi apparatus, and lysosomes). The inability of V-type ATPases to form at adequate levels in these membranes can cause the organelle to have limited effectiveness in some functions, as described in more detail below.

[0015]

[0004] With further reference to Figure 1, in an attempt to maintain an optimal pH level within a cell, cells may contain proton pumps integrated into their membranes to actively transport protons into and out of the cell. In some embodiments, the pH level within a cell may be very high (i.e., high [H+]), causing an imbalance for organelles within the cell. The proton pump may then be used to pump protons out of the cell, diluting the [H+] and lowering the pH level to make it more optimal for cellular functionality. The proton pump may use energy in the form of adenosine triphosphate (ATP) to phosphorylate subunits and assemble the functional pump. The pump may alter the acidity to create the appropriate pH environment needed to carry out a wide range of cellular processes within the organelle space or outside the cell.

[0016]

[0003] With further reference to Figure 1, ATP6AP2 (116) deficiency can cause pH changes within organelle spaces, including lysosomes, or outside the cell. The ATP6AP2 gene encodes a subunit of the V-type ATPase protein complex. This protein is also known as an endoplasmic reticulum (ER)-localized transmembrane adaptor and lysosome-interacting protein due to its high level of expression in the endoplasmic reticulum and lysosomal membranes. Genetic mutations in ATP6AP2 can cause multiple disorders, including parkinsonism with spasticity, nephrotoxicity, epilepsy, neurodevelopmental problems, brain atrophy, encephalopathy, and X-linked or congenital glycosylation disorder type IIr. In some embodiments, an individual may have multiple dysfunctions (e.g., mutations) in the V-type ATPase protein complex. In some embodiments, the compositions and methods described herein can be used to treat one or more dysfunctions in V-type ATPase.

[0017]

[0003] Still referring to Figure 1, in some embodiments, mutations in V-ATPase subunits (e.g., ATP6AP2) can cause V-ATPases to fail to assemble properly in sufficient quantities in lysosomes. Lysosomes are important for the clearance of cellular waste products due to their function in autophagy. In autophagy, autophagosomes containing waste products fuse with lysosomes, which, under normal circumstances, contain enzymes and a low pH. This combination causes the waste products in the autophagosomes to be degraded. However, if the lysosomal membrane does not contain enough properly functioning V-ATPase complexes, the pH of the lysosomes may be too high, preventing the waste products from being efficiently degraded when the autophagosomes fuse with the lysosomes.

[0018]

[0004] Still referring to Figure 1, organelle acidity is crucial to the waste management system in animal cells. Lysosomes and peroxisomes break down waste products through the use of digestive enzymes that are active in specific pH environments. A decrease in lysosomal pH is observed in a category of diseases called lysosomal storage diseases.

[0019] Referring further to Figure 1, autophagy is a metabolic process that selectively or globally collects and degrades cellular waste or improperly synthesized macromolecules. Autophagy involves many signaling and scaffolding proteins, generating membrane structures called autophagosomes that collect waste products. Lysosomes are membrane-bound organelles containing pH and enzymes that degrade, destroy, and / or denature waste products. During autophagy, autophagosomes may fuse with lysosomes, allowing their contents to be degraded. In this way, autophagy is a regulated process in cells that disposes of errors and mistakes in cellular synthesis. As a metabolic process, the degradation of waste products is necessary for new, appropriate synthesis to occur. In autophagy disorders, the congestion of waste products that cannot be degraded affects the regeneration and synthesis of new substances. TFEB, a lysosomal synthesis pathway protein, promotes autophagy by binding to the promoter regions of several autophagy genes.

[0020] Referring further to FIG. 1, enzymes in lysosomes may include cathepsins, collagenases, peptidases, nucleases, phosphatases, glycoprotein / lipid / oligosaccharide-osidases, and lipases. This organelle can form degradation by regulating signaling pathways known as autophagy regulatory pathways (e.g., mTOR). Lysosomes may interact with the AMPK pathway, which inhibits mTORC1. Lysosomes may regulate their own production through the TFEB pathway, a transcription factor pathway that induces lysosomal biogenesis. Lysosomal function may be regulated by a V-ATPase proton pump. Lysosomes can perform many functions, including waste degradation and autophagy regulation. Lysosomes may require a specific pH and / or a pH within a specific range to function optimally; as a non-limiting example, the range may include a pH anywhere between approximately 4.5 and approximately 5. If the pH of lysosomes is outside this ideal range, one or more of the functions performed by the lysosomes may be altered. The optimal pH of lysosomes may differ from that of other components of the cell (e.g., the cytosol, whose pH may typically be around 7.2). This pH difference can generate a membrane potential that can support proton transport across the cell, between organelles, and the like. If the pH of the cytosol and / or lysosomes changes, this can lead to a cascade of problems, including accumulation of cellular components inside the cell, inability to deliver macromolecular components of the cell, loss of neuronal function, and many other problems. Lysosomal function can also help regulate autophagy as part of a feedback pathway by providing macromolecular components. In some cases, this can ultimately cause the transcription factor EB (TFEB) pathway to shut down lysosomal production, ultimately leading to disorders, including, but not limited to, cancer, cardiovascular disease, neurodegeneration, infectious diseases, and / or aging.

[0021] With further reference to FIG. 1 , V-ATPase deficiency can cause altered pH levels that can interfere with protein synthesis and / or glycosylation. Proteins can be synthesized using RNA in the endoplasmic reticulum. The synthesized proteins can be modified for stability, transport, and function by the Golgi apparatus. Under normal pH conditions in the Golgi apparatus, sialyltransferases can attach sialic acid to proteins in a process known as glycosylation. Proper glycosylation can be necessary for proper protein function, for example, by enabling proteins to attach to specific targets in the body. In some cases, glycosylation may be required for correct protein folding. However, if the pH is abnormal due to a dysfunctional V-ATPase, proteins may not be efficiently glycosylated. In some embodiments, a higher-than-normal pH in the Golgi apparatus can affect the distribution of sialyltransferases in the Golgi apparatus, which can affect the rate of glycosylation. Inappropriate glycosylation is known as a category of diseases called congenital disorders of glycosylation (CDGs).

[0022] With further reference to Figure 1, congenital glycosylation disorders (e.g., congenital glycosylation disorders resulting from V-type ATPase deficiency) can also affect a subject's ability to properly form glycolipids. While glycolipids act as cellular recognition units, glycoproteins act as receptors for chemical signals. Reduction or loss of these macromolecules can result in a lack of proper cellular communication. There are a diverse set of glycoproteins present or expressed at different levels throughout various organs throughout the body. The type of glycosylation disorder a person inherits can influence the disease phenotype observed.

[0023] 2, a block diagram of an exemplary composition 200 for treating V-type ATPase dysfunction is shown. Composition 200 can be used to correct a dysfunction in V-type ATPase 100. Composition 200 can include a glycosylated precursor 204, a modulator 208, and a delivery vehicle 216. As described further below, in some embodiments, composition 200 can be used to treat a subject in need thereof (e.g., a subject suffering from a genetic disorder that causes insufficient production of functional V-type ATPase).

[0024] 2, composition 200 may include glycosylation precursors. In some embodiments, glycosylation precursors may modulate glycosylation. In some embodiments, glycosylation precursors may promote glycosylation. In some embodiments, glycosylation precursors may modulate lysosomal pH. In some embodiments, glycosylation precursors may increase lysosomal pH. In some embodiments, glycosylation precursors may decrease lysosomal pH. In some embodiments, glycosylation precursors may include sialic acid precursors. The sialic acid precursors may include any sialic acid precursor described herein (including, but not limited to, ManNR, ManNAc, UDP-GlcNac, UDP-GlcNR, ManNac-6-P, and / or SiaNR). In some embodiments, glycosylation precursors 204 may comprise at least 1 percent of composition 200.

[0025] With further reference to FIG. 2 , composition 200 may include a modulator. As used herein, a "modulator" is a substance that modulates glycosylation, modulates lysosomal pH, or both. In some embodiments, a modulator may include a lysosomal pH modulator. In some embodiments, a lysosomal pH modulator may increase lysosomal pH. In some embodiments, a lysosomal pH modulator may decrease lysosomal pH. In some embodiments, a modulator may include a glycosylation modulator. In some embodiments, a glycosylation modulator may increase the glycosylation rate. In some embodiments, a modulator may include a P2Y12 inhibitor. As used herein, a "P2Y12 inhibitor" is a substance that reduces ADP binding to P2Y12. In some embodiments, a P2Y12 inhibitor may modulate pH in lysosomes. In some embodiments, a P2Y12 inhibitor may decrease pH in lysosomes. In some embodiments, P2Y12 inhibitors can regulate pH in lysosomes by increasing cAMP levels. In some embodiments, drugs other than P2Y12 inhibitors that increase cAMP levels can be used as regulators. In some embodiments, regulating lysosomal pH in subjects lacking sufficient functional V-type ATPase complexes can allow lysosomes to more effectively degrade waste products through autophagy. In some embodiments, lysosomal pH regulators can also regulate pH in the Golgi apparatus and endoplasmic reticulum. In some embodiments, lysosomal pH regulators can be used to regulate organelle pH in the liver, kidney, heart, brain, and / or spinal cord. In some embodiments, the combination of glycosylation precursor 204 and regulator 208 can have a synergistic effect in increasing glycosylation rates. In some embodiments, the combination of glycosylation precursor 204 and regulator 208 can increase glycosylation rates more than either glycosylation precursor 204 or regulator 208 alone.In some embodiments, the combination of glycosylation precursor 204 and regulating agent 208 may have a synergistic effect in reducing lysosomal pH. In some embodiments, the combination of glycosylation precursor 204 and regulating agent 208 may reduce lysosomal pH more than either glycosylation precursor 204 or regulating agent 208 alone. Glycosylation precursor 204 and / or regulating agent 208, individually and / or in combination, may synergistically enhance each other's effectiveness in reducing and / or treating any of the pathologies described herein.

[0026] 2, the P2Y12 inhibitor inhibits the binding of ADP to the P2Y12 receptor. The P2Y12 inhibitor can attenuate platelet aggregation. In some cases, the P2Y12 inhibitor can include any of the P2Y12 inhibitors described herein, including but not limited to clopidogrel (Plavix®), prasugrel (Effient®), ticlopidine (Ticlid®), and / or ticagrelor (Brilinta®). In some embodiments, the lysosomal pH regulator can include a suitable anticoagulant. The P2Y12 inhibitor can comprise at least 1 percent of the composition 200. The P2Y12 inhibitor can be used to improve the pH in retinal cell lysosomes, the pH environment around the Golgi apparatus (GA), the pH environment around the endoplasmic reticulum (ER), and the lysosomal pH in multiple tissues. P2Y12 inhibitors can affect the restoration of lysosomal pH in retinal cells in age-related macular degeneration. Macular degeneration can have similar characteristics to many autophagy-related diseases, including the accumulation of deposits in Bruch's membrane of the eye. These deposits can have a toxic "blocking" effect that leads to the degeneration of the retinal pigment endothelium (RPE). In a non-limiting example, composition 100 can manipulate the lysosomes of other cell types, correct the defective pathways of the inventors, and improve the lysosomal accumulation phenotype seen in ATP6AP2 mutations.

[0027] 2 , glycosylated precursor 204 and modulator 208 (e.g., P2Y12 inhibitor) can be combined to produce cocktail 212. Various concentrations and amounts of glycosylated precursor 204 and modulator 208 (e.g., P2Y12 inhibitor) can be chemically combined in laboratory glassware. As used in this disclosure, a “novel cocktail” is a chemical combination of at least a glycosylated precursor and a P2Y12 inhibitor used as a treatment for a specified dysfunction of the V-type ATPase protein. Novel cocktail 212 can be used to deliver composition 200 to V-type ATPase 100. In some cases, novel cocktail 212 can be used to treat an ATP6AP2(116) deficiency. However, in some embodiments, novel cocktail 212 can be used to treat other defects within cells and / or organelles that can negatively affect the pH of the cells and / or organelles. In some embodiments, the modulator 208 may be used individually to treat a V-type ATPase dysfunction. In some embodiments, the glycosylation precursor 204 may be used individually to treat a V-type ATPase dysfunction. In some embodiments, the modulator 208 and the glycosylation precursor 204 may be used in combination to treat a V-type ATPase dysfunction. In some embodiments, the combination of the modulator 208 and the glycosylation precursor 204 may have a synergistic effect, e.g., may increase the glycosylation rate more than one element of the combination alone and / or may decrease lysosomal pH more than one element of the combination alone. The modulator 208 and the glycosylation precursor 204 may be tested together and / or in combination.

[0028] With further reference to FIG. 2 , composition 200 may include a delivery vehicle 216. As used herein, a “delivery vehicle” is an agent suitable for delivering a payload to a target site (including, but not limited to, an organ, cell, and / or organelle) in a subject. In some embodiments, the delivery vehicle may include a prodrug delivery system. In some embodiments, the prodrug delivery system may include a prodrug. As used in this disclosure, a “prodrug” is a biologically inactive compound that can be converted, metabolized, or excreted in the body, leaving the active drug or therapeutic agent in the desired organ / region. Prodrug strategies may enhance the pharmacokinetic properties of the payload. The application method for this prodrug carrier may enhance the efficacy of the administered dose. Prodrugs may be selected to target specific tissues or organs, particularly reducing off-target effects. In some embodiments, the prodrug delivery system may be capable of delivering a payload across the blood-brain barrier. In some embodiments, the prodrug delivery system may enable the glycosylated precursor 204 and / or the modulator 208 to cross the blood-brain barrier without crossing it itself. The prodrug may contain a removable unit or a bioreversible version of the active drug that can be converted in vivo to release the active drug by a chemical reaction that should occur in the predicted system. The prodrug may be used as a delivery system in "hard-to-reach" organs, including the central nervous system (CNS) and skeletal muscle. In CNS delivery constructs, the prodrug may contain a lipophilic carbon chain that can be bound to endogenous proteins, carried in the blood, and ultimately cross the blood-brain barrier (BBB). In some embodiments, a first prodrug delivery system is used to deliver the lysosomal pH modulator, and a second prodrug delivery system is used to deliver the glycosylated precursor. In some embodiments, a first prodrug delivery system is used to deliver both the lysosomal pH modulator and the glycosylated precursor.

[0029] Further referring to Figure 2, prodrugs can include ProTide prodrugs. ProTide prodrugs are a category of prodrugs that are linked to phosphate. Prodrugs can be used with another glycosylated precursor, ManNAc-6-phosphate. As a non-limiting example, masked lipophilic ManNAc-6-P can undergo enzymatic activation and deacetylation to yield ManNAc-6-P, a precursor to sialic acid. Glycosylated precursors can be used in the treatment of ATP6AP2, as discussed further in this disclosure.

[0030] With further reference to Figure 2, a prodrug delivery system can be a more efficient transport mechanism once sialic acid is added. As used in this disclosure, a "transport mechanism" is a process that moves a substance into or out of a cell. A prodrug delivery system can be more efficient because it can inactivate a drug during biodistribution so that the drug is specifically delivered to its target. As described herein, ManNAc can be a glycosylation precursor 204. Therefore, including ManNAc in a prodrug can provide a precursor of sialic acid so that the prodrug can yield sialic acid when metabolized, and therefore have a more efficient transport mechanism. Furthermore, including ManNAc in a prodrug can enable specific delivery of the prodrug (including ManNAc) to its target. It should be noted that the structure of a prodrug can introduce delivery constraints. As a non-limiting example, a prodrug with 16 carbons can cross the blood-brain barrier (BBB). As another non-limiting example, a prodrug with only 12 carbons may not cross the BBB.

[0031] 2, administration of glycosylation precursors 204 using prodrugs can increase glycosylation. In some embodiments, V-ATPase 100 dysfunction can cause cells and / or organelles to have a pH environment that is not sufficiently acidic or is too acidic for sialic acid transfer. By using a prodrug to deliver glycosylation precursors 204, the glycosylation precursors can still be delivered to the cell by the prodrug and can promote the final stage of glycosylation in proteins.

[0032] Further referring to Figure 2, the prodrug may comprise any of the prodrug delivery systems described herein. In some embodiments, g-dopamine receptors and / or g-adenosine receptors may be part of the novel cocktail. G-dopamine receptors may be G protein-coupled receptors involved in regulating motor activity and various neurological disorders (including, but not limited to, schizophrenia, bipolar disorder, Alzheimer's disease, Parkinson's disease, etc.). G-adenosine receptors may be G protein-coupled receptors involved in mediating the physiological effects of adenosine.

[0033] With further reference to Figure 2, G-adenosine receptors and / or G-dopamine receptors may alternatively or additionally be utilized to phosphorylate assembly factors of the V-ATPase 100. In some cases, it may not be desirable to use the P2Y12 receptor to initiate a pathway to phosphorylate assembly factors of the V-ATPase 100. In certain embodiments, other G protein receptors (e.g., G-adenosine, G-dopamine) may be used to phosphorylate ATP6AP2(116) to correct an ATP6AP2(116) deficiency. In other embodiments, G-adenosine and / or G-dopamine may be used to correct a defect in any other assembly factor of the V-ATPase 100. Upon reading this disclosure, one skilled in the art will understand that assembly factors that may have a defect can be corrected by G protein receptor-induced phosphorylation.

[0034]

[0023] Still referring to Figure 2, in some embodiments, the delivery vehicle may include an organelle targeting moiety. The organelle targeting moiety may direct the payload to a desired organelle (e.g., lysosomes or the Golgi apparatus). In a non-limiting example, a compound contained in the composition of the desired organelle may be attached to the payload. In another non-limiting example, techniques common to enzyme replacement therapy (e.g., interaction with mannose-6-phosphate) may be used to target the lysosome.

[0035] 2, composition 200 can be formulated in any dosage form, including, but not limited to, oral, ophthalmic, inhalation, injection, topical, vaginal, and / or rectal administration. For example, but not limited to, composition 200 can be formulated as a pill, capsule, syrup, liquid, elixir, emulsion, tincture, orally disintegrating tablet, lozenge, thin film, powder, edible, eye drop, lotion, ointment, aerosolized medication, metered dose, nebulizer, smoking, vaporizer, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal, intravenous, cream, gel, hydrogel, ear drops, skin patch, powder, etc. In some embodiments, composition 200 can be administered using a dose schedule. As used in this disclosure, a "dose schedule" is the amount of a composition to be used for a subject at a given time. In some embodiments, the administration schedule may include frequency, timing, duration, dosage, etc. As a non-limiting example, the administration schedule may include the time at which administration of composition 200 is to be given, the time between administrations of composition 200, the length of time for which composition 200 is to be given, and / or the amount of composition 200 to be given at each particular time point. In a non-limiting example, composition 200 may be administered daily, twice daily, three times daily, once every six months, etc. In another non-limiting example, a dose of glycosylation precursor 204 may be administered daily and a dose of P2Y12 inhibitor may be administered twice daily. In another non-limiting example, a dose of between 0.2 g and 10 g of glycosylation precursor 204 may be administered orally, intravenously, or by injection. In a non-limiting example, glycosylation precursor 204 may increase 60% transferrin glycosylation to 85% transferrin (+25%) glycosylation. Additionally and / or alternatively, glycosylation precursor 204 can increase glycosylation from 5% to 75%. In another non-limiting example, a P2Y12 inhibitor can increase the pH in lysosomes from about 7 to about 4.8 (-2.2). Additionally and / or alternatively, a P2Y12 inhibitor can decrease the pH in lysosomes by between 0.3 and 4.

[0036] 2, composition 200 can be used to address symptomatic protein synthesis and ameliorate glycosylation effects on the liver. Additionally, composition 200 can be utilized for glycosylation effects on protein synthesis, protein translation, and glycoprotein interactions with various organelles in the liver, kidney, heart, brain, and spinal cord.

[0037]

[0023] With further reference to Figure 2, composition 200 can be utilized to address insufficient protein transport, degradation, and waste removal. Composition 200 can improve the pH in retinal cell lysosomes. In some embodiments, composition 200 can downregulate enzymes, acidification, or both. For purposes of this disclosure, "downregulation" refers to a decrease in the expression, production, or activity of a particular gene, protein, or receptor in a biological system. In some embodiments, composition 200 can upregulate enzymes, acidification, or both. For purposes of this disclosure, "upregulation" refers to an increase in the expression, production, or activity of a particular gene, protein, or receptor in a biological system. In a non-limiting example, the composition can upregulate acidification to treat not only ATP6AP mutations, but also mutations in lysosomal enzyme genes and mutations in glycosylation enzyme genes. Composition 200 can be utilized to improve the pH of the Golgi apparatus (GA), endoplasmic reticulum (ER), and lysosomes in multiple tissues, including those listed above. Composition 200 can be utilized for congenital glycosylation disorders (CDGs) and lysosomal storage disorders (LSCs). For purposes of this disclosure, "congenital glycosylation disorders" refers to a set of rare genetic disorders that affect a person's ability to construct some or all of their glycoproteins and glycolipids. While glycolipids can act as cellular recognition units, glycoproteins can act as receptors for chemical signals. The reduction or loss of these macromolecules can result in a lack of proper cellular communication. There are a diverse set of glycoproteins that can be present or expressed at various levels throughout various organs in the body. The type of glycosylation disorder a person inherits can influence the disease phenotype observed. Generally, most patients with CDG experience the following: hypotonia, failure to thrive, developmental delay, liver disease, bleeding or clotting disorders, seizures, stroke, cardiomyopathy, ataxia, hormonal imbalances, muscle diseases, muscular dystrophy, Duchenne muscular dystrophy, neuromuscular scoliosis, poor vision, muscle degeneration, dysarthria, and kidney failure.Therapies targeted at improving glycosylation may have beneficial therapeutic effects in these patients. In a non-limiting example, the therapeutic agent may serve as a glycosylation treatment by adding precursor molecules to improve proteinopathy by correcting protein synthesis and translation at the beginning of the pathway. In a non-limiting example, composition 200 may affect ATP6AP2 asp107asp CT, ATP6AP2 ser115ser CT, ATP6AP2 IVS2DS T-A+6, ATP6AP2 leu98ser, ATP6AP2 arg71his, ATP6AP2 IVS3AS TT del, ATP6AP2 ile21met, etc. to treat ATP6AP2 deficiency.

[0038]

[0023] Referring further to Figure 2, in some embodiments, this therapeutic agent can serve as a treatment for lysosomal acidification. Lysosomal degradation is the final step in removing toxic proteins caused by improper synthesis or misfolding. Improving lysosomal acidification corrects proteinopathy at the final checkpoint of the pathway before cell death occurs. Lipid-based prodrug delivery can be used to deliver composition 200. The lipid-based delivery system can be a multi-carbon chain construct that can penetrate muscle, tissue, and even the brain. Defects in lysosomes can result in a category of diseases called lysosomal storage diseases. These disorders are characterized by the loss of enzymes or the loss of their functionality. In acidification disorders, enzyme function in lysosomes can be impaired. This dysfunction can lead to toxic accumulation in cells. These diseases may include, but are not limited to, Fabry disease, Niemann-Pick disease, Krabbe disease, Gaucher disease, metachromatic leukodystrophy, Sandhoff disease, Tay-Sachs disease, Batten disease, cystinosis, Danon disease, Pompe disease, etc. Symptoms of these diseases may include abnormally large organs, changes in skeletal muscles, coarse facial features, and developmental problems. In some embodiments, composition 200 may be configured to treat lysosomal storage diseases (e.g., but not limited to, mucopolysaccharidosis (MPS) types I, II, IIA, IIIB, IIIC, IIID, IVA, IVB, VI, VII, mucolipidosis IV (ML4), a neurodegenerative lysosomal storage disease caused by TRPML1 mutations, multiple sulfatase deficiency, Fabry disease, Farber lipogranulomatosis, Gaucher disease, Krabbe disease, Niemann-Pick disease types A, B, and C, GM1 gangliosidosis, GM2 gangliosidosis, aspartylglucosaminuria, fucosidosis, α-mannosidosis, β-mannosidosis, sialidosis, Schindler disease, Pompe disease, etc., as described above).In a non-limiting example, composition 200 may be selected from the group consisting of GBA 1q21, GLA Xq22, GAA 17q25.3, IDUA 4p16.3, IDS Xq28, SGSH 17q25.3, NAGLU 17q21.2, HGSNAT 8p11.21, HGSNAT 8p11.21, GNS 12q14.3, GALNS 16p24.3, GLB1 3p22.3, ARSB 5q14.1, GUSB 7q11.21, SMPD1 11p15.4, NPC1, NPC 2, ASAH1 10q22.1, HEXA 15q23, HEXB 5q13, GALC 14q31.3, ARSA 22q13.33, SUFM1 3p26.1, AGA 4q34.3, FUCA1 1p36.11, MANSA 19p13.2, NAGA 22q13.2, NEU1 6p21.33, GNPTAB 12q23.2, TRMPL1 19p13.2, LIPA 10q23.31, CTSA 20q13.12, LAMP2A Xq24 6'-(R)-methyl-5-O-(5-amino-5,6-dideoxy-α-L-talofuranosyl)-paromamine sulfate, gemfibrozil, N-butylhydroxylamine, modified cholera toxin, pyrimethamine, ambroxol, N-acetyl-glucosamine thiazoline, migalastat hydrochloride. * , miglustat, odiparcil, lucerastat, benglustat, (3S)-1-azabicyclo[2.2.2]oct-3-yl{2-[2-(4-fluorophenyl)-1,3-thiazol-4-yl]propan-2-yl}carbamate, 2-hydroxypropyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, eliglustat, cysteamine, 1,5-(butylimino)-1,5 dideoxy, D-glucitol, L-cycloserine, clenbuterol, ursodeoxycholic acid, gemfibrozil and vitamin A, ibudilast, pentosan polysulfate sodium, triheptanoin, etc.

[0039] 2, composition 200 can be utilized for other proton pump defects (e.g., but not limited to, liver inflammation and necrosis, neurogenesis, myogenesis, immune system response, blood glucose and blood pressure, nerve maintenance, cardiac function, renal fibrosis, and renal insufficiency). Composition 200 can be utilized for defective proton pump function that affects autophagy and lysosomes and manifests as degenerative diseases (including, but not limited to, Alzheimer's disease, multiple sclerosis, spinal muscular atrophy, Parkinson's disease, Huntington's disease, cancer, Parkinsonism with spasticity, congenital glycosylation disorder type IIr, X-linked syndrome intellectual developmental disorder type Hedera, etc.). Composition 200 can be utilized for defective proton pump function that affects glycosylation and manifests as congenital glycosylation disorders (including, but not limited to, 170 CDG subtypes, type 2 diabetes, metabolism, and / or obesity). Composition 200 may be utilized for defective proton pump function that affects immune and inflammatory responses and manifests as infectious diseases (including, but not limited to, bacterial defense, COVID-19, COPD, and / or cancer).

[0040] Still referring to FIG. 2, in some embodiments, composition 200 can be configured to treat CDGs (e.g., but not limited to, congenital deglycosylation disorders 1 and 2, congenital glycosylation disorders Ia-Iz, IIa-IIz, etc.). In a non-limiting example, composition 200 may contain ALG6 ALA333VAL, ALG6 SER478PRO, ALG6 IVS3DS GA, ALG6 3bp del 895ATA, ALG6 3bp del 897AAT, ALG6 IVS7DS TG, ALG6 TYR131HIS, NGLY1 ARG401TER, NGLY1 1-bp dup 1370G, NGLY1 3bp del 1205TTC, NGLY1 ARG542TER, NGLY1 CYS283TRP, NGLY1 GLU356GLY, NGLY1 1bp del NT1837, PMM2 ARG141HIS, PMM2 ASN216ILE, PMM2 VAL129MET, PMM2 ARG162TRP, PMM2 ASP65TYR, PMM2, PHE119LEU, PMM2, ASP188GLY, PMM2 GLY117ARG, PMM2 ASP223GLU, PMM2 357C-A, PMM2 THR237ARG, PMM2 CYS241SER, PMM2 ILE132THR, PMM2 VAL231MET, PMM2 CYS9TYR, PMM2 LEU32ARG, PMM2 THR226SER, PMM2 PRO113LEU, PMM2 IVS7 CT, PMM2 VAL44ALA, PMM2, 28-kb del, PMM2 IVS3ASAS GC, PMM2 TYR106PHE,It may affect MGAT2 SER290PHE, MGAT2 HIS262ARG, MGAT2 ASN318ASP, MGAT2 CYS339TER, MGAT2 LYS237ASN, ALG12 PHE142VAL, ALG12 THR61MET, ALG12 ARG146GLN, ALG12 GLY101ARG, ALG12 LEU158PRO, ALG12 TYR414TER, ALG12 THR224MET, ALG12 1bp del 1001A, ALG12 1bp del 117G, SLC35C1 ARG147CYS, SLC35C1 THR308ARG, SLC35C1 GLU31TER, SLC35C1 3bp del 501CTT, ALG1 SER258LEU, ALG1 GLU342PRO, ALG1 SER150ARG, ALG1 MET377VAL, ALG1 GLY145ASP, ALG1 CYS396TER, ALG1 ARG276TRP, MPI ARG219GLN, MPI SER102LEU, MPI MET138THR, MPI 1bp ins 166C, MPI ARG295HIS, DPM1 ARG92GLY, DPM1 13bp del, DPM1 1bp del 628C, DPM1 SER248PRO, DPM1 IVS4AS T-A, DPM1 GLY152VAL, DPM1 100kb del, STT3A VAL626ALA, STT3A THR546ILE, STT3A TYR530SER, STT3A HIS46ARG, STT3A ARG160GLN, STT3A ARG405CYS, STT3A ARG405HIS, STT3A ARG329CYS, etc.

[0041] Still referring to FIG. 2 , in some embodiments, composition 200 may be configured to treat other V-ATPase deficiencies (e.g., but not limited to, cutis laxa type 2, developmental and epileptic encephalopathy 93, immunodeficiency 47, X-linked myopathy with excessive autophagy, distal renal tubular acidosis 3, developmental and epileptic encephalopathy 104, neurodevelopmental disorders with epilepsy and brain atrophy, wrinkly skin syndrome, distal renal tubular acidosis with progressive sensorineural hearing loss 2, congenital deafness with onychodystrophy, Zimmermann-Laband syndrome 2, etc.). In a non-limiting example, composition 200 may contain ATP6AP1 met428ile, ATP6AP1 leu144pro, ATP6AP1 glu346lys, ATP6AP1 tyr313cys, ATP6AP1 leu181arg, ATP6AP1 leu47pro, ATP6AP1 leu311gln, ATP6AP1 tyr217asn, ATP6V1A ARG338CYS, ATP6V1A GLY72ASP, ATP6V1A ASP100TYR, ATP6V1A ASP349ASN, ATP6V1A PRO27ARG, ATP6V1A ASP371GLY, VMA21 IVS1 AC, VMA21 IVS1 AT, VMA21 IVS2 AG, VMA21 IVS2 TG, VMA21 272G-C, VMA21 TER+6, VMA21 IVS2 TG, VMA21 92-bp del, VMA21 9-bp del, ATP6V1E1 LEU128PRO, ATP6V1E1 ARG212TRP, ATP6V0A4 GLU753TER, ATP6V0A4 GLY820ARG, ATP6V0A4 IVS17 GA, ATP6V0A4 1 bp deVAL35l, ATP6V0A4 MET580THR, ATP6V0A4 IVS6 GA, ATP6V0A4 1bp de GLN276l, ATP6V0A4 PRO524LEU, ATP6V0A4 TYR502TER, ATP6V0A4 ARG807GLN, ATP6V0A4, 1bp del 2137G, ATP6V0A1ARG741GLN, ATP6V0A1 ALA512PRO, ATP6V0A1 50-kb del, ATP6V0A1 ASN534ASP, ATP6V0A1, IVS2 GA, ATP6V0A1 ARG740GLN, ATP6V0A1 GLY551GLU, ATP6V0A1 ARG804HIS, ATP6V0A1 1bp del 445G, AP6V0A1 ARG495TRP,ATP6V0A2 1bp ins 100A, ATP6V0A2 7bp del NT2355, ATP6V0A2 10132G-A, ATP6V0A2 ARG63TER, ATP6V0A2 GLN765TER, ATP6V1B1 IVS12DS GC, ATP6V1B1 GLY78ARG, ATP6V1B1 It may affect LEU81PRO, ATP6V1B1 IVS6DS GA, ATP6V1B1 1bp del, ATP6V1B1 ARG31TER, ATP6V1B2 ARG485PRO, ATP6V1B2 ARG506TER, etc.

[0042] Referring further to Figure 2, abnormal upregulation or downregulation can be a hallmark of multiple pathologies. In non-limiting examples, autophagy-related diseases can include neurodegenerative diseases, aging, and metabolic diseases. Metabolic disorders associated with autophagy can include diabetes, obesity, nonalcoholic steatohepatitis, and atherosclerosis. Factors such as overnutrition, high-fat diets, insulin resistance, and high cholesterol can generate or result in dysfunction in autophagy. Impaired autophagy can be found in Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and aging. Upregulating autophagy against toxic proteins can be a therapeutic solution for these neurodegenerative diseases. Such upregulators of autophagy may include starvation conditions, ALFY overexpression, Afinitor, Torisel, Zotres, Rapamune, Hiftor, Fyarro, Afinitor Disperz, ridaforolimus, umirolimus, zotarolimus, Torin-1, Torin-2, vistusertib, etc. In some embodiments, composition 200 may be configured to treat autophagy disorders (e.g., but not limited to, Paget's disease of bone, frontotemporal dementia, amyotrophic lateral sclerosis, distal myopathy with rimmed vacuoles, childhood-onset neurodegeneration with ataxia, dystonia, gaze palsy, Alzheimer's disease, Parkinson's disease, etc.). In non-limiting examples, composition 200 may affect SQSTM1 PRO392LEU, SQSTM1 1bp ins 1224T, SQSTM1 IVS7DS GA, SQSTM1 LYS378TER, SQSTM1 PRO387LEU, SQSTM1 ALA33VAL, SQSTM1 3 bp del 714GAA, SQSTM1 2T-A, SQSTM1 2bp del NT311, SQSTM1 ARG96TER, SQSTM1 IVS2DS TA, SQSTM1 ARG312TER, SQSTM1 1bp ins 875T, and the like.

[0043] Still referring to FIG. 2, in some embodiments, composition 200 may be configured to treat aging and / or neurodegeneration (e.g., but not limited to, macular degeneration, cerebral arteriopathy, Stargardt's disease, retinitis pigmentosa, cone-rod dystrophy, fundus flavimaculatus, etc.).Additional ingredients include C2 GLY444ARG, C2 GLU318ASP, C2 IVS10 GT, CFB LEU9HIS, CFB ARG32GLN, and CFB PHE286LEU, CFB PHE286LEU, CFB LYS323GLU, HTRA1 512G-A, HTRA1 ARG370TER, HTRA1 ARG302TER, HTRA1 VAL297MET, HTRA1 ALA252THR, HTRA1 GLY295ARG, HTRA1 ALA321THR, HTRA1 1bp of 126G, HTRA1 ARG166LEU, HTRA1 ALA173PRO, HTRA1 SER284ARG, HTRA1 IVS4AS GA, ABCA4 GLY863ALA, ABCA4 VAL931MET, ABCA4 ALA1028VAL, ABCA4 LEU2027PHE, ABCA4 VAL2050LEU, ABCA4 ASP2177ASN, ABCA4 GLY1961GLU, ABCA4 1-bp of 1847A, ABCA4 IVS30DS GT, ABCA4 IVS40DS GA, ABCA4 TRP855TER, ABCA4 GLU1036LYS, ABCA4 2bp ins 3211GT, ABCA4 LEU1970PHE, ABCA4 LEU1971ARG, ABCA4 ALA1038VAL, ABCA4 IVS13AS GA, ABCA4 TYR340ASP, ABCA4 IVS5AS AG, ABCA4 ARG212CYS, ABCA4 ARG18TRP, ABCA4 ARG572GLN, ABCA4 LEU541PRO&ALA1038VAL, ABCA4 2bp of the 2617CT, ABCA4,LEU1201ARG, ABCA4 PRO1380LEU, ABCA4 1bp of the 2888G, ABCA4 1bp of the 1225A, ABCA4 ARG2030TER, ABCA4 IVS39AS TC, ABCA4 ALA1762ASP, ABCA4 15bp of the NT3539, ABCA4 LEU1940PRO, ABCA4 PRO1780ALA, ABCA4 ARG943GLN, ABCA4 TRP821ARG, ABCA4 GLU1122LYS are also available.

[0044] Still referring to FIG. 2, in some embodiments, composition 200 may be configured to treat metabolic disorders (e.g., but not limited to, thrombophilia due to HRG deficiency, leukocyte adhesion deficiency, etc.). In non-limiting examples, composition 200 may affect HRG GLY85GLU, HRG CYS223ARG, HRG PRO73SER, ITGB2 ARG593CYS, ITGB2 LYS196THR, ITGB2 LEU149PRO, ITGB2 GLY169ARG, ITGB2 ATG-AAG, ITGB2 ARG586TRP&12bp ins, ITGB2 ASN351SER, ITGB2 PRO178LEU, ITGB2 ASP128ASN, ITGB2 IVSDS GA, ITGB2 GLY284SER, ITGB2 SER138PRO, ITGB2 GLY273ARG, ITGB2 IVS4AS 169bp del, etc.

[0045] 2, in some embodiments, inappropriate pH in lysosomes as a result of insufficient functional V-type ATPase can cause B cells to be unable to produce sufficient antigen and / or can cause reduced B cell numbers. This can reduce the effectiveness of the immune system of a subject suffering from such a disorder. In some embodiments, composition 200 can be used to improve the immune system of such a subject. In some embodiments, composition 200 can be administered to a subject with an infection (e.g., a bacterial or viral infection) or a subject at increased risk of infection, where the subject has a disorder involving insufficient functional V-type ATPase.

[0046] 2, in some embodiments, composition 200 can be used to treat viral infections in subjects with insufficient functional V-type ATPase. Such infections can include, but are not limited to, influenza, encephalomyocarditis virus, hepatitis C virus, respiratory syncytial virus, human immunodeficiency virus-1, human rhinovirus, Zika virus, dengue virus, Rift Valley fever virus, measles, Sendai virus, enterovirus 71, coronavirus, Helicobacter pylori infection, or Mycobacterium tuberculosis infection. In a non-limiting example, composition 200 can affect NSP6, NLRP3 activation, and the like.

[0047] 2, in some embodiments, composition 200 may be used to treat bacterial infections in subjects with insufficient functional V-type ATPase. Such infections may include, but are not limited to, bacterially activated thrombosis, immune thrombocytopenia, and the like. In non-limiting examples, composition 200 may affect platelet adhesion, platelet aggregation, and the like. In some embodiments, composition 200 may be configured to treat arteriosclerotic diseases, inflammatory bowel diseases, oncological diseases, and the like. In some embodiments, composition 200 may be configured to treat transient receptor potential mucolipin-1 (TRPML1), two-pore channel (TPC), transient receptor potential mucolipin-3 (TRPML3), P2X 4 As used in the present disclosure, "transient receptor potential mucolipin-1" is a Ca2+ receptor agonist that regulates certain aspects of lysosomal trafficking, including autophagy. 2+ TRPML1 is an inward rectifying channel that transports cations from the lysosomal lumen to the cytosol. Ca2+ release from lysosomes using TRPML1 can regulate transcription factor terminal binding activity using local calcineurin activation, which can induce autophagy and lysosomal biogenesis.

[0048] 2, composition 200 can be utilized to address pH in metabolic pathways (e.g., but not limited to, myopathy, hypertension, diabetes, renal tubular acidosis, chronic kidney disease, fibrosis, and / or cirrhosis). In a non-limiting example, composition 200 can be utilized to address pH in CNS pathways by acidification using V-type ATPase 100 through organelles (e.g., but not limited to, lysosomes and Golgi apparatus) using waste removal and protein synthesis machinery throughout organ systems (e.g., but not limited to, heart, kidney, liver, etc.) to address the diseases disclosed above. By way of non-limiting example, affected metabolic pathways can include interleukin secretion, CD36 translocation, inflammasome activity, endocytic trafficking, HGFR interaction, mTORC1 activation, etc.

[0049] 2, composition 200 can be utilized to address pH in central nervous system (CNS) pathways, including, but not limited to, retinopathy, degeneration, gangliosidosis, encephalopathy, development, ataxia, stenosis, and / or sclerosis. In a non-limiting example, composition 200 can be utilized to address pH in CNS pathways by acidification using V-type ATPase 100 through organelles (e.g., but not limited to, lysosomes and Golgi apparatus) using waste removal and protein synthesis machinery throughout organ systems (e.g., but not limited to, the eye, brain, spinal cord, etc.) to address the diseases disclosed above. By way of non-limiting example, affected CNS pathways can include β / γ-crystallin expression, Notch signaling, Ac45RP neurite outgrowth, catalytic palmitoyltransferase, neuronal polarization, etc.

[0050] 2, composition 200 can be utilized to address pH in immunological pathways (including, but not limited to, coronavirus, RSV, influenza, HIV, staphylococcus, cancer immunogenicity, thrombocytopenia, and / or encephalomyocarditis). In a non-limiting example, composition 200 can be utilized to address pH in CNS pathways by acidification using V-type ATPase 100 through organelles (e.g., but not limited to, lysosomes and Golgi apparatus) using waste removal and protein synthesis machinery through organ systems (e.g., but not limited to, heart, kidney, liver, etc.) to address the diseases disclosed above. By way of non-limiting example, affected immunological pathways can include platelet aggregation, inflammasome activity, mTOR interaction, etc.

[0051]

[0023] With further reference to Figure 2, composition 200 may address additional pH imbalances for other pathways. As a non-limiting example, composition 200 may address additional pH imbalances for the ubiquitin-proteasome pathway. For purposes of this disclosure, the "ubiquitin-proteasome pathway" is an important cellular mechanism responsible for regulating protein degradation and turnover within eukaryotic cells. The ubiquitin-proteasome pathway may maintain cellular homeostasis, remove damaged or misfolded proteins, and control the levels of various proteins involved in cell cycle regulation, signal transduction, and other cellular processes.

[0052] Referring further to Figure 2, in some embodiments, ATP6AP2(116) deficiency may include, but is not limited to, Parkinsonism with spasticity, congenital glycosylation disorder type Iir, and X-linked syndrome intellectual disability (IDD). Lysosomes may serve many functions, including waste degradation and autophagy regulation. Lysosomes may require a specific pH (e.g., somewhere between 4.5 and 5) to function optimally. If the pH of lysosomes is outside this ideal range, one or more of the functions performed by the lysosomes may be altered. The optimal pH of lysosomes may differ from other components of the cell (e.g., the cytosol, whose pH may typically be around 7.2). This pH difference may generate a membrane potential that supports proton transport across the cell. If the pH of the cytosol and / or lysosomes is altered, this can lead to a cascade of problems, including accumulation of cellular components inside the cell, an inability to deliver macromolecular components of the cell, loss of neuronal function, and many other issues. Lysosomal function may also help regulate autophagy as part of a feedback pathway by providing macromolecular building blocks. In some cases, this may ultimately cause the transcription factor EB (TFEB) pathway to shut down lysosomal production, ultimately leading to disorders including, but not limited to, cancer, cardiovascular disease, neurodegeneration, infectious diseases, and / or aging.

[0053] With further reference to FIG. 2 , in some embodiments, composition 200 may comprise 0.001% of the modulator. In some embodiments, composition 200 may comprise about 0.01% by weight of the modulator. In some embodiments, composition 200 may comprise about 0.1% by weight of the modulator. In some embodiments, composition 200 may comprise about 1% by weight of the modulator. In some embodiments, composition 200 may comprise about 5% by weight of the modulator. In some embodiments, composition 200 may comprise about 10% by weight of the modulator. In some embodiments, composition 200 may comprise about 15% by weight of the modulator. In some embodiments, composition 200 may comprise about 20% by weight of the modulator. In some embodiments, composition 200 may comprise about 30% by weight of the modulator. In some embodiments, composition 200 may comprise about 40% by weight of the modulator. In some embodiments, composition 200 may comprise about 50% by weight of the modulator. In some embodiments, composition 200 may comprise about 60% by weight of the modulator. In some embodiments, composition 200 may comprise about 70% by weight of the modulator. In some embodiments, composition 200 may comprise about 80% by weight of the modulator. In some embodiments, composition 200 may comprise about 90% by weight of the modulator. In some embodiments, composition 200 may comprise about 99% by weight of the modulator. In some embodiments, the percentage of the modulator in composition 200 may fall within a range between the above values.

[0054] 2, in some embodiments, composition 200 may comprise 0.001% of the modulator. In some embodiments, composition 200 may comprise about 0.01 mol%. In some embodiments, composition 200 may comprise about 0.1 mol% of the modulator. In some embodiments, composition 200 may comprise about 1 mol% of the modulator. In some embodiments, composition 200 may comprise about 5 mol% of the modulator. In some embodiments, composition 200 may comprise about 10 mol% of the modulator. In some embodiments, composition 200 may comprise about 15 mol% of the modulator. In some embodiments, composition 200 may comprise about 20 mol% of the modulator. In some embodiments, composition 200 may comprise about 30 mol% of the modulator. In some embodiments, composition 200 may comprise about 40 mol% of the modulator. In some embodiments, composition 200 may comprise about 50 mol% of the modulator. In some embodiments, composition 200 may comprise about 60 mol% of the modulator. In some embodiments, composition 200 may comprise about 70 mol% of the modulator. In some embodiments, composition 200 may comprise about 80 mole % of the modulator. In some embodiments, composition 200 may comprise about 90 mole % of the modulator. In some embodiments, composition 200 may comprise about 99 mole % of the modulator. In some embodiments, the percentage of the modulator in composition 200 may fall within a range between the above values.

[0055] With further reference to FIG. 2 , in some embodiments, composition 200 may comprise 0.001% glycosylation precursors. In some embodiments, composition 200 may comprise about 0.01% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 0.1% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 1% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 5% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 10% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 15% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 20% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 30% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 40% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 50% glycosylation precursors by weight. In some embodiments, composition 200 may comprise about 60% by weight of glycosylated precursors. In some embodiments, composition 200 may comprise about 70% by weight of glycosylated precursors. In some embodiments, composition 200 may comprise about 80% by weight of glycosylated precursors. In some embodiments, composition 200 may comprise about 90% by weight of glycosylated precursors. In some embodiments, composition 200 may comprise about 99% by weight of glycosylated precursors. In some embodiments, the percentage of glycosylated precursors in composition 200 may fall within a range between the above values.

[0056] With further reference to FIG. 2 , in some embodiments, composition 200 may comprise 0.001% glycosylation precursors. In some embodiments, composition 200 may comprise about 0.01 mol%. In some embodiments, composition 200 may comprise about 0.1 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 1 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 5 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 10 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 15 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 20 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 30 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 40 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 50 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 60 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 70 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 80 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 90 mol% glycosylation precursors. In some embodiments, composition 200 may comprise about 99 mol% glycosylation precursors. In some embodiments, the percentage of glycosylation precursors in composition 200 may fall within a range between the above values.

[0057]

[0023] Still referring to Figure 2, in some embodiments, composition 200 may comprise 0.001% delivery vehicle. In some embodiments, composition 200 may comprise about 0.01% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 0.1% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 1% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 5% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 10% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 15% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 20% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 30% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 40% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 50% delivery vehicle by weight. In some embodiments, composition 200 may comprise about 60% by weight of delivery vehicle. In some embodiments, composition 200 may comprise about 70% by weight of delivery vehicle. In some embodiments, composition 200 may comprise about 80% by weight of delivery vehicle. In some embodiments, composition 200 may comprise about 90% by weight of delivery vehicle. In some embodiments, composition 200 may comprise about 99% by weight of delivery vehicle. In some embodiments, the percentage of delivery vehicle in composition 200 may be within a range between the above values.

[0058]

[0023] Still referring to Figure 2, in some embodiments, composition 200 may comprise 0.001% of a delivery vehicle. In some embodiments, composition 200 may comprise about 0.01 mol%. In some embodiments, composition 200 may comprise about 0.1 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 1 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 5 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 10 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 15 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 20 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 30 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 40 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 50 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 60 mol% of a delivery vehicle. In some embodiments, composition 200 may comprise about 70 mol% delivery vehicle. In some embodiments, composition 200 may comprise about 80 mol% delivery vehicle. In some embodiments, composition 200 may comprise about 90 mol% delivery vehicle. In some embodiments, composition 200 may comprise about 99 mol% delivery vehicle. In some embodiments, the percentage of delivery vehicle in composition 200 may fall within a range between the above values.

[0059]

[0023] Referring further to Figure 2, in some embodiments, the composition can be customized according to the characteristics of the subject to which the composition is to be administered. For example, different V-type ATPase dysfunctions can cause different symptoms in the subject and require different treatments. In some embodiments, the percentages of the modulator, glycosylated precursor, and delivery vehicle in the composition can be customized according to the subject and / or the specific V-type ATPase dysfunction. For example, if the specific V-type ATPase dysfunction primarily affects glycosylation, the percentage of glycosylated precursor can be increased. In another example, if the specific V-type ATPase dysfunction primarily affects lysosomal pH, the percentage of the modulator can be increased.

[0060] 2, in some embodiments, a pharmaceutical composition can be produced by receiving a glycosylated precursor, receiving a modulating agent, and combining the glycosylated precursor and the modulating agent with a delivery vehicle. In some embodiments, such a pharmaceutical composition can be suitable for treating V-type ATPase dysfunction.

[0061] Referring now to FIG. 3, a method 300 for treating V-type ATPase dysfunction is shown. In step 305, method 300 may include identifying dysfunction of at least a portion of the V-type ATPase protein. The identification may be performed by a computer device as described herein. It should be noted that a test sample may be used to identify the dysfunction. In some cases, the identification may be performed in real time by using a non-invasive test. The portion of the V-type ATPase protein may be any portion of the V-type ATPase protein as described herein. Step 305 may be performed according to, but is not limited to, FIG. 1 and FIG. 2.

[0062] With further reference to FIG. 3 , method 300 can include, in step 310, preparing a glycosylated precursor. It should be noted that the preparation of the glycosylated precursor can be any suitable laboratory preparation. Those skilled in the art will understand, after reading this disclosure, suitable laboratory preparation methods for glycosylated precursors. The glycosylated precursor can be any of the glycosylated precursors described herein. Step 310 can be performed according to, but is not limited to, FIGS. 1 and 2 . Three enzymatic steps can convert glucose to uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a biochemical precursor for the biosynthesis of Neu5Ac and other carbohydrates. The bifunctional enzyme UDP-GlcNAc 2-epimerase / ManNAc kinase (GNE) can convert UDP-GlcNAc to N-acetylmannosamine (ManNAc) using its epimerase domain (GNE), and then generate ManNAc-6-phosphate using its kinase activity (MNK). ManNAc-6-phosphate can be converted to Neu5Ac-9-phosphate by N-acetylneuraminic acid synthase (NANS) and dephosphorylated by Neu5Ac-9-P-phosphatase (NANP) to produce Neu5Ac. In the nucleus, Neu5Ac can be conjugated to cytidine monophosphate (CMP) by CMP-sialic acid synthase (CMAS) and transported into the Golgi system by the CMP-sialic acid transporter SLC35A1. Cytosolic CMP-sialic acid levels can regulate sialic acid synthesis through feedback inhibition of GNE. In the Golgi, 20 sialyltransferase isozymes link sialic acid to glycans. Sialoglycans present on the cell surface or in intracellular compartments (such as lysosomes) can be cleaved by sialidases to release free sialic acid. Free lysosomal sialic acid can be transported into the cytosol by the sialin (SLC17A5) transporter for recycling in the sialic acid biosynthetic pathway or for degradation to ManNAc and pyruvate by N-acetylneuraminic acid pyruvate lyase (NPL).

[0063]

[0033] Still referring to Figure 3, method 300 may include, in step 315, preparing a modulator. In some embodiments, the modulator may include a P2Y12 inhibitor. It should be noted that the preparation of the P2Y12 inhibitor may be any suitable laboratory preparation. Those skilled in the art will understand, after reading this disclosure, suitable laboratory preparation methods for P2Y12 inhibitors. The P2Y12 inhibitor may be any P2Y12 inhibitor described herein. Step 315 may be performed according to, but is not limited to, Figures 1 and 2.

[0064]

[0033] With further reference to Figure 3, method 300 may include, in step 320, combining a glycosylated precursor with a modulator to generate a novel cocktail. After reading this disclosure, those skilled in the art will understand the appropriate laboratory preparation methods and conditions for combining a glycosylated precursor with P2Y12. Conditions may include, but are not limited to, pH environment, temperature, pressure, volume, and the like. The P2Y12 inhibitor and glycosylated precursor may be any of the P2Y12 inhibitors and glycosylated precursors described herein. Step 320 may be performed according to, but is not limited to, Figures 1 and 2.

[0065] With further reference to FIG. 3 , method 300 may include, at step 325, delivering the novel cocktail. The delivering the novel cocktail may be performed in a controlled laboratory environment using a test sample. In some embodiments, the delivering the novel cocktail may be performed non-invasively. The novel cocktail may be any novel cocktail described herein. In some cases, the delivering of the novel cocktail may be performed by a computing device. The computing device may receive at least input from a user indicating test parameters. Test parameters may include time, temperature, pH, etc. The computing device may be any computing device described herein. Step 325 may be performed according to, but is not limited to, FIGS. 1 and 2 . It should be noted that, upon reading this disclosure, one skilled in the art will understand that the steps shown in FIG. 3 may be performed in any order according to, but are not limited to, FIGS. 1 and 2 .

[0066]

[0014] Still referring to Figure 3, in some embodiments, the method may include administering to a subject in need thereof a composition comprising a glycosylated precursor, a modulator, and a delivery vehicle. In some embodiments, the modulator may include a P2Y12 inhibitor. In some embodiments, the delivery vehicle may include a prodrug delivery system. In some embodiments, the method may further include identifying a subject having insufficient functional V-type ATPase. In some embodiments, the subject has a genetic mutation in a V-type ATPase subunit gene. In some embodiments, the subject has a genetic mutation in ATP6AP2. In some embodiments, the glycosylated precursor includes UDP-GLcNR (N-acetylglucosamine). In some embodiments, the glycosylated precursor may include ManNR (N-acetylmannosamine). In some embodiments, the glycosylated precursor may include an item selected from the list consisting of ManNR, ManNAc, UDP-GlcNac, UDP-GlcNR, ManNac-6-P, and SiaNR. In some embodiments, the prodrug delivery system may comprise a prodrug, wherein the prodrug of the prodrug delivery system is physiologically activated. In some embodiments, the prodrug delivery system may comprise a prodrug, wherein the prodrug of the prodrug delivery system is externally activated. In some embodiments, the prodrug delivery system may comprise a retrosynthetic design. In some embodiments, the prodrug delivery system may be capable of crossing the blood-brain barrier.

[0067] Referring now to FIG. 4, an illustrative example of a lysosomal process 400 is shown. The autophagy-lysosomal pathway (ALP) is the primary mechanism for degrading macromolecules. As used in this disclosure, "autophagy" refers to the conserved degradation of cells, removing unnecessary or dysfunctional components through lysosome-dependent regulatory mechanisms. Autophagy is a transport and waste management system found in nearly all cell types. Autophagy regulates and is regulated by a continuously expanding list of pathways throughout the body. Autophagosomes 404 are formed by numerous scaffolding and transport proteins and phosphorylation factors and can ultimately engulf damaged cargo (e.g., inappropriate proteins). The autophagy machinery can distinguish between dysfunctional and healthy cellular components and initiate the biogenesis of autophagosomes 404 around the dysfunctional component (i.e., pre-autophagic structures 412). Signaling pathways send proteins to escort scaffolding proteins 408 that bind to the pre-autophagy structures 412. By way of non-limiting example, the pre-autophagy structures 412 can contain cellular components as small as a single protein or as large and complex as mitochondria, protein aggregates, and pathogens. This cascade of binding and interactions can initiate elongation, nucleation, and maturation to form the autophagosome 404. The first stage of autophagy can involve the degradation process, which is initiated by the fusion of the autophagosome with a lysosome 416 to generate an autolysosome 420. The fusion of the lysosome 416 with the autophagosome 404 can deliver specific pH and key enzymes to complete the degradation. As the final stage of autophagy, lysosomal function can be an essential factor in waste management in cells in multiple organs. Byproducts of degradation can be recycled back into the cell. Defects in lysosomal pH disrupt autophagy, resulting in the accumulation of undegraded waste products that are toxic to cells. Defects in autophagy have been linked to many age-related diseases (e.g., Parkinson's disease, Alzheimer's disease, frontotemporal dementia, and Huntington's disease).

[0068] 4, in addition to membrane repair, metabolic component sequestration, waste sequestration, and transcriptional regulation, lysosomes 416 degrade nearly all harmful waste products in our cells. The pH of lysosomes 416 has been measured to be less than 5, while the pH of the cytosol has been measured to be approximately 7.2. The ability of lysosomes to degrade waste products and fuse with autophagosomes 404 is highly dependent on their low pH or high acidity and a number of enzymes.

[0069] Continuing with Figure 4, lysosomes 416 are the final step in multiple cellular pathways, including the TFEB lysosomal signaling pathway. Lysosomal health is also a regulator of upstream autophagy as part of a feedback pathway by providing macromolecular building blocks. Waste accumulation leading to cell death is found in a vast number of diseases, including cancer, neurodegeneration, cardiovascular disease, infectious diseases, and aging.

[0070] 4, PKA and cAMP regulate the lysosomal 416 pathway. P2Y12 inhibitors (antiplatelet drugs) (including but not limited to clopidogrel (Plavix®), prasugrel (Effient®), ticlopidine (Ticlid®), ticagrelor (Brilinta®), etc.) can be used to activate the cAMP and PKA pathways by forcing phosphorylation of V-type ATPase and restoring lysosomal acidification. V 0 Phosphorylating the subunit mimics the interaction of ATP6AP2 with ATP6AP1 as assembly factors for the V-ATPase, and this restoration mechanism alleviates some of the autophagy symptoms described herein.

[0071] Referring now to FIG. 5, an exemplary diagram of the pH environment within a cell 500 is shown. Cellular acidity (pH) is determined by H+ (cation / proton) concentration and affects metabolism, membrane potential, cell growth, membrane trafficking, muscle function, and many other factors. Organelles (e.g., lysosomes 504, Golgi apparatus 508, and endoplasmic reticulum 512) maintain specific organelle pH to generate, modify, and degrade important proteins. Golgi 508 glycosylation, lysosomal degradation, and autophagy are three important pathways dramatically affected by mutations in V-ATPase assembly factors. As used in this disclosure, "glycosylation" refers to the process by which carbohydrates are covalently attached to target macromolecules (e.g., proteins and lipids).

[0072] Still referring to Figure 5, the Golgi 508 and endoplasmic reticulum 512 synthesize and package proteins. The pH in the endoplasmic reticulum (ER) (512) and Golgi 508 is precisely maintained for enzymes to carry out protein synthesis. Sialyltransferase is an enzyme that attaches sialic acid to proteins during glycosylation under specific pH conditions. Glycosylation is a post-translational (final) modification of proteins that stabilizes and designates them for specific required functions. Glycosylation results in a properly synthesized protein, which contains a sialic acid moiety. In mutated V-type ATPases, glycosylation is impaired, and the protein does not acquire its functional unit and eventually misfolds, generating harmful waste products inside the cell.

[0073] Continuing with Figure 5, in an impaired pH environment, sialic acid binding is disrupted. Restoration can be achieved, in part, by adding one or more compounds that act as glycosylation precursors (i.e., including, but not limited to, UDP-GlcNR (N-acetylglucosamine) and / or ManNR (N-acetylmannosamine)). This potential therapeutic agent can improve the synthesis and modification of all proteins in the Golgi and endoplasmic reticulum.

[0074] Referring now to Figures 6A-6D, an exemplary embodiment of a prodrug structure 600 is shown. A common problem in drug delivery is low permeability. Prodrug strategies have enhanced pharmacokinetic properties in various applications, including chemotherapy. The use of this prodrug carrier application method increases the efficacy of the administered dose. Prodrugs can be designed to be specific to the target tissue or cellular environment. Reducing off-target effects is a common goal in drug safety. Prodrug delivery systems can assist in crossing the blood-brain barrier. In a non-limiting example, a prodrug can include a carrier prodrug as shown in Figure 6A. As a non-limiting example, a carrier prodrug can include a drug, a linker, and a carrier. For example, but not limited to, carrier prodrugs can include folic acid, RGD peptide, sugars, and the like. In some embodiments, the carrier prodrug can be physiologically activated. In another non-limiting example, a prodrug can include a decaging prodrug as shown in Figure 6B. In some embodiments, the decaging prodrug can be externally activated. In another non-limiting example, a prodrug can include a bioprecursor prodrug, such as that shown in FIG. 6C. In some embodiments, a bioprecursor prodrug can be physiologically activated. In another non-limiting example, a prodrug can include a synthetic prodrug, such as that shown in FIG. 6D. In some embodiments, a prodrug can be designed by finding an appropriate reaction (e.g., metathesis, aromatization, etc.). The structure of the prodrug can then be optimized, in non-limiting examples, by, for example, using scaffold selection and leaving groups to increase cascade reactivity, increasing activity in biocatalysis using hydrophobic esters, increasing hydrolytic stability using pivalates, reducing the impact of the prodrug using bulky esters, etc.

[0075] 6A-6D, unique front-end and back-end therapeutic approaches can be implemented using prodrug strategies. In particular, a front-end approach can include supplementation with N-acetylmannosamine, which can ameliorate the effects of glycosylation on the liver. Furthermore, the effects of glycosylation on protein synthesis, protein translation, and glycoprotein interactions with various organelles in the liver, kidney, heart, brain, and spinal cord can be exploited. Furthermore, a back-end approach can include the addition of a P2Y12 antiplatelet drug, which can improve pH in retinal cell lysosomes and improve Golgi, endoplasmic reticulum (ER), and lysosomal pH in multiple tissues, including those listed above.

[0076] Referring now to FIG. 7 , a diagram of an exemplary prodrug delivery system 700 using ManNAc is shown. The prodrug delivery system 700 can be consistent with any of the prodrug delivery systems 700 described in this disclosure. In some embodiments, a prodrug asset can be included in a cocktail treatment for ATP6AP2 deficiency. The prodrug asset can be a ProTide prodrug, a category of prodrugs that just attach to a phosphate. The prodrug asset can be used with another glycosylated precursor, ManNAc-6-phosphate. As shown in FIG. 7 , the masked lipophilic ManNAc-6-P (704) can undergo enzymatic activation 708 and enzymatic deacetylation 712 to generate ManNAc-6-P (716), a precursor to sialic acid. In a non-limiting example, this can restore sialic acid at 1 mM in a GNE-deficient cell line. Glycosylated precursors can be used in the treatment of ATP6AP2, as further discussed in this disclosure.

[0077] Referring now to Figure 8, an illustrative embodiment of the structure of a V-type ATPase is shown. In some embodiments, the V 1 is widely responsible for hydrolyzing ATP, and V 0 V can be widely responsible for transporting H+ across membranes. 1 V-ATPase complexes can be composed of subunits A through H. Each V-ATPase complex can contain three A subunits and three B subunits. ATP hydrolysis can occur at catalytic sites on the A and B subunits. The A and B subunits can also provide nucleotide binding sites to regulate V-ATPase activity. 0 V can be composed of subunits a, c, c'', d, e and Ac45 in mammals, and a, c, c', c'', d and e in yeast. 1 and V 0 The ATP hydrolysis pathway is connected to the V pathway by a central stalk composed of subunits D, F, and d, and three peripheral stalks composed of subunits C, E, G, and part of subunit a. 0 This rotation can cause a rotation of the components of this V 0 Components of the ring can be caused to receive protons and deliver them to a channel on the other side of the membrane, where they are released.

[0078] Referring now to Figure 9, a diagram of the function of a V-type ATPase (proton pump in Figure 9) is presented. ATP is used to transport H+ from one side of a membrane to the other. This can be used by the cell to create a pH imbalance across the membrane. While the V-type ATPase in this diagram is present in the outer cell membrane, V-type ATPases can also be located in membranes of various organelles (e.g., the Golgi apparatus and lysosomes).

[0079] Referring now to Figure 10, an exemplary glycosylation process is shown. For example, Man I, GnT1, Man II, GnTII, and GnTV can perform steps in the glycosylation process. In some embodiments, glycosylation is pH-dependent and may not occur properly when the pH of the Golgi apparatus is abnormal due to the lack of a functional V-type ATPase.

[0080] 11, an exemplary process for converting glycosylation precursors to sialic acid is shown. In some embodiments, the addition of glycosylation precursors can improve glycosylation in situations where the pH of the Golgi apparatus is abnormal due to the lack of a functional V-type ATPase.

[0081] Referring now to Figure 12, the autophagy-lysosome pathway is shown as it relates to lysosomal fusion and waste degradation. In some embodiments, the lack of functional V-type ATPase can cause lysosomal pH to be higher than normal. This can cause lysosome fusion with autophagosomes to not sufficiently reduce the lysosomal pH. This can cause the autolysosomes to not be able to effectively degrade their contents.

[0082] Referring now to Figure 13, the interaction of TFEB, AMPK, and V-type ATPase is shown as it relates to lysosomal function and biogenesis.

[0083] Referring now to FIG. 14, the chemical structures and pathways of one or more glycosylation precursors, including ManNR, UDP-GlcNR, and SiaNR, are disclosed therein.

[0084] The foregoing has been a detailed description of exemplary embodiments of the present invention. Various modifications and additions may be made without departing from the spirit and scope of the present invention. Features of each of the various embodiments described above may be combined as appropriate with features of other embodiments described to provide numerous feature combinations in related novel embodiments. Moreover, while the above describes many separate embodiments, what has been described herein is merely illustrative of the application of the principles of the present invention. Furthermore, while certain methods herein may be shown and / or described as being performed in a particular order, that order is highly variable within the ordinary skill in the art to accomplish methods, systems, and software according to the present disclosure. Accordingly, the present specification is intended to be illustrative only and is not intended to otherwise limit the scope of the present invention.

[0085] Exemplary embodiments are disclosed above and shown in the accompanying drawings. It will be understood by those skilled in the art that various modifications, omissions, and additions may be made to that specifically disclosed herein without departing from the spirit and scope of the invention.

Claims

1. a method of treating a dysfunction of a V-ATPase, the method comprising identifying a dysfunction of at least a portion of a V-ATPase protein in a subject; preparing a composition comprising a glycosylated precursor, a modulator, and a delivery vehicle; and The composition is administered to the subject.

2. The method of claim 1 , wherein the modulator comprises a P2Y12 inhibitor.

3. 2. The method of claim 1, wherein the P2Y12 inhibitor is selected from the list consisting of clopidogrel, prasugrel, ticlopidine, and ticagrelor.

4. The method of claim 1 , wherein the modulator comprises ticagrelor.

5. The method of claim 1, wherein the subject has a genetic mutation in a V-ATPase subunit gene.

6. The method of claim 1, wherein the subject has a genetic mutation in ATP6AP2.

7. 2. The method of claim 1, wherein the glycosylated precursor comprises UDP-GLcNR (N-acetylglucosamine).

8. The method of claim 1, wherein the glycosylated precursor comprises ManNR (N-acetylmannosamine).

9. The method of claim 1, wherein the glycosylation precursor comprises a sialic acid precursor.

10. 10. The method of claim 9, wherein the sialic acid precursor comprises an item selected from the list consisting of ManNR, ManNAc, UDP-GlcNac, UDP-GlcNR, ManNac-6-P, and SiaNR.

11. 10. The method of claim 9, wherein the delivery vehicle comprises a prodrug delivery system.

12. 12. The method of claim 11, wherein the prodrug delivery system comprises a prodrug, and the prodrug of the prodrug delivery system is physiologically active.

13. 12. The method of claim 11, wherein the prodrug delivery system comprises a prodrug, and the prodrug of the prodrug delivery system is externally activated.

14. 12. The method of claim 11, wherein the prodrug delivery system comprises a retrosynthetic design.

15. 12. The method of claim 11, wherein the prodrug delivery system is capable of facilitating delivery of the glycosylated precursor, the modulator, or both, across the blood-brain barrier.

16. The method of claim , wherein the composition increases glycosylation relative to the glycosylation precursor alone.

17. The method of claim , wherein the composition improves lysosomal function more than the modulator alone.

18. A pharmaceutical composition, the composition comprising: a glycosylated precursor; a modulator; and a delivery vehicle.

19. 19. The pharmaceutical composition of claim 18, wherein the modulator comprises a P2Y12 inhibitor.

20. 19. The pharmaceutical composition of claim 18, wherein the P2Y12 inhibitor is selected from the list consisting of clopidogrel, prasugrel, ticlopidine, and ticagrelor.

21. 19. The pharmaceutical composition of claim 18, wherein the modulator comprises ticagrelor.

22. 20. The pharmaceutical composition of claim 18, wherein the delivery vehicle comprises a prodrug delivery system.

23. 20. The pharmaceutical composition of claim 18, wherein the prodrug delivery system comprises a prodrug, and the prodrug of the prodrug delivery system is physiologically active.

24. 19. The pharmaceutical composition of claim 18, wherein the glycosylated precursor comprises UDP-GLcNR (N-acetylglucosamine).

25. 19. The pharmaceutical composition of claim 18, wherein the glycosylated precursor comprises ManNR (N-acetylmannosamine).

26. 19. The pharmaceutical composition of claim 18, wherein the glycosylation precursor comprises an item selected from the list consisting of ManNR, ManNAc, UDP-GlcNac, UDP-GlcNR, ManNac-6-P, and SiaNR.

27. 20. The pharmaceutical composition of claim 18, wherein the prodrug delivery system is capable of facilitating delivery of the glycosylated precursor, the modulator, or both, across the blood-brain barrier.

28. 19. The pharmaceutical composition of claim 18, wherein the modulator comprises from 0.01% to 99% of the composition.

29. 19. The pharmaceutical composition of claim 18, wherein the glycosylated precursor comprises 0.01% to 99% of the composition.

30. 20. The pharmaceutical composition of claim 18, wherein the delivery vehicle comprises: 0.01% to 99% of the composition.

31. A method for producing a pharmaceutical composition, the method comprising receiving a glycosylated precursor; receiving a modulator; The glycosylated precursor and modulator are combined with a delivery vehicle.

32. 32. The method of claim 31 , wherein the modulator comprises a P2Y12 inhibitor.

33. 32. The method of claim 31, wherein the modulator comprises ticagrelor.

34. 32. The method of claim 31, wherein the P2Y12 inhibitor is selected from the list consisting of clopidogrel, prasugrel, ticlopidine, and ticagrelor.

35. 32. The method of claim 31, wherein the glycosylation precursor comprises an item selected from the list consisting of ManNR, ManNAc, UDP-GlcNac, UDP-GlcNR, ManNac-6-P, and SiaNR.

36. 32. The method of claim 31 , wherein the delivery vehicle comprises a prodrug delivery system.

37. A method for treating a condition of V-ATPase dysfunction, comprising the steps of: a glycosylated precursor, a modulator, and a method for treating a condition of V-ATPase dysfunction. Delivery vehicle.

38. 38. The method of claim 37, wherein the modulator comprises a P2Y12 inhibitor.

39. 38. The method of claim 37, wherein the P2Y12 inhibitor is selected from the list consisting of clopidogrel, prasugrel, ticlopidine, and ticagrelor.

40. 38. The method of claim 37, wherein the modulator comprises ticagrelor.

41. 38. The method of claim 37, wherein the glycosylation precursor comprises an item selected from the list consisting of ManNR, ManNAc, UDP-GlcNac, UDP-GlcNR, ManNac-6-P, and SiaNR.

42. 38. The method of claim 37, wherein the delivery vehicle comprises a prodrug delivery system.

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