Nanoparticle formulations for a non-opioid drug

Abstract

This disclosure is directed to a nanoparticle formulation for N,N-Diethyl-2-(N-(2-(4-hydroxyphenylamino)-2-oxoethyl)sulfamoyl)benzamide (SRP-3D (DA)), an active pharmaceutical ingredient that is insoluble in water and cannot be dissolved in any injection-safe vehicle to a sufficient concentration for dosing. Also encompassed by the disclosure are nanoparticulate suspension comprising a nanoparticulate SRP-3D (DA) (or SRP-3D (DA) nanoparticles), a wetting agent and a cryoprotectant. The nanoparticulate compositions comprise SRP-3D (DA) particles having an average particle size of less than about 500 nm or less than about 300 nm.

Description

NANOPARTICLE FORMULATIONS FOR A NON-OPIOID DRUGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to US Provisional Patent Application No. 63 / 451,481, filed March 10, 2023, the contents of which are hereby incorporated by reference in its entirety for all purposes.FIELD OF THE DISCLOSURE

[0002] This disclosure relates to solid forms of N,N-Di ethyl -2-(N-(2-(4- hydroxyphenylamino)-2-oxoethyl)sulfamoyl)benzamide (SRP-3D (DA)), a drug substance; nanoparticle formulations, methods for the preparation thereof and dispersions containing such nanoparticles. This disclosure further relates to the use of such nanoparticles in pharmaceutical compositions and methods of treating mammals.BACKGROUND OF THE DISCLOSURE

[0003] SRP-3D (DA) is a drug molecule having a MW of 405.5 g / mol and a structure as shown FIG 1. SRP-3D (DA) is a non-narcotic analgesic for acute and chronic pain with an expected daily human dose in the 100 - 1,000 mg range. The desired routes of administration for SRP-3D (DA) are intravenous (IV) injection and oral intake (PO).

[0004] SRP-3D (DA) is insoluble in water. It is difficult to inject SRP-3D (DA) IV due to its lack of solubility. To develop an IV injection formulation, various excipients have been tried to solubilize SRP-3D (DA), including organic solvents, surfactants, emulsifiers, suspending agents, and solubilizing agents, but to no avail. These commonly used solubilizers are either too toxic or unable to solubilize SRP-3D (DA) to a sufficient concentration for dosing.

[0005] Any injected drug must be sterile. To sterilize a drug, filtration through a 0.2-micron filter is commonly used. However, a suspension is generally not filterable through such a small pore filter membrane. Therefore, an injectable suspension drug must be formulated such that it can be sterilized by either filtration (e.g., with drug particles small enough to pass freely through a 0.2 pm filter) or other means of sterilization such as gamma irradiation.

[0006] In addition, an injectable suspension formulation must be stable so it can provide the desired shelf life for commercial use. The stability of a suspension is often measured by the ability of the suspended particles to maintain their particle size. A suspension that exhibits growth in particle size or aggregation of particles where the particles stick together to form a cake (“caking”) is deemed unstable and therefore not acceptable for pharmaceutical use.

[0007] SRP-3D (DA) has poor oral bioavailability due to its lack of solubility (e.g., 0.3 mg / mL in either fed- or fasted-state simulated intestinal fluid), making its oral administration unacceptable. Therefore, there is a need for an SRP-3D (DA) formulation to allow its administration by injection and oral intake. The present disclosure satisfies this need and offers other advantages as well.BRIEF SUMMARY

[0008] This disclosure relates to new nanoparticle formulations of SRP-3D (DA) where the SRP-3D (DA) nanoparticles are safe for IV injection or oral administration. Moreover, the SRP-3D (DA) nanoparticles disclosed in this application also provide a greatly increased oral bioavailability.

[0009] For IV injection, the formulation must not contain any large particles that can block the capillary vessels and cause embolism. The United States Pharmacopeia has set a specification for an injected drugs as follow (USP <788> Method 1): “the average number of particles present in the units tested does not exceed 6000 per container equal to or greater than 10 micron and does not exceed 600 per container equal to or greater than 25 micron.”

[0010] The present disclosure provides an injectable, stable, dispersible SRP-3D (DA) nanoparticle formulation and a method for preparing such nanoparticles by wet milling in the presence of grinding media in conjunction with a wetting agent. The nanoparticles can be formulated for either safe injection or oral administration with enhanced oral bioavailability.

[0011] More specifically, in accordance with this disclosure, there are provided dry solid nanoparticles comprising or consisting essentially of crystalline SRP-3D (DA) having an average particle size of less than about 400 nm and sufficiently stable for use in a pharmaceutical dosage form.

[0012] This disclosure also provides an injectable suspension dosage form comprising or consisting essentially of a liquid or suspension vehicle or medium and the above-described nanoparticles dispersed in the liquid.

[0013] This disclosure also provides a nanosuspension composition comprising or consisting essentially of the above-described SRP-3D (DA) nanoparticles in an aqueous suspension vehicle.

[0014] In one embodiment, about the nanosuspension can pass through a 10-micron filter.

[0015] In another embodiment, the nanosuspension can pass through a 0.2-micron filter.

[0016] In another embodiment, the nanosuspension can be sterilized by gamma irradiation.

[0017] In a particularly relevant embodiment, the nanosuspension is stable.

[0018] In a particularly relevant embodiment of the disclosure, the nanosuspension disclosed in this disclosure is safe for IV injection.

[0019] In another embodiment of the disclosure, the nanosuspension disclosed in this disclosure can provide faster in vitro dissolution for SRP-3D (DA) than the SRP-3D (DA) of a larger particle size.

[0020] In another particularly relevant embodiment of the disclosure, the nanosuspensions or nanoparticles disclosed in this disclosure provide increased oral bioavailability for SRP-3D (DA).

[0021] In another embodiment of the disclosure, there is provided a wet milling method of preparing the above-described particles comprising the steps of dispersing SRP-3D (DA) drug substance in a liquid suspension vehicle and applying mechanical attrition in the presence of grinding media (balls or beads) and a surface modifier or wetting agent to reduce the particle size of SRP-3D (DA) to an effective average particle size of less than about 400 nm.

[0022] In a particular embodiment of the disclosure, there is provided a pharmaceutical composition comprising the dry nanoparticles or a nanosuspension wherein the nanoparticles are dispersed in a pharmaceutically acceptable suspension vehicle. Such pharmaceutical compositions are useful for treating mammals.

[0023] In still yet another embodiment, the present disclosure provides a method for treating, reducing, or ameliorating pain in a subject in need thereof, the method comprising: administering a compound of Formula I to the subject, to thereby treat, reduce or ameliorate pain.

[0024] These and other aspects, objects and embodiments will become more apparent when read with the detailed description and drawings that follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 illustrates the SRP-3D (DA) chemical structure.

[0026] FIG. 2 illustrates the optical microscopic images of regular un-milled SRP-3D (DA) particles (Left) versus SRP-3D (DA) nanoparticles of 400 nm average size (Right).

[0027] FIG. 3 illustrates a method for synthesizing SRP-3D (DA).

[0028] FIG. 4 illustrates the1H NMR spectrum and structure correlation of SRP-3D (DA).

[0029] FIG. 5 illustrates the13C NMR spectrum and structure correlation of SRP-3D (DA).

[0030] FIG. 6 illustrates the FT-IR spectrum of SRP-3D (DA).

[0031] FIG. 7 illustrates a mass spectrum of SRP-3D (DA).

[0032] FIG. 8 illustrates an expanded view of the mass spectrum of the FIG. 7 mass spectrum of SRP-3D (DA).

[0033] FIG. 9 illustrates the daughter peaks of m / z 406.1 of SRP-3D (DA).

[0034] FIG. 10 illustrates the X-ray powder diffraction pattern of SRP-3D (DA) Form A, Specimen 1.

[0035] FIG. 11 illustrates the X-ray powder diffraction pattern of SRP-3D (DA) Form A, Specimen 2 (unground specimen).

[0036] FIG. 12 illustrates the X-ray powder diffraction pattern of SRP-3D (DA) Form A, Specimen 3 (ground specimen).

[0037] FIG. 13 illustrates a sample chromatogram of the C-4 Identification Standard for the HPLC Method for In-Process Monitoring of Steps 1~3 and Recrystallization.

[0038] FIG. 14 illustrates a sample chromatogram of the Step 2 endpointing standard for the HPLC Method for In-Process Monitoring of Steps 1~3 and Recrystallization.

[0039] FIG. 15 illustrates a sample chromatogram of the Step 3 endpointing standard for the HPLC Method for In-Process Monitoring of Steps 1~3 and Recrystallization.

[0040] FIG. 16 illustrates a Differential Scanning Calorimetry (DSC) curve of SRP-3D (DA).

[0041] FIG. 17 illustrates the asymmetric unit from the crystal structure of SRP-3D (DA) Form A determined by X-ray diffraction. Carbon atoms are gray, hydrogen atoms are white, oxygen atoms are red (3), nitrogen atoms are blue (2), and sulfur atoms are yellow (4).

[0042] FIG. 18 illustrates a Thermogravimetric Analysis Curve (TGA) of SRP-3D (DA).DETAILED DESCRIPTION OF THE DISCLOSURE

[0043] Definitions1. Nanoparticles

[0044] Nanosuspensions or nanoparticulate compositions referred to herein are formulations containing nanoparticles, i.e., particles having at least one external dimension of less than about 1 pm, of a poorly soluble drug or a non-opioid and one or more than one wetting agent.2. Wetting agent

[0045] The compositions of the disclosure include one or more wetting agents or surface stabilizers. The wetting agents of the disclosure are preferably adsorbed on, or associated with, the surface of the drug particles. The wetting agents especially useful herein preferably do not chemically react with the drug or its particles. Suitable wetting agents can preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients may include various polymers, low molecular weight oligomers, natural products, and surfactants. Preferred wetting agents include nonionic, anionic, cationic and zwitterionic surfactants, hydrophilic polymers, and polyether-derivatized polysaccharides. A more preferred wetting agent is a bile salt.

[0046] A wetting agent can be the same chemical as a drug solubilizer. For example, sodium deoxycholate which is a preferred wetting agent for this disclosure, can also be usedas a solubilizer to solubilize water-insoluble drugs, such as amphotericin B, resveratrol, and oxaprozin. However, the function of a wetting agent is different from that of a solubilizer. A solubilizer is used to solubilize an insoluble drug while a wetting agent is not intended to solubilize a drug. A wetting agent is added to reduce the surface tension of the solid drug particles so they can form a physically more stable suspension in water and enhance the efficiency of wet milling to the desired particle size. The solid particles must remain undissolved in the presence of a wetting agent in a nanosuspension of this disclosure.

[0047] Moreover, a wetting agent is generally used at a lower concentration than a typical concentration used for a drug solubilizer (>10%). The typical wetting agent concentration in a nanosuspension of this disclosure is less than 10%, preferably less than 5%, more preferably less than 2% and most preferably less than 1%, based on the weight of the nanosuspension.

[0048] Furthermore, the wetting agent-to-drug weight ratio in a nanosuspension of the current disclosure is much lower than a typical solubilizer-to-drug weight ratio. For example, each vial of Fungizone® contains 50 mg insoluble drug amphotericin B and 40 mg sodium deoxy cholate as the solubilizer, or a solubilizer-to-drug weight ratio of 5:4. The typical wetting agent-to-drug weight ratio in a nanosuspension of this disclosure is less than 1 : 1, preferably less than 0.5: 1, more preferably less than 0.2: 1 and most preferably less than 0.1 : 1.3. Bile salt

[0049] Bile salts are one of the primary components of bile. Bile is a greenish-yellow fluid made by the liver and stored in the gallbladder. Bile salts have been exploited to improve hydrophilicity of water-insoluble drugs (such as amphotericin B, resveratrol, and oxaprozin) mainly by the wetting effect. Also, bile salts have been employed as permeation enhancers in topical dosage forms including buccal, ocular, nasal, and transdermal routes of administration.

[0050] As used herein, a “bile salt” used in the compositions of the current disclosure as a wetting agent refers to an alkali salt, such as sodium salt, of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glyco- or taurodeoxycholic acid, chenodeoxycholic acid and glyco- or taurochenoxydeoxycholic acid. Sodium glycocholate and sodium deoxycholate are preferred bile salts. Other cations including, but not limited to, potassium, lithium, calcium, arginine, lysine or ammonium may also be used in a bile salt. To prepare a wettingagent, either a bile acid or a bile salt can be used as a starting material. If a bile acid is used as the starting material, it is usually converted (completely or partially) to its bile salt (i.e., the conjugate base) by adjusting the pH upward before or during the preparation of a wetting agent.

[0051] If a bile salt is used as the starting material, it may be converted (completely or partially) to its bile acid (i.e., the conjugate acid) by adjusting the pH downward during the preparation of a wetting agent. In one aspect, the term “bile salt” or “B” used in this application also refers to a mixture of a bile salt and its conjugate acid. For example, the pKa of deoxycholic acid is 6.58 (The Merck Index, 12thEd., Entry #12946). According to the Henderson-Hasselbalch equation, a solution of deoxycholic acid in water at pH 4.58 would contain a mixture of 99% protonated deoxy cholic acid (bile acid) and 1% deprotonated deoxycholate (bile salt). Such a bile acid / bile salt mixture is well suited for use as a wetting agent.4. Cryoprotectant

[0052] “Cryoprotectants”, as used in the present disclosure refers to those ingredients which are added to maintain the discrete and nanosized particles of SRP-3D (DA) in the nanosuspension during a freeze-thawing, freeze-drying, air-drying, vacuum-drying, or spraydrying process. Cryoprotectants that may be used in the nanosuspension compositions of this disclosure include, but are not limited to, polyols, monosaccharides, disaccharides, polysaccharides, amino acids, peptides, proteins, and hydrophilic polymers, or mixtures thereof. Polyols that may be used in the present disclosure include, but are not limited to, glycerin, mannitol, erythritol, maltitol, xylitol, sorbitol, polyglycitol or mixtures thereof. Monosaccharides that may be used in this disclosure include, but are not limited to, glucose, mannose, fructose, lactulose, allose, altrose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose or mixtures thereof. Disaccharides that may be used in this disclosure include, but are not limited to, sucrose, lactose, maltose, isomaltose, trehalose, cellobiose or mixtures thereof. Polysaccharides that may be used in this disclosure include, but are not limited to, cellulose, amylose, inulin, chitin, chitosan, amylopectin, glycogen, pectin, hyaluronic acid or mixtures thereof. Amino acids that may be used in this disclosure include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine or mixtures thereof.Peptides that may be used in this disclosure include, but are not limited to, diglycine and triglycine. Proteins that may be used in this disclosure include, but are not limited to, albumin, collagen, casein, and gelatin. Hydrophilic polymers that may be used in this disclosure include, but are not limited to, cellulose or its derivatives such as microcrystalline cellulose, methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polyethylene glycols, povidones, poloxamers, polyvinyl alcohols or mixtures thereof. The most preferred hydrophilic polymers are polyethylene glycols and povidones. The concentration of the cryoprotectants used in the nanosuspension compositions may be in the range of about 2% to about 40% w / w, such as about 5% to about 20% w / w and about 10% to about 15% w / w.

[0053] As used herein, the term “impurity” refers to extraneous matter included in a compound or a pharmaceutically acceptable salt, or polymorph thereof. Extraneous matter includes one or more substances that are different from the compound, or the pharmaceutically acceptable salt, or polymorph thereof. In certain embodiments, the extraneous matter is undesired extraneous matter. For example, when an anhydrous compound is desired, a solvent (e.g., water) included in the compound is an impurity. When a crystalline compound is desired, an amorphous form of the compound included in the compound is an impurity. When a certain polymorph of a compound is desired, a different polymorph of the compound included in the compound is an impurity. The term “substantially free of impurities” means that a compound (e.g., a compound of Formula I), or a pharmaceutically acceptable salt, or polymorph thereof, contains no significant amount of extraneous matter (e.g., undesired extraneous matter). What amount of the extraneous matter constitutes a significant amount depends on the subject matter and is understood in the art. In certain embodiments, greater than about 1% to about 10% is a significant amount or about 1 wt %, about 2 wt %, about 3 wt %, about 5 wt %, about 7 wt %, or about 10 wt % of extraneous matter in a compound, or a pharmaceutically acceptable salt, or polymorph thereof, is a significant amount of extraneous matter. The compound is substantially pure if the sample contains between about 90-100% w / w, such as about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% of the compound or a pharmaceutically acceptable salt, or polymorph thereof.

[0054] The present disclosure is directed to new nanoparticle formulations of SRP-3D (DA) where the SRP-3D (DA) nanoparticles are safe for injection. Moreover, the SRP-3D (DA) nanoparticles disclosed in this application also provide greatly increased oral bioavailability.

[0055] SRP-3D (DA) is being developed for use as an analgesic. SRP-3D (DA) is an acetaminophen (ApAP) derivative with the chemical structure shown in FIG 1. SRP-3D (DA) has an empirical formula of C19H23N3O5S and a molecular weight of 405.5 g / mol. SRP-3D (DA) exhibits anti-acute and chronic pain activities.

[0056] Because SRP-3D (DA) is practically insoluble in water, attaining an injectable formulation or sufficient bioavailability by oral administration of this drug is problematic. Efforts to solubilize SRP-3D (DA) to form a solution formulation using polysorbates, propylene glycol, polyethylene glycol (PEG), DMSO, Labrasol, methylcellulose and cottonseed oil failed to produce a safe, bioavailable, or stable formulation for injection or oral administration.5. Compositions / Formulations

[0057] In one embodiment, the present disclosure is directed to formulation compositions comprising nanoparticulate SRP-3D (DA) or SRP-3D (DA) nanoparticles. The compositions comprise nanoparticulate SRP-3D (DA) and at least one wetting agent.

[0058] The SRP-3D (DA) nanoparticles of this disclosure have an average particle diameter less than 1 micron, preferably less than 500-400 nm, more preferably less than 200 nm, and most preferably less than 100 nm. Smaller particles (<80 nm) are desired but are difficult to achieve by the milling method disclosed in this application. In certain instances, the particles are about 50 nm to 1000 nm, such as for example, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, and / or about 1000 nm. In certain instances, the particles are less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 75 nm, or less than about 50 nm.

[0059] The size of SRP-3D (DA) nanoparticles of this disclosure can be measured using various analytical instruments including but not limited to dynamic light scattering, laser diffraction, electron microscope, and field flow fractionation techniques.

[0060] The nanoparticulate SRP-3D (DA) of this disclosure may be provided in a dry powder form or in a suspension form in which the nanoparticles are suspended in a liquidsuch as water. Such suspension is also referred to as SRP-3D (DA) nanosuspension or SRP- 3D (DA) nanoparticulate suspension.

[0061] Nanoparticulate SRP-3D (DA) formulations suitable for parenteral injection (e.g., intravenous, intramuscular, or subcutaneous) for the treatment of acute and chronic pain are highly superior to conventional SRP-3D (DA) formulations because they are expected to have much faster onset of action due to the nanoparticulate size of the active agent.

[0062] Nanoparticulate SRP-3D (DA) formulations combine fast onset with long duration of action following IV injection.

[0063] Nanoparticulate formulations of SRP-3D (DA) also provide an increased oral bioavailability as compared to other formulations containing larger SRP-3D (DA) particles (e.g., > 1 micron).

[0064] Additionally, nanoparticulate SRP-3D (DA) formulations do not possess the toxic or undesired side effects of solubilizers used in solution formulations. For example, nanoparticulate formulations of SRP-3D (DA) do not cause the hypersensitivity reaction of many detergent-like drug solubilizers (polysorbate 80, Cremophor, Labrasol, etc.), kidney toxicity of the cyclodextrins, or acute toxicity of organic solvents such as ethanol, DMSO, PEG, etc. A nanoparticulate SRP-3D (DA) formulation of this disclosure is safer than a solution formulation.

[0065] The nanoparticulate SRP-3D (DA) particles of this disclosure surprisingly exhibit superior safety for injection as compared to the solution formulations.

[0066] The nanoparticulate SRP-3D (DA) particles of this disclosure surprisingly exhibit enhanced oral bioavailability as compared to other SRP-3D (DA) formulations containing larger SRP-3D (DA) particles (e.g., > 1 micron).

[0067] In one embodiment, the nanoparticulate SRP-3D (DA) formulation comprises SRP- 3D (DA) and a bile salt as the wetting agent.

[0068] In one embodiment, the nanoparticulate SRP-3D (DA) formulation comprises sodium deoxycholate as the wetting agent.

[0069] In one embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprise SRP-3D (DA) and a wetting agent in a weight ratio between 5 to 1 and 40 to 1 in a nanosuspension form.

[0070] In a preferred embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprise SRP-3D (DA) and sodium deoxy cholate in a weight ratio between 10 to 1 and 30 to 1 in a nanosuspension form.

[0071] In a more preferred embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprise SRP-3D (DA) and sodium deoxy cholate in a weight ratio between 15 to 1 and 25 to 1 in a nanosuspension form.

[0072] In the most preferred embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprises SRP-3D (DA) and sodium deoxycholate in a weight ratio between 18 to 1 and 20 to 1 in a nanosuspension form.

[0073] In one embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprise SRP-3D (DA) and a wetting agent in a weight ratio between 10 to 1 and 30 to 1 in a dry powder form.

[0074] In a preferred embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprise SRP-3D (DA) and sodium deoxy cholate in a weight ratio between 15 to 1 and 25 to 1 in a nanosuspension form.

[0075] In a more preferred embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprise SRP-3D (DA) and sodium deoxy cholate in a weight ratio between 15 to 1 and 20 to 1 in a dry powder form.

[0076] In the most preferred embodiment, the nanoparticulate SRP-3D (DA) particles of this disclosure comprise SRP-3D (DA) and sodium deoxycholate in a weight ratio between 18 to 1 and 20 to 1 in a dry powder form.

[0077] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles, a wetting agent and a suspending vehicle.

[0078] In another embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles, a wetting agent and water.

[0079] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles, a wetting agent and an organic solvent selected from the group consisting of medium chain triglyceride, vegetable oil, mineral oil and silicone oil.

[0080] hi one embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles and a wetting agent selected from the group consisting of sodium deoxycholate, sodium cholate, and sodium glycocholate.

[0081] In one embodiment, the disclosure provides SRP-3D (DA), which is substantially pure, or contains no significant amount of extraneous matter (e.g. , undesired extraneous matter). The molecular structure of SRP-3D(DA) exists as Formula I. In one embodiment, the disclosure provides a compound of Formula I:wherein the compound is substantially free of impurities.

[0082] hi certain instances, the compound is greater than 90% w / w pure, or greater than 95% w / w pure, or greater than 99% w / w pure.

[0083] In one embodiment, the disclosure provides a solid form of Formula I:wherein the solid form is a crystalline phase, an amorphous phase, a semi-crystalline phase, a semi-amorphous phase, and mixtures thereof.

[0084] hi one embodiment, the solid form is crystalline form such as a solid polymorphic form, which is characterized by an X-ray powder (XRPD) pattern comprising the following 20 peaks measured using CuKa radiation 9.0 ± 0.2, 9.2 ± 0.2, 13.2 ± 0.2, 21.9 ± 0.2;alternatively, or further comprising the solid form is characterized by an X-ray powder (XRPD) pattern comprising the following 20 peaks measured using CuKa radiation 12.2 ± 0.2, 15.4 ± 0.2, 18.3 ± 0.2, 19.2 ± 0.2, 20.7 ± 0.2, 22.5 ± 0.2; alternatively, the solid polymorphic form is characterized by an X-ray powder (XRPD) pattern comprising the 20 peaks measured using CuKa radiation substantially similar to FIGs. 10, 11 or 12.

[0085] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles in a crystalline form, wherein the crystalline compound is optionally substantially pure.

[0086] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles in a crystalline form having an X-ray powder diffraction (XRPD) pattern comprising the following 20 peaks measured using CuKa radiation: 9.0 ± 0.2, 9.2 ± 0.2, 13.2 ± 0.2, 21.9 ± 0.2.

[0087] In another embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles in a crystalline form having an X-ray powder diffraction (XRPD) pattern further comprising the following 20 peaks measured using CuKa radiation: 12.2 ± 0.2, 15.4 ± 0.2, 18.3 ± 0.2, 19.2 ± 0.2, 20.7 ± 0.2, 22.5 ± 0.2.

[0088] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure comprises SRP-3D (DA) nanoparticles in a crystalline form having an XRPD pattern substantially similar to that set forth in FIGs. 10, 11 or 12 as measured using CuKa radiation.

[0089] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure has an average particle diameter less than 1 micron, preferably less than 900-600 nm, more preferably less than 500-400 nm, and most preferably less than 300 nm.

[0090] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure has a narrow particle size distribution with a poly dispersity index value less than 1, preferably less than 0.8, more preferably less than 0.6, and most preferably less than 0.4.

[0091] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure is free of any particles of size greater than 20 micron, preferably greater than 10 micron, and more preferably greater than 7 micron.

[0092] In one embodiment, the SRP-3D (DA) nanosuspension of this disclosure is free of any particles of size greater than 15 micron, preferably greater than 8 micron, and more preferably greater than 5 micron.

[0093] In one embodiment, the SRP-3D (DA) nanoparticles or nanosuspension of this disclosure is packaged in a container such as a vial, a syringe or a bottle, with each container or unit containing Img - 10,000 mg SRP-3D (DA).

[0094] In one embodiment, the SRP-3D (DA) nanoparticles or nanosuspension of this disclosure is packaged in a container such as a vial, a syringe or a bottle, with each container or unit containing a dose of SRP-3D (DA) of between 0.1 g and 10 g.

[0095] In one embodiment, the SRP-3D (DA) nanoparticles or nanosuspension as packaged in a container unit in which the number of particles of size equal to or greater than 10 microns does not exceed 6000 per container and the number of particles of size greater than 25 microns does not exceed 600 per container.

[0096] In one embodiment, the SRP-3D (DA) nanoparticles or nanosuspension of this disclosure meets the USP <788> Particulate Matter requirements for an injectable drug.

[0097] In one embodiment, the compound of Formula I is amorphous synthesized using the method such as the method of Example 13.

[0098] In one embodiment, the SRP-3D (DA) nanoparticles or nanosuspension of this disclosure may contain other pharmaceutical excipients selected from the group consisting of cryoprotectants, lyoprotectants, antioxidants, stabilizers, buffers, tonicity modifiers, viscosity modifiers, bulking agents, disintegrants, lubricants, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, wetting agents, effervescent agents, or other excipients known in the art.6. Method of Making SRP-3D (DA)

[0099] SRP-3D (DA) can be made following the scheme outlined in FIG. 3 and Example 1. Briefly, 4-aminophenol and chloroacetic anhydride are reacted to produce 2-chloro-N-(4- hydroxyphenyl)acetamide (C-4). Next, C-4 is reacted with sodium saccharin hydrate (C-5) to generate C-l:which is 2-(l,l-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)-N-(4-hydroxyphenyl)acetamide (C-l). C-l is then reacted with diethyl amine to generate SRP-3D (DA).

[0100] In one embodiment, as shown in FIG. 3, the disclosure provides a method for making SRP-3D (DA), the method comprising:(a) admixing 4-aminophenol (C-3) and chloroacetic anhydride to produce 2-chloro-N-(4- hydroxyphenyl)acetamide (C-4);(b) admixing (C-4) with sodium saccharin hydrate (C-5) to generate C-l; and(c) admixing C-l with diethyl amine to generate SRP-3D (DA).7. Making the Nanosuspension

[0101] While the nanosuspension is the desired form for injection, both dry powder and suspension forms are suitable for oral administration. The dry powder may be incorporated in a capsule or tablet dosage form and the nanosuspension can be dosed orally directly.

[0102] The nanoparticulate SRP-3D (DA) of this disclosure is prepared by wet mill, i.e., being milled in a form of suspension. For this disclosure, a wet mill is used to grind a suspension containing coarse SRP-3D (DA) particles (>2 micron) to form a nanosuspension.

[0103] For this disclosure, any wet mill designed to break down solid particles to a submicron size may be used. A wet mill useful for this disclosure includes, but is not limited to, a netzsch mill, media mill, colloidal mill, ball mill, high shear homogenizer, high pressure homogenizer, high pressure extruder and microfluidizer. The preferred mill is a microfluidizer or a ball mill using grinding balls (beads).

[0104] For a microfluidizer used in this disclosure, either a Y-chamber or Z-chamber with a pore size of 75-300 micron may be used. The operation pressure may vary from 10K to 30Kpsi. The number of processing passes may be up to 100 passes. The preferred microfluidizer set up consists of a Y-chamber with an operation pressure of greater than 20K psi.

[0105] For a ball mill used in this disclosure, beads made with zirconium oxide or polystyrene with size from ranging from 300 to 100 microns may be used. The preferred beads are the yttrium-stabilized zirconium oxide beads.

[0106] The suspending liquid for the wet mill or for the final nanosuspension can be water or a non-aqueous liquid. Water is the preferred suspending liquid for the wet mill process.

[0107] The non-aqueous liquid for the wet mill may include but is not limited to medium chain triglycerides, vegetable oil, silicone oil, mineral oil or other SRP-3D (DA)-compatible and non-toxic organic solvent. The preferred non-aqueous liquid is medium chain triglycerides.

[0108] A nanosuspension produced by a wet mill can subsequently be separated into the nanoparticles and the suspending liquid. The separation can be performed using a filtration membrane with pore size smaller than that of the nanoparticles to separate the nanoparticles from the suspending liquid, centrifugal force to settle down the nanoparticles into a pellet from the suspending liquid, evaporation, or drying. The preferred separation method is the evaporation or drying method for an aqueous nanosuspension or the centrifugal method for a non-aqueous nanosuspension. The evaporation / drying method may include spray drying, freeze-drying or vacuum drying at ambient or elevated temperature to remove the water.

[0109] While the selection of a mill may affect the particle reduction rate and final particle size somewhat, the selection of a wetting agent seems more critical in dictating the final particle size and the physical stability of the nanoparticles after they are formed.

[0110] A wetting agent used for this disclosure can be water soluble or water insoluble. If it is water soluble, it would remain preferentially in the aqueous phase of the suspension, otherwise it may preferentially adhere to the surface of the solid particles. In either case, it would reduce the surface tension between the solid particles and water by allowing them to have better contact with water or be wetted by water.

[0111] SRP-3D (DA) is hydrophobic with a calculated log P of 0.760 ± 0.823. It is difficult to reduce SRP-3D (DA) particle size by milling without a wetting agent because the hydrophobic particles tend to stick together or lump together and are unable to disperse in water to form a uniform suspension. With a carefully selected wetting agent, the wettedparticles have less tendency to lump together. They form a uniform suspension where the particles are suspended as separated or individual particles instead of lumps.

[0112] A wetting agent affords obtaining SRP-3D (DA) nanoparticles or nanosuspension by a milling technique. As the particle size decreases, the particle surface area increases, and particle size reduction becomes more difficult. For example, only micron size (1-100 micron) of particles can be achieved by milling without a wetting agent, e.g., by a Jet Mill. Therefore, a wetting agent which reduces the surface tension of the solid particles is needed to enable the production of SRP-3D (DA) nanoparticles by milling. To date, there is no clear guidance or teaching in the prior art as to how to select a wetting agent for a new chemical such as SRP-3D (DA).

[0113] Furthermore, the smaller the particle size, the greater the surface tension, and the stronger the tendency for the particle to aggregate or exhibit instability. A wetting agent is also desired to maintain the particle size or the physical stability of the nanoparticles or nanosuspension after they are formed.

[0114] It was discovered by the inventors that not every wetting agent will result in a stable nanoparticulate composition for SRP-3D (DA). It was surprisingly discovered that stable nanoparticulate SRP-3D (DA) formulations can be made with, as described in more detail below, a bile salt selected from a group consisting of sodium salt of cholic acid, Glycocholic acid, Taurocholic acid, Deoxy cholic acid, Chenodeoxy cholic acid, Glycochenodeoxy cholic and Taurochenodeoxycholic acid with the preferred wetting agent being sodium deoxycholate.

[0115] On the other hand, other commonly used wetting agents such as docusate sodium, polyvinyl pyrrolidones (e.g., PVP K-12 and PVP K 29 / 32), poloxamers (e.g., Poloxamer 188 and Poloxamer 407, which are block copolymers of polyethylene glycol and polypropylene glycol), polyethylene sorbitan monooleate (polysorbate 80), failed to produce or stabilize SRP-3D (DA) nanoparticles.

[0116] In certain instances, sodium deoxy cholate is particularly suited for injectable nanoparticulate SRP-3D (DA) formulations. This is significant, and surprising, as for injectable formulations it is critical that very small SRP-3D (DA) particles be obtained. Moreover, the composition is stable, with very little or no particle size growth observed, as injectable formulations having large particles can cause embolism.8. Methods of Treatment

[0117] The compounds and formulations herein described are useful in treating acute, chronic, and / or neuropathic pain. Current medications, including opioids, acetaminophen / paracetamol, and nonsteroidal anti-inflammatory drugs (NSAIDs), offer limited relief and have significant drawbacks, such as addiction and organ toxicity. The opioid crisis has further complicated the landscape, with 8.7 million people aged 12 or older misusing prescription opioids in the U.S. alone. These forms of pain degrade the quality of life and pose a significant economic burden.

[0118] Embodiments can be used for the treatment or amelioration of pain, non-limiting examples of which comprise post-surgical, neuropathic, dental, ophthalmic, arthritic, acute, chronic, inflammatory, and / or traumatic pain.

[0119] In embodiments, compounds and compositions as described herein, can be used as the only pharmacologically active compound in the treatment of neuropathic pain without a second active agent, such as a GABA analogue, such as Gabapentin (Neurontin). In other embodiments, the compounds and compositions as described herein can be administered to a subject concurrently with and / or in combination with a second active ingredient, such as an opioid or a non-steroidal anti-inflammatory drug (NSAID). Opioid drugs work by binding to opioid receptors in the brain and spinal cord. Non-limiting examples of such opioids comprise codeine, fentanyl, hydrocodone, hydrocodone / ApAP, hydromorphone, meperidine, methadone, morphine, oxycodone, oxycodone and ApAP, oxycodone and naloxone. Nonsteroidal anti-inflammatory drugs (NSAIDs) block the COX enzymes and reduce prostaglandins throughout the body. As a consequence, ongoing inflammation, pain, and fever are reduced. Non-limiting examples of NSAIDs comprise aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, and tolmetin.

[0120] Compounds as described herein, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions can comprise a compound as described herein and a pharmaceutically acceptable carrier. Thus, in some embodiments, the compounds of the invention are present in a pharmaceutical composition.

[0121] For example, a pharmaceutical composition comprising a compound as described herein can be used for preventing and / or treating pain, such as a therapeutically effectiveamount of a compound of Formula I in admixture with a pharmaceutical acceptable carrier or excipient. For example, a therapeutically effective amount of a compound of Formula I can be administered to a subject so as to prevent or reduce the onset of pain or prevent the severity of pain from increasing.

[0122] “ Treatment” can refer to an approach for obtaining beneficial or desired clinical results, for example improvement or alleviation of any aspect of pain, such as post-surgical, neuropathic, dental, ophthalmic, arthritic, acute, chronic, inflammatory, and / or traumatic pain. Beneficial or desired clinical results comprise, but are not limited to, one or more of the following: including lessening severity, alleviation of one or more symptoms associated with pain including any aspect of pain (such as shortening duration of pain, and / or reduction of pain sensitivity or sensation).

[0123] “Ameliorating” pain or one or more symptoms of pain can refer to a lessening or improvement of one or more symptoms of a pain as compared to not administering a composition as described herein. “Ameliorating” can also comprise shortening or reduction in duration of a symptom. For example, a therapeutically effective amount of a compound of Formula I can be administered to a subject afflicted with pain so as to ameliorate, or lessen, the pain.

[0124] The term “alleviate” or “alleviating” can refer to lightening, reducing or lessening the severity of a symptom, condition, or disorder. For example, a treatment, such as a compound of Formula I, that reduces the severity of pain in a subject can be said to alleviate pain. For example, a therapeutically effective amount of a compound of Formula I can be administered to a subject afflicted with pain, wherein the severity of the pain is lessened. It is understood that, in certain circumstances, a treatment can alleviate a symptom or condition without treating the underlying disorder. In certain aspects, this term can be synonymous with the language “palliative treatment.”

[0125] Embodiments can be used for reducing the incidence of or delaying pain, nonlimiting examples of which comprise post-surgical, neuropathic, dental, ophthalmic, arthritic, acute, chronic, inflammatory, and / or traumatic pain. “Reducing incidence” of pain can refer to any of reducing severity (which can include reducing need for and / or amount of (e.g., exposure to) other drugs and / or therapies generally used for this conditions), duration, and / or frequency (including, for example, delaying or increasing time to pain in an individual). As is understood by those skilled in the art, individuals can vary in terms of their response totreatment, and, as such, for example, a “method of reducing incidence of pain in an individual” reflects administering compositions as described herein, based on a reasonable expectation that such administration can cause such a reduction in incidence in that particular individual.

[0126] “Delaying” the development of pain can refer to deferring, hindering, slowing, retarding, stabilizing, and / or postponing progression of pain. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop pain. A method that “delays” development of the symptom is a method that reduces the probability of developing the symptoms in a given time frame and / or reduces the extent of the symptoms in a given time frame, when compared to not using the method.

[0127] “Development” or “progression” of pain can refer to initial manifestations and / or ensuing progression of the disorder. Development of pain can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this invention, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of pain includes initial onset and / or recurrence. For example, embodiments as described herein can be used to prevent the development of pain or prevent the progression of pain.

[0128] Embodiments can be used for palliating pain, non-limiting examples of which comprise post-surgical, neuropathic, dental, ophthalmic, arthritic, acute, chronic, inflammatory, and / or traumatic pain.

[0129] “Palliating” pain or one or more symptoms of pain can refer to lessening the extent of one or more undesirable clinical manifestations of pain in an individual or population of individuals treated with a composition as described herein.

[0130] Embodiments comprise administering to a subject an effective amount of a composition as described herein, for the treatment of pain.

[0131] An “effective amount”, “sufficient amount” or “therapeutically effective amount” can refer to an amount sufficient to effect beneficial or desired clinical results including alleviation or reduction in the pain sensation. For purposes of this invention, an effectiveamount of a composition as described herein, can comprise an amount sufficient to treat, ameliorate, reduce the intensity of or prevent pain of any sort, such as post-surgical, neuropathic, dental, ophthalmic, arthritic, acute, chronic, inflammatory, and / or traumatic pain. In some embodiments, an effective amount of compositions as described herein can modulate the sensitivity threshold to external stimuli to a level comparable to that observed in healthy subjects. In other embodiments, this level is not comparable to that observed in healthy subjects but is reduced compared to not receiving the therapy.

[0132] Specific compositions as described herein, can be administered to a subject by any suitable means, such as oral, intravenous, parenteral, subcutaneous, intrapulmonary, topical, intravitreal, dermal, transmucosal, rectal, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, or intraperitoneal administration. The compounds can also be administered transdermally, for example in the form of a slow-release subcutaneous implant or as a transdermal patch. They can also be administered by inhalation. Although direct oral administration may cause some loss of desired activity, for example pain relieving activity, the analgesics can be packaged in such a way to protect the active ingredient(s) from digestion by use of enteric coatings, capsules or other methods known in the art.

[0133] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. The use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.

[0134] Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

[0135] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of anactive ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

[0136] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; anti-inflammatory agents; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH value can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

[0137] Compositions as described herein can be administered to the subject one time (e.g., as a single injection or deposition). While the term for administering of at least one compound to prevent pain varies depending on species, and the nature and severity of the condition to be prevented or treated, the compound can be administered to humans for a short term or a long term, e.g. for 1 week to 1 year. For example, administration can be once or twice daily to a subject in need thereof for a period of time, such as one week or one month.

[0138] The dosage can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.

[0139] A therapeutically effective dose can depend upon a number of factors known to those of ordinary skill in the art. The dose(s) can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires. These amounts can be readily determined by the skilled artisan.

[0140] In some embodiments, the therapeutically effective amount is at least about 0.1 mg / kg body weight, at least about 0.25 mg / kg body weight, at least about 0.5 mg / kg body weight, at least about 0.75 mg / kg body weight, at least about 1 mg / kg body weight, at least about 2 mg / kg body weight, at least about 3 mg / kg body weight, at least about 4 mg / kg body weight, at least about 5 mg / kg body weight, at least about 6 mg / kg body weight, at least about7 mg / kg body weight, at least about 8 mg / kg body weight, at least about 9 mg / kg body weight, at least about 10 mg / kg body weight, at least about 15 mg / kg body weight, at least about 20 mg / kg body weight, at least about 25 mg / kg body weight, at least about 30 mg / kg body weight, at least about 40 mg / kg body weight, at least about 50 mg / kg body weight, at least about 75 mg / kg body weight, at least about 100 mg / kg body weight, at least about 150 mg / kg body weight, at least about 200 mg / kg body weight, at least about 250 mg / kg body weight, at least about 300 mg / kg body weight, at least about 350 mg / kg body weight, at least about 400 mg / kg body weight, at least about 450 mg / kg body weight, at least about 500 mg / kg body weight, at least about 550 mg / kg body weight, at least about 600 mg / kg body weight, at least about 650 mg / kg body weight, at least about 700 mg / kg body weight, at least about 750 mg / kg body weight, at least about 800 mg / kg body weight, at least about 900 mg / kg body weight, or at least about 1000 mg / kg body weight.

[0141] A therapeutically effective dose can depend upon a number of factors known to those of ordinary skill in the art. The dose(s) can vary, for example, depending upon the identity, size, and condition of the subject being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires. These amounts can be readily determined by the skilled artisan.

[0142] In an embodiment, the recommended daily dose range of a compound as described herein for pain as described herein lies within the range of from about, a daily dose of about 1 mg / body to about 10 g / body, for example about 5 mg / body to about 5 g / body, or for example about 10 mg / body to about 2 g / body of the active ingredient is generally given for treating this disease, and an average single dose of about 0.5 mg to about 1 mg, about 5 mg, about 10 mg, about 50 mg, about 100 mg, about 250 mg, about 500 mg, about 1 g, about 2 g and about 3 g is generally administered. Daily dose for administration in humans for treating or ameliorating pain could be in the range of about 1 mg / kg to about 300 mg / kg.

[0143] A compound as described herein, for example a compound of Formula I, or composition comprising the same can be administered to the subject one time (e.g., as a single injection or deposition). Alternatively, administration can be once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0144] Single unit dosage forms of the disclosure are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial), topical (e.g., eye drops or other ophthalmic preparations), transdermal (e.g., cream, lotion, or dermal spray) or transcutaneous administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; powders; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions or solutions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; eye drops or other ophthalmic preparations suitable for topical administration; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms for parenteral administration to a subject.

[0145] The composition, shape, and type of dosage forms of the disclosure will typically vary depending on their use. Further, the dosage can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.

[0146] For example, a dosage form used in the acute treatment of a disease can contain larger amounts of one or more of the active agents it comprises than a dosage form used in the chronic treatment of the same disease. Similarly, a parenteral dosage form can contain smaller amounts of one or more of the active agents it comprises than an oral dosage form used to treat the same disease. These and other ways in which specific dosage forms encompassed by this disclosure will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990). The methods are non-cytotoxic to the liver.

[0147] Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a mouse, a rat, a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human. In some embodiments, the subject is a mouse, rat, pig, or human. In some embodiments, the subject is a mouse. Insome embodiments, the subject is a rat. In some embodiments, the subject is a pig. In some embodiments, the subject is a human.9. Methods Useful in the Disclosure

[0148] METHOD 1 : GC Method for the TAN Purity Analysis of 4-Aminophenol, 2- Chloroacetic Anhydride, and Compound 4, as well as In-Process Reaction Monitoring for Step 1 During the Synthesis of SRP-3D (DA)

[0149] Preparation of Identification and Endpointing Markers

[0150] Analytes are calculated by total area normalization; therefore, quantitatively accurate standards are not necessary. Qualitative markers that verify the current retention times of the target compounds should be run with each analysis. For chemical structures, see FIG. 3.4-Aminophenol Identification Standard: (0.33 mg / mL 4-aminophenol in acetonitrile)1. Add approximately 20 mg of the 4-aminophenol standard and 60 mL of acetonitrile into a scintillation vial.2. Mix by inversion.2-Chloroacetic Acid Anhydride Identification Standard: (2 mg / mL 2 -chloroacetic acid anhydride in acetonitrile)1. Add approximately 20 mg of the 2-chloroacetic acid anhydride standard and 10 mL of acetonitrile into a scintillation.2. Mix by inversion.Step 1 Endpoint Standard: (0.03 mg / mL 4-aminophenol and 0.68 mg / mL compound 4 in acetonitrile)1. Add approximately 34 mg of the compound 4 standard, 4.5 mL of the 4-aminophenol identification standard, and 45.5 mL of acetonitrile into a scintillation vial.2. Mix by inversion.Note: Retention time marker solutions can be prepared by using proportional equivalents to the stated amounts and may be used as long as the components of interest remain detectable.

[0151] Preparation of SamplesReaction MonitoringStep 1 (target concentration 0.68 mg / mL):1. Allow quenched reaction mixture to set for a minimum of 15 minutes.2. Transfer approximately 575 pL of the quenched reaction mixture and 10 mL of acetonitrile into a scintillation vial.3. Mix by inversion.

[0152] Actual concentrations of the reaction solutions may be obtained from the lead chemist or engineer and sample preparations may be adjusted as needed to achieve the specified target concentration.Regulatory Starting Materials and Isolated Intermediates

[0153] TAN purity checks of 4-aminophenol, 2-chloroacetic acid anhydride, and compound 4 can be performed by preparing and analyzing samples at 0.33 mg / mL, 2 mg / mL, and 0.68 mg / mL in acetonitrile, respectively. It is recommended that release samples be prepared in duplicate.Note: Since this method is a TAN analysis, quantitative sample preparations are not required.System Suitability1. Inject a blank to ensure no peaks are present at the retention times of the analytes of interest. If an interfering peak is present, evaluate the level of impact that it may contribute to the analysis and take corrective action if necessary.2. All peaks must be on-scale to assure proper integration.

[0154] Procedure1. Set-up the gas chromatographic system as follows:GC Column: RTX-5 Amine, 30 m x 0.32 mm ID, 1.5 μm FTNeedle Wash: AcetonitrileInjection Vol.: 2 μLInjector Temp.: 300oC (Base Deactivated Liner)Carrier Gas: H2Column Pressure: 12 PSI (Constant Pressure)Split Ratio: 10:1 (adjust as needed to ensure appropriate response)Oven Program: 115oC to 300 C @15C / min (5 min hold)Detector: FID @ 300oC2. Run a blank at least once prior to initiating an analysis.3. Set up data system and load autosampler using the following example format for the sequence of injections:Injection# Injection «> Test .1 Blank Column Condition2 Blank Acceptability of Diluent3 Identification / Endpoint Standard Verification of Retention Times4 Sample _ Sample Analysis _5 identification / Endpoint Standard Verification of Retention Times6 Blank Carryover4. Verify that all chromatograms have been integrated appropriately. If not, it may be necessary for the analyst to perform manual integration (set peak starts, stops, and baselines). System peaks (i.e., peaks observed in the blank) should be excluded from the sample profile.5. Verify the system suitability requirements were met. If the criteria are not met, the ran is invalid. Take corrective action and repeat the analysis.6. Calculate and report results. Calculations and Reporting Results

[0155] Confirmation of Identity

[0156] The presence of the analytes of interest in the sample is confirmed if the peaks in the chr omatogram of the sample correspond with the peaks produced by authentic material within a 5% retention time window. The expected retention times for the various reaction components are as follows:Expected RetentionComponent Time (min)2-Chioroacetic Acid Anhydride 2.84-Aminophenol 4.1Compound 4 8.8TAN Analysis of Endpointing SamplesReport the TAN value of the starting material and product to two decimal places.TAN PurityReport the TAN value of isolated intermediates and regulated starting materials to two decimal places.

[0157] METHOD 2: HPLC Method for In-Process Monitoring of Steps 1-3 andRecrystallization HPLC Column: YMC PACK-PRO C18, 4.6x150 mm, 5 μmColumn Temp.: 35oCSample Temp,: Ambient or 20 °CInjection Volume: 10 pLFlow Rate: 1.2 mL / min UV Detection: 240 nmRun Time: 40 minutesAutosampler Flush Solvent: 1:1 H2O:ACNMobile Phase: mpA - H2O with 0.1% TFA mpB - ACN with 0.1%TFAGradient Pump Program*:Gradient Step Tim* Elapsed. Time% mp A % mp B CurveStep (minutes ) (minutes)0 0.5 0.0 95 5 01 20.0 20.0 52 48 1_2 10.0 30.0 0_ 100 13 10.0 40.0 95 5 0*Developed on a Perkin Elmer Series 200 pump. Curve I is a linear change and curve 0 is an immediate change. Gradient may need to be adjusted depending on the dwell volume of the HPLC being used.

[0158] For a sample chromatogram of the C-4 Identification Standard, see FIG. 13.

[0159] For a sample chromalogram of the Step 2 Endpointing Standard, see FIG. 14.

[0160] For a sample chromatogram of the Step 3 Endpointing Standard, see FIG. 15.

[0161] METHOD 3: Determination of the Molar Ratio of Dichloromethane and Ethanol to n-Heptane bylH NMRPurpose

[0162] This procedure can be used to calculate the molar ratio of both dichloromethane (DCM) and ethanol (EtOH) to n-heptane.Materials• 1 clean 5 mm NMR tube• CDCh, >99.8 atom % D containing 0.03% v / v TMS, or its equivalent.Procedure• Quantities of all chemicals are very qualitative and not critical factors in this experiment. Dissolve about 5 drops of supernatant after solvent exchange in 0.5-1 mL of CDCb (containing tetramethylsilane as a reference) in a clean 5 mm NMR tube. Cap the tube and mix thoroughly by inversion.• Analyze by1H NMR using the standard operating parameters.Analysis• Ensure that the spectrum is referenced properly to TMS at 0.00 ppm.• Integrate the triplet at approximately 0.89 ppm, which is the signal for the methyl groups of n-heptane. Calibrate the integral to a value of 6.00.• Integrate the singlet at approximately 5.27 ppm, which are the hydrogens of DCM (2 protons). Call the resulting integral value "A".• Integrate the quartet at approximately 3.75 ppm which is the methylene group of ethanol. Call the resulting integral value "B".Calculation of the DCM to n-heptane molar ratio as mole%:DCM / n-heptane (mole%) = (A / 2) * 100Calculation of the ethanol to n-heptane molar ratio as mole%:Ethanol / n-heptane (mole%) = (B / 2) * 100

[0163] Print the spectrum with the integral regions, have the spectrum reviewed, and attach it along with notebook pages for sample preparation to the batch record.

[0164] METHOD 4: Determination of the Molar Ratio of Diethylamine to Acetonitrile by 'H NMRPurposeThis procedure can be used to calculate the molar ratio of diethylamine to acetonitrile (ACN).Materials• 1 clean 5 mm NMR tube• DMSO-de, >99.8 atom % D containing 0.03% v / v TMS, or its equivalentProcedure• Quantities of all chemicals are very qualitative and not critical factors in this experiment. Dissolve about 5 drops of supernatant after solvent exchange in 0.5-1 mL of DMSO-de (containing tetramethylsilane as a reference) in a clean 5 mm NMR tube. Cap the tube and mix thoroughly by inversion.• Analyze by1H NMR using the standard operating parameters.Analysis• Ensure that the spectrum is referenced properly to TMS at 0.00 ppm.• The appropriate signals for diethylamine tend to shift slightly depending on its concentration as the desired compound of this reaction step, SRP-3D (DA), contains an acidic phenolic functionality. Ensure that the signal is baseline separated from the DMSO peak.• Integrate the singlet at approximately 2.05 ppm, which is the methyl group of ACN (3 protons). Calibrate this value to 3.00.• Integrate the quartet at approximately 2.6-2.8 ppm which is the methylene group of di ethylamine (4 protons). Call the resulting integral value "A".Calculation of the di ethylamine to ACN molar ratio as mole%:Diethylamine / ACN (mole%) = (A / 4) * 100

[0165] Print the spectrum with the integral regions, have the spectrum reviewed, and attach it along with notebook pages for sample preparation to the batch record.

[0166] METHOD 5: Determination of the Molar Ratio of Water to ACN by 'H NMRPurpose

[0167] This procedure can be used to calculate the molar ratio of water to ACN.Materials• 1 clean 5 mm NMR tube• DMSO-de, >99.8 atom % D containing 0.03% v / v TMS, or its equivalentProcedure• Quantities of all chemicals are very qualitative and not critical factors in this experiment. Dissolve about 10 drops of supernatant after water addition in 0.5-1 mL of DMSO-de (containing tetramethylsilane as a reference) in a clean 5 mm NMR tube. Cap the tube and mix thoroughly by inversion.• Analyze by1H NMR using the standard operating parameters.

[0168] Analysis• Ensure that the spectrum is referenced properly to TMS at 0.00 ppm.• The appropriate signal for water tends to shift slightly depending on water to ACN ratio. Use only supernatant in this experiment to exclude any potentially interfering product peak.• Integrate the singlet at approximately 3.4-3.8 ppm, which is the signal for water (2 protons). Calibrate this value to 2.00.• Integrate the singlet at approximately 2.05 ppm which is the methyl group of ACN (3 protons). Call the resulting integral value "A".Calculation of the water to ACN molar ratio:Water / ACN (mole%) = 1 to (A / 3)Calculation of the water to ACN volume ratio:Water / ACN (vol / vol) = 1 to (A / 3)*41.05 / (18.02*0.786)

[0169] Print the spectrum with the integral regions, have the spectrum reviewed, and attach it along with notebook pages for sample preparation to the batch record.

[0170] METHOD 6: Stability Indicating Method for Identity, Assay and Purity of SRP-3D (DA) by HPLC-UVPreparation of Mobile Phase1. 10 mM Tetrabutylammonium acetate a. Add 1.0 mL of 1.0 M Tetrabutylammonium acetate to a 100 mL volumetric flask. b. Dilute to volume with water and mix well. c. Expires in 2 weeks.2. Mobile Phase “A”: 20 mM Ammonium acetate and 0.1 mM Tetrabutylammonium acetate, pH 5.8 in Water a. Weigh 1.6 g of ammonium acetate into a 1 L reservoir. b. Add 1 L of Water using a graduated cylinder. c. Add 10.0 mL of 10 mM Tetrabutylammonium acetate. d. Adjust the pH to 5.8 using Acetic acid e. Stir the solution well to ensure thorough mixing. f. Expires in 2 weeks.3. Mobile Phase “B”: 20 mM Ammonium acetate and 0.1 mM Tetrabutylammonium acetate, pH 5.8 in 30% Water and 70% Acetonitrile a. Weigh 1.6 g of ammonium acetate into a 1 L reservoir. b. Add 300 mL of Water using a graduated cylinder. c. Add 10.0 mL of 10 mM Tetrabutylammonium acetate. d. Adjust the pH to 5.8 using Acetic acid e. Add 700 mL of Acetonitrile using a graduated cylinder f. Stir the solution well to ensure thorough mixing. g. Expires in 4 weeks.

[0171] Preparation of Sample / Standard Diluent / Autosampler Flush1. Sample / Standard Diluent: 20 mM Ammonium acetate, pH 5.8 in 50% Water and 50% Acetonitrile a. Weigh 1.6 g of ammonium acetate into a 1 L reservoir. b. Add 500 mL of Water using a graduated cylinder. c. Adjust the pH to 5.8 using Acetic acid d. Add 500 mL of Acetonitrile e. Stir the solution well to ensure thorough mixing. f. Expires in 4 weeks.2. Autosampler Flush: 50% Water and 50% Acetonitrile a. Add 500 mL of Water to a 1 L reservoir using a graduated cylinder. b. Add 500 mL of Acetonitrile c. Stir the solution well to ensure thorough mixing. d. Expires in 4 weeks.

[0172] Preparation of Calibration and Check Standards1. In duplicate, prepare SRP-3D (DA) reference standard stock solutions at a concentration of 1.0 mg / mL in a 25-mL volumetric flask by accurately weighing 25 mg (± 2 mg) of the reference standard.2. Partially fill each flask with the diluent and mix well to dissolve. Mild sonication (~1 min) may be needed for completion of dissolution.3. Dilute to volume with the diluent and mix by inversion.4. Transfer 2.5 mL of each stock solution into separate 25 mL volumetric flasks and bring to volume with Diluent (10% solution, concentration 0.1 mg / mL). Label solutions as Standard 1 and Standard 2.

[0173] Preparation of LOQ Solution1. Transfer 2.5 mL of one of the standard solutions prepared in Section F into a 25-mL volumetric flask, dilute to volume with the Diluent, and mix well. This intermediate LOQ solution contains approximately 10 pg / mL SRP-3D (DA).2. Transfer 2.5 mL of the intermediate LOQ solution into a 50-mL volumetric flask, dilute to volume with the diluent, and mix well. This LOQ solution contains approximately 0.5 pg / mL SRP-3D (DA) (i.e. 0.05%w / w of SRP-3D (DA))

[0174] Preparation of Retention Time Marker Solution1. This solution is not required for each analysis. Only prepare if peak identification is uncertain. Due to the instability of Compound 3, this solution should be injected within 1 day of preparation.2. Weigh approximately 10 mg of Compound 3, 4, 5 and 1 into a 50-mL volumetric flask. Dissolve and dilute to volume with the Diluent. Mix well.3. Transfer 2.5 mL of above solution into a 50-mL volumetric flask. Dilute with one the reference standard solution to volume. Mix well.

[0175] Preparation of Samples1. Impurities Determination Solution (1 mg / mL) a. In duplicate, prepare SRP-3D (DA) impurities sample at a target concentration of 1.0 mg / mL by weighing 25 mg (± 2 mg) of sample into 25 mL volumetric flasks. b. Partially fill each flask with the diluent and mix well to dissolve. If required, sonication may be employed for completion of dissolution. c. Allow the solution to attain room temperature. d. Dilute to volume with the Diluent and mix by inversion. e. Only the first impurity sample preparation should be analyzed.2. Assay Determination Solution (0.1 mg / mL or 10% of the Impurities Solution) a. Transfer 2.5 mL of each impurities solution into a 25 mL volumetric flask and bring to volume with Diluent. b. Mix well.

[0176] System Suitability1. The diluent blank should not contain peaks that may interfere with the determination of the sample assay or impurity profile. If an interfering peak is present, evaluate the level of impact that it may contribute to the analysis. If the impact is significant, investigate the potential source of the peak. If the source of the peak is determined to be from the column, condition the HPLC column as follows: Flush the system with 50% Acetonitrile and 50% Water to remove the buffer and modifier. Switch the flow to 100% Acetonitrile at a flow rate of 0.2 mL / min through the system overnight.2. Inject the SRP-3D (DA) LOQ solution one time. The average calibration factor from the first six injections of SRP-3D DA reference standard is used to calculate the percent recovery of SRP-3D (DA) in the LOQ solution. The percent recovery for the LOQ solution should be between 50-150%.RFLOQ50% < < 150%RFSTD (n=6)Where:RFLOQ = Response Factor of LOQ injectionRFsTD(n=6) = Average Response Factor of the first six injections of Reference Standard3. All peaks must be on-scale to assure proper integration.4. Inject one of the SRP-3D (DA) reference standards six consecutive times. The following system suitability criteria for these six injections must be obtained: a. % RSD API peak area: < 2.0% b. % RSD API retention time: < 2.0% c. Average API tailing factor: 0.5 < x < 2.0The mean calibration factor from the first six injections of reference standard is used to calculate the recovery of SRP-3D (DA) in the duplicate standard preparation and each bracketing standard injection throughout the sequence. The recovery of SRP-3D (DA) must be between 98% and 102%.RF98%< < 102%RFSTD (n=6)Where:RF = Response Factor of Check Standard or Bracket Standard InjectionRFsTD(n=6) = Average Response Factor of the first six injections of Reference Standard6. The diluent blanks used to evaluate carryover should not show significant quantities of SRP-3D (DA) (i.e. < LOQ). If a carryover is present, evaluate the level of impact that it may contribute to the analysis.If the system suitability criteria are not met, additional injections of the standards should be made to determine if the problem is the solution or the HPLC system. If the problem is determined to be the solutions, new solutions will be prepared and analyzed. If the problem is the HPLC system, the analyst will troubleshoot the problem, take corrective action, and repeat the analysis.

[0177] Procedure1. Set-up liquid chromatographic system as follows: HPLC Column: Phenomenex, PS C18, 3 x 150 mm, and 2.6 pm,PN OOF-4780-YOColumn Temp.: 40 ± 1 °CSample Temp.: AmbientAutosampler Flush: 50% ACNFlow Rate: 1.0 mL / minInjection Volume: 5 pLUV Detection: 225 nm (4 nm bandwidth, 400 nm reference wavelength, as applicable)Run Time: 25 minutesMobile Phase: A - 20 mM Ammonium acetate and 0.1 mMTetrabutylammonium acetate, pH 5.8 in WaterB - 20 mM Ammonium acetate and 0.1 mM Tetrabutylammonium acetate, pH 5.8 in 30% Water and 70% AcetonitrileGradient Pump Program:2. Equilibrate the column with the mobile phase prior to commencing any analysis. For a gradient analysis, it is necessary to r un the system through the gradient program at least one time prior to initiating an analysis. The autosampler and pump should be flushed with autosampler flush and mobile phase on a daily basis prior to analysis. After use, the column should be washed with 50% Acetonitrile and 50% Water. It is recommended to store the column for long periods in 100% Acetonitrile.3. Set up data system and load autosampler using the following example format for the sequence of injections:InjectionINjection ID Test #Sample 3; Weight 1 (100%20 _ Impurity level) _ Sample 4; Weight 1 (10% Assay21 _ level) _ Sample 4; Weight 2 (10% Assay22 _ level) _Sample 4; Weight 1 (100%23 _ Impurity level) _24 _ Standard, W1 _ Agreement of Standards25 Blank (Dilution Solvent) Carryover26 Blank (Dilution Solvent)4. Review the following chromatograms for each injection of standard or sample:• Full scale: X scale range = 0 minutes to 25 minutesY scale range should be set to review entire SRP-3D (DA) peak• Expanded response scale: X scale range = 0 minutes to 25 minutesY scale range should be set to review impurities baseline5. Verify that all standards and samples have been integrated appropriately. If not, the analyst may need to perform manual integration (set peak starts, stops, and baselines) to include all peaks other than system peaks. System peaks (i.e., peaks observed in the blank sample diluent) should be excluded from the sample impurity profile.6. Verify the system suitability requirements were met. If the criteria are not met, tire run is invalid. Take corrective action and repeat the analysis. Calculate and report th e results.

[0178] Calculations and ReportingConfirmation of Identity

[0179] The identity of SRP-3D (DA) in the sample is confirmed if the principal peak in the chromatogram of the sample corresponds to the average retention time of the peak produced by th e reference material within ± 2.5%.SRP-3D (DA) Content1. SRP-3D (DA) Content of SampleWhere:ASPL = Area of SRP-3D (DA) peak in sample solutionASTD = Area of SRP-3D (DA) peak in Reference Standard solutionCSTD= Concentration of Reference Standard (mg'mL)PSTD= Potency of Reference Standard, in decimalSS= Sample Size (mg)If agreement of replicate sample preparations is acceptable (< 2% absolute difference), average the two values to obtain the test result.Where: KF% = Water content from Karl Fischer per release testing / stability test point TS% = Total solvent content from GCReport result to one decimal place.Note: AU values used in calculations should include enough significant figures to avoid rounding errors. ted Substances . For each HPLC injection of an impurity sample solution, calculate the impurity content. The expected and relative retention times for die various known related substances are as follows:Expected RetentionAPI and Related Time Relative . Substances (min Retention Time3 1.5 0.125 4.6 0.374 7.1 0.561 10.7 0.84SRP-3D (DA) 12.7 1.00 . Calculate the individual impurity content for impurities > LOQ (0.05%, excluding system peaks) as follows:Where: ASTD = Average Area of SRP-3D (DA) in six (6) injections of STD1 Standard solution.CSTD = Concentration of SRP-3D (DA) in 10% Standard solution (mg / mL).PSTD = Purity of SRP-3D (DA) Reference standard (as decimal)AIMP = Area of individual impurity in Impurities Determination Solution (100%)CIMP = Concentration of SRP-3D (DA) in Impurities Solution (mg / mL). Calculate the Total impurities content using the individual impurities >LOQ (0.05%) as follows:Total impurities (% Area) = ∑ each individual impurity > LOQrting . For Specified Imnurities: Report all specified impurities by name. Specified impurities not observed in a sample are reported as < LOD. Values that are LOD but < LOQ (0.05 % w / w) are reported as < LOQ. Numerical values rounded to two decimal places are reported for specified impurities that are ≥ LOQ.2. For All Unspecified Impurities: Report all unspecified (includes unidentified and potential) impurities that are > LOQ (0.05% w / w) referenced to their RRT. Values that are > LOD but < LOQ are reported as < LOQ. Numerical values rounded to two decimal places are reported for unspecified impurities that are > LOQ.3. For Total Impurities: Sum all specified and unspecified individual impurities that are >LOQ. Individual impurity values which are < LOQ are not included in the calculation of total impurities content. Report the total % of impurities present to two decimal places (e.g., 1.23%).Chromatographic Purity of SRP-3D (DA) (for Characterization)1. When determining the chromatographic purity, the material being characterized is prepared in duplicate at 1 mg / mL (100%) and each solution is further diluted down to 10%. The Area Response of SRP-3D in the 10% solution is used as the reference response.2. Calculate the individual impurity content and total impurity content as in the “Related Substances” section above.3. Examine the chromatograms of the replicate sample preparations. If the chromatograms reveal the same profile and the relative peak sizes are similar, average the values to obtain one test result.4. Chromatographic Purity (CP (%)) is determined as:CP (%) = 100% - Total Impurities > 0.05% (%)5. Report result to one decimal place.

[0180] METHOD 7: Identification of SRP-3D (DA) by Proton and Carbon-13 NMR Spectroscopy

[0181] Characterization of a qualified SRP-3D (DA) authentic reference standard by 'H,13C, COSY, HSQC and HMBC NMR was performed on a Bruker Avance III 400 MHz instrument using Bruker TopSpin 2.1.8 software. Spectra were obtained in deuterated DMSO (DMSO-de) and referenced to tetramethylsilane. For representative 'H and13C data and peak assignments, see FIG. 4 and FIG. 5, respectively. Subsequent batches were identified by comparison of their1H and13C spectra to the corresponding reference spectra.

[0182] METHOD 8: Infrared Analysis of SRP-3D (DA)Samples of SRP-3D (DA) were analyzed on a Nicolet 6700 FTIR equipped with a Thermo Fisher Smart iTX accessory and OMNIC 9.8 software. The wavelength range 4000 to 400 cm’1was scanned 128 times at 4 cm’1resolution. A representative spectrum is shown in FIG. 6. It is consistent with the proposed structure.METHOD 9: Identification of SRP-3D (DA) by Mass Spectrometry

[0183] SRP-3D (DA) was identified by Positive Ion Mode Electrospray Ionization Mass Spectrometry (+ESI MS) including Full Spectrum Scan Q3 Mass Spectrometry and Tandem Mass Spectrometry (MS / MS). The identification was performed by comparing the MS data of the test article against the MS data of a qualified SRP-3D (DA) authentic reference material. Alternatively, the identification was performed by comparing the MS data of the test article against reference MS data, previously generated through validation activities, using a qualified SRP-3D (DA) authentic reference material using the same MS instrument. Reference mass spectra are presented in FIG. 7, FIG. 8 and FIG. 9. Test solutions were introduced into the mass spectrometer ion source via the direct infusion technique. The overall method and instrument suitability were assessed through the evaluation of the accuracy and consistency of the MS data when compared to reference data. SRP-3D (DA) was detected in Full Scan Q3 mode. The identification of SRP-3D (DA) took into account three aspects of the MS data: the exact mass of the molecular ion [M+H]+, the isotopic pattern, and the MS / MS pattern of the fragmented monoisotopic pseudomolecular ion at m / z = 406.1.

[0184] The method was developed and validated on an AB Sciex 4000 Q-Trap mass spectrometer system. Some mass parameters or scan types might be associated with capabilities of the 4000 Q-Trap only. The analysis may be performed on different models of mass spectrometers as long as the system suitability criteria are met. Method validation was performed according to current Good Manufacturing Practices.Diluent: 0.1% FA in Acetonitrile: Water (1 : 1 v / v)Sample or reference standard stock solution: 35-65 pg / mL in diluentSystem Suitability

[0185] The following criteria should be met in order to deem the test suitable.

[0186] The diluent (blank) should exhibit no or negligible interference ions at the m / z range of interest (<5% of the ion intensity of interest). If a substantial interference is observed, evaluate the impact on selectivity considering the intensity (counts) of the signals. If the impact is significant, run diluent until a stable and low-noise MS spectrum baseline is achieved.39Accuracy

[0187] The following ions should be observed when analyzing the solution of the SRP-3D (DA) authentic material. The m / z value of each ion should be accurate to within± 0.2 amu when using the centroid data presentation option.Full Scan Mass Spectrum (03 Positive) Ion m / z (Centroid)406.1 amuProduct Ion Mass Spectrum (MS2 Positive) Ions m / z (Centroid)Parent Ion 406.1 amuFrag ment 240.1 amuNote: When comparing against the reference MS data, the MS spectra of the SRP-3D (DA) Test Article should meet tire Accuracy criteria described above.

[0188] In any case, if the generated MS data does not exhibit the expected pattern and m / z values and / or does not meet the accuracy criteria, the calibration of the mass spectrometer needs to be verified using the Applied Biosystems Sciex Standards Chemical KitProcedureSystem Set-Up

[0189] MS Instrument: AB Sciex 4000 Q-Trap Mass Spectrometer or similar Mass Spectrometer with triple-quadrupole capabilities able to work under full spectrum scan and MS / MS mode. Note: Instrument parameters may need to be adjusted to meet system suitability criteria and avoid signal saturation, such as CUR, IS, GS, Nozzle position etc. Since the MS condition on different MS systems varies, the MS parameter modification is acceptable.

[0190] Injection Method: Direct InfusionSyringe Pump Parameters: Syringe Diameter (mm): 4.6Flow Rate: lO.OpL / minSyringe Size: 1000μLSoftware: Analyst1. Full Scan spectra (Q3 Positive)4000 Q-Trap Mass Spectrometer Method ParametersNozzle positions: X-Axis: 5.0: Y-Axis: 5.0Scan Type: Q3 MSPolarity: PositiveScan Mode : Profile39Ion Source: Turbo SprayStart (amu) Stop (amu) Time (Sec) Cycles120.00 850.00 8.00 30 _Parameter Value Parameter ValueCUR 15.00 TEM 300.00GS1 30.00 GS2 30.00 ihe ON IS 2800.00DP 0.00 EP 10.00CXP 8.002. Product Ion (MS2) Mass Spectra4000 Q-Trap Mass Spectrometer Method Parameters Nozzle positions: X-Axis: 5.0; Y-Axis: 5.0 Scan Type: Product Ion (MS2)Polarity: PositiveScan Mode: Profile Ion Source: Turbo Spray Product of: 406.1 amuStart (amu) Stop (amu) Time (Sec) Cycles50.00 500.00 8.00 30Parameter Value Parameter ValueCUR 15.00 TEM 300.00GS1 30.00 GS2 30.00 ihe _ ON IS _ 2800.00CAD Medium (or 2~5) CXP 8.00DP _ 0.00 EP 10.00CE 20.00Sample Analysis

[0191] Analyze samples using above MS parameters. Q3 MS is for full scan to obtain general views of ions in the samples; Product ion (MS2) is to obtain fragmentation information from the precursor ion.Analyze the samples following the example sequence below.Samples PurposeDiluent Full scan to obtain background from blankStandard* Full scan / MS2 scan to obtain MS spectra of the standardDiluent Full scan to obtain background from blankSample Full scan / MS2 scan to obtain MS spectra of the sample* Sequence may be altered to allow analyzing more samples.

[0192] Alternately, compare the MS spectra (Full spectrum Scan and MS2) of SRP-3D (DA) test article against the reference MS spectra generated during the validation of this test method.Reporting ResultConfirmation of Identity

[0193] The identity of SRP-3D (DA) test article is confirmed if the following criteria were met. a. The full scan mass data (Q3 Positive) of the test article should match that of the reference spectra. Accuracy of m / z values for the molecular ion and similar isotopic pattern should be ± 0.2 amu. See example MS spectra in FIG. 7 and FIG. 8. b. The MS / MS data (Product Ion, MS2 Positive) of the test article should match that of the reference substance / reference spectra (± 0.2 amu). See example MS / MS spectrum in FIG. 9.

[0194] METHOD 10: Characterization of SRP-3D (DA) by X-Ray Powder Diffraction

[0195] X-ray powder diffraction patterns were obtained using a Bruker D8 Discovery diffractometer equipped with an XYZ stage, laser video microscope for positioning, and a two dimensional VANTEC-500 area detector. A Cu Ka radiation 1.5406 A source operating at 40 kV and 40 mA was used to irradiate samples. The X-ray optics consisted of a Gobel mirror coupled with a pinhole collimator of 0.5 mm. Theta-theta continuous scans were employed with a sample-detector distance of approximately 30 cm, which gave an effective 29 range of 4-40°. The samples were mounted in low background quartz plates. Major XRPD peaks (relative intensity >10%) for three specimens of SRP-3D (DA) Form A of the present disclosure are tabulated below.Table 1. XRPD Peaks for SRP-3D (DA) Form A, Specimen 1Table 2. XRPD Peaks for SRP-3D (DA) Form A, Specimen 2 (Unground Specimen)Table 3. XRPD Peaks for SRP-3D (DA) Form A, Specimen 3 (Ground Specimen)

[0196] A variety of factors, including instrument design, maintenance, calibration and mode of operation, specimen preparation and presentation, particle size and inhomogeneity, preferred orientation, and environmental conditions can cause variability in the measurement of diffraction angles and peak intensities. Angle measurements are presented herein as ±0.2° 29. Relative intensities can vary widely. As used in the description and the appended claims, the phrase “an XRPD pattern substantially similar to” a reference pattern takes into account these sources of experimental variability. For example, the XRPD patterns for SRP-3D (DA) Form A, Specimen 1, , Specimen 2 (unground specimen), and Specimen 3 (ground specimen) presented in FIG. 10 (with peaks listed in Table 1), FIG. 11 (with peaks listed in Table 2), and FIG. 12 (with peaks listed in Table 3), respectively, are “substantially similar” as defined herein, even though the corresponding 29 values are offset by as much as 0.2° and the relative intensities for certain peaks (e.g., -12.2°, -18.3° and -21.9° 29) are very different. These differences in relative intensities may be due to “preferred orientation” in one or more of the specimens, as is well understood in the art.

[0197] METHOD 11 : Characterization of SRP-3D (DA) by Differential Scanning CalorimetryInstrument: TA Instruments DSC Q2000 Software: Thermal Advantage V 5.0.0 Sample pans and lids: aluminum, crimped Sample size: 2~10mg Heating range: 25° to 250°C Heating rate: 10°C / min Purge: N2

[0198] A representative DSC curve for a qualified authentic reference standard of SRP-3D(DA) Form A is presented in FIG. 16. It shows a single sharp endotherm:Melt onset: 172.47°CMelt peak: 174.47°CAHf: 101.9 J / g

[0199] METHOD 12: Characterization of SRP-3D (DA) by Thermogravimetric AnalysisInstrument: TA Instruments TGA Q500Software: Thermal Advantage V 5.2.5Sample pans: platinumSample size: 2~20mgHeating range: 25° to 250°CHeating rate: 10°C / minPurge: N2

[0200] A representative TGA curve for a qualified authentic reference standard of SRP-3D(DA) Form A is presented in FIG. 18. It shows 0% weight loss at 175°C.

[0201] This result is consistent with Form A being anhydrous and unsolvated.

[0202] METHOD 13: Determination of Identification, Assay and Purity of SRP-3D (DA)Nanoparticle Suspension by HPLCMobile phase, standard and buffer preparation:1) 10 mM Tetrabutylammonium acetate buffer preparation a. Weigh and transfer 301.50 mg Tetrabutylammonium acetate in a 100 mL volumetric flask. Add about 60 mL DI water, mix well, then dilute to 100 mL with DI water. b. Expires in 2 weeks2) Mobile phase A: 20 mM Ammonium acetate and 0.1 mM Tetrabutylammonium acetate, pH 5.8 in Water a. Weigh 1.6 g of ammonium acetate into a 1 L reservoir. b. Add 1 L of DI water using a graduated cylinder. c. Add 10.0 mL of 10 mM Tetrabutylammonium acetate. d. Adjust the pH to 5.8 using glacial acetic acid, Stir the solution well to ensure thorough mixing. e. Expires in 2 weeks3) Mobile phase B: 20 mM Ammonium acetate and 0.1 mM Tetrabutylammonium acetate, pH 5.8, in 30% water and 70% ACN a. Weigh 1.6 g of ammonium acetate into a 1 L reservoir. b. Add 300 mL of DI water using a graduated cylinder. c. Add 10.0 mL of 10 mM Tetrabutylammonium acetate. d. Adjust the pH to 5.8 using glacial acetic acid e. Add 700 mL of Acetonitrile using a graduated cylinder f. Stir the solution well to ensure thorough mixing.g. Expires in 4 weeks.4) Sample / Standard Diluent:20 mM Ammonium acetate, pH 5.8 in 50% Water and 50% Acetonitrile a. Weigh 1.6 g of ammonium acetate into a IL reservoir. b. Add 500 mL of Water using a graduated cylinder. c. Adjust the pH to 5.8 using glacial acetic acid d. Add 500 mL of Acetonitrile e. Stir the solution well to ensure thorough mixing. f. Expires in 4 weeks.HPLC method:Sample preparation:1. Reference standard (RS) preparation (0.1 mg / mL):1) Accurately weigh 20 ± 2 mg of SRP-3D (DA) on a weighing paper, then transfer it into 200 mL volumetric flask, record the weight of SRP-3D (DA).2) Add ~ 120 mL of prepared diluent, then sonicate to dissolve. Fill it up to the volume 200 mL using the diluent. Mix well. DO NOT use the prepared reference standard for longer than 7 days.2. Impurity sample preparation (1 mg / mL):25 °C sample:1) Tare the weight of a 100-mL volumetric flask to zero. Transfer 1 g of the SRP-3D (DA) ball-milling nanosuspension product (stored at 25 °C) into the 100-mL volumetric flask, record the weight.2) Add ~ 50 mL of the diluent to dilute it. Mix well and fill it up to the volume using prepared diluent2-8 °C sample:3) Tare the weight of a 100-mL volumetric flask to zero. Transfer 1 g of the SRP-3D (DA) ball-milling nanosuspension product (stored at 2-8 °C) into the 100-mL volumetric flask, record the weight.4) Add ~ 50 mL of the diluent to dilute it. Mix well and fill it up to the volume using prepared diluent3. ID and Assay samples preparation (0.1 mg / mL):25 °C sample:1) Tare the weight of a 100-mL volumetric flask to zero. Transfer 100 mg of the ballmilling product (stored at 25 °C) to the VF, record the weight.2) Add ~ 50 mL of the diluent to dilute it. Mix well and fill it up to the volume using prepared diluent2-8 °C sample:3) Tare the weight of a 100-mL volumetric flask to zero. Transfer 100 mg of the ballmilling product (stored at 2-8 °C) to the VF, record the weight.4) Add ~ 50 mL of the diluent to dilute it. Mix well and fill it up to the volume using prepared diluent.

[0203] SRP-3D (DA) Reference Standard Concentration (Cs) calculation:„ Ws PurityCs = — XV 100Ws = weight of standard (mg) V = dilution volume (mL) Purity: use Co A valueImpurity (%w / w) calculation:

[0204] Impurity 100Ri = Peak area from each impurity in impurity sample > LOQ peak area (0.05%)Rs = Peak area of SRP-3D (DA) from all RS injections (use average value)Cs = Concentration of the RS (see above for calculation)Cu = Label concentration of SRP-3D (DA) suspension (e.g., 100 mg / mL)F = Dilution factor = — X D wVf = Dilution volume (mL)W = Weight (g) of the 1 gram of SRP-3D (DA) suspension in impurity sample preparationD = Bulk density of SRP-3D (DA) suspension = 0.990 g / mL

[0205] Assay (%w / w) calculation:Assay 100Ru = Peak area of SRP-3D (DA) from sample solutionRs = Peak area of SRP-3D (DA) from all RS injections (use average value) Cs = Concentration of the RS (see above for calculation)Ci = Concentration of assay sample solution (see above)W = Weight of SRP-3D (DA) suspension in impurity sample preparation D = Bulk density of SRP-3D (DA) suspension = 0.990 g / mL L = Label concentration of SRP-3D (DA) suspensionVi = Dilution volume in impurity sample preparation (e.g., 100 mL)V2 = Volume of impurity sample solution used to prepare assay solution (e.g., 5 mL) V3 = Dilution volume in assay sample preparation (e.g., 50 mL)Total impurities (%w / w) calculation:Total impurities (%w / w) = S each individual impurity > LOQLOQ = 0.5 pg / mL or 0.05% peak area of the impurity sample

[0206] Identification by retention time:The retention time of SRP-3D (DA) in sample is comparable to that of the retention time of the SRP-3D (DA) reference standard.Reporting of results:• Identification of SRP-3D (DA)• SRP-3D (DA) assay (%w / w)• Individual impurities (%w / w)• Total impurities (%w / w)

[0207] METHOD 14: Determination of Particle Size Distribution and Poly dispersity Index (PDI) of SRP-3D (DA) Nanoparticle Suspension

[0208] The particle size distribution and poly dispersity index of SRP-3D (DA) nanoparticle suspensions were determined according to USP <729> Method I Measurement of Mean Droplet Diameter by Dynamic Light Scattering or Classical Light Scattering (USP 45-NF 40 2022-2023) using a Malvern Zetasizer Nano with 0.2 pm filtered DI water as diluent.

[0209] METHOD 15 : Determination of Zeta Potential of SRP-3D (DA) Nanoparticle Suspension

[0210] The zeta potential of SRP-3D (DA) nanoparticle suspensions was determined according to USP <432> Determination of Zeta Potential by Electrophoretic Light Scattering (USP 45-NF 40 2022-2023) using a Malvern Zetasizer Nano with 0.2 pm filtered DI water as diluent.

[0211] METHOD 16: Determination of Viscosity of SRP-3D (DA) Nanoparticle SuspensionThe viscosity of SRP-3D (DA) nanoparticle suspensions was determined according to USP <912> Method III for Cone-and-Plate Rheometer (USP 45-NF 40 2022-2023) using a Brookfield DV-III Ultra Rheometer. The temperature of the sample was controlled with a circulating water bath at 25°C ± 0.5°C.

[0212] EXAMPLE 1

[0213] Synthesis of SRP-3D (DA) [SRP-3D (DA)]The drug substance was manufactured by the process depicted in FIG. 3. SRP-3D (DA) was obtained as 9.38 kg of white crystalline solid (Form A) in 46.2% overall yield having an HPLC assay value of 100.6% on anhydrous, solvent free basis, and total impurities of 0.13%.

[0214] The manufacturing process involved three chemical steps and one recrystallization. Each of the three isolated intermediates was subjected to in-process controls, including loss on drying (LOD) and HPLC total area normalization (TAN), before use in the next step. In- process control test methods are presented in Table 4.Step 1 : Preparation of C-41. Into a 50-L glass lined steel reactor was charged 4-aminophenol (5.6 kg, 1.00 equiv).2. Dichloromethane (110.9 kg) was charged, and agitation of the mixture was begun.3. The batch temperature was adjusted to 20 °C and phosphoric acid (263.4 g, 0.05 equiv) was charged.4. Chloroacetic anhydride (9.2 kg, 1.05 equiv) was charged, and the batch was mixed at 20 °C for at least 10 hr until C-3 < 1% (IPC-1, GC).5. Ethanol (4.4 kg) was charged and the batch was agitated for at least 1 hour.6. The solvent was distilled under vacuum at a jacket temperature of <35 °C until 74.4 kg ± 10 kg of distillate was collected.7. ^-Heptane (39.2 kg) was charged, and then the solvent was distilled under vacuum at a jacket temperature of <60 °C until 39.2 kg ± 10 kg of distillate was collected.8. ^-Heptane (39.2 kg) was charged, and then the solvent was distilled under vacuum at a jacket temperature of <60 °C until 52.1 kg ± 10 kg of distillate was collected and DCM / ^-heptane < 1% and ethanol / ^-heptane < 1% (IPC-2, 'H NMR).9. The batch was cooled to 20 °C and process water (7.4 gal) was charged. The batch was mixed for at least 1 hour, then cooled to 0 °C and mixed for at least 2 hours.10. The batch was filtered, and the cake was washed with process water (5.9 gal) and n- heptane (15.3 kg).11. The cake was dried under vacuum with a nitrogen sweep at 45 °C (jacket inlet temperature) for at least 12 hours until loss on drying (LOD) < 1% (IPC-3) to afford 8.72 kg (91.6% yield) of off-white solid, which was 99.35% pure (IPC-4, HPLC, for information only).Step 2: Preparation of C-l1. Into a 50-L glass lined steel reactor was charged DMF (16.2 kg). Agitation was initiated and the batch temperature was adjusted to 25 °C.2. Compound C-4 (8.6 kg, 1.00 equiv) was charged, resulting in a beige / brown solution.3. Sodium iodide (208.4 g, 0.03 equiv) was charged.4. Sodium saccharin hydrate (11.4 kg, 1.10 equiv) was charged. The batch was heated to 80 °C for at least 12 hours until C-4 < 2% (IPC-5, HPLC, target: C-4 < 1%).5. The batch was cooled to 25 °C. Ethyl acetate (38.7 kg) was charged, followed by process water (22.7 gal). The batch was mixed for at least 4 hours at 25 °C until sampling revealed the presence of solids (IPC-6, visual test). If no solids had been present after holding at least 24 hours, then step 6 would have been executed. However, solids were present, so step 6 was skipped and step 7 was executed.6. Slowly charge / / -heptane (10% of the charge indicated in step 7) over at least 20 minutes. Mix for at least 1 hour at 25 °C until sampling reveals the presence of solids (IPC-6, visual test). Proceed to step 7, but reduce the / / -heptane charge indicated there by 10%.7. / / -Heptane (29.2 kg) was slowly charged over at least 90 minutes. The batch was mixed for at least 18 hours at 25 °C and filtered. The filtration was slower than expected, as compared to previous batches. Poor filtration was known to lead to poor deliquoring of the wet cake. Therefore, the decision was made to analyze the wet cake in step 9.8. The filter cake was washed with process water (6.8 gal), followed by n-heptane (3 x 5.8 kg)-9. The cake was dried under vacuum with a nitrogen sweep at 50 °C (jacket inlet temperature) for 12.6 hours. Analysis of the cake showed purity 96.2% (HPLC) and LOD 27.95%. This purity was lower than the expected value of 99+%. Therefore, corrective action was taken in the following supplemental batch process.Step 2 Supplemental Batch Process:10. Into a 50-L glass lined steel reactor was charged DMF (17.3 kg). Agitation was initiated and the batch temperature was adjusted to 25 °C.11. The C-l wetcake (18.4 kg) from step 9 was charged slowly, resulting in a beige / brown solution. The batch was mixed at 25 °C for at least 1 hour.12. Ethyl acetate (33.9 kg) was charged, followed by process water (22.4 gal). The batch was mixed for at least 16 hours at 25 °C until sampling revealed the presence of solids (IPC-6, visual test). If no solids had been present, then step 13 would have been executed. However, solids were present, so step 13 was skipped and step 14 was executed.13. Slowly charge / / -heptane (10% of the charge indicated in step 14) over at least 10 minutes. Mix for at least 1 hour at 25 °C until sampling reveals the presence of solids (IPC-6, visual test). Proceed to step 14, but reduce the / / -heptane charge indicated there by 10%.14. / / -Heptane (25.8 kg) was slowly charged over at least 90 minutes. The batch was mixed for at least 6 hours at 25 °C and filtered.15. The filter cake was washed sequentially with process water (4.9 gal), / / -heptane (12.6 kg), process water (9.8 gal), and / / -heptane (2 x 12.6 kg).16. The cake was dried under vacuum with a nitrogen sweep at 50 °C (jacket inlet temperature) for at least 12 hours until LOD < 1.0% (IPC-7) to afford 10.50 kg (68.2% based on C-4) of white to off-white solid, which was 99.33% pure (IPC-8, HPLC, for information only) and contained 457 ppm moisture by Karl Fischer (IPC-9, for information only).Step 3 : Preparation of Crude SRP-3D (diethylamide)1. Into a 50-L glass lined steel reactor at a jacket temperature setpoint of 20 °C was charged C-l (10.5 kg).2. Acetonitrile (33.1 kg) was charged and agitation was initiated.3. Diethylamine (9.2 kg) was charged and chased with acetonitrile (3.9 kg). Mixing was continued for at least 10 minutes, and then the batch was heated to reflux (65-75 °C) for at least 12 hours until C-l < 1% (IPC-10, HPLC).4. The batch was cooled to 22 °C, drained through a filter cartridge into a new 55-gal HDPE drum, and chased with ACN (10.3 kg). The reactor was cleaned.5. The reactor jacket was adjusted to 25 °C, and the combined ACN solution from step 4 was charged through a 0.25 pm filter into the clean reactor. Agitation was begun. All subsequent solvent charges were passed through the 0.25 pm filter.6. Distillation was carried out under vacuum at < 45 °C jacket temperature until 19.8 ± 5 kg of solvent was collected.7. Acetonitrile (36.4 kg) was charged. Distillation was continued until 36.4 ± 5 kg of distillate was collected.8. Acetonitrile (36.4 kg) was charged. Distillation was continued until 36.4 ± 5 kg of distillate was collected.9. Acetonitrile (36.4 kg) was charged. Distillation was continued until 42.8 ± 5 kg of distillate was collected and diethylamine / ACN mole ratio < 1% (IPC-11, 'H NMR) in the cooled (20 °C) batch.10. WFI quality water was charged until water / ACN - 3: 1 mol / mol (2 x 31.7 kg; IPC-12, 'H NMR).11. The batch was filtered, and the wetcake was washed with WFI quality water (20.8 kg), and n-heptane (21.3 kg).12. The cake was dried under vacuum with a nitrogen sweep at 50 °C (jacket inlet temperature) for at least 12 hours until LOD < 1% (IPC-13) to afford 10.64 kg (83.1%) of light yellow to light tan to off-white solid, which was 99.68% pure (IPC- 14, HPLC, for information only).Step 4: Preparation of SRP-3D (diethylamide) Form A1. All solvent charges were passed through a 0.25 pm filter.2. Into a 50-L glass lined steel reactor at a jacket temperature setpoint of 25 °C was charged Crude SRP-3D (diethylamide) (10.53 kg).3. Acetone (33.5 kg) was charged and agitation was begun.4. WFI quality water (42.4 kg) was charged.5. The batch was heated to 60 °C and held for 10 minutes.6. WFI quality water (42.4 kg) was charged over at least 2 hours.7. The batch was cooled to 22 °C over > 3 hours. The presence of a slurry was confirmed (IPC-15, visual test). Mixing was continued for > 8 hours at 22 °C.8. The batch was filtered, and the wetcake washed with WFI quality water (2 x 15.9 kg) and / / -heptane (14.5 kg).9. The cake was dried under vacuum with a nitrogen sweep at 50 °C (jacket inlet temperature) for at least 12 hours until LOD < 1.0% (IPC-16). Optional analysis for residual solvents showed ACN not detected (ND), n-heptane ND, DMF ND and acetone <2446 ppm (IPC-17, GC). Optional HPLC analysis showed purity 100.6% (IPC-18). The dried batch was de-lumped by passing through a comill to afford 9.38 kg (89.1%) of white solid.

[0215] Analysis results for the product of Step 4 are presented in Table 5.Table 4. In-Process Controls for SRP-3D (diethylamide)Table 5. Analysis Results for SRP-3D (DA) Manufactured According to Example 1

[0216] EXAMPLE 2

[0217] Single Crystal X-ray Structure Determination of SRP-3D (DA) Form A

[0218] A sample of SRP-3D (DA) Form A prepared according to Example 1 was subjected to single crystal X-ray structure determination. A colorless crystal fragment having approximate dimensions of 0.45 x 0.40 x 0.21 mm was mounted on a Mitegen micromesh mount in a random orientation. Preliminary examination and data collection were performed using Mo Ka radiation (Z. = 0.71073 A) on a Bruker AXS D8 Quest CMOS diffractometer with a fixed chi angle, a sealed tube fine focus X-ray tube, single crystal curved graphite incident beam monochromator, a Photon 100 CMOS area detector and an Oxford Cryosystems low temperature device. The initial unit cell was determined and data were collected using Apex3 v2018.3-0 at a temperature of 150 K. Frames were integrated using SAINT V8.38A.

[0219] A total of 96,505 reflections were collected, of which 7,679 were unique. Cell constants for data collection were obtained from least-squares refinement using 9,996 reflections between 2.4086 and 33.1648°. The orthorhombic cell parameters and calculated volume are a = 9.3858(5) A, b = 10.9470(6) A, c = 19.4881(11) A, V = 2002.33(19) A3. For Z = 4 and a formula weight of 405.46 the calculated density is 1.345 g / cm3. The linear absorption coefficient is 0.197 / mm for Mo Ka radiation. Scaling and a multi-scan absorption correction using SADABS (Apex3 v2017 / 3) was applied. Transmission coefficients rangedfrom 0.7195 to 0.7465. Intensities of equivalent reflections were not averaged during data processing.

[0220] The space group was determined by the program XPREP as embedded in SHELXTL. Systematic absences and intensity statistics indicated the space group to be F212121 (# 19). The structure was solved by direct methods using SHELXS and refined by full matrix least squares against F2with all reflections using SHELXL-2018 and the graphical user interface ShelXle. Additional atoms were located in succeeding difference Fourier syntheses.

[0221] The structure was refined using full-matrix least-squares where the function minimized was Sw(|F0|2-|Fc|2)2and the weight w is defined as w = l / [o2(F02) + (0.0384F)2+ 0.3049F] where P = (Fo2+ 2Fc2) / 3. Scattering factors were taken from the International Tables for Crystallography (Vol C Tables 4.2.6.8 and 6.1.1.4). A total of 7,679 independent reflections were used in the refinements. A total of 7,251 reflections with F2> '1<5(F2) were used in the calculation of Rl.

[0222] Hydrogen atoms attached to carbon atoms were positioned geometrically and constrained to ride on their parent atoms, with carbon-hydrogen bond distances of 0.95 A for aromatic C-H, and 0.99 and 0.98 A for aliphatic CH2 and CH3 moieties, respectively.

[0223] The final cycle of refinement included 265 variable parameters and 0 restraints and converged (largest parameter shift was 0.002 times its standard uncertainty) with unweighted and weighted agreement factors of:Rl = E |F0| - |FC| / S |F0| = 0.0267 wR2= {E [w (Fo2- Fc2)2] / S [w(Fo2)2]}0 5= 0.0720

[0224] The goodness-of-fit parameter was 1.085. The highest peak in the final difference Fourier had a height of 0.295 e / A3. The minimum negative peak had a height of -0.303 e / A3.

[0225] Crystal data and data collection parameters are given in Table 6.

[0226] The asymmetric unit from the crystal structure of SRP-3D (DA) Form A determined by X-ray diffraction is shown in FIG.17.Table 6. Crystal Data and Data Collection and Refinement Parameters for SRP-3D (DA) Form A

[0227] EXAMPLE 3

[0228] Preparation of an SRP-3D (DA) nanoparticle suspension for oral intake by wet millingThe batch formula for SRP-3D (DA) Oral Suspension, 100 mg / mL is presented in Table 7. Table 7. Batch Formula for SRP-3D (diethylamide) Oral Suspension, 100 mg / mL“Batch size may be adjusted as needed QS = quantity sufficient“Density = 0.990 g / mL; 4,950 g = 5,000 mL

[0229] The SRP-3D (diethylamide) Oral Suspension, 100 mg / mL was manufactured by the process described below. The process can be scaled to different batch sizes and vial sizes as needed.1. All equipment, vessels and supplies expected to come in direct contact with the batch were purchased sterile, autoclaved or cleaned with alcohol before use.Production Procedure: Step 12. A 10-L stainless steel container was labeled as Container #1 and tared.3. To Container #1 was added hydroxypropyl cellulose, USP (62.48 g). 4. To Container #1 was added sodium benzoate, NF (6.25 g).5. To Container #1 was added purified USP water (5931.3 g).6. The contents of Container #1 were mixed with a Silverson mixer at 5,000 rpm until a clear solution (1% HPC solution) was obtained. Production Procedure: Step 27. A second 10-L stainless steel container was labeled as Container #2 and tared.8. To Container #2 was added SRP-3D (diethylamide) (500.0 g).9. Using a peristaltic pump, 1% HPC solution (3,955.7 g) was transferred from Container #1 to Container #2. 10. The contents of Container #2 were mixed with a Silverson mixer at 10,000 rpm for 10 minutes or until a uniform suspension formed.Production Procedure: Step 311. A 5-L Nalgene bottle was labeled as Container #3 and tared.12. A Netzsch MiniCer ball mill was set up per the user manual with 140 mL of 0.4 mm Zetabeads in the mixing chamber. The outlet feed was replaced with a tri-clamp 5 / 8” hose barb fitting, to which a 36”-long sterile tube was attached. A 48”-long sterile tube was attached to the inlet. The chiller was turned on and set to 5 °C, and coolant was allowed to circulate through the ball mill to equilibrate it before use.13. The contents of Container #2 were stirred at 400 rpm with an overhead mixer to prevent the suspension from settling. If necessary, the bottom of Container #2 was scraped with a spatula to resuspend any large particles.14. The inlet and outlet tubes of the ball mixer were inserted into Container #2 and Container #3, respectively.15. The peristaltic pump of the ball mill apparatus was turned on with minimal flow rate and the direction set to pump the suspension into the milling chamber.16. The ball mill rotor was turned on at minimal speed (10 Hz or 600 rpm), then sped up to 2,400 rpm within 1 minute.17. The flow rate of the pump was gradually increased so as to keep the pressure of the milling chamber below 15 psi. If the pressure exceeded 15 psi, the pump was turned off and the pressure slowly released.18. The pressure, pump flow setting, temperature, and rotor speed were recorded.19. When all of the suspension had passed through the milling chamber, the mill and pump were stopped and a 1-mL sample was withdrawn using a sterile pipette.20. The particle size distribution (PSD) of the collected sample was measured using a Malvern Zeta-Sizer.21. The cycle # and PSD results were recorded.22. Container #3 was gently shaken and the contents poured into Container #2. The outlet tubing was placed back in Container #3.23. Steps 15 - 22 were repeated and a 1-mL sample withdrawn for PSD analysis after every 4 cycles until 12 cycles had been completed.24. Both the inlet and outlet tubes were placed in Container #2.25. The suspension was recirculated through the ball mill with the same parameters as above.26. The pressure, pump flow setting, temperature, and rotor speed were recorded every 60 minutes.27. Every 60 minutes, a 1-mL sample was withdrawn and analyzed for PSD until Dso was below 300 nm.28. Finally, the suspension was pumped through the ball mill into Container #3, weighed (W13 = 4,255.8 g), and analyzed by HPLC for API concentration. Based on these results, the amount of additional 1% HPC solution needed to adjust the final API concentration to 100 mg / mL (10% w / v) was calculated.29. The calculated amount (W14 = 474.5 g) of 1% HPC solution was added.30. A 1-mL sample was withdrawn and analyzed for PSD.Production Procedure: Step 431. The nanosuspension was transferred from Container #3 to Container #2 so that mixing with the overhead mixer could be continued.32. Using the previously determined density (0.99 g / mL) of SRP-3D (diethylamide) suspension, 100 mg / mL, and the acceptable filling volume range of 20.2 - 21.8 mL (target 21.0 mL), the acceptable filling weight range was calculated as 20.0 - 21.6 g.33. The fill volume on a Baxter Baxa Repeater pump was calibrated by receiving one fill into a tared, sterile 50-mL Falcon tube, recording the weight, and adjusting the fillvolume as needed for a 20.8-g fill. All weight check samples were recycled back into Container #2. When three successive fills were within the acceptance criteria (20.0 - 21.6 g), the next step was executed.34. While mixing of the suspension in Container #2 was maintained with the overhead mixer, the Repeater pump was used to fill 20-mL type 1 glass vials to the target fill weight. Weight checks were performed before and after the filling run.35. After filling, the vials were closed with sterile 18-mm rubber stoppers.36. Caps were screwed on to seal tightly.37. Trays containing the vials were appropriately labeled and stored at the recommended storage condition.38. The Repeater pump was reversed and any residue in the tubing pushed back into Container #2 as much as possible.39. The weight of bulk suspension available for filling was calculated as W13 + W14 = 4,730.3 g.40. The weight of unfilled bulk suspension in Container #2 was determined (18.7 g).41. The weight of filled bulk was calculated as 226 filled vials x 20.8 g / vial (target fill weight) = 4700.8 g.42. The total bulk accounted for was 4719.5 g (99.8%).43. The % yield of filled vials was calculated as (number of vials filled) / [(bulk suspension available for filling) / (target fill weight)] x 100% = (226 vials filled) / [(4,730.3 g) / (20.8 g / vial)] x 100% = 99.6%.

[0230] Release and stability results for the batch produced in Example 3 through 24 months at 5 °C, 25 °C / 60% RH and 6 months at 40 °C / 75% RH are presented in Tables 8, 9 and 10. This batch met all specifications at all of the above conditions. It exhibited a trend of slightly increasing particle size at 5 °C and 25 °C / 60% RH, which was more pronounced at 40 °C / 75% RH.Table 8. Stability Results for SRP-3D (diethylamide) Oral Suspension, 100 mg / mL at 5± 3 °C“ Vial 1: RRT 0.97 0.22%, RRT 1.12 0.09%. Vial 2: none detected.Table 9. Stability Results for SRP-3D (diethylamide) Oral Suspension, 100 mg / mL at25 ± 2 °C / 60% RH ± 5%Table 10: Stability Results for SRP-3D (diethylamide) Oral Suspension, 100 mg / mL at40 ± 2 °C / 75% RH ± 5%

[0231] EXAMPLE 4

[0232] Preparation of an SRP-3D (DA) nanoparticle suspension with IV-compatible excipients by wet milling

[0233] A slurry or thick suspension containing 10% (w / w) SRP-3D (DA), 0.5% (w / w) sodium deoxy cholate and 10% (w / w) sucrose was wet milled for 1 hour using a Netzsch MiniCer® ball mill with 0.4 mm grinding zirconium beads.

[0234] The nanoparticle suspension produced was a translucent liquid, with no visual particles seen under an optical microscope. Dynamic light scattering measurement revealed an average particle size of 199.3 nm and polydispersity index of 0.084.

[0235] EXAMPLE 5

[0236] Comparison of wetting agents for preparation of SRP-3D (DA) nanoparticle suspensions by wet milling

[0237] The following aqueous suspending liquids containing various wetting agents were used to wet mill SRP-3D (DA) using similar milling conditions as shown in Example 4. The particle size of the resulting SRP-3D (DA) nanosuspensions were as indicated below:

[0238] The data indicated that sodium deoxycholate is the preferred wetting agent.

[0239] EXAMPLE 6

[0240] Preparation of other SRP-3D (DA) nanoparticle suspension formulations

[0241] The following formulations have been designed for wet milling to produce SRP-3D(DA) aqueous nanoparticle suspensions:SRP-3D (DA) nanosuspension formulations

[0242] EXAMPLE 7

[0243] Freeze-drying of an SRP-3D (DA) nanoparticle suspension

[0244] The SRP-3D (DA) nanoparticle suspension of Example 4 was freeze-dried or lyophilized using the following conditions:■ Primary drying step: -40 °C for 48 hours■ Secondary drying step: From -40 °C to 25 °C for 12 hours

[0245] The particle size of the nanoparticle formulation was measured before and after lyophilization, giving the following results:deoxy cholate, and 10% _ sucrose __ *Lyophilized samples were reconstituted with DI water before testing

[0246] The lyophilized nanoparticle formulation was tested for the presence of large particles (> 10-20 micron) or Particulate Matter and passed the USP Particulate Matter specification (USP <788>), indicating it was free of large particles and suitable for injection.

[0247] The SRP-3D (DA) nanoparticle formulation of Example 4 was stable physically during the lyophilization process with no significant increase in particle size detected.

[0248] EXAMPLE 8

[0249] Stability of a lyophilized SRP-3D (DA) nanoparticle formulation

[0250] The lyophilized SRP-3D (DA) nanoparticle formulation of Example 7 was stored at5°C, 25°C and 40°C for stability testing. After 7 and 14 days, it was tested for average particle size (Zaverage) by dynamic light scattering. The results are presented below.*Lyophilized samples were reconstituted with DI water before testing

[0251] The lyophilized SRP-3D (DA) nanoparticle formulation of Example 7 was stable physically, with no significant increase in particle size detected at all 3 storage temperatures.

[0252] EXAMPLE 9

[0253] Sterilization of a lyophilized SRP-3D (DA) nanoparticle formulation

[0254] Another batch of SRP-3D (DA) nanoparticle suspension was prepared using the same composition and process as described in Example 4. The nanoparticle suspension containing 10% (w / w) SRP-3D (DA), 0.5% (w / w) sodium deoxy cholate and 10% (w / w) sucrose in water was prepared by wet milling and subsequently lyophilized.

[0255] Gamma irradiation was applied to the lyophilized nanoparticle formulation at dose range 15.0 - 19.5 kGy. This dose would render the nanoparticle formulation sterile. Survival of the nanoparticle formulation after such radiation indicates that the gamma irradiation is a suitable method to sterilize an SRP-3D (DA) nanoparticle formulation for injection.

[0256] The test conditions and results are summarized below:aLyophilized samples were reconstituted with DI water before testingbMETHOD 13cMETHOD 14dMicrobial testing according to USP <71>NA = not applicable; ND = none detected; NT = not tested; NG = no growth

[0257] EXAMPLE 10

[0258] Oral dosing of SRP-3D (DA) nanoparticle suspension in dogs

[0259] A 28-day repeat dose oral toxicity study with an SRP-3D (DA) nanoparticle suspension prepared as described in Example 3 (mean diameter 258.4 nm) in beagle dogs was carried out, including the following treatment groups:Three anima Is / group were euthanized on Day 29. i he remaining two animals / grou in Groups 1 and 4 remained on study, untreated, for a 14-day recovery period.

[0260] The toxicology observation and tests concluded that the no observed adverse effect level (NOAEL) of SRP-3D (DA) in male Beagle dogs dosed three times daily for 28 days was 495 mg / kg / day.

[0261] Toxicokinetics (TK) findings are summarized as follow:

[0262] Day 1

[0263] On Day 1, Cmax values of SRP-3D (DA) appeared to increase with SRP-3D (DA) dose from 165 to 495 mg / kg / day while AUC (0-24) values increased with SRP-3D (DA) dose from 165 to 330 mg / kg / day and did not appear to change from 330 to 495 mg / kg / day. On Day 1, mean Cmax values were 4,380, 8,650 and 9,420 ng / mL and the mean AUC (0-24) values were 12,500, 36,700 and 34,700 ng h / mL for SRP-3D (DA) doses of 165, 330 and 495 mg / kg, respectively.

[0264] Day 14

[0265] On Day 14, Cmax and AUC (0-24) values of SRP-3D (DA) appeared to increase with SRP-3D (DA) dose from 165 to 330 mg / kg / day and did not appear to change from 330 to 495 mg / kg / day. On Day 14, mean Cmax values were 2,330, 4,240 and 3,830 ng / mL andthe mean AUC (0-24) values were 7,090, 17,700 and 17,700 ng h / mL for SRP-3D (DA) doses of 165, 330 and 495 mg / kg, respectively.

[0266] Day 28

[0267] On Day 28, Cmax and AUC (0-24) values of SRP-3D (DA) appeared to increase with SRP-3D (DA) dose from 165 to 495 mg / kg / day. Day 28 mean Cmax values were 3,030, 5,580 and 7,710 ng / mL and the mean AUC (0-24) values were 7,360, 18,000 and 20,300 ng h / mL for SRP-3D (DA) doses of 165, 330 and 495 mg / kg / day, respectively.

[0268] The SRP-3D (DA) nanoparticle suspension is orally available in dogs. It provides a systemic exposure (AUC) that is proportional to the dose up to 330 to 495 mg / kg / day.

[0269] EXAMPLE 11

[0270] Reduction of the particle size of SRP-3D (DA) to <1 pm resulted in a dramatic increase in plasma AUCo-24 / (dose / day) after oral administration in dogs, as compared to unmilled particles. Plasma levels of SRP-3D (DA) were determined by LC-MSMS using a validated method.

[0271] Single oral administration of SRP-3D (diethylamide) powder (Dio 50.22 pm; Dso 217.26 pm; D90 538.54 pm) in gelatin capsules at 300 mg / kg gave the following results:AUCo-24 1,993 ng.hr / mLAUCo-24 / (dose / day) 6.64 (ng.hr / mL) / [(mg / kg) / day]

[0272] Oral administration of an SRP-3D (DA) nanoparticle suspension prepared as described in Example 3 (mean diameter 258.4 nm) at 110 mg / kg / dose three times daily, 4 hours apart (330 mg / kg / day) gave the following results:AUCO-24 36,700 ng.hr / mLAUCo-24 / (dose / day) 111 (ng.hr / mL) / [(mg / kg) / day]

[0273] The above results indicate a 16.7-fold increase in AUCo-24 / (dose / day) for the nanoparticle suspension vs. un-micronized powder in capsules.EXAMPLE 12Oral dosing of SRP-3D (DA) nanosuspension in healthy human volunteers

[0274] In a single ascending dose study, an SRP-3D (DA) nanoparticle suspension prepared as described in Example 3 (mean diameter 301 nm) was administered as a single oral dose of5 300 mg, 600 mg, 900 mg, or 2000 mg to healthy human volunteers. The effect of food on pharmacokinetics was assessed in Cohort 3 by administering a single 900 mg dose to volunteers who were either fasted (Regimen C) or fed (Regimen D) prior to dosing. Volunteers in all other cohorts were fasted prior to dosing. Plasma levels of SRP-3D (DA) were determined by LC-MSMS using a validated method. The pharmacokinetic and safety10 findings are summarized in Table 11.Table 11. SRP-3D (DA) Single- Ascending Dose (SAD) Pharmacokinetic and SafetyFindings*geometric mean (geometric CV%)15

[0275] In a multiple ascending dose study, the SRP-3D (DA) nanoparticle suspension prepared as described in Example 3 (mean diameter 301 nm) was administered once daily (QD) at either 500 mg / day or 1000 mg / day for five days to healthy human volunteers in the5 fasted state. The pharmacokinetic and safety findings are summarized in Table 12.Table 12. SRP-3D (DA) Multiple-Ascending Dose (MAD) Pharmacokinetic and Safety Findings*geometric mean (geometric CV%) 0

[0276] When administered orally to healthy human volunteers, the SRP-3D (DA) nanoparticle suspension prepared as described in Example 3 was absorbed rapidly. The resulting plasma levels of SRP-3D (DA) increased approximately in proportion to dose. No serious adverse events or discontinuations for safety reasons were observed, indicating that the drug administered in this manner was well-tolerated.EXAMPLE 13

[0277] Preparation of Amorphous SRP-3D (DA)

[0278] A saturated solution was prepared by agitating excess SRP-3D (DA), prepared according to Example 1, in contact with either toluene or trifluoroethanol (2-10 mL) at ambient temperature. The mother liquor was then heated above the saturation temperature to dissolve any remaining solids. The temperature of the solution was then adjusted to ambient and a controlled nitrogen shear flow was introduced to begin solvent evaporation. Analysis of the resulting solid by XRPD according to Method 10 showed no discernible diffraction peaks, indicating that the sample was non-crystalline.

Claims

WHAT IS CLAIMED IS:

1. A stable SRP-3D (DA) nanoparticulate composition, the composition comprising:(a) particles of SRP-3D (DA) or a salt thereof, and(b) at least one wetting agent, wherein the SRP-3D (DA) particles have an average particle size of less than about 500 nm or less than about 300 nm.

2. The composition of claim 1, wherein the SRP-3D (DA) is a member selected from the group consisting of a crystalline phase, an amorphous phase, a semi-crystalline phase, a semi-amorphous phase, and mixtures thereof.

3. The composition of any one of claims 1-2, wherein the average particle size of the nanoparticulate SRP-3D (DA) particles is a member selected from the group consisting of less than about 500 nm, or less than about 400 nm, or less than about 300 nm, or less than about 250 nm, or less than about 200 nm, or less than about 150 nm, or less than about 100 nm, or less than about 75 nm, and less than about 50 nm.

4. The composition of any one of claims 1-3, wherein the composition is formulated for administration which is a member selected from the group consisting of oral administration and injection.

5. The composition of any one of claims 1-4, wherein the composition is formulated into a dosage form which is a member selected from the group which is a member selected from the group consisting of a liquid dispersion, a liquid suspension, a solution, a powder, a capsule, and a tablet.

6. The composition of any one of claims 1-5, wherein the composition further comprises one or more pharmaceutically acceptable excipients, carriers, or a combination thereof.

7. The composition of any one of claims 1-6, wherein the SRP-3D (DA) is present in an amount which is a member selected from the group consisting of from about 99.5% to about 0.001%, from about 95% to about 0.1%, and from about 90% to about 0.5%,by weight, based on the total combined dry weight of the SRP-3D (DA) and at least one wetting agent, not including other excipients.

8. The composition of any one of claims 1-7, wherein the at least one wetting agent is present in an amount which is a member selected from the group consisting of from about 0.01% to about 99.5% by weight, from about 0.1% to about 95% by weight, and from about 0.5% to about 90% by weight, and from about 0.5 % to about 2%, based on the total combined dry weight of SRP-3D (DA) and at least one wetting agent, not including other excipients.

9. The composition of any one of claims 1-8, wherein the at least one wetting agent is present in an amount which is a member selected from the group consisting of less than about 10%, less than about 5%, less than about 2% and less than about 1%, based on the weight of the nanoparticulate composition.

10. The composition of any one of claims 1-9, wherein the at least one wetting agent is a member selected from the group consisting of a bile salt and an alkali salt of a bile acid.

11. The composition of claim 10, wherein the bile salt is a member selected from the group consisting of sodium, potassium, lithium, calcium, arginine, lysine and ammonium salt of a bile acid.

12. The composition of claim 10, comprising a salt of a bile acid which is a member selected from the group consisting of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid glycochenoxydeoxycholic acid and taurochenoxydeoxycholic acid.

13. The composition of any one of claims 1-12, further comprising a secondary wetting agent which is a member selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, and cellulose derivatives.

14. The composition of any one of claims 1-13, further comprising a cryo- or lyo- protectant which is a member selected from the group consisting of sucrose, mannitol, trehalose, lactose, and altodextrin.7315. A method of making a nanoparticulate composition comprising wet milling SRP-3D (DA) particles with at least one wetting agent for a time and under conditions sufficient to provide a SRP-3D (DA) nanoparticulate suspension having an average particle size of less than about 500 nm, or less than about 300 nm.

16. The method of claim 15, wherein said wet milling comprises a method which is a member selected from the group consisting of ball milling and microfluidization.

17. The method of any one of claims 15-16, wherein said wet milling comprises: (a) adding SRP-3D (DA) particles to a solution comprising a wetting agent to obtain a suspension and (b) wet milling tire suspension to obtain a nanoparticulate suspension having the desired final particle size of less than about 500 nm or less than about 300 nm.

18. The method of any one of claims 15-17, wherein the final particle size of the nanoparticulate SRP-3D (DA) particles is selected from the group which is a member consisting of less than about 500 nm. less than about 300 mm, less than about 250 nm, less than about 200 nm less than about 150 nm, less than about 100 nm, less than about 75 nm, and less than about 50 nm19. The method of any one of claims 15-18, wherein said wet milling process further comprises a drying step to remove the water from the nanoparticulate suspension to obtain dry powder by one or more methods which is a member selected from the group consisting of filtration, centrifugation, freeze-drying, spray drying and vacuum drying.

20. A compound of Formula I:wherein the compound is substantially free of impurities.

21. The compound of claim 20, wherein the compound is greater than 90% w / w pure.

22. The compound of any one of claims 20-21, wherein the compound is greater than 95% w / w pure.

23. The compound of any one of claims 20-22, wherein the compound is greater than 99% w / w pure.

24. The compound of any one of claims 20-23, wherein the compound is amorphous.

25. The compound of any one of claims 20-23, wherein the compound is crystalline.

26. A solid form of Formula I:wherein the solid form is characterized by an X-ray powder diffraction (XRPD) pattern comprising the following 20 peaks measured using CuKa radiation: 9.0 ± 0.2, 9.2 ± 0.2, 13.2 ± 0.2, 21.9 ± 0.2.

27. A solid form of Formula I according to claim 26, wherein the solid form is characterized by an X-ray powder diffraction (XRPD) pattern furflier comprising the following 20 peaks measured using CuKa radiation: 12.2 ± 0.2, 15.4 ± 0.2, 18.3 ± 0.2, 19.2 ± 0.2, 20.7 ± 0.2, 22.5 ± 0.2.

28. A solid form of Formula I according to claim 26, wherein the solid form is characterized by an X-ray powder diffraction (XRPD) pattern substantially similar to that set forth in FIG. 10, 11 or 12 as measured using CuKa radiation.

29. A nanoparticle suspension for oral administration, the nanoparticle suspension comprising:(a) SRP-3D (DA); and(b) at least one wetting agent.

30. The nanoparticle suspension of claim 29, wherein the wetting agent is hydroxypropyl cellulose.

31. The nanoparticle suspension of any one of claims 29-30, further comprising a preservative.

32. The nanoparticle suspension of any one of claims 29-31, wherein the suspension is aqueous.

33. A method for treating, reducing, or ameliorating pain in a subject in need thereof, the method comprising: administering a compound of Formula I to the subject, to thereby treat, reduce or ameliorate pain.

34. The method of claim 33, wherein the pain which is a member selected from the group consisting of acute, chronic, and neuropathic pain.

35. The method of any one of claims 33-34, wherein the compound of Formula I is formulated as a nanoparticle suspension for oral administration, the nanoparticle suspension comprising:(a) a compound of Formula I; and(b) at least one wetting agent.

36. The method of any one of claims 33-35, wherein the method is non-cytotoxic to the liver of the subject.

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