Nanoparticle formulations of non-opioid drugs

Abstract

The present disclosure relates to nanoparticle formulations of N, N-diethyl-2-(N-(2-(4-hydroxyphenylamino)-2-oxoethyl) sulfamoyl) benzamide (SRP-3D (DA)), which is an active pharmaceutical ingredient that is insoluble in water and cannot be dissolved in any injection safe carrier to a sufficient administration concentration. The present disclosure also includes nanoparticle suspensions comprising SRP-3D (DA) (or SRP-3D (DA) nanoparticles) in the form of nanoparticles, a wetting agent, and a cryoprotectant. The nanoparticle composition includes SRP-3D (DA) particles having an average particle size of less than about 500 nm or less than about 300 nm.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 451,481, filed March 10, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This disclosure relates to N,N-diethyl-2-(N-(2-(4-hydroxyphenylamino)-2-oxoethyl)aminosulfonyl)benzamide (SRP-3D(DA), a pharmaceutical ingredient) in solid form; nanoparticle formulations, methods for preparing the same, and dispersions comprising said nanoparticles. This disclosure also relates to the use of said nanoparticles in pharmaceutical compositions and methods of treating mammals. Background Technology

[0004] SRP-3D(DA) is a drug molecule with a molecular weight of 405.5 g / mol, and its structure is as follows: Figure 1 As shown. SRP-3D(DA) is a non-narcotic analgesic used to treat acute and chronic pain, with a projected daily human dose in the range of 100–1000 mg. The intended routes of administration for SRP-3D(DA) are intravenous (IV) injection and oral (PO).

[0005] SRP-3D(DA) is insoluble in water. Due to its poor solubility, it is difficult to administer SRP-3D(DA) intravenously. Various excipients have been attempted to solubilize SRP-3D(DA) in order to develop an intravenous formulation, including organic solvents, surfactants, emulsifiers, suspending agents, and solubilizers, but without success. These commonly used solubilizers are either too toxic or cannot solubilize SRP-3D(DA) to a sufficient dosage concentration.

[0006] All injectable drugs must be sterile. Sterilization of drugs is typically achieved through filtration using a 0.2-micron filter membrane. However, suspensions generally cannot pass through such small-pore membranes. Therefore, formulations of injectable suspension drugs must be sterilizable by filtration (e.g., drug particles small enough to freely pass through a 0.2 μm filter membrane) or by other sterilization methods (such as gamma irradiation).

[0007] Furthermore, injectable suspension formulations must be stable to meet the shelf life required for commercial use. The stability of a suspension is typically measured by the ability of suspended particles to maintain their particle size. If particles in a suspension increase in size or aggregate (particles clump together to form lumps, i.e., "caking"), the suspension is considered unstable and therefore unsuitable for pharmaceutical use.

[0008] SRP-3D(DA) has low oral bioavailability due to its poor solubility (e.g., 0.3 mg / mL in simulated intestinal fluid during eating or fasting), making its oral administration less than ideal. Therefore, there is a need for an SRP-3D(DA) formulation that allows for both injection and oral administration. This disclosure addresses this need and offers additional advantages. Summary of the Invention

[0009] This disclosure relates to novel nanoparticle formulations of SRP-3D(DA), wherein the SRP-3D(DA) nanoparticles are safe for intravenous or oral administration. Furthermore, the SRP-3D(DA) nanoparticles disclosed in this application can significantly improve oral bioavailability.

[0010] For intravenous injection, the formulation must not contain any large particles that could obstruct capillaries and cause embolism. The United States Pharmacopeia (USP) specifies the following for injectable drugs (USP). <788> Method 1): "In the tested unit, the average number of particles equal to or greater than 10 micrometers shall not exceed 6,000 per container, and the average number of particles equal to or greater than 25 micrometers shall not exceed 600 per container."

[0011] This disclosure provides an injectable, stable, and dispersible SRP-3D(DA) nanoparticle formulation, and a method for preparing the nanoparticles by wet milling in the presence of a grinding medium and a wetting agent. The nanoparticles can be formulated into safe injectable or oral formulations with higher oral bioavailability.

[0012] More specifically, according to this disclosure, dry solid nanoparticles are provided that comprise or consist substantially of crystalline SRP-3D(DA) with an average particle size of less than about 400 nm and sufficient stability for use in pharmaceutical dosage forms.

[0013] This disclosure also provides an injectable suspension formulation comprising or substantially consisting of a liquid or suspension carrier or medium and the aforementioned nanoparticles dispersed in the liquid.

[0014] This disclosure also provides a nanosuspension composition comprising or substantially consisting of the above-described SRP-3D(DA) nanoparticles in an aqueous suspension carrier.

[0015] In one embodiment, the nanosuspension can be passed through a 10-micron filter.

[0016] In another embodiment, the nanosuspension can be filtered through a 0.2-micron filter.

[0017] In another embodiment, the nanosuspension can be sterilized by gamma irradiation.

[0018] In a particularly relevant embodiment, the nanosuspension is stable.

[0019] In a particularly relevant embodiment of this disclosure, the nanosuspension described herein is safe for intravenous injection.

[0020] In another embodiment of this disclosure, the nanosuspension described herein provides faster in vitro dissolution of SRP-3D(DA) compared to larger particle sizes.

[0021] In another particularly relevant embodiment of this disclosure, the nanosuspension or nanoparticles described herein can improve the oral bioavailability of SRP-3D(DA).

[0022] In another embodiment of this disclosure, a wet milling method for preparing the above-mentioned particles is provided, the method comprising the steps of: dispersing SRP-3D(DA) active pharmaceutical ingredient in a liquid suspension carrier, and applying mechanical milling in the presence of a milling medium (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.

[0023] In one specific embodiment of this disclosure, a pharmaceutical composition is provided comprising dried nanoparticles or a nanosuspension, wherein the nanoparticles are dispersed in a pharmaceutically acceptable suspension. The pharmaceutical composition may be used to treat mammals.

[0024] In yet another embodiment, this disclosure provides a method for treating, reducing, or improving pain in an individual in need, the method comprising: administering a compound of formula I to the individual to treat, reduce, or improve the pain.

[0025] These and other aspects, objectives, and implementation schemes will become clearer when read in conjunction with the following detailed description and accompanying figures. Attached Figure Description

[0026] Figure 1 The chemical structure of SRP-3D(DA) is shown.

[0027] Figure 2 Optical microscope images showing conventional unground SRP-3D(DA) particles (left) and SRP-3D(DA) nanoparticles with an average particle size of 400 nm (right).

[0028] Figure 3 The method for synthesizing SRP-3D(DA) is shown.

[0029] Figure 4 The SRP-3D(DA) is shown. 1H NMR spectroscopy and structural correlation.

[0030] Figure 5 The SRP-3D(DA) is shown. 13 C10 NMR spectroscopy and structural correlation.

[0031] Figure 6 The FT-IR spectrum of SRP-3D(DA) is shown.

[0032] Figure 7 The mass spectrum of SRP-3D(DA) is shown.

[0033] Figure 8 Show Figure 7 A magnified view of the SRP-3D(DA) mass spectrometer.

[0034] Figure 9 The peak of the daughter ion at m / z 406.1 is shown for SRP-3D(DA).

[0035] Figure 10 The X-ray powder diffraction pattern of SRP-3D(DA) crystal form A sample 1 is shown.

[0036] Figure 11 The X-ray powder diffraction pattern of sample 2 (unground sample) of SRP-3D(DA) crystal form A is shown.

[0037] Figure 12 The X-ray powder diffraction pattern of sample 3 (ground sample) of SRP-3D(DA) crystal form A is shown.

[0038] Figure 13 The sample chromatograms of the C-4 identification standard are shown in the HPLC method used for monitoring steps 1–3 and the recrystallization process.

[0039] Figure 14 The sample chromatogram of the endpoint standard in step 2 of the HPLC method used for monitoring steps 1-3 and the recrystallization process is shown.

[0040] Figure 15 The sample chromatograms of the endpoint standard in step 3 of the HPLC method used for monitoring steps 1-3 and the recrystallization process are shown.

[0041] Figure 16 The differential scanning calorimetry (DSC) curve of SRP-3D(DA) is shown.

[0042] Figure 17 The asymmetric unit cell in the crystal structure of SRP-3D(DA) crystal form A, as determined by X-ray diffraction, is shown. Carbon atoms are gray, hydrogen atoms are white, oxygen atoms are red (3), nitrogen atoms are blue (2), and sulfur atoms are yellow (4).

[0043] Figure 18 The thermogravimetric analysis (TGA) curves of SRP-3D(DA) are shown. Detailed Implementation

[0044] definition

[0045] 1. Nanoparticles

[0046] The nanosuspension or nanoparticle composition described herein refers to a formulation containing nanoparticles (i.e., at least one particle with an external dimension of less than about 1 μm), said nanoparticles being composed of a poorly soluble drug or a non-opioid drug and one or more wetting agents.

[0047] 2. Wetting agent

[0048] The compositions disclosed herein comprise one or more wetting agents or surface stabilizers. The wetting agents of this disclosure are preferably adsorbed onto or associated with the surface of drug particles. Wetting agents particularly useful herein preferably do not chemically react with the drug or its particles. Suitable wetting agents are preferably 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 amphoteric surfactants, hydrophilic polymers, and polyether-derived polysaccharides. More preferred wetting agents are bile salts.

[0049] Wetting agents can be the same chemical substance as drug solubilizers. For example, sodium deoxycholate is a preferred wetting agent of this disclosure and can also be used as a solubilizer to solubilize water-insoluble drugs such as amphotericin B, resveratrol, and oxapzin. However, the function of wetting agents differs from that of solubilizers. Solubilizers are used to solubilize insoluble drugs, while wetting agents are not intended to solubilize drugs. Wetting agents are added to reduce the surface tension of solid drug particles, enabling them to form suspensions with higher physical stability in water and improving the efficiency of wet milling to the desired particle size. In the nanosuspensions of this disclosure, the solid particles must remain in an insoluble state in the presence of a wetting agent.

[0050] Furthermore, the concentration of the wetting agent used is typically lower than the typical concentration of the drug solubilizer (>10%). Based on the weight of the nanosuspension, the typical concentration of the wetting agent in the nanosuspension of this disclosure is less than 10%, preferably less than 5%, more preferably less than 2%, and most preferably less than 1%.

[0051] Furthermore, the weight ratio of wetting agent to drug in the nanosuspension disclosed herein is significantly lower than the typical weight ratio of solubilizer to drug. For example, per bottle The nanosuspension contains 50 mg of the insoluble drug amphotericin B and 40 mg of sodium deoxycholate as a solubilizer, or the weight ratio of the solubilizer to the drug is 5:4. The typical weight ratio of the wetting agent to the drug in the 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.

[0052] 3. Bile salts

[0053] Bile salts are one of the main components of bile, a yellowish-green liquid produced by the liver and stored in the gallbladder. Bile salts have been used to improve the hydrophilicity of water-insoluble drugs (such as amphotericin B, resveratrol, and oxaprazin), primarily through wetting. In addition, bile salts are used as penetration enhancers in topical dosage forms, including buccal, ocular, nasal, and transdermal routes of administration.

[0054] In this document, "bile salt" used as a wetting agent in the compositions disclosed herein refers to alkali metal salts of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid or taurodeoxycholic acid, chenodeoxycholic acid, and glycochenodeoxycholic acid or taurochenodeoxycholic acid, such as sodium salts. Preferred bile salts are sodium glycocholate and sodium deoxycholate. Other cations, including but not limited to potassium, lithium, calcium, arginine, lysine, or ammonium, may also be used in bile salts. Bile acids or bile salts can be used as starting materials for the preparation of the wetting agent. If bile acids are used as starting materials, they are typically converted (completely or partially) to their bile salts (i.e., conjugate bases) by adjusting the pH before or during the preparation of the wetting agent.

[0055] If bile salts are used as starting materials, they can be converted (completely or partially) to their bile acids (i.e., conjugate acids) during the preparation of wetting agents by lowering the pH. In one aspect, the term "bile salt" or "B" as used in this application also refers to a mixture of bile salt and its conjugate acid. For example, deoxycholic acid has a pKa of 6.58 (Merck Index, 12th edition, entry #12946). According to the Henderson-Hasselbalch equation, deoxycholic acid in an aqueous solution at pH 4.58 will contain a mixture of 99% protonated deoxycholic acid (bile acid) and 1% deprotonated deoxycholate (bile salt). This bile acid / bile salt mixture is well-suited for use as a wetting agent.

[0056] 4. Cryoprotectant

[0057] As used in this disclosure, "cryoprotectant" refers to a component added during freeze-thaw, freeze-drying, air-drying, vacuum-drying, or spray-drying processes to maintain the discrete nanoparticle state of SRP-3D(DA) in a nanosuspension. Cryoprotectants that can 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 can be used in this disclosure include, but are not limited to, glycerol, mannitol, erythritol, maltitol, xylitol, sorbitol, polyglucan, or mixtures thereof. Monosaccharides that can be used in this disclosure include, but are not limited to, glucose, mannose, fructose, lactulose, allose, azoose, gulose, idole, galactose, tarose, ribose, arabinose, xylose, lysolose, or mixtures thereof. Disaccharides that can be used in this disclosure include, but are not limited to, sucrose, lactose, maltose, isomaltose, trehalose, cellobiose, or mixtures thereof. Polysaccharides that can 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 can 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 can be used in this disclosure include, but are not limited to, diglycine and triglycine. Proteins that can be used in this disclosure include, but are not limited to, albumin, collagen, casein, and gelatin. Hydrophilic polymers that can 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 glycol, povidone, poloxamer, polyvinyl alcohol, or mixtures thereof. The most preferred hydrophilic polymers are polyethylene glycol and povidone. The concentration of cryoprotectant used in the nanosuspension composition can be in the range of about 2% to about 40% w / w, for example about 5% to about 20% w / w and about 10% to about 15% w / w.

[0058] In this document, the term "impurity" refers to a foreign substance contained in a compound or its pharmaceutically acceptable salt or polymorph. A foreign substance includes one or more substances that are different from the compound or its pharmaceutically acceptable salt or polymorph. In some embodiments, the foreign substance is an undesirable foreign substance. For example, when an anhydrous compound is required, the solvent (such as water) contained in the compound is an impurity. When a crystalline compound is required, the amorphous form of the compound contained in the compound is an impurity. When a certain polymorph of the compound is required, other polymorphs of the compound contained in the compound are impurities. The term "substantially free of impurities" means that the compound (e.g., compound of formula I) or its pharmaceutically acceptable salt or polymorph does not contain a significant amount of a foreign substance (e.g., an undesirable foreign substance). How much of a foreign substance constitutes a significant amount depends on the specific substance and will be understood by those skilled in the art. In some embodiments, a significant amount is greater than about 1% to about 10%, or about 1 wt%, about 2 wt%, about 3 wt%, about 5 wt%, about 7 wt%, or about 10 wt% of a foreign substance in the compound or its pharmaceutically acceptable salt or polymorph is a significant amount of foreign substance. If a sample contains about 90-100% w / w of the compound or its pharmaceutically acceptable salt or polymorph, such as about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%, the compound is substantially pure.

[0059] This disclosure relates to novel nanoparticle formulations of SRP-3D(DA), wherein the SRP-3D(DA) nanoparticles are safe for injection. Furthermore, the SRP-3D(DA) nanoparticles disclosed in this application can significantly improve oral bioavailability.

[0060] SRP-3D(DA) is being developed for use as an analgesic. SRP-3D(DA) is an acetaminophen (ApAP) derivative, and its chemical structure is as follows: Figure 1 As shown. The empirical formula for SRP-3D(DA) is C. 19 H 23 N3O5S, with a molecular weight of 405.5 g / mol, SRP-3D(DA) exhibits activity against both acute and chronic pain.

[0061] Because SRP-3D(DA) is practically insoluble in water, obtaining injectable formulations or achieving adequate bioavailability via oral administration presents challenges. Attempts have been made to solubilize SRP-3D(DA) with polysorbate, propylene glycol, polyethylene glycol (PEG), DMSO, Labrasol, methylcellulose, and cottonseed oil to form solution formulations, but safe, bioavailable, or stable formulations for injection or oral administration have not been obtained.

[0062] 5. Composition / Formulation

[0063] In one embodiment, this disclosure relates to a formulation composition comprising SRP-3D(DA) or SRP-3D(DA) nanoparticles in the form of nanoparticles. The composition comprises SRP-3D(DA) in the form of nanoparticles and at least one wetting agent.

[0064] The SRP-3D(DA) nanoparticles disclosed herein have an average particle size of less than 1 micrometer, preferably less than 500-400 nm, more preferably less than 200 nm, and most preferably less than 100 nm. Smaller particles (<80 nm) are desirable, but difficult to achieve using the grinding method disclosed in this application. In some cases, the particle size is from about 50 nm to 1000 nm, 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 some cases, the particle size is 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.

[0065] The particle size of the SRP-3D(DA) nanoparticles disclosed herein can be measured using various analytical instruments, including but not limited to dynamic light scattering, laser diffraction, electron microscopy, and field flow classification techniques.

[0066] The SRP-3D(DA) nanoparticles disclosed herein can be provided in dry powder or suspension form, wherein the nanoparticles are suspended in a liquid such as water. Such suspensions are also referred to as SRP-3D(DA) nanoparticle suspensions or SRP-3D(DA) nanoparticle suspensions.

[0067] SRP-3D(DA) formulations in nanoparticle form are superior to conventional SRP-3D(DA) formulations for parenteral (e.g., intravenous, intramuscular, or subcutaneous) administration of the active ingredient for the treatment of acute and chronic pain because they are expected to have a faster onset of action due to the nanoparticle size of the active ingredient.

[0068] The SRP-3D(DA) formulation in nanoparticle form has the advantages of rapid onset of action and long duration of action after intravenous injection.

[0069] Compared to other formulations containing larger SRP-3D(DA) particles (e.g., >1 micrometer), SRP-3D(DA) nanoparticle formulations also improve oral bioavailability.

[0070] Furthermore, the nanoparticle form of SRP-3D(DA) formulations does not possess the toxicity or adverse side effects of the solubilizers used in solution formulations. For example, SRP-3D(DA) nanoparticle formulations do not cause allergic reactions to many detergent-like drug solubilizers (polysorbate 80, Cremophor, Labrasol, etc.), nephrotoxicity of cyclodextrin, or acute toxicity to organic solvents such as ethanol, DMSO, and polyethylene glycol. The SRP-3D(DA) nanoparticle formulations disclosed herein are safer than solution formulations.

[0071] Compared to solution formulations, the SRP-3D(DA) particles in nanoparticle form disclosed herein unexpectedly exhibit superior injection safety.

[0072] Compared to other SRP-3D(DA) formulations containing larger SRP-3D(DA) particles (e.g., >1 micrometer), the SRP-3D(DA) particles in nanoparticle form disclosed herein unexpectedly exhibit higher oral bioavailability.

[0073] In one embodiment, the SRP-3D(DA) formulation in nanoparticle form comprises SRP-3D(DA) and bile salt as a wetting agent.

[0074] In one embodiment, the SRP-3D(DA) formulation in nanoparticle form comprises sodium deoxycholate as a wetting agent.

[0075] In one embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in nanosuspension form and a wetting agent in a weight ratio of 5:1 to 40:1.

[0076] In a preferred embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in nanosuspension form and sodium deoxycholate in a weight ratio of 10:1 to 30:1.

[0077] In a more preferred embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in nanosuspension form and sodium deoxycholate in a weight ratio of 15:1 to 25:1.

[0078] In the most preferred embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in nanosuspension form and sodium deoxycholate in a weight ratio of 18:1 to 20:1.

[0079] In one embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in dry powder form and a wetting agent in a weight ratio of 10:1 to 30:1.

[0080] In a preferred embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in nanosuspension form and sodium deoxycholate in a weight ratio of 15:1 to 25:1.

[0081] In a more preferred embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in dry powder form and sodium deoxycholate in a weight ratio of 15:1 to 20:1.

[0082] In the most preferred embodiment, the SRP-3D(DA) particles in nanoparticle form disclosed herein comprise SRP-3D(DA) in dry powder form and sodium deoxycholate in a weight ratio of 18:1 to 20:1.

[0083] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles, a wetting agent, and a suspension carrier.

[0084] In another embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles, a wetting agent, and water.

[0085] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles and a wetting agent, as well as an organic solvent selected from medium-chain triglycerides, vegetable oils, mineral oils and silicone oils.

[0086] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles and a wetting agent selected from sodium deoxycholate, sodium cholate, and sodium glycocholate.

[0087] In one embodiment, the SRP-3D(DA) provided in this disclosure is substantially pure or does not contain significant amounts of foreign substances (e.g., undesirable foreign substances). The molecular structure of SRP-3D(DA) exists as Formula I. In one embodiment, this disclosure provides a compound of Formula I:

[0088]

[0089] The compound in question is essentially free of impurities.

[0090] In some cases, the purity of the compound is greater than 90 w / w%, or greater than 95 w / w%, or greater than 99 w / w%.

[0091] In one embodiment, this disclosure provides Formula I in solid form:

[0092]

[0093] The solid form mentioned therein is a crystalline phase, an amorphous phase, a semi-crystalline phase, a semi-amorphous phase, or a mixture thereof.

[0094] In one embodiment, the solid form is a crystalline form, such as a polymorphic solid, characterized in that the X-ray powder (XRPD) spectrum measured using CuKα radiation contains the following 2θ peaks: 9.0±0.2, 9.2±0.2, 13.2±0.2, 21.9±0.2; or, further characterized in that the solid form is characterized in that the X-ray powder (XRPD) spectrum measured using CuKα radiation contains the following 2θ peaks: 12.2±0.2, 15.4±0.2, 18.3±0.2, 19.2±0.2, 20.7±0.2, 22.5±0.2; or, characterized in that the X-ray powder (XRPD) spectrum measured using CuKα radiation and... Figure 10 , Figure 11 or Figure 12 They are basically similar.

[0095] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles in crystalline form, wherein the crystalline compound is optionally substantially pure.

[0096] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles in crystalline form, the X-ray powder diffraction (XRPD) pattern of which contains the following 2θ peaks measured using CuKα radiation: 9.0±0.2, 9.2±0.2, 13.2±0.2, 21.9±0.2.

[0097] In another embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles in crystalline form, the X-ray powder diffraction (XRPD) pattern of which also contains the following 2θ peaks measured using CuKα 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.

[0098] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure comprises SRP-3D(DA) nanoparticles in crystalline form, and its XRPD spectrum, measured using CuKα radiation, is consistent with... Figure 10 , Figure 11 or Figure 12 The diagrams shown are basically similar.

[0099] In one embodiment, the average particle size of the SRP-3D(DA) nanosuspension disclosed herein is less than 1 micrometer, preferably less than 900-600 nm, more preferably less than 500-400 nm, and most preferably less than 300 nm.

[0100] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure has a narrow particle size distribution, wherein the polydispersity index value is less than 1, preferably less than 0.8, more preferably less than 0.6, and most preferably less than 0.4.

[0101] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure is free of any particles with a diameter greater than 20 micrometers, preferably free of particles with a diameter greater than 10 micrometers, and more preferably free of particles with a diameter greater than 7 micrometers.

[0102] In one embodiment, the SRP-3D(DA) nanosuspension of this disclosure is free of any particles with a diameter greater than 15 micrometers, preferably free of particles larger than 8 micrometers, and more preferably free of particles larger than 5 micrometers.

[0103] In one embodiment, the SRP-3D(DA) nanoparticles or nanosuspensions of this disclosure are packaged in containers, such as vials, syringes, or bottles, wherein each container or unit contains 1 mg to 10,000 mg of SRP-3D(DA).

[0104] In one embodiment, the SRP-3D(DA) nanoparticles or nanosuspensions of this disclosure are packaged in containers, such as vials, syringes, or bottles, wherein each container or unit contains a dose of 0.1 g to 10 g of SRP-3D(DA).

[0105] In one embodiment, SRP-3D(DA) nanoparticles or nanosuspensions are packaged in container units, wherein the number of particles with a diameter equal to or greater than 10 micrometers in each container does not exceed 6,000, and the number of particles with a diameter greater than 25 micrometers does not exceed 600.

[0106] In one embodiment, the SRP-3D(DA) nanoparticles or nanosuspensions of this disclosure conform to USP <788> Requirements for particulate matter in injectable drugs.

[0107] In one embodiment, the compound of formula I is amorphous and is synthesized using, for example, the method of Example 13.

[0108] In one embodiment, the SRP-3D(DA) nanoparticles or nanosuspensions of this disclosure may contain other pharmaceutical excipients selected from the group consisting of: cryoprotectants, lyophilization protectants, antioxidants, stabilizers, buffers, osmotic pressure regulators, viscosity regulators, fillers, disintegrants, lubricants, suspending agents, sweeteners, flavorings, preservatives, wetting agents, foaming agents, or other excipients known in the art.

[0109] 6. Preparation method of SRP-3D(DA)

[0110] According to Figure 3 SRP-3D(DA) was prepared according to the scheme described in Example 1. Briefly, 4-aminophenol reacts with chloroacetic anhydride to generate 2-chloro-N-(4-hydroxyphenyl)acetamide (C-4). C-4 then reacts with sodium saccharin hydrate (C-5) to generate C-1:

[0111]

[0112] That is, 2-(1,1-dioxide-3-oxobenzo[d]isothiazolyl-2(3H)-yl)-N-(4-hydroxyphenyl)acetamide (C-1). Then C-1 reacts with diethylamine to generate SRP-3D (DA).

[0113] In one implementation scheme, such as Figure 3 As shown, this disclosure provides a method for preparing SRP-3D(DA), the method comprising:

[0114] (a) 4-Aminophenol (C-3) is mixed with chloroacetic anhydride to generate 2-chloro-N-(4-hydroxyphenyl)acetamide (C-4);

[0115] (b) Mixing (C-4) with sodium saccharin hydrate (C-5) yields C-1; and

[0116] (c) C-1 is mixed with diethylamine to generate SRP-3D(DA).

[0117] 7. Preparation of Nano-Suspensions

[0118] While nanosuspensions are the preferred form for injection, both dry powder and suspension forms are suitable for oral administration. Dry powders can be incorporated into capsule or tablet formulations, while nanosuspensions can be administered directly orally.

[0119] The SRP-3D(DA) nanoparticles of this disclosure are prepared by wet milling, i.e., milling in the form of a suspension. For this disclosure, a wet milling machine is used to mill a suspension containing coarse SRP-3D(DA) particles (>2 micrometers) to form a nanosuspension.

[0120] For the purposes of this disclosure, any wet mill designed to break solid particles down to submicron sizes can be used. Wet mills that can be used with respect to this disclosure include, but are not limited to, Netzsch mills, media mills, colloid mills, ball mills, high-shear homogenizers, high-pressure homogenizers, high-pressure extruders, and microfluidizers. Preferred mills are microfluidizers or ball mills using grinding balls (beads).

[0121] The microjets used in this disclosure can employ Y-cavities or Z-cavities with pore sizes of 75-300 micrometers. Operating pressures can vary between 10K and 30K psi. Processing cycles can be up to 100. A preferred microjets setup includes a Y-cavity with an operating pressure greater than 20K psi.

[0122] For the ball mill used in this disclosure, beads made of zirconium oxide or polystyrene, ranging in size from 300 to 100 micrometers, can be used. Preferred beads are yttrium-stabilized zirconium oxide beads.

[0123] The suspension used for wet milling or the final nano-suspension can be water or a non-aqueous liquid. Water is the preferred suspension liquid in wet milling processes.

[0124] Non-aqueous liquids used for wet milling may include, but are not limited to, medium-chain triglycerides, vegetable oils, silicone oils, mineral oils, or other organic solvents that are compatible with SRP-3D(DA) and are non-toxic. Preferred non-aqueous liquids are medium-chain triglycerides.

[0125] The nanoparticle suspension produced by wet milling can then be separated into nanoparticles and a liquid suspension. Separation can be achieved using a filter membrane with a pore size smaller than the nanoparticles to separate the nanoparticles from the liquid suspension, centrifugation to settle the nanoparticles into pellets, evaporation, or drying. For aqueous nanoparticle suspensions, evaporation or drying is the preferred separation method; for non-aqueous nanoparticle suspensions, centrifugation is preferred. Evaporation / drying methods may include spray drying, freeze drying, or vacuum drying at ambient temperature or elevated temperatures to remove water.

[0126] While the choice of mill may affect the particle reduction rate and final particle size to some extent, the choice of wetting agent appears to be more critical in determining the final particle size and the physical stability of nanoparticles after formation.

[0127] The wetting agent used in this disclosure can be water-soluble or water-insoluble. If it is water-soluble, it will preferentially remain in the aqueous phase of the suspension; otherwise, it may preferentially adhere to the surface of the solid particles. In either case, it can reduce the surface tension between the solid particles and water by improving the contact between the solid particles and water or by wetting the solid particles with water.

[0128] SRP-3D(DA) is hydrophobic, with a calculated log P of 0.760 ± 0.823. Without a wetting agent, it is difficult to reduce the particle size of SRP-3D(DA) through grinding because the hydrophobic particles tend to clump together or agglomerate, failing to disperse in water to form a homogeneous suspension. With a carefully selected wetting agent, the wetted particles exhibit less tendency to agglomerate. They form a homogeneous suspension in which the particles are suspended as separate or individual particles rather than clumps.

[0129] Wetting agents enable the production of SRP-3D(DA) nanoparticles or nanosuspensions via grinding techniques. As particle size decreases, the particle surface area increases, making particle size reduction more difficult. For example, without the use of a wetting agent, grinding (e.g., using an air jet mill) can only yield micron-sized (1-100 micrometers) particles. Therefore, a wetting agent that can reduce the surface tension of solid particles is needed to produce SRP-3D(DA) nanoparticles via grinding. To date, there is no clear guidance or teaching in the prior art on how to select a wetting agent for novel chemicals such as SRP-3D(DA).

[0130] Furthermore, the smaller the particle size, the greater the surface tension, and the stronger the tendency for particles to aggregate or exhibit instability. Wetting agents are also required to maintain the particle size or physical stability of nanoparticles or nanosuspensions after formation.

[0131] The inventors discovered that not every wetting agent can produce stable SRP-3D(DA) nanoparticle compositions. Surprisingly, it was found that, as described in more detail below, stable SRP-3D(DA) formulations in nanoparticle form can be prepared using bile salts selected from sodium salts of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid, with sodium deoxycholate being the preferred wetting agent.

[0132] On the other hand, other commonly used wetting agents, such as sodium docusate, povidone (e.g., PVP K-12 and PVP K 29 / 32), poloxamer (e.g., poloxamer 188 and poloxamer 407, which are block copolymers of polyethylene glycol and polypropylene glycol), and polyoxyethylene sorbitan monooleate (polysorbate 80), have failed to produce or stabilize SRP-3D(DA) nanoparticles.

[0133] In certain cases, sodium deoxycholate is particularly suitable for use in injectable nanoparticle forms of SRP-3D(DA) formulations. This is significant and surprising because obtaining very small SRP-3D(DA) particles is crucial for injectable formulations. Furthermore, the composition is stable, with little or no particle size growth observed, as injectable formulations with large particles can lead to embolism.

[0134] 8. Treatment methods

[0135] The compounds and formulations described in this article are intended for the treatment of acute, chronic, and / or neuropathic pain. Current medications, including opioids, acetaminophen (ApAP), and nonsteroidal anti-inflammatory drugs (NSAIDs), offer limited relief and have significant drawbacks such as addiction and organ toxicity. The opioid crisis further complicates matters, with 8.7 million people aged 12 and older abusing prescription opioids in the United States alone. These forms of pain reduce quality of life and impose a substantial economic burden.

[0136] The implementation plan can be used to treat or improve pain, and non-limiting examples include postoperative pain, neuropathic pain, dental pain, eye pain, arthritis pain, acute pain, chronic pain, inflammatory pain, and / or traumatic pain.

[0137] In embodiments, the compounds and compositions described herein may be used as the sole pharmacologically active compound for the treatment of neuropathic pain, without the need for a second active agent, such as a GABA analogue, like gabapentin (Neurontin). In other embodiments, the compounds and compositions described herein may be administered to an individual concurrently and / or in combination with a second active ingredient, such as an opioid or a nonsteroidal anti-inflammatory drug (NSAID). Opioids exert their effects by binding to opioid receptors in the brain and spinal cord. Non-limiting examples of opioids include codeine, fentanyl, hydrocodone, hydrocodone / ApAP, hydromorphone, meperidine, methadone, morphine, oxycodone, oxycodone and ApAP, oxycodone and naloxone. Nonsteroidal anti-inflammatory drugs (NSAIDs) block the COX enzyme and reduce systemic prostaglandins. Therefore, persistent inflammation, pain and fever are relieved. Non-limiting examples of NSAIDs include aspirin, celecoxib, diclofenac, diflunisal, etodoxacin, ibuprofen, indomethacin, ketoprofen, ketoroxyprofen, nabumetone, naproxen, oxapazine, piroxicam, magnesium salicylate, sulindac, and tometidine.

[0138] The compounds described herein can be incorporated into pharmaceutical compositions suitable for administration. Such compositions may comprise the compounds described herein and a pharmaceutically acceptable carrier. Thus, in some embodiments, the compounds of the present invention are present in the pharmaceutical composition.

[0139] For example, pharmaceutical compositions comprising the compounds described herein can be used to prevent and / or treat pain, such as a therapeutically effective amount of a compound of formula I mixed with a pharmaceutically acceptable carrier or excipient. For instance, a therapeutically effective amount of a compound of formula I can be administered to an individual to prevent or reduce the onset of pain or to prevent an increase in the severity of pain.

[0140] "Treatment" can refer to methods that achieve beneficial or desired clinical outcomes, such as improving or alleviating any aspect of pain, including postoperative pain, neuropathic pain, dental pain, ophthalmic pain, arthritis pain, acute pain, chronic pain, inflammatory pain, and / or traumatic pain. Beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: including reduction in severity, relief of one or more pain-related symptoms, including any aspect of pain (such as shortening the duration of pain, and / or reducing pain sensitivity or sensation).

[0141] "Improvement" of pain or one or more symptoms of pain can mean a reduction or improvement in one or more symptoms of pain compared to not using the composition described herein. "Improvement" may also include a reduction in the duration of symptoms or a reduction in the degree of symptom relief. For example, a therapeutically effective amount of a compound of formula I may be administered to an individual suffering from pain to improve or reduce pain.

[0142] The term "relief" can refer to a reduction in the severity of symptoms, condition, or disease. For example, a treatment that reduces the severity of pain in an individual, such as a compound of formula I, can be called pain relief. For instance, a therapeutically effective amount of a compound of formula I can be administered to an individual suffering from pain, wherein the severity of the pain is reduced. It should be understood that in some cases, treatment may relieve symptoms or conditions without treating the underlying disorder. In some respects, the term may be synonymous with "palliative care."

[0143] The implementation scheme can be used to reduce the incidence of pain or delay pain, and non-limiting examples include postoperative pain, neuropathic pain, dental pain, ophthalmic pain, arthritis pain, acute pain, chronic pain, inflammatory pain, and / or traumatic pain. "Reducing the incidence of pain" can refer to any reduction in severity (including reduced need and / or dosage (e.g., exposure) of other medications and / or therapies commonly used for such conditions), duration, and / or frequency (including, for example, delaying or increasing the time of onset of pain in an individual). As those skilled in the art will understand, individuals may respond differently to treatment; therefore, for example, "a method of reducing the incidence of pain in an individual" reflects the administration of the composition described herein, based on reasonable expectation that such administration will result in a reduction in the incidence in that particular individual.

[0144] "Delayed" pain development can refer to postponing, hindering, slowing, delaying, stabilizing, and / or postponing the progression of pain. The length of this delay can vary depending on the medical history and / or the individual receiving treatment. As those skilled in the art will appreciate, sufficient or significant delay can indeed include prevention, as the individual will not develop pain. Methods of "delayed" symptom development involve reducing the probability of symptom occurrence and / or the severity of symptoms within a given timeframe compared to not using this method.

[0145] The “development” or “progression” of pain can refer to the initial manifestations and / or subsequent progression of a disease. The development of pain can be detected and assessed using standard clinical techniques well-known in the art. However, development can also refer to progression that may go undetected. For the purposes of this invention, development or progression refers to the biological process of symptoms. “Development” includes occurrence, recurrence, and onset. As used herein, an “onset” or “occurrence” of pain includes an initial onset and / or recurrence. For example, the embodiments described herein can be used to prevent the development or progression of pain.

[0146] The implementation scheme can be used to relieve pain, and non-limiting examples include postoperative pain, neuropathic pain, dental pain, eye pain, arthritis pain, acute pain, chronic pain, inflammatory pain, and / or traumatic pain.

[0147] "Relief" of pain or one or more symptoms of pain can refer to the degree to which one or more undesirable clinical manifestations of pain are reduced in an individual or group of individuals treated with the compositions described herein.

[0148] The implementation scheme includes administering an effective amount of the composition described herein to an individual for the treatment of pain.

[0149] "Effective amount," "sufficient amount," or "therapeutic effective amount" can refer to an amount sufficient to achieve a beneficial or desired clinical outcome, including relief or reduction of pain. For the purposes of this invention, an effective amount of the compositions described herein may include an amount sufficient to treat, improve, reduce the intensity of any type of pain, or prevent pain, such as postoperative pain, neuropathic pain, dental pain, ophthalmic pain, arthritis pain, acute pain, chronic pain, inflammatory pain, and / or traumatic pain. In some embodiments, an effective amount of the compositions described herein may adjust the sensitivity threshold to external stimuli to a level comparable to that observed in healthy individuals. In other embodiments, this level may not be comparable to that observed in healthy individuals, but may be reduced compared to not receiving the treatment.

[0150] The specific compositions described herein may be administered to an individual in any suitable manner, such as orally, intravenously, parenterally, subcutaneously, intrapulmonaryly, topically, intravitreally, through the skin, mucous membranes, rectum, and nasally. Parenteral administration includes intramuscular, intravenous, intra-arterial, or intraperitoneal administration. The compounds may also be administered transdermally, for example, in the form of sustained-release subcutaneous implants or transdermal patches. They may also be administered by inhalation. Although direct oral administration may result in some loss of desired activity (e.g., analgesic activity), analgesics can be packaged using enteric coating, capsules, or other methods known in the art to protect the active ingredient from digestion.

[0151] The common goal of controlled-release drug products is to improve drug therapy and make it superior to non-controlled-release drugs. The characteristic of using optimally designed controlled-release formulations in medical treatment is to cure or control the disease in the shortest possible time using the least amount of drug substance.

[0152] The advantages of controlled-release formulations include prolonged drug activity, reduced dosing frequency, and improved patient compliance. Furthermore, controlled-release formulations can influence on onset time or other characteristics, such as drug plasma concentrations, and thereby affect the occurrence of side effects (e.g., adverse reactions).

[0153] Most controlled-release formulations are designed to initially release a certain amount of drug (active ingredient) to rapidly produce the desired therapeutic effect, followed by a gradual and sustained release of additional amounts of drug to maintain this therapeutic or preventative effect over an extended period. To maintain a constant level of drug in the body, the drug must be released from the dosage form at a controlled rate to replace the amount metabolized and excreted from the body. Controlled release of the active ingredient can be stimulated by a variety of conditions, including but not limited to pH, temperature, enzymes, water, or other physiological conditions or compounds.

[0154] Solutions or suspensions intended for parenteral, intradermal, or subcutaneous administration may include, for example, the following components: sterile diluents, such as water, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents for injection; 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 reagents for adjusting osmotic pressure, such as sodium chloride or glucose. pH may be adjusted by acid or base, such as hydrochloric acid or sodium hydroxide.

[0155] The compositions described herein can be administered to an individual once (e.g., as a single injection or deposition). While the duration for which at least one compound is administered to prevent pain varies by species and the nature and severity of the condition to be prevented or treated, the compound can be administered to humans for short or long periods, such as from one week to one year. For example, it can be administered to an individual in need once or twice daily for a period of time, such as one week or one month.

[0156] Dosage can vary depending on known factors such as the pharmacodynamic characteristics of the active ingredient and its route of administration; the timing of administration of the active ingredient; the recipient's age, sex, health status, and weight; the nature and severity of symptoms; the type, frequency, and expected effect of concurrent treatments; and the rate of excretion.

[0157] The effective therapeutic dose can depend on many factors known to those skilled in the art. For example, the dose can vary depending on the identity, size, and condition of the individual or sample being treated, and further depends on the route of administration of the composition (if applicable) and the desired effect by the practitioner. These amounts can be readily determined by those skilled in the art.

[0158] In some implementations, the therapeutically effective dose 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 about 7 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. g 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 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.

[0159] The effective therapeutic dose can depend on many factors known to those skilled in the art. For example, the dose can vary depending on the identity, size, and condition of the individual or sample being treated, and further depends on the route of administration of the composition (if applicable) and the desired effect by the practitioner. These amounts can be readily determined by those skilled in the art.

[0160] In one embodiment, the recommended daily dose range of the compound described herein for treating the pain described herein is from about 1 mg / kg to about 10 g / kg per day, for example from about 5 mg / kg to about 5 g / kg per day, or from about 10 mg / kg to about 2 g / kg per day. The active ingredient is typically used to treat the condition, and is usually administered as 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. Daily doses for treating or improving human pain may range from about 1 mg / kg to about 300 mg / kg.

[0161] The compounds described herein, such as compounds of formula I, or compositions comprising them, may be administered to an individual once (e.g., as a single injection or deposition). Alternatively, they may be administered to an individual in need once or twice daily for about 2 to about 28 days, or about 7 to about 10 days, or about 7 to about 15 days. They may also be administered to an individual once or twice daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or combinations thereof.

[0162] The single-unit dosage forms disclosed herein are suitable for oral, mucosal (e.g., intranasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intra-arterial), topical (e.g., eye drops or other ophthalmic preparations), transdermal (e.g., creams, lotions, or skin sprays), or percutaneous administration to patients. Examples of dosage forms include, but are not limited to: tablets; capsules; capsules, such as soft elastic gelatin capsules; flat capsules; lozenges; sugar lozenges; dispersions; suppositories; powders; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to patients, including suspensions (e.g., aqueous or non-aqueous liquid suspensions or solutions, oil-in-water emulsions, or water-in-oil emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to patients; eye drops or other ophthalmic preparations suitable for topical administration; and sterile solids (e.g., crystalline or amorphous solids) reconfigurable to provide liquid dosage forms for parenteral administration to individuals.

[0163] The composition, shape, and type of the dosage forms disclosed herein will generally vary depending on their intended use. Furthermore, the dosage may vary based on known factors such as the pharmacodynamic characteristics of the active ingredient and its route of administration; the timing of administration of the active ingredient; the recipient's age, sex, health status, and weight; the nature and severity of symptoms; the type, frequency, and desired effect of concurrent treatments; and the rate of excretion.

[0164] For example, a dosage form for the acute treatment of a disease may contain a greater amount of one or more of its contained active agents than a dosage form for the chronic treatment of the same disease. Similarly, a parenteral dosage form may contain fewer of one or more of its contained active agents than an oral dosage form for the treatment of the same disease. These and other variations between the specific dosage forms included in this disclosure will be readily understood by those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990). These methods are not cytotoxic to the liver.

[0165] Any therapeutic application described herein can be applied to any individual requiring such treatment, including, for example, mammals such as mice, rats, dogs, cats, cattle, horses, rabbits, monkeys, pigs, sheep, goats, or humans. In some embodiments, the individual is a mouse, rat, pig, or human. In some embodiments, the individual is a mouse. In some embodiments, the individual is a rat. In some embodiments, the individual is a pig. In some embodiments, the individual is a human.

[0166] 9. Useful methods in this disclosure

[0167] Method 1: GC method for TAN purity analysis of 4-aminophenol, 2-chloroacetic anhydride and compound 4, and for process reaction monitoring in step 1 of the SRP-3D(DA) synthesis.

[0168] Identification and preparation of endpoint markers

[0169] The analyte is calculated using total area normalization; therefore, accurately quantified standards are not required. Qualitative markers validating the current retention time of the target compound should be run with each analysis. See chemical structure below. Figure 3 .

[0170] 4-Aminophenol identification standard: (0.33 mg / mL 4-aminophenol in acetonitrile solution)

[0171] 1. Add approximately 20 mg of 4-aminophenol standard and 60 mL of acetonitrile to the scintillation bottle.

[0172] 2. Mix by inverting.

[0173] 2-Chloroacetic anhydride identification standard: (2 mg / mL acetonitrile solution of 2-chloroacetic anhydride)

[0174] 1. Add approximately 20 mg of 2-chloroacetic anhydride standard and 10 mL of acetonitrile to the scintillation bottle.

[0175] 2. Mix by inverting.

[0176] Endpoint standard for step 1: (acetonitrile solution of 0.03 mg / mL 4-aminophenol and 0.68 mg / mL compound 4)

[0177] 1. Add approximately 34 mg of Compound 4 standard, 4.5 mL of 4-aminophenol identification standard, and 45.5 mL of acetonitrile to the scintillation vial.

[0178] 2. Mix by inverting.

[0179] Note: The retention time marker solution can be prepared in equivalent amounts proportional to the stated amount and can be used as long as the target component is detectable.

[0180] Sample preparation

[0181] Reaction monitoring

[0182] Step 1 (Target concentration 0.68 mg / mL):

[0183] 1. Allow the quenched reaction mixture to stand for at least 15 minutes.

[0184] 2. Transfer approximately 575 μL of the quenched reaction mixture and 10 mL of acetonitrile to the scintillation bottle.

[0185] 3. Mix by inverting.

[0186] The actual concentration of the reaction solution can be obtained from the chief chemist or engineer, and the sample preparation can be adjusted as needed to achieve the specified target concentration.

[0187] Regulatory starting materials and separated intermediates

[0188] The TAN purity of 4-aminophenol, 2-chloroacetic anhydride, and compound 4 can be determined by preparing and analyzing acetonitrile solutions at concentrations of 0.33 mg / mL, 2 mg / mL, and 0.68 mg / mL, respectively. It is recommended that duplicate release samples be prepared.

[0189] Note: Since this method is a TAN analysis, quantitative sample preparation is not required.

[0190] System Applicability

[0191] 1. Inject a blank sample to ensure no peak is observed at the retention time of the target analyte. If interfering peaks are present, assess their potential impact on the analysis and take corrective action if necessary.

[0192] 2. All peaks must be within the range to ensure proper integration.

[0193] step

[0194] 1. Set up the gas chromatography system as follows:

[0195] GC column: RTX-5Amine, 30m × 0.32mm inner diameter, 1.5μm FT

[0196] Needle wash: Acetonitrile

[0197] Injection volume: 2 μL

[0198] Inlet temperature: 300℃ (substrate devitrified liner)

[0199] Carrier gas: H2

[0200] Column pressure: 12 PSI (constant pressure)

[0201] Split ratio: 10:1 (adjustable as needed to ensure proper response)

[0202] Furnace program: Increase from 115℃ to 300℃ at a rate of 15℃ / min (hold for 5 minutes).

[0203] Detector: FID, 300℃

[0204] 2. Run a blank sample at least once before starting the analysis.

[0205] 3. Configure the data system and load the autosampler using the following sample injection sequence format:

[0206] Sample injection # Sample ID test 1 blank column conditions 2 blank Acceptability of diluent 3 Identification / Endpoint Standard Verification retention time 4 sample Residue 5 Identification / Endpoint Standard Verification retention time 6 blank Residue

[0207] 4. Verify that all chromatograms have been properly integrated. If not, the analyst may need to perform manual integration (setting the peak start, end, and baseline). Systematic peaks (i.e., peaks observed in blank samples) should be excluded from the sample chromatograms.

[0208] 5. Verify whether the system suitability requirements are met. If the standards are not met, the run is invalid. Take corrective action and repeat the analysis.

[0209] 6. Calculate and report the results.

[0210] Calculate and report results

[0211] Identity verification

[0212] If the peaks in the sample chromatogram match the peaks produced by the actual substance within a 5% retention time window, the presence of the target analyte in the sample is confirmed. The expected retention times for various reaction components are as follows:

[0213] Components Expected retention time (min) 2-Chloroacetic anhydride 2.8 4-Aminophenol 4.1 Compound 4 8.8

[0214] TAN analysis of the endpoint sample

[0215] Report the TAN values ​​of starting materials and products to two decimal places.

[0216] Percentage of starting material = (100 × peak area of ​​starting material) / (total peak area)

[0217] TAN purity

[0218] Report the TAN values ​​of the separated intermediates and regulatory starting materials to two decimal places.

[0219] TAN purity = (peak area of ​​intermediate / starting material according to regulations) / (total peak area) × 100

[0220] Method 2: HPLC method for monitoring steps 1-3 and the recrystallization process

[0221]

[0222]

[0223] Gradient pump program*:

[0224]

[0225] *Developed on a Perkin Elmer Series 200 pump. Curve 1 represents a linear change, and curve 0 represents an instantaneous change. The gradient may need to be adjusted according to the dead volume of the HPLC used.

[0226] The chromatogram of the C-4 identification standard sample is shown below. Figure 13 .

[0227] The chromatogram of the endpoint standard sample in step 2 is shown below. Figure 14 .

[0228] The chromatogram of the endpoint standard sample in step 3 is shown below. Figure 15 .

[0229] Method 3: Through 1 H NMR determination of the molar ratio of dichloromethane and ethanol to n-heptane

[0230] Purpose

[0231] This program can be used to calculate the molar ratio of dichloromethane (DCM) and ethanol (EtOH) to n-heptane.

[0232] Material

[0233] • One clean 5mm NMR tube

[0234] • CDCl3, >99.8 atomic%D, containing 0.03% v / v TMS, or its equivalent.

[0235] step

[0236] • The amounts of all chemicals used in this experiment are qualitative and not critical factors. Dissolve approximately 5 drops of the supernatant after solvent exchange in 0.5–1 mL of CDCl3 (containing tetramethylsilane as a reference) and place in a clean 5 mm NMR tube. Cap the tube and invert it to mix thoroughly.

[0237] • Analyzed by 1H NMR using standard operating parameters.

[0238] analyze

[0239] • Ensure the spectrum is correctly referenced to 0.00 ppm of TMS.

[0240] • The triplet at approximately 0.89 ppm is the signal for the methyl group in n-heptane. The integral is calibrated to 6.00.

[0241] The singlet at approximately 5.27 ppm is the hydrogen (2 protons) of the DCM. The resulting integral value is referred to as "A".

[0242] The quartet at approximately 3.75 ppm is the methylene group of ethanol. The resulting integral value is referred to as "B".

[0243] Calculation of the molar ratio (mol%) of dichloromethane to n-heptane:

[0244] Dichloromethane / n-heptane (mol%) = (A / 2) × 100

[0245] Calculation of the molar ratio (mol%) of ethanol to n-heptane:

[0246] Ethanol / n-heptane (mol%) = (B / 2) × 100

[0247] Print the spectrum with the integration region, and after review, attach it to the batch record along with the experimental record page of the sample preparation.

[0248] Method 4: Through 1 H NMR determination of the molar ratio of diethylamine to acetonitrile

[0249] Purpose

[0250] This program can be used to calculate the molar ratio of diethylamine to acetonitrile (ACN).

[0251] Material:

[0252] • One clean 5mm NMR tube

[0253] DMSO-d6, >99.8 atomic%D, containing 0.03% v / v TMS, or its equivalent.

[0254] step

[0255] • The quantities of all chemicals used in this experiment are qualitative and not critical factors. Dissolve approximately 5 drops of the supernatant after solvent exchange in 0.5–1 mL of DMSO-d6 (containing tetramethylsilane as a reference) and place in a clean 5 mm NMR tube. Cap the tube and invert it to mix thoroughly.

[0256] • Using standard operating parameters 1 Analysis was performed using H NMR.

[0257] analyze

[0258] • Ensure the spectrum is correctly referenced to 0.00 ppm of TMS.

[0259] The corresponding signal for diethylamine will be slightly shifted due to its concentration because the target compound SRP-3D(DA) in this reaction step contains an acidic phenolic functional group. Ensure that this signal is baseline separated from the DMSO peak.

[0260] • The singlet at approximately 2.05 ppm is the methyl group (3 protons) of ACN. Calibrate this value to 3.00.

[0261] The quartet at approximately 2.6–2.8 ppm is the methylene group (4 protons) of diethylamine. The resulting integral value is referred to as "A".

[0262] Calculation of the molar ratio (mol%) of diethylamine to ACN:

[0263] Diethylamine / ACN (mol%) = (A / 4) × 100

[0264] Print the spectrum with the integration region, and after review, attach it to the batch record along with the experimental record page of the sample preparation.

[0265] Method 5: Through 1 H NMR determination of the molar ratio of water to ACN

[0266] Purpose

[0267] This program can be used to calculate the molar ratio of water to ACN.

[0268] Material

[0269] • One clean 5mm NMR tube

[0270] DMSO-d6, >99.8 atomic%D, containing 0.03% v / v TMS, or its equivalent.

[0271] step

[0272] • The amounts of all chemicals used in this experiment are qualitative and not critical factors. Dissolve approximately 10 drops of the supernatant after adding water in 0.5–1 mL of DMSO-d6 (containing tetramethylsilane as a reference) and place it in a clean 5 mm NMR tube. Cap the tube and invert it to mix thoroughly.

[0273] • Using standard operating parameters 1 Analysis was performed using H NMR.

[0274] analyze

[0275] • Ensure the spectrum is correctly referenced to 0.00 ppm of TMS.

[0276] The corresponding signal from water will be slightly shifted depending on the water-to-ACN ratio. Only the supernatant was used in this experiment to eliminate any possible interfering product peaks.

[0277] The single peak at approximately 3.4–3.8 ppm indicates the presence of water (2 protons). Calibrate this value to 2.00.

[0278] The singlet at approximately 2.05 ppm is the methyl group (3 protons) of ACN. The resulting integral value is referred to as "A".

[0279] Calculation of the water to ACN molar ratio:

[0280] Water / ACN (mol%) = 1:(A / 3)

[0281] Calculation of the water to ACN volume ratio:

[0282] Water / ACN (volume / volume) = 1: (A / 3) × 41.05 / (18.02 × 0.786)

[0283] Print the spectrum with the integration region, and after review, attach it to the batch record along with the experimental record page of the sample preparation.

[0284] Method 6: Stability indicator method for determining the identity, content, and purity of SRP-3D(DA) by HPLC-UV

[0285] Preparation of mobile phase

[0286] 1. 10mM Tetrabutylammonium Acetate

[0287] a. Add 1.0 mL of 1.0 M tetrabutylammonium acetate to a 100 mL volumetric flask.

[0288] b. Dilute with water to the mark and mix thoroughly.

[0289] c. It will expire within 2 weeks.

[0290] 2. Mobile phase "A": Water containing 20 mM ammonium acetate and 0.1 mM tetrabutylacetic acid ammonium, pH 5.8

[0291] a. Weigh 1.6g of ammonium acetate and place it in a 1L volumetric flask.

[0292] b. Add 1L of water using a graduated cylinder.

[0293] c. Add 10.0 mL of 10 mM tetrabutylammonium acetate.

[0294] d. Adjust the pH to 5.8 with acetic acid.

[0295] e. Stir the solution thoroughly to ensure a uniform mixture.

[0296] f. It will expire within 2 weeks.

[0297] 3. Mobile phase "B": A solution of 30% water and 70% acetonitrile containing 20 mM ammonium acetate and 0.1 mM tetrabutylammonium acetate, pH 5.8.

[0298] a. Weigh 1.6g of ammonium acetate and place it in a 1L volumetric flask.

[0299] b. Add 300mL of water using a graduated cylinder.

[0300] c. Add 10.0 mL of 10 mM tetrabutylammonium acetate.

[0301] d. Adjust the pH to 5.8 with acetic acid.

[0302] e. Add 700 mL of acetonitrile using a graduated cylinder.

[0303] f. Stir the solution thoroughly to ensure a uniform mixture.

[0304] g. It will expire within 4 weeks.

[0305] Preparation of sample / standard diluent / autosampler rinse solution

[0306] 1. Sample / Standard Diluent: A 50% water and 50% acetonitrile solution containing 20mM ammonium acetate, pH 5.8

[0307] a. Weigh 1.6g of ammonium acetate and place it in a 1L volumetric flask.

[0308] b. Add 500mL of water using a graduated cylinder.

[0309] c. Adjust the pH to 5.8 with acetic acid.

[0310] d. Add 500 mL of acetonitrile.

[0311] e. Stir the solution thoroughly to ensure a uniform mixture.

[0312] f. It will expire within 4 weeks.

[0313] 2. Automatic sampler flushing solution: 50% water and 50% acetonitrile

[0314] a. Add 500 mL of water to a 1 L volumetric flask using a graduated cylinder.

[0315] b. Add 500 mL of acetonitrile.

[0316] c. Stir the solution thoroughly to ensure a uniform mixture.

[0317] d. It will expire within 4 weeks.

[0318] Preparation of calibration and control standards

[0319] 1. Prepare a 1.0 mg / mL SRP-3D(DA) reference standard stock solution by accurately weighing 25 mg (±2 mg) of reference standard into two 25 mL volumetric flasks.

[0320] 2. Add a portion of the diluent to each volumetric flask and mix thoroughly to dissolve. Mild sonication (approximately 1 minute) may be necessary for complete dissolution.

[0321] 3. Dilute to the mark with diluent and invert to mix.

[0322] 4. Transfer 2.5 mL of each stock solution to a separate 25 mL volumetric flask and dilute to the mark with diluent (10% solution, concentration 0.1 mg / mL). Label the solutions Standard 1 and Standard 2.

[0323] Preparation of LOQ solution

[0324] 1. Transfer 2.5 mL of one of the standard solutions prepared in Part F to a 25 mL volumetric flask, dilute to the mark with diluent, and mix thoroughly. This intermediate LOQ solution contains approximately 10 μg / mL of SRP-3D(DA).

[0325] 2. Transfer 2.5 mL of the intermediate LOQ solution to a 50 mL volumetric flask, dilute to the mark with diluent, and mix thoroughly. This LOQ solution contains approximately 0.5 μg / mL of SRP-3D(DA) (i.e., 0.05% w / w SRP-3D(DA)).

[0326] Preparation of retention time marker solutions

[0327] 1. This solution is not required for each analysis. Prepare only when peak identification is uncertain. Due to the instability of compound 3, this solution should be injected within one day of preparation.

[0328] 2. Weigh approximately 10 mg of compounds 3, 4, 5, and 1 into a 50 mL volumetric flask. Dissolve and dilute to the mark with diluent. Mix thoroughly.

[0329] 3. Transfer 2.5 mL of the above solution to a 50 mL volumetric flask. Dilute to the mark with one of the reference standard solutions. Mix thoroughly.

[0330] Sample preparation

[0331] 1. Impurity assay solution (1 mg / mL)

[0332] a. Prepare two copies of the SRP-3D(DA) impurity sample with a target concentration of 1.0 mg / mL by weighing 25 mg (±2 mg) of the sample into a 25 mL volumetric flask.

[0333] b. Add a portion of the diluent to each volumetric flask and mix thoroughly to dissolve. If necessary, sonicate to ensure complete dissolution.

[0334] c. Allow the solution to reach room temperature.

[0335] d. Dilute to the mark with diluent and invert to mix.

[0336] e. Only the preparation solution of the first impurity sample should be analyzed.

[0337] 2. Assay solution (0.1 mg / mL or 10% impurity solution)

[0338] a. Transfer 2.5 mL of each impurity solution to a 25 mL volumetric flask and dilute to the mark with diluent.

[0339] b. Mix thoroughly.

[0340] System Applicability

[0341] 1. The diluent blank should not contain peaks that may interfere with the determination of sample content or impurity spectra. If interfering peaks are present, assess their potential impact on the analysis. If the impact is significant, investigate the potential source of the peak. If the source of the peak is determined to be the column, treat the HPLC column as follows: Wash the system with 50% acetonitrile and 50% water to remove buffer and modifiers. Switch the flow rate to 100% acetonitrile at 0.2 mL / min and allow it to flow through the system overnight.

[0342] 2. Inject the SRP-3D(DA) LOQ solution once. Calculate the recovery rate of SRP-3D(DA) in the LOQ solution using the average calibration factor from the previous six SRP-3D(DA) reference standard injections. The recovery rate of the LOQ solution should be between 50% and 150%.

[0343]

[0344] Wherein:

[0345] RF LOQ = Response factor for LOQ injection

[0346] RF STD(n＝6) = Average response factor for the first six injections of reference standards.

[0347] 3. All peaks must be within the range to ensure proper integration.

[0348] 4. Inject one of the SRP-3D(DA) reference standards six times consecutively. These six injections must meet the following system suitability criteria:

[0349] a. %RSD of API peak area: ≤2.0%

[0350] b. %RSD of API retention time: ≤2.0%

[0351] c. Average API tailing factor: 0.5 ≤ x ≤ 2.0

[0352] 5. The average calibration factor for the first six injections of reference standards is used to calculate the recovery rate of SRP-3D(DA) in the repeated standard preparation and each intermediate standard injection throughout the sequence. The recovery rate of SRP-3D(DA) must be between 98% and 102%.

[0353]

[0354] Wherein:

[0355] RF = Response factor for the injection of control standard or intermediate standard

[0356] RF STD(n＝6) = Average response factor for the first six injections of reference standards.

[0357] 6. The diluent blank used to evaluate residues should not show a significant amount of SRP-3D(DA) (i.e., < LOQ). If residues are present, evaluate the possible impact on the analysis.

[0358] 7. If the system suitability criteria are not met, additional standard injections should be made to determine whether the problem is from the solution or the HPLC system. If it is determined that the problem is from the solution, a new solution will be prepared and analyzed. If the problem lies in the HPLC system, the analyst will troubleshoot the problem, take corrective measures, and repeat the analysis.

[0359] Procedure

[0360] 1. Set up the liquid chromatography system as follows:

[0361]

[0362] Gradient pump program:

[0363]

[0364] 2. Equilibrate the column with the mobile phase before starting any analysis. For gradient analyses, the system gradient program must be run at least once before starting the analysis. The autosampler and pump should be flushed daily with autosampler flush buffer and mobile phase before analysis.

[0365] After use, the column should be rinsed with 50% acetonitrile and 50% water. For long-term storage of the column, 100% acetonitrile is recommended.

[0366] 3. Configure the data system and load the autosampler using the following sample injection sequence format:

[0367]

[0368] 4. Review the following chromatograms for each injection of the standard or sample:

[0369] • Full range: X-axis range = 0 minutes to 25 minutes

[0370] The Y-axis range should be set to allow viewing of the entire SRP-3D(DA) peak.

[0371] • Extended response range: X-axis range = 0 minutes to 25 minutes

[0372] The Y-axis range should be set to allow viewing of the impurity baseline.

[0373] 5. Verify that all standards and samples have been properly integrated. If not, the analyst may need to perform manual integration (setting the peak start, end, and baseline) to include all peaks except for the systematic peak. Systematic peaks (i.e., peaks observed in the blank sample diluent) should be excluded from the sample impurity spectrum.

[0374] 6. Verify that the system suitability requirements are met. If the standards are not met, the run is invalid. Take corrective action and repeat the analysis. Calculate and report the results.

[0375] Calculation and Reporting

[0376] Identity verification

[0377] If the average retention time of the main peak in the sample chromatogram is consistent with that of the peak produced by the reference substance within ±2.5%, the sample SRP-3D(DA) is confirmed.

[0378] SRP-3D(DA) content

[0379] 1. SRP-3D(DA) content of the sample

[0380]

[0381] in:

[0382] A SPL = Area of ​​the SRP-3D(DA) peak in the sample solution

[0383] A STD = Area of ​​the SRP-3D(DA) peak in the reference standard solution

[0384] C STD =Concentration of reference standard (mg / mL)

[0385] P STD =Valence of the reference standard (expressed as a decimal)

[0386] DF = Dilution Factor (mL)

[0387] SS = Sample volume (mg)

[0388] If the consistency of the repeated sample preparation is acceptable (<2% absolute difference), the average of the two values ​​is taken as the test result.

[0389]

[0390] Where: KF% = Moisture content measured by Karl Fischer method (per batch release test / stability test point)

[0391] TS% = Total solvent content determined by gas chromatography

[0392] Results are reported to one decimal place.

[0393] Note: All values ​​used in the calculations should contain sufficient significant figures to avoid rounding errors.

[0394] Related substances

[0395] 1. For each HPLC injection of the impurity sample solution, calculate the impurity content. The expected retention times and relative retention times of various known relevant substances are as follows:

[0396]

[0397] 2. Calculate the individual impurity content of impurities with a LOQ (0.05%, excluding system peaks) using the following formula:

[0398]

[0399] Where: ASTD = average area of SRP-3D(DA) in six (6) injections of STD1 standard solution

[0400] CSTD = concentration of SRP-3D(DA) in 10% standard solution (mg / mL)

[0401] PSTD = purity of SRP-3D(DA) reference standard (expressed as a decimal)

[0402] AIMP = area of a single impurity in the impurity determination solution (100%)

[0403] CIMP = concentration of SRP-3D(DA) in the impurity solution (mg / mL)

[0404] 3. For single impurities with > LOQ (0.05%), calculate the total impurity content using the following formula:

[0405] Total impurities (% area) = ∑ for each single impurity > LOQ

[0406] Report

[0407] 1. For specific impurities : Report all specific impurities by name. Specific impurities not observed in the sample are reported as < LOD. Values ≥ LOD but < LOQ (0.05% w / w) are reported as < LOQ. For specific impurities ≥ LOQ, report the value rounded to two decimal places.

[0408] 2. For all non-specific impurities : Report all non-specific (including unidentified and potential) impurities ≥ LOQ (0.05% w / w), and refer to their relative retention times. Values ≥ LOD but < LOQ are reported as < LOQ. For non-specific impurities ≥ LOQ, report the value rounded to two decimal places.

[0409] 3. For total impurities : Add up all specific and non-specific single impurities ≥ LOQ. Single impurity values < LOQ are not included in the calculation of the total impurity content. Report the total percentage of impurities present to two decimal places (e.g., 1.23%).

[0410] Chromatographic purity of SRP-3D(DA) (for characterization)

[0411] 1. When determining the chromatographic purity, the substance to be characterized is prepared in duplicate at 1 mg / mL (100%), and each solution is further diluted to 10%. The area response of SRP-3D in this 10% solution is used as the reference response.

[0412] 2. Calculate the single impurity content and total impurity content as described in the "Related Substances" section above.

[0413] 3. Examine the chromatograms of replicate samples. If the chromatograms show the same pattern and similar relative peak areas, take the average value as a test result.

[0414] 4. Chromatographic purity (CP (%)) determination:

[0415] CP (%) = 100% - Total impurities ≥ 0.05% (%)

[0416] 5. Report results to one decimal place.

[0417] Method 7: Identification of SRP-3D(DA) by proton and carbon-13 NMR spectroscopy

[0418] Using Bruker TopSpin 2.1.8 software on a Bruker Avance III 400MHz instrument, via... 1 H, 13 C, COSY, HSQC, and HMBC NMR characterization were performed on qualified SRP-3D(DA) authentic reference standards. Spectra were obtained in deuterated DMSO (DMSO-d6) with reference to tetramethylsilane. Representative... 1 H and 13 C data and peak attribution are respectively shown in [reference]. Figure 4 and Figure 5 Subsequent batches will pass through. 1 H and 13 The C-spectrum was compared with the corresponding reference spectrum for identification.

[0419] Method 8: Infrared analysis of SRP-3D(DA)

[0420] SRP-3D(DA) samples were analyzed on a Nicolet 6700FTIR equipped with a Thermo Fisher Smart iTX accessory and OMNIC 9.8 software. The temperature range was 4000 to 400 cm⁻¹. -1 Within the wavelength range of 4cm -1 128 resolution scans. Representative spectra are shown below. Figure 6 As shown. The spectrum is consistent with the proposed structure.

[0421] Method 9: Identification of SRP-3D(DA) by mass spectrometry

[0422] SRP-3D(DA) was identified using positive ion mode electrospray ionization mass spectrometry (+ESIMS), including full-spectrum scan Q3 mass spectrometry and tandem mass spectrometry (MS / MS). Identification was performed by comparing the MS data of the test article with the MS data of a qualified SRP-3D(DA) real reference material. Alternatively, identification was performed by comparing the MS data of the test article with reference MS data previously generated through validation activities using a qualified SRP-3D(DA) real reference material, using the same MS instrument. Reference mass spectra are shown below. Figure 7 , Figure 8 and Figure 9 As shown. The test solution was introduced into the mass spectrometer ion source using a direct infusion technique. The accuracy and consistency of the MS data were evaluated by comparing them with reference data, thereby assessing the overall method and instrument suitability. SRP-3D(DA) was detected in full-scan Q3 mode. The identification of SRP-3D(DA) considered three aspects of the MS data: the precise mass of the molecular ion [M+H]+, the isotopic mode, and the MS / MS mode of the monoisotopic pseudomolecular ion fragment at m / z = 406.1.

[0423] This method was developed and validated on the AB Sciex 4000Q-Trap mass spectrometer system. Some mass parameters or scan types may be specific to the 4000Q-Trap's capabilities. Analysis can be performed on different mass spectrometer models, provided system suitability criteria are met. Method validation was conducted according to current Good Manufacturing Practices (GMP).

[0424] Diluent: Acetonitrile containing 0.1% formic acid: water (1:1 v / v)

[0425] Sample or reference standard stock solution: 35–65 μg / mL (in diluent)

[0426] System Applicability

[0427] For the test to be valid, the following criteria should be met.

[0428] The diluent (blank) should be free of interfering ions or have negligible interfering ions (less than 5% of the target ion intensity) within the target m / z range. If significant interference is observed, assess the impact on selectivity by considering the signal intensity (count). If the effect is significant, run the diluent until a stable and low-noise MS spectral baseline is obtained.

[0429] Accuracy

[0430] When analyzing solutions of real SRP-3D(DA) material, the following ions should be observed. When using the centroid data presentation option, the accuracy of the m / z value for each ion should be within ±0.2 amu.

[0431] Full scan mass spectrometry (Q3 positive ion) ion m / z (center of mass) <![CDATA[[M+H] + ]]> 406.1amu Product ion mass spectrometry (MS2 positive ion) ions m / z (center of mass) Mother ion 406.1amu fragments 240.1amu

[0432] Note: When comparing with reference MS data, the MS spectrum of the SRP-3D(DA) test item should meet the above accuracy standards.

[0433] In any case, if the generated MS data does not show the expected mode and m / z values ​​and / or does not meet the accuracy criteria, the mass spectrometer calibration needs to be verified using the Applied Biosystems Sciex Standard Chemistry Kit.

[0434] step

[0435] System Settings

[0436] MS Instrument: AB Sciex 4000Q-Trap mass spectrometer or a similar mass spectrometer with triple quadrupole capability, capable of operating in full-spectrum scanning and MS / MS modes. 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. MS parameter modifications are acceptable due to varying MS conditions across different MS systems.

[0437] Injection method: direct infusion

[0438] Injection pump parameters: Syringe diameter (mm): 4.6

[0439] Flow rate: 10.0 μL / min

[0440] Syringe size: 1000μL

[0441] Software: Analyst

[0442] 1. Full scan spectrum (Q3 positive ion)

[0443] 4000Q-Trap Mass Spectrometer Method Parameters

[0444] Nozzle position: X-axis: 5.0; Y-axis: 5.0

[0445] Scan type: Q3 MS

[0446] Polarity: Positive ion

[0447] Scanning mode: Contour map

[0448] Ion source: Turbo Spray

[0449] Start (amu) Termination (amu) Time (seconds) Loop count 120.00 850.00 8.00 30 parameter value parameter value CUR 15.00 TEM 300.00 GS1 30.00 GS2 30.00 ihe ON IS 2800.00 DP 0.00 EP 10.00 CXP 8.00

[0450] 2. Product ion (MS2) mass spectrometry

[0451] 4000Q-Trap Mass Spectrometer Method Parameters

[0452] Nozzle position: X-axis: 5.0; Y-axis: 5.0

[0453] Scan type: Product ions (MS2)

[0454] Polarity: Positive ion

[0455] Scanning mode: Contour map

[0456] Ion source: Turbo Spray

[0457] Product source: 406.1amu

[0458] Start (amu) Termination (amu) Time (seconds) Loop count 50.00 500.00 8.00 30 parameter value parameter value CUR 15.00 TEM 300.00 GS1 30.00 GS2 30.00 ihe ON IS 2800.00 CAD Medium (or 2-5) CXP 8.00 DP 0.00 EP 10.00 CE 20.00

[0459] Sample Analysis

[0460] The sample was analyzed using the MS parameters described above. Q3 MS was used for a full scan to obtain an overall picture of the ions in the sample; product ions (MS2) were used to obtain fragmentation information from the parent ions.

[0461] Analyze the sample according to the following example sequence.

[0462] sample Purpose diluent Full scan to obtain a blank background Standard product* Full scan / MS2 scan to obtain the MS spectrum of the standard. diluent Full scan to obtain a blank background sample Full scan / MS2 scan to obtain the MS spectrum of the sample

[0463] *The sequence can be adjusted to analyze more samples.

[0464] Alternatively, the MS spectra (full spectrum scan and MS2) of the SRP-3D(DA) test item can be compared with the reference MS spectra generated during the validation of the test method.

[0465] Results Report

[0466] Identity verification

[0467] The identity of an SRP-3D(DA) test item is confirmed if the following criteria are met.

[0468] a. The full scan mass data (Q3 positive ion) of the test item should be consistent with the reference spectrum. The accuracy of the m / z values ​​of the molecular ion and similar isotopic modes should be within ±0.2 amu. See also Figure 7 and Figure 8 Example MS spectra in the image.

[0469] b. The MS / MS data (product ions, MS2 positive ions) of the test items should be consistent with the reference material / reference spectrum (±0.2 amu).

[0470] See Figure 9 Example MS / MS spectra.

[0471] Method 10: Characterization of SRP-3D(DA) by X-ray powder diffraction

[0472] X-ray powder diffraction patterns were obtained using a Bruker D8 Discovery diffractometer, which is equipped with an XYZ platform, a laser video microscope for positioning, and a two-dimensional... Area detector. Using Cu Kα radiation. The X-ray source, operating at 40 kV and 40 mA, was used to irradiate the sample. The X-ray optical system consisted of a Gobel lens with a 0.5 mm pinhole collimator. θ-θ continuous scanning was employed, with a sample-detector distance of approximately 30 cm and an effective 2θ range of 4–40°. The sample was mounted in a low-background quartz plate. The main XRPD peaks (relative intensity ≥10%) of the three SRP-3D(DA) crystal form A samples of this disclosure are shown in the table below.

[0473] Table 1: XRPD peaks of SRP-3D(DA) crystal form A, Sample 1

[0474] Position (°2θ) Relative strength (%) 9.0±0.2 41.5 9.2±0.2 56.0 12.2±0.2 32.7 13.2±0.2 77.3 15.4±0.2 16.6 16.5±0.2 16.8 18.3±0.2 22.2 19.2±0.2 38.2 20.7±0.2 36.4 21.9±0.2 100.0 22.5±0.2 46.5 26.0±0.2 10.4 26.5±0.2 10.1 30.8±0.2 12.7 31.2±0.2 13.7

[0475] Table 2: XRPD peaks of SRP-3D(DA) crystal form A, Sample 2 (unground sample)

[0476] Position (°2θ) Relative strength (%) 13.3±0.2 19.0 18.1±0.2 100.0 19.3±0.2 14.9 20.6±0.2 10.4 20.8±0.2 30.4 22.5±0.2 13.0 24.3±0.2 19.3 25.5±0.2 53.5 26.0±0.2 12.9 26.6±0.2 39.7 33.0±0.2 25.6 35.3±0.2 55.2 38.7±0.2 11.3 39.0±0.2 16.0

[0477] Table 3: XRPD peaks of SRP-3D(DA) crystal form A, Sample 3 (ground sample)

[0478] Position (°2θ) Relative strength (%) 9.0±0.2 62.5 9.2±0.2 31.9 13.2±0.2 100.0 15.3±0.2 19.4 16.5±0.2 11.7 18.3±0.2 19.3 19.2±0.2 20.1 20.7±0.2 24.4 21.9±0.2 19.3 22.5±0.2 29.1 25.3±0.2 14.6 26.5±0.2 10.2

[0479] Several factors can cause variability in diffraction angle and peak intensity measurements, including instrument design, maintenance, calibration and operating mode, sample preparation and presentation, particle size and inhomogeneity, preferred orientation, and environmental conditions. Angle measurements in this document are expressed as ±0.2°2θ. Relative intensities can vary considerably. As used in the specification and appended claims, the phrase "XRPD spectrum substantially similar to a reference spectrum" accounts for the sources of these experimental variability. For example, Figure 10 (Peaks are listed in Table 1) Figure 11 (Peaks are listed in Table 2) and Figure 12 The XRPD spectra of SRP-3D(DA) crystal form samples 1, 2 (unground sample), and 3 (ground sample) presented in Table 3 (peaks listed) show that, although the corresponding 2θ values ​​deviate by up to 0.2° and the relative intensities of some peaks (e.g., ~12.2°, ~18.3°, and ~21.9°2θ) differ significantly, they are “fundamentally similar” as defined herein. These differences in relative intensity may be due to “preferred orientations” in one or more samples, which is well known in the art.

[0480] Method 11: Characterization of SRP-3D(DA) by Differential Scanning Calorimetry

[0481] Instrument: TA Instruments DSC Q2000

[0482] Software: Thermal Advantage V 5.0.0

[0483] Sample tray and cover: aluminum, press-fit

[0484] Sample size: 2–10 mg

[0485] Heating range: 25°C to 250°C

[0486] Heating rate: 10℃ / minute

[0487] Purge gas: Nitrogen

[0488] A representative DSC curve of a qualified SRP-3D(DA) crystal form A true reference standard is shown below. Figure 16 As shown, it exhibits a single, sharp endothermic peak:

[0489] Melting point onset temperature: 172.47℃

[0490] Peak melting temperature: 174.47℃

[0491] Enthalpy of fusion (ΔHf): 101.9 J / g

[0492] Method 12: Characterization of SRP-3D(DA) by thermogravimetric analysis

[0493] Instrument: TA Instruments TGA Q500

[0494] Software: Thermal Advantage V 5.2.5

[0495] Sample tray: Platinum

[0496] Sample size: 2–20 mg

[0497] Heating range: 25°C to 250°C

[0498] Heating rate: 10℃ / minute

[0499] Purge gas: Nitrogen

[0500] A representative TGA curve of a qualified SRP-3D(DA) crystal form A true reference standard is shown below. Figure 18 As shown, it indicates a 0% weight loss at 175°C.

[0501] This result is consistent with the characteristics of crystal form A being anhydrous and free of solvates.

[0502] Method 13: Identification, content and purity of SRP-3D(DA) nanoparticle suspension determined by HPLC

[0503] Preparation of mobile phase, standards and buffer solutions:

[0504] 1) Preparation of 10mM tetrabutylacetic acid ammonium buffer

[0505] a. Weigh 301.50 mg of tetrabutylammonium acetate and transfer it to a 100 mL volumetric flask. Add approximately 60 mL of deionized water, mix thoroughly, and then dilute to 100 mL with deionized water.

[0506] b. Expires within 2 weeks

[0507] 2) Mobile phase A: Water containing 20 mM ammonium acetate and 0.1 mM tetrabutylacetic acid, pH 5.8

[0508] a. Weigh 1.6g of ammonium acetate and place it in a 1L volumetric flask.

[0509] b. Add 1L of deionized water using a graduated cylinder.

[0510] c. Add 10.0 mL of 10 mM tetrabutylammonium acetate.

[0511] d. Adjust the pH to 5.8 with glacial acetic acid and stir the solution thoroughly to ensure a uniform mixture.

[0512] e. Valid within 2 weeks

[0513] 3) Mobile phase B: A solution of 30% water and 70% acetonitrile containing 20 mM ammonium acetate and 0.1 mM tetrabutylammonium acetate, pH 5.8.

[0514] a. Weigh 1.6g of ammonium acetate and place it in a 1L volumetric flask.

[0515] b. Add 300 mL of deionized water using a graduated cylinder.

[0516] c. Add 10.0 mL of 10 mM tetrabutylammonium acetate.

[0517] d. Adjust the pH to 5.8 using glacial acetic acid.

[0518] e. Add 700 mL of acetonitrile using a graduated cylinder.

[0519] f. Stir the solution thoroughly to ensure a uniform mixture.

[0520] g. It will expire within 4 weeks.

[0521] 4) Sample / standard diluent: a 50% water and 50% acetonitrile solution containing 20mM ammonium acetate, pH 5.8.

[0522] a. Weigh 1.6g of ammonium acetate and place it in a 1L volumetric flask.

[0523] b. Add 500mL of water using a graduated cylinder.

[0524] c. Adjust the pH to 5.8 using glacial acetic acid.

[0525] d. Add 500 mL of acetonitrile

[0526] e. Stir the solution thoroughly to ensure a uniform mixture.

[0527] f. It will expire within 4 weeks.

[0528] HPLC method:

[0529]

[0530]

[0531] Sample preparation:

[0532] 1. Preparation of Reference Standard (RS) (0.1 mg / mL):

[0533] 1) Accurately weigh 20±2 mg of SRP-3D(DA) on weighing paper, then transfer it to a 200 mL volumetric flask and record the weight of SRP-3D(DA).

[0534] 2) Add approximately 120 mL of the prepared diluent, then sonicate to dissolve. Make up to 200 mL with the diluent. Mix thoroughly. The prepared reference standard should not be used for more than 7 days.

[0535] 2. Impurity sample preparation (1 mg / mL): Sample at 25℃:

[0536] 1) Peel the 100mL volumetric flask to zero. Transfer 1g of SRP-3D(DA) ball-milled nano-suspension product (stored at 25°C) to the 100mL volumetric flask and record the weight.

[0537] 2) Add approximately 50 mL of diluent to dilute. Mix thoroughly and bring to volume with the prepared diluent. Samples at 2-8℃:

[0538] 3) Peel the 100mL volumetric flask to zero. Transfer 1g of SRP-3D(DA) ball-milled nano-suspension product (stored at 2-8℃) to a 100mL volumetric flask and record the weight.

[0539] 4) Add approximately 50 mL of diluent to dilute. Mix thoroughly and bring to volume with the prepared diluent.

[0540] 3. Identification and content sample preparation (0.1 mg / mL): Sample at 25℃:

[0541] 1) Peel the 100mL volumetric flask to zero. Transfer 100mg of the ball-milled product (stored at 25°C) into the volumetric flask and record the weight.

[0542] 2) Add approximately 50 mL of diluent to dilute. Mix thoroughly and bring to volume with the prepared diluent. Samples at 2-8℃:

[0543] 3) Peel the 100mL volumetric flask to zero. Transfer 100mg of the ball-milled product (stored at 2-8℃) into the volumetric flask and record the weight.

[0544] 4) Add approximately 50 mL of diluent to dilute. Mix thoroughly and bring to volume with the prepared diluent.

[0545] SRP-3D(DA) reference standard concentration (Cs) calculation:

[0546]

[0547] Ws = Weight of standard sample (mg)

[0548] V = Dilution volume (mL)

[0549] Purity: Use CoA value

[0550] Impurity (%w / w) calculation:

[0551]

[0552] Ri = the area of ​​each impurity peak ≥ LOQ (0.05%) in the impurity sample.

[0553] Rs = Peak area of ​​SRP-3D(DA) in all reference standard injections (using the average value)

[0554] Cs = Concentration of reference standard (calculation explained above)

[0555] The labeled concentration of Cu=SRP-3D(DA) suspension (e.g., 100 mg / mL).

[0556]

[0557] Vf = Dilution volume (mL)

[0558] W = Weight (g) of 1 gram of SRP-3D(DA) suspension used in the preparation of the impurity sample.

[0559] The bulk density of the SRP-3D(DA) suspension is 0.990 g / mL.

[0560] Content (%w / w) calculation:

[0561]

[0562] Ru = Peak area of ​​SRP-3D(DA) in the sample solution

[0563] Rs = Peak area of ​​SRP-3D(DA) in all reference standard injections (using the average value)

[0564] Cs = Concentration of reference standard (calculation explained above)

[0565] Ci = Concentration of the sample solution for content determination (see above).

[0566]

[0567] W = Weight of SRP-3D(DA) suspension during impurity sample preparation

[0568] The bulk density of the SRP-3D(DA) suspension is 0.990 g / mL.

[0569] L = labeled concentration of SRP-3D(DA) suspension

[0570] V1 = Dilution volume during impurity sample preparation (e.g., 100 mL)

[0571] V2 = The volume of the impurity sample solution used to prepare the assay solution (e.g., 5 mL).

[0572] V3 = Dilution volume (e.g., 50 mL) used in sample preparation for content determination.

[0573] Total impurities (%w / w) calculation:

[0574] Total impurities (% w / w) = Σ each individual impurity ≥ LOQ

[0575] LOQ = 0.5 μg / mL or 0.05% of the peak area of ​​the impurity sample.

[0576] Identification based on retention time:

[0577] The retention time of SRP-3D(DA) in the sample was comparable to that of the SRP-3D(DA) reference standard.

[0578] Results Report:

[0579] Identification of SRP-3D(DA)

[0580] • SRP-3D(DA) content (% w / w)

[0581] • Individual impurity (% w / w)

[0582] Total impurities (% w / w)

[0583] Method 14: Determination of particle size distribution of SRP-3D(DA) nanoparticle suspension

[0584] According to USP <729> Method I (Measuring the average droplet diameter by dynamic light scattering or classical light scattering, USP 45-NF 40 2022-2023) was used to determine the particle size distribution of SRP-3D(DA) nanoparticle suspensions using Malvern Zetasizer Nano with 0.2 μm filtered deionized water as a diluent.

[0585] Method 15: Determination of the zeta potential of SRP-3D(DA) nanoparticle suspension

[0586] According to USP <432> (Z-potential determined by electrophoretic light scattering, USP 45-NF 40 2022-2023) The z-potential of SRP-3D(DA) nanoparticle suspension was determined using Malvern Zetasizer Nano with 0.2 μm filtered deionized water as diluent.

[0587] Method 16: Determination of the viscosity of SRP-3D(DA) nanoparticle suspension

[0588] According to USP <912> Method III (cone-plate rheometer method, USP 45-NF 40 2022-2023) was used to determine the viscosity of the SRP-3D(DA) nanoparticle suspension using a Brookfield DV-III Ultra rheometer. The sample temperature was controlled at 25℃±0.5℃ using a circulating water bath.

[0589] Example 1

[0590] Synthesis of SRP-3D(DA)

[0591] raw materials for pharmaceuticals Figure 3 The process shown was used to prepare SRP-3D(DA). 9.38 kg of white crystalline solid (crystal form A) was obtained, with an overall yield of 46.2%, an HPLC purity of 100.6% based on anhydrous and solvent-free conditions, and total impurities of 0.13%.

[0592] The preparation process comprises three chemical steps and one recrystallization. All three isolated intermediates underwent process control before being used in the next step, including loss on drying (LOD) and HPLC total area normalization (TAN). The process control test methods are listed in Table 4.

[0593] Step 1: Preparation of C-4

[0594] 1. Add 5.6 kg of 4-aminophenol (1.00 equivalent) to a 50-liter glass-lined steel reactor.

[0595] 2. Add dichloromethane (110.9 kg) and start stirring the mixture.

[0596] 3. Adjust the batch temperature to 20℃ and add phosphoric acid (263.4 g, 0.05 equivalent).

[0597] 4. Add chloroacetic anhydride (9.2 kg, 1.05 equivalent) and mix the batch at 20°C for at least 10 hours until C-3 ≤ 1% (process control-1, gas chromatography).

[0598] 5. Add ethanol (4.4 kg) and stir the batch for at least 1 hour.

[0599] 6. Vacuum distill the solvent at a jacket temperature ≤35℃ until 74.4 kg ± 10 kg of distillate is collected.

[0600] 7. Add n-heptane (39.2 kg), and then vacuum distill the solvent at a jacket temperature ≤60℃ until 39.2 kg ± 10 kg of distillate is collected.

[0601] 8. Add n-heptane (39.2 kg), and then vacuum distill the solvent at a jacket temperature ≤60℃ until 52.1 kg ± 10 kg of distillate is collected, and the dichloromethane / n-heptane ≤1% and ethanol / n-heptane ≤1% (process control - 2, 1H NMR).

[0602] 9. Cool the batch to 20°C and add process water (7.4 gallons). Mix the batch for at least 1 hour, then cool to 0°C and mix for at least 2 hours.

[0603] 10. Filter the batch, and wash the filter cake with process water (5.9 gallons) and n-heptane (15.3 kg).

[0604] 11. The filter cake was vacuum dried under nitrogen at 45°C (jacket inlet temperature) for at least 12 hours until the loss on drying (LOD) was ≤1% (process control-3), yielding 8.72 kg (yield 91.6%) of grayish-white solid with a purity of 99.35% (process control-4, HPLC, for reference only).

[0605] Step 2: Preparation of C-1

[0606] 1. Add 16.2 kg of DMF to a 50-liter glass-lined steel reactor. Start stirring and adjust the batch temperature to 25°C.

[0607] 2. Add compound C-4 (8.6 kg, 1.00 equivalent) to obtain a beige / brown solution.

[0608] 3. Add sodium iodide (208.4 g, 0.03 equivalent).

[0609] 4. Add sodium saccharin hydrate (11.4 kg, 1.10 equivalents). Heat the batch to 80°C for at least 12 hours until C-4 ≤ 2% (Process Control-5, HPLC, Target: C-4 ≤ 1%).

[0610] 5. Cool the batch to 25°C. Add ethyl acetate (38.7 kg), followed by process water (22.7 gallons). Mix the batch at 25°C for at least 4 hours until a sample shows the presence of solids (Process Control-6, Visual Inspection). If no solids are found after at least 24 hours, proceed to step 6. However, if solids are present, skip step 6 and proceed to step 7.

[0611] 6. Slowly add n-heptane (10% of the amount shown in step 7) over at least 20 minutes. Mix at 25°C for at least 1 hour until a sample shows the presence of solids (Process Control-6, Visual Inspection). Proceed to step 7, but reduce the amount of n-heptane added as shown therein by 10%.

[0612] 7. Slowly add n-heptane (29.2 kg) over at least 90 minutes. Mix the batch at 25°C for at least 18 hours and filter. The filtration rate was slower than expected compared to previous batches. Poor filtration is known to lead to poor wet filter cake removal. Therefore, it was decided to analyze the wet filter cake in step 9.

[0613] 8. The filter cake was washed with process water (6.8 gallons) and then with n-heptane (3 x 5.8 kg).

[0614] 9. The filter cake was vacuum dried at 50°C (jacket inlet temperature) with nitrogen purging for 12.6 hours. Filter cake analysis showed a purity of 96.2% (HPLC) and a loss on drying (LOD) of 27.95%. This purity is lower than the expected 99%. Therefore, corrective actions were taken in the following supplementary batch process.

[0615] Step 2: Supplementing the batch process:

[0616] 10. Add 17.3 kg of DMF to a 50-liter glass-lined steel reactor. Begin stirring and adjust the batch temperature to 25°C.

[0617] 11. Slowly add the C-1 wet filter cake (18.4 kg) from step 9 to obtain a beige / brown solution. Mix the batch at 25°C for at least 1 hour.

[0618] 12. Add ethyl acetate (33.9 kg), followed by process water (22.4 gallons). Mix the batch at 25°C for at least 16 hours until a sample shows the presence of solids (Process Control-6, Visual Inspection). If no solids are found, proceed to step 13. However, if solids are present, skip step 13 and proceed to step 14.

[0619] 13. Slowly add n-heptane (10% of the amount shown in step 14) over at least 10 minutes. Mix at 25°C for at least 1 hour until a sample shows the presence of solids (Process Control-6, Visual Inspection). Proceed to step 14, but reduce the amount of n-heptane added as shown therein by 10%.

[0620] 14. Slowly add n-heptane (25.8 kg) over at least 90 minutes. Mix the batch at 25°C for at least 6 hours and then filter.

[0621] 15. The filter cake was washed sequentially with process water (4.9 gallons), n-heptane (12.6 kg), process water (9.8 gallons), and n-heptane (2 × 12.6 kg).

[0622] 16. The filter cake was vacuum dried under nitrogen at 50°C (jacket inlet temperature) for at least 12 hours until the loss on drying was ≤1.0% (Process Control-7), yielding 10.50 kg (68.2% yield based on C-4) of white to off-white solid with a purity of 99.33% (Process Control-8, HPLC, for reference only). The moisture content was determined to be 457 ppm by Karl Fisher (Process Control-9, for reference only).

[0623] Step 3: Preparation of crude SRP-3D (diacetamide)

[0624] 1. Add C-1 (10.5 kg) to a 50-liter glass-lined steel reactor with the jacket temperature set at 20°C.

[0625] 2. Add acetonitrile (33.1 kg) and start stirring.

[0626] 3. Add diethylamine (9.2 kg) and rinse with acetonitrile (3.9 kg). Continue mixing for at least 10 minutes, then heat the batch to reflux (65–75 °C) for at least 12 hours until C⁻¹ ≤ 1% (Process Control-10, HPLC).

[0627] 4. Cool the batch to 22°C, drain it through a filter into a new 55-gallon high-density polyethylene drum, and rinse with acetonitrile (10.3 kg). Clean the reactor.

[0628] 5. Adjust the reactor jacket to 25°C, and add the combined acetonitrile solution from step 4 through a 0.25μm filter into the clean reactor. Start stirring. All subsequent solvent additions should be filtered through a 0.25μm filter.

[0629] 6. Vacuum distillation at a jacket temperature of ≤45℃ until 19.8±5 kg of solvent is collected.

[0630] 7. Add acetonitrile (36.4 kg). Continue distillation until 36.4 ± 5 ​​kg of distillate is collected.

[0631] 8. Add acetonitrile (36.4 kg). Continue distillation until 36.4 ± 5 ​​kg of distillate is collected.

[0632] 9. Add acetonitrile (36.4 kg). Continue distillation until 42.8 ± 5 kg of distillate is collected, and the molar ratio of diethylamine to acetonitrile in the cooled (20 °C) batch is ≤1% (process control - 1H NMR).

[0633] 10. Add water for injection (WFI) grade until the water / acetonitrile molar ratio is approximately 3:1 (2 × 31.7 kg; Process Control -12). 1 HNMR).

[0634] 11. Filter the batch, and wash the wet filter cake with water for injection (20.8 kg) and n-heptane (21.3 kg).

[0635] 12. The filter cake was vacuum dried under nitrogen at 50°C (jacket inlet temperature) for at least 12 hours until the weight loss was ≤1% (process control-13), yielding 10.64 kg (yield 83.1%) of a light yellow to light brown to grayish-white solid with a purity of 99.68% (process control-14, HPLC, for reference only).

[0636] Step 4: Preparation of SRP-3D (diacetamide) crystal form A

[0637] 1. All solvents were added through a 0.25μm filter.

[0638] 2. Add crude SRP-3D (diacetamide) (10.53 kg) to a 50-liter glass-lined steel reactor with the jacket temperature set at 25℃.

[0639] 3. Add acetone (33.5 kg) and start stirring.

[0640] 4. Add water for injection (42.4 kg).

[0641] 5. Heat the batch to 60℃ and maintain for 10 minutes.

[0642] 6. Add water for injection (42.4 kg) within at least 2 hours.

[0643] 7. Cool the batch to 22°C within ≥3 hours. Confirm the presence of slurry (Process Control-15, visual inspection). Continue mixing at 22°C for ≥8 hours.

[0644] 8. Filter the batch, and wash the wet filter cake with water for injection (2 × 15.9 kg) and n-heptane (14.5 kg).

[0645] 9. The filter cake was vacuum dried at 50°C (jacket inlet temperature) with nitrogen purging for at least 12 hours until the loss on drying was ≤1.0% (Process Control-16). Optional analysis of residual solvents showed no (ND) acetonitrile, no n-heptane, no DMF, and acetone <2446 ppm (Process Control-17, Gas Chromatography). Optional HPLC analysis showed a purity of 100.6% (Process Control-18). The dried batch was pulverized to obtain 9.38 kg (yield 89.1%) of white solid.

[0646] The analytical results of the product from step 4 are listed in Table 5.

[0647] Table 4: Process control of SRP-3D (diethylamine)

[0648]

[0649]

[0650] Table 5: Analytical results of SRP-3D(DA) prepared according to Example 1

[0651]

[0652] Example 2

[0653] Single-crystal X-ray structure determination of SRP-3D(DA) crystal form A

[0654] Single-crystal X-ray structure determination was performed on the SRP-3D(DA) crystal form A sample prepared according to Example 1. A colorless crystal fragment measuring approximately 0.45 × 0.40 × 0.21 mm was mounted on a Mitegen microgrid support with random orientation. Preliminary examination and data collection were performed using Mo Kα radiation. The measurements were performed on a Bruker AXSD8 Quest CMOS diffractometer equipped with a fixed χ-angle, a sealed-tube fine-focusing X-ray tube, a single-crystal bent graphite incident beam monochromator, a Photon100 CMOS surface detector, and Oxford Cryosystems cryogenic equipment. Initial unit cells were determined and data collected at 150 K using Apex3 v2018.3-0. Frames were integrated using SAINT V8.38A.

[0655] A total of 96,505 reflections were collected, of which 7,679 were unique. The cell constants for the collected data were obtained by least-squares refinement of 9,996 reflections between 2.4086 and 33.1648°. The orthorhombic cell parameters and computational volume are... For Z = 4 and formula weight 405.46, the calculated density is 1.345 g / cm³. 3 For Mo Kα radiation, the linear absorption coefficient is 0.197 / mm. Scaling and multi-scan absorption correction were applied using SADABS (Apex3 v2017 / 3). The transmittance ranges from 0.7195 to 0.7465. The intensity of the equivalent reflection was not averaged during data processing.

[0656] The space group was determined by the program XPREP embedded in SHELXTL. Systematic extinction and intensity statistics indicate that the space group is P212121(#19). The structure was resolved using SHELXS via the direct method, and SHELXL-2018 and the graphical user interface ShelXle were used for F... 2 All reflections were refined using full-matrix least squares. Additional atoms were located in subsequent difference Fourier synthesis.

[0657] The structure is refined using full matrix least squares, where the minimized function is Σw(|Fo| 2 -|Fc| 2 ) 2 And the weight w is defined as w = 1 / [σ] 2 (Fo 2 )+(0.0384P) 2 +0.3049P], where P = (Fo 2 +2Fc 2 ) / 3. Scattering factors are taken from the International Tables for Crystallography (Volume C, Tables 4.2.6.8 and 6.1.1.4). A total of 7,679 independent reflections were used in the refinement. A total of 7,251 F 2 >2σ(F 2The reflection of ) is used to calculate R1.

[0658] Hydrogen atoms bonded to carbon atoms are placed in geometric positions and constrained to their parent atoms. The carbon-hydrogen bond distance of aromatic CH atoms is... The carbon-hydrogen bond distances of the aliphatic CH2 and CH3 moieties are respectively and

[0659] The final refinement cycle included 265 variable parameters and 0 constraints, and converged (the maximum parameter displacement was 0.002 times its standard uncertainty). The unweighted and weighted consistency factors were:

[0660] R1=Σ|F o |-|F c | / Σ|F o |=0.0267

[0661] wR 2 ={Σ[w(F o 2 -F c 2 ) 2 ] / Σ[w(F o 2 ) 2 ]} 0.5 =0.0720

[0662] The goodness-of-fit parameter is 1.085. The highest peak height in the final difference Fourier plot is... The lowest negative peak height is -0.303.

[0663] Crystal data and data collection parameters are listed in Table 6.

[0664] The asymmetric unit cells of the crystal structure of SRP-3D(DA) crystal form A, as determined by X-ray diffraction, are as follows: Figure 17 As shown.

[0665] Table 6: Crystal data, data collection and refinement parameters of SRP 3D(DA) crystal form A

[0666]

[0667] Example 3

[0668] SRP-3D(DA) nanoparticle suspensions for oral administration were prepared by wet milling.

[0669] The batch formulation of 100 mg / mL SRP-3D(DA) oral suspension is shown in Table 7.

[0670] Table 7: Batch Formulation of 100 mg / mL SRP-3D (Diacetamide) Oral Suspension

[0671]

[0672] The batch size can be adjusted as needed.

[0673] b QS = sufficient quantity

[0674] c density = 0.990 g / mL; 4,950 g = 5,000 mL

[0675] A 100 mg / mL SRP-3D (diacetamide) oral suspension is prepared using the process described below. This process can be scaled up to different batch sizes and vial specifications as needed.

[0676] 1. All equipment, containers, and supplies that are expected to come into direct contact with the batch are purchased as sterile, autoclaved, or cleaned with alcohol before use.

[0677] Production steps: Step 1

[0678] 2. Label a 10-liter stainless steel container as Container #1 and peel it.

[0679] 3. Add hydroxypropyl cellulose USP (62.48g) to container #1.

[0680] 4. Add sodium benzoate NF (6.25g) to container #1.

[0681] 5. Add purified USP water (5931.3g) to container #1.

[0682] 6. Stir the contents of container #1 with a Silverson mixer at 5,000 rpm until a clear solution (1% HPC solution) is obtained.

[0683] Production steps: Step 2

[0684] 7. Label the second 10-liter stainless steel container as container #2 and peel it.

[0685] 8. Add SRP-3D (diacetamide) (500.0g) to container #2.

[0686] 9. Using a peristaltic pump, transfer 1% HPC solution (3,955.7 g) from container #1 to container #2.

[0687] 10. Stir the contents of container #2 at 10,000 rpm for 10 minutes using a Silverson mixer, or until a homogeneous suspension is formed.

[0688] Production steps: Step 3

[0689] 11. Label a 5-liter Nalgene bottle as container #3 and peel it.

[0690] 12. Set up the Netzsch MiniCer ball mill according to the user manual. Add 140 mL of 0.4 mm Zeta beads to the mixing chamber. Replace the outlet feed with a three-clamp 5 / 8 inch hose barbed connector, to which a 36-inch sterile tube is attached. Connect a 48-inch sterile tube to the inlet. Turn on the cooler and set it to 5°C, then allow the coolant to circulate in the ball mill to reach equilibrium before use.

[0691] 13. Use an overhead agitator to stir the contents of container #2 at 400 rpm to prevent the suspension from settling. If necessary, scrape the bottom of container #2 with a scraper to resuspend any large particles.

[0692] 14. Insert the inlet and outlet pipes of the ball mill into containers #2 and #3, respectively.

[0693] 15. Turn on the peristaltic pump of the ball mill equipment, use the minimum flow rate, and set the direction to pump the suspension into the grinding chamber.

[0694] 16. Start the ball mill rotor at the minimum speed (10Hz or 600rpm), and then accelerate to 2,400rpm within 1 minute.

[0695] 17. Gradually increase the pump flow rate to keep the pressure in the grinding chamber below 15 psi. If the pressure exceeds 15 psi, turn off the pump and slowly release the pressure.

[0696] 18. Record the pressure, pump flow setting, temperature, and rotor speed.

[0697] 19. After all the suspension has passed through the grinding chamber, stop the grinder and pump, and then use a sterile pipette to remove 1 mL of sample.

[0698] 20. Use a Malvern Zeta sieve to measure the particle size distribution (PSD) of the collected samples.

[0699] 21. Record the number of loops and the PSD result.

[0700] 22. Gently shake container #3 and pour the contents into container #2. Place the outlet tube back into container #3.

[0701] 23. Repeat steps 15-22, taking out 1 mL of sample for PSD analysis after every 4 cycles, until 12 cycles are completed.

[0702] 24. Place both the inlet and outlet pipes into container #2.

[0703] 25. Circulate the suspension through the ball mill using the same parameters as described above.

[0704] 26. Record the pressure, pump flow setting, temperature, and rotor speed every 60 minutes.

[0705] 27. Take 1 mL of sample every 60 minutes and analyze the PSD until D. 50 Below 300nm.

[0706] 28. Finally, the suspension was pumped into container #3 using a ball mill, weighed (W13 = 4,255.8 g), and the API concentration was analyzed by HPLC. Based on these results, the amount of additional 1% HPC solution required to adjust the final API concentration to 100 mg / mL (10% w / v) was calculated.

[0707] 29. Add the calculated amount (W14 = 474.5 g) of 1% HPC solution.

[0708] 30. Take out 1 mL of sample and analyze PSD.

[0709] Production steps: Step 4

[0710] 31. Transfer the nano suspension from container #3 to container #2 for continued mixing with an overhead stirrer.

[0711] 32. Using the previously determined density of the 100 mg / mL SRP-3D (diacetamide) suspension (0.99 g / mL) and an acceptable fill volume range of 20.2–21.8 mL (target 21.0 mL), the acceptable fill weight range was calculated to be 20.0–21.6 g.

[0712] 33. Receive the fill volume into a tare sterile 50 mL Falcon tube, record the weight, and adjust the fill volume as needed to obtain a fill volume of 20.8 g, thereby calibrating the fill volume on the Baxter Baxa Repeater pump. All weighted samples are circulated back into container #2. When three consecutive fills are within the acceptable range (20.0–21.6 g), proceed to the next step.

[0713] 34. While keeping the suspension in container #2 stirred with an overhead stirrer, use a repeater pump to fill 20 mL Type 1 glass vials to the target filling weight. Check the weight before and after the filling run.

[0714] 35. After filling, seal the vial with a sterile 18mm rubber stopper.

[0715] 36. Tighten the cap to seal.

[0716] 37. Label the trays containing the vials appropriately and store them under the recommended storage conditions.

[0717] 38. Reverse the Repeater pump to push any residue in the tubing back into container #2.

[0718] 39. Calculate the weight of the bulk suspension that can be used for filling as W13 + W14 = 4,730.3 g.

[0719] 40. Determine the weight (18.7g) of the unfilled bulk suspension in container #2.

[0720] 41. The calculated bulk weight of the filling is 226 filled vials × 20.8g / via (target filling weight) = 4700.8g.

[0721] 42. The total bulk quantity is 4719.5g (99.8%).

[0722] 43. The yield percentage of the filled vials is calculated as (number of filled vials) / [(bulk suspension available for filling) / (target filling weight)] × 100% = (226 filled vials) / [(4,730.3g) / (20.8g / via)] × 100% = 99.6%.

[0723] The release and stability results of the batch produced in Example 3 after 12 months at 5°C, 12 months at 25°C / 60% relative humidity, and 6 months at 40°C / 75% relative humidity are shown in Tables 8, 9, and 10. This batch met all specifications under all the above conditions. A slight increase in particle size was observed at 5°C, which was slightly more pronounced at 25°C / 60% relative humidity, and even more pronounced at 40°C / 75% relative humidity.

[0724] Table 8: Stability results of SRP-3D (diethylamide) oral suspension 100 mg / mL at 5±3℃

[0725]

[0726] Table 9: Stability results of SRP-3D (diethylamide) oral suspension 100 mg / mL at 25±2℃ / 60% RH±5%.

[0727]

[0728] Table 10: Stability results of SRP-3D (diethylamide) oral suspension 100 mg / mL at 40±2℃ / 75% RH±5%.

[0729]

[0730] Example 4

[0731] SRP-3D(DA) nanoparticle suspensions containing intravenously compatible excipients were prepared by wet milling.

[0732] A slurry or concentrated suspension containing 10% (w / w) SRP-3D (DA), 0.5% (w / w) sodium deoxycholate and 10% (w / w) sucrose, using Netzsch Wet grinding for 1 hour using a ball mill with 0.4mm zirconia grinding beads.

[0733] The resulting nanoparticle suspension was a semi-transparent liquid, with no visible particles under an optical microscope. Dynamic light scattering measurements showed an average particle size of 199.3 nm and a polydispersity index of 0.084.

[0734] Example 5

[0735] Comparison of wetting agents used in the preparation of SRP-3D(DA) nanoparticle suspensions by wet milling

[0736] SRP-3D(DA) was wet-milled using aqueous suspensions containing various wetting agents under milling conditions similar to those in Example 4. The particle sizes of the resulting SRP-3D(DA) nanosuspensions are shown below:

[0737]

[0738] Data indicate that sodium deoxycholate is the preferred wetting agent.

[0739] Example 6

[0740] Preparation of other SRP-3D(DA) nanoparticle suspension formulations

[0741] The following formulations are designed for wet milling to prepare SRP-3D(DA) aqueous nanoparticle suspensions:

[0742]

[0743] Example 7

[0744] Freeze-drying of SRP-3D(DA) nanoparticle suspension

[0745] The SRP-3D(DA) nanoparticle suspension of Example 4 was freeze-dried (lyophilized) under the following conditions:

[0746] Primary drying step: -40℃ for 48 hours

[0747] Secondary drying step: Increase from -40℃ to 25℃ and continue for 12 hours.

[0748] The particle size of the nanoparticle formulation was measured before and after lyophilization, and the results are as follows:

[0749]

[0750] *The lyophilized sample was reconstituted with deionized water before testing.

[0751] The lyophilized nanoparticle formulation was examined for large particles (>10-20 micrometers) or particulate matter, and the results met the USP particulate matter specifications (USP). <788> The presence of this ingredient indicates that it does not contain large particles and is suitable for injection.

[0752] The SRP-3D(DA) nanoparticle formulation in Example 4 exhibited stable physical properties during freeze-drying, and no significant increase in particle size was detected.

[0753] Example 8

[0754] Stability of lyophilized SRP-3D(DA) nanoparticle formulations

[0755] The freeze-dried SRP-3D(DA) nanoparticle formulation from Example 7 was stored at 5°C, 25°C, and 40°C for stability testing. The average particle size (Z-mean) was determined by dynamic light scattering after 7 and 14 days. The results are as follows:

[0756] *The lyophilized sample was reconstituted with deionized water before testing.

[0757] The freeze-dried SRP-3D(DA) nanoparticle formulation in Example 7 was physically stable, and no significant increase in particle size was detected at any of the three storage temperatures.

[0758] Example 9

[0759] Sterilization of lyophilized SRP-3D(DA) nanoparticle formulations

[0760] Another batch of SRP-3D(DA) nanoparticle suspension was prepared using the same composition and process as described in Example 4. An aqueous nanoparticle suspension containing 10% (w / w) SRP-3D(DA), 0.5% (w / w) sodium deoxycholate, and 10% (w / w) sucrose was prepared by wet milling, followed by lyophilization.

[0761] Lyophilized nanoparticle formulations were irradiated with gamma rays at doses ranging from 15.0 to 19.5 kGy. This dose was sufficient to sterilize the nanoparticle formulations. The nanoparticle formulations remained stable after this irradiation, indicating that gamma ray irradiation is a suitable method for sterilizing injectable SRP-3D(DA) nanoparticle formulations.

[0762] The test conditions and results are summarized as follows:

[0763]

[0764] The lyophilized sample was reconstituted with deionized water before testing.

[0765] Method b 13

[0766] Method c 14

[0767] d According to USP <17> Microbial testing

[0768] NA = Not applicable, ND = Not detected, NT = Not detected; NG = No growth

[0769] Example 10

[0770] Canine oral administration study of SRP-3D(DA) nanoparticle suspension

[0771] A 28-day repeated-dose oral toxicity study was conducted in beagle dogs using an SRP-3D(DA) nanoparticle suspension (average diameter 258.4 nm) prepared according to Example 3, including the following treatment groups:

[0772]

[0773] *Three animals in each group were euthanized on day 29. In groups 1 and 4, the remaining two animals from each group were deprived of medication and underwent a 14-day recovery period (untreated).

[0774] Toxicological observations and tests concluded that, in male beagle dogs, the no-observed adverse effects level (NOAEL) of SRP-3D(DA) was 495 mg / kg / day when administered three times daily for 28 consecutive days.

[0775] The results of the toxicokinetics (TK) study are summarized as follows:

[0776] Day 1

[0777] On day 1, the Cmax value increased as the SRP-3D(DA) dose increased from 165 mg / kg / day to 495 mg / kg / day; while the AUC(0-24) value increased when the dose increased from 165 mg / kg / day to 330 mg / kg / day, but did not change when increasing from 330 mg / kg / day to 495 mg / kg / day. On day 1, the mean Cmax values ​​were 4,380, 8,650, and 9,420 ng / mL for SRP-3D(DA) doses of 165, 330, and 495 mg / kg, respectively, and the mean AUC(0-24) values ​​were 12,500, 36,700, and 34,700 ng·h / mL, respectively.

[0778] Day 14

[0779] On day 14, when the SRP-3D(DA) dose increased from 165 mg / kg / day to 330 mg / kg / day, its Cmax and AUC(0-24) values ​​increased accordingly, but no change was observed when the dose increased from 330 mg / kg / day to 495 mg / kg / day. On day 14, the mean Cmax values ​​at SRP-3D(DA) doses of 165, 330, and 495 mg / kg were 2,330, 4,240, and 3,830 ng / mL, respectively, and the mean AUC(0-24) values ​​were 7,090, 17,700, and 17,700 ng·h / mL, respectively.

[0780] Day 28

[0781] On day 28, as the SRP-3D(DA) dose increased from 165 mg / kg / day to 495 mg / kg / day, its Cmax and AUC(0-24) values ​​increased accordingly. On day 28, at SRP-3D(DA) doses of 165, 330, and 495 mg / kg / day, the mean Cmax values ​​were 3,030, 5,580, and 7,710 ng / mL, respectively, and the mean AUC(0-24) values ​​were 7,360, 18,000, and 20,300 ng·h / mL, respectively.

[0782] SRP-3D(DA) nanoparticle suspension has oral bioavailability in dogs. At doses ranging from 330 to 495 mg / kg / day, the systemic exposure (AUC) is dose-dependent.

[0783] Example 11

[0784] Compared to unground particles, the particle size of SRP-3D(DA) was reduced to <1 μm, resulting in a decrease in plasma AUC after oral administration in dogs. 0-24 / (dose / day) significantly increased. Plasma levels of SRP-3D(DA) were determined by LC-MS / MS using a validated method.

[0785] SRP-3D (diacetamide) powder in gelatin capsules was administered orally in a single dose of 300 mg / kg. 10 It is 50.22 μm; D 50 It is 217.26 μm; D 90 (538.54 μm), the results are as follows:

[0786] AUC 0-24 1,993 ng·hr / mL

[0787] AUC 0-24 / (Dosage / day): 6.64(ng·hr / mL) / [(mg / kg) / day]

[0788] The SRP-3D(DA) nanoparticle suspension (average diameter 258.4 nm) prepared according to the method described in Example 3 was orally administered at a dose of 110 mg / kg three times daily at 4-hour intervals (330 mg / kg / day). The results are as follows:

[0789] AUC 0-24 36,700 ng·hr / mL

[0790] AUC0-24 / (dosage / day): 111(ng·hr / mL) / [(mg / kg) / day]

[0791] The above results indicate that, compared with the unmicronized powder in the capsule, the AUC of the nanoparticle suspension is higher. 0-24 The dose per day increased by 16.7 times.

[0792] Example 12

[0793] Oral administration trial of SRP-3D(DA) nanosuspension in healthy human volunteers

[0794] In a single-dose escalation study, SRP-3D(DA) nanoparticle suspensions (average particle size 301 nm) prepared according to the method of Example 3 were administered to healthy human volunteers in single oral doses of 300 mg, 600 mg, 900 mg, or 2000 mg. In Cohort 3, volunteers who fasted before administration (Program C) or ate before administration (Program D) were given a single 900 mg dose to assess the effect of food on pharmacokinetics. Volunteers in all other cohorts remained fasted before administration. Plasma concentrations of SRP-3D(DA) were determined by LC-MSMS using a validated method. The pharmacokinetic and safety results are summarized in Table 11.

[0795] Table 11: Pharmacokinetic and safety results of single-incremental doses (SAD) of SRP-3D(DA)

[0796]

[0797] *Geometric Mean (Geometric CV%)

[0798]

[0799] In a multiple escalation dose study, SRP-3D(DA) nanoparticle suspension (average particle size 301 nm) prepared according to the method of Example 3 was administered once daily (QD) at doses of 500 mg / day or 1000 mg / day to fasting healthy human volunteers for five consecutive days. The pharmacokinetic and safety results are summarized in Table 12.

[0800] Table 12: Pharmacokinetic and safety results of SRP-3D (DA) multiple escalation doses (MAD)

[0801]

[0802] *Geometric Mean (Geometric CV%)

[0803]

[0804] When administered orally to healthy human volunteers, the SRP-3D(DA) nanosuspension prepared according to the method of Example 3 was rapidly absorbed. The corresponding increase in SRP-3D(DA) plasma concentration was approximately proportional to the administered dose. No serious adverse events or trial discontinuation due to safety concerns were observed, indicating that this route of administration was well tolerable.

[0805] Example 13

[0806] Preparation of amorphous SRP-3D (DA)

[0807] An excess of SRP-3D(DA) prepared according to the method of Example 1 was continuously stirred with toluene or trifluoroethanol (2-10 mL) at ambient temperature to prepare a saturated solution. The mother liquor was then heated above the saturation temperature to dissolve any residual solids. After adjusting the solution temperature to ambient temperature, a controlled nitrogen shear flow was introduced to initiate solvent evaporation. The resulting solid was analyzed by XRPD according to Method 10, and the results showed indistinguishable diffraction peaks, indicating that the sample was amorphous.

Claims

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

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

3. The composition according to any one of claims 1-2, wherein the average particle size of the SRP-3D(DA) particles in the form of nanoparticles is selected from less than about 500 nm, less than about 400 nm, 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 and less than about 50 nm.

4. The composition according to any one of claims 1-3, wherein the composition is formulated for administration by a route selected from oral administration and injection.

5. The composition according to any one of claims 1-4, wherein the composition is formulated into a dosage form selected from liquid dispersions, liquid suspensions, solutions, powders, capsules and tablets.

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

7. The composition according to any one of claims 1-6, wherein based on the total dry weight of SRP-3D(DA) and at least one wetting agent, wherein no other excipients are included, and the amount of SRP-3D(DA) present is selected from about 99.5% by weight to about 0.001% by weight, about 95% by weight to about 0.1% by weight, and about 90% by weight to about 0.5% by weight.

8. The composition according to any one of claims 1-7, wherein the total dry weight of SRP-3D (DA) and at least one wetting agent is excluded, wherein other excipients are not included, and the at least one wetting agent is present in an amount selected from about 0.01 wt% to about 99.5 wt%, about 0.1 wt% to about 95 wt%, about 0.5 wt% to about 90 wt%, and about 0.5 wt% to about 2 wt%.

9. The composition according to any one of claims 1-8, wherein the amount of the at least one wetting agent is selected from less than about 10%, less than about 5%, less than about 2%, and less than about 1%, based on the weight of the nanoparticle composition.

10. The composition according to any one of claims 1-9, wherein the at least one wetting agent is selected from alkali metal salts of bile salts and bile acids.

11. The composition according to claim 10, wherein the bile salt is selected from sodium, potassium, lithium, calcium, arginine, lysine, and ammonium salts of bile acids.

12. The composition according to claim 10, comprising a salt of bile acids selected from the group consisting of: cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycodeoxycholic acid, and taurodeoxycholic acid.

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

14. The composition according to any one of claims 1-13, further comprising a cryoprotectant or lyophilization protectant selected from the group consisting of sucrose, mannitol, trehalose, lactose, or maltodextrin.

15. A method for preparing a nanoparticle composition, comprising wet milling SRP-3D(DA) particles with at least one wetting agent, wherein the milling time and conditions are sufficient to provide a suspension of SRP-3D(DA) nanoparticles with an average particle size of less than about 500 nm or less than about 300 nm.

16. The method of claim 15, wherein the wet milling comprises a method selected from ball milling and microfluidization.

17. The method according to any one of claims 15-16, wherein the wet milling comprises: (a) SRP-3D(DA) particles are added to a solution containing a wetting agent to obtain a suspension, and (b) the suspension is wet-milled to obtain a nanoparticle suspension having a desired final particle size of less than about 500 nm or less than about 300 nm.

18. The method according to any one of claims 15-17, wherein the final particle size of the SRP-3D(DA) particles in the form of nanoparticles is selected from less than about 500 nm, 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, and less than about 50 nm.

19. The method according to any one of claims 15-18, wherein the wet milling process further comprises a drying step, wherein water is removed from the nanoparticle suspension by one or more methods selected from filtration, centrifugation, freeze drying, spray drying and vacuum drying to obtain dry powder.

20. A compound of formula I: The compound in question is essentially free of impurities.

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

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

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

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

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

26. A solid form of Formula I: The solid form is characterized by X-ray powder diffraction (XRPD) patterns obtained using CuKα radiation containing the following 2θ peaks: 9.0±0.2, 9.2±0.2, 13.2±0.2, and 21.9±0.

2.

27. The solid form of Formula I according to claim 26, characterized in that... The X-ray powder diffraction (XRPD) patterns obtained using CuKα radiation also contain the following 2θ peaks: 12.2±0.2, 15.4±0.2, 18.3±0.2, 19.2±0.2, 20.7±0.2, and 22.5±0.

2.

28. The solid form of Formula I according to claim 26, wherein the solid form is characterized in that the X-ray powder (XRPD) pattern obtained by CuKα radiation is substantially similar to that of Figure 10, Figure 11 or Figure 12.

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

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

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

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

33. A method for treating, reducing, or improving pain in an individual in need, the method comprising: The individual was given a compound of formula I to treat, reduce, or improve pain.

34. The method of claim 33, wherein the pain is selected from acute, chronic, and neuropathic pain.

35. The method of claim 33, wherein the compound of formula I is formulated into a nanoparticle suspension for oral administration, the nanoparticle suspension comprising: (a) Compound of Formula I; and (b) At least one wetting agent.

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

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