Salt-supported solid lipid nanoparticles containing activators

Abstract

Solid lipid nanoparticles (SLNs) containing (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoylphosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a drug containing a phosphate, phenolate, or carboxylate encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt of the solid lipid nanoparticles (SLNs); SLNs containing an anionic form of a drug containing a phosphate, phenolate, or carboxylate encapsulated in dimethyldidodecylammonium bromide or 1,2-dioleoyl-3-trimethylammonium-propane; pharmaceutical compositions containing SLNs; methods for producing SLNs.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the priority of U.S. Provisional Application No. 63 / 532,230, filed on August 11, 2023, and U.S. Provisional Application No. 63 / 653,261, filed on May 30, 2024, and incorporates the entire contents of both applications herein by reference.

[0002] (Technical Field) The present disclosure relates to solid lipid nanoparticles containing an anionic - form drug containing phosphate, phenolate, or carboxylate encapsulated in a cationic - form lipid as an ion complex or salt, a pharmaceutical composition containing the same, and a method for producing the same.

Background Art

[0003] Nanopharmaceuticals using liposomes and lipids have been important drug delivery vehicles for many years [1 - 4]. Nanosystems based on solid lipids loaded with drugs have been successfully used for treating various cancers since Doxil was introduced more than 20 years ago. Abraxane, an albumin - coated nanoparticle of the neutral drug paclitaxel, shows improved activity compared to paclitaxel (25% in combination with carboplatin), (33% in combination with carboplatin). Doxil, a liposomal form of doxorubicin, is less cardiotoxic than doxorubicin (Gyongyosi, Card. Res. 116: 970 (2020)).

[0004] The structures of liposomes, solid lipid nanoparticles (SLNs), and nanolipid carriers are not well understood. Numerous diagrams and cartoons illustrating the structures of these species exist, including simple bilayer liposomes with nucleic acids inside, double-loaded bilayers containing an outer bilayer and an internal compartment with nucleic acids, and even bilayers with nucleic acids attached to the outside. Significant effort has been invested in nucleic acid and RNA-loaded carriers, including, of course, the COVID-19 vaccine, which contains carriers composed of cationic lipids, helper lipids, cholesterol, and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) lipids on which mRNA is loaded. Surprisingly, little effort has been invested in drug-loaded carriers, and the properties and characterization of COVID-19 carriers loaded with anionic drugs, including phosphates, carboxylates, and phenolates, have not been reported at all. This is surprising because anionic drugs can form ion complexes / salts with cationic lipids, which make up the majority of the composition of SLN carriers (for example, SLN carriers used in anti-COVID-19 vaccines).

[0005] Based on the above, the object of this disclosure is to provide solid lipid nanoparticles (SLNs) supported with anionic surfactants. This object, as well as other objects and advantages and features of the invention, are evident herein. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Gyongyosi, Card. Res. 116: 970 (2020) [Overview of the project]

[0007] Solid lipid nanoparticles (SLNs) are provided, containing (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoyl phosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt. The ionic complex or salt is formed in ethanol or another non-aqueous solvent is used for the reaction. The anionic form of the drug may form a salt with sodium or triethylamine hydrochloride before being encapsulated in the carrier. The SLN carrier may be stable for at least about 29 days in a refrigerator at about 1.7–3.3°C. The drug may be an anticancer agent such as fludarabine phosphate, etoposide phosphate, or a flavone anion. If the drug is fludarabine phosphate, presaturation is performed, and in the proton NMR spectrum of SLN in suspension using ethanol-d6+D2O with tetramethylsilane (TMS) as the reference material, no signal for fludarabine phosphate is shown at approximately 6-6.5 ppm or approximately 8-8.5 ppm. The drug may also be an antiinfective agent, such as an antiinfective agent containing a phenolic anion.

[0008] Pharmaceutical compositions are also provided. The pharmaceutical compositions include the above-mentioned SLN and a pharmaceutically acceptable carrier.

[0009] A method for producing an SLN is further provided, comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) DSPC, (c) cholesterol, and (d) an anionic form of a drug containing a phosphate, phenolate, or carboxylate encapsulated in DMG-PEG-2000 or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt. The method comprises: (i) mixing an anionic form of the drug dissolved in 100% ethanol with a cationic salt of an ethoxide (e.g., sodium ethoxide) dissolved in ethanol, then stirring, filtering, evaporating as necessary, and powdering as necessary to obtain a powder or residue; (ii) (a) mixing the powder or residue obtained in (i) with ethanol and either SM-102 or ALC-0315 in a drug-lipid molar ratio of about 0.7:1 to about 1.5:1 until dissolved; (b) separately mixing DSPC, cholesterol, and either DMG-PEG-2000 or ALC-0159 in ethanol until dissolved; and (c) mixing (a) and (b) together to obtain a solution; and (iii) adding the solution obtained in (ii) to water, or a solution containing water and a buffer, thereby obtaining an SLN containing an anionic form of the drug encapsulated in either SM-102 or ALC-0315. The cationic form may be sodium or triethylamine. The drug may be an anticancer agent such as fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. The drug may also be an antiinfective agent, such as an antiinfective agent containing a phenolic anion.

[0010] A further method for producing an SLN is also provided, comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) DSPC, (c) cholesterol, and (d) an anionic form of phosphate drug encapsulated in DMG-PEG-2000 or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt. The method comprises (i) mixing a phosphate drug dissolved in 100% ethanol in ethanol with (a) SM-102 or ALC-0315, (b) DSPC, (c) cholesterol, and (d) DMG-PEG-2000 or ALC-0159 until dissolved to obtain a solution; and (ii) adding the solution obtained in (i) to water, or a solution containing water and a buffer, thereby obtaining an SLN containing the anionic form of the drug encapsulated in either SM-102 or ALC-0315. The cationic form may be sodium or triethylamine. The drug may be an anticancer agent such as fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. The drug may also be an antiinfective agent, such as an antiinfective agent containing a phenolic anion.

[0011] Further provided are SLNs containing an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in dimethyldidodecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The anionic form of the drug may form a salt with sodium or triethylamine hydrochloride. The SLN may be stable for at least about 29 days in a refrigerator at about 1.7–3.3°C. The drug may be an anticancer agent such as fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. The drug may also be an antiinfective agent, such as an antiinfective agent containing a phenolic anion. The antiinfective agent may be quercetin. For example, if the drug is quercetin, a peak can be produced at 46+ / -3°C in differential scanning calorimetry.

[0012] Pharmaceutical compositions are also provided. The pharmaceutical compositions include the above-mentioned SLN and a pharmaceutically acceptable carrier.

[0013] A method for producing SLN containing an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in DDAB or DOTAP is also provided. The method comprises (i) mixing the anionic form of the drug with either (a) DDAB / ethanol or (b) DOTAP / ethanol, then stirring, filtering, evaporating as necessary, and powdering as necessary; and (ii) adding the solution to water, or a second solution containing water and buffer (or dissolving the powder to make a solution), thereby obtaining SLN containing an anionic form of the drug encapsulated in DDAB or DOTAP. The anionic form of the drug may be salted with sodium or triethylamine hydrochloride. The drug may be an anticancer agent such as fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. The drug may also be an antiinfective agent, such as an antiinfective agent containing a phenolic anion. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows the reaction that forms the salt of the nanoparticles. During the reaction, the ratio of quercetin to SM-102 was 1:1. The ratio of lipids was 50:10:38.5:1.5. [Figure 2] Figure 2 shows a TEM image of nanoparticles formed by the reaction of a lipid mixture containing the cationic lipid SM-102 with sodium quercetin. [Figure 3] Figure 3 shows the reaction between the ionizable lipid SM-102 and quercetin triethylamine salt. [Figure 4] Figure 4 shows TEM images of the dispersion and supernatant formed by the reaction of quercetin triethylamine salt with SM-102 (using other lipids). [Figure 5]Figure 5 shows the reaction between quercetin and benzoyl chloride, which forms quercetin benzoate. [Figure 6] Figure 6 shows a TEM image of nanoparticles supported with quercetin benzoate. [Figure 7] Figure 7 shows the reaction between sodium quercetin and dimethyldidodecylammonium bromide (DDAB) via salt exchange. [Figure 8] Figure 8 shows a TEM image of quercetin sodium DDAB+ nanoparticles. [Figure 9A] Figure 9A shows the structure of etoposide phosphate. [Figure 9B] Figure 9B shows the structure of fludarabine phosphate. [Figure 9C] Figure 9C shows the structure of niclosamide. [Figure 9D] Figure 9D shows the structure of niclosamide phosphate. [Figure 9E] Figure 9E shows the structures of niclosamide phosphate and cationic lipid salts. [Figure 10] Figure 10 shows differential scanning calorimetry (DSC) of quercetin (Q)-DDAB nanoparticles at various quercetin:DDAB ratios. A is QNa-DDAB(1:1.25) nanoparticles with an enthalpy (normalized) of 69.384 J / g, a peak temperature of 51.84 °C, and an onset of 45.08 °C. B is QNa-DDAB(1:1.5) nanoparticles with an enthalpy (normalized) of 75.440 J / g, a peak temperature of 50.66 °C, and an onset of 43.99 °C. C is QNa-DDAB(1:1.75) nanoparticles with an enthalpy (normalized) of 74.541 J / g, a peak temperature of 49.68 °C, and an onset of 43.53 °C. D is a DDAB nanoparticle with an enthalpy (normalized) of 14.422 J / g, a peak temperature of 53.81 °C, and an onset of 50.76 °C, and an enthalpy (normalized) of 46.442 J / g, a peak temperature of 67.62 °C, and an onset of 65.07 °C. E is a DDAB with an enthalpy (normalized) of 136.23 J / g, a peak temperature of 89.26 °C, and an onset of 85.43 °C. [Figure 11] Figure 11 shows the solution nuclear magnetic resonance (NMR) spectra of full darabin phosphate (FP)-COVID-19 nanoparticles and FP-SM-102 nanoparticles. A is FP + SM-102 + (distearoyl phosphatidylcholine) DSPC + cholesterol + 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) (FP-SM-102 = 1:1.5). B is FP + SM-102 (1:1.5). C is SM-102, and D is FP.

Modes for Carrying Out the Invention

[0015] This disclosure is based on the discovery that activators can be supported and stabilized on solid lipid nanoparticles (SLNs) (e.g., COVID-19 vaccine carriers) using salt chemistry. As an example, sodium quercetin was formed in pure ethanol (weak acid) by reacting sodium ethoxide with quercetin. The sodium quercetin salt was then used to form nanoparticles with the cationic lipid SM-102 (a cationic lipid used in Moderna's SARS-CoV-2 vaccine) and dimethyldioctadecylammonium (bromide salt) (DDAB; a cationic lipid). The sodium quercetin salt and DDAB formed lipid nanoparticles without the use of other lipids. Quercetin salt also formed nanoparticles with a COVID-19 type mixture of SM-102, cholesterol, 1,2-distearoyl-s,n-glycero-3-phosphocholine (DSPC; neutral lipid), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMP-PEG-2000) in a ratio of 50:38.5:10:1.5. Nanoparticle formation using this salt chemistry approach was confirmed by particle size measurement, transmission electron microscopy (TEM) analysis, nuclear magnetic resonance (NMR) analysis, and differential scanning calorimetry (DSC) analysis, and encapsulation efficiency was measured using high-performance liquid chromatography (HPLC). Quercetin sodium also reacted with benzoyl chloride to form benzoylquercetin. The reaction product could be encapsulated in nanoparticles, but the encapsulation efficiency was 28%. This is likely due to incomplete reaction with benzoyl chloride, resulting in some of the ionic drug being encapsulated.

[0016] As a second example, fludarabine phosphate and SM-102 reacted in ethanol to form a salt / ion complex detected by the dissolution of fludarabine phosphate. DSPC, cholesterol, and DMG-PEG-2000 were dissolved in a separate pure ethanol, and the two ethanol solutions were mixed. This drug-lipid / ethanol solution was added to an aqueous layer (deionized water or 10 mM, pH 5.0 acetate buffer) with stirring, and since it contained fludarabine phosphate, it formed COVID-19 type SLNs with a different composition from COVID-19 nanoparticles. This solution was dialyzed, sucrose was added, and then lyophilized to obtain solid SLNs. The formation of the nanoparticles using this salt chemistry approach was confirmed by particle size measurement and NMR analysis.

[0017] Additional examples are provided in more detail in the Examples section.

[0018] Advantages of the SLN salts loaded with the active agent may include one or more of the following: improved solubility, increased absorption, delayed metabolism and excretion, maintenance of a more stable plasma concentration, improved drug stability, high drug payload, enhanced activity or efficacy, reduced toxicity, improved tolerance, large-scale production, and the possibility of manufacturing sustained-release formulations.

[0019] Based on the above, an SLN (e.g., a COVID-19 vaccine-type carrier) is provided, containing (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoylphosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a drug comprising a phosphate, phenolate, or carboxylate encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt. The ionic complex or salt is formed in ethanol or another non-aqueous solvent (e.g., acetone or tetrahydrofuran (THF)) is used for the reaction. The anionic form of the drug may form a salt with, for example, sodium or triethylamine hydrochloride before encapsulation in the carrier. SLN carriers can be stable for at least about 29 days in a refrigerator at approximately 1.7–3.3°C. The drug may be an anticancer agent such as fludarabine phosphate, etoposide phosphate, or a flavone anion.Examples of flavonoid anions include, but are not limited to, those based on plant-derived flavones (e.g., Tsimogiannis and Oreopoulou, “Classification of Phenolic Compounds in Plants,” in Polyphenols in Plants, 2nd ed., Isolation, Purification, and Extract Preparation, pp. 263-284, Academic Press). (See 2019) This includes apigenin, apigenin C-glycoside, apigenin O-glycoside, luteolin, luteolin C-glycoside, luteolin O-glycoside, diosmetin C-glycoside, diosmetin O-glycoside, chrysoeriol C-glycoside, chrysoeriol O-glycoside, acacetin O-glycoside, myricetin, chrysin, baicalein, scutellain, hispizuline, tricetin, sinensetin, tangeretin, serpylin, nobiletin, scaposin, and genkwanin. If the drug is fludarabine phosphate, presaturation is performed, and in the proton NMR spectrum of SLN in suspension using ethanol-d6+D2O with tetramethylsilane (TMS) as the reference material, no signal for fludarabine phosphate is shown at approximately 6-6.5 ppm or approximately 8-8.5 ppm. The drug may also be an anti-infective agent, for example, an anti-infective agent containing a phenolic anion.Phenolic anions are plant-derived phenolic acids, such as flavonoids, galangin, campaverol, cinnamic acid, p-coumaric acid, ferulic acid, sinapic acid, caffeic acid, benzoic acid, 4-hydroxybenzoic acid, vanillic acid, silicic acid, protocatechuic acid, tannic acid, epicatechin, epigallocatechin, gallic acid, resveratrol, and triacetylresveratrol. It may be based on polyphenol compounds such as triacetate, catechol, catechin, epicatechin-3-gallate, epigallocatechin gallate, ellagic acid, punicalagin, protocatecaldehyde, afzelin, formononetin, quercetin, pyrogallol, capsaicin, wrightiadione, thespesin, chamazulene / matricin, silymarin, hydroxytyrosol, chlorogenic acid, luteolin, EGCG, hesperetin, daidzein, myricetin, apigenin, curcumin, stilbene, benzoic acid, and kaempferol. Cationic lipid SM-102 has already been used in pharmaceuticals (e.g., COVID-19 vaccines), and this use will facilitate regulatory approval of new drugs containing SM-102. Another anti-infective drug, niclosamide, may require the addition of agents to delay or prevent aggregation of nanoparticles, one example being mannitol.

[0020] Pharmaceutical compositions are also provided. The pharmaceutical compositions include the above-mentioned SLN and a pharmaceutically acceptable carrier.

[0021] A method for producing an SLN is further provided, comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) DSPC, (c) cholesterol, and (d) an anionic form of a drug containing a phosphate, phenolate, or carboxylate encapsulated in DMG-PEG-2000 or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt. The method comprises (i) mixing an anionic form of the drug dissolved in 100% ethanol with a cationic salt of an ethoxide (e.g., sodium ethoxide) dissolved in ethanol, then stirring, filtering, evaporating as necessary, and powdering as necessary to obtain a powder or residue; (ii) (a) mixing the powder or residue obtained in (i) with ethanol and either SM-102 or ALC-0315 until dissolved, with a drug-to-lipid molar ratio of about 0.7:1 to about 1.5:1; (b) separately mixing DSPC, cholesterol, and either DMG-PEG-2000 or ALC-0159 in ethanol until dissolved; and (c) mixing (a) and (b) together to obtain a solution; and (iii) adding the solution obtained in (ii) to water, or a solution containing water and buffer (e.g., pH about 5), thereby obtaining an SLN containing an anionic form of the drug encapsulated in either SM-102 or ALC-0315. The cationic form may be, for example, sodium or triethylamine. The drug may be an anticancer agent such as fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion.Examples of flavonoid anions include, but are not limited to, those based on plant-derived flavones (e.g., Tsimogiannis and Oreopoulou, “Classification of Phenolic Compounds in Plants,” in Polyphenols in Plants, 2nd ed., Isolation, Purification, and Extract Preparation, pp. 263-284, Academic Press). (See 2019), these include apigenin, apigenin C-glycoside, apigenin O-glycoside, luteolin, luteolin C-glycoside, luteolin O-glycoside, diosmetine C-glycoside, diosmetine O-glycoside, chrysoeriol C-glycoside, chrysoeriol O-glycoside, acacetin O-glycoside, myricetin, chrysin, baicalein, scutellain, hispizuline, tricetin, sinensetin, tangeretin, serpyrin, nobiletin, scaposin, and genkwanin. The drugs may also be antiinfective agents, for example, antiinfective agents containing phenolic anions. Phenolic anions are plant-derived phenolic acids, such as flavonoids, galangin, campaberol, cinnamic acid, p-coumaric acid, ferulic acid, sinapic acid, caffeic acid, benzoic acid, 4-hydroxybenzoic acid, vanillic acid, silicic acid, protocatechuic acid, tannic acid, epicatechin, epigallocatechin, gallic acid, resveratrol, triacetylresveratrol, catechol, catechin, epicatechin-3-gallate, epigallocatechin gallate This may be based on polyphenol compounds such as tetraphosphate, ellagic acid, punicalagin, protocatecaldehyde, afzelin, formononetin, quercetin, pyrogallol, capsaicin, lythiazion, tespesin, chamazulene / matricin, silymarin, hydroxytyrosol, chlorogenic acid, luteolin, EGCG, hesperetin, daidzein, myricetin, apigenin, curcumin, stilbene, benzoic acid, and kaempferol. Another anti-infective drug, niclosamide, may require the addition of an agent to delay / prevent aggregation, such as mannitol.

[0022] The reaction steps shown below illustrate this process using etoposide phosphate. [ka]

[0023] Further provided is another method for producing an SLN, comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) DSPC, (c) cholesterol, and (d) an anionic form of phosphate drug encapsulated in DMG-PEG-2000 or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt. The method comprises (i) mixing a phosphate ester drug dissolved in 100% ethanol with (a) SM-102 or ALC-0315, (b) DSPC, (c) cholesterol, and (d) DMG-PEG-2000 or ALC-0159 in ethanol until dissolved to obtain a solution; and (ii) adding the solution obtained in (i) to water, or a solution containing water and buffer (e.g., pH about 5), thereby obtaining an SLN containing the anionic form of the drug encapsulated in either SM-102 or ALC-0315. The cationic form may be, for example, sodium or triethylamine. The drug may be an anticancer agent such as, for example, fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion.Examples of flavonoid anions include, but are not limited to, those based on plant-derived flavones (e.g., Tsimogiannis and Oreopoulou, “Classification of Phenolic Compounds in Plants,” in Polyphenols in Plants, 2nd ed., Isolation, Purification, and Extract Preparation, pp. 263-284, Academic Press). (See 2019), these include apigenin, apigenin C-glycoside, apigenin O-glycoside, luteolin, luteolin C-glycoside, luteolin O-glycoside, diosmetine C-glycoside, diosmetine O-glycoside, chrysoeriol C-glycoside, chrysoeriol O-glycoside, acacetin O-glycoside, myricetin, chrysin, baicalein, scutellain, hispizuline, tricetin, sinensetin, tangeretin, serpyrin, nobiletin, scaposin, and genkwanin. The drugs may also be antiinfective agents, for example, antiinfective agents containing phenolic anions. Phenolic anions are plant-derived phenolic acids, such as flavonoids, galangin, campaberol, cinnamic acid, p-coumaric acid, ferulic acid, sinapic acid, caffeic acid, benzoic acid, 4-hydroxybenzoic acid, vanillic acid, silicic acid, protocatechuic acid, tannic acid, epicatechin, epigallocatechin, gallic acid, resveratrol, triacetylresveratrol, catechol, catechin, epicatechin-3-gallate, epigallocatechin gallate These may be based on polyphenol compounds such as tetraphosphate, ellagic acid, punicalagin, protocatecaldehyde, afzelin, formononetin, quercetin, pyrogallol, capsaicin, lythiadione, tespesin, chamazulene / matricin, silymarin, hydroxytyrosol, chlorogenic acid, luteolin, EGCG, hesperetin, daidzein, myricetin, apigenin, curcumin, stilbene, benzoic acid, and kaempferol. Another anti-infective drug, niclosamide, may require the addition of an agent to delay / prevent aggregation (e.g., mannitol).

[0024] Further SLNs are also provided that contain an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in dimethyldidodecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The anionic form of the drug may be salted with sodium or triethylamine hydrochloride. The SLN may be stable for at least about 29 days in a refrigerator at about 1.7–3.3°C. The drug may be an anticancer agent such as, for example, fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. Examples of flavonoid anions include, but are not limited to, those based on plant-derived flavones (e.g., Tsimogiannis and Oreopoulou, “Classification of Phenolic Compounds in Plants,” in Polyphenols in Plants, 2nd ed., Isolation, Purification, and Extract Preparation, pp. 263-284, Academic Press). (See 2019) These include apigenin, apigenin C-glycoside, apigenin O-glycoside, luteolin, luteolin C-glycoside, luteolin O-glycoside, diosmetine C-glycoside, diosmetine O-glycoside, chrysoeriol C-glycoside, chrysoeriol O-glycoside, acacetin O-glycoside, myricetin, chrysin, baicalein, scutellain, hispizuline, tricetin, sinensetin, tangeretin, serpyrin, nobiletin, scaposin, and genkwanin. The drugs may also be anti-infective agents, for example, phenolic anions.Phenolic anions are plant-derived phenolic acids, such as flavonoids, galangin, campaberol, cinnamic acid, p-coumaric acid, ferulic acid, sinapic acid, caffeic acid, benzoic acid, 4-hydroxybenzoic acid, vanillic acid, silicic acid, protocatechuic acid, tannic acid, epicatechin, epigallocatechin, gallic acid, resveratrol, triacetylresveratrol, catechol, catechin, epicatechin-3-gallate, epigallocatechin gallate These may be based on polyphenol compounds such as tetraphosphate, ellagic acid, punicalagin, protocatecaldehyde, afzelin, formononetin, quercetin, pyrogallol, capsaicin, lythiadione, tespesin, chamazulene / matricin, silymarin, hydroxytyrosol, chlorogenic acid, luteolin, EGCG, hesperetin, daidzein, myricetin, apigenin, curcumin, stilbene, benzoic acid, and kaempferol. The anti-infective agent may also be quercetin. For example, if the drug is quercetin, a peak can be produced at 46+ / -3°C in differential scanning calorimetry.

[0025] Pharmaceutical compositions are also provided. The pharmaceutical compositions include the above-mentioned SLN and a pharmaceutically acceptable carrier.

[0026] A method for producing SLN containing an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in DDAB or DOTAP is also provided. The method comprises (i) mixing the anionic form of the drug with either (a) DDAB / ethanol or (b) DOTAP / ethanol, then stirring, filtering, evaporating as necessary, and powdering as necessary; and (ii) adding the solution to water, or a second solution (e.g., pH about 5) containing water and buffer (or dissolving the powder to make a solution), thereby obtaining SLN containing an anionic form of the drug encapsulated in DDAB or DOTAP. The anionic form of the drug may be salted with sodium or triethylamine hydrochloride. The drug may also be an anticancer agent such as fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. Examples of flavonoid anions include, but are not limited to, those based on plant-derived flavones (e.g., Tsimogiannis and Oreopoulou, "Classification of Phenolic Compounds in Plants," in Polyphenols in Plants, 2nd ed., Isolation, Purification, and Extract Preparation, pp. 263-284, Academic Press). (See 2019), these include apigenin, apigenin C-glycoside, apigenin O-glycoside, luteolin, luteolin C-glycoside, luteolin O-glycoside, diosmetine C-glycoside, diosmetine O-glycoside, chrysoeriol C-glycoside, chrysoeriol O-glycoside, acacetin O-glycoside, myricetin, chrysin, baicalein, scutellain, hispizuline, tricetin, sinensetin, tangeretin, serpyrin, nobiletin, scaposin, and genkwanin. The drugs may also be antiinfective agents, for example, antiinfective agents containing phenolic anions.Phenolic anions are plant-derived phenolic acids, such as flavonoids, galangin, campaberol, cinnamic acid, p-coumaric acid, ferulic acid, sinapic acid, caffeic acid, benzoic acid, 4-hydroxybenzoic acid, vanillic acid, silicic acid, protocatechuic acid, tannic acid, epicatechin, epigallocatechin, gallic acid, resveratrol, triacetylresveratrol, catechol, catechin, epicatechin-3-gallate, epigallocatechin gallate These may be based on polyphenol compounds such as tetraphosphate, ellagic acid, punicalagin, protocatecaldehyde, afzelin, formononetin, quercetin, pyrogallol, capsaicin, lythiadione, tespesin, chamazulene / matricin, silymarin, hydroxytyrosol, chlorogenic acid, luteolin, EGCG, hesperetin, daidzein, myricetin, apigenin, curcumin, stilbene, benzoic acid, and kaempferol. Another anti-infective drug, niclosamide, may require the addition of an agent to delay / prevent aggregation (e.g., mannitol).

[0027] "Cationic lipids" refer to lipid species that have a positive net charge at a selected pH, such as physiological pH (e.g., pH approximately 7.0). Cationic lipids containing alkyl chains with multiple unsaturated sites, such as at least two or three unsaturated sites, can be particularly useful in forming lipid particles with increased membrane fluidity.

[0028] "Neutral lipids" refer to lipid species that exist either as uncharged or as neutral zwitterionic forms at a selected pH, such as physiological pH (approximately pH 7.0).

[0029] A "salt" refers to an association or ionic complex of cationic and anionic molecules. Salts are formed by the reaction of acids and bases.

[0030] Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0031] The drug may be any anionic activator, such as an anticancer agent or an anti-infective agent (e.g., antiviral, antibacterial, or antiparasitic agent). Examples of anionic anticancer agents include, but are not limited to, etoposide, etoposide phosphate, and fludarabine phosphate (see Figure 11 for structure). Examples of anionic antiviral agents include, but are not limited to, niclosamide (which may require the addition of an agent to delay / prevent aggregation, such as mannitol), acyclovir, salicylamides, curcumin, quercetin, flavonoids, and any of the aforementioned phosphates.

[0032] Homogenization, sonication, and poor solvent mixing methods can be used for formulation (Mehnert et al., Adv Drug Deliv Rev 64: 83-101 (2012)). Hot homogenization has been used for formulations containing stearic acid and tristearin that melt during the process. Higher temperatures reduce viscosity, potentially resulting in smaller particles. Solvent emulsification is also a powerful method, involving precipitation of the oil / water emulsion due to solvent evaporation. In some cases, the emulsion is formed using high-pressure homogenization. Naturally, lipids, emulsifiers, and manufacturing procedures all affect the properties of the resulting SLN. Spray drying can be a powerful method in SLN production, as it usually yields a powder that can be filled into capsules. In addition, if the product is heat-sterilized, the effect of this step on the SLN needs to be evaluated. Sterilization by gamma irradiation can also cause changes. Sterilization methods should be developed after the final structure and formulation composition of the SLN have been determined.

[0033] A crucial part of SLN production is the measurement of the structure and quality of SLNs. Typical characterization methods include: (1) particle size; (2) zeta potential; (3) crystallinity of components; (4) thermal analysis; and (5) nuclear magnetic resonance (NMR). Particle size is usually measured using dynamic light scattering. Zeta potential measures the movement of particles when exposed to an electric field. Crystallinity is measured using X-ray diffraction and pair correlation analysis, as detailed below. Thermal analysis is performed using differential scanning calorimeters and can be used to determine crystallinity. NMR is a powerful method for measuring the bonding and mobility of molecules. By combining X-ray pair correlation analysis and NMR, the domain structure of solid lipid nanoparticles can be elucidated.

[0034] Typically, SLNs are less toxic than polymer nanoparticles. Generally, SLNs are desirable to be non-toxic because they contain physiological compounds and have metabolic pathways for the formulation components. SLNs are usually phagocytosed.

[0035] Pharmaceutical compositions can be prepared for any suitable route of administration. Examples of suitable routes include, but are not limited to, oral, parenteral, subcutaneous, inhalation, depot, and topical administration. While the properties of SLN may differ depending on the route of administration, a single SLN composition can be administered via multiple routes if the SLN is stable. Lipases can degrade SLN and may be present in multiple organs in the body (Mehnert et al. (2012), see above). The SLN degradation route is determined empirically. [Examples]

[0036] The following examples are for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

[0037] (Example 1: Preparation of Solid Lipid Nanoparticles (SLNs)) SLN was prepared according to the injection procedure described in reference [7,8]. Alternatively, SLN can be prepared using a confined impinging jet mixer-CIJ mixer (Holland Applied Technologies, P0288404).

[0038] Lipids and drugs were purchased commercially. Cholesterol, quercetin, and pure ethanol were sourced from the laboratory.

[0039] Particle size was measured using a Malvern Zetasizer Nano ZX (Malvern Panalytical). Transmission electron microscopy (TEM) imaging was performed using a 200kV transmission electron microscope (Tecnai G2 T20, FEI). Drug encapsulation efficiency was measured using Agilent 1100 series high-performance liquid chromatography (HPLC). Nuclear magnetic resonance (NMR) spectra were measured using a Bruker Avance-III 800MHz NMR with a QCI cryoprobe. Quercetin encapsulation efficiency within nanoparticles was measured using HPLC based on the chromatography conditions in Table 1 [9]. [Table 1]

[0040] Drug stock solutions were prepared in methanol at a concentration of 100 mg / ml. Calibration plots were prepared in the range of 2–50 μg / ml. Drug encapsulation efficiency (%EE) was measured by analyzing the drug content in the total nanoparticle dispersion and supernatant using an Agilent 1100 series HPLC. The supernatant was analyzed using Beckman Coulter® Optima TMThe nanoparticle dispersion was ultracentrifuged using a MAX-XP ultracentrifuge and then recovered. One ml of the nanoparticle dispersion was added to a 1.5 ml centrifuge tube and centrifuged at 100,000 rpm (rcf 543,000 xg) for 15 minutes at 4°C. The entire dispersion and the supernatant were analyzed by HPLC. Injection samples were prepared by diluting the nanoparticle dispersion 10-fold with methanol and mixing until a clear solution was obtained. Supernatant samples were prepared by diluting the dispersion 5-fold with methanol. The drug encapsulation efficiency is given by the following formula: [ka] For imaging of nanoparticles, a Tecnai G2 20 transmission electron microscope and a 200KV LaB6 filament transmission electron microscope, both equipped with a Fischione HAADF detector and automated electron tomography software (FEI and SerialEM), were used in conjunction with a Fischione 2020 high-tilt holder. The CCD camera used was a bottom-mounted Gatan US1000 2Kx2K. Samples were stained with 2% uranyl acetate on a 400-mesh copper grid coated with a carbon film.

[0041] Solid lipid nanoparticles were lyophilized using a 1:1 ratio of nanoparticles-mannitol / sucrose, with mannitol or sucrose as a cytoprotective agent. Mannitol was dissolved in the nanoparticle dispersion by stirring. The dispersion was frozen at -80°C for 48 hours. The frozen samples were subjected to a freeze-drying cycle (-80°C, <0.05 mBar for 96 hours). The lyophilized products were stored at -20°C.

[0042] (Example 2: Preparation of quercetin sodium-DDAB nanoparticles) Sodium quercetin (~90% pure) and triethylamine hydrochloride (molar ratio 1:4) were added to pure ethanol and sonicated until the sodium quercetin dissolved. Separately, dimethyldidodecylammonium bromide (DDAB) was dissolved in pure ethanol and added to the quercetin solution. The drug-lipid solution was injected into an aqueous layer (deionized water or 10 mM, pH 5.0 acetate buffer) while stirring with a magnetic stirrer. Ethanol in the formulation (and buffer salts when prepared in 10 mM, pH 5.0 acetate buffer as a poor solvent) was removed by dialysis against deionized water using a 10K MWCO Slide-A-Lyzer dialysis cassette (ThermoFisher Scientific). The drug loading rate of the sodium quercetin-DDAB nanoparticles was 15-25%, and the encapsulation efficiency was 75-100%. The aqueous nanoparticle suspension was freeze-dried in a Labconco® benchtop freeze-dryer at -80°C and less than 0.1 mbar (freeze-drying time: 5 days). Dry nanoparticles were obtained. Dialysis and freeze-drying did not reduce the encapsulation efficiency.

[0043] (Example 3: Preparation of quercetin sodium-DOTAP nanoparticles) The procedure of Example 2 was used to prepare quercetin sodium-1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) nanoparticles.

[0044] (Example 4: Preparation of Quercetin Sodium-DDAB / DOTAP-DSPC-Cholesterol-DMG-PEG-2000 Nanoparticles) Sodium quercetin (~90% pure) and triethylamine hydrochloride (molar ratio 1:4) were added to pure ethanol and sonicated until the sodium quercetin was dissolved. Separately, DDAB / DOTAP was dissolved in pure ethanol and added to the sodium quercetin solution. 1,2-Distearoyl-s,n-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-Dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) were dissolved in another pure ethanol and added to the sodium quercetin solution. The drug-lipid solution was injected into an aqueous layer (deionized water or 10 mM, pH 5.0 acetate buffer) while stirring with a magnetic stirrer.

[0045] (Example 5: Preparation of quercetin-type COVID-19 lipid nanoparticles using sodium quercetin instead of quercetin as a starting material) Quercetin sodium (prepared as described above) and SM-102 (ionizable synthetic aminolipid) were stirred in pure ethanol at a 1:1 drug-to-lipid molar ratio until the drugs were completely dissolved. Separately, DSPC, cholesterol, and DMG-PEG-2000 were dissolved in pure ethanol, maintaining a molar ratio of 50:10:38.5:1.5 by weight for the SM-102, DSPC, cholesterol, and DMG-PEG-2000 lipid mixture. The two solutions were mixed. The total solid content was 6 mg / ml, and the drug-total lipid weight ratio was 19:81. The volumes of the solvent phase and the poor solvent phase (acetic acid buffer, pH 5.0, 10 mM) were measured, maintaining a solvent:poor solvent ratio of 1:3. The solvent phase was injected into the poor solvent phase with a 20 G syringe while stirring, keeping the syringe below the liquid surface of the poor solvent phase during injection. Stirring was continued for 15 minutes.

[0046] The reaction for forming the nanoparticle salt is shown in Figure 1. In the reaction, the ratio of quercetin:SM-102 was 1:1. The ratio of lipids was 50:10:38.5:1.5. These nanoparticles contained quercetin sodium:SM102:cholesterol:DSPC:DMG-PEG-2000 in the proportions of 18.6%:40.7%:8.1%:31.3%:1.2%. Unlike quercetin nanoparticles, quercetin sodium nanoparticles were stable. This is likely due to the strong negative charge on the phenol group oxygen atom of quercetin caused by the formation of the sodium salt, which readily loses sodium ions at the acidic pH used in the manufacturing of the formulation. This results in a stronger interaction with cationic SM-102 compared to free quercetin. Particle size, PDI, zeta potential, and encapsulation efficiency are shown in Table 2. [Table 2]

[0047] TEM images of the formed nanoparticles are shown in Figure 2.

[0048] (Example 6: Results for drug loading, %EE, stability, size, and PDI) The drug loading, encapsulation efficiency (EE), particle size, and polydispersity index (PDI) of QS are summarized in the table below (Table 3). [Table 3]

[0049] (Example 7: NMR spectroscopy of quercetin sodium-DDAB-SLN dispersion) SLN dispersions (drug-DDAB or pure DDAB) prepared using the procedure outlined above were then subjected to three ultrafiltration cycles using 4 ml Amicon Ultra Centrifugal Filters (MilliporeSigma). In each step, the concentrated dispersion was diluted with deutherium oxide (D2O) (99.9%) until virtually all ethanol was removed and water was replaced with D2O. For pure drug spectra, sodium quercetin was dissolved in a 1:3 mixture of ethanol-d6 (99%) and D2O (99.9%). Bruker Avance-III 800 MHz NMR with a QCI cryoprobe was used for all analyses. The disappearance or near-disappearance of several drug and lipid peaks in the nanoparticle spectra was observed, indicating quercetin sodium-DDAB interaction. The disappearance of the quercetin signal in the salt suggests that the quercetin anion may be immobilized within the lipid bilayer formed by DDAB.

[0050] (Example 8: Differential scanning calorimetry of freeze-dried quercetin-DDAB nanoparticles) Because the freeze-dried pure nanoparticles were completely dry, their thermal motion could be investigated by DSC. Pure lipid (DDAB) had a single endothermic melting peak at 89°C, while pure DDAB nanoparticles (prepared in the same manner as the drug-lipid nanoparticles) showed two endothermic melting peaks at 54°C and 68°C. The drug-lipid nanoparticles showed neither of these peaks, while a new single endothermic melting peak was observed around 50°C. The melting peaks gradually shifted to lower melting points with decreasing drug load (51.84°C at a quercetin sodium-DDAB molar ratio of 1:1.25, 50.66°C at 1:1.5, and 49.68°C). The disappearance of the pure lipid melting peak indicates that the drug is contained as an ion complex with a cationic lipid. Figure 10 and Table 4 show the DSC data for this ion complex / salt. In this ionic complex (salt), the sodium originally bound to the quercetin anion was replaced by a DDAB cation, resulting in a different composition from the original molecules mixed together. [Table 4]

[0051] (Example 9: X-ray data of quercetin sodium DDAB nanoparticles) Powder X-ray diffraction patterns of DDAB:quercetin SLN and its components showed that sodium quercetin has an amorphous pattern and is non-crystalline. DDAB nanoparticles have a crystalline pattern consistent with endothermic DSC traces. Sodium quercetin:DDAB nanoparticles exhibit an amorphous pattern, which is different from the starting material, sodium quercetin.

[0052] (Example 10: Stability of quercetin sodium nanoparticles) Since the two SLN-based COVID-19 vaccines were stored at -80°C, the stability of the SLNs is important. Initial studies on the stability of quercetin:SLNs were conducted using particle size increase as a marker of instability. Particle size is an excellent marker for liposome and nanoparticle instability, as adverse events due to particle growth in liposome formulations have been known for over 20 years. Table 5 reports the stability of quercetin SLNs using SM-102, DSPC, cholesterol, and DMG-PEG. Table 5 also reports the stability of quercetin:DDAB nanoparticles. In all cases, the SLN-quercetin compositions were stable for 29 days in a refrigerator. [Table 5]

[0053] (Example 11: Preparation plan for COVID-19 type nanoparticles using the neutral drug quercetin) Quercetin-filled nanoparticles were prepared containing quercetin:SM-102:DSPC:cholesterol:DMG-PEG-2000 in concentrations of 17.5%:41.2%:8.2%:31.7%:1.2%. These nanoparticles were not stable, as evidenced by their large average particle size and high polydispersity index (PDI). The average particle size, PDI, zeta potential, and encapsulation efficiency for these nanoparticles are shown in Table 6. [Table 6]

[0054] The formulation showed numerous precipitates after 2 hours.

[0055] Quercetin sodium appeared to have less than 100% solubility in pure ethanol in several experiments. To investigate whether higher encapsulation efficiency of the drug could be obtained using different cations, quercetin sodium was converted to the triethylamine salt of quercetin. This triethylamine salt was then reacted in a 1:1 ratio with the same lipid mixture containing SM-102. The total lipid ratio in this reaction was 50:10:38.5:1.5 (SM-102:DSPC:cholesterol:DMG-PEG-2000). The chemical reaction is shown in Figure 3. In this reaction, the encapsulation efficiency was higher than that of the sodium salt of quercetin. Table 7 shows the particle size, polydispersity, zeta potential, and encapsulation efficiency of 70% or higher. This is higher than the encapsulation efficiency of either quercetin or quercetin sodium without reaction with triethylamine hydrochloride. This is likely because the amine improved the solubility of sodium quercetin in pure ethanol, thereby improving the efficiency of its interaction with SM-102. [Table 7]

[0056] The dispersion and supernatant formed by the reaction of quercetin triethylamine salt with SM-102 (using other lipids) are shown in Figure 4.

[0057] (Example 12: Preparation plan for quercetin benzoate nanoparticles) Quercetin benzoate was prepared, and its encapsulation efficiency of neutral quercetin molecules in SM-102 cationic solid lipid nanoparticles containing other lipids was tested. For this study, sodium quercetin was reacted with benzoyl chloride as shown in Figure 5. Then, quercetin benzoate was reacted with SM-102 and other lipids in the same ratio (50:10:38.5:1.50) to form nanoparticles. The drug-to-lipid weight ratio was 19:81. The volumes of the solvent phase and the poor solvent phase (pH 5.0 acetate buffer) were maintained at a solvent:poor solvent ratio of 1:3. The solvent phase was injected into the poor solvent phase with agitation using a 20G syringe, and the syringe was kept below the liquid surface of the poor solvent phase during injection. Particle size, polydispersity, zeta potential, and encapsulation efficiency are shown in Table 8. It should be noted that the encapsulation efficiency was considerably low in this case, which is probably because the neutral charge of the ester reduced the proportion of drug available for ionic interaction with the lipid.

[0058] Sodium quercetin was stirred in pure ethanol, and benzoyl chloride was added in a 1:1 molar ratio. The mixture was stirred until the drug was completely dissolved. A product with a distinct fruity odor was formed. The product was a yellow rather than reddish-brown solution of sodium quercetin. [Table 8]

[0059] The TEM of the quercetin benzoate-supported particles is shown in Figure 6.

[0060] (Example 13: Preparation of fludarabine phosphate-DDAB nanoparticles) Fludarabine phosphate and triethylamine (1:2 molar ratio) were added to pure ethanol and sonicated until the fludarabine phosphate dissolved. Separately, DDAB was dissolved in pure ethanol and added to the fludarabine phosphate solution. The drug-lipid solution was injected into an aqueous layer (deionized water or 10 mM, pH 5.0 acetate buffer) while stirring with a magnetic stirrer. Ethanol in the formulation (and buffer salts when prepared in 10 mM, pH 5.0 acetate buffer as a poor solvent) was removed by dialysis against deionized water using a 10K MWCO Slide-A-Lyzer dialysis cassette (ThermoFisher Scientific). The drug loading rate of fludarabine phosphate-DDAB nanoparticles was 15-25%, and the encapsulation efficiency was 75-100%.

[0061] (Example 14: Preparation of fludarabine phosphate-DOTAP nanoparticles) Fludarabine phosphate-DOTAP nanoparticles were prepared using the procedure of Example 13.

[0062] (Example 15: Preparation of fludarabine phosphate-DOTAP-DSPC-cholesterol-DMG-PEG-2000 nanoparticles) Fludarabine phosphate and triethylamine (1:2 molar ratio) were added to pure ethanol and sonicated until the fludarabine phosphate dissolved. Separately, DOTAP was dissolved in pure ethanol and added to the fludarabine phosphate solution. DSPC, cholesterol, and DMG-PEG-2000 were dissolved in separate pure ethanol and added to the fludarabine phosphate solution. The drug-lipid solutions were injected into an aqueous layer (deionized water or 10 mM, pH 5.0 acetate buffer) while stirring with a magnetic stirrer.

[0063] (Example 16: Preparation of fludarabine phosphate-SM-102-DSPC-cholesterol-DMG-PEG-2000 nanoparticles) Fludarabine phosphate and SM-102 were added to pure ethanol and sonicated until the fludarabine phosphate dissolved. DSPC, cholesterol, and DMG-PEG-2000 were dissolved in separate pure ethanol and added to the fludarabine phosphate solution. The drug-lipid solution was injected into an aqueous layer (deionized water or 10 mM, pH 5.0 acetate buffer) while stirring with a magnetic stirrer. The resulting nanoparticles had a drug loading rate of 10–17% and an encapsulation efficiency of 52–83%. Ethanol in the formulation (and buffer salts when prepared in 10 mM, pH 5.0 acetate buffer as a poor solvent) was removed by dialysis against deionized water using a 10K MWCO Slide-A-Lyzer dialysis cassette (ThermoFisher Scientific). The aqueous nanoparticle suspension was freeze-dried in a Labconco® benchtop freeze-dryer at -80°C and less than 0.1 mbar with the addition of 2.5% w / v or 5% w / v sucrose (freeze-drying time: 7 days).

[0064] (Example 17: Freeze-drying of SLN) SLNs containing fludarabine phosphate-SM-102(1:1.5)-DSPC-Chol-DMG-PEG-2000 were lyophilized and reconstituted with 2.5% sucrose, 2.5% sucrose, and 5% sucrose, without sucrose. As shown in Table 5, the nanoparticles showed only slight particle growth. The drug loading (DL), encapsulation efficiency (EE), particle size, and polydispersity index (PDI) after lyophilization are shown in Table 9. [Table 9]

[0065] (Example 18: NMR spectroscopy of fludarabine phosphate-SM-102 SLN dispersion) SLN dispersions (drug-SM-102, pure SM-102, and drug-SM-102-DSPC-cholesterol-DMG-PEG-2000) were prepared according to the outlined procedure, replacing the usual solvent with a deuterated solvent, using ethanol-d6 (99%) as the solvent phase and D2O (99.9%) as the poor solvent phase. All samples were prepared while maintaining the drug concentration at 1 mM and the SM-102 concentration at 1.6 mM. The SLN dispersions were analyzed directly after preparation. For pure drug spectra, fludarabine phosphate was dissolved in a 1:3 mixture of ethanol-d6 (99%) and D2O (99.9%). Bruker Avance-III 800 MHz NMR with a QCI cryoprobe was used for all analyses. Disappearance or decay peaks of components (drug or lipid) were observed, indicating drug-lipid interactions.

[0066] (Example 19: Preparation of etoposide phosphate-SM-102-DSPC-cholesterol-DMG-PEG-2000 nanoparticles) SM-102 (50 mg) and etoposide phosphate (8 mg) were dissolved in ethanol (2 mL). Cholesterol (38.5 mg), DSPC (10 mg), and DMG-PEG2000 (1.5 mg) were added to the ethanol solution. The lipid-etoposide phosphate solution was slowly added to rapidly agitated water (8 mL, 1,200 rpm) via a syringe and a 22-gauge needle to obtain solid lipid nanoparticles.

[0067] Alternatively, SLN was prepared using a constrained impingement jet mixer (CIJ) and a syringe pump. The lipid-etoposide phosphate solution prepared as described above was rapidly mixed with an equal volume of water using a CIJ mixer (Holland Applied Technologies, P0288404). The mixed stream of solid lipid nanoparticles was collected in water (6 mL). [Table 10]

[0068] Salts in pharmaceuticals are typically thought to form when the pKa of a basic species (cationic lipid, DDAB, or ionizable lipid SM-102) differs from the pKa of an acidic species (quercetin) by more than two orders of magnitude (units). The pKa of quercetin is listed as 6.3

[12] . SM-102 and related cationic lipids have been the subject of extensive research. Tilstra reported that ionizable lipids containing a pure ethanolamine core with an apparent pKa between 6.6 and 6.9 maximize intramuscular mRNA delivery

[13] . In a screening study of novel ionizable lipids, Lam reported that the pKa of SM-102 is 6.3 and that of ALC-0315 is 6.09

[14] . Good inclusion was observed because quercetin anions and SM-102 as a cation readily form complexes under acidic pH. DDAB is a quaternary ammonium ion that does not have a pKa, but it permanently possesses a positive charge on its nitrogen atom. This also contributed to its good encapsulation.

[0069] The above examples demonstrate that stable SLNs containing a drug and a cationic lipid can be formed by forming a sodium salt of phenol using sodium ethoxide in ethanol, and then reacting this sodium salt with an ionizable / cationic lipid in an acidic environment, either with or without other lipids. The ionic interaction between the drug and the lipid results in high encapsulation efficiency and improved stability. This approach is attractive for designing stable SLNs for drugs containing phenol groups. This approach and related approaches provide a novel approach for forming drug-containing SLNs, which is considered broadly applicable to the formation of solid lipid nanoparticles of weak acids such as phenols and carboxylic acids. Salts formed by this method are expected to be injectable and readily testable in investigational new drug (IND) trials.

[0070] (Enumeration of embodiments) The embodiments listed below represent claims that include multiple dependent claims dependent on multiple dependent claims, and are intended for examination in jurisdictions where such dependency relationships are permitted. (Claim 1) Solid lipid nanoparticles (SLNs) containing (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoyl phosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a drug comprising a phosphate, phenolate, or carboxylate encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt. (Claim 2) Anionic forms of drugs form salts with sodium. The SLN according to claim 1. (Claim 3) The anionic form of the drug forms a salt of triethylamine hydrochloride. The SLN according to claim 1. (Claim 4) It remains stable for at least approximately 29 days in a refrigerator at approximately 1.7-3.3°C. The SLN according to any one of claims 1 to 3. (Claim 5) The drug is an anti-cancer drug. The SLN according to any one of claims 1 to 4. (Claim 6) The anticancer drug is fludarabine phosphate, etoposide, etoposide phosphate, or flavone anion. The SLN according to claim 5. (Claim 7) The anticancer drug is fludarabine phosphate, and after presaturation, the proton NMR spectrum of SLN in the suspension, using ethanol-d6+D2O with tetramethylsilane (TMS) as the reference material, does not show a signal for fludarabine phosphate at approximately 6-6.5 ppm or approximately 8-8.5 ppm. The SLN according to claim 6. (Claim 8) The drug is an anti-infective drug. The SLN according to any one of claims 1 to 4. (Claim 9) Antiinfective drugs contain phenolic anions, The SLN according to claim 8. (Claim 10) A pharmaceutical composition comprising an SLN according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier. (Claim 11) A method for producing solid lipid nanoparticles (SLNs) comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoyl phosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a drug containing a phosphate, phenolate, or carboxylate encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt, (i) Mix a drug dissolved in 100% ethanol with a cationic salt of an ethoxide (e.g., sodium ethoxide) dissolved in ethanol, then stir, filter, evaporate as necessary, and powder as necessary to obtain a powder or residue; (ii) (a) Mix the powder or residue obtained in (i) with ethanol and either SM-102 or ALC-0315 in a drug-lipid molar ratio of approximately 0.7:1 to approximately 1.5:1 until dissolved; (b) Separately, mix DSPC, cholesterol, and either DMG-PEG-2000 or ALC-0159 in ethanol until dissolved; and (c) Mix (a) and (b) together to obtain a solution; and A method comprising adding the solution obtained in (iii)(ii) to water, or a solution comprising water and a buffer, thereby obtaining an SLN containing an anionic form of the drug enclosed in SM-102 or ALC-0315. (Claim 12) The cationic form is sodium. The method according to claim 11. (Claim 13) The cationic form is triethylamine. The method according to claim 11. (Claim 14) The drug is an anti-cancer drug. The method according to any one of claims 11 to 13. (Claim 15) The method according to claim 14, wherein the anticancer agent is fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. (Claim 16) The drug is an anti-infective drug. The method according to any one of claims 11 to 13. (Claim 17) Antiinfective drugs contain phenolic anions, The method according to claim 16. (Claim 18) A method for producing solid lipid nanoparticles (SLNs) comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoyl phosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a phosphate drug encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt, (i) Mixing a phosphate drug dissolved in 100% ethanol with (a) SM-102 or ALC-0315, (b) DSPC, (c) cholesterol, and (d) DMG-PEG-2000 or ALC-0159 in ethanol until dissolved to obtain a solution; and (ii) A method comprising adding the solution obtained in (i) to water, or a solution comprising water and a buffer, thereby obtaining an SLN containing an anionic form of a phosphate drug encapsulated in either SM-102 or ALC-0315. (Claim 19) The cationic form is sodium. The method according to claim 18. (Claim 20) The cationic form is triethylamine. The method according to claim 18. (Claim 21) The drug is an anti-cancer drug. The method according to any one of claims 18 to 20. (Claim 22) The anticancer drug is fludarabine phosphate, etoposide, etoposide phosphate, or flavone anion. The method according to claim 22. (Claim 23) The drug is an anti-infective drug. The method according to any one of claims 18 to 20. (Claim 24) The anti-infective drug is niclosamide, or an anti-infective drug containing a phenolic anion. The method according to claim 23. (Claim 25) Solid lipid nanoparticles (SLNs) containing an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in dimethyldidodecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). (Claim 26) Anionic forms of drugs form salts with sodium. The SLN according to claim 25. (Claim 27) The anionic form of the drug forms a salt with triethylamine hydrochloride. The SLN according to claim 25. (Claim 28) It remains stable for at least approximately 29 days in a refrigerator at a temperature of approximately 1.7°C to 3.3°C. The SLN according to any one of claims 25 to 27. (Claim 29) The drug is an anti-cancer drug. The SLN according to any one of claims 25 to 28. (Claim 30) The anticancer drug is fludarabine phosphate, etoposide, etoposide phosphate, or flavone anion. The SLN according to claim 29. (Claim 31) The drug is an anti-infective drug. The SLN according to any one of claims 25 to 28. (Claim 32) Antiinfective drugs contain phenolic anions, The SLN according to claim 31. (Claim 33) The drug is quercetin. The SLN according to claim 32. (Claim 34) In differential scanning calorimetry, a peak occurs at 46 ± 3°C. The SLN according to claim 33. (Claim 35) A pharmaceutical composition comprising an SLN according to any one of claims 25 to 34 and a pharmaceutically acceptable carrier. (Claim 36) A method for producing solid lipid nanoparticles (SLNs) containing an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in dimethyldidodecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), (i) Mix the anionic form of the drug with either (a) DDAB / ethanol or (b) DOTAP / ethanol, then stir, filter, evaporate as necessary, and powder as necessary; and (ii) A method comprising adding the solution to water, or a second solution comprising water and a buffer (or dissolving the powder to make a solution), thereby obtaining an SLN containing the anionic form of the drug encapsulated in the DDAB. (Claim 37) Anionic forms of drugs form salts with sodium. The method according to claim 36. (Claim 38) The anionic form of the drug forms a salt with triethylamine hydrochloride. The method according to claim 36. (Claim 39) The drug is an anti-cancer drug. The method according to any one of claims 36 to 38. (Claim 40) The method according to claim 39, wherein the anticancer agent is fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion. (Claim 41) The drug is an anti-infective drug. The method according to any one of claims 36 to 38. (Claim 42) Antiinfective drugs contain phenolic anions, The method according to claim 41.

[0071] (References) TIFF2026528833000014.tif218164

[0072] All patent documents, patent application documents, academic papers, textbooks, and other documents referenced herein represent the state of the art for those skilled in the art to which this disclosure pertains. All such documents are incorporated herein by reference to the same extent as when each individual document is specifically and individually indicated to be incorporated by reference. In the event of any inconsistency between the usage herein and the incorporated documents, the usage in the incorporated document shall be deemed to supplement the usage herein; in the event of an irreconcilable inconsistency, the usage herein shall prevail.

[0073] The inventions described exemplary herein may be appropriately implemented without any elements or limitations not specifically disclosed herein. Accordingly, for example, the terms “including,” “substantially consisting of,” and “consisting of” may each be replaced in each case herein by any of the other two terms. Similarly, the singular forms “a,” “an,” and “the” include the plural form unless the context clearly indicates otherwise. Accordingly, for example, the term “method” includes one or more methods and / or steps of that type which are described herein and / or which are evident from this disclosure to those skilled in the art. The term “or” is used to mean nonexclusive “or” unless otherwise specified.

[0074] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. The following terms and phrases should have the meanings indicated.

[0075] The term "approximately" means that, when referring to a number or numerical value or range of numerical values ​​(e.g., all numbers, fractions, and percentages), the number or numerical range indicated is an approximation within the range of experimental variability (or statistical experimental error), and therefore the numerical value or numerical range may vary by 1% to 15% of the stated number or numerical range (e.g., + / - 5% to 15% of the stated value, e.g., within 10%, 5%, or 1% of the stated value or stated range limit), provided that a person skilled in the art would consider it equivalent to the stated value (e.g., having the same function or result). The term "substantially" means that some variation in a value or range is permissible, e.g., within 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, or at least approximately 99.999% of the stated value or stated range limit.

[0076] In addition, any phrases or terms used herein should be understood to be for illustrative purposes only, and not for limitation, unless otherwise specified. Any use of section headings is intended to aid in reading the document and should not be construed as limitation. Furthermore, information related to section headings may also be found within or outside of that particular section.

Claims

1. Solid lipid nanoparticles (SLNs) containing (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoylphosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a drug comprising a phosphate, phenolate, or carboxylate encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt.

2. Anionic forms of drugs form salts with sodium. The SLN according to claim 1.

3. The anionic form of the drug forms a salt with triethylamine hydrochloride. The SLN according to claim 1.

4. SLN is stable for at least approximately 29 days in a refrigerator at approximately 1.7 to 3.3°C. The SLN according to claim 1.

5. The drug is an anti-cancer drug. The SLN according to any one of claims 1 to 4.

6. The anticancer drug is fludarabine phosphate, etoposide, etoposide phosphate, or flavone anion. The SLN according to claim 5.

7. The anticancer drug is fludarabine phosphate, and presaturation is performed, followed by ethanol-d ６ +D ２ In O, the proton NMR spectrum of SLN in the suspension, using tetramethylsilane (TMS) as a reference substance, does not show a signal for fludarabine phosphate at approximately 6–6.5 ppm or approximately 8–8.5 ppm. The SLN according to claim 6.

8. The drug is an anti-infective drug. The SLN according to any one of claims 1 to 4.

9. Antiinfective drugs contain phenolic anions, The SLN according to claim 8.

10. A pharmaceutical composition comprising an SLN according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising the SLN described in claim 5 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition comprising the SLN described in claim 6 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising the SLN described in claim 7 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition comprising the SLN described in claim 8 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising the SLN described in claim 9 and a pharmaceutically acceptable carrier.

16. A method for producing solid lipid nanoparticles (SLNs) comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoylphosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a drug containing a phosphate, phenolate, or carboxylate encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt, (i) Mixing a drug dissolved in 100% ethanol with a cationic salt of an ethoxide dissolved in ethanol (for example, sodium ethoxide, where sodium is the cation and ethoxide is the anion), then stirring, filtering, evaporating as necessary, and powdering as necessary to obtain a powder or residue; (ii) (a) Mix the powder or residue obtained in (i) with ethanol and either SM-102 or ALC-0315 in a drug-lipid molar ratio of about 0.7:1 to about 1.5:1 until dissolved; (b) Separately, mix DSPC, cholesterol, and either DMG-PEG-2000 or ALC-0159 in ethanol until dissolved; and (c) Mix (a) and (b) together to obtain a solution; and A method comprising adding the solution obtained in (iii) to water, or a solution containing water and a buffer, thereby obtaining an SLN containing an anionic form of the drug encapsulated in either SM-102 or ALC-0315.

17. The cationic form is sodium. The method according to claim 16.

18. The cationic form is triethylamine. The method according to claim 16.

19. The drug is an anti-cancer drug. The method according to any one of claims 16 to 18.

20. The method according to claim 19, wherein the anticancer agent is fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion.

21. The drug is an anti-infective drug. The method according to any one of claims 16 to 18.

22. Antiinfective drugs contain phenolic anions, The method according to claim 21.

23. A method for producing solid lipid nanoparticles (SLNs) comprising (a) a cationic form of lipid SM-102 or a cationic form of lipid ALC-0315, (b) distearoylphosphatidylcholine (DSPC), (c) cholesterol, and (d) an anionic form of a phosphate drug encapsulated in 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) or ALC-0159, wherein the anionic form of the drug and the cationic form of lipid (a) form an ionic complex or salt, (i) Mixing a phosphate drug dissolved in 100% ethanol with (a) SM-102 or ALC-0315, (b) DSPC, (c) cholesterol, and (d) DMG-PEG-2000 or ALC-0159 in ethanol until dissolved to obtain a solution; and (ii) A method comprising adding the solution obtained in (i) to water, or a solution comprising water and a buffer, thereby obtaining an SLN containing an anionic form of a phosphate drug encapsulated in either SM-102 or ALC-0315.

24. The cationic form is sodium. The method according to claim 23.

25. The cationic form is triethylamine. The method according to claim 23.

26. The drug is an anti-cancer drug. The method according to any one of claims 23 to 25.

27. The drug is fludarabine phosphate, etoposide, etoposide phosphate, or flavone anion. The method according to claim 26.

28. The drug is an anti-infective drug. The method according to any one of claims 23 to 25.

29. The anti-infective drug is niclosamide, or an anti-infective drug containing a phenolic anion. The method according to claim 28.

30. Solid lipid nanoparticles (SLNs) containing an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in dimethyldidodecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

31. Anionic forms of drugs form salts with sodium. The SLN according to claim 30.

32. The anionic form of the drug forms a salt with triethylamine hydrochloride. The SLN according to claim 30.

33. It remains stable for at least approximately 29 days in a refrigerator at a temperature of approximately 1.7°C to 3.3°C. The SLN according to claim 30.

34. The drug is an anti-cancer drug. The SLN according to any one of claims 30 to 33.

35. The anticancer drug is fludarabine phosphate, etoposide, etoposide phosphate, or flavone anion. The SLN according to claim 34.

36. The drug is an anti-infective drug. The SLN according to any one of claims 30 to 33.

37. Antiinfective drugs contain phenolic anions, The SLN according to claim 36.

38. The drug is quercetin. The SLN according to claim 37.

39. In differential scanning calorimetry, a peak is produced at 46 ± 3°C. The SLN according to claim 38.

40. A pharmaceutical composition comprising an SLN according to any one of claims 30 to 33 and a pharmaceutically acceptable carrier.

41. A pharmaceutical composition comprising the SLN described in claim 34 and a pharmaceutically acceptable carrier.

42. A pharmaceutical composition comprising the SLN described in claim 35 and a pharmaceutically acceptable carrier.

43. A pharmaceutical composition comprising the SLN described in claim 36 and a pharmaceutically acceptable carrier.

44. A pharmaceutical composition comprising the SLN described in claim 37 and a pharmaceutically acceptable carrier.

45. A pharmaceutical composition comprising the SLN described in claim 38 and a pharmaceutically acceptable carrier.

46. A pharmaceutical composition comprising the SLN described in claim 39 and a pharmaceutically acceptable carrier.

47. A method for producing solid lipid nanoparticles (SLNs) containing an anionic form of a drug, including a phosphate, phenolate, or carboxylate, encapsulated in dimethyldidodecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), (i) mixing an anionic form of the drug with either (a) DDAB / ethanol or (b) DOTAP / ethanol, then stirring, filtering, evaporating as necessary, and powdering as necessary; and (ii) A method comprising adding the solution to water, or a second solution comprising water and a buffer (or dissolving the powder to make a solution), thereby obtaining an SLN containing the drug in an anionic form encapsulated in DDAB.

48. Anionic forms of drugs form salts with sodium. The method according to claim 47.

49. The anionic form of the drug forms a salt with triethylamine hydrochloride. The method according to claim 47.

50. The drug is an anti-cancer drug. The method according to any one of claims 47 to 49.

51. The method according to claim 50, wherein the anticancer agent is fludarabine phosphate, etoposide, etoposide phosphate, or a flavone anion.

52. The drug is an anti-infective drug. The method according to any one of claims 47 to 49.

53. Antiinfective drugs contain phenolic anions, The method according to claim 52.

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