Lipid compound, lipid nanoparticle and pharmaceutical composition

Abstract

Provided herein are lipid compounds, such as compounds of Formula (I). Also provided are lipid nanoparticles and pharmaceutical compositions, each comprising a lipid compound, such as a compound of the formula.

Description

1. Cross-application of related applications

[0001] This application claims priority to PCT / CN2025 / 074222, filed on January 23, 2025, the disclosures of each of which are incorporated herein by reference in their entirety. 2. Related sequence list

[0002] This application incorporates by reference the sequence listing submitted with this application, which is an XML file entitled "TPI04652 Sequence Listing.XML", created on July 10, 2025, and is 75,014 bytes in size. Technical Field

[0003] Provided herein are lipid compounds, lipid nanoparticles, and pharmaceutical compositions thereof. Background Art

[0004] Lipid nanoparticles (LNPs) are promising delivery vehicles for a variety of therapeutic agents, including small molecule drugs, proteins, and nucleic acids. See, Akinc et al., Nat. Nanotechnol. 2019, 14, 1084-87; Hou et al., Nat. Rev. Mater. 2021, 6, 1078-94. Two FDA-approved COVID-19 mRNA vaccines (mRNA-1273 and BNT162b) use lipid nanoparticles for antigen mRNA delivery. See, Polack et al., N. Engl. J. Med. 2020, 383, 2603-15; Baden et al., N. Engl. J. Med. 2021, 384, 403-16. However, effectively delivering highly charged nucleic acids as therapeutic agents remains challenging. See, Weng et al., Biotechnol. Adv. 2020, 40, 107534; Hou et al., Nat. Rev. Mater. 2021, 6, 1078-94. To achieve therapeutic effects, for example, highly charged nucleic acids must reach their intracellular targets. See, Torres-Canegas et al., Pharmaceutics 2021, 13, 428; Hou et al., Nat. Rev. Mater. 2021, 6, 1078-94. Therefore, lipid compounds for the efficient delivery of therapeutic agents are needed. Application Overview

[0005] The present application provides compounds of the following formula: or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0006] In addition, the present application provides lipid nanoparticles comprising: a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0007] Further, the present application provides lipid nanoparticles comprising: (i) a nucleic acid, and (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0008] The present application provides a pharmaceutical composition comprising lipid nanoparticles, wherein the lipid nanoparticles comprise: (i) a nucleic acid, and (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof. Exemplary embodiments

[0009] Compounds having the formula: or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0010] The lipid nanoparticle comprises the compound of paragraph

[0009] , or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0011] The pharmaceutical composition comprises a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a nucleic acid; (ii) a compound of paragraph

[0009] , or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (iii) a phospholipid; and (iv) cholesterol.

[0012] The pharmaceutical composition of paragraph

[0011] further comprises a conjugated lipid.

[0013] In the pharmaceutical composition of paragraph

[0012] , the coupled lipid comprises a polyethylene glycol-coupled lipid.

[0014] In the pharmaceutical composition of any of paragraphs

[0011] to

[0013] , the nucleic acid is mRNA.

[0015] In the pharmaceutical composition of any of paragraphs

[0011] to

[0014] , the nucleic acid is a circular RNA.

[0016] In the pharmaceutical composition of any of paragraphs

[0011] to

[0015] , the pharmaceutical composition is preferentially distributed to a target organ of a subject in need thereof.

[0017] In the pharmaceutical composition of paragraph

[0016] , the target organ is the eye, heart, lung, kidney, liver or spleen of the subject.

[0018] In the pharmaceutical composition of any of paragraphs

[0011] to

[0015] , the pharmaceutical composition is preferentially distributed to liver cells.

[0019] In the pharmaceutical composition of paragraph

[0018] , the liver cells are hepatocytes.

[0020] In the pharmaceutical composition of any of paragraphs

[0011] to

[0015] , the pharmaceutical composition preferentially partitions to spleen cells.

[0021] In the pharmaceutical composition of paragraph

[0020] , wherein the spleen cells are splenocytes.

[0022] In the pharmaceutical composition of any of paragraphs

[0011] to

[0021] , the nucleic acid encodes a biologically active protein or enzyme.

[0023] In the pharmaceutical composition of paragraph

[0022] , the biologically active protein or enzyme is produced in or systemically secreted from liver cells.

[0024] In the pharmaceutical composition of paragraph

[0022] , the biologically active protein or enzyme is produced in or systemically secreted from cells of the lungs, heart or eyes, or the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The NMR spectrum of compound 4a is shown.

[0026] Figure 2 The NMR spectrum of compound E-1-1755 is shown.

[0027] Figure 3 A schematic diagram showing the in vitro cytotoxicity results of E-1-1755 is shown.

[0028] Figure 4 Figure 24 shows the in vitro evaluation of luciferase expression of the circular RNA encoding luciferase. This experiment adopts human embryonic kidney 293T cells (HEK 293T cells), non-small cell lung cancer cell line (A549), human hepatocarcinoma cell line (HepG2) and mouse myoblast cell line (c2c12). The figure is processed by the mean value of the corresponding expression of calculating LNP. The relative value of the expression of LNP is divided by the mean value of the expression of reference 1, to obtain the consistency of data.

[0029] See further Figure 4 , Figure 5 In vitro expression of cationic liposomes with different tail types is shown.

[0030] Figure 6 Figure 2 shows the in vivo expression of cationic liposomes of different tail types. The circular RNA encoding luciferase encapsulated by LNP was systemically administered to female BALB / cJ mice of 6-8 weeks of age by tail vein injection or intramuscular injection. 2 mice / group were injected with a predetermined dose by tail vein injection. 1 mouse / group was injected with a predetermined dose by intramuscular injection into the left and right legs. At 6 hours and 24 hours, mice were placed on an imaging platform. Bioluminescence imaging was performed with an IVIS spectrum, and data were obtained. The figure was processed by calculating the mean value of the corresponding expression of LNP.

[0031] See further Figure 6 , Figure 7 Expression with five cationic liposomes is shown. Detailed Description of the Invention 8.1 Definitions

[0032] To facilitate understanding of the disclosure described herein, a number of terms are defined below.

[0033] Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, biochemistry, biology and pharmacology described herein are those known and commonly used in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art.

[0034] The term "subject" refers to an animal, including but not limited to a primate (e.g., human), cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein, e.g., with reference to a mammalian subject, such as a human subject. In one embodiment, the subject is a human.

[0035] The terms "treat," "treating," and "treatment" are intended to include alleviating or eliminating a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause of the disorder, disease, or condition itself.

[0036] The terms "prevent," "preventing," and "prevention" are intended to include a method of delaying and / or excluding the onset of a disorder, disease, or condition, and / or its attendant symptoms; preventing a subject from acquiring a disorder, disease, or condition; or reducing a subject's risk of developing a disorder, disease, or condition.

[0037] The terms "alleviate" and "alleviating" refer to relieving or alleviating one or more symptoms (e.g., pain) of a disorder, disease, or condition. These terms may also refer to reducing an adverse effect associated with an active ingredient. Sometimes, the beneficial effects a subject experiences from a preventive or therapeutic drug do not cure the disorder, disease, or condition.

[0038] The term "contacting" or "contact" refers to the bringing together of a therapeutic agent and a biomolecule (such as a protein, enzyme, RNA or DNA), cell or tissue so that the contact produces a physiological and / or chemical effect. Contact can be performed in vitro, ex vivo or in vivo. In one embodiment, the therapeutic agent is contacted with a biomolecule in vitro to determine the effect of the therapeutic agent on the biomolecule. In another embodiment, the therapeutic agent is contacted with cells in cell culture (in vitro) to determine the effect of the therapeutic agent on the cell. In yet another embodiment, contacting the therapeutic agent with the biomolecule, cell or tissue comprises administering the therapeutic agent to a subject having the biomolecule, cell or tissue to be contacted.

[0039] The term "therapeutically effective amount" or "effective amount" refers to an amount of a compound that, upon administration, is sufficient to prevent the development of, or alleviate to some extent, one or more symptoms of the disorder, disease, or condition being treated. The term "therapeutically effective amount" or "effective amount" also refers to an amount of a compound that is sufficient to elicit the biological or medical response of a biomolecule (such as a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician.

[0040] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with tissues or organs of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, and commensurate with a reasonable benefit / risk ratio. See, for example, Remington: The Science and Practice of Pharmacy, 23rd ed.; AdejareEd.; Academic Press, 2020; Handbook of Pharmaceutical Excipients, 9th ed.; Sheskey et al., Eds.; Pharmaceutical Press, 2020; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Synapse Information Resources,2007;PharmaceuticalPreformulation and Formulation,1st ed.;Gibson Ed.;CRC Press,2015.

[0041] The term "about" or "approximately" refers to the acceptable error for a particular value as determined by one of ordinary skill in the art, which depends to some extent on how the value is measured or determined. In some embodiments, the term "about" or "approximately" refers to within 1, 2, or 3 standard deviations. In some embodiments, the term "about" or "approximately" refers to within 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0042] As used herein, the terms "a / an" in this specification may refer to one or more. The terms "a / an" used in the claims, when used with the term "comprising", may refer to one or more.

[0043] In some embodiments, "optically active" and "enantiomeric activity" refer to a collection of molecules having an enantiomeric excess of not less than about 80%, not less than about 90%, not less than about 91%, not less than about 92%, not less than about 93%, not less than about 94%, not less than about 95%, not less than about 96%, not less than about 97%, not less than about 98%, not less than about 99%, not less than about 99.5%, or not less than about 99.8%. In some embodiments, the optically active compound comprises about 95% or more of one enantiomer and about 5% or less of the other enantiomer, based on the total weight of the enantiomeric mixture. In some embodiments, the optically active compound comprises about 98% or more of one enantiomer and about 2% or less of the other enantiomer, based on the total weight of the enantiomeric mixture. In some embodiments, the optically active compound comprises about 99% or more of one enantiomer and about 1% or less of the other enantiomer, based on the total weight of the enantiomeric mixture.

[0044] When describing an optically active compound, the prefixes R and S are used to denote the absolute configuration of the compound about its chiral center. The prefixes (+) and (-) are used to denote the compound's optical rotation, which is the direction in which the optically active compound rotates the plane of polarized light. The prefix (-) indicates that the compound is levorotatory, meaning that the compound rotates the plane of polarized light to the left, or counterclockwise. The prefix (+) indicates that the compound is dextrorotatory, meaning that the compound rotates the plane of polarized light to the right, or clockwise. However, the signs of optical rotation (+) and (-) are unrelated to the absolute configuration, R or S, of the compound.

[0045] The term "isotopically enriched" refers to a compound that contains an unnatural proportion of isotopes on one or more atoms comprising the compound. In some embodiments, an isotopically enriched compound contains an unnatural proportion of one or more isotopes, including but not limited to: hydrogen ( 1 H), deuterium ( 2 H), tritium ( 3 H), carbon-11 ( 11 C), carbon-12 ( 12 C), carbon-13 ( 13 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-14 ( 14 O), oxygen-15 (15 O), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), fluorine-18 ( 18 F), phosphorus-31 ( 31 P), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-35 ( 35 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-36 ( 36 Cl), chlorine-37( 37 Cl), bromine-79( 79 Br), bromine-81( 81 Br), iodine-123 ( 123 I), iodine-125( 125 I), iodine-127( 127 I), iodine-129( 129 I) and iodine-131( 131 I). In some embodiments, the isotopically enriched compound is in a stable form, i.e., non-radioactive. In some embodiments, the isotopically enriched compound contains an unnatural proportion of one or more isotopes, including but not limited to: hydrogen ( 1 H), deuterium ( 2 H), carbon-12 ( 12 C), carbon-13 ( 13 C), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), phosphorus-31 ( 31 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-37( 37 Cl), bromine-79( 79 Br), bromine-81( 81 Br) and iodine-127( 127I). In some embodiments, the isotopically enriched compound is in an unstable form, i.e., radioactive. In some embodiments, the isotopically enriched compound contains an unnatural proportion of one or more isotopes, including but not limited to: tritium ( 3 H), carbon-11 ( 11 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), fluorine-18 ( 18 F), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-35( 35 S), chlorine-36 ( 36 Cl), iodine-123 ( 123 I), iodine-125( 125 I), iodine-129( 129 I) and iodine-131( 131 I) It should be understood that in the compounds of the present application, according to the judgment of those skilled in the art, any hydrogen, for example, can be 2 H, or any carbon such as 13 C, or any nitrogen such as 15 N, or any oxygen such as 18 O.

[0046] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably herein to refer to polymers or oligomers of nucleotides of any length. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides (e.g., methylated, hydroxymethylated, or glycosylated), non-natural nucleotides, non-nucleotide building blocks that exhibit similar structures and / or functions to natural nucleotides (i.e., "nucleotide analogs"), and / or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The composition of nucleic acids or polynucleotides can be heterogeneous or homogeneous, can be isolated from natural sources, or can be artificially or synthetically produced. In addition, nucleic acids can be DNA or RNA, or mixtures thereof, and can exist permanently or transiently in single-stranded or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states. Nucleic acid structures also include, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, for example, Braasch and Corey, Biochemistry 2002, 4, 4503-10; and US 5,034,506), locked nucleic acids (LNAs; see, for example, Wahlestedt et al., Proc. Natl. Acad. Sci. USA 2000, 97, 5633-8), cyclohexenyl nucleic acids (see, for example, Wang, Am. Chem. Soc. 2000, 122, 8595-602), and / or ribozymes.

[0047] As used herein, "substantially free" with respect to a particular ingredient means that none of the particular ingredient is intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular ingredient due to any unintentional contamination of the composition is well below 0.1%, preferably below 0.05%, and more preferably below 0.01%. Most preferred are compositions in which the amount of the particular ingredient cannot be detected by standard analytical methods.

[0048] The terms "substantially pure" and "substantially homogeneous" refer to a substance that is sufficiently homogeneous to be free of readily detectable impurities as determined by standard analytical methods used by one of ordinary skill in the art, including but not limited to thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC), gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectrometry (MS); or sufficiently pure such that further purification does not detectably alter the physical, chemical, biological, and / or pharmacological properties of the substance, such as enzymatic and biological activity. In some embodiments, "substantially pure" or "substantially homogeneous" refers to a collection of molecules wherein at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% by weight of the molecules are a single compound, including a single enantiomer, a racemic mixture, or a mixture of enantiomers, as determined by standard analytical methods. As used herein, when an atom at a particular position in an isotopically enriched molecule is designated as a particular less common isotope, the molecule is an impurity relative to the isotopically enriched compound if other isotopes are contained at that particular position. Thus, for a deuterated compound where a particular position is designated as deuterium, a compound containing protium at the same position is an impurity.

[0049] The term "solvate" refers to a complex or aggregate formed by one or more solute molecules (e.g., a compound of the present application) and one or more solvent molecules, which are present in stoichiometric or non-stoichiometric amounts. Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid. In some embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in a crystalline form. In another embodiment, the complex or aggregate is in a non-crystalline form. When the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, hemihydrates, monohydrates, dihydrates, trihydrates, tetrahydrates, and pentahydrates.

[0050] The phrase "enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variation thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof" has the same meaning as the phrases: "(i) an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variation of a compound mentioned herein; (ii) a pharmaceutically acceptable salt, solvate, or hydrate of a compound mentioned herein; or (iii) a pharmaceutically acceptable salt, solvate, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or a hydrate of an isotopic variation of a compound mentioned herein." 8.2 Lipid compounds

[0051] In one embodiment, the present application provides a compound of the formula: or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0052] In some embodiments, the compounds provided herein are isolated or purified. In some embodiments, the purity of the compounds provided herein is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% by weight.

[0053] The compound provided herein is intended to include all possible stereoisomers, unless specific stereochemistry is specified. In the case where the compound of the present application contains alkenyl, the compound can exist as a kind of or mixture of geometric cis / trans (or Z / E) isomers. In the case where structural isomers are mutually convertible, the compound can exist as a mixture of single tautomers or tautomers. This can take the form of proton tautomerism in compounds containing, for example, imino, keto or oxime groups; or the so-called valence tautomeric form in compounds containing aromatic moieties. Therefore, a single compound may show more than one type of isomerism.

[0054] The compounds provided herein may be enantiomerically pure, such as a single enantiomer or a single diastereomer, or may be stereoisomeric mixtures, such as a mixture of enantiomers, for example, a racemic mixture of two enantiomers; or a mixture of two or more diastereomers. Thus, one of ordinary skill in the art will recognize that, for compounds that undergo epimerization in vivo, administration of a compound in its (R) form is equivalent to administration of the compound in its (S) form. Conventional techniques for preparing / isolating individual enantiomers include synthesis from suitable optically pure precursors, asymmetric synthesis from achiral starting materials, or resolution of enantiomeric mixtures, such as chiral chromatography, recrystallization, resolution, formation of diastereomeric salts, or derivatization into diastereomeric adducts followed by separation.

[0055] When the compounds provided herein contain an acidic or basic moiety, they can also be provided as pharmaceutically acceptable salts. See Berge et al., J. Pharm. Sci. 1977, 66, 1-19; Handbook of Pharmaceutical Salts: Properties, Selection, and Use, 2nd ed.; Stahl and Wermuth Eds.; John Wiley & Sons, 2011. In some embodiments, the pharmaceutically acceptable salts of the compounds provided herein are solvates. In some embodiments, the pharmaceutically acceptable salts of the compounds provided herein are hydrates.

[0056] Suitable acids for preparing pharmaceutically acceptable salts of the compounds of the present application include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfonamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glucoheptanoic acid, D-gluconic acid, D-glucuronic acid, L -Glutamic acid, α-ketoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid.

[0057] Suitable bases for preparing pharmaceutically acceptable salts of the compounds of the present application include, but are not limited to, inorganic bases such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, and sodium hydroxide; and organic bases such as primary, secondary, tertiary, and quaternary aliphatic and aromatic amines, including, but not limited to, L-arginine, phenethylbenzylamine, benzophenone, choline, dimethylethanolamine, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)ethanol, Ethanolamine, ethylamine, ethylenediamine, isopropylamine, N-methyl-glucamine, hydroxylamine, 1H-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine. 8.3 Lipid Nanoparticles

[0058] In one embodiment, provided herein are lipid nanoparticles comprising a compound of the present application, e.g., a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0059] In another embodiment, provided herein are lipid nanoparticles comprising: (i) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; (ii) a phospholipid; and (iii) cholesterol.

[0060] In yet another embodiment, provided herein are lipid nanoparticles comprising: (i) a nucleic acid, and (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0061] In yet another embodiment, provided herein are lipid nanoparticles comprising: (i) a nucleic acid; (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (iii) a phospholipid; and (iv) cholesterol.

[0062] In some embodiments, the lipid nanoparticles of the present application further comprise coupled lipids. In some embodiments, the coupled lipids comprise polyethylene glycol (PEG)-coupled lipids. In some embodiments, the coupled lipids are PEG-coupled lipids.

[0063] Thus, in one embodiment, provided herein are lipid nanoparticles comprising: (i) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; (ii) a phospholipid; (iii) cholesterol; and (iv) a PEG-conjugated lipid.

[0064] In another embodiment, provided herein are lipid nanoparticles comprising: (i) a nucleic acid; (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (iii) a phospholipid; (iv) cholesterol; and (v) a PEG-conjugated lipid.

[0065] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is a circular nucleic acid. In some embodiments, the nucleic acid is a circular RNA. In some embodiments, the nucleic acid is a nucleic acid described in the following documents: US2022 / 0177898 A1; or PCT application No. PCT / CN2022 / 095749 filed on May 27, 2022; or PCT / CN2022 / 095949 filed on May 30, 2022, the disclosures of each of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is an antisense oligonucleotide. In some embodiments, the nucleic acid comprises a sequence of any one of SEQ ID NOs: 1-29 and 33-36, or a nucleic acid sequence encoding an amino acid sequence of any one of SEQ ID NOs: 30-32. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is an mRNA having the sequence of SEQ ID NO:36.

[0066] In some embodiments, the phospholipid is dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), 1,2-dielaidylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-dipalmitoylphosphatidyl-N-methylethanolamine, 1,2-dipalmitoylphosphatidyl-N,N-dimethylethanolamine, dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), or a combination thereof.

[0067] In some embodiments, the PEG-coupled lipid is PEG diacylglycerol (DAG), PEG dialkoxypropyl (DAA), PEG-phospholipid, PEG ceramide, or a combination thereof. In some embodiments, the PEG-coupled lipid is 2-(polyethylene glycol)-N,N-ditetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG), PEG-c-DMG, PEG-DSG, PEG-DSPE, or a combination thereof. 8.4 Pharmaceutical Compositions

[0068] In one embodiment, provided herein are pharmaceutical compositions comprising a compound of the present application, e.g., a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and a pharmaceutically acceptable excipient.

[0069] In another embodiment, provided herein is a pharmaceutical composition comprising lipid nanoparticles, wherein the lipid nanoparticles comprise a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; and a pharmaceutically acceptable excipient.

[0070] In yet another embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (ii) a phospholipid; and (iii) cholesterol.

[0071] In yet another embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a therapeutic agent, and (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0072] In yet another embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a therapeutic agent; (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (iii) a phospholipid; and (iv) cholesterol.

[0073] In yet another embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a nucleic acid, and (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0074] In yet another embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a nucleic acid; (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (iii) a phospholipid; and (iv) cholesterol.

[0075] In some embodiments, the pharmaceutical composition of the present application further comprises a coupled lipid. In some embodiments, the coupled lipid comprises a PEG-coupled lipid.

[0076] Thus, in one embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; (ii) a phospholipid; (iii) cholesterol; and (iv) a PEG-conjugated lipid.

[0077] In another embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a nucleic acid; (ii) a compound of formula (I), or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (iii) a phospholipid; (iv) cholesterol; and (v) a PEG-conjugated lipid.

[0078] In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is a circular nucleic acid. In some embodiments, the nucleic acid is a circular RNA. In some embodiments, the nucleic acid is a nucleic acid described in the following documents: US2022 / 0177898 A1; or PCT application No. PCT / CN2022 / 095749 filed on May 27, 2022; or PCT / CN2022 / 095949 filed on May 30, 2022, the disclosures of each of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid is an siRNA. In some embodiments, the nucleic acid is an antisense oligonucleotide. In some embodiments, the nucleic acid comprises a sequence of any one of SEQ ID NOs: 1-29 and 33-36, or a nucleic acid sequence encoding an amino acid sequence of any one of SEQ ID NOs: 30-32. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is an mRNA having a sequence of SEQ ID NO: 36.

[0079] In some embodiments, the phospholipid is dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), 1,2-dielaidylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-dipalmitoylphosphatidyl-N-methylethanolamine, 1,2-dipalmitoylphosphatidyl-N,N-dimethylethanolamine, dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), or a combination thereof.

[0080] In some embodiments, the PEG-coupled lipid is PEG diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide, or a combination thereof. In some embodiments, the PEG-coupled lipid is 2-(polyethylene glycol)-N,N-ditetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG), PEG-c-DMG, PEG-DSG, PEG-DSPE, or a combination thereof.

[0081] In some embodiments, the pharmaceutical composition of the present application is distributed to the target organ of a subject in need. In some embodiments, the pharmaceutical composition of the present application is preferentially distributed to the target organ of a subject in need. In some embodiments, the target organ is the subject's eye, heart, lung, kidney, liver, or spleen. In some embodiments, the target organ is the subject's eye. In some embodiments, the target organ is the subject's heart. In some embodiments, the target organ is the subject's lung. In some embodiments, the target organ is the subject's kidney. In some embodiments, the target organ is the subject's liver. In some embodiments, the target organ is the subject's spleen.

[0082] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0083] In some embodiments, the pharmaceutical composition of the present application is distributed to liver cells. In some embodiments, the pharmaceutical composition of the present application is preferentially distributed to liver cells. In some embodiments, the liver cells are hepatocytes.

[0084] In some embodiments, the pharmaceutical composition of the present application is distributed to spleen cells. In some embodiments, the pharmaceutical composition of the present application is preferentially distributed to spleen cells. In some embodiments, the spleen cells are splenocytes.

[0085] In some embodiments, the nucleic acid encodes a biologically active protein or enzyme. In some embodiments, the biologically active protein or enzyme is produced in hepatocytes and / or systemically excreted from hepatocytes. In some embodiments, the biologically active protein or enzyme is produced in cells of the lungs, heart, eyes, or central nervous system and / or systemically excreted therefrom.

[0086] The pharmaceutical compositions of the present application can be formulated in various dosage forms, including but not limited to oral, parenteral, and topical dosage forms. The pharmaceutical compositions can also be formulated as modified-release dosage forms, including delayed, extended, long-term, sustained, pulsatile, controlled, accelerated, rapid, targeted, programmed release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology, 2nd ed.; Rathbone et al., Eds.; Drugs and the Pharmaceutical Sciences 184; CRC Press: Boca Raton, FL, 2008.

[0087] In one embodiment, the pharmaceutical composition of the present application is formulated as an oral dosage form. In another embodiment, the pharmaceutical composition of the present application is formulated as a parenteral dosage form. In yet another embodiment, the pharmaceutical composition of the present application is formulated as an intravenous dosage form. In yet another embodiment, the pharmaceutical composition of the present application is formulated as an intramuscular dosage form. In yet another embodiment, the pharmaceutical composition of the present application is formulated as a subcutaneous dosage form. In yet another embodiment, the pharmaceutical composition of the present application is formulated as a topical dosage form.

[0088] The pharmaceutical composition of the present application can be provided in unit dosage form or multiple dosage form. Unit dosage form used herein refers to a physically discrete unit suitable for application to a subject, and is individually packaged as known in the art. Each unit dose contains a predetermined amount of active ingredient (such as the compound of the present application) sufficient to produce the desired therapeutic effect, associated with the required pharmaceutical excipients. Examples of unit dosage forms include, but are not limited to, ampoules, syringes, and individually packaged tablets and capsules. Unit dosage forms can be administered in small portions or multiples thereof. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container, administered in a separate unit dosage form. Examples of multiple dosage forms include, but are not limited to, vials, tablet or capsule bottles, or pint or gallon bottles.

[0089] The pharmaceutical composition of the present application can be administered once or repeatedly within certain time intervals. It should be understood that the precise dosage and duration for the treatment can vary with the age, weight and condition of the treated subject, and can be determined empirically using known testing protocols or by inferring from in vivo or in vitro tests or diagnostic data. It is further understood that for any specific individual, a specific administration regimen should be adjusted according to the needs of the subject and the professional judgment of the person administering or supervising the administration of the pharmaceutical composition. 8.4.1 Oral Administration

[0090] The present application pharmaceutical composition for oral administration can be provided for oral administration with solid, semisolid or liquid dosage form.As used herein, oral administration also comprises through cheek, tongue and sublingual administration.Suitable oral dosage form includes but is not limited to: tablet, quick melt, chewable tablet, capsule, pill, strip, lozenge, lozenge, soft pastille, flat capsule, micropill, medicinal chewing gum, bulk powder, effervescent or non-effervescent powder or granule, oral spray, solution, emulsion, suspension, thin slice, spray, elixir and syrup.Except active ingredient, pharmaceutical composition can also contain one or more pharmaceutically acceptable carriers or excipients, include but are not limited to adhesive, filler, diluent, disintegrant, wetting agent, lubricant, glidant, coloring agent, dye migration inhibitor, sweetener, flavoring, emulsifying agent, suspending agent and dispersant, preservative, solvent, non-aqueous liquid, organic acid and carbon dioxide source.

[0091] Binders or granulating agents impart cohesiveness to the tablet to ensure that the tablet remains intact after compression. Suitable binders or granulating agents include, but are not limited to, starches such as corn starch, potato starch, and pregelatinized starch (e.g., STARCH ); gelatin; sugars such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums such as gum arabic, alginic acid, alginates, Irish moss extract, Panwar gum, Ghatti gum, isabgol husk gum, carboxymethyl cellulose, methyl cellulose, polyvinyl pyrrolidone (PVP), larch arabinogalactan, tragacanth gum powder, and guar gum; celluloses, such as ethylcellulose, cellulose acetate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC); and microcrystalline cellulose, such as PH-101, PH-103, PH-105 and RC-581. Suitable fillers include, but are not limited to, talc, calcium carbonate, microcrystalline cellulose, powdered cellulose, dextran, kaolin, mannitol, silicic acid, sorbitol, starch, and pregelatinized starch. The amount of binder or filler in the pharmaceutical compositions of the present application varies depending on the type of formulation and is readily discernible to one of ordinary skill in the art. The binder or filler in the pharmaceutical compositions of the present application may comprise from about 50% to about 99% by weight.

[0092] Suitable diluents include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar. Certain diluents, such as mannitol, lactose, sorbitol, sucrose, and inositol, when present in sufficient amounts, can impart properties to some compressed tablets, i.e., disintegrate in the oral cavity by chewing. Such compressed tablets can be used as chewable tablets. The amount of diluent in the pharmaceutical composition of the present application varies according to the type of preparation and is readily discernible to one of ordinary skill in the art.

[0093] Suitable disintegrants include, but are not limited to, agar; bentonite; celluloses such as methylcellulose and carboxymethylcellulose; wood products; natural sponges; cation exchange resins; alginic acid; gums such as guar gum and Citrus pulp; cross-linked cellulose, such as cross-linked carboxymethyl cellulose; cross-linked polymers, such as cross-linked povidone; cross-linked starch; calcium carbonate; microcrystalline cellulose, such as sodium starch glycolate; polyclinic potassium; starches, such as corn starch, potato starch, tapioca starch, and pregelatinized starch; clay; and algin. The amount of disintegrant in the pharmaceutical composition of the present application varies depending on the type of formulation and is readily discernible to one of ordinary skill in the art. The pharmaceutical composition of the present application may contain from about 0.5% to about 15%, or from about 1% to about 5%, by weight, of a disintegrant.

[0094] Suitable lubricants include, but are not limited to, calcium stearate; magnesium stearate; mineral oil; light mineral oil; glycerin; sorbitol; mannitol; glycols such as glyceryl behenate and polyethylene glycol (PEG); stearic acid; sodium lauryl sulfate; talc; hydrogenated vegetable oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; zinc stearate; ethyl oleate; ethyl laurate; agar; starch; lycopodium powder; and silicon dioxide or silica gel, such as 200 and The amount of lubricant in the pharmaceutical composition of the present application varies with the type of formulation and is readily discernible to one of ordinary skill in the art. The pharmaceutical composition of the present application may contain from about 0.1% to about 5% lubricant by weight.

[0095] Suitable glidants include, but are not limited to, colloidal silicon dioxide, and asbestos-free talc. Suitable colorants include, but are not limited to, any approved, certified water-soluble FD&C dye, water-insoluble FD&C dye suspended on aluminum hydroxide hydrate, and lakes. Lakes refer to combinations of water-soluble dyes adsorbed onto hydrous oxides of heavy metals to form insoluble dyes. Suitable flavoring agents include, but are not limited to, natural flavors extracted from plants (such as fruits), and synthetic mixtures of compounds that produce pleasant tastes, such as mint and methyl salicylate. Suitable sweeteners include, but are not limited to, sucrose, lactose, mannitol, syrups, glycerin, and artificial sweeteners, such as saccharin and aspartame. Suitable emulsifiers include, but are not limited to, gelatin, gum arabic, gum tragacanth, bentonite, and surfactants, such as polyoxyethylene sorbitan monooleate. Polyoxyethylene sorbitan monooleate 80 and triethanolamine oleate. Suitable suspending and dispersing agents include, but are not limited to, sodium carboxymethylcellulose, pectin, tragacanth, Gum arabic, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinyl pyrrolidone. Suitable preservatives include, but are not limited to, glycerin, methyl and propyl parabens, benzoic acid additives, sodium benzoate, and alcohol. Suitable wetting agents include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Suitable solvents include, but are not limited to, glycerin, sorbitol, ethanol, and syrup. Suitable non-aqueous liquids for use in emulsions include, but are not limited to, mineral oil and cottonseed oil. Suitable organic acids include, but are not limited to, citric acid and tartaric acid. Suitable sources of carbon dioxide include, but are not limited to, sodium bicarbonate and sodium carbonate.

[0096] It will be appreciated that many carriers and excipients may serve multiple functions, even within the same formulation.

[0097] The pharmaceutical compositions of the present application for oral administration may be provided as compressed tablets, milled tablets, chewable tablets, fast-dissolving tablets, multilayer compressed tablets, or enteric-coated tablets, sugar-coated tablets, or film-coated tablets. Enteric-coated tablets are compressed tablets coated with a substance that resists the effects of gastric acid but dissolves or decomposes in the intestines, thereby protecting the active ingredient from the acidic environment of the stomach. Enteric coatings include, but are not limited to, fatty acids, fats, phenyl salicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. Sugar-coated tablets are compressed tablets coated with a sugar coating that helps mask unpleasant tastes or odors and protect the tablets from oxidation. Film-coated tablets are compressed tablets covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Film coatings have the same general properties as sugar coatings. Multi-compressed tablets are tablets produced through more than one compression cycle, including layered tablets, press-coated tablets, or dry-coated tablets.

[0098] Tablet dosage forms can be prepared from the active ingredient in powder, crystalline or granular form alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled release polymers, lubricants, diluents and / or colorants. Flavorings and sweeteners are particularly useful in the formation of chewable tablets and lozenges.

[0099] The pharmaceutical compositions of the present application for oral administration can be provided as soft capsules or hard capsules, which can be made of gelatin, methylcellulose, starch or calcium alginate. Hard gelatin capsules, also known as dry-filled capsules (DFCs), consist of two parts, one part sliding on the other, thereby completely encapsulating the active ingredient. Soft elastic capsules (SECs) are a soft, spherical shell, such as a gelatin shell, which is plasticized by adding glycerol, sorbitol or similar polyols. The soft gelatin shell can contain a preservative to prevent microbial growth. Suitable preservatives are the preservatives described herein, including methyl and propyl parabens and sorbic acid. The liquid, semisolid and solid dosage forms of the present application can be encapsulated in capsules. Suitable liquid and semisolid dosage forms include solutions and suspensions of propylene carbonate, vegetable oils or triglycerides. Capsules containing such solutions can be prepared as described in the following documents: U.S. Patents 4,328,245; 4,409,239; and 4,410,545. The capsules may also be coated as known to those skilled in the art to modify or sustain dissolution of the active ingredient.

[0100] The pharmaceutical compositions of the present application for oral administration can be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system in which one liquid is dispersed in another liquid in the form of small globules, which can be oil-in-water or water-in-oil. An emulsion can include a pharmaceutically acceptable non-aqueous liquid or solvent, an emulsifier, and a preservative. A suspension can include a pharmaceutically acceptable suspending agent and a preservative. An alcoholic aqueous solution can include a pharmaceutically acceptable acetal, such as a di(lower alkyl) acetal of a lower alkyl aldehyde, for example, acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxyl groups, such as propylene glycol and ethanol. An elixir is a clear, sweetened, alcoholic aqueous solution. A syrup is a concentrated aqueous solution of a sugar (e.g., sucrose) that may also contain a preservative. For liquid dosage forms, for example, a solution in polyethylene glycol can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier (e.g., water) to facilitate metered administration.

[0101] Other useful liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient and dialkylated mono- or polyalkylene glycols, including 1,2-dimethoxymethane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, and polyethylene glycol-750-dimethyl ether, wherein the numbers 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol. These dosage forms may further contain one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.

[0102] The pharmaceutical composition of the present application for oral administration can also be provided in the form of liposomes, micelles, microspheres or nanosystems, etc. Micellar dosage forms can be prepared as described in US Pat. No. 6,350,458.

[0103] The pharmaceutical compositions of the present application for oral administration can be provided as non-effervescent or effervescent formulations, granules, and powders for reconstitution into liquid dosage forms. Pharmaceutically acceptable carriers and excipients used in non-effervescent granules or powders may include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable carriers and excipients used in effervescent granules or powders may include organic acids and carbon dioxide sources.

[0104] Coloring and flavoring agents can be used in all dosage forms described herein.

[0105] The pharmaceutical compositions of the present application for oral administration can be formulated as immediate-release or extended-release dosage forms, including delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release, and programmed-release dosage forms. 8.4.2 Parenteral Administration

[0106] The pharmaceutical composition of the present application can be administered parenterally by injection, infusion or implantation for local or systemic administration. Parenteral administration as used herein includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intrasynovial, intravesical and subcutaneous administration.

[0107] The pharmaceutical compositions of the present application for parenteral administration can be formulated in any dosage form suitable for parenteral administration, including but not limited to solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and pre-injectable solid forms suitable for solutions or suspensions in liquids. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmacy. See, for example, Remington: The Science and Practice of Pharmacy, supra.

[0108] The pharmaceutical compositions of the present application for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including but not limited to: aqueous carriers, water-miscible carriers, non-aqueous carriers, antimicrobial agents or preservatives to prevent the growth of microorganisms, stabilizers, solubility enhancers, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting agents or emulsifying agents, complexing agents, sequestering agents or chelating agents, cryoprotectants, lyophilization support agents, thickening agents, pH adjusters and inert gases.

[0109] Suitable aqueous carriers include, but are not limited to, water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, glucose and lactated Ringer's injection. Suitable non-aqueous carriers include, but are not limited to, fixed oils of plant origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, medium-chain triglycerides of coconut oil, and palm seed oil. Suitable water-miscible carriers include, but are not limited to, ethanol, 1,3-butylene glycol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerol, N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0110] Suitable antimicrobial agents or preservatives include, but are not limited to, phenol, cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl and propyl parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerol, and glucose. Suitable buffers include, but are not limited to, phosphates and citrates. Suitable antioxidants include those described herein, such as bisulfites and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents include those described herein, such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifiers include those described herein, such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable masking agents or chelating agents include, but are not limited to, EDTA. Suitable pH adjusters include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, including α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cycloglucan, and sulfobutyl ether 7-β-cycloglucan.

[0111] When the pharmaceutical composition of the present application is formulated for multiple dose administration, the multiple dose parenteral formulation must contain an antimicrobial agent at a bacteriostatic or fungistatic concentration. As is known and practiced in the art, all parenteral formulations must be sterile.

[0112] In one embodiment, the pharmaceutical composition for parenteral administration is provided as a ready-to-use sterile solution. In another embodiment, the pharmaceutical composition is provided as a sterile dry soluble product, including a lyophilized powder and a subcutaneous tablet, which is reconstituted with a solvent before use. In yet another embodiment, the pharmaceutical composition is provided as a ready-to-use sterile suspension. In yet another embodiment, the pharmaceutical composition is provided as a sterile dry insoluble product, which is reconstituted with a carrier before use. In yet another embodiment, the pharmaceutical composition is provided as a ready-to-use sterile emulsion.

[0113] The pharmaceutical compositions of the present application for parenteral administration can be formulated as immediate-release or extended-release dosage forms, including delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release, and programmed-release dosage forms.

[0114] The pharmaceutical composition of the present application for parenteral administration can be formulated as a suspension, solid, semisolid or thixotropic liquid and administered as an implanted reservoir. In one embodiment, the pharmaceutical composition of the present application is dispersed in a solid inner matrix, which is surrounded by an outer polymer membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical composition to diffuse through.

[0115] Suitable inner matrices include, but are not limited to, polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicic acid ester copolymers, hydrophilic polymers (such as hydrogels of acrylic acid and methacrylate), collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate. Suitable outer polymer films include, but are not limited to, polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber-epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers, and ethylene / vinyloxyethanol copolymers. 8.4.3 Topical application

[0116] The pharmaceutical compositions of the present application can be topically applied to the skin, orifices or mucous membranes. As used herein, topical administration includes dermal (intra), conjunctival, intracorneal, intraocular, ocular, ear, transdermal, nasal, vaginal, urethral, ​​respiratory and rectal administration.

[0117] The pharmaceutical compositions of the present application can be formulated into any dosage form suitable for topical administration for local or systemic effect, including but not limited to: emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, lavages, sprays, suppositories, bandages, and skin patches. Topical formulations of the pharmaceutical compositions of the present application can also include liposomes, micelles, microspheres, and nanosystems.

[0118] Pharmaceutically acceptable carriers and excipients suitable for use in topical formulations include, but are not limited to, aqueous carriers, water-miscible carriers, non-aqueous carriers, bacteriostatic agents or preservatives to prevent the growth of microorganisms, stabilizers, solubilizers, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyophilization support agents, thickening agents, and inert gases.

[0119] The pharmaceutical composition can also be injected via electroporation, iontophoresis, phonophoresis, sonophoresis, or microneedle or needle-free injection (e.g., POWDERJECT TM and BIOJECT TM ) for local administration.

[0120] The pharmaceutical composition of the present application can be provided as an ointment, cream and gel dosage form. Suitable ointment bases include oily or hydrocarbon bases, including lard, benzoated lard, olive oil, cottonseed oil and other oils, white petrolatum; emulsifiable or absorbable bases, such as water-soluble petrolatum, hydroxystearic acid sulfate and anhydrous lanolin; water-removable bases, such as hydrophilic ointments; water-soluble ointment bases, including polyethylene glycols of different molecular weights; emulsion bases, water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. See, for example, Remington: The Science and Practice of Pharmacy, supra. These bases have an emollient effect, but usually require the addition of antioxidants and preservatives.

[0121] Suitable cream bases can be oil-in-water or water-in-oil. Suitable cream bases can be washable and comprise an oil phase, an emulsifier and an aqueous phase. The oil phase is also referred to as the "inner" phase and is generally composed of vaseline and a fatty alcohol such as cetyl alcohol or stearyl alcohol. The volume of the aqueous phase generally (although not necessarily) exceeds the oil phase and generally contains a wetting agent. The emulsifier in the cream formulation can be a nonionic, anionic, cationic or amphoteric surfactant.

[0122] Gels are semisolid suspension systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout a liquid matrix. Suitable gelling agents include, but are not limited to, cross-linked acrylic acid polymers such as carbomers, carboxypolyalkylenes and Hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a uniform gel, a dispersant such as alcohol or glycerin may be added, or the gelling agent may be dispersed by trituration, mechanical mixing, and / or stirring.

[0123] The pharmaceutical composition of the present application can be administered through the rectum, urethra, vagina or perivalvulva in the form of a suppository, vaginal suppository, strip, ointment or cataplasm, paste, powder, dressing, cream, plaster, contraceptive, ointment, solution, emulsion, suspension, tampon, gel, foam, spray or enema. These dosage forms can be prepared using conventional methods as described in Remington: The Science and Practice of Pharmacy (supra).

[0124] Rectal, urethral, ​​and vaginal suppositories are solid pharmaceutical preparations that are inserted into body orifices. They are solid at room temperature but melt or soften at body temperature, releasing the active ingredient within the orifice. Pharmaceutically acceptable carriers used in rectal and vaginal suppositories include a base or carrier, such as a hardening agent, which, when formulated with the active ingredient, produces a melting point close to body temperature; and antioxidants as described herein, including bisulfites and sodium metabisulfite. Suitable carriers include, but are not limited to, cocoa butter (theobroma oil), glycerol-gelatin, carbowax (polyoxyethylene glycol), spermaceti, paraffin, white wax, and yellow wax, as well as suitable mixtures of mono-, di-, and triglycerides of fatty acids, and hydrogels such as polyvinyl alcohol, hydroxyethyl methacrylate, and polyacrylic acid. Combinations of various carriers may also be used. Rectal and vaginal suppositories can be prepared by compression or molding. Typical weights of rectal and vaginal suppositories are from about 2 g to about 3 g.

[0125] The pharmaceutical compositions of the present application can be ophthalmologically administered in the form of solutions, suspensions, ointments, emulsions, gel-forming solutions, powders for solutions, gels, ophthalmic implants, and implants.

[0126] The pharmaceutical composition of the present application can be administered intranasally or by inhalation to the respiratory tract. The pharmaceutical composition can be provided in the form of an aerosol or solution for delivery using a pressurized container, a pump, a spray, a nebulizer (such as a nebulizer that uses electrohydrodynamics to generate a fine mist) or a nebulizer alone or in combination with a suitable propellant (such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane). The pharmaceutical composition can also be provided as a dry powder for insufflation alone or in combination with an inert carrier such as lactose or a phospholipid; and nasal drops. For intranasal use, the powder can include a bioadhesive, including chitosan or cyclodextrin.

[0127] Solutions or suspensions for use in a pressurized container, pump, spray, atomizer, or nebulizer can be formulated to contain ethanol, aqueous ethanol, or a suitable alternative for dispersing, solubilizing, or prolonging the release of the active ingredient; a propellant as a solvent; and / or a surfactant, such as sorbitan trioleate, oleic acid, or oligolactic acid.

[0128] The pharmaceutical composition of the present application can be micronized to a size suitable for delivery by inhalation, such as about 50 microns or less, or about 10 microns or less. Particles of this size can be prepared using comminution methods known to those skilled in the art, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization or spray drying.

[0129] Capsules, blisters, and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the pharmaceutical composition of the present application; a suitable powder base, such as lactose or starch; and a performance modifier, such as L-leucine, mannitol, or magnesium stearate. Lactose can be anhydrous or present in the form of a monohydrate. Other suitable excipients or carriers include, but are not limited to, dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. The pharmaceutical composition for inhalation / intranasal administration of the present application can further include a suitable flavoring, such as menthol and levomenthol; and / or a sweetener, such as saccharin and saccharin sodium.

[0130] The pharmaceutical compositions of the present application for topical administration can be formulated for immediate release or extended release, including delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. 8.4.4 Modified Release

[0131] The pharmaceutical compositions of the present application can be formulated as modified release dosage forms. As used herein, the term "modified release" refers to a dosage form in which the release rate or release location of the active ingredient is different from that of an immediate release dosage form when administered by the same route. Modified release dosage forms include, but are not limited to, delayed release, extended release, long-term release, sustained release, pulsatile release, controlled release, accelerated release and rapid release, targeted release, programmed release, and gastric retention dosage forms. Pharmaceutical compositions in modified release dosage forms can be prepared using various modified release devices and methods known to those skilled in the art, including, but not limited to, matrix controlled release devices, osmotic controlled release devices, multi-particulate controlled release devices and ion exchange resins, enteric coatings, multi-layer coatings, microspheres, liposomes, and combinations thereof. The release rate of the active ingredient can also be changed by changing the particle size and polymorphism of the active ingredient. 8.4.4.1. Matrix Controlled Release Devices

[0132] The modified-release pharmaceutical composition of the present application can be prepared using a matrix controlled-release device known to those skilled in the art, see, for example, Takada et al. in Encyclopedia of Controlled Drug Delivery, Mathiowitz Ed.; Wiley, 1999; Vol. 2.

[0133] In some embodiments, the modified release pharmaceutical compositions of the present application are formulated using an erodible matrix device, which is a water-swellable, erodible, or soluble polymer, including but not limited to: synthetic polymers and naturally occurring polymers and derivatives, such as polysaccharides and proteins.

[0134] Materials that can be used to form the erodible matrix include, but are not limited to, chitin, chitosan, dextran, and pullulan; agar, gum arabic, karaya gum, locust bean gum, tragacanth gum, carrageenan, Ghatti gum, guar gum, xanthan gum, and scleroglucan; starches such as dextrin and maltodextrin; hydrophilic colloids such as pectin; phospholipids such as lecithin; alginates; propylene glycol alginate; gelatin; collagen; celluloses such as ethylcellulose (EC), methylethylcellulose (MEC), carboxymethylcellulose (CMC), CMEC, hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methylcellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methylcellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC); polyvinyl pyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acid esters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or methacrylic acid Poly(2-hydroxyethyl methacrylate); polylactic acid; copolymers of L-glutamic acid and ethyl L-glutamate; degradable lactic acid-glycolic acid copolymer; poly-D-(-)-3-hydroxybutyric acid; and other acrylic acid derivatives, such as homopolymers and copolymers of butyl methacrylate, methyl methacrylate, ethyl methacrylate, ethyl acrylate, 2-dimethylaminoethyl methacrylate, and (trimethylaminoethyl) methacrylate chloride.

[0135] In some embodiments, the pharmaceutical compositions of the present application are formulated using a non-corrodible matrix device. The active ingredient is dissolved or dispersed in an inert matrix and released after administration primarily by diffusion through the inert matrix. Suitable materials for use as the non-erodible matrix device include, but are not limited to, insoluble plastics such as polyethylene, polypropylene, polyisoprene, polyisobutylene, polybutadiene, polymethyl methacrylate, polybutyl methacrylate, chlorinated polyethylene, polyvinyl chloride, methyl acrylate-methyl methacrylate copolymers, ethylene-vinyl acetate copolymers, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers, ethylene / vinyloxyethanol copolymers, polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, silicone rubber, polydimethylsiloxane, and silicon carbonate copolymers; hydrophilic polymers such as ethylcellulose, cellulose acetate, crospovidone, and cross-linked partially hydrolyzed polyvinyl acetate; and fatty compounds such as carnauba wax, microcrystalline wax, and triglycerides.

[0136] In matrix controlled release systems, the desired release kinetics can be controlled by, for example, the type of polymer used, polymer viscosity, particle size of the polymer and / or active ingredient, the ratio of active ingredient to polymer, and other excipients or carriers in the composition.

[0137] The pharmaceutical composition of the modified release dosage form of the present application can be prepared by methods known to those skilled in the art, including direct tableting, dry granulation or wet granulation followed by tableting, and melt granulation followed by tableting. 8.4.4.2. Osmotic Controlled Release Devices

[0138] The pharmaceutical compositions of the modified release dosage form of the present application can be prepared using an osmotic controlled release device, including but not limited to: a single chamber system, a dual chamber system, asymmetric membrane technology (AMT) and an extruded core system (ECS). Generally speaking, such a device has at least two components: (a) a core containing the active ingredient; and (b) a semipermeable membrane having at least one delivery port that encapsulates the core. The semipermeable membrane controls the flow of water from the aqueous environment of use into the core, thereby causing drug release through extrusion of the delivery port.

[0139] In addition to the active ingredient, the core of the osmotic device optionally includes an osmotic agent that creates a driving force for transporting water from the environment of use into the device core. One class of osmotic agents are water-swellable hydrophilic polymers, also known as "osmopolymers" and "hydrogels." Suitable water-swellable hydrophilic polymers as osmotic agents include, but are not limited to, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate), poly(acrylic acid), polymethacrylic acid, polyvinyl pyrrolidone (PVP), cross-linked PVP, polyvinyl alcohol (PVA), PVA / PVP copolymers, PVA / PVP-copolymers with hydrophobic monomers such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, cross-linked sodium carboxymethylcellulose, carrageenan, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC) and carboxyethylcellulose (CEC), sodium alginate, polycarboxyl, gelatin, xanthan gum and sodium carboxymethyl starch.

[0140] Another class of osmotic agents is osmolytes, which are capable of absorbing water, thereby affecting an osmotic pressure gradient across the surrounding coating barrier. Suitable osmolytes include, but are not limited to, inorganic salts such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium phosphate, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, and sodium sulfate; sugars such as dextrose, fructose, glucose, inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose, and xylitol; organic acids such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid, sebacic acid, sorbic acid, adipic acid, ethylenediaminetetraacetic acid, glutamic acid, p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; and mixtures thereof.

[0141] Osmotic agents with different dissolution rates can be used to affect the initial release rate of the active ingredient from the dosage form. For example, amorphous sugars such as MANNOGEM can be used. TM EZ, provides faster delivery in the first few hours to quickly produce the desired therapeutic effect, and gradually releases the remaining amount to maintain the desired level of therapeutic or preventive effect over a longer period of time. In this case, the active ingredient is released at such a rate to replace the amount of active ingredient metabolized and excreted.

[0142] The core may also include various other excipients and carriers as described herein to enhance the performance of the dosage form or to facilitate stability or processing.

[0143] Materials that can be used to form the semipermeable membrane include various grades of acrylic, vinyl, ether, polyamide, polyester, and cellulose derivatives that are water permeable and water-insoluble at physiologically relevant pH or that are readily rendered water-insoluble by chemical alteration (e.g., cross-linking). Examples of suitable polymers that can be used to form the coating include plasticized, unplasticized, and reinforced cellulose acetate (CA), cellulose diacetate, cellulose triacetate, CA propionate, cellulose nitrate, cellulose acetate butyrate (CAB), ethyl carbamate, CAP, methyl carbamate, CA succinate, trimethylcellulose acetate (CAT), calcium chloroacetate, ethyl oxalate, calcium methanesulfonate, calcium butylsulfonate, calcium p-toluenesulfonate, agar acetate, amylose triacetate, β-glucan acetate, β-glucan triacetate, acetaldehyde dimethyl ester, and the like. Acetate, locust bean gum triacetate, hydroxylated ethylene vinyl acetate, EC, PEG, PPG, PEG / PPG copolymer, PVP, HEC, HPC, CMC, CMEC, HPMC, HPMCP, HPMCAS, HPMCAT, poly(acrylic) acids and esters and poly(methacrylic) acids and esters and copolymers thereof, starch, dextran, dextrin, chitosan, collagen, gelatin, polyolefins, polyethers, polysulfones, polyethersulfones, polystyrene, polyvinyl halides, polyvinyl esters and ethers, natural waxes and synthetic waxes.

[0144] The semipermeable membrane can also be a hydrophobic microporous membrane in which the pores are substantially filled with a gas and are not wetted by aqueous media but are permeable to water vapor, as disclosed in U.S. Patent No. 5,798,119. Such hydrophobic but water vapor permeable membranes are typically composed of hydrophobic polymers such as polyolefins, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylic acid derivatives, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidene fluoride, polyvinyl esters and ethers, natural waxes, and synthetic waxes.

[0145] The delivery port on the semipermeable membrane can be formed by mechanical or laser drilling after the coating is completed. The delivery port can also be formed in situ by etching a water-soluble material plug or a thinner portion of the membrane at the indentation on the core. In addition, the delivery port can be formed during the coating process, as disclosed in U.S. Patent Nos. 5,612,059 and 5,698,220.

[0146] The total amount of active ingredient released and the rate of release can be essentially adjusted by the thickness and porosity of the semipermeable membrane, the composition of the core, and the number, size, and location of the delivery openings.

[0147] The pharmaceutical composition in the form of an osmotic controlled-release dosage form may further comprise additional conventional excipients or carriers as described herein to enhance the performance or processability of the formulation.

[0148] Osmotic controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, supra; Santus and Baker, J. Controlled Release, 1995, 35, 1-21; Verma et al., Drug Dev. Ind. Pharm., 2000, 26, 695-708; Verma et al., J. Controlled Release, 2002, 79, 7-27.

[0149] In some embodiments, the pharmaceutical compositions of the present application are formulated as AMT controlled-release dosage forms, which comprise an asymmetric osmotic membrane coating a core comprising the active ingredient and other pharmaceutically acceptable excipients or carriers. See, for example, U.S. Pat. No. 5,612,059 and WO 2002 / 17918. AMT controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art, including direct compression, dry granulation, wet granulation, and dip coating.

[0150] In some embodiments, the pharmaceutical composition of the present application is formulated as an ESC controlled-release dosage form, which includes a permeable membrane covering a core comprising the active ingredient, hydroxyethylcellulose and other pharmaceutically acceptable excipients or carriers. 8.4.4.3. Multi-element controlled-release devices

[0151] The pharmaceutical compositions of the modified release dosage form of the present application can be prepared as multiparticulate controlled release devices, which include a plurality of particles, microparticles or pellets with a diameter ranging from about 10 μm to about 3 mm, about 50 μm to about 2.5 mm, or about 100 μm to 1 mm. Such multiparticulates can be prepared by methods known to those skilled in the art, including wet and dry granulation, extrusion / spheronization, roller compaction, melt congealing, and by spray coating seed cores. See, for example, Multiparticulate Oral Drug Delivery; Ghebre-Sellassie Eds.; Drugs and the Pharmaceutical Sciences 65; CRC Press: 1994; and Pharmaceutical Palletization Technology; Ghebre-Sellassie Eds.; Drugs and the Pharmaceutical Sciences 37; CRC Press: 1989.

[0152] Other excipients or carriers described herein can be mixed with the pharmaceutical composition to aid processing and formation of multiparticulates. The resulting particles themselves can constitute a multiparticulate dosage form, or can be coated with various film-forming materials, such as enteric polymers, water-swellable polymers, and water-soluble polymers. The multiparticulates can be further processed into capsules or tablets. 8.4.4.4. Targeted delivery

[0153] The pharmaceutical compositions of the present application can also be formulated to target specific tissues, receptors, or other areas in the subject to be treated, including liposome-based, resealed erythrocyte-based, and antibody-based delivery systems. Examples include, but are not limited to, those disclosed in U.S. Patents 6,316,652; 6,274,552; 6,271,359; 6,253,872; 6,139,865; 6,131,570; 6,120,751; 6,071,495; 6,060,082; 6,048,736; 6,039,975; 6,004,534; 5,985,307; 5,972,366; 5,900,252; 5,840,674; 5,759,542; and 5,709,874. 8.5 Products or Kits

[0154] The pharmaceutical compositions of the present application can also be manufactured using packaging materials well known to those skilled in the art. See, for example, U.S. Patents 5,525,907; 5,052,558; and 5,055,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0155] In some embodiments, a kit is provided herein that, when used by a physician, can simplify administration of an appropriate amount of a pharmaceutical composition as an active ingredient provided herein to a subject. In some embodiments, the kit of the present application includes a container and a dosage form of the pharmaceutical composition provided herein.

[0156] The kit of the present application may further include a device for administering the active ingredient. Examples of such devices include, but are not limited to, a syringe, a needle-free syringe drip bag, a patch, and an inhaler. The kit of the present application may also include a condom for administering the active ingredient.

[0157] The kit of the present application may further include a pharmaceutically acceptable carrier that can be used to administer one or more active ingredients. For example, if the active ingredient is provided in solid form and must be reconstituted for parenteral administration, the kit may include a sealed container containing a suitable carrier in which the active ingredient can be dissolved to form a particle-free sterile solution suitable for parenteral administration. Examples of pharmaceutically acceptable carriers include, but are not limited to: aqueous carriers, including but not limited to Water for Injection USP, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible carriers, including but not limited to ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous carriers, including but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. 8.6 Measurement and Analysis 8.6.1. Characterization of Lipid Nanoparticles

[0158] According to the procedure described in Roces et al., Pharmaceutics 2020, 12, 1095 (the disclosure of which is incorporated herein by reference in its entirety), the NANOSYSTEMS from PRECISION NANOSYSTEMS can be used. The lipid nanoparticles (LNPs) of the present application were prepared on a desktop machine.

[0159] The particle size (Z-average diameter), polydispersity index (PDI) and zeta potential (ZP) of the LNPs of the present application can be determined by dynamic light scattering (DLS) using a ZETASIZER NANO ZS equipped with a 633 nm laser and a 173° detection angle according to the procedure described in Roceset al., Pharmaceutics 2020, 12, 1095.

[0160] According to the procedure described in Roces et al., Pharmaceutics 2020, 12, 1095, the surface pKa values ​​of the compounds of the present application in LNPs can be determined by measuring the fluorescence of 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS) in buffer solutions (10 mM HEPES, 130 mM NaCl, 10 mM NH4OAc and 10 mM MES) with a pH range of 3.5 to 9, with an increment of 0.5 pH units.

[0161] INVITROGEN can be used according to the procedure described in Roces et al., Pharmaceutics 2020, 12, 1095. TM QUANT-IT TM RIBOGREEN TMRNA detection kit, used to measure the nucleic acid encapsulation efficiency (EE%) of the LNP of the present application.

[0162] The HPLC retention time of LNPs can be measured using the following procedure. The retention times relative to the retention time of cholesterol are reported in the table below. The HPLC buffer used was a mixture of two solutions (Solution #1 and Solution #2). Solution #1: 80% methanol / 20% 10 mM NH₄HCO₃; Solution #2: 80% methanol / 20% isopropanol. The ratio of the two solutions in the mixture varied over time, as shown in the table below.

[0163] The LCMS results of LNP can be measured using the following procedure. The LCMS method used was derived from two methods (Method A and method B). Method A (5 minutes): Instrument: Agilent 1290 Column: Poroshell 120EC-C18, 2.1*50 mm, 1.9 μm; Oven temperature: 45°C; Flow rate (ml / min): 0.8; Inj V (ul): 0.2; Run time (minutes): 5; Mobile phase: Mobile phase A: H2O (0.01% TFA), Mobile phase B: Acetonitrile (0.01% TFA); Elution gradient:

[0164] Method B (10 minutes): Instrument: Agilent 1290; Column: Poroshell 120EC-C18, 2.1*50 mm, 1.9 μm; Oven temperature: 45°C; Flow rate (ml / min): 0.8; Inj V (ul): 0.2; Run time (minutes): 10; Mobile phase: Mobile phase A: H2O (0.01% TFA), Mobile phase B: acetonitrile (0.01% TFA); Elution gradient:

[0165] NMR measurements of LNPs can be performed using the following procedure. Prepare a concentrated solution of the compound in a deuterated solvent, ensuring that the sample is free of impurities and completely dissolved. Place the sample in the NMR instrument and initiate data acquisition by running the selected pulse sequence. The acquired NMR data is processed using appropriate software, such as NMR data processing software or a specialized NMR analysis package. Instrument: Bruker AVANCE NEO 400 MHz; Solvent: CDCl3; Oven temperature: 45°C; Nuclei: 1H.

[0166] TLC results for LNPs can be measured using the following procedure. Prepare the sample in a solvent and apply a small drop of the sample solution to the starting line of the TLC plate. In the TLC chamber, add the developing solvent to a depth of several millimeters, ensuring that the liquid level is below the starting line. Iodine staining is then used to detect and locate the separated compounds. The developing solvent is methanol / dichloromethane (1 / 20); the iodine stain is iodine-silica gel 100-200 mesh. 8.6.2 Evaluation of Expression Levels and / or Activity of Therapeutic Products

[0167] In some embodiments, the nucleic acid sequences of the present application include expressed sequences encoding therapeutic products. In some embodiments, the expressed sequences encode reporter proteins. In some embodiments, the expressed sequences encode therapeutic proteins.

[0168] In some embodiments, the RNA polynucleotide comprises one expressed sequence.In some embodiments, the polynucleotide comprises more than one expressed sequence, such as 2, 3, 4 or 5 expressed sequences.

[0169] In some embodiments, the RNA polynucleotide encodes a protein composed of subunits encoded by more than one gene. For example, the protein can be a heterodimer, wherein each chain or subunit of the protein is encoded by a separate gene. It is possible to deliver more than one RNA polynucleotide in a transfer vector, and each RNA polynucleotide encodes a separate subunit of the protein. Alternatively, a single RNA polynucleotide can be engineered to encode more than one subunit. In some embodiments, the separate RNA polynucleotide molecules encoding each subunit can be placed in different transfer vectors for administration.

[0170] The level of therapeutic product (such as polypeptide, protein, antibody or enzyme) can be determined by any method known in the art or described herein. In some embodiments, the level of therapeutic product (such as polypeptide, protein, antibody or enzyme) can be determined by assessing (such as quantifying) the RNA transcribed in the lipid nanoparticles of the present invention in a sample from a subject, and the assessment method uses, for example, Northern blot, PCR analysis, real-time PCR analysis, or any other technology known in the art or described herein. In some embodiments, the level of therapeutic product (such as polypeptide, protein, antibody or enzyme) can be determined by assessing (such as quantifying) the mRNA produced by the lipid nanoparticles of the present invention in a sample from a subject. In some embodiments, the level of therapeutic product (such as polypeptide, protein, antibody or enzyme) can be determined by assessing (such as quantifying) the polypeptide or protein expression level of the therapeutic product produced by the lipid nanoparticles of the present invention in a sample from a subject, by using, for example, immunohistochemical analysis, Western blot, ELISA, immunoprecipitation, flow cytometry analysis or any other technology known in the art or described herein.

[0171] In some embodiments, sample can be collected from various tissues or organs of the experimenter producing or suspecting the production of therapeutic products. In some embodiments, sample can be collected from one or more tissues selected from epithelial tissue (for example, skin tissue, gastrointestinal lining), connective tissue (for example blood, fat, bone, tendon), muscle tissue (for example cardiac muscle, smooth muscle, skeletal muscle) and neural tissue (for example brain tissue, spinal cord tissue, neural tissue). In some embodiments, sample can be collected from one or more organs, and the one or more organs are selected from head, neck, brain, eyes, ears, nose, tongue, throat, heart, lung, pancreas, spleen, thyroid, esophagus, stomach, intestine, liver, gall bladder, colon, bladder, kidney, breast, ovary, uterus, testicle, cervix, skin, blood and lymph node and a part thereof. In certain embodiments, experimenter is any experimenter as described herein. In certain embodiments, experimenter is people. In some embodiments, experimenter is the people suffering from disease or illness, for example, therapeutic products participate in the disease or illness that disease or illness occurs, develops and / or manifests.

[0172] In some embodiments, the sample can be a primary cell, a cell line derived from a primary cell, an immortalized cell, or a mixture thereof.

[0173] In some embodiments, the level of therapeutic products is directly measured, i.e., without label or mark. In some embodiments, the level of therapeutic products is indirectly measured, i.e., by label or marker. In some embodiments, therapeutic products are fused with label or marker. Any label or mark for measuring gene / protein expression as known in the art can be used. In some embodiments, label or marker are selected from fluorescent protein (e.g., GFP, BFP, YFP, RFP), enzyme (e.g., luciferase), HA tag, His tag, FLAG tag, Myc tag and combination thereof.

[0174] In certain embodiments, protein levels are determined by methods capable of quantifying the amount of therapeutic product present in a patient tissue sample (e.g., human serum) and / or capable of detecting correction of protein levels following treatment with an RNA polynucleotide, polypeptide, DNA vector, or cell described herein.

[0175] In some embodiments, the level of a therapeutic product in a tissue sample is determined by assessing (eg, quantifying) protein expression of the therapeutic product in the sample using an ELISA, Western blot, or luciferase assay.

[0176] In some embodiments, RNA encoding a reporter protein (e.g., luciferase) is used to determine the in vitro protein expression efficiency of RNA encapsulated in LNP. Briefly, mRNA or circular RNA encoding luciferase is encapsulated in LNPs containing compounds. Cells are seeded into 24-well plates and incubated at 37°C for 24 hours with LNPs encapsulated with mRNA or circular RNA encoding luciferase. Cell lysates and supernatants are collected separately and used Reporter detection systems analyze them in luminescence assays.

[0177] In some embodiments, according to the process described in WO 2017 / 049245A2 or WO 2018 / 081480A1 (its respective disclosed contents are incorporated herein by reference in their entirety), RNA encoding reporter protein (e.g., luciferase) is used to determine the in vivo protein expression efficiency of the RNA encapsulated in LNP. In brief, mRNA or circular RNA encoding luciferase is encapsulated in LNP comprising compound. A predetermined dose of LNP encapsulated with mRNA or circular RNA encoding luciferase is injected into the tail vein, and systemic administration is performed to female C57BL / 6 mice of 6-8 weeks of age. At a predetermined time point (e.g., 4 hours, 6 hours, 12 hours, ... 14 days) after administration, mice are euthanized. The liver, spleen, brain, lung, heart, kidney, pancreas, and muscle tissue of mice are collected, immediately frozen rapidly in liquid nitrogen, and stored at -80 ° C for analysis. Each type of tissue collected is homogenized. According to the manufacturer's protocol, the homogenate was analyzed using the STEADY-GLO luciferase assay system. The amount of the detected protein was determined using the BCA protein assay kit. Relative luminescence units (RLU) can be standardized to the total μg protein detected and compared with a standard curve. In brief, mRNA or circular RNA encoding luciferase was encapsulated in LNPs comprising a compound. LNPs encapsulated with mRNA or circular RNA encoding luciferase were systemically administered to female C57BL / 6 mice of 6-8 weeks of age via tail vein injection with a predetermined dose. At a predetermined time point (e.g., 4h, 6h, 12h, ... 14 days) after administration, mice were euthanized and placed on an imaging platform while continuously administering isoflurane via a nose cone. Bioluminescence imaging was performed using an IVIS Spectrum (Roper Scientific). Mice were imaged 5 minutes after injection with an exposure time of 30 seconds to ensure effective and sufficient signal acquisition.

[0178] In some embodiments, immunogenicity is assessed in 6-week-old female BALB / cJ, C57BL / 6J and / or B6C3F1 / J mice by intramuscular immunization twice with a predetermined dose (e.g., 0.01, 0.1, or 1 μg) of mRNA or circular RNA LNP, with an inoculation interval of 2 weeks or 3 weeks. Serum is collected 2 weeks after the initial immunization and 2 weeks after the booster immunization, and target-specific IgG is assessed by enzyme-linked immunosorbent assay (ELISA). Mouse spleens are also harvested at the endpoint (2 weeks after the booster immunization or 3 weeks after the booster immunization) for immunostaining and flow cytometry. The time points within each dose level are compared by statistical methods (e.g., two-sided Wilcoxon signed rank test), and the doses are compared after the booster immunization (e.g., by Kruskal–Wallis ANOVA and Dunn's multiple comparison test).

[0179] In some embodiments, protective immunity is assessed in young adult BALB / cJ mice stimulated with a predetermined dose of mRNA or circular RNA LNP. A mononuclear single cell suspension is generated from each immunized mouse using a RWD tissue dissociator, followed by 70 μM filtration and density gradient centrifugation using FICO / LITE-LM medium. Splenocytes are cryopreserved for further analysis. Splenocytes are cultured in R10 medium and incubated with mRNA or circular RNA LNP for 6 hours at 37°C prior to T cell flow cytometry analysis. The cells are then incubated with ZOMBIE NIR TM The cells were washed before solution staining. The cells were washed and permeabilized using BD CYTOFIX / CYTOPERM fixation / permeabilization solution according to the manufacturer's instructions. Before staining with the antibody mixture, the cells were washed in permeabilization / washing solution and resuspended in FC BLOCK, wherein the antibody mixture contained one or more antibodies selected from the following: Ia / IE PE, CD4 BV421, CD8a BUV510, CD44 PE-Cy7, CD62L BB515, CD3e AF700, IFN-γ PE / Dazzle, TNF BB700, IL-2BV605, IL-4BV650, and IL-5APC, and 1× permeabilization / washing solution diluted with high-brightness staining buffer. After washing with the permeabilization / washing solution, the cells were resuspended in FC buffer (PBS supplemented with 2% heat-inactivated FBS and 0.05% NaN3) and then detected on a full-spectrum flow cytometer system. Analyses were performed using FLOWJO software. Example

[0180] As used herein, the symbols and conventions used in these procedures, schemes, and examples, whether or not specific abbreviations are specifically defined, are consistent with those used in contemporary scientific literature, for example, the Journal of the American Chemical Society, the Journal of Medicinal Chemistry, or the Journal of Biological Chemistry. Specifically, but not limited to, the following abbreviations may be used in the examples and throughout the specification: g (gram); mg (milligram); mL (milliliter); mL (microliter); mM (millimole); μM (micromole); mmol (millimolar); min (minute or minute); h (hour); Boc (tert-butyloxycarbonyl); TEA (triethylamine); TFA (trifluoroacetic acid); Ts (tosyl); LCMS (liquid chromatography-mass spectrometry); MS (mass spectrometry); NMR (nuclear magnetic resonance); and preparative HPLC (preparative high performance liquid chromatography).

[0181] For all the following examples, standard operations and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are expressed in ° C (degrees Celsius). Unless otherwise specified, all reactions are carried out at room temperature. The synthetic methods shown herein are intended to illustrate applicable chemistry by using specific examples, rather than indicating the scope of this disclosure. The purification of the compound can be carried out by conventional methods in the art, such as extracting the product obtained by the reaction using an organic solvent such as DCM, and concentrating and purifying the extracted organic layer, such as the residue obtained by silica gel column chromatography, optionally eluting with MeOH / DCM to obtain the compound. 8.7.1. Example 1. Preparation of key intermediates Compound 2a was added to a THF solution of compound 1a (30 g), and the mixture was reacted at rt for 20 h to obtain compound 3a; compound 2a was added to a NaOH solution of compound 3a (30 g), and the mixture was reacted at 40° C. for 1 h to obtain compound 4a, wherein the NMR spectrum of compound 4a is as follows: Figure 1 shown. 8.7.2. Example 2. Preparation of Compound E-1-1755 Compound E-1-1755 was prepared as described above. Compound 2 and K2CO3 were added to a solution of compound 1 (6 g) in MeCN, and the reaction was carried out at 80°C to obtain compound 3; HCl and 1,4-dioxane were added to a solution of compound 3 (4.3 g) in DCM, and the reaction was carried out at 20°C to obtain compound 4; Key INT-12, DIEA and MeCN were added to compound 4 (3.0 g), and the reaction was carried out at 60°C for 20 h to obtain compound 5; Pd / C and H2 were added to a solution of compound 5 (2.2 g) in EtOH, and the reaction was carried out at 70°C for 72 h to obtain compound Key INTB57; 2-octyldecanoic acid, EDCI and DMAP were added to a DCM solution of compound Key INTB57 (1.3 g), and the reaction was carried out for 24 h, followed by extraction with DCM. The combined organic layers were concentrated, and the resulting residue was purified by silica gel column chromatography using MeOH / DCM as eluent to obtain compound E-1-1755 (purity: 95%). 1H NMR (400MHz, CDCl3) δ5.64(s,1H),4.74(d,J=5.8Hz,1H),3.99(t,J=6.2Hz,4H),3.60(s,2H),3.48(t,J=4.9Hz,2H),3.30(d,J=4.9Hz,2H),2.71(s, 4H),2.27–2.16(m,2H),1.66–1.46(m,14H),1.35(dd,J=13.8,6.4Hz,4H) ,1.20(d,J=15.1Hz,40H),1.09(d,J=11.3Hz,6H),0.81(t,J=6.7Hz,12H). The preparation of compound Key INT-12 is as follows: Compound 4 and pyridine were added to a DCM solution of compound 4a (3 g), and the mixture was reacted at 0° C. for 2 h to obtain compound Key INT-12. The NMR spectrum of the compound E-1-1755 is as follows Figure 2 shown. 8.7.3. Example B-1. Characterization of lipid nanoparticles

[0182] The PCR products were prepared using a PCR product from PRECISION NANOSYSTEMS according to the procedure described in Roces et al., Pharmaceutics 2020, 12, 1095 (the disclosure of which is incorporated herein by reference in its entirety). Benchtop preparation of lipid nanoparticles (LNPs) containing compounds provided herein.

[0183] The particle size (Z average diameter), polydispersity index (PDI) and zeta potential (ZP) of LNP are determined by dynamic light scattering (DLS) using a ZETASIZER NANO ZS equipped with a 633 nm laser and a 173 ° detection angle according to the process described in Roces et al., Pharmaceutics 2020, 12, 1095. In brief, LNPs were diluted to a LNP concentration of 0.2 mg / mL in filtered (0.22 μm) ultrapure water. The diluted LNPs (1 mL) were then loaded into a 4 mL cuvette for measurement.

[0184] The surface pKa of compound provided herein in LNP, according to the process described in Roces et al., Pharmaceutics 2020,12,1095, is measured by measuring the fluorescence of 2- (p-toluidinyl) -6- naphthalenesulfonic acid (TNS) in the buffer solution of pH 3.5-9 with 0.5 pH unit increments (10mM HEPES, 130mM NaCl, 10mM NH4OAc and 10mM MES). In brief, LNP is diluted to 0.78mM with MILLIQ water, and the diluted LNP (6.4 μL) is added to a black 96-well plate containing pH buffer (233.6 μL) and 25 μM TNS solution (10 μL), and fluorescence is quantified at λem=445nm and λex=321nm. Determine pKa value, which is defined as the pH value at half the maximum fluorescence intensity.

[0185] INVITROGEN was used according to the protocol described in Roces et al., Pharmaceutics 2020, 12, 1095. TM QUANT-IT TM RIBOGREEN TM RNA detection kit to measure the nucleic acid encapsulation efficiency (EE%) of LNPs.

[0186] The nucleic acid encapsulation efficiency (EE%) of LNP is determined using RNA encoding a reporter protein (e.g., luciferase). Exemplary expressed RNAs include, but are not limited to, SEQ ID NOs: 2, 4, 6, 11, 12, 13, 14, 16, 17, 19, 21, 22, 23, 24, 25, 27, 29, and 33-36, or nucleic acid sequences encoding any one of the amino acid sequences in SEQ ID NOs: 30-32. Exemplary circular RNAs include those described in the following PCT applications: PCT / CN2020 / 077026, filed February 27, 2020; PCT / CN2022 / 095749, filed May 27, 2022; and PCT / CN2022 / 095949, filed May 30, 2022; the contents of each of which are incorporated herein by reference in their entirety. Exemplary mRNAs include SEQ ID NO: 36. 8.7.4. Example B-2. In vitro evaluation of protein expression using mRNA or circular RNA

[0187] The in vitro protein expression efficiency of the RNA encapsulated in LNP is determined using RNA encoding a reporter protein (e.g., luciferase). Exemplary expression RNAs include, but are not limited to, SEQ ID NOs: 2, 4, 6, 11, 12, 13, 14, 16, 17, 19, 21, 22, 23, 24, 25, 27, 29, and 33-36, or nucleic acid sequences encoding any one of the amino acid sequences in SEQ ID NOs: 30-32. Exemplary circular RNAs include those described in the following PCT applications: PCT / CN2020 / 077026, filed February 27, 2020; PCT / CN2022 / 095749, filed May 27, 2022; and PCT / CN2022 / 095949, filed May 30, 2022; each of which is incorporated herein by reference in its entirety. Exemplary mRNAs include SEQ ID NO: 36.

[0188] Briefly, mRNA or circular RNA encoding luciferase was encapsulated in LNPs containing a compound provided herein as described in Example B-1. Cells were seeded into 24-well plates and incubated with LNP-encapsulated luciferase encoding mRNA or circular RNA at 37°C for 24 hours. Cell lysates and supernatants were collected and used Reporter assay systems were analyzed in luminescence assays. 8.7.5. Example B-3. mRNA or circular RNA for in vivo assessment of protein expression

[0189] According to the process described in WO2017 / 049245A2 or WO2018 / 081480A1 (the disclosures of each of which are incorporated herein by reference in their entirety), RNA encoding a reporter protein (e.g., luciferase) is used to determine the in vivo protein expression efficiency of RNA encapsulated in LNP. Exemplary expression RNAs include, but are not limited to, SEQ ID NOs: 2, 4, 6, 11, 12, 13, 14, 16, 17, 19, 21, 22, 23, 24, 25, 27, 29, and 33-36, or a nucleic acid sequence encoding any one of the amino acid sequences in SEQ ID NOs: 30-32. Exemplary circular RNAs include those described in the following PCT applications: PCT / CN2020 / 077026, filed February 27, 2020; PCT / CN2022 / 095749, filed May 27, 2022; and PCT / CN2022 / 095949, filed May 30, 2022; the disclosures of each of which are incorporated herein by reference in their entirety. Exemplary mRNAs include SEQ ID NO: 36.

[0190] In brief, mRNA or circular RNA encoding luciferase is encapsulated in LNP, and the LNP comprises the compound provided herein as described in Example B-1. LNPs encapsulated with mRNA or circular RNA encoding luciferase are systemically administered to female C57BL / 6 mice of 6-8 weeks of age by tail vein injection at a predetermined dose. At a predetermined time point (e.g., 4 hours, 6 hours, 12 hours, ... 14 days) after administration, mice are euthanized. The liver, spleen, brain, lung, heart, kidney, pancreas, and muscle tissue of mice are collected, immediately frozen rapidly in liquid nitrogen, and stored at -80 ° C for analysis. Each type of tissue collected is homogenized. According to the manufacturer's protocol, the homogenate is analyzed using the STEADY-GLO luciferase assay system. Using the BCA protein detection kit, the amount of the protein measured is determined. The relative luminescence unit (RLU) can be standardized to the total μg number of the protein measured and compared with the standard curve.

[0191] In brief, mRNA or circular RNA encoding luciferase is encapsulated in LNP, and the LNP includes the compound provided herein as described in Example B-1. The LNP encapsulated with mRNA or circular RNA encoding luciferase is systemically administered to female C57BL / 6 mice of 6-8 weeks of age by tail vein injection at a predetermined dose. At a predetermined time point (e.g., 4h, 6h, 12h, ... 14 days) after administration, mice are euthanized and placed on an imaging platform while continuously inhaling isoflurane through a nose cone. Bioluminescent imaging is performed using IVIS spectroscopy (Roper Scientific). Mice are imaged 5 minutes after injection with an exposure time of 30 seconds to ensure effective and sufficient signal acquisition. 8.7.6. Example B-4. Immunogenicity Assessment of mRNA or CircRNA

[0192] Immunogenicity was assessed in 6-week-old female BALB / cJ, C57BL / 6J, and / or B6C3F1 / J mice by intramuscular immunization with two predetermined doses (e.g., 0.01, 0.1, or 1 μg) of mRNA or circular RNA LNPs, administered 2 or 3 weeks apart. Serum was collected 2 weeks after the primary immunization and 2 weeks after the boost immunization and assessed for target-specific IgG by enzyme-linked immunosorbent assay (ELISA). Spleens were also harvested at endpoint (2 or 3 weeks after the boost immunization) for immunostaining and flow cytometry. Time points within each dose level were compared by statistical methods (e.g., two-sided Wilcoxon signed-rank test) and doses were compared after the boost immunization (e.g., Kruskal–Wallis ANOVA with Dunn's multiple comparison test). 8.7.7. Example B-5. Immunization with mRNA or circular RNA

[0193] Protective immunity was assessed in young adult BALB / cJ mice stimulated with a predetermined dose of mRNA or circular RNA LNPs. Mononuclear single cell suspensions were generated from each immunized mouse using a RWD tissue dissociator, followed by 70 μM filtration and density gradient centrifugation using FICO / LITE-LM medium. Splenocytes were cryopreserved for further analysis. Splenocytes were cultured in R10 medium and incubated with mRNA or circular RNA LNPs at 37°C for 6 hours prior to T cell flow cytometric analysis. They were then cultured in ZOMBIE NIR TMBefore staining with the solution, the cells were washed. According to the manufacturer's instructions, the cells were washed and permeabilized using BD CYTOFIX / CYTOPERM fixation / permeabilization solution. Before staining with the antibody mixture, the cells were washed in permeabilization / washing solution and resuspended in FC BLOCK. The antibody mixture contained one or more antibodies selected from the following: Ia / IE PE, CD4 BV421, CD8a BUV510, CD44 PE-Cy7, CD62L BB515, CD3e AF700, IFN-γ PE / Dazzle, TNF BB700, IL-2BV605, IL-4BV650 and IL-5APC, and 1× permeabilization / washing solution diluted with high-brightness staining buffer. Before running on the full spectrum flow cytometer system, the cells were washed in permeabilization / washing solution and resuspended in FC buffer (PBS supplemented with 2% heat-inactivated FBS and 0.05% NaN3). Analyses were performed using FLOWJO software. 8.7.8. Example B-6. Measurement of apparent pKa of LNP

[0194] According to the process described in Buschmann et al., Communications Biology 2021,4,956, the apparent pKa value of LNP was determined by measuring the fluorescence of 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS) in a buffer (20mM boric acid, 10mM imidazole, 10mM sodium acetate, 10mM glycylglycine, and 25mM NaCl) with a pH value ranging from 3 to 10 (in 0.5 pH unit increments). Briefly, LNP was diluted to 3mM with ethanol, and the diluted LNP (2.5μL) was added to a black 96-well plate containing pH buffer (95.5μL) and 300μM TNS solution (2μL). Fluorescence was quantified at λem=445nm and λex=321nm. The pKa value was determined, which is defined as the pH value at half the maximum fluorescence intensity. In the same way, the pKa of ionizable lipids was detected. 8.7.9. Example B-7. In vitro cytotoxicity assay by ATP luminescence

[0195] ((4-Hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 2-((polyethylene glycol)-2000)-N,N-ditetradecylacetamide (ALC-0159) were purchased from AVITO. cRNA was obtained from Shanghai Huanma Biopharmaceutical Co., Ltd. 2.0 Cell viability assay (Cell Viability Assay) was purchased from Promega. Human embryonic kidney 293T cells (HEK 293T cells), non-small cell lung cancer cell line (A549), human hepatocellular carcinoma (HepG2), and mouse muscle myoblast cell line (C2C12) were purchased from ATCC. Dulbecco's modified Eagle's medium (DMEM), Ham's F-12K (Kaighn's), and fetal bovine serum (FBS) were purchased from ThermoFisher.

[0196] 293T, HepG2, and c2c12 cells were cultured in DMEM supplemented with 10% FBS. A549 cells were cultured in Ham's F12K supplemented with 10% FB. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. One day before co-culture, 1.0×10 4 Cells were seeded in 100 μL of complete cell culture medium. After 24 hours, the original culture medium of the 96-well plate was replaced with new culture medium, and LNPs containing 100 ng of cRNA were added to the 96-well plate. The 96-well plate was centrifuged at 900 rpm for 1 hour and returned to the incubator for culture. After 24 hours, the cells were ready for toxicity assay. Briefly, the original culture medium was discarded. 50 μL of fresh DMEM and 50 μL of CellTiter Reagents were added to each well and chemiluminescence was detected by a Synergy H1 Hybrid Multimode Reader at λ = 560 nm.

[0197] 10×10 4 293t cells were cultured in a cell culture incubator overnight. The next day, 1 μg of the prepared cFluc-LNP was transfected into the cells and co-incubated. After incubation for 4 hours, the medium was changed and the cells were cultured overnight for 20 hours. The cells were then detected by microplate reader for luminescence. The results are shown in Figure 2. Figure 3 shown. 8.7.10. Example B-8. Circular RNA Encoding Luciferase for In Vitro Assessment of Luciferase Expression

[0198] ((4-Hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 2-((polyethylene glycol)-2000)-N,N-ditetradecylacetamide (ALC-0159) were purchased from Avito. Circular RNA was obtained from Shanghai Ringcode Biopharmaceutical Co., Ltd. 2.0 Cell viability assay (Cell Viability Assay) was purchased from Promega. Human embryonic kidney 293T cells (HEK 293T cells), non-small cell lung cancer cell line (A549), human hepatocellular carcinoma (HepG2), and mouse muscle myoblast cell line (C2C12) were purchased from ATCC. Dulbecco's modified Eagle's medium (DMEM), Ham's F-12K (Kaighn's), and fetal bovine serum (FBS) were purchased from ThermoFisher.

[0199] 293T, HepG2, and c2c12 cells were cultured in DMEM supplemented with 10% FBS. A549 cells were cultured in Ham's F12K supplemented with 10% FB. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2. One day before co-culture, 1.0×10 4 Cells were seeded in 100 μL of complete cell culture medium. The cells were cultured in 96-well transparent polystyrene microplates. After 24 hours, the original culture medium of the 96-well plate was replaced with new culture medium, and LNPs containing 100 ng of cRNA were added to the 96-well plate. The 96-well plate was centrifuged at 900 rpm for 1 hour and returned to the incubator for culture. After 24 hours, the cells were ready for firefly luciferase (Fluc) assay. Briefly, the original culture medium was discarded. 100 μL of britelite plus reagent was added to each well, and firefly luciferase was detected at λ = 560 nm using a Synergy H1 hybrid multimode reader.

[0200] The data were processed by calculating the mean of the corresponding expression of LNP. The relative value of LNP expression was divided by the mean of the expression of reference 1 to obtain data consistency. Figure 4-5 . 8.7.11. Example B-9. Animal Experiment

[0201] ((4-Hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 2-((polyethylene glycol)-2000)-N,N-ditetradecylacetamide (ALC-0159) were purchased from Avito. Circular RNA was obtained from Shanghai Huanma Biopharmaceutical Co., Ltd. BALB / cJ mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. All mice were 6-8 weeks old at the time of experiment.

[0202] LNPs encapsulated with circular RNA encoding luciferase were systemically administered to female BALB / cJ mice aged 6-8 weeks via tail vein injection or intramuscular injection. Two mice / group were injected via tail vein at a predetermined dose. One mouse / group was injected intramuscularly into the left and right legs at a predetermined dose. Six and 24 hours after injection, the mice were placed on an imaging platform. Bioluminescence imaging was performed using an IVIS Spectrum (Roper Scientific) and data were acquired.

[0203] Data were processed by calculating the mean of the corresponding LNP expression. The relative value of LNP expression was divided by the mean of the expression of reference 1 to obtain data consistency. The difference in LNP expression was normalized. The data were obtained by dividing the relative value of LNP expression by the maximum relative value of LNP expression. Figure 6-7 . 8.7.12. Example B-10. HPLC

[0204] The HPLC retention time of lipid nanoparticles prepared from each cationic lipid was measured. The lipid nanoparticles were prepared as described in Example B-1. The retention time of each lipid nanoparticle relative to the retention time of cholesterol is reported in the table below.

[0205] The HPLC buffer used was a mixture of two solutions (solution #1 and solution #2).

[0206] Solution #1: 80% methanol / 20% 10 mM NH4HCO3

[0207] Solution #2: 80% methanol / 20% isopropanol

[0208] As shown in the table below, the percentages of the two solutions in the mixture changed over time. 8.7.13. Example B-11. LCMS method

[0209] LCMS analysis results were performed on lipid nanoparticles prepared from each cationic lipid.Lipid nanoparticles were prepared as described in Example B-1.

[0210] The LCMS method used was selected from two methods (Method A and Method B).

[0211] Method A (5 min): Instrument: Agilent 1290 Column: Poroshell 120EC-C18, 2.1*50mm, 1.9um Oven temperature: 45°C Flow rate (ml / min): 0.8 Inj V(ul):0.2 Run time (min): 5 Mobile phase: Mobile phase A: H2O (0.01% TFA), Mobile phase B: Acetonitrile (0.01% TFA) Elution gradient:

[0212] Method B (10 min): Instrument: Agilent 1290 Column: Poroshell 120EC-C18, 2.1*50mm, 1.9um Oven temperature: 45°C Flow rate (ml / min): 0.8 Inj V(ul):0.2 Run time (min): 10 Mobile phase: Mobile phase A: H2O (0.01% TFA), Mobile phase B: Acetonitrile (0.01% TFA) Elution gradient: 8.7.14. Example B-12. NMR Method

[0213] NMR measurements were performed on lipid nanoparticles prepared from each cationic lipid.Lipid nanoparticles were prepared as described in Example B-1.

[0214] Prepare a concentrated solution of the compound in a deuterated solvent, ensuring that the sample is free of impurities and properly dissolved. Place the sample in the NMR instrument and start the data acquisition process by running the selected pulse sequence. Process the acquired NMR data using appropriate software, such as NMR data processing software or a specialized NMR analysis package. Instrument: Bruker AVANCE NEO 400MHz Solvent: CDCl3 Oven temperature: 45°C Nucleus: 1H 8.7.15. Example B-13. TLC method

[0215] In lipid nanoparticles prepared therefrom, TLC results of each cationic lipid were measured.Lipid nanoparticles were prepared as described in Example B-1.

[0216] Prepare the sample in a solvent and apply a small drop of the sample solution to the starting line of the TLC plate. In the TLC chamber, add the developing solvent to a depth of several millimeters, ensuring that the liquid level is below the starting line. Iodine staining is then used to detect and locate the separated compounds. Developing solvent: methanol / dichloromethane (1 / 20) Iodine stain: iodine silica gel 100-200 mesh 9. Sequence Listing

[0217] The sequences described herein are provided in the sequence listing below. Sequence Listing *****

[0218] The above examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the claimed embodiments and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art are intended to fall within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.

Claims

1. A compound of formula (I): or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

2. A lipid nanoparticle comprising: a compound of claim 1, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

3. A pharmaceutical composition comprising: a lipid nanoparticle and a pharmaceutically acceptable excipient; wherein the lipid nanoparticle comprises: (i) a nucleic acid; (ii) a compound of claim 1, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (iii) a phospholipid; and (iv) cholesterol.

4. The pharmaceutical composition of claim 3, further comprising a coupled lipid.

5. The pharmaceutical composition of claim 4, wherein the coupled lipid comprises a polyethylene glycol-coupled lipid.

6. The pharmaceutical composition of any one of claims 3 to 5, wherein the nucleic acid is mRNA.

7. The pharmaceutical composition of any one of claims 3 to 6, wherein the nucleic acid is a circular RNA.

8. The pharmaceutical composition of any one of claims 3-7, wherein the pharmaceutical composition is preferentially distributed to a target organ of a subject in need thereof.

9. The pharmaceutical composition of claim 8, wherein the target organ is an eye, heart, lung, kidney, liver or spleen of a subject.

10. The pharmaceutical composition of any one of claims 3-7, wherein the pharmaceutical composition preferentially partitions into liver cells. The pharmaceutical composition of claim 10 , wherein the liver cells are hepatocytes.

12. The pharmaceutical composition of any one of claims 3-7, wherein the pharmaceutical composition preferentially partitions to spleen cells.

13. The pharmaceutical composition of claim 12, wherein the spleen cells are splenocytes.

14. The pharmaceutical composition of any one of claims 3 to 13, wherein the nucleic acid encodes a biologically active protein or enzyme.

15. The pharmaceutical composition of claim 14, wherein the biologically active protein or enzyme is produced in or systemically secreted from liver cells.

16. The pharmaceutical composition of claim 14, wherein the biologically active protein or enzyme is produced in or systemically secreted from cells of the lungs, heart, eyes, or central nervous system.

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