WO2024183497A1 - Intrathecal delivery formulation and use thereof - Google Patents

Abstract

Provided in the present invention is an intrathecal delivery formulation for treating brain neurodegenerative diseases. The intrathecal delivery formulation comprises lipid nanoparticles, the lipid nanoparticles comprising an intrathecal delivery lipid. The intrathecal delivery formulation avoids the problem that drugs are liable to block by the blood-brain barrier during conventional intravenous injection and leads to low delivery efficiencies, can deliver nucleic acid drugs to the brain, and is universal for ASO, siRNA and mRNA drugs.

Description

An intrathecal delivery preparation and its use Technical Field

The present invention relates to the field of preparations for central nervous system delivery, in particular to an intrathecal administration preparation and use thereof.

Background Art

Nucleic acid drugs can be used to treat protein-related diseases by regulating the expression of target proteins. Since the discovery of mRNA in the early 1960s, after decades of development, antisense oligonucleotides (ASO), siRNA and mRNA nucleic acid treatment strategies have finally shown clinical benefits, and a number of nucleic acid drugs including the new crown vaccine have been approved for marketing worldwide. However, the development of nucleic acid drug delivery vectors has always been one of the core barriers and technical difficulties recognized by the industry. As exogenous substances, nucleic acid drugs may be degraded by nucleases in the blood circulation, and due to their large molecular weight and negative charge, they are difficult to cross the cell membrane to exert their therapeutic effects.

Lipid nanoparticles (LNPs) are one of the effective carriers for safely and efficiently delivering nucleic acid drugs (mRNA, siRNA, etc.) to specific target organs and protecting them from degradation. They have many advantages, such as high encapsulation rate, good cell transfection efficiency, strong tissue penetration, low cytotoxicity and immunogenicity, etc. They have been successfully used in multiple commercial products. Taking the FDA-approved drugs as an example, the mRNA COVID-19 vaccine developed by Moderna and Pfizer-BioNTech, and the siRNA drug Onpattro developed by Alnylam, both use lipid nanoparticle drug delivery systems.

The blood-brain barrier refers to the barrier between the plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between the plasma and cerebrospinal fluid formed by the choroid plexus, which can prevent certain substances from entering the brain tissue from the blood. Due to the large size of lipid nanoparticles, it is difficult to cross the blood-brain barrier if common systemic administration, such as intravenous administration, is used, and the efficacy for central nervous system diseases is very limited. For this reason, some studies have used the method of intrathecal injection of mRNA-LNP, and the expression of related proteins was mainly detected in the dorsal root ganglia after administration (Nabhan, J.F.et al. Intrathecal delivery of frataxin mRNA encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for Friedreich's ataxia. Sci.Rep.2016,6,20019). However, there is still a lack of effective means for the selective delivery of nucleic acid drugs to the brain region using lipid nanoparticles.

In addition, the structures of different types of nucleic acid drugs may be quite different. For example, siRNA usually has only about 20 pairs of nucleotides, while mRNA is a single-stranded nucleotide of thousands of kb in length. The different structures lead to obvious differences in the morphology of LNP encapsulation and the interaction between lipids and drugs. Different LNP carriers need to be developed for different types of nucleic acid drugs, which increases the R&D cycle of nucleic acid drugs. Therefore, there is a need for a universal LNP carrier that can efficiently deliver ASO, siRNA and mRNA (Kloczewiak, M. et al. A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines. Mol. Pharmaceutics 2022, 19, 7, 2022–2031), (Sebastiani, F. et al. Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles. ACS Nano 2021, 15, 4, 6709–6722.).

Summary of the invention

In one aspect, the present invention provides a lipid nanoparticle suitable for intrathecal administration, which comprises intrathecal delivery lipids, structural lipids, neutral lipids, and polymer lipids.

In another aspect, the present invention provides a lipid nanoparticle composition comprising the above-mentioned lipid nanoparticle and a load.

In another aspect, the present invention provides a method for preparing the above-mentioned lipid nanoparticle composition, comprising: mixing the various lipid components and then mixing with a load.

In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned lipid nanoparticle composition and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides use of the lipid nanoparticle composition or the pharmaceutical composition in preparing a drug for treating, diagnosing or preventing a disease.

In a specific embodiment, the above-mentioned disease is a central nervous system disease, preferably a brain neurodegenerative disease, more preferably a brain degenerative disease related to autonomous movement, and further preferably Huntington's disease, ALS (amyotrophic lateral sclerosis) or Parkinson's disease.

In another aspect, the present invention provides use of the lipid nanoparticle composition or the pharmaceutical composition in preparing a drug for delivering a load, wherein the load is selected from one or more therapeutic agents, preventive agents or diagnostic agents.

In another aspect, the present invention provides a method for treating, diagnosing or preventing a disease in a subject, comprising intrathecally administering the above-mentioned lipid nanoparticle composition or the above-mentioned pharmaceutical composition to the subject.

In another aspect, the present invention provides the above-mentioned lipid nanoparticle composition or the above-mentioned pharmaceutical composition for use in treating, diagnosing or preventing a disease.

In another aspect, the present invention provides a method for delivering a load into a subject, comprising intrathecally administering the above-mentioned lipid nanoparticle composition or the above-mentioned pharmaceutical composition to the subject.

In another aspect, the present invention provides the above-mentioned lipid nanoparticle composition or the above-mentioned pharmaceutical composition for delivering a load.

In a specific embodiment, the cargo is selected from one or more of a therapeutic agent, a prophylactic agent or a diagnostic agent; preferably, the therapeutic agent, the prophylactic agent or the diagnostic agent is a nucleic acid.

In more specific embodiments, the nucleic acid is selected from one or more of ASO, RNA or DNA.

In a more specific embodiment, the RNA is selected from one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), micro RNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme; preferably guide RNA (gRNA), CRISPR RNA (crRNA), mRNA, ASO or siRNA.

In a specific embodiment, the intrathecal delivery lipid compound has the general formula (I):

in,

G ₁ , G ₂ , G ₃ or G ₄ are each independently selected from a bond, a C _1-20 alkyl group, a C _2-20 alkenyl group or a C _2-20 alkynyl group;

_G5 or _G6 are each independently selected from a bond or a _C1-8 alkyl group;

_M1 or _M2 are each independently selected from a biodegradable group;

Q is selected from a bond or a biodegradable group;

_R1 or _R2 are each independently selected from _C4-28 alkyl, _C4-28 alkenyl or _C4-28 alkynyl;

R ₃ or R ₄ are each independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

or R ₃ , R ₄ and the N atom to which they are connected together form a 3-14 membered heterocyclic group;

or R ₃ , G ₆ and the atoms to which they are attached together form a 3-10 membered heterocyclic group;

R ₅ , R ₆ , R ₇ or R ₈ are each independently selected from C _1-8 alkyl;

Each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl groups is independently optionally further substituted.

Preferably, the intrathecal delivery lipid compound has the general formula (I), wherein:

G ₁ , G ₂ , G ₃ or G ₄ are each independently selected from a bond, a C _1-20 alkyl, a C _2-20 alkenyl or a C _2-20 alkynyl; the C _1-20 alkyl, C _2-20 alkenyl or C _2-20 alkynyl is optionally substituted with one or more substituents selected from H, -OH, alkyl, hydroxyalkyl, alkoxy, amino, alkylamino, dialkylamino;

_G5 or _G6 are each independently selected from a bond or a _C1-8 alkyl group; the _C1-6 alkyl group is optionally substituted by one or more substituents selected from H, OH, alkyl, hydroxyalkyl, alkoxy, amino, alkylamino, dialkylamino;

_M1 or _M2 is each independently selected from -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -O-, -OC(O)O-, -SC(O)O-, -OC(O)S-, _-NRa- , -C(O) _NRa- , -NRaC(O)-, -NRaC(O)O-, _-OC (O) _{NRa- ,} -NRaC(O) _S- , -SC(O) _{NRa- ,} -NRaC(O)NRa- _, -C(O)-, -OC(S)-, -C(S)O-, -OC(S) _NRa- , _-NRaC (S)O-, -SS- or -S(O) _m- ;

Q is selected from a bond, -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -O-, -OC ₍ O)O-, -SC(O)O-, -OC(O) _S- , -NRb-, -C(O)NRb- _, -NRbC(O)-, -NRbC(O _{) O-} , -OC ₍ O)NRb-, -NRbC(O)S-, -SC(O)NRb- _, -NRbC(O) _NRb-, -C(O)-, _-OC (S)-, -C(S)O-, -OC(S) _NRb- , _-NRaC (S)O-, -SS-, -S(O) _n- , phenyl or pyridinyl; said phenyl or pyridinyl is optionally substituted with one or more substituents selected from H, hydroxy, halogen, cyano, alkyl, hydroxyalkyl, haloalkyl or alkoxy;

R ₁ or R ₂ is each independently selected from C _4-28 alkyl, C _4-28 alkenyl or C _4-28 alkynyl; the C _4-28 alkyl, C _4-28 alkenyl or C _4-28 alkynyl is optionally substituted with one or more substituents selected from H, OH, alkyl, hydroxyalkyl, alkoxy, amino, alkylamino, dialkylamino;

R ₃ or R ₄ are each independently selected from H or C _1-20 alkyl; the C _1-20 alkyl is optionally substituted by one or more substituents selected from H, OH, alkyl, hydroxyalkyl, alkoxy, amino, alkylamino, dialkylamino;

or R ₃ , R ₄ and the N atom to which they are attached together form a 3-14 membered heterocyclic group; or R3 , G6 and the atom to which they are attached together form a 3-10 membered heterocyclic group;

The 3-14 membered heterocyclic group is optionally further substituted by a substituent selected from halogen, cyano, OH, alkyl, hydroxyalkyl, haloalkyl, alkoxy, amino, alkylamino, dialkylamino;

R ₅ , R ₆ , R ₇ or R ₈ are each independently selected from C _1-8 alkyl;

Each _Ra or _Rb is independently selected from H, _C1-20 alkyl or _C3-14 cycloalkyl; the _C1-20 alkyl or _C3-14 cycloalkyl is optionally substituted by one or more selected from H, OH, alkyl, hydroxyalkyl, alkoxy, amino, alkylamino, dioxane substituted by a substituent of an alkylamino group;

m or n is each independently selected from 0, 1 or 2.

Preferably, _G1 and _G3 are both selected from _C2-8 alkyl, and _G2 and _G4 are both selected from a bond; preferably, _G1 and _G3 are both selected from _C5 alkyl, and _G2 and _G4 are both selected from a bond.

Preferably, G ₅ is selected from a bond.

Preferably, _G6 is selected from a bond or _C1-8 alkyl.

Preferably, _M1 or _M2 is each independently selected from -C(O)O-, -OC(O), -C(O)S-, -SC(O)-, -NRaC(O)- or -C(O)NRa-, and Ra is selected from H or _C4-24 alkyl; preferably, _M1 or _M2 is each independently selected from -C(O)O-, -OC(O) or -C(O)S-.

Preferably, Q is selected from a bond, -O-, -OC(O)-, -C(O)O-, -OC(O)O- or -OC(O)NH-, -NHC(O)O-, -NHC(O)NH-, -OC(O)S-, -SC(O)O-; preferably Q is selected from -C(O)O- or -OC(O).

Preferably, R ₁ or R ₂ is each independently selected from C _4-28 alkyl, C _4-28 alkenyl or C _4-28 alkynyl, and the C _4-28 alkyl, C _4-28 alkenyl or C _4-28 alkynyl is optionally substituted with one or more substituents selected from H, hydroxyl or C _2-14 alkyl; preferably R ₁ or R ₂ is each independently selected from C _4-28 alkyl, C _4-28 alkenyl or C _4-28 alkynyl, more preferably C _6-8 alkyl, C _6-8 alkenyl or C _6-8 alkynyl.

Preferably, R ₅ , R ₆ , R ₇ or R ₈ are each independently selected from C _1-3 alkyl; preferably, R ₅ , R ₆ , R ₇ or R ₈ are all methyl.

Preferably, the intrathecal delivery lipid is selected from one or more of the following compounds 1-30, compounds 32-34, compounds 36-37, compounds 39-75, compounds 77-131:

In a preferred embodiment, the intrathecal delivery lipid compound is selected from one or more of compounds 18, 20, 23, 26, 27, 34, 41, 46, 98, 100, 102, 104, 106, 107, 108, 109, 111, 112, 115, 116, 118, 119, 120, 121, 122, 123, 125, 126, 127, and 128, more preferably one or more of compounds 20, 26, 46, 108, 116, 121, 122, and 123.

In a preferred embodiment, the molar percentage of the intrathecal delivery lipid to the total lipid is 40.0-65.0%, preferably 47.5-62.5%.

In a preferred embodiment, the molar percentage of intrathecal delivery lipids to total lipids is 40%, 42.5%, 45%, 47.5%, 47.6%, 50.0%, 52.5%, 55.0%, 57.5%, 60.0%, 62.5% or 65.0%.

In a specific embodiment, the structured lipid is selected from one or more of the following: cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol and campesterol; preferably, the structured lipid is selected from cholesterol and/or β-sitosterol; more preferably, the structured lipid is cholesterol.

In a preferred embodiment, the molar percentage of the structural lipids to the total lipids is 5.0-50.0%, preferably 5.0-47.0%, and more preferably 15.5-47.0%.

In a preferred embodiment, the molar percentage of the structured lipids to the total lipids is 5.0%, 10.0%, 15.0%, 15.5%, 20.0%, 22.0%, 25.0%, 26.5%, 30.0%, 32.0%, 35.0%, 35.6%, 37%, 37.5%, 41%, 45.5%, 46%, 46.5%, 47.0% or 50.0%.

In a specific embodiment, the polymer lipid is a pegylated lipid.

Optionally, the PEGylated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol;

Preferably, the PEGylated lipid comprises a PEG moiety of 1000Da to 20kDa, preferably a PEG moiety of about 1000Da to about 5000Da;

Preferably, the PEGylated lipid is selected from one or more of the following: DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, DSPE-PEG5000, DSPE-PEG2000amine and ALC-0159, preferably DMG-PEG2000 and/or ALC-0159.

In a preferred embodiment, the molar percentage of the PEGylated lipids to the total lipids is 0.5-45.0%, preferably 0.5-41.5%, and more preferably 0.5-2.0%.

In a preferred embodiment, the molar percentage of the PEGylated lipids to the total lipids is 0.5%, 1%, 1.5%, 1.8%, 2% or 41.5%.

In a specific embodiment, the neutral lipid is selected from phosphatidylcholine and/or phosphatidylethanolamine.

Preferably, the phosphatidylcholine is selected from one or more of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

Preferably, the phosphatidylethanolamine is selected from one or more of the following: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleyloxy)propyl))dimethylammonio)ethyl hydrogen phosphate (DOCP), dimyristoylphosphatidylethanolamine (DMPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and dipalmitoylphosphatidylethanolamine (DPPE).

In a preferred embodiment, the molar percentage of the neutral lipids to the total lipids is 1.0-30.0%, preferably 1.0-20.0%, and more preferably 5.0-20.0%.

In a preferred embodiment, the molar percentage of the neutral lipids to the total lipids is 1.0%, 5.0%, 10.0%, 15.0% or 20.0%.

Preferably, the lipid fraction comprises, in molar percentage:

Preferably, the lipid fraction comprises, in molar percentage:

More preferably, the lipid fraction comprises, in molar percentage:

In a specific embodiment, the lipid nanoparticles contain compound 46, DOPE, cholesterol and DMG-PEG2000.

The present invention provides a lipid nanoparticle suitable for intrathecal administration to deliver nucleic acid drugs to the brain, which can successfully deliver nucleic acid drugs to the brain and is commonly used for ASO, siRNA and mRNA drugs, avoiding the problem of low delivery efficiency caused by the drug being easily blocked by the blood-brain barrier under conventional intravenous injection. It is particularly noteworthy that the delivery area of the lipid nanoparticles provided by the present invention not only includes the cerebral cortex area, but also includes the basal ganglia closely related to autonomous motor function, such as the striatum, which is crucial for the treatment of Huntington's disease, ALS, Parkinson's disease and other diseases (Drori, E. et al. Mapping microstructural gradients of the human striatum in normal aging and Parkinson's disease. Sci Adv. 2022, 8(28): eabm1971.), (Tabrizi, S. J. et al. Huntingtin Lowering Strategies for Disease Modification in Huntington's Disease. Neuron. 2019, 101(5): 801-819.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the fluorescence expression of Cy7-labeled ASO encapsulated by lipid nanoparticle 1-1 in Example 3 of the present invention in the corresponding organ parts of mice;

FIG. 2 is a mouse brain of a Cy7-labeled ASO encapsulated by lipid nanoparticles 1-1 in Example 3-A of the present invention. anatomical imaging findings;

Figures 3-1 to 3-4 are the observation results of fluorescence microscopy imaging of the ASO monomer and the Cy3-labeled ASO encapsulated by the lipid nanoparticle 1-1 in Example 3-B of the present invention after staining the mouse brain slices;

FIG4 is the mouse fluorescence real-time quantitative PCR result of ASO encapsulated by lipid nanoparticle 1-1 in Example 3-C of the present invention;

FIG5 shows the fluorescence expression of Cy7-labeled siRNA encapsulated by different lipid nanoparticles 2-1 and 2-2 in Example 5-A of the present invention in the corresponding organ parts of mice;

FIG6 is the observation result of fluorescence microscope imaging of Cy3-labeled siRNA encapsulated by lipid nanoparticles 2-1 in Example 5-B of the present invention after staining in mouse brain slices;

FIG7 is the mouse fluorescent real-time quantitative PCR result of siRNA encapsulated by lipid nanoparticles 2-1 in Example 5-C of the present invention;

FIG8 is the anatomical imaging result of the mouse brain of mRNA of different lipid nanoparticles 3-1 and 3-2 in Example 7-B of the present invention.

DETAILED DESCRIPTION

The present invention is further described below by means of specific examples. The examples described in the present invention are only used to illustrate the present invention and do not limit the scope of the present invention.

Lipid nanoparticles suitable for intrathecal administration include intrathecal delivery lipids, structural lipids, neutral lipids, and polymer lipids.

The "central nervous system" or "CNS" includes all cells and tissues of the brain and spinal cord of vertebrates. It includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bones, cartilage, etc. Each region of the CNS is associated with different behaviors and/or functions. For example, the basal ganglia of the brain are associated with motor functions, especially voluntary movements. The basal ganglia are composed of 6 pairs of nuclei: the caudate nucleus, putamen, globus pallidus, nucleus accumbens, nucleus at the base of the thalamus, and substantia nigra. Although separated by the internal capsule, the caudate nucleus and putamen share cytoarchitectonic, chemical and physiological characteristics, commonly referred to as the striatum.

In a specific embodiment, the central nervous system disease is a brain neurodegenerative disease, preferably a brain degenerative disease related to voluntary movement, more preferably Huntington's disease, ALS (amyotrophic lateral sclerosis) or Parkinson's disease.

The spinal cavity is a space filled with cerebrospinal fluid around the spinal cord, and the space is surrounded by two membranes, the arachnoid and the dura mater. The spinal cavity is a space inside the arachnoid that exists in the two surrounding membranes and is located inside the inner side. Intrathecal administration means administration in the subarachnoid space. The surroundings of the brain and spinal cord are filled with cerebrospinal fluid, and the ventricles inside the brain are also filled with cerebrospinal fluid. The ventricles, the surroundings of the brain and the spinal cavity form a connected space, and the cerebrospinal fluid circulates in the space. Therefore, intraventricular administration and intrathecal administration are both to administer the agent into the cerebrospinal fluid, and usually, intraventricular administration and intrathecal administration are substantially the same route of administration. In addition, the preparation of the present invention can also be administered into the brain parenchyma or the spinal cord parenchyma. When administering intraventricularly or intrathecally, or administering intraparenchymally or intraparenchymally, the injection solution can be administered by intravenous push, or continuous injection such as an injection pump can be used.

"Nucleic acid" refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and hybrid molecules thereof. Examples of nucleic acid molecules include, but are not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), and antisense oligonucleotides (ASOs). Nucleic acids can be further chemotherapeutic. Chemical modification, chemical modification is selected from pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-methylcytosine or a combination thereof. The mRNA molecule contains a protein coding region and may further contain an expression regulatory sequence, typical expression regulatory sequences include but are not limited to a 5' cap (5'cap), a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), a polyadenylic acid sequence (PolyA), and a miRNA binding site.

mRNA includes modified RNA and unmodified RNA. The term "modified mRNA" refers to mRNA comprising at least one chemically modified nucleotide. mRNA may comprise one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using a recombinant expression system, and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, mRNA may comprise nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); a nucleoside analog (e.g., pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methyl oxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose); and/or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds).

"Lipids" refers to a group of organic compounds including, but not limited to, esters of fatty acids, and is characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derivative lipids" such as steroids.

"Lipid nanoparticle" or "LNP" refers to a particle made of lipids and nucleic acids, wherein the nucleic acid is encapsulated in the lipids. When present in the lipid particles of the invention, the nucleic acid is resistant to degradation in aqueous solution using nucleases.

Intrathecal delivery lipids are lipids that can specifically deliver nucleic acid drugs to the central nervous system by intrathecal injection of LNPs containing the lipids. In the present invention, they are ionizable cationic lipids that can exist in a positively charged form or a neutral form depending on the pH value. The ionization of ionizable cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions.

"Structured lipids" refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids, such as steroids. Steroids are compounds with a cyclopentane pyrophenanthrene carbon skeleton. In a specific embodiment, the structured lipids are selected from one or more of the following: cholesterol, sitosterol, coprostanol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol and campesterol; preferably, the structured lipids are selected from cholesterol and/or β-sitosterol; more preferably, the structured lipids are cholesterol.

"Polymer conjugated lipid" refers to a molecule containing a polymer portion and a lipid portion. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid. PEG lipid refers to any complex of polyethylene glycol (PEG) and a lipid. There is no particular limitation on the PEG lipid, as long as it has the effect of inhibiting the aggregation of the lipid nanoparticles of the present invention. Other lipids that can reduce aggregation, such as products of lipid coupling with compounds having uncharged, hydrophilic, steric barrier portions, can also be used.

Optionally, the PEGylated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol;

Preferably, the PEGylated lipid comprises a PEG moiety of 1000Da to 20kDa, preferably a PEG moiety of about 1000Da to about 5000Da;

Preferably, the PEGylated lipid is selected from one or more of the following: DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, DSPE-PEG5000, DSPE-PEG2000amine and ALC-0159, preferably DMG-PEG2000 and/or ALC-0159.

In a specific embodiment, the neutral lipid is selected from phosphatidylcholine and/or phosphatidylethanolamine.

Preferably, the phosphatidylcholine is selected from one or more of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

Preferably, the phosphatidylethanolamine is selected from one or more of the following: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleyloxy)propyl))dimethylammonio)ethyl hydrogen phosphate (DOCP), dimyristoylphosphatidylethanolamine (DMPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and dipalmitoylphosphatidylethanolamine (DPPE).

The RNA is selected from one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), micro RNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme, preferably guide RNA (gRNA), CRISPR RNA (crRNA), mRNA or siRNA.

The intrathecal delivery preparation of the present invention may further include a pharmaceutically acceptable diluent or excipient.

Other definitions

Terminology used in this article

"Treatment" refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such a disorder or condition. As used herein, the noun "treat" refers to the action of the verb treat, the latter being as just defined.

"Subjects" for administration include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

"Disease," "disorder," and "condition" are used interchangeably herein.

As used herein, and unless otherwise indicated, the terms "treatment,""treating,""treating,""treating," and "treatment" include actions that occur while a subject has a particular disease, disorder, or condition, that reduce the severity of, or delay or slow the development of, the disease, disorder, or condition ("therapeutic treatment"), and also include actions that occur before a subject develops a particular disease, disorder, or condition ("prophylactic treatment").

Generally, the "effective amount" of a pharmaceutical composition refers to an amount sufficient to cause a target biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of the pharmaceutical composition of the present invention may vary depending on factors such as the biological target, the pharmacokinetics of the pharmaceutical composition, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes a therapeutically effective amount and a prophylactically effective amount.

Unless otherwise specified, a "therapeutically effective amount" of a pharmaceutical composition as used herein is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of a pharmaceutical composition refers to an amount of a therapeutic agent, when used alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effect of other therapeutic agents.

Unless otherwise specified, a "prophylactically effective amount" of a pharmaceutical composition as used herein is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a pharmaceutical composition refers to an amount of a therapeutic agent that provides a prophylactic benefit in the process of preventing a disease, disorder, or condition when used alone or in combination with other agents. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic agents.

"Combination" and related terms refer to the simultaneous or sequential administration of a pharmaceutical composition of the present invention and other therapeutic agents. For example, a pharmaceutical composition of the present invention can be administered simultaneously or sequentially with other therapeutic agents in separate unit dosage forms, or can be administered simultaneously with other therapeutic agents in a single unit dosage form.

Descriptions related to intrathecal delivery of lipids, including methods for their preparation, can be found in CN 115850104A, the entire contents of which are incorporated into this application by reference.

The RNA sequences and liposomes involved in the present invention are shown in Table 1.

Table 1

Example 1: Synthesis of compounds

Synthesis of compound 20:

The tetrahydrofuran (800 mL) solution of compound 1-1 (100 g, 979 mmol) was cooled to -40°C, and LDA (2M, 490 mL) was slowly added to the solution. After the addition was completed, stirring was continued for 1 hour. At the same temperature, a tetrahydrofuran (100 mL) solution of 1-2 (315 g, 1.37 mol) was added to the reaction system, and the reaction system was stirred overnight. The reaction system was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain a crude product. The crude product was separated and purified by silica gel column to obtain compound 1-3 (115 g). 1H NMR (400MHz, CDCl3): δppm1.06-1.11(m,6H),1.13-1.22(m,2H),1.29-1.39(m,2H),1.42-1.49(m,2H),1.73-1.82(m,2H),3.28-3.40(m,2H),3.55-3 .66(m,3H).

A solution of compound 1-3 (100 g, 398 mmol), TosMIC (TsCH ₂ CN) (38.9 g, 199 mmol) and TBAI (14.7 g, 39.8 mmol) in dimethyl sulfoxide (800 mL) was cooled to 0°C, and sodium hydride (20.7 g, 517 mmol) was slowly added in batches, and the mixture was reacted at room temperature overnight. The reaction system was quenched with a saturated aqueous sodium chloride solution, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain 115 g of crude compound 1-4, which was directly used in the next step without separation and purification.

Add 330 mL of concentrated hydrochloric acid to a solution of the crude compound 1-4 (110 g, 205 mmol) in dichloromethane (880 mL), react at room temperature for 2 hours, and monitor the substrate reaction completely by TLC. The reaction system is quenched with a saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phases are combined and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate to dryness to obtain the crude product. The crude product is separated and purified by a silica gel column to obtain compound 1-5 (30.0 g, 80.9 mmol, yield 39.4%).

TMSOK (11.0 g, 86.4 mmol) was added to a tetrahydrofuran (35.0 mL) solution of compound 1-5 (8.0 g, 21.6 mmol) at room temperature, and the reaction system was heated to 70°C for stirring. TLC monitored the complete consumption of the reaction raw materials. The reaction solution was cooled to room temperature, and the organic solvent was removed by rotary evaporation. 20 mL of water was added to the crude product and extracted with dichloromethane. The aqueous phase was collected, and the pH value of the solution was adjusted to less than 5 with 1M hydrochloric acid, and extracted with dichloromethane. The organic phases were combined and dried with anhydrous sodium sulfate, and the filtrate was collected by filtration and concentrated to obtain compound 1-6 (7.0 g).

1H NMR (400MHz, CDCl ₃ ): δppm 1.03 (s, 12H), 1.08-1.17 (m, 8H), 1.34-1.45 (m, 8H), 2.21 (t, J = 7.2Hz, 4H).

Potassium carbonate (482 mg, 3.48 mmol) was added to compound 1-6 (294 mg, 0.87 mmol) and 1-7 (721 mg, 3.48 mmol) in DMF solution, then the reaction temperature was raised to 60°C for 6 hours, and the reactant 1-6 disappeared completely after monitoring, and then cooled to room temperature, the reaction system was quenched with saturated sodium chloride aqueous solution, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain a crude product, which was purified by silica gel column to obtain compound 1-8 (318 mg).

Compound 1-8 (318 mg, 0.53 mmol) was dissolved in 4.0 mL of methanol, sodium borohydride (30 mg, 0.80 mmol) was added to the reaction system, and the reaction was carried out at room temperature. TLC monitored the complete disappearance of the reactant, and the reaction system was quenched with a saturated sodium chloride aqueous solution, extracted with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain a crude compound 1-9 (242 mg), which was directly used in the next step without purification.

The crude compound 1-9 (242 mg, 0.41 mmol), 1-10 (81.3 mg, 0.62 mmol), EDCI (236 mg, 1.23 mmol), triethylamine (0.17 mL, 1.23 mmol) and DMAP (50 mg, 0.41 mmol) were dissolved in 5.0 mL of dichloromethane, and the reaction solution was stirred at room temperature for 12 hours. The reaction solution was quenched with a saturated sodium chloride aqueous solution, extracted with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by a rotary evaporator to obtain a crude product, which was purified by preparative high performance liquid chromatography to obtain compound 20 (130 mg).

¹ H NMR (400MHz, CDCl ₃ ): δppm 0.89 (t, J = 7.2Hz, 6H), 1.15 (s, 12H), 1.27 (m, 34H), 1.47 (m, 8H), 1.51 (m, 6H) ,1.79(m,2H),2.23(s,6H),2.33(m,4H),4.04(t,J＝6.8Hz,4H),4.85(m,1H); ESI-MS m/z:710.7[ M+H] ⁺ .

Synthesis of compound 26:

Compound 2-5 was prepared by referring to the synthesis method of compound 20 to obtain 300 mg of an oily product.

¹ H NMR (400MHz, CDCl ₃ ): δppm 0.87 (t, J=7.2Hz, 6H), 1.16 (s, 12H), 1.20-1.39 (m, 28H), 1.45-1.54 (m, 12H), 1.74- 1.82(m,2H),2.12-2.35(m,14),4.63(t,J＝2.4Hz,4H),4.79-4.88(m,1H); ESI-MS m/z:730.6[M+H] ⁺ .

Compound 2-5 (300 mg, 0.41 mmol) and quinoline (106 mg, 0.82 mmol) were dissolved in 3.0 mL of ethyl acetate. The air in the reaction system was exchanged with nitrogen at room temperature for 2 to 3 minutes. Then, Lindela catalyst (16.9 mg) was added. Hydrogen was introduced into the reaction solution and gas exchange was performed for 2 to 3 minutes. The reaction system was kept under a hydrogen atmosphere (15 psi) and stirred at room temperature for 30 minutes. LC-MS monitored the complete disappearance of the reactants. The reaction solution was filtered, the filter cake was rinsed with ethyl acetate 3 to 4 times, the ethyl acetate was collected and combined, and the organic solvent was removed by a rotary evaporator to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography to obtain compound 26 (31.3 mg).

¹ H NMR (400MHz, CDCl ₃ ): δppm 0.81 (t, J = 7.2Hz, 6H), 1.08 (s, 12H), 1.15-1.28 (m, 32H), 1.38-1.44 (m, 8H), 1.70- 1.79(m,2H),2.01(m,4H),2.15(s,6H),2.16-2.28(m,4H),4.54(d,J＝12.0Hz,4H),4.75(m,1H),5.39 -5.59(m,4H); ESI-MS m/z:734.6[M+H] ⁺ .

Synthesis of compound 46:

Potassium carbonate (1.55 g, 11.2 mmol, 4.0 eq.) was added to a DMF solution of compound 1-6 (959 mg, 2.8 mmol, 1.0 eq.) and 3-1 (638 mg, 3.08 mmol, 1.1 eq.), and then the reaction temperature was raised to 60°C for 4 hours, cooled to room temperature, the reaction system was quenched with a saturated sodium chloride aqueous solution, extracted with ethyl acetate, the organic phases were combined, and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain a crude product, which was purified by silica gel column to obtain compound 3-2 (682 mg).

Compound 3-2 (324 mg, 0.69 mmol, 1.0 eq.) was dissolved in 5.0 mL of dichloromethane, and the reaction system was cooled to 0°C in an ice bath, 2 drops of DMF were added, and then oxalyl chloride (0.24 mL, 2.8 mmol, 4.0 eq.) was added dropwise to the reaction solution. After the addition was completed, the ice bath was removed and stirred at room temperature for 1 hour. The solvent was removed by a rotary evaporator to obtain an oily crude acyl chloride (309 mg), which was directly used in the next step.

3-3 (407 mg, 1.9 mmol, 3.0 eq) was added to a solution of crude acyl chloride (309 mg) in DCE (3.0 mL), and the reaction was heated to 70°C overnight. The reaction solution was cooled to room temperature, and the solvent was removed by a rotary evaporator to obtain a crude product, which was purified by a silica gel column to obtain compound 3-4 (325 mg).

Compound 3-4 (325 mg) was dissolved in 4.0 mL of methanol, sodium borohydride (28 mg, 0.73 mmol) was added to the reaction system, and the reaction was allowed to proceed at room temperature. TLC monitored the complete disappearance of the reactant, and the reaction system was quenched with a saturated aqueous sodium chloride solution, extracted with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain a crude compound 3-5 (260 mg), which was used directly in the next step without purification.

The crude compound 3-5 (260 mg, 0.39 mmol), 1-10 (77.4 mg, 0.59 mmol), EDCI (224 mg, 1.17 mmol), triethylamine (0.16 mL, 1.17 mmol) and DMAP (48 mg, 0.39 mmol) were dissolved in 5.0 mL of dichloromethane, and the reaction solution was stirred at room temperature for 12 hours. The reaction solution was quenched with a saturated sodium chloride aqueous solution, extracted with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by a rotary evaporator to obtain a crude product, which was purified by preparative high performance liquid chromatography to obtain compound 46 (32.6 mg).

¹ H NMR (400MHz, CDCl ₃ ): δppm 0.88 (t, J=7.2Hz, 9H), 1.14 (s, 12H), 1.15-1.28 (m, 37H), 1.47-1.59 (m, 18H), 1.75- 1.84(m,2H),2.24-2.35(m,10H),3.95(d,J＝5.6Hz,2H),4.03(t,J＝6.8Hz,2H),4.80-4.87(m,1H); ESI -MS m/z:780.7[M+H] ⁺ .

Synthesis of compound 108:

Compound 1-6 (448 mg, 1.3 mmol) was dissolved in 5.0 mL of dichloromethane and the reaction system was cooled to 40°C in an ice bath. At 0°C, DMF (10 μL, 0.13 mmol) was added, and then oxalyl chloride (0.44 mL, 5.2 mmol) was added dropwise to the reaction solution. After the addition was complete, the ice bath was removed and stirred at room temperature for 1 hour. The solvent was removed by rotary evaporator to obtain an oily crude acyl chloride (330 mg), which was directly used in the next step.

1-Nonanethiol 4-1 (418 mg, 2.61 mmol) was added to a DCE (3.0 mL) solution of the crude acyl chloride (330 mg, 0.87 mmol), and the reaction was heated to 70°C overnight. The reaction solution was cooled to room temperature, and the solvent was removed by a rotary evaporator to obtain a crude product, which was purified by a silica gel column to obtain compound 4-2 (400 mg). 1HNMR (400 MHz, CDCl ₃ ): δ ppm.

Compound 4-2 (288 mg, 0.46 mmol) was dissolved in 3.0 mL methanol, and NaBH4 (52.5 mg, 1.38 mmol) was added in batches. The reaction solution was stirred at room temperature for 2 hours under a nitrogen atmosphere. TLC monitoring showed that the reaction raw materials completely disappeared. Saturated ammonium chloride solution was added to the reaction solution to quench, and the mixture was extracted with ethyl acetate. The organic phases were combined and dried with anhydrous sodium sulfate. The filtrate was collected by filtration and concentrated to obtain 288 mg of crude compound 4-3. It was used directly in the next step without further purification.

The crude compound 4-3 (288 mg, 0.46 mmol), 4-4 (108 mg, 0.69 mmol), EDCI (264.5 mg, 1.38 mmol), triethylamine (0.19 mL, 1.38 mmol) and DMAP (56.2 mg, 0.46 mmol) were dissolved in 8.0 mL of dichloromethane, and the reaction solution was stirred at room temperature until the reaction raw material 4-3 was completely consumed, and the reaction solution was quenched with a saturated sodium chloride aqueous solution, extracted with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by a rotary evaporator. The crude product was purified by preparative high performance liquid chromatography to obtain compound 108 (67.3 mg).

¹ H NMR (400MHz, CDCl ₃ ): δppm 0.88 (t, J=6.80Hz, 6H), 1.18 (s, 12H), 1.20-1.39 (m, 38H), 1.40-1.62 (m, 14H), 1.66- 1.86(m,3H),1.89-2.10(m,2H),2.19-2.27(m,3H),2.28(br s,2H),2.79-2.83(m,4H),4.79-4.88(m,1H) ;ESI-MS m/z:768.5[M+H] ⁺ .

Example 2: Delivery of LNPs loaded with oligonucleotide chains (ASOs) to the brain via intrathecal injection

1. Lipid Nanoparticle Assembly:

The materials used for lipid nanoparticle assembly are: (1) intrathecal delivery lipids: such as the ionizable liposomes designed and synthesized by the present invention, Compound 20, Compound 26, Compound 46 and Compound 108; (2) structural liposomes: such as cholesterol (purchased from AVT); (3) neutral lipids: such as DSPC, which is 1,2-distearoyl-SN-glycero-3-phosphocholine (distearoylphosphatidylcholine, purchased from AVT); (4) polymer lipids: such as DMG-PEG2000, which is dimyristoylglycerol-polyethylene glycol 2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, purchased from AVT); (5) nucleic acid parts: such as Cy7-ASO, Cy3-ASO. The names and structural formulas of lipid nanoparticle assembly materials are shown in Table 1.

The method for preparing lipid nanoparticles comprises: (1) dissolving one or more of the intrathecal delivery lipids, structured liposomes, such as cholesterol, neutral lipids, such as phospholipids, and polymer lipids, such as polymer lipid PEGylated lipids, in ethanol in sequence; (2) adding HTT Cy7-ASO or LRRK2 Cy3-ASO is dissolved in 25mM sodium acetate solution (pH=4.5); (3) an automated high-throughput microfluidic system is used to mix the organic phase containing the lipid mixture and the aqueous phase containing the ASO component at a flow rate ratio of 1:1 to 1:4, and the mixing speed is between 10mL/min and 18mL/min to prepare lipid nanoparticles; (4) the prepared lipid nanoparticles are diluted with phosphate buffered saline, and the nanoparticle solution is subjected to a first ultrafiltration to the original preparation volume using an ultrafiltration tube (purchased from Millipore); (5) the nanoparticles obtained by the first ultrafiltration are sterilized by filtration through a 0.2μm sterile filter membrane and stored at low temperature in a sealed glass bottle; (6) the lipid nanoparticles after filtration and sterilization are continued to be subjected to a second ultrafiltration to the nanoparticle solution using an ultrafiltration tube (purchased from Millipore) to one-tenth to one-third of the original preparation volume. The preparation method of lipid nanoparticles includes a microfluidic mixing system, but is not limited to this method, and also includes a T-type mixer and ethanol injection method, etc.

The composition of lipid nanoparticles loaded with HTT Cy7-ASO and LRRK2 Cy3-ASO is shown in Table 2:

Table 2

Example 3 Animal Experiment

The experimental mice were SPF-grade BALB/c female mice, 6-8 weeks old, weighing 18-22 g, purchased from Beijing Sibeifu Biotechnology Co., Ltd. All animals were adaptively raised for more than 7 days before the experiment. During the experiment, they were free to eat and drink, with 12/12h light and dark alternation, indoor temperature of 20-26°C, and humidity of 40-70%. The mice were randomly divided into groups, with three or more mice in each group.

The Cy7-ASO lipid nanoparticles 1-1 prepared by the method described in Example 2 were used as intrathecal delivery preparations and injected into mice by intrathecal injection at a single dose of 0.25 mg/kg ASO. The intrathecal injection method was as follows:

1) Shave the mouse from the tail to the head to visualize the spine of the mouse;

2) Aspirate the drug into a 25 μL microsyringe;

3) Spray 75% alcohol on the back of the mouse to visualize the spinal segments, pinch the hip bone of the mouse with two fingers to make the spine protrude, and insert the needle into the gap between the L5 and L6 joints of the mouse spine. Polarization of the mouse's tail indicates that the needle insertion is successful;

4) Inject the drug into the mouse spine with an injection volume between 5-15 μL.

Three hours after administration, the mice were subjected to in vivo bioluminescence detection using a small animal in vivo imaging system (IVIS LUMINA III, purchased from PerkinElmer) to obtain the fluorescence status of their brains.

The specific operation steps of the detection are as follows: placing the mouse in an anesthesia box and anesthetizing it with 2.5% isoflurane, placing the anesthetized mouse in IVIS, performing fluorescence imaging, and collecting and analyzing data on the part where the fluorescence is concentrated.

Cy7-labeled ASO monomers without lipid nanoparticle encapsulation were used as controls. The horizontal axis represents different lipid nanoparticles, and the vertical axis represents the total fluorescence intensity. Each column represents the average value of the fluorescence expression in the corresponding organ part of 3 mice (n=3). The test results are shown in Figure 1 and Table 3.

Table 3

The results showed that the fluorescence signal of Cy7-labeled ASO encapsulated by lipid nanoparticles 1-1 in the brain was stronger than that of unencapsulated ASO monomer.

3-A Brain anatomical imaging results

Mice with fluorescent signals expressed in their brains were dissected at 6 h, and the brains were taken for imaging. The results are shown in Figure 2.

The results showed that Cy7-labeled ASO encapsulated in lipid nanoparticles 1-1 had a stronger fluorescence signal in the brain than ASO monomers. And unlike ASO monomers, which mostly accumulate in the hindbrain, Cy7-labeled ASO delivered by lipid nanoparticles is distributed in almost all areas of the brain, with a wider delivery range.

3-B Fluorescence microscope imaging results

Brain sections of mice injected with Cy3-ASO were stained 24 hours later. DAPI dye mainly stained the nucleus to show the location of the cells. Cy3 has red fluorescence and does not require additional staining. After staining, the sections were observed under a fluorescence microscope. As shown in Figures 3-1 to 3-4, it can be clearly observed that the use of lipid nanoparticles 1-1 not only delivered Cy3-labeled ASO to the cells of the mouse cerebral cortex, but also detected Cy3-labeled ASO in deep brain regions such as the striatum and hippocampus. However, ASO not delivered using lipid nanoparticles was not detected in the striatum.

3-C fluorescence real-time quantitative PCR results

After successful delivery, ASO will knock out the target mRNA sequence. By measuring the target mRNA content in the brain, the lower the mRNA content, the better the delivery effect. Figure 4 and Table 4 show the extent to which the target mRNA in the brain is knocked out when lipid nanoparticles 1-1 and ASO monomers are delivered. The lower the value, the higher the knockout efficiency and the better the delivery efficiency. The results show that the ASO delivery efficiency in lipid nanoparticles 1-1 is significantly better than that of ASO monomers at the same dose.

Table 4

Example 4: Delivery of LNPs loaded with small interfering RNA (siRNA) to the brain via intrathecal injection

1. Lipid Nanoparticle Assembly:

Preparations 2-1 to 2-8 were prepared according to the method of Example 2.

The composition of lipid nanoparticles loaded with HTT Cy7-siRNA is shown in Table 5:

Table 5

Example 5 Animal Experiment

5-A In vivo imaging results

The experimental method is as in Example 3, and the results are shown in Figure 5 and Table 6. It can be seen that when different lipid nanoparticles 2-1 and 2-2 are used as intrathecal delivery preparations for experiments, the Cy7-labeled siRNAs encapsulated therein all have fluorescent signals in the brain, and the signals are significantly stronger than those of siRNA monomers.

Table 6

5-B Fluorescence microscope imaging results

Brain sections of mice injected with Cy3-siRNA were stained 24 hours later. DAPI dye mainly stained the nucleus to show the location of cells. Cy3 has red fluorescence and does not require additional staining. After staining, the cells were observed under a fluorescence microscope. As shown in Figure 6, it can be clearly observed that lipid nanoparticles delivered Cy3-labeled siRNA to mouse cerebellar cells, while siRNA monomers could not be delivered to the corresponding locations.

5-C fluorescence real-time quantitative PCR results

After successful delivery, siRNA will knock out the target mRNA sequence. By measuring the target mRNA content in the brain, the lower the mRNA content, the better the delivery effect; Figure 7 and Table 7 show the extent to which the target mRNA in the brain is knocked out when lipid nanoparticles encapsulate siRNA and siRNA monomers are delivered; the lower the value, the higher the knockout efficiency, and the better the delivery efficiency. The results show that the siRNA delivery efficiency of all lipid nanoparticles is significantly better than that of siRNA monomers at the same dose.

Table 7

Example 6 Delivery of LNPs loaded with mRNA to the brain by intrathecal injection

1. Lipid Nanoparticle Assembly:

Preparations 3-1 and 3-2 were prepared according to the method of Example 2.

The composition of lipid nanoparticles encapsulating Luciferase mRNA is shown in Table 8:

Table 8

Example 7 Animal Experiment

7-A In vivo imaging results

The experimental method is as shown in Example 3, and the results are shown in Table 9. It can be seen that when different lipid nanoparticles are used as intrathecal delivery preparations for experiments, the Luciferase mRNA encapsulated therein has luminescent signals in the brain, and the signals are significantly stronger than the Luciferase mRNA monomer.

Table 9

7-B Brain anatomical imaging results

The mice expressing luminescent signals in the brain were dissected after 6 hours, and the brains were taken for imaging. As shown in Figure 8, the Luciferase mRNA in the lipid nanoparticles had a stronger luminescent signal in the brain than the unencapsulated Luciferase mRNA. The preparations can successfully deliver Luciferase mRNA to the mouse brain.

Example 8 Optimization of formulation for delivery of mRNA

Based on preparation 3-1, 6 formulations were selected based on expert experience, and preparations of different proportions were prepared according to the method of Example 2, and the intrathecal delivery efficiency of these formulations was measured according to the method of Example 3. AI modeling was performed based on the measured results, new prescriptions were recommended, and iterative screening began. During the screening process, the AI model recommended 3-5 formulations in each round based on the accumulated experimental formulation data currently available. After the intrathecal delivery efficiency was measured experimentally, the data was fed back to the AI model. After 7 rounds of iterations, a formulation that met expectations was found. The composition prescriptions and in vivo delivery results of lipid nanoparticles encapsulating Luciferase mRNA used in each round of screening are shown in Table 10. The results show that according to this experimental-AI model iterative screening, the formulation prescription obtained can achieve a delivery efficiency equivalent to 3.9 times that of the prescription before screening.

Table 10

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims (16)

A lipid nanoparticle suitable for intrathecal administration comprises intrathecal delivery lipids, structural lipids, neutral lipids and polymer lipids. The lipid nanoparticle according to claim 1, wherein the intrathecal delivery lipid has the general formula (I):
in, G ₁ , G ₂ , G ₃ or G ₄ are each independently selected from a bond, a C _1-20 alkyl group, a C _2-20 alkenyl group or a C _2-20 alkynyl group; _G5 or _G6 are each independently selected from a bond or a _C1-8 alkyl group; _M1 or _M2 are each independently selected from a biodegradable group; Q is selected from a bond or a biodegradable group; _R1 or _R2 are each independently selected from _C4-28 alkyl, _C4-28 alkenyl or _C4-28 alkynyl; R ₃ or R ₄ are each independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; or R ₃ , R ₄ and the N atom to which they are connected together form a 3-14 membered heterocyclic group; or R ₃ , G ₆ and the atoms to which they are attached together form a 3-10 membered heterocyclic group; R ₅ , R ₆ , R ₇ or R ₈ are each independently selected from C _1-8 alkyl; Each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl groups is independently optionally further substituted; Preferably, the intrathecal delivery lipid is selected from one or more of the following compounds 1-30, compounds 32-34, compounds 36-37, compounds 39-75, compounds 77-131:











The lipid nanoparticle according to claim 1, wherein the intrathecal delivery lipid is selected from one or more of compound 20, compound 26, compound 46, and compound 108. The lipid nanoparticles according to any one of claims 1 to 3, wherein the structured lipids are selected from one or more of the following: cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol and campesterol; preferably, the structured lipids are selected from cholesterol and/or β-sitosterol; more preferably, the structured lipids are cholesterol. The lipid nanoparticle according to any one of claims 1 to 4, wherein the polymer lipid is a pegylated lipid, preferably DMG-PEG2000 and/or ALC-0159. The lipid nanoparticle according to any one of claims 1 to 5, wherein the neutral lipid is selected from phosphatidylcholine and/or phosphatidylethanolamine; preferably one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The lipid nanoparticle according to any one of claims 1 to 6, wherein the lipid portion comprises, in molar percentage:
A lipid nanoparticle composition, comprising the lipid nanoparticle according to any one of claims 1 to 7 and a load. The lipid nanoparticle composition according to claim 8, wherein the load is a nucleic acid, and the nucleic acid is selected from one or more of ASO, RNA or DNA; the RNA is selected from one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), micro RNA (miRNA), small activating RNA (saRNA), poly-coding nucleic acid (MCNA), poly-coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme; preferably guide RNA (gRNA), CRISPR RNA (crRNA), mRNA, ASO or siRNA. A pharmaceutical composition comprising the lipid nanoparticles according to any one of claims 1 to 7 or the lipid nanoparticle composition according to any one of claims 8 to 9, and a pharmaceutically acceptable excipient. Use of the lipid nanoparticles according to any one of claims 1 to 7, the lipid nanoparticle composition according to any one of claims 8 to 9, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating, diagnosing or preventing a disease. The use according to claim 11, wherein the disease is a central nervous system disease, preferably a brain neurodegenerative disease; more preferably a brain degenerative disease associated with autonomous movement; further preferably Huntington's disease, ALS or Parkinson's disease. The use according to claim 11, wherein the lipid nanoparticle, the lipid nanoparticle composition or the pharmaceutical composition is administered intrathecally. Use of the lipid nanoparticles according to any one of claims 1 to 7, the lipid nanoparticle composition according to any one of claims 8 to 9, or the pharmaceutical composition according to claim 10 in the preparation of a drug for delivering a load, wherein the load is selected from one or more of a therapeutic agent, a preventive agent or a diagnostic agent. A method for treating, diagnosing or preventing a disease in a subject, comprising intrathecally administering to the subject the lipid nanoparticle of any one of claims 1 to 7, the lipid nanoparticle composition of any one of claims 8 to 9, or the pharmaceutical composition of claim 10. A method for delivering a load into a subject, comprising intrathecally administering the lipid nanoparticle of any one of claims 1 to 7, the lipid nanoparticle composition of any one of claims 8 to 9, or the pharmaceutical composition of claim 10 to the subject.