WO2024094194A1 - 一种用于雾化吸入脂质纳米粒的通用型处方 - Google Patents
一种用于雾化吸入脂质纳米粒的通用型处方 Download PDFInfo
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- WO2024094194A1 WO2024094194A1 PCT/CN2023/129753 CN2023129753W WO2024094194A1 WO 2024094194 A1 WO2024094194 A1 WO 2024094194A1 CN 2023129753 W CN2023129753 W CN 2023129753W WO 2024094194 A1 WO2024094194 A1 WO 2024094194A1
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- lipid
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- lnp
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- ethanol
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- 229940096998 ursolic acid Drugs 0.000 description 1
- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000002888 zwitterionic surfactant Substances 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to the field of nucleic acid delivery, in particular to a universal prescription for atomized inhalation of lipid nanoparticles.
- Nucleic acid drugs prevent and treat diseases by regulating the expression of target genes.
- Nucleic acid drugs have the advantages of high specificity and high efficiency, and are expected to overcome the drug-making limitations of existing targets, and have great potential for treating "untargetable” and “undruggable” diseases.
- efficient and safe delivery carrier technology is essential, especially the key technical difficulties of organ/cell-specific delivery of nucleic acid therapeutic molecules need to be solved, while improving the stability of nucleic acid drugs and improving the bioavailability and in vivo pharmacokinetic properties of nucleic acid drugs.
- the most effective nucleic acid drug delivery carrier is lipid nanoparticle (LNP), which is formed by self-assembly of ionizable lipid compounds, neutral co-lipids, structural lipids and PEG lipids.
- LNP lipid nanoparticle
- the key role is played by ionizable lipids, whose pKa is generally between 5.5 and 6.8. This feature makes its surface charge basically neutral in the serum environment, which is conducive to the cell swallowing the LNP with nucleic acid fragments into the cell as a whole to form endosomes.
- the acidic environment of the endosome makes the head of the ionizable lipid protonated and positively charged, thereby fusing with the inner membrane of the endosome, allowing the target nucleic acid drug to escape from the endosome, and then exert its efficacy by expressing the target protein or inhibiting the expression of the target gene.
- nucleic acid drugs have been approved and marketed using LNP as a delivery carrier, including: Onpattro for delivering siRNA, which is administered by intravenous injection, and LNP is enriched in the liver for RNAi treatment; mRNA vaccines for preventing the new coronavirus SARS-CoV-2 are administered by intramuscular injection, etc.
- Nucleic acid drugs are bringing changes to the development of a new generation of drugs. Faced with the global COVID-19 pandemic, people have gradually realized the importance and necessity of developing effective prevention and treatment measures for respiratory diseases (especially the development of transformative nucleic acid drugs).
- nucleic acid drugs that can be effectively delivered to the lungs for lung diseases, including pulmonary fibrosis, lung cancer, asthma, bronchial diseases, etc.
- respiratory diseases such as the new crown
- pulmonary inhalation administration can not only form effective systemic immunity (producing IgG antibodies), but also form local mucosal immunity (producing IgA antibodies), forming a combined cross-protection effect. Therefore, inhalation nucleic acid delivery carriers are particularly important for the development of vaccines and drugs for respiratory diseases.
- Inhalation aerosol preparations can deliver drugs directly to the lungs, which can greatly increase the concentration of drugs in the lungs and reduce systemic toxicity, which is an inherent advantage of respiratory administration.
- the development of inhalation preparations, especially inhalation nucleic acid LNP preparations faces the challenge of poor aerosolization, that is, the inability to withstand the shear damage during the aerosolization process, resulting in leakage of nucleic acid drugs during aerosolization, a significant increase in particle size and dispersity (PDI, polydispersity index) after aerosolization, and a significant decrease in transfection efficiency after aerosolization. Therefore, the primary key to the development of aerosol inhalation LNP nucleic acid drugs is to overcome the destructive effect of the aerosolization process on LNP.
- LNP will experience the destructive effect of shear force during atomization, which will lead to an increase in LNP particle size, an increase in PDI and a decrease in encapsulation efficiency (EE) caused by the leakage of nucleic acid molecules, which will ultimately affect its transfection effect.
- EE encapsulation efficiency
- the particle size after atomization usually increases by at least one time, and the EE decreases by at least one time, which is not ideal. How to ensure that LNP can withstand the shearing effect during atomization and achieve the consistency and integrity of its structure before and after atomization is a technical problem that the market urgently needs to solve.
- the present invention provides a universal prescription, so that the prepared LNP can withstand the atomization shear force, and the particle size, PDI and EE of the LNP can remain unchanged before and after atomization, breaking through the technical barriers of the market atomized inhalation LNP that cannot withstand the atomization shear force and has poor stability.
- this prescription is suitable for different types of LNPs and LNPs with different ratios, and is a universal method. In vivo mouse imaging experiments have verified that the LNP nucleic acid drug using this prescription can achieve effective lung deposition compared to the LNP not using this prescription, thereby increasing the efficiency of lung gene transfection by multiple orders of magnitude.
- the present invention aims to provide a universal formulation for aerosol inhalation of lipid nanoparticles (LNPs), so that the LNPs can withstand shear damage during the aerosolization process, improve the stability of the LNPs during the aerosolization process, and achieve efficient delivery.
- LNPs lipid nanoparticles
- the present invention adopts the following technical solution:
- a universal formulation for aerosol inhalation of LNP wherein a surface tension reducing regulator is added to the LNP dispersion system to enhance the tolerance of LNP to aerosol shear. It should be noted that any compound or composition that adds a surface tension reducing regulator to the LNP dispersion system to increase the aerosol shear tolerance of LNP and improves the consistency of particle size, PDI and EE before and after aerosolization is within the scope of protection of the present invention and is inspired by the present invention.
- the aforementioned universal prescription for aerosol inhalation of LNP comprises: an LNP dispersion system and a surface tension reducing regulator added to the LNP dispersion system; the particle size range of the LNP is 70 to 160 nm; the mass volume percentage range of the surface tension reducing regulator in the LNP dispersion system is 0.01% to 30%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%,
- the LNP dispersion system comprises: LNP, system solution;
- the mass volume percentage range of the LNP in its dispersed system is 0.0025% to 10%, and the concentration range of the system solution is 0 to 1000 mM.
- the LNP dispersion system includes: LNP, system solution, and cryoprotectant; the mass volume percentage range of the LNP in its dispersion system is 0.0025% to 10%, the concentration range of the system solution is 0 to 1000mM, and the mass volume percentage concentration range of the cryoprotectant is 0% to 20%; it should be noted that: the LNP dispersion system may or may not contain a cryoprotectant.
- Biological barriers refer to: the mucus layer of epithelial cells in tracheal and alveolar tissue, mucociliary and alveolar surfactant, etc.; intracellular barriers refer to the difficulty of cell uptake and insufficient lysosomal escape caused by the high negative charge and large relative molecular mass of nucleic acids themselves.
- the system solution includes: physiological saline, 4-hydroxyethylpiperazineethanesulfonic acid HEPEs buffer, tris(hydroxymethylaminomethane) Tris buffer, Tris-EDTA buffer, phosphate PB and phosphate PBS buffer, Dulbecco's phosphate DPBS buffer, citrate buffer, sulfate buffer, carbonate buffer, acetate buffer, Tris buffer containing Tween TBST, buffer containing EDTA and its sodium salt, or a combination of several thereof; it should be noted that the system solution here is not exhaustive, but only a preferred one. As long as the solution has a regulating or buffering effect on the system osmotic pressure or pH, it is within the protection scope of the present invention.
- the surface tension reducing regulator includes: ethanol, propylene glycol, phenylethyl alcohol, poloxamer 188 (Poloxamer 188), Tween-80 (Tween-80 or Polysorbate 80), glycerol, polyoxy-15 hydroxystearate or a mixture of several thereof; the above is not exhaustive, and whether known or unknown surface tension reducing regulators, as long as they are regulators that can reduce the surface tension of the dispersed system, they are within the protection scope of the present invention.
- the surface tension reducing regulator is ethanol
- the mass volume percentage of ethanol in the LNP dispersion system is in the range of 0.05 to 30%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 30%.
- the surface tension reducing agent is propylene glycol
- the mass volume percentage of propylene glycol in the LNP dispersion system is in the range of 0.05 to 30%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 30%.
- the surface tension reducing regulator is a composition of propylene glycol and ethanol
- the mass volume percentage of propylene glycol in the LNP dispersion system is in the range of 0.05 to 30%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 30%
- the mass volume percentage of ethanol in the LNP dispersion system ranges from 0.05 to 30%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 0.5%, 0.
- the surface tension reducing regulator is poloxamer 188
- the concentration range of poloxamer 188 in the LNP dispersion system is 0.5 to 10 mg/mL, such as 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, and 10 mg/mL.
- the surface tension reducing regulator is a mixture of poloxamer 188 and ethanol
- the concentration range of poloxamer 188 in the LNP dispersion system is 0.5-10 mg/mL
- the mass volume percentage range of ethanol in the LNP dispersion system is 0.05-30%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 30%.
- the surface tension reducing regulator is Tween-80
- the volume percentage range of Tween-80 in the LNP dispersion system is 0.01-2%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%.
- the surface tension reducing regulator is a mixture of Tween-80 and ethanol
- the volume percentage of Tween-80 in the LNP dispersion system is in the range of 0.01 to 2%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%
- the mass volume percentage of ethanol in the LNP dispersion system ranges from 0.05 to 30%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 30%.
- the surface tension reducing regulator is polyoxy-15 hydroxystearate
- the concentration range of polyoxy-15 hydroxystearate in the lipid nanoparticle dispersion system is 0.05-20 mg/ml, preferably 0.1-10 mg/ml, and further preferably 3-7.5 mg/mL, such as 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 7.5mg/mL, 8mg/mL, 9mg/mL, 10mg/mL.
- the present invention is beneficial in that:
- the present invention introduces molecules that can reduce the surface tension of water into the dispersion system of LNP, and unexpectedly finds that the LNP prepared using such a prescription can withstand the shear force during atomization, and the particle size and dispersion index (PDI) remain unchanged before and after atomization; breaking through the technical barrier that the commercially available atomized inhalation LNP cannot withstand the atomization shear force and has poor stability; achieving unexpected technical effects;
- the present invention unexpectedly overcomes the problem of nucleic acid leakage of LNP during atomization by introducing molecules that can reduce the surface tension of water into the dispersion system of LNP, and can achieve that the encapsulation efficiency (EE) of LNP before and after atomization remains almost unchanged.
- EE encapsulation efficiency
- the present invention introduces molecules that can reduce the surface tension of water into the dispersion system of LNP, and unexpectedly finds that the LNP before and after atomization can have a higher deposition rate in the lungs and a higher gene transfection effect.
- the prescription of the present invention has high versatility. When applied to different LNP lipid components or formulations, it can still ensure that it can withstand the shear force during the atomization process and achieve consistency in particle size, PDI and EE before and after atomization;
- FIG1 is a schematic diagram of the particle size of LNP before and after atomization in a dispersion system with different ethanol contents in Experiment 1 of the present invention
- FIG2 is a schematic diagram of the particle sizes of LNPs of different particle sizes before and after atomization in a dispersion system with a fixed ethanol content in Experiment 1 of the present invention
- FIG3 is a schematic diagram of the particle sizes of LNPs of different concentrations before and after atomization in a 7% ethanol system in Experiment 2 of the present invention
- FIG4 is an imaging result of atomization of C12LNP in a dispersion system without ethanol and a dispersion system containing 8% ethanol in mice in Experiment 3 of the present invention
- FIG5 is a graph showing the particle size of each LNP before and after atomization in a dispersion system with different ethanol contents in Experiment 4 of the present invention.
- FIG6 is a graph showing the particle size of LNP before and after atomization when the dispersion system contains 2 mg/mL of poloxamer-188, or 2 mg/mL of poloxamer and 4% ethanol in Experiment 4 of the present invention;
- FIG. 7 is a graph showing the particle size of LNP before and after atomization when the dispersion system contains different concentrations of Tween-80 in Experiment 4 of the present invention.
- the term "surface tension reduction modifier” refers to a substance added to the LNP dispersion system for increasing the atomization shear tolerance of the LNP, generally refers to a chemical substance that can reduce the surface tension of water after adding water, but is not limited to such substances.
- Such substances can be surfactants common in the art, such as anionic surfactants, cationic surfactants, zwitterionic surfactants, nonionic surfactants, exemplary representatives such as poloxamers, tweens, benzyls, and can also be ethanol, propylene glycol, phenylethyl alcohol, glycerol, polyoxy-15 hydroxystearate and other substances.
- the surface tension reduction modifier is added to the LNP dispersion system to increase the atomization shear tolerance of the LNP, the compound or composition that improves the particle size before and after atomization, the consistency of PDI and EE is within the scope of protection of the present invention, and is inspired by the present invention.
- mass volume percentage and mass volume percentage concentration described herein all represent w/v%, that is, 1% w/v means 1 g/100 ml mass/volume (w: mass, v: volume).
- Nucleic acid is a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and is a biological macromolecule composed of multiple nucleotide monomers; nucleic acid is composed of nucleotides, and nucleotide monomers are composed of pentose, phosphate, nitrogenous base, or any modified group. If the pentose is ribose, the polymer formed is RNA; if the pentose is deoxyribose, the polymer formed is DNA.
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the drugs that need to be delivered include: nucleic acid molecules, small molecule compounds, peptides, proteins and other medicinal ingredients.
- Nucleic acid molecules include single-stranded DNA, double-stranded DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro non-coding RNA (miRNA), small interfering RNA (siRNA), telomerase RNA (Telomerase RNA Component), small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense DNA, antisense RNA, ribozyme, asymmetric interfering RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), gRNA, sgRNA, crRNA or tracrRNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotide, morpholin
- mRNA messenger RNA
- Chinese translation: messenger ribonucleic acid is a type of single-stranded ribonucleic acid transcribed from a strand of DNA as a template, carrying genetic information and guiding protein synthesis.
- mRNA can be monocistronic mRNA or It can be a polycistronic mRNA.
- the mRNA can also contain one or more functional nucleotide analogs, examples of which include pseudouridine, 1-methyl-pseudouridine or 5-methylcytosine. The examples here are not exhaustive, and any modified mRNA or its derivatives can be applied to the present invention.
- siRNA Small interfering RNA
- siRNA is a type of double-stranded RNA molecule.
- siRNA is cut into double-stranded RNA of more than 20 bp in size, generally not exceeding 30 bp, by RNase III (such as Dicer) in cells.
- dsRNA can be exogenous, such as viral RNA replication intermediates or artificially introduced dsRNA; it can also be endogenous, such as dsRNA formed by single-stranded RNA in cells under the action of RNA-dependent RNA polymerase.
- siRNA is a short dsRNA with a phosphorylated 5' end and a hydroxylated 3' end with two protruding nucleotides.
- siRNA binds to a ribonucleoprotein called RNA-induced silencing complex (RISC), and siRNA begins to unwind.
- RISC RNA-induced silencing complex
- guide strand sense strand
- the guide strand is complementary to the target mRNA and guides the RISC complex to bind to the mRNA, inducing mRNA degradation. Since siRNA can be designed and synthesized in principle to silence any gene, it is an important tool for verifying gene function and drug targeting in the post-genomic era, and also marks the advent of a transformative era of biomedicine.
- Small molecule compounds can be active ingredients in therapeutic or preventive agents, such as anti-tumor drugs, anti-infective drugs, local anesthetics, antidepressants, anticonvulsants, antibiotics/antibacterial agents, antifungal drugs, antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or imaging agents, etc., which are not exhaustive.
- therapeutic or preventive agents such as anti-tumor drugs, anti-infective drugs, local anesthetics, antidepressants, anticonvulsants, antibiotics/antibacterial agents, antifungal drugs, antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or imaging agents, etc., which are not exhaustive.
- Polypeptides are compounds formed by ⁇ -amino acids linked together by peptide bonds and are intermediate products of protein hydrolysis.
- Protein is a substance with a certain spatial structure formed by the coiling and folding of polypeptide chains composed of amino acids in a "dehydration condensation" manner; protein can be interferon, protein hormone, cytokine, chemokine or enzyme, etc.
- the diluent is any pharmaceutically acceptable water-soluble excipient known to those skilled in the art, including: amino acids, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, other oligosaccharides, mannitol, dextran, sodium chloride, sorbitol, polyethylene glycol, phosphates, or derivatives thereof or combinations thereof.
- Charged lipid compounds refer to a class of lipid compounds that exist in the form of positive or negative charges; their charge is independent of pH within the physiological range, such as pH 3-9, and is not affected by pH.
- Charged lipids can be synthetic or naturally derived. Examples of charged lipids include, but are not limited to, DOTAP, DOTMA, and 18PA. This is not an exhaustive list, and any LNP composition using the prescription of the present invention is within the scope of protection of the present invention and is inspired by the present invention.
- the lipid aids include: one or a combination of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), sterols and their derivatives, ceramides, and charged lipids;
- phosphatidylcholine as a preferred embodiment includes: DSPC, DPPC, DMPC, DOPC, POPC;
- phosphatidylethanolamine as a preferred embodiment includes DOPE;
- sterol as a preferred embodiment includes cholesterol; this is not an exhaustive list, as long as the LNP composition using the prescription of the present invention is within the protection scope of the present invention, all are inspired by the present invention.
- Structural lipids include: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, One or more of solanine, tomatine, ursolic acid, ⁇ -tocopherol or corticosteroids. This is not an exhaustive list, and the choice of structural lipids is not limited.
- the polymer conjugated lipid is a PEGylated lipid; as an example, the PEGylated lipid includes: one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol. This is not exhaustive, and the selection of polymer conjugated lipids is not limited.
- DSPC English name: distearoyl Phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine; Chinese name: Distearoyl Phosphatidylcholine, CAS number: 816-94-4.
- DPPC Chinese name: Dipalmitoylphosphatidylcholine; English name: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, CAS number: 63-89-8.
- DMPC Chinese name: dimyristoylphosphatidylcholine; English name: 1,2-dimyristoyl-sn-glycero-3-phosphocholine, CAS number: 18194-24-6.
- DOPC Chinese name: 1,2-dioleoyl-sn-glycero-3-phosphocholine; English name: 1,2-dioleoyl-sn-glycero-3-phosphocholine, CAS number: 4235-95-4.
- POPC Chinese name: 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine; English name: 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, CAS number: 26853-31-6.
- DOPE Chinese name: 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine; English name: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, CAS number: 4004-05-1.
- DOTAP Chinese name: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt); English name: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt), CAS number: 132172-61-3; the chemical structure is as follows:
- DOTMA Chinese name: N,N,N-trimethyl-2,3-bis(octadec-9-en-1-yloxy)propan-1-ammonium chloride, CAS number: 1325214-86-5, chemical structure is as follows:
- SM Chinese name: sphingomyelin (SM); English name: sphingomyelin.
- PEG Chinese name: polyethylene glycol; English name: Polyethylene glycol.
- Buffer solution refers to a mixed solution composed of weak acids and their salts, weak bases and their salts, which can offset and reduce the effects of external strong acids or strong bases on the pH of the solution to a certain extent, thereby keeping the pH value of the solution relatively stable; examples include: phosphoric acid, citric acid, carbonic acid, acetic acid, acetic acid-sodium acetate, acetic acid-ammonium acetate, barbituric acid, Tris, PBS, HEPEs, EDTA and its sodium salt, etc., or combinations thereof, which are not exhaustive.
- HEPEs buffer Chinese name: 4-hydroxyethylpiperazineethanesulfonic acid; English name: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; CAS number: 7365-45-9.
- Tris buffer The Chinese product name is tris(hydroxymethyl)aminomethane; tromethamine; tromethamine; 2-amino-2-(hydroxymethyl)-1,3-propanediol; CAS number: 77-86-1.
- Citrate Buffer The main components of citrate buffer are citric acid and sodium hydrogen phosphate.
- DPBS Dulbecco's phosphate buffered saline.
- TBST composed of Tris-HCl, NaCl, and tween20.
- EDTA sodium salt EDTA disodium dihydrate is ethylenediaminetetraacetic acid disodium dihydrate, English name: EDTA disodium salt dihydrate.
- PBS buffer The main components are Na 2 HPO 4 , KH 2 PO 4 , NaCl and KCl.
- Cryoprotectants refer to substances (usually solutions) that can protect cells from freezing damage; they can be divided into: sugars, alcohols, amino acids, salts, etc.; specifically include: sucrose, mannitol, trehalose, lactose, glucose, maltose, polyvinyl pyrrolidone (PVP), polyethylene glycol, dextran, albumin and hydroxyethyl starch.
- sucrose, mannitol, trehalose, lactose, glucose, maltose, polyvinyl pyrrolidone (PVP), polyethylene glycol, dextran, albumin and hydroxyethyl starch PVP
- a vibrating mesh atomizer model Aerogen solo Nebulizer System
- Table 1 Changes in particle size, PDI and EE of LNP before and after atomization in dispersion systems with different ethanol contents
- the dispersion system preferably contains 0.01% to 30% ethanol.
- Example 2 For the LNP of Example 1, we selected the ethanol content in the dispersion system to be 6.5%, and further studied the applicable particle size range in which the particle size, PDI and EE did not change before and after atomization.
- Example 1 LNP was prepared using a microfluidic device, wherein the lipid formulation of Example 1 LNP was dissolved with anhydrous ethanol, mRNA was diluted with sodium acetate buffer, the volume ratio of alcohol-water phase was 1 to 3, and LNP with a particle size distribution of 70 to 160 nm was prepared by adjusting the flow rate.
- Table 3 Particle size, PDI and EE of LNPs of different concentrations before and after atomization in a 10% ethanol system.
- the nebulization is stable when the mRNA concentration is as high as 2.5 ⁇ g/ ⁇ L (the corresponding LNP mass volume fraction is 10%). If it is diluted, the nebulization will be more stable. When the mRNA concentration is 0.5 ⁇ g/ ⁇ L, it is as stable as when it is 0.005 ⁇ g/ ⁇ L.
- the mass volume fraction of LNP corresponding to the mRNA concentration of 0.005 ⁇ g/ ⁇ L is 0.0025%, that is, when the mass volume percentage of LNP in the dispersed system is 0.0025% ⁇ 10%, the nebulization is stable.
- the formula of the ionizable lipid compound and the composition was changed as shown in Examples 2 to 4 LNP, and the commercial DLin-MC3-DMA (abbreviated as MC3) was selected as the comparative example LNP.
- the molar ratio of the prescription was shown in Table 4.
- the microfluidic preparation parameters were: 0.3 ml of front-end waste liquid, 0.05 mL of terminal waste liquid, 0.75 mL of total volume, 2 to 8 mL/min of total flow rate, and volume ratio: 3 to 1.
- the particle size distribution was 70 to 160 nm, dialyzed in HEPEs, taken out, 50 ⁇ L of LNP was taken, and then 100 ⁇ L of ethanol-containing HEPEs buffer was added to dilute it so that the system contained different concentrations of ethanol, atomized, and the particle size, PDI and EE before and after atomization were tested.
- Table 5 Particle size, PDI and EE of each LNP before and after atomization in dispersion systems with different ethanol contents.
- a represents the ethanol content in the dispersion system, b represents not detected.
- Example 1 LNP was prepared by microfluidics method, Luciferase mRNA was encapsulated, and then mice were allowed to inhale by aerosolization. After 6 hours, potassium luciferin substrate was injected intraperitoneally. After 5 minutes, the mice were killed and the heart, liver, spleen, lungs and kidneys were imaged as shown in Figure 4.
- Example 1 LNP was prepared using a microfluidic device, wherein the lipid formulation was dissolved with anhydrous ethanol, the mRNA was diluted with sodium acetate buffer, and the volume ratio of the alcohol-water phase was 1 to 3. By adjusting the flow rate, LNP with a particle size of about 130 nm was prepared.
- poloxamer 188 a surface tension reducing regulator
- the concentration range of 0.5-10 mg/mL the concentration range of 0.5-10 mg/mL
- the changes in various parameters of LNP before and after atomization are minimal.
- 4% ethanol is introduced into the system, the concentration is stable at 5 mg/mL and 2 mg/mL. Therefore, the combination of ethanol and poloxamer 188 has an unexpected synergistic effect in improving the stability of particle size and PDI before and after atomization.
- Table 7 Changes in particle size and PDI of LNP before and after atomization when Tween-80 is added to the dispersion system
- Tween-80 added to the LNP dispersion system as a surface tension regulator can also keep the LNP particle size stable before and after atomization.
- the volume concentration of Tween-80 is in the range of 0.01% to 2%, the particle size does not increase significantly after atomization. Since Tween-80 itself will self-assemble to form micelles, the PDI detected by the particle size analyzer is larger, but the PDI of LNP itself and There is almost no change compared to before atomization. In addition, as the content of Tween-80 increases, the increase in particle size becomes less obvious.
- the dispersion system does not contain sucrose, the viscosity of the dispersion system can be reduced, and the consistency of particle size and EE before and after atomization can be achieved at a lower surface tension reducing agent concentration.
- C12 was used as an ionizable lipid to encapsulate luc mRNA, and LNPs were prepared using a microfluidic preparation instrument for atomized LNP research.
- Polyoxyl-15 hydroxystearate (abbreviated as HS15, commercially available) was added, and the experimental steps and methods were the same as those in Experiment 1.
- pre and post refer to samples before and after nebulization, respectively.
- concentrations of HS 15 were 0.1 mg/ml, 2 mg/ml, 5 mg/ml and 10 mg/ml, respectively. No HS15 was added to the control group.
- the samples prepared in Table 11 were subjected to a stability study and stored in a refrigerator at 4° C. The particle size of the samples was tested after 7 days of storage.
- the present invention introduces molecules that can reduce the surface tension of water into the dispersion system of LNP, and unexpectedly finds that the LNP prepared using such a prescription can withstand the shear force during the atomization process, and the particle size and PDI remain unchanged before and after atomization.
- the nucleic acid encapsulated in the LNP will not leak, and the EE of the LNP before and after atomization can be maintained unchanged; the LNP before and after atomization has a higher deposition rate and a higher gene transfection effect in the lungs, breaking through the technical barrier of the market that atomized inhalation LNP cannot withstand the atomization shear force and has poor stability; and achieving unexpected technical effects.
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Abstract
本发明公开了一种用于雾化吸入脂质纳米粒的通用型处方,属于生物医药领域,处方包括:脂质纳米粒(LNP)分散体系和加入在所述LNP分散体系中的表面张力下降调节剂;LNP的粒度范围为70~160nm;表面张力下降调节剂在LNP分散体系的质量体积百分比在0.01~30%;本发明意外的发现在LNP分散体系中加入表面张力下降调节剂能增加LNP雾化剪切的耐受能力,解决了雾化的剪切力使得LNP在雾化前后粒度、分散度(PDI)和包封率(EE)稳定性差的问题,确保雾化吸入减轻病人痛苦的同时,实现高效递送。
Description
本发明涉及核酸递送领域,特别是一种用于雾化吸入脂质纳米粒的通用型处方。
核酸药物通过调节目标基因的表达,达到预防和治疗疾病的目的。核酸药物具有高特异性、高效性的优势,并有望攻克现有靶点的成药局限性,具备治疗“不可靶向”、“不可成药”疾病的巨大潜力。然而,要想实现核酸药物的巨大潜能,高效安全的递送载体技术必不可少,尤其是需要解决针对器官/细胞特异性递送核酸治疗分子的关键技术难题,在提高核酸药物稳定性的同时,改善核酸药物的生物利用度和体内药代动力学性质。
目前,最有效的核酸药物递送载体是脂质纳米粒(LNP),由可离子化脂质化合物、中性助脂质、结构脂质和PEG脂质等自组装形成,其中起关键作用的是可离子化脂质,其pKa一般介于5.5~6.8之间,这个特性让它在血清的环境中表面电荷基本为中性,有利于细胞将带有核酸片段的LNP整个吞进细胞内,形成胞内体(endosome)。一旦进入细胞后,胞内体的酸性环境使可离子化脂质的头部质子化并带正电荷,从而与胞内体的内膜融合,使目标核酸药物从胞内体中逃逸出来,进而通过表达目的蛋白或抑制靶基因的表达来发挥药效。
目前,已有多款核酸药物使用LNP作为递送载体获批并上市,其中包括:用于递送siRNA的Onpattro,通过静脉注射给药,LNP富集于肝部进行RNAi治疗;通过肌肉注射给药,预防新型冠状病毒SARS-CoV-2的mRNA疫苗等。核酸药物正为新一代药物研发带来变革。面对全球肆虐的新冠疫情,人们逐步认识到研发针对呼吸道疾病的有效防治手段(尤其是核酸变革性药物的研发)的重要性和必要性。然而,由于目前的核酸药物缺乏肺组织特异性的递送系统,针对肺部的疾病,包括肺纤维化、肺癌、哮喘、支气管疾病等,还没有可有效递送到肺部的核酸药物上市。此外,研究发现,对于类似于新冠这样的呼吸道传播疾病,采用模拟病毒感染的给药路径,即肺部吸入给药,不仅可以形成有效的系统免疫(产生lgG抗体),而且还可以形成局部粘膜免疫(产生lgA抗体),形成联合交叉保护效力。因此,吸入式核酸递送载体对于研发针对呼吸系统疾病的疫苗和药物显得尤为重要。
吸入式雾化制剂可以将药物直接递送到肺部,可以大大提高药物在肺部的富集浓度并减少系统毒性,是呼吸系统给药的先天优势。然而,吸入制剂研发特别是吸入式核酸LNP制剂,面临着可雾化性差的挑战,即因无法耐受雾化过程中的剪切破坏作用而导致雾化时核酸药物泄露、雾化后粒度和分散度(PDI,polydispersity index)明显增加、雾化后转染效率明显下降等,因此,研发雾化吸入型LNP核酸药物的首要关键是克服雾化过程对LNP的破坏作用。
无论哪种雾化形式,包括压缩式雾化、超声波雾化、网式雾化、软雾剂和压力定量气雾剂等,雾化过程中,LNP均要经历剪切力的破坏作用,该剪切破坏作用会导致LNP粒度增大、PDI增大和因核酸分子泄露而引起的包封率(EE,encapsulation efficiency)下降,最终影响其转染效果。目前关于雾化吸入型LNP核酸药物的专利和文献寥寥无几,而且大部分的报道是通过改变LNP的四个组分配比,以筛选出雾化前后粒度、PDI和EE变化较小的处方。但他们筛选出的最佳处方中,雾化后的粒度通常都会增加至少一倍以上,EE至少下降一倍,结果并不理想。如何保证LNP可以耐受雾化时的剪切作用,实现雾化前后其结构的一致性和完整性,是市场急需解决的技术问题。
本发明通过提供一种通用型的处方,实现制备得到的LNP能够耐受雾化剪切力,可以使得LNP在雾化前后粒度、PDI和EE保持不变,突破了市场雾化吸入LNP无法耐受雾化剪切力,稳定性差的技术壁垒。且本处方适用于不同种类的LNP及不同配比的LNP,是一种通用型的方法。通过体内小鼠成像实验验证,使用该处方的LNP核酸药物相比于未使用该处方的LNP而言,可以实现肺部的有效沉积,使肺部基因转染效率提高了多个数量级。
发明内容
为解决现有技术的不足,本发明的目的在于提供一种用于雾化吸入脂质纳米粒(LNP)的通用型处方,使得LNP可以耐受雾化过程中的剪切破坏作用,提高LNP在雾化过程中的稳定性,实现高效递送。
为了实现上述目标,本发明采用如下的技术方案:
一种用于雾化吸入LNP的通用型处方,在LNP分散体系中加入表面张力下降调节剂能增强LNP对雾化剪切的耐受能力。需要说明的是:只要是将表面张力下降调节剂加入LNP分散体系中用于增加LNP雾化剪切耐受能力,提高雾化前后粒度、PDI和EE一致性的化合物或组合物均在本发明的保护范围内,且都受本发明的启发。
前述的一种用于雾化吸入LNP的通用型处方,作为一种优选,包括:LNP分散体系和加入在所述LNP分散体系中的表面张力下降调节剂;所述LNP的粒度范围为70~160nm;所述表面张力下降调节剂在LNP分散体系中的质量体积百分比范围为0.01%~30%,例如0.01%、0.02%、0.03%、0.04%、0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%或30%;需要说明的是:粒度分布在70~160nm之间的LNP可以更好的耐受雾化过程的破坏作用。
前述的一种用于雾化吸入LNP的通用型处方,LNP分散体系包括:LNP、体系溶液;所
述LNP在其分散体系中的质量体积百分比范围为0.0025%~10%,所述体系溶液的浓度范围为0~1000mM。
前述的一种用于雾化吸入LNP的通用型处方,所述LNP分散体系包括:LNP、体系溶液、冻存保护剂;所述LNP在其分散体系中的质量体积百分比范围为0.0025%~10%,所述体系溶液的浓度范围为0~1000mM,所述冻存保护剂的质量体积百分比浓度范围为0%~20%;需要说明的是:LNP分散体系中可以包含冻存保护剂也可以不包含冻存保护剂。
在另一优选例中,所述的LNP按照摩尔比包括:可离子化脂质化合物:助脂质:结构脂质:聚合物缀合脂质=30~60:5~40:20~40:0.1~15。进一步优选的,所述的LNP按照摩尔比包括:可离子化脂质化合物:助脂质:结构脂质:聚合物缀合脂质=40~60:5~30:25~40:1~10。需要说明的是:本发明的纳米粒子可以克服肺部递送所面临的生物学屏障和细胞内屏障,大量积累在肺部;其中聚合物缀合脂质的含量对LNP渗透到肺部细胞内部具有更深的研究价值,基于本发明的处方做的研究也都受本发明的启发。生物学屏障指:气管及肺泡组织上皮细胞的黏液层、黏膜纤毛和肺泡的表面活性物质等;细胞内屏障指由核酸自身的高负电性和较大的相对分子质量所造成的细胞摄取困难和溶酶体逃逸不足。
在另一优选例中,所述的体系溶液包括:生理盐水、4-羟乙基哌嗪乙磺酸HEPEs缓冲液、三羟甲基氨基甲烷Tris缓冲液、Tris-EDTA缓冲液、磷酸PB和磷酸盐PBS缓冲液、Dulbecco's磷酸盐DPBS缓冲液、柠檬酸盐缓冲液、硫酸盐缓冲液、碳酸盐缓冲液、醋酸盐缓冲液、含吐温的Tris缓冲液TBST、含EDTA及其钠盐的缓冲液中的一种或几种的组合;需要说明的是此处的体系溶液并非穷举,只是一种优选,只要对体系渗透压或pH起到调节或缓冲作用的溶液均在本发明的保护范围内。
在另一优选例中,所述的表面张力下降调节剂包括:乙醇、丙二醇、苯乙醇、泊洛沙姆188(Poloxamer 188)、吐温-80(Tween-80或Polysorbate 80)、甘油、聚氧基-15羟基硬脂酸酯中一种或几种的混合物;以上并非穷举,无论已知或未知的表面张力下降调节剂,只要是能够让分散体系表面张力下降的调节剂均在本发明的保护范围内。
在另一优选例中,所述的表面张力下降调节剂为乙醇,乙醇在LNP分散体系的质量体积百分比范围为0.05~30%,例如0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%或30%。
在另一优选例中,所述的表面张力下降调节剂为丙二醇,丙二醇在LNP分散体系的质量体积百分比范围为0.05~30%,例如0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、
0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%或30%。
在另一优选例中,所述表面张力下降调节剂为丙二醇和乙醇的组合物,丙二醇在LNP分散体系的质量体积百分比范围为0.05~30%,例如0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%或30%,乙醇在LNP分散体系的质量体积百分比范围为0.05~30%,例如0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%或30%。
在另一优选例中,所述的表面张力下降调节剂为泊洛沙姆188,泊洛沙姆188在LNP分散体系的浓度范围为0.5~10mg/mL,如0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL。
在另一优选例中,所述的表面张力下降调节剂为泊洛沙姆188和乙醇的混合物,泊洛沙姆188在LNP分散体系的浓度范围为0.5~10mg/mL,乙醇在LNP分散体系的质量体积百分比范围为0.05~30%,例如0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%或30%。
在另一优选例中,所述的表面张力下降调节剂为吐温-80,吐温-80在LNP分散体系的体积百分比范围为0.01~2%,例如0.01%、0.02%、0.03%、0.04%、0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、1.5%、2%。
在另一优选例中,所述的表面张力下降调节剂为吐温-80和乙醇的混合物,吐温-80在LNP分散体系的体积百分比范围为0.01~2%,例如0.01%、0.02%、0.03%、0.04%、0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、1.5%、2%,乙醇在LNP分散体系的质量体积百分比范围为0.05~30%,例如0.05%、0.06%、0.07%、0.08%、0.09%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、15%、20%、25%或30%。
在另一优选例中,所述的表面张力下降调节剂为聚氧基-15羟基硬脂酸酯,聚氧基-15羟基硬脂酸酯在脂质纳米粒分散体系的浓度范围为0.05-20mg/ml,优选的为0.1-10mg/ml,进一步优选的为3-7.5mg/mL,如0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、
0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、7.5mg/mL、8mg/mL、9mg/mL、10mg/mL。
本发明的有益之处在于:
1、本发明通过在LNP的分散体系中引入可以降低水的表面张力的分子,意外的发现,使用这样的处方制备得到的LNP能够耐受雾化过程中的剪切力,在雾化前后粒度和分散度(PDI)保持不变;突破了市售的雾化吸入LNP无法耐受雾化剪切力,稳定性差的技术壁垒;实现了意想不到的技术效果;
2、本发明通过在LNP的分散体系中引入可以降低水的表面张力的分子,意外地克服了LNP的核酸在雾化过程中泄露的问题,可以实现雾化前后LNP的包封率(EE)几乎保持不变。
3、本发明通过在LNP的分散体系中引入可以降低水的表面张力的分子,意外的发现,可以实现雾化前后的LNP在肺部具有较高的沉积率和较高的基因转染效果。
4、本发明的处方具有很高的通用性,当应用于不同LNP脂质成分或配方时,仍然可以保证其可以耐受雾化过程中的剪切力作用,实现其雾化前后粒度、PDI和EE的一致性;
5、在对可以降低水的表面张力的分子与LNP雾化稳定性的研究中发现,使用乙醇作为表面张力下降调节剂对提高雾化前后LNP的粒度、PDI和EE的稳定性具有优秀的效果;
6、在对可以降低水的表面张力的分子与LNP雾化稳定性的研究中发现,使用泊洛沙姆188和乙醇组合作为表面张力下降调节剂在提高雾化前后LNP的粒度、PDI和EE的稳定性具有进一步协同的效果。
7、在对可以降低水的表面张力的分子与LNP雾化稳定性的研究中发现,使用丙二醇和乙醇组合作为表面张力下降调节剂在提高雾化前后LNP的粒度、PDI和EE的稳定性具有进一步协同的效果。
8、通过体内小鼠成像实验验证,使用该处方的LNP核酸药物相比于未使用该处方的LNP而言,可以实现肺部的有效沉积,使肺部基因转染效率提高了数个数量级。
图1是本发明实验一中LNP在不同乙醇含量的分散体系中雾化前后的粒度示意图;
图2是本发明实验一中在固定乙醇含量的分散体系中,不同粒度的LNP在雾化前后的粒度示意图;
图3是本发明实验二在7%的乙醇体系中,不同浓度的LNP雾化前后的粒度示意图;
图4是本发明实验三中C12LNP在不含乙醇的分散体系和含8%乙醇的分散体系中进行小鼠雾化的成像结果;
图5是本发明实验四中各个LNP在不同含量乙醇的分散体系中雾化前后的粒度图;
图6是本发明实验四中当分散体系中含有2mg/mL的泊洛沙姆-188时,或2mg/mL泊洛沙姆及4%的乙醇时,LNP雾化前后的粒度图;
图7是本发明实验四中当分散体系中含有不同浓度的吐温-80时,LNP雾化前后的粒度图。
专业名词释义:
如本文所用,术语“表面张力下降调节剂”是指加入LNP分散体系中用于增加LNP雾化剪切耐受能力的物质,通常是指在加入水之后可以降低水的表面张力的化学物质,但不局限于该类物质。该类物质可以为本领域常见的表面活性剂,如阴离子表面活性剂、阳离子表面活性剂、两性离子表面活性剂、非离子表面活性剂,示例性的代表如泊洛沙姆类、吐温类、苄泽类,也可以是乙醇、丙二醇、苯乙醇、甘油、聚氧基-15羟基硬脂酸酯等其他物质。需要说明的是只要是将表面张力下降调节剂加入LNP分散体系中用于增加LNP雾化剪切耐受能力,提高雾化前后粒度、PDI和EE一致性的化合物或组合物均在本发明的保护范围内,且都受本发明的启发。
本文所述质量体积百分比、质量体积百分比浓度均表示w/v%,即1%w/v意为1g/100ml质量/体积(w:质量,v:体积)。
核酸是脱氧核糖核酸(DNA)和核糖核酸(RNA)的总称,是由多个核苷酸单体组成的生物大分子;核酸由核苷酸组成,核苷酸单体由五碳糖、磷酸基、含氮碱基、或任何修饰基团组成。如果五碳糖是核糖,则形成的聚合物是RNA;如果五碳糖是脱氧核糖,则形成的聚合物是DNA。
需要递送的药物包括:核酸分子、小分子化合物、多肽、蛋白质等可以药用的成分。
核酸分子包括单链DNA、双链DNA、短异构体、mRNA、tRNA、rRNA、长链非编码RNA(lncRNA)、微小非编码RNA(miRNA)、小干扰RNA(siRNA)、端粒酶RNA(Telomerase RNAComponent)、小分子RNA(snRNA和scRNA)、环状RNA(circRNA)、合成miRNA(miRNA mimics、miRNA agomir、miRNA antagomir)、反义DNA、反义RNA、核酶(ribozyme)、不对称干扰RNA(aiRNA)、Dicer~substrate RNA(dsRNA)、小发夹RNA(shRNA)、转移RNA(tRNA)、信使RNA(mRNA)、gRNA、sgRNA、crRNA或tracrRNA、锁核酸(LNA)、肽核酸(PNA)、吗啉反义寡核苷酸、吗啉代寡核苷酸或生物定制寡核苷酸等或其组合。这里的举例也并非穷举,只要是由核苷酸单体聚合成的都可以应用于本发明。
mRNA,信使RNA,中文译名:信使核糖核酸,是由DNA的一条链作为模板转录而来的、携带遗传信息能指导蛋白质合成的一类单链核糖核酸。mRNA可以是单顺反子mRNA也
可以是多顺反子mRNA。mRNA也可以包含一种或多种功能性核苷酸类似物,功能性核苷酸类似物举例包括:假尿嘧啶核苷、1~甲基~假尿嘧啶核苷或5~甲基胞嘧啶等。这里的举例也并非穷举,任何修饰的mRNA或其衍生物都可以应用于本发明。
小干扰RNA(siRNA),称为短干扰RNA或沉默RNA,是一类双链RNA分子。siRNA由双链RNA(double strand RNA,dsRNA)在细胞内被RNase III(如Dicer)切割成20多bp大小的双链RNA,一般不超过30bp。dsRNA可以是外源的,如病毒RNA复制中间体或人工导入的dsRNA;也可以是内源的,如细胞中单链RNA在RNA依赖的RNA聚合酶的作用下形成的dsRNA。siRNA具有磷酸化5'末端和具有两个突出核苷酸的羟基化3'末端的短dsRNA。在细胞质内,siRNA与一种叫做RNA诱导沉默复合体(RISC)的核糖核蛋白结合,siRNA开始解链,其引导链(正义链)由RISC中的阿尔古(Argonaute)蛋白保留,随从链(反义链)被降解,从而形成单链结构。引导链和目标mRNA互补并引导RISC复合体与mRNA结合,诱导mRNA降解。由于原则上siRNA可以被设计合成,沉默任何基因,因此siRNA是在后基因组时代验证基因功能和药物靶向的重要工具,也标志着变革性生物医药时代的来临。
小分子化合物可以是用于治疗或预防的试剂中的有效成分,例如:抗肿瘤药、抗感染药、局部麻醉药、抗抑郁药、抗惊厥药、抗生素/抗菌剂、抗真菌药、抗寄生虫药、激素、激素拮抗剂、免疫调节剂、神经递质拮抗剂、抗青光眼剂、麻醉剂、或成像剂等,这里并非穷举。
多肽是α-氨基酸以肽键连接在一起而形成的化合物,是蛋白质水解的中间产物。
蛋白质是由氨基酸以“脱水缩合”的方式组成的多肽链经过盘曲折叠形成的具有一定空间结构的物质;蛋白质可以是干扰素、蛋白质激素、细胞因子、趋化因子或者酶类等。
稀释剂是本领域技术人员可知的任意可以药用的水溶性辅料,包括:氨基酸、单糖、二糖、三糖、四糖、五糖、其它寡聚糖、甘露醇、右旋糖苷、氯化钠、山梨醇、聚乙二醇、磷酸盐,或其衍生物等或其组合。
带电脂质化合物是指一类脂质化合物以带正电荷或带负电荷的形式存在;其所带电荷不依赖于生理学范围内的pH,例如pH 3~9,不受pH的影响。带电脂质可以是合成的或天然来源的。带电脂质的实例包括但不限于DOTAP、DOTMA、18PA。这里并非穷举,只要是采用本发明处方的LNP组合物均在本发明的保护范围内,均受本发明启示。
助脂质包括:磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂(SM)、甾醇及其衍生物、神经酰胺、带电脂质中的一种或几种的组合;磷脂酰胆碱作为一种优选包括:DSPC,DPPC,DMPC,DOPC,POPC;磷脂酰乙醇胺作为一种优选为DOPE;甾醇作为一种优选为胆固醇;这里并非穷举,只要是采用本发明处方的LNP组合物均在本发明的保护范围内,均受本发明启示。
结构脂质包括:胆固醇、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番
茄碱、番茄碱、熊果酸、α~生育酚或皮质类固醇中的一种或几种。这里并非穷举,结构脂质的选择不受限制。
聚合物缀合脂质为聚乙二醇化脂质;作为一种实施例,聚乙二醇化脂质包括:PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油或PEG修饰的二烷基甘油中的一种或多种。这里并非穷举,聚合物缀合脂质的选择不受限制。
DSPC:英文名称:distearoyl Phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine;中文名称:二硬脂酰基卵磷脂,CAS号:816-94-4。
DPPC:中文名称:二棕榈酸磷脂酰胆碱;英文名称:1,2-dipalmitoyl-sn-glycero-3-phosphocholine,CAS号:63-89-8。
DMPC:中文名称:二肉豆蔻酰磷脂酰胆碱;英文名称:1,2-dimyristoyl-sn-glycero-3-phosphocholine,CAS号:18194-24-6。
DOPC:中文名称:1,2-二油酰基-sn-甘油-3-磷酸胆碱;英文名称:1,2-dioleoyl-sn-glycero-3-phosphocholine,CAS号:4235-95-4。
POPC:中文名称:2-油酰-1-棕榈锡甘油-3-磷酸胆碱;英文名称:2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine,CAS号:26853-31-6。
DOPE:中文名称:1,2-二油酰-SN-甘油-3-磷酰乙醇胺;英文名称:1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,CAS号:4004-05-1。
DOTAP:中文名称:1,2-二油酰基-3-三甲基铵-丙烷(氯盐);英文名称:1,2-dioleoyl-3-trimethylammonium-propane(chloride salt),CAS号:132172-61-3;化学结构式如下所示:
DOTMA:中文名称:N,N,N-三甲基-2,3-双(十八碳-9-烯-1-基氧基)丙-1-铵氯化物,CAS号:1325214-86-5,化学结构式如下所示:
18PA:CAS号:108392-02-5,化学结构式如下所示:
SM:中文名称:鞘磷脂(SM);英文名称:sphingomyelin。
PEG:中文名称:聚乙二醇;英文名称:Polyethylene glycol。
缓冲溶液:缓冲溶液指的是由弱酸及其盐、弱碱及其盐组成的混合溶液,能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响,从而保持溶液的pH值相对稳定;举例包括:磷酸、柠檬酸、碳酸、醋酸、醋酸-醋酸钠、醋酸-醋酸铵、巴比妥酸、Tris、PBS、HEPEs、EDTA及其钠盐等,或其组合,此处并非穷举。
HEPEs缓冲液:中文名称:4-羟乙基哌嗪乙磺酸;英文名称:2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;CAS号:7365-45-9。
Tris缓冲液:中文品名为三羟甲基氨基甲烷;氨基丁三醇;缓血酸胺;2-氨基-2-(羟甲基)-1,3-丙二醇;CAS号:77-86-1。
柠檬酸盐缓冲液:柠檬酸盐缓冲液主要成分为柠檬酸和磷酸氢钠。
DPBS:杜氏磷酸盐缓冲液。
TBST:由Tris-HCl,NaCl,tween20组成而成。
EDTA钠盐:其中EDTA二钠二水合物为乙二胺四乙酸二钠二水合物,英文名:EDTA disodium salt dihydrate。
PBS缓冲液:主要成分为Na2HPO4、KH2PO4、NaCl和KCl。
冻存保护剂:冻存保护剂是指可以保护细胞免受冷冻损伤的物质(常为溶液);可以分为:糖、醇类、氨基酸类、盐类等;具体包括:蔗糖、甘露醇、海藻糖、乳糖、葡萄糖、麦芽糖、聚乙烯吡咯烷酮(PVP)、聚乙二醇、葡聚糖、白蛋白以及羟乙基淀粉。
除非特别说明,本申请中的术语“粒度”和“水动力学直径”、“水动力学粒径”可以互换使用,均指水动力学直径。
以下结合附图和具体实施例对本发明作具体的介绍。
本发明通过采用以下实验验证本发明通用型处方的技术效果:
实验材料:
实验一:乙醇作为表面张力下降调节剂加入脂质纳米粒(LNP)分散体系中对雾化前后LNP的粒度、分散度(PDI)和包封率(EE)变化的影响实验:
1.1 LNP在不同乙醇含量的分散体系中雾化前后的粒度、PDI和EE变化实验:
实施例1LNP处方(摩尔比):以下LNP的配比为可离子化脂质化合物C12:DSPC:胆固醇:DMG-PEG=46.3:9.4:42.7:1.6。
实验方法:用微流控设备制备实施例1LNP,其中实施例1LNP处方用无水乙醇溶解,mRNA用醋酸钠buffer稀释,醇水相的体积比为1比3,通过调节流速,使制备出的LNP粒度分布在110~140nm。制备结束后,孵育20min,然后置于含10%蔗糖的pH=6的20mM HEPEs缓冲溶液(buffer)中透析,然后分别取出50μL,用不同乙醇含量的上述buffer稀释3倍,用振动网筛式雾化装置雾化(型号Aerogen solo Nebulizer System),测试雾化前后的粒度、PDI和EE。
实验结果如表1和图1所示:
表1 LNP在不同乙醇含量的分散体系中雾化前后的粒度、PDI和EE变化
实验结论:对于实施例1的LNP,若分散体系中不含乙醇,则雾化后粒度增大近一倍,PDI增大至0.5以上,且EE下降明显,若体系中包含乙醇,则可有效降低雾化的破坏作用,可保证LNP雾化前后粒度、PDI和EE变化较小,且随着乙醇含量的增加,变化的也更小。对于该处方,
分散体系中含有0.01%~30%的乙醇均合适。
1.2对于实施例1的LNP,我们选定分散体系中乙醇含量为6.5%,进一步研究了雾化前后粒度、PDI和EE没有变化的适用的粒度范围。
实验方法:用微流控设备制备实施例1LNP,其中实施例1LNP的脂质处方用无水乙醇溶解,mRNA用醋酸钠buffer稀释,醇水相的体积比为1比3,通过调节流速,制备出不同粒度大小的LNP。制备结束后,孵育20min,然后置于含10%蔗糖的pH=6的20mM HEPEs buffer中透析,然后分别取出50μL,用含乙醇的buffer稀释3倍,雾化,测试雾化前后的粒度、PDI和EE。
实验结果如表2和图2所示:
表2在固定乙醇含量的分散体系中,不同粒度的LNP在雾化前后其粒度、PDI和EE变化
实验结论:不同粒度的LNP在乙醇含量为6.5%的HEPEs分散体系中雾化前后粒度、PDI和EE有差别,其中粒度在70~160nm之间各个参数变化较小,小于70nm时,例如65.3nm时,雾化后粒度增加近一倍,PDI增加至0.349,且EE降低明显,所以当LNP粒度分布在70~160nm之间时,可以更好的保持雾化后的稳定性。
1.3固定体系中乙醇含量,研究不同浓度的LNP在雾化前后的粒度、PDI和EE变化。
实验方法:用微流控设备制备实施例1LNP,其中实施例1LNP的脂质处方用无水乙醇溶解,mRNA用醋酸钠buffer稀释,醇水相的体积比为1比3,通过调节流速,制备出粒度大小分布在70~160nm的LNP。制备结束后,孵育20min,然后置于含10%蔗糖和7%乙醇的pH=6的20mM HEPEs buffer中透析,然后分别取出150μL,不稀释雾化,或用含乙醇7%的buffer稀释不同的倍数雾化,测试雾化前后的粒度、PDI和EE。
实验结果如表3和图3所示:
表3在10%的乙醇体系中,不同浓度的LNP雾化前后的粒度、PDI和EE。
实验结论:在7%乙醇含量体系,在mRNA浓度高达2.5μg/μL(对应的LNP质量体积分数为10%)时雾化是稳定的,若稀释一下雾化会更稳定,其中在mRNA浓度为0.5μg/μL时和0.005μg/μL时一样稳定,其中mRNA浓度为0.005μg/μL对应的LNP的质量体积分数为0.0025%,即当分散体系中LNP的质量体积百分比为0.0025%~10%时雾化均是稳定的。
实验二:验证本发明处方的通用性实验
通过更换LNP组合物中的组分,但其分散体系仍然是添加了表面张力下降调节剂,研究LNP雾化的耐受性,证明本处方的通用性。
实验方法:更换可离子脂质化合物和组合物的配方如实施例2~4LNP所示,选择商业化的DLin-MC3-DMA(简写为MC3)作为对比实施例LNP,处方摩尔比如表4所示,微流控制备参数为:前端废液0.3ml,末端废液0.05mL,总体积0.75mL,总流速2~8mL/min,体积比:3比1。使其粒度分布在70~160nm,在HEPEs中透析,取出,各取50μL LNP,然后加入100μL含乙醇的HEPEs buffer稀释,使其体系中含有不同浓度的乙醇,雾化,测试雾化前后的粒度、PDI和EE。
表4各个实施例LNP的配方摩尔比
实验结果如表5和图5所示:
表5各个LNP在不同含量乙醇的分散体系中雾化前后的粒度、PDI和EE。
备注:a代表分散体系中乙醇含量,b代表未检测。
实验结论:对于不同种类的LNP或不同的LNP处方,只要粒度在70~160nm范围,都可以通过调节分散体系中乙醇的含量实现雾化前后粒度、PDI和EE保持不变;对于不同的LNP,保持雾化前后一致所需的乙醇含量不同,一般不超过30%。
实验三:小鼠雾化吸入LNP后肺部的表达情况
实验方法:用微流控的方法制备实施例1LNP,包载Luciferase mRNA,然后让小鼠雾化吸入,6小时候后腹腔注射荧光素钾底物,5分钟后将小鼠处死,取其心肝脾肺肾成像如图4所示。
实验结论:LNP在引入了乙醇的buffer中进行雾化其转染效果明显高于不含乙醇的buffer体系,含乙醇buffer的LNP的荧光定量值高于不含乙醇buffer的LNP多个数量级,说明该处方可以在肺部实现有效沉积且可以耐受雾化过程对其的剪切破坏,非常有效。
实验四:其他表面张力下降调节剂通过降低水的表面张力对LNP雾化前后粒度和PDI稳定性的影响。
实验方法:用微流控设备制备实施例1LNP,其中脂质处方用无水乙醇溶解,mRNA用醋酸钠buffer稀释,醇水相的体积比为1比3,通过调节流速,制备出粒度大小为130nm左右的LNP。制备结束后,孵育20min,然后置于含10%蔗糖的pH=6的20mM HEPEs buffer中透析,然后分别取出50μL,通过用含泊洛沙姆188、吐温-80、苯乙醇、丙二醇、甘油或乙醇的HEPEs buffer的一种或两种组合稀释3倍,雾化,测试雾化前后的粒度和PDI,表面张力下降调节剂为泊洛沙姆188或乙醇时,实验结果如表6和图6,表面张力下降调节剂为吐温或乙醇时,实验结果如
表7和图7。
表6当分散体系中添加泊洛沙姆188时,LNP雾化前后粒度和PDI的变化
实验结论:引入表面张力下降调节剂泊洛沙姆188可以提高雾化的稳定性,对于C12~LNP,当表面张力下降调节剂为泊洛沙姆188时,在浓度为0.5~10mg/mL范围内,粒度、PDI和EE均没有发生巨大变化,当泊洛沙姆188的浓度为5~10mg/mL时,雾化前后的LNP各个参数变化最小,且若体系中引入4%的乙醇,则浓度在5mg/mL和2mg/mL均是稳定的,所以乙醇和泊洛沙姆188的联用在提高雾化前后粒度和PDI的稳定性上具有意想不到的协同作用。
表7当分散体系中添加吐温-80时,LNP雾化前后粒度和PDI的变化
实验结论:作为表面张力下降调节剂加入LNP分散体系中的吐温-80,也会使雾化前后LNP粒度保持稳定,在吐温-80体积浓度为0.01%~2%范围内,雾化后粒度没有明显增大,由于本身吐温-80会自组装形成胶束,所以造成粒度仪检测的PDI较大,但LNP本身的PDI和
雾化前相比几乎没有改变。另外,且随着吐温-80含量的增加,粒度增大的也越不明显。
表8当用4mL/min的总流速制备较大粒度的LNP时,在不同浓度的吐温-80或吐温-80与乙醇
联用的体系雾化前后粒度、PDI和EE的表征
表9当用12mL/min的总流速制备较小粒度的LNP时,在不同浓度的吐温-80或吐温-80与乙醇联用的体系雾化前后粒度、PDI和EE的表征
实验结论:对于两个不同粒度的LNP,例如表8中雾化前LNP的粒度为112.2nm,表9中LNP的粒度为64.5nm,加入吐温-80雾化前均发生粒度减少现象;对于较大粒度的LNP,即112.2nm的LNP,引入吐温或吐温与乙醇联用雾化后的结果相对于小粒度的LNP,即64.5nm,其雾化前后变化较小,所以粒度分布在70~160nm之间的LNP可以更好的耐受雾化过程的破坏作用,另外,乙醇和吐温-80的联用也可以起到增强雾化稳定性的作用。
实验五:在不含蔗糖的HEPEs分散体系,引入表面张力下降调节剂,看对其雾化稳定性的影响。
表10在不含蔗糖的HEPEs buffer中引入各个表面张力下降调节剂对LNP雾化稳定性的影响。
实验结论:1)采用除了乙醇以外的其他表面张力下降调节剂,比如:泊洛沙姆188、吐温-80、丙二醇、苯乙醇、甘油,或者几种表面张力下降调节剂的组合也能够提高雾化前后LNP粒度和PDI稳定性的影响,当丙二醇的质量体积浓度在0.05%~30%的范围内,均可以保持雾化前后粒度、PDI和EE的较小变化。
2)若分散体系中不含蔗糖,可以降低分散体系的粘度,可以在更低的表面张力下降调节剂浓度下实现雾化前后粒度和EE的一致性。
实验六:引入表面张力下降调节剂HS15情况下雾化稳定性的影响
1)实验目的
C12作为可电离脂质,包封luc mRNA,采用微流控制备仪制备LNP,用于雾化LNP研究。添加聚氧基-15羟基硬脂酸酯(简称为HS15,市售来源),实验步骤及方法同实验一。
2)实验结果
表11 LNP雾化前后的粒径和包封率
备注:“pre”和“post”分别表示雾化前和雾化后的样品。HS 15浓度分别为0.1mg/ml、2mg/ml、5mg/ml和10mg/ml,对照组不加入HS15。
结论:若分散体系中不含HS15,则雾化前后粒径明显增大,PDI、EE及完整性均发生大幅度下降,明显受到雾化的破坏作用。而添加0.1-10mg/ml的HS15后,雾化前后的各值均未发生明显变化,尤其是mRNA的完整性几乎不受影响,从而保证递送的mRNA可以在体内更好地发挥治疗效果。
3)稳定性研究
对表11中制备的样品进行稳定性研究,存放于4℃冰箱中,检测样品放置时间为7天时的粒径。
表12稳定性样品信息
结果显示,在存放一段时间后,样品的粒径没有明显变化,说明引入HS15后不会影响所得到的LNP的存放稳定性。
4)雾化时间研究
我们进一步考察了HS15的加入对雾化时间和过程的影响,其中,2%乙醇+10%丙二醇在表格中简写为2+10,即为表10中的2%乙醇+10%丙二醇处方。
表13雾化现象
结论:在雾化过程中发现,HS15的加入几乎对雾化时间没有影响,且整个雾化过程比较顺畅。和其它含表面张力调节剂的体系如2%乙醇+10%丙二醇相比,雾化时间缩短了近3倍。因雾化时间过长,会对制剂稳定性造成不良影响,且在开发成雾化制剂后容易有患者依从性不佳的问题,而加入HS15后可以克服上述缺陷,因此具备优异的技术效果。
本发明通过在LNP的分散体系中引入可以降低水的表面张力的分子,意外的发现,使用这样的处方制备得到的LNP能够耐受雾化过程中的剪切力,在雾化前后粒度和PDI保持不变,同时LNP包载的核酸不会泄露,可以实现雾化前后LNP的EE保持不变;雾化前后的LNP在肺部具有较高的沉积率和较高的基因转染效果,突破了市场雾化吸入LNP无法耐受雾化剪切力,稳定性差的技术壁垒;实现了意想不到的技术效果。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解,上述实施例不以任何形式限制本发明,凡采用等同替换或等效变换的方式所获得的技术方案,均落在本发明的保护范围内。
Claims (15)
- 一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,在脂质纳米粒分散体系中加入表面张力下降调节剂能增加脂质纳米粒耐受雾化剪切的能力。
- 根据权利要求1所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,包括:脂质纳米粒分散体系和加入在所述脂质纳米粒分散体系中的表面张力下降调节剂;所述脂质纳米粒的粒度范围为70~160nm;所述表面张力下降调节剂在脂质纳米粒分散体系中的质量体积百分比范围为0.01%~30%。
- 根据权利要求2所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述脂质纳米粒分散体系包括:脂质纳米粒、体系溶液;所述脂质纳米粒在分散体系中的质量体积百分比范围为0.0025%~10%,所述体系溶液的浓度范围为0~1000mM。
- 根据权利要求3所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述脂质纳米粒分散体系包括:脂质纳米粒、体系溶液、冻存保护剂;所述脂质纳米粒在分散体系中的质量体积百分比范围为0.0025%~10%,所述体系溶液的浓度范围为0~1000mM,所述冻存保护剂的质量体积百分比范围为0%~20%。
- 根据权利要求3所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述脂质纳米粒按照摩尔比包括:可离子化脂质化合物:助脂质:结构脂质:聚合物缀合脂质=30~60:5~40:20~40:0.1~15。
- 根据权利要求3所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述体系溶液包括:生理盐水、4-羟乙基哌嗪乙磺酸HEPEs缓冲液、三羟甲基氨基甲烷Tris缓冲液、Tris-EDTA缓冲液、磷酸PB和磷酸盐PBS缓冲液、Dulbecco's磷酸盐DPBS缓冲液、柠檬酸盐缓冲液、硫酸盐缓冲液、碳酸盐缓冲液、醋酸盐缓冲液、含吐温的Tris缓冲液TBST、含EDTA及其钠盐的缓冲液,及上述中的一种或几种的组合。
- 根据权利要求1所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂包括:乙醇、丙二醇、苯乙醇、泊洛沙姆188、吐温-80、甘油、聚氧基-15羟基硬脂酸酯中一种或几种的混合物。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为乙醇,乙醇在脂质纳米粒分散体系的质量体积百分比范围为0.05~30%。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为丙二醇,丙二醇在脂质纳米粒分散体系中的质量体积百分比范围为 0.05~30%。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为丙二醇和乙醇的组合物,丙二醇在脂质纳米粒分散体系的质量体积百分比范围为0.05~30%,乙醇在脂质纳米粒分散体系的质量体积百分比范围为0.05~30%。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为泊洛沙姆188,泊洛沙姆188在脂质纳米粒分散体系的浓度范围为0.5~10mg/mL。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为泊洛沙姆188和乙醇的混合物,泊洛沙姆188在脂质纳米粒分散体系的浓度范围为0.5~10mg/mL,乙醇在脂质纳米粒分散体系的质量体积百分比范围为0.05~30%。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为吐温-80,吐温-80在脂质纳米粒分散体系的质量体积百分比范围为0.01~2%。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为吐温-80和乙醇的混合物,吐温-80在脂质纳米粒分散体系的质量体积百分比范围为0.01~2%,乙醇在脂质纳米粒分散体系的质量体积百分比范围为0.05~30%。
- 根据权利要求7所述的一种用于雾化吸入脂质纳米粒的通用型处方,其特征在于,所述表面张力下降调节剂为聚氧基-15羟基硬脂酸酯,聚氧基-15羟基硬脂酸酯在脂质纳米粒分散体系的浓度范围为0.05-20mg/ml,优选的为0.1-10mg/ml,进一步优选的为3-7.5mg/ml。
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