Patent Application
20240099993
A copy of the Sequence Listing is submitted with the specification electronically via EFS-Web as an ASCII formatted sequence listing with a file name of “074844-8006US02-SL-20201208_ST25”, a creation date of Dec. 8, 2020, and a size of 1,601 bytes. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
The present application generally relates to a delivery means of nucleic acid therapy, more particularly, relates to a more efficient delivery carrier and delivery means.
Nucleic acid, as the main genetic material of living organisms, also has its unique potential for drug development. Currently, there are 6 types of nucleic acid drugs approved by the FDA, including Formavirsen (ISIS PHARMS INC, NDA: 20-961), mainly for the treatment of cytomegalovirus retinitis; Pagatanib (VALEANT PHARMS LLC, NDA: 21-756), mainly for neovascular age-related macular degeneration; Mipomethan (KASTLE THERAPS LLC, NDA: 203458), commonly for Homozygous familial hypercholesterolemia; Exondys 51 (SAREPTA THERAPS INC, NDA: 206488), for Duchenne muscular dystrophy; defibrotide-sodium (JAZZ PHAEMS INC, NDA: 208114), for the treatment of hepatic vein occlusive disease with renal or pulmonary dysfunction after hematopoiesis; Nusinersen (BIOGEN IDEC, NDA: 209531), for the treatment of spinal muscular atrophy; Patisiran (ALNYLAM PHARMS INC, NDA: 210922), for amyloidosis related to hereditary hyperthyroidism protein. These drugs are all administered by injection, and the drug delivery efficiency is relatively low.
From our previous research, there are hundreds of single lipids in the herbal decoctosome. In the further experiments of the present application, it was found that sphingosine lipids had better efficiency in the delivery process. Therefore, a series of sphingosine lipids and related derivatives had been artificially synthesized to specifically explore the delivery effect of sphingosine lipids. It was concluded that sphingosines and derivative lipids thereof could effectively deliver small RNAs.
The present application is partly based on the inventors' discovery of a series of single sphingosine lipid. It was found that single sphingosine can be used as a carrier to efficiently deliver small RNA into cells.
The present application discovered that single sphingosine lipids can efficiently deliver small RNAs as drugs into cells without any toxicity to the body and significantly improve the delivery efficiency and medicinal value of nucleic acid drugs.
The sphingosine lipids and its single derivative can effectively deliver sRNA into cells. Functional small RNA with fluorescent FAM label (sRNA-PGY6) was used as a marker to detect the fluorescence shift after delivery to cells. Compared with free uptake, the experimental group of sphingosine lipid and its single derivative delivering small RNA had significant fluorescence shift, indicating that bencaosome formed by the sphingosine lipid and its single derivative can more effectively deliver small RNAs into cells to perform corresponding functions.
The present application provides the following contents:
The term as used herein may have a single dash “-” (or horizontal line) or a double dash “=” in front of and/or behind it to indicate the bond level of the bond between the mentioned substituent and its parent moiety; a single dash “-” (or horizontal line) refers to a single bond, and a double dash refers to a double bond; in the absence of single or double dash, it is understood that a single bond is formed between the substituent and its parent moiety; in addition, the substituent is to be construed “from left to right” unless otherwise indicated.
A dashed line (“-”) that is not between two letters or symbols is used to represent the linking points of substituents. For example, —C(O)NH2 is linked through a carbon atom. The dashed line before or after a chemical group is for convenience. The chemical group can be depicted with or without one or more dashed lines without losing its usual meaning. The wavy line drawn through the lines in the structure represents the linking points of the groups.
When a range of values is listed, it is intended to include every value and subrange within the range. For example, “C1-6alkyl” is intended to include C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5 and C5-6alkyl groups.
The term “alkyl” as used herein refers to a straight or branched saturated hydrocarbon chain. As described herein, an alkyl group has 1 to 24 carbon atoms (i.e., C1-24alkyl), 1 to 20 carbon atoms (i.e., C1-20alkyl), 1 to 8 carbon atoms (i.e., C1-8alkyl), 1 to 6 carbon atoms (i.e., C1-6alkyl), or 1 to 4 carbon atoms (i.e., C1-4alkyl). In one embodiment, the alkyl group is a C1-6alkyl group. In one embodiment, the alkyl group is a C14-20alkyl group. In one embodiment, the alkyl group is a straight C14, C15, C16, C17, C18, C19, C20alkyl group.
The term “alkenyl” as used herein refers to an aliphatic hydrocarbon chain having specified number of carbon atoms and containing at least one carbon-carbon double bond. As described herein, an alkenyl group has 2 to 24 carbon atoms (i.e., C2-24alkenyl), 2 to 20 carbon atoms (i.e., C2-20alkenyl), 2 to 8 carbon atoms (i.e., C2-8alkenyl), 2 to 6 carbon atoms (i.e., C2-6alkenyl), or 2 to 4 carbon atoms (i.e., C2-4alkenyl). In one embodiment, the alkenyl group is a C14-20alkenyl group. In one embodiment, the alkenyl group is a C14, C15, C16, C17, C18, C19, C20alkenyl group.
As used herein, the term “acyl” refers to group —CO—.
As used herein, the term “phosphate ester” refers to group
As used herein, the term “biotin acyl” refers to group
As used herein, the term “glycosyl” refers to a monovalent substituent obtained by removing the hemiacetal hydroxyl group from the cyclic form of a monosaccharide. For example, the term “1-β-D-glucosyl” refers to group
Examples of glycosyl include, but are not limited to, glucosyl, lactosyl, galactosyl, mannosyl, fructosyl, and sorbosyl. In one embodiment, the glycosyl is β-D-glucosyl.
The term “pharmaceutically acceptable salt” refers to the salt, within the scope of reasonable medical judgment, suitable for use in contact with tissues of human and lower animals without improper toxicity, stimulation, allergic reactions, etc. and commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, pages 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed from amino with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by using other methods known in the art (for example, ion exchange methods). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentane propionate, digluconate, lauryl sulfate, ethane sulfonate, formate, fumarate, gluceptate, glycerophosphate, gluconate, hemisulfate, enanthate, caproate, hydroiodide, 2-hydroxy-ethane sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, methyl sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from appropriate bases include alkali metal salts, alkaline-earth metal salts, ammonium salts, and N+(C1-4alkyl)4−salts. Representative alkali metal or alkaline-earth metal salts include sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt and the like. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halogen, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkylsulfonate and arylsulphonate.
The term reverse evaporation method as described herein refers to adding an aqueous solution of nucleic acid to a solution of lipid in an organic solvent, ultrasonicating, evaporating to remove the organic solvent, and then hydrating to obtain a lipid nucleic acid mixture.
The term “boiling method” (also refers to “heating method”) as described herein refers to adding an organic solvent solution of lipid to an aqueous solution of nucleic acid and boiling at about 100° C. for 30 minutes to obtain a lipid nucleic acid mixture. The method is not limited to heating by boiling, and other means of heating or raising temperature known in the art can also be used.
Reverse evaporation method and boiling method are carried out under controlled temperature and mixing conditions. Suitable processing times, and temperatures can be readily determined by a person skilled in the art. For example, the temperature of reverse evaporation method is ranged preferably from about 25° C. to about 70° C., more preferably from about 30° C. to about 65° C., and more preferably from about 40° C. to about 60° C., especially about 55° C. The temperature of boiling method is ranged preferably from about 25° C. to about 100° C., more preferably from about 50° C. to about 100° C., and more preferably from about 95° C. to about 10° C., especially, preferably from about 80° C. to 100° C.
The nucleic acid as described herein comprises DNA and RNA, preferably small RNA, for example, the small RNA having a length of 14-32 bp, 16-28 bp, 18-24 bp, and particularly, a length of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 bp.
In the present application, a nucleic acid can be delivered to a cell or a subject by mixing compounds or combination or composition containing one or more of the compounds and the nucleic acid. In one embodiment, the compound has a structure of Formula (I), a stereoisomer thereof or a pharmaceutical acceptable salt thereof,
In one embodiment, the substituents of Formula (I) are as defined above. The compound may be a compound in Table 1. The combination or composition comprising the compound may include any one or more of the compounds in Table 1. Those skilled in the art can add or delete one or more compounds in the combination or composition as needed.
In one embodiment, the combination or composition herein is a combination comprising any one or more of No. 41, No. 38, No. 48. In one embodiment, a combination comprising any one or more of No. 41, No. 38, No. 48 and any one or more of the compound selecting from item 11. In one embodiment, a combination comprising the following: No. 41+No. 38+sphingosine derivative So-1; No. 41+No. 38+sphingosine derivative So-2; No. 41+No. 38+sphingosine derivative So-3; No. 41+No. 38+sphingosine derivative So-4; No. 41+No. 38+sphingosine derivative So-5; No. 41+No. 38+sphingosine derivative So-6; No. 41+No. 38+sphingosine derivative So-7; No. 41+No. 38+sphingosine derivative So-8; No. 41+No. 38+sphingosine derivative So-9; No. 41+No. 38+sphingosine derivative So-10; No. 41+No. 38+sphingosine derivative So-11; No. 41+No. 38+sphingosine derivative So-12; No. 41+No. 38+sphingosine derivative So-13; No. 41+No. 38+sphingosine derivative So-14; No. 41+No. 38+sphingosine derivative So-15; No. 41+No. 38+sphingosine derivative So-45; No. 41+No. 38+sphingosine derivative So-46; No. 41+No. 38+sphingosine derivative So-47; No. 41+No. 38+sphingosine derivative So-48; No. 41+No. 38+sphingosine derivative So-49; No. 41+No. 38+sphingosine derivative So-50; No. 41+No. 38+sphingosine derivative So-51; No. 41+No. 38+sphingosine derivative So-52; No. 41+No. 38+sphingosine derivative So-53; No. 41+No. 38+sphingosine derivative So-54; No. 41+No. 38+sphingosine derivative So-55; No. 41+No. 38+sphingosine derivative So-56; No. 41+No. 38+sphingosine derivative So-57; No. 41+No. 38+sphingosine derivative So-58; No. 41+No. 38+sphingosine derivative So-59; No. 41+No. 38+sphingosine derivative So-60; No. 41+No. 38+sphingosine derivative So-61; No. 41+No. 38+sphingosine derivative So-62; No. 41+No. 38+sphingosine derivative So-63; No. 41+No. 38+sphingosine derivative So-64; No. 41+No. 38+sphingosine derivative So-65; No. 41+No. 38+sphingosine derivative So-66; No. 41+No. 38+sphingosine derivative So-67; No. 41+No. 38+sphingosine derivative So-68; No. 41+No. 38+sphingosine derivative So-69; No. 41+No. 38+sphingosine derivative So-70; No. 41+No. 38+sphingosine derivative So-71; No. 41+No. 38+sphingosine derivative So-72; No. 41+No. 38+sphingosine derivative So-73; No. 41+No. 38+sphingosine derivative So-74; No. 41+No. 38+sphingosine derivative So-75; No. 41+No. 38+No. 48+sphingosine derivative So-42; No. 41+No. 38+No. 48+sphingosine derivative So-43; No. 41+No. 38+No. 48+sphingosine derivative So-44; No. 41+No. 38+No. 48+sphingosine derivative So-45; No. 41+No. 38+No. 48+sphingosine derivative So-46; No. 41+No. 38+No. 48+sphingosine derivative So-47; No. 41+No. 38+No. 48+sphingosine derivative So-52; No. 41+No. 38+No. 48+sphingosine derivative So-56; No. 41+No. 38+No. 48+sphingosine derivative So-57; No. 41+No. 38+No. 48+sphingosine derivative So-58; No. 41+No. 38+No. 48+sphingosine derivative So-59; No. 41+No. 38+No. 48+sphingosine derivative So-60; No. 41+No. 38+No. 48+sphingosine derivative So-61; No. 41+No. 38+No. 48+sphingosine derivative So-62; No. 41+No. 38+No. 48+sphingosine derivative So-63; No. 41+No. 38+No. 48+sphingosine derivative So-64; No. 41+No. 38+No. 48+sphingosine derivative So-67; No. 41+No. 38+No. 48+sphingosine derivative So-68; No. 41+No. 38+No. 48+sphingosine derivative So-69; No. 41+No. 38+No. 48+sphingosine derivative So-70; No. 41+No. 38+any one or more of sphingosine derivative So-23, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 70, 71 or 73. Those skilled in the art can select the appropriate concentration and use volume. Preferably, the compounds above are used at concentrations as shown in Table 1. The ratio of the compounds above is 0.1-10:0.1-10, 0.2-9:0.2-9, 0.3-8:0.3-8, 0.4-7:0.4-7, 0.5-6:0.5-6, 0.6-5:0.6-5, 0.7-4:0.7-4, 0.8-3:0.8-3, 0.9-2:0.9-2; more preferably 1:1, or any ratio between them. Those skilled in the art can appropriately adjust the ratio of various compounds according to the concentration of the mother liquor of the compounds. In addition, the present application proved that a specific lipid combination can effectively promote the delivery of nucleic acids, and the effect is better than that of a single lipid.
In one embodiment, the nucleic acid is synthetic or purified, therapeutic or non-therapeutic, and/or diagnostic or non-diagnostic, for example selected from RNA or DNA, for example selected from single-stranded or double-stranded or partially double-stranded RNA or DNA. When the nucleic acid is therapeutic or diagnostic, the nucleic acid is used to treat or diagnose a disease selected from the group consisting of: inflammatory diseases, pneumonia, myocarditis, acute and chronic gastritis, acute and chronic enteritis, acute and chronic hepatitis, acute and chronic nephritis, dermatitis, encephalitis, lymphitis, conjunctivitis, keratitis, iridocyclitis, otitis media, allergic rhinitis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease, allergic dermatitis, sickle cell disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, lung cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cervical cancer, breast cancer, leukemia, multiple myeloma, diabetes, and gout. Those skilled in the art can select a suitable nucleic acid according to the specific situation. For example, the RNA can be messenger RNA (mRNA), rRNA (ribosomal RNA), tRNA (transfer RNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small cytoplasmic RNA, small RNA, transfer-messenger RNA (tmRNA), telomerase RNA and antisense RNA, preferably small RNA.
In one embodiment, the delivery comprises treating the compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof or a combination comprising them by heating method, reverse evaporation method, direct mixing, repeated freeze-thaw and/or thin film dispersion. Those skilled in the art can select a suitable method for delivery. In one embodiment, the heating method is conducted at a temperature of about 0° C. to about 100° C., about 25° C. to about 100° C., preferably about 80° C. to about 100° C., for example 4° C., 37° C., 60° C., 80° C. or 100° C.; the reverse evaporation method is conducted at a temperature of about 25° C. to about 70° C., preferably about 55° C. for a heating time of about 0 minutes to about 24 hours, about 5 minutes to about 20 hours, about 5 minutes to about 16 hours, about 5 minutes to about 10 hours, about 5 minutes to about 4 hours, or about 10 hours to about 1 hour, preferably 15 minutes.
In one embodiment, the nucleic acid is treated with the compound, and the treated mixture can be directly administered to the subject via oral administration. In addition, the subject can also be administered by other means, for example, intravenous administration such as injection or infusion, subcutaneous administration. intramuscular administration, intraarterial administration, intraperitoneal administration, intrapulmonary administration, intracerebral and intraspinal administration, intra-articular administration, intrasynovial administration, intrathecal administration, intra-trauma administration, and/or administration via inhalation paths such as intranasal, typically intravenous or subcutaneous administration.
The invention also provides combinations, compositions or kits of compounds, which comprise any compound described herein, such as any one or more of the compounds in Table 1. Those skilled in the art can add other compounds to the combinations, compositions or kits of compounds as needed, as long as the function of delivering nucleic acid is retained.
The human embryonic kidney cell line HEK293T and human monocyte THP-1 used in the experiment were purchased from the Cell Culture Center of Peking Union Medical College. The cells were cultured in a 37° C., 5% CO2 incubator, wherein HEK293T cells were cultured in DMEM medium (HyClone) and THP-1 cells were cultured in RPMI-1640 medium (HyClone), each medium containing 10% fetal bovine serum and a certain proportion of antibiotics (penicillin 100 U/ml & streptomycin 100 mg/ml). The cells were cultured to logarithmic growth phase, and the cell density was 6×105/1 mL medium/well. The 12-well plate (1 mL medium/well) was incubated overnight (12 h) at 37° C., followed by subsequent experiments.
To the cells cultured in a 12-well plate (about 1×106cells/well) was added 1 mL TRIzol lysis buffer in each well, and then placed on ice. After all the samples were added TRIzol, they were allowed to stand at room temperature for 5 min to be fully lysed.
Centrifugation was conducted at 4° C., 12,000 rpm for 5 min. The precipitates were discarded and TRIzol was transferred to a fresh centrifuge tube;
Chloroform was added at a ratio of 200 μL chloroform/mL TRIzol, shaken well and mixed. The mixture was placed at room temperature for 5 min;
Centrifugation was conducted at 4° C., 12,000 rpm for 15 min;
The upper aqueous phase was pipetted into another centrifuge tube, and isopropanol was added at a ratio of 0.5 mL isopropanol/mL TRIzol. The mixture was placed at room temperature for 5-10 min;
Centrifugation was conducted at 4° C., 12,000 rpm for 15 min. The supernatant was discarded, and the RNA precipitates to the bottom of the tube;
The tube was added 1 mL 75% ethanol and gently shaken to suspend the precipitates;
Centrifugation was conducted at 4° C., 12,000 rpm for 10 min. The supernatant was discarded. The tube was added 1 mL 75% ethanol acid gently shaken to suspend the precipitate;
Centrifugation was conducted at 4° C., 12,000 rpm for 10 min, and the supernatant was discarded. The DNA sample was dried at room temperature and dissolved with 50 μL RNase-free H2O. The RNA concentration was quantified by measuring the OD value.
Model building and transfection: artificially synthesized single lipid shown in Table 1, artificially synthesized 5′-FAM labeled sRNAs (single-stranded, Ribobio) shown in Table 2;
Conclusions: The results indicated that as compared to the free uptake group, the fluorescence value showed significant shift in the experimental group after delivery of fluorescently labeled nucleic acids by sphingosine lipids, indicating that different single sphingosine lipid derivatives were effective in delivering single-stranded small RNA into cells (see
Conclusions: The results indicated that as compared to the free uptake group, the fluorescence value showed significant shift in the experimental group after delivery of nucleic acids by sphingosine lipids (see
Conclusions: The results indicated that as compared to the free uptake group, the fluorescence value showed significant shift in the experimental group after delivery of nucleic acids by sphingosine lipids (see
Conclusions: The results indicated that as compared to the Naïve group and the negative control group, a significant increase in relative uptake amount of small RNA in cells was detected after delivery of 0.1 nmol nucleic acids by 12.5 μg of the lipid combination (No. 41: No. 38: So=1:1:1) containing sphingosine derivative in the experimental group, indicating that the addition of multiple single sphingosine lipid derivatives could greatly improve the efficiency of the delivery of lipid combinations.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/081155 | Mar 2018 | WO | international |
This application is a continuation application of U.S. application Ser. No. 17/042,931 filed Sep. 29, 2020, which is a continuation of PCT Patent Application No. PCT/CN2019/080519, filed on Mar. 29, 2019, which claims priority to PCT Application No. PCT/CN2018/081155, filed on Mar. 29, 2018, each of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17042931 | Sep 2020 | US |
| Child | 18506138 | US |
