Bio-inspired lipid derivatives and their use

Abstract

The present disclosure relates to compounds, compositions, and methods for delivering therapeutic, diagnostic, or prophylactic agents (eg, nucleic acids).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 067,030, filed on 18 August 2020, which is incorporated herein by reference in its entirety.

[0002] Statement on federally funded research This invention was made with the assistance of the United States Government through grant R35GM119679 from the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] This disclosure relates to compounds, compositions, and methods for the delivery of therapeutic, diagnostic, or prophylactic agents (e.g., nucleic acids). [Background technology]

[0004] Messenger RNA (mRNA)-based therapies have shown great promise for the expression of functional antibodies and proteins. Clinical studies are exploring mRNAs for use in gene therapy, immunotherapy, and vaccines. Efficient mRNA delivery is a critical step and challenge in mRNA therapy. Despite the promising data from ongoing clinical trials, the discovery and development of more efficient delivery systems is necessary for the clinical use of mRNA.

[0005] The compounds, compositions, and methods disclosed herein address these and other needs. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Provisional Application No. 63 / 067,030 [Overview of the project]

[0007] This disclosure provides compounds, compositions, and methods of using them. It also provides compositions containing the compounds and drugs (e.g., mRNA) of the present invention. Furthermore, this disclosure provides methods of using the compositions for target delivery of drugs.

[0008] In one embodiment, the present disclosure relates to a compound of formula A, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. Each R 8 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol. R 9 is hydrogen, alkyl, alkenyl, alkynyl, ester, alkyl ester, or [ka] Selected from, X is selected from O or N. m is an integer between 0 and 10.

[0009] Several embodiments, each R 8 is alkyl. In some embodiments, each R 8 is methyl. In some embodiments, each R 8 It is an alkyl alcohol.

[0010] In some embodiments, R 9 is hydrogen. In some embodiments, R 9 teeth [ka] That is the case.

[0011] In some embodiments, m is an integer from 1 to 3. In some embodiments, X is O. In some embodiments, X is N, and R 9 is hydrogen. In some embodiments, X is N, and R 9 is [Chemical formula] as follows.

[0012] In one aspect, the present disclosure provides a compound of formula I, namely, [[ID=)18]] [Chemical formula] or a salt thereof, wherein, each R 7 is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate, X is selected from O or N.

[0013] In one aspect, the present disclosure provides a compound of formula II, namely, [Chemical formula] or a salt thereof, wherein, each R 7 is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate, X is selected from O or N.

[0014] In one aspect, the present disclosure provides a compound of formula III, namely, [Chemical formula] or a salt thereof, wherein, each R 7This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate.

[0015] In one embodiment, the present disclosure relates to a compound of formula IV, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate.

[0016] In one embodiment, the present disclosure relates to a compound of formula V, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate.

[0017] In one embodiment, the present disclosure relates to a compound of formula VI, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. Each R 8 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol. X is selected from O or N.

[0018] In one embodiment, the present disclosure relates to a compound of formula VII, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. Each R 8 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol. X is selected from O or N.

[0019] In one embodiment, the present disclosure relates to a compound of formula VIII, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. Each R 8 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol.

[0020] In one embodiment, the present disclosure relates to a compound of formula IX, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. Each R 8This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol.

[0021] In one embodiment, the present disclosure relates to a compound of formula X, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. Each R 8 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol.

[0022] In some embodiments, R 7 Independently, [ka] Selected from.

[0023] In some embodiments, the compound is given by formula: [ka] It has, In the formula, each R 7 Independently, [ka] Selected from.

[0024] In some embodiments, the compound is given by formula: [ka] It has, In the formula, each R 7 teeth, [ka] That is the case.

[0025] In some embodiments, the compound is given by formula: [ka] It has, in the formula, each R 7 teeth, [ka] That is the case.

[0026] In some embodiments, this disclosure is, Compound of formula A, and medication The present invention provides a composition containing the following:

[0027] In some embodiments, this disclosure is, Compounds of formulas I, II, III, IV, or V, and medication The present invention provides a composition containing the following:

[0028] In some embodiments, this disclosure is, Compounds of formulas VI, VII, VIII, IX, or X, and medication The present invention provides a composition containing the following:

[0029] In some embodiments, this disclosure is, Compound of formula A, Noncationic lipids, Polyethylene glycol lipids, and Sterols The present invention provides nanoparticles containing the following:

[0030] In some embodiments, this disclosure is, Compounds of formula I, II, III, IV, or V, Noncationic lipids, Polyethylene glycol lipids, and Sterols The present invention provides nanoparticles containing the following:

[0031] In some embodiments, this disclosure is, Compounds of formulas VI, VII, VIII, IX, or X, Noncationic lipids, Polyethylene glycol lipids, and Sterols The present invention provides nanoparticles containing the following:

[0032] In some embodiments, the nanoparticles further contain a drug.

[0033] In some embodiments, the drug is a polynucleotide. In some embodiments, the drug is RNA. In some embodiments, the drug is mRNA.

[0034] In some embodiments, the Specified Pharmaceutical Compositions are disclosed comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X.

[0035] In some embodiments, a method for delivering a drug into a cell is provided. In some embodiments, a method for delivering nucleic acids is provided. In some embodiments, a method for delivering polynucleotides is provided herein.

[0036] The attached drawings are incorporated herein by reference and constitute part of this specification, illustrating several embodiments described below. [Brief explanation of the drawing]

[0037] [Figure 1] The results of in vitro mRNA delivery screening of ionic lipids in Hep3b cells at a ratio of 20:30:40:0.75 (compound / DOPE / cholesterol / PEG lipids) are shown. [Figure 2]The results of in vitro mRNA delivery screening of ionic lipids in Hep3b cells at a ratio of 40:30:40:0.75 (compound / DOPE / cholesterol / PEG lipids) are shown. [Figure 3] The results of in vitro mRNA delivery screening of ionic lipids in Hep3b cells at a ratio of 60:30:40:0.75 (compound / DOPE / cholesterol / PEG lipids) are shown. [Modes for carrying out the invention]

[0038] This disclosure provides novel compounds, bio-inspired lipid derivatives, compositions, nanoparticles, and methods for using them. It also provides compositions containing the compounds and drugs (e.g., mRNA) of the present invention. Furthermore, this disclosure provides methods for using the compositions to deliver drugs to cells or targets. These bio-inspired compounds are used in applications such as gene therapy, drug delivery, and vaccines.

[0039] Embodiments of the present invention will be described in detail, examples of which are shown in the drawings and embodiments. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments described herein.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs. The following definitions are provided for a complete understanding of the terms used herein.

[0041] definition General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. The terms “comprising” and its variations as used herein are synonymous with the terms “including” and its variations and are open, non-restrictive terms. While the terms “comprising” and “including” are used herein to describe various embodiments, the terms “essentially consisting of” and “consisting of” may be used instead of “comprising” and “including,” and are disclosed herein. The following definitions are provided for a full understanding of the terms used herein.

[0042] As used herein, the articles "a," "an," and "the" mean "at least one" unless the context in which the article is used clearly indicates otherwise.

[0043] As used herein, the term "nucleic acid" means a polymer consisting of nucleotides, such as deoxyribonucleotides or ribonucleotides.

[0044] As used herein, the terms "ribonucleic acid" and "RNA" refer to polymers composed of ribonucleotides.

[0045] As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to polymers composed of deoxyribonucleotides.

[0046] The term “oligonucleotide” refers to a single-stranded or double-stranded nucleotide polymer with a length of approximately 2 to 100 nucleotides. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method described by Matteucci, et al., J.Am. Chem. Soc., 103:3185 (1981) (both incorporated herein by reference), or by other chemical methods using either a commercially available automated oligonucleotide synthesizer or VLSIPS® technology. When an oligonucleotide is referred to as “double-stranded,” those skilled in the art will understand that a pair of oligonucleotides typically exist in a hydrogen-bonded helical sequence, for example, associated with DNA. In addition to the 100% complementary forms of double-stranded oligonucleotides, the term “double-stranded” as used herein also means forms that include structural features such as bulges and loops, which are better described in biochemistry textbooks such as Stryer, Biochemistry, Third Ed., (1988), which are incorporated herein by reference for all purposes.

[0047] The term "polynucleotide" refers to a single-chain or double-chain polymer composed of nucleotide monomers.

[0048] The term "polypeptide" refers to a compound composed of a single chain of D-amino acids or L-amino acids, or a mixture of D-amino acids and L-amino acids linked by peptide bonds.

[0049] The term "complementary" refers to the topological compatibility or agreement of the interaction surfaces of a probe molecule and its target. Therefore, it can be said that the target and its probe are complementary, and furthermore, the properties of their contact surfaces are complementary to each other.

[0050] The term "hybridization" refers to the process of establishing non-covalent sequence-specific interactions between two or more complementary strands of nucleic acids to form a single hybrid; in the case of two strands, it is called a double-stranded hybrid.

[0051] The term "annealing" refers to the process by which single-stranded nucleic acid sequences pair with a complementary sequence through hydrogen bonding, including the reformation (regeneration) of the complementary strand separated by heat (thermal denaturation), to form a double-stranded nucleic acid sequence.

[0052] The term "melting" refers to the denaturation of a double-stranded nucleic acid sequence due to high temperatures, where the hydrogen bonds between the strands are broken, causing the double strand to separate into two single strands.

[0053] The terms “promoter” or “regulatory element” refer to a region or sequencer located upstream or downstream of the transcription start that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters do not need to be of bacterial origin; for example, promoters derived from viruses or other organisms may be used in the compositions, systems, or methods described herein. The term “regulatory element” is intended to include promoters, enhancers, intra-sequence ribosome entry sites (IRESs), and other expression regulatory elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 1990). Regulatory elements include those that direct the constitutive expression of nucleotide sequences in many types of host cells, and those that direct the expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory elements). Tissue-specific promoters can primarily direct expression in desired target tissues such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell-type specific. In some embodiments, the vector includes one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters.Examples of pol II promoters include, but are not limited to, the retroviral rust sarcoma virus (RSV) LTR promoter (optionally including an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally including a CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" also includes enhancer elements such as WPRE, the CMV enhancer; the R-U5' segment within the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It is understood by those skilled in the art that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired expression level.

[0054] The term “recombinant” refers to a human engineered nucleic acid (e.g., polynucleotide) or a replica or complement of a human engineered nucleic acid (e.g., polynucleotide), or, in the case of a protein (i.e., “recombinant protein”), to a protein (e.g., polynucleotide) encoded by a recombinant nucleic acid. In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human engineering (e.g., by the methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may include a nucleic acid (e.g., polynucleotide) that is combined in such a way that the nucleic acid (e.g., polynucleotide) is very unlikely to be found in nature. For example, the restriction enzyme site or plasmid vector sequence manipulated by a human may be positioned adjacent to or separated from the promoter of a second nucleic acid (e.g., a polynucleotide). Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be manipulated in many ways, and are not limited to the examples given above.

[0055] The term “expression cassette” refers to a nucleic acid construct that, when introduced into a host cell, results in the transcription and / or translation of RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably ligated to a second nucleic acid (e.g., polynucleotide) may include a promoter that is heterogeneous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably ligated to a second nucleic acid (e.g., polynucleotide) may include a terminator that is heterogeneous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette includes a promoter operably ligated to a second nucleic acid (e.g., a polynucleotide) and a terminator operably ligated to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette includes an endogenous promoter. In some embodiments, the expression cassette includes an endogenous terminator. In some embodiments, the expression cassette includes a synthetic (or unnatural) promoter. In some embodiments, the expression cassette includes a synthetic (or unnatural) terminator.

[0056] The term "identity" or percentage "identity" for two or more nucleic acid or polypeptide sequences was measured using the BLAST or BLAST2.0 sequence comparison algorithm with the default parameters listed below, or by manual alignment and visual inspection (e.g., NCBI). Refers to two or more sequences or subsequences having identical amino acid residues or nucleotides (i.e., when compared and aligned to obtain the maximum correspondence in a comparison window or specified region, in a specified region, approximately 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity). Such sequences are said to be "substantially identical". This definition refers to or may apply to the complement of the test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Preferred algorithms, as described below, can account for gaps, etc. Preferably, identity resides in a region of at least about 10 amino acids or 20 nucleotides in length, or more preferably, in a region of 10 to 50 amino acids or 20 to 50 nucleotides in length. As used herein, amino acid sequence identity percentage (%) is defined as the percentage of amino acids in a candidate sequence that are identical to amino acids in a reference sequence after aligning the sequences to obtain the maximum sequence identity percentage and introducing gaps as necessary. Alignment for determining sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithm required to achieve the maximum alignment across the entire length of the sequences being compared, can be determined by known methods.

[0057] For sequence comparison, typically one sequence is used as a reference sequence, and the sequence under comparison is performed against it. When using a sequence comparison algorithm, the sequence under comparison and the reference sequence are input into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the sequence under comparison relative to the reference sequence based on the program parameters.

[0058] Preferred examples of algorithms suitable for determining sequence identity percentage and sequence similarity are the BLAST and BLAST 2.0 algorithms (described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively). Software for performing BLAST analysis is generally available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying shorter words W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighbor word score threshold (Altschul et al. (1990) J.Mol.Biol.215:403-410). These first neighbor word hits serve as seed values ​​to initiate the search for longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs, always greater than 0) and N (penalty score for mismatched residues, always less than 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction stops when the cumulative alignment score decreases by a factor X from its maximum actual value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a default word length (W), expected value (E), or 10, M=5, N=-4, and comparison of both strands.For amino acid sequences, the BLASTP program defaults to using a word length of 3 and an expected value of 10 (E), as well as a BLOSUM62 scoring matrix (Henikoff and Henikoff (1989), Proc Natl Acad Sci USA, 89:10915) alignment (B), an expected value of 10 (E), M=5, N=-4, and comparison of both strands.

[0059] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences could occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0060] The term "codon optimization," in the context of genes or coding regions of nucleic acid molecules for transformation into various hosts, refers to modifying codons in the genes or coding regions of a polynucleic acid molecule to reflect the typical codon usage frequencies of a selected organism, without altering the polypeptide encoded by DNA. Such optimization may involve replacing at least one, two or more, or a significant number of codons with one or more codons that are more frequently used in the genes of the selected organism.

[0061] Nucleic acids are "operably linked" if they are placed in a functional relationship with another nucleic acid sequence. For example, a pre-sequence or secretion leader DNA is operably linked to the polypeptide DNA if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in close proximity to each other, and in the case of a secretion leader, they are contiguous and located in the reading phase. However, operably linked nucleic acids (e.g., enhancers and coding sequences) do not need to be contiguous. Linking is achieved by ligation at a convenient restriction enzyme site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to conventional practice. In embodiments, a promoter is operably linked to a coding sequence if it can affect the expression of a protein from that coding sequence (e.g., modulate in the absence of the promoter) (i.e., the coding sequence is under the transcriptional control of the promoter).

[0062] The term "nucleic acid base" refers to the portion of a nucleotide that possesses the function of Watson / Crick base pairing. The most common natural nucleic acid bases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), possess the function of hydrogen bonding, which sequence-specifically links one nucleic acid chain to another.

[0063] As used throughout, “subject” (or “host”) means an individual. Therefore, “subject” can include, for example, domesticated animals such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. A subject may be a mammal such as a primate or a human.

[0064] As used herein, the term "approximately" when referring to measurable values ​​such as quantities or proportions means that the measurable value is subject to variation of ±20%, ±10%, ±5%, or ±1%.

[0065] As used herein, the terms “to treat” or “to treat” include administering a drug to a subject for the purpose of curing, restoring, reducing, alleviating, altering, treating, relieving, improving, stabilizing, or influencing a disease or disorder, or the symptoms of a disease or disorder. The terms “to treat” and “to treat” may also mean reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying causes, and improving or repairing damage.

[0066] As used herein, the term “prevention” of a disease, disorder, or undesirable physiological event in the subject matter means prevention of a disease, disorder, or undesirable physiological event, or prevention of symptoms of a disease, disorder, or undesirable physiological event.

[0067] The “effective dose” of a drug refers to the amount of drug sufficient to provide the desired effect. The amount of drug that is “effective” will vary from subject to subject, depending on many factors such as the subject’s age and general condition, and the specific drug(s) involved. Therefore, it is not always possible to specify a quantifiable “effective dose.” However, an appropriate “effective dose” for any subject can be determined by those skilled in the art using routine experiments. Also, as used herein, unless otherwise specified, the “effective dose” of a drug may refer to an amount that encompasses both the therapeutic effective dose and the prophylactic effective dose. The “effective dose” of a drug required to obtain a therapeutic effect may vary depending on factors such as the subject’s age, sex, and weight. The dose regimen can also be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the urgency of the treatment situation.

[0068] A “pharmaceutically acceptable” ingredient can mean an ingredient that is not biologically or otherwise undesirable, i.e., the ingredient is incorporated into the pharmaceutical formulation of the present invention and administered to the subjects described herein without causing any significant undesirable biological effects or without adverse interactions with any of the other ingredients in the formulation containing the ingredient. When used in reference to administration to humans, this term generally means that the ingredient meets the required standards of toxicological and manufacturing testing, or that the ingredient is included in the Inactive Ingredients Guide created by the U.S. Food and Drug Administration.

[0069] A “pharmaceutically acceptable carrier” (sometimes referred to as “carrier”) generally means a carrier or excipient useful in preparing safe and non-toxic pharmaceutical or therapeutic compositions, and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term “carrier” or “pharmaceutically acceptable carrier” may include, but is not limited to, phosphate-buffered saline, water, emulsions (such as oil / water or water / oil emulsions) and / or various types of wetting agents. As used herein, the term “carrier” includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art and further described herein for use in pharmaceutical formulations.

[0070] "Therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include both therapeutic effects, such as the treatment of a disorder or other undesirable physiological condition, and preventive effects, such as the prevention of a disorder or other undesirable physiological condition. These terms also include, but are not limited to, pharmaceutically acceptable pharmacologically active derivatives of beneficial agents specifically referred to herein, such as salts, esters, amides, precursors, active metabolites, isomers, fragments, and analogs. Where the term "therapeutic agent" is used, or where a particular agent is specifically identified, the term includes the agent itself, as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, precursors, conjugates, active metabolites, isomers, fragments, and analogs.

[0071] As used herein, the terms “controlled release,” “controlled release drug delivery,” or “sustained release” refer to releasing or administering a drug from a given dosage form in a controlled manner to achieve desired pharmacokinetic properties in vivo. A “controlled” aspect of drug delivery is the ability to manipulate the formulation and / or dosage form to establish the desired kinetics of drug release.

[0072] As used herein, the terms “concurrent administration,” “combined administration,” “simultaneous administration,” or “administer at the same time” mean administering compounds at the same time or immediately after each other.

[0073] The term “antibody” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term “antibody” also includes fragments or polymers of those immunoglobulin molecules, as well as humanized or humanized forms of immunoglobulin molecules or fragments thereof. Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic and / or prophylactic activity can be tested according to known clinical trial methods. There are five major classes of human immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize the equivalent classes in mice. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0074] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, individual antibodies within the population are identical except for possible naturally occurring variations that may exist in a small subset of antibody molecules. Monoclonal antibodies as used herein specifically include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, as well as in a fragment of such an antibody, insofar as they exhibit the desired antagonistic activity.

[0075] The disclosed monoclonal antibodies can be prepared using any procedure for producing monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, e.g., the method described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice or other suitable host animals are usually immunized with an immunizer to induce lymphocytes that produce antibodies that specifically bind to the immunizer, or lymphocytes capable of producing such antibodies. Alternatively, lymphocytes may be immunized in vitro.

[0076] Monoclonal antibodies may also be produced by recombinant DNA methods. The DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the mouse antibody). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display methods, for example, as described in U.S. Patent No. 5,804,440 by Burton et al. and U.S. Patent No. 6,096,441 by Barbas et al.

[0077] The in vitro method is also suitable for the preparation of monovalent antibodies. Digestion of antibodies to produce fragments, particularly Fab fragments, can be carried out using routine methods known in the art. For example, digestion can be carried out using papain. Examples of papain digestion are described in WO94 / 29348, published December 22, 1994, and U.S. Patent No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments called Fab fragments, each having a single antigen-binding site, and a residual Fc fragment. Pepsin treatment yields a fragment having two antigen-binding sites and still capable of crosslinking antigens.

[0078] As used herein, the terms “antibody or its antigen-binding fragment” or “antibody or its fragment” encompass fragments such as F(ab')2, Fab', Fab, Fv, sFv, scFv, and hybrid fragments, including chimeric antibodies and hybrid antibodies having dual or multiple antigen or epitope specificity. Thus, we provide antibody fragments that retain the ability to bind to specific antigens. For example, antibody fragments that maintain binding activity are included within the meaning of the term “antibody or its antibody-binding fragment.” Such antibodies and fragments can be manufactured by techniques well known in the art and can be screened for specificity and activity according to the methods described in the examples and general methods for generating antibodies and screening them for specificity and activity (see Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0079] The term "antibody or its antigen-binding fragment" includes both antibody fragments and conjugates of antigen-binding proteins (single-chain antibodies). It also includes, for example, immunoglobulin single variable domains such as nanobodies.

[0080] The fragment may also include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not they are bound to other sequences, as long as the activity of the antibody or fragment is not significantly altered or impaired compared to an unmodified antibody or antibody fragment. These modifications may provide several additional properties, such as removing / adding disulfide-bondable amino acids, extending lifespan, or altering secretory properties. In any case, the antibody or antibody fragment must possess bioactive properties, such as specific binding to a congener antigen. The functional or active region of the antibody or antibody fragment may be identified by introducing a mutation into a specific region of the protein, subsequently expressing it, and testing the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis into the nucleic acid encoding the antibody or antibody fragment (Zoller, MJCurr.Opin.Biotechnol.3:348-354,1992).

[0081] As used herein, the term “antibody” (plural) may also mean human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and, therefore, can cause undesirable immune responses when administered to humans. Thus, using human antibodies or humanized antibodies in this method helps reduce the likelihood that antibodies administered to humans will cause undesirable immune responses.

[0082] chemical definition As used herein, the term “substituted” is intended to include all permissible substituents of an organic compound. In broad embodiments, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of an organic compound. Exemplary substituents include, for example, those listed below. There are one or more permissible substituents for a given organic compound, and they may be the same or different. For the purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of the organic compounds described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any way by the permissible substituents of an organic compound. Furthermore, the terms “substituted” or “substituted with” imply that such substitutions conform to the permissible valencies of the substituted atom and substituent, and that the substitution results in a compound (e.g., a stable compound that does not naturally undergo transformation by rearrangement, cyclization, exclusion, etc.).

[0083] "Z 1 "Z 2 "Z 3 " and "Z 4 In this specification, the symbols '' are used as a general term to represent various specific substituents. These symbols can represent any substituents, not limited to those disclosed herein, and where they are defined as specific substituents in one example, they may be defined as several other substituents in another example.

[0084] As used herein, the term "aliphatic" refers to a non-aromatic hydrocarbon group, including branched and unbranched, alkyl, alkenyl, or alkynyl groups.

[0085] As used herein, the term “alkyl” refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, and tetracosyl. Alkyl groups may also be substituted or unsubstituted. Alkyl groups may be substituted with one or more groups, including but not limited to alkyl, alkyl halides, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0086] Throughout this specification, the term “alkyl” is generally used to refer to both unsubstituted and substituted alkyl groups, but here, substituted alkyl groups are specifically referred to by identifying the specific substituent(s) in that alkyl group. For example, the term “alkyl halide” specifically means an alkyl group substituted with one or more halogens, such as fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically means an alkyl group substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically means an alkyl group substituted with one or more amino groups, as described below. If “alkyl” is used in one example and a specific term such as “alkyl alcohol” is used in another example, it is not intended that the term “alkyl” does not also mean the specific term such as “alkyl alcohol.”

[0087] This practice is also applied to other groups described herein. That is, terms such as “cycloalkyl” refer to both unsubstituted and substituted cycloalkyl moieties, but substituted moieties can be further specifically identified herein; for example, certain substituted cycloalkyls may be referred to as, for example, “alkylcycloalkyl.” Similarly, substituted alkoxys may be specifically referred to as, for example, “halogenated alkoxy,” and certain substituted alkenyls may be referred to as, for example, “alkenyl alcohols.” Again, the practice of using general terms such as “cycloalkyl” and specific terms such as “alkylcycloalkyl” is not intended to mean that the general terms do not include specific terms.

[0088] As used herein, the term "alkoxy" refers to an alkyl group linked via a single terminal ether bond, i.e., the "alkoxy" group is -OZ 1 It can be defined as, in the formula, Z 1 This is the alkyl group defined above.

[0089] As used herein, the term "alkenyl" refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon double bond. (Z 1 Z 2 )C=C(Z 3 Z 4 Asymmetric structures such as ) are intended to include both E and Z isomers. This can be inferred from the structural formulas herein where the asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C=C. The alkenyl group may be substituted with one or more groups, including but not limited to alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as listed below.

[0090] As used herein, the term "alkynyl" refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. Alkynyl groups can be substituted with one or more groups, including but not limited to alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0091] As used herein, the term "aryl" refers to a group containing any carbon-based aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, and phenoxybenzene. The term "heteroaryl" is defined as a group containing an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term "nonheteroaryl," which is included in the term "aryl," defines a group containing an aromatic group that does not contain a heteroatom. Aryl groups or heteroaryl groups may be substituted or unsubstituted. Aryl groups or heteroaryl groups may be substituted with one or more groups, including but not limited to alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein. The term "biaryl" is a special type of aryl group and is included in the definition of aryl. A biaryl refers to two aryl groups that are linked via a fused ring structure, as in naphthalene, or linked via one or more carbon-carbon bonds, as in biphenyl.

[0092] As used herein, the term “cycloalkyl” refers to a non-aromatic carbon-carbon ring consisting of at least three carbon atoms. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “heterocycloalkyl” refers to a cycloalkyl group as defined above, wherein at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl groups and heterocycloalkyl groups may be substituted with or unsubstituted groups, as described herein, including but not limited to alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0093] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbon-carbon ring consisting of at least three carbon atoms and containing at least one double bond, i.e., a C=C bond. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, and cyclohexadienyl. The term "heterocycloalkenyl" is a type of cycloalkenyl group as defined above and is included in the meaning of the term "cycloalkenyl," wherein at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups may be substituted or unsubstituted. Cycloalkenyl and heterocycloalkenyl groups may be substituted with one or more groups, including but not limited to alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol groups, as described herein.

[0094] The term “cyclic group” is used herein to refer to aryl groups, nonaryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. A cyclic group has one or more ring systems, which may be substituted or unsubstituted. A cyclic group may consist of one or more aryl groups, one or more nonaryl groups, or one or more aryl groups and one or more nonaryl groups.

[0095] As used herein, the term "aldehyde" is represented by the formula -C(O)H. Throughout this specification, "C(O)" or "CO" is an abbreviation for C=O.

[0096] As used herein, the terms "amine" or "amino" refer to formula -NZ 1 Z 2 It is expressed as, in the formula, Z 1 and Z 2Each of these substituents may be one of the substituents described herein, for example, the hydrogen, alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0097] As used herein, the term "carboxylic acid" is represented by the formula -C(O)OH. As used herein, the "carboxylate" or "carboxyl" group is represented by the formula -C(O)OH - It is represented as follows.

[0098] As used herein, the term "ester" refers to the formula -OC(O)Z 1 Or -C(O)OZ 1 It is expressed as, in the formula, Z 1 This can be the alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0099] As used herein, the term "ether" is derived from formula Z 1 OZ 2 It is expressed as, in the formula, Z 1 and Z 2 These can independently be the alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups.

[0100] As used herein, the term "ketone" refers to the formula Z 1 C(O)Z 2 It is expressed as, in the formula, Z 1 and Z 2 These can independently be the alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups.

[0101] As used herein, the terms "halogen" or "halogen" refer to fluorine, chlorine, bromine, and iodine.

[0102] As used herein, the term "hydroxyl" is represented by the formula -OH.

[0103] As used herein, the term "nitro" is represented by the formula -NO2.

[0104] As used herein, the term "silyl" is derived from the formula -SiZ 1 Z 2 Z 3 It is expressed as, in the formula, Z 1 , Z 2 , and Z 3 This can independently be hydrogen, the alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0105] The term "sulfonyl" is derived from the formula -S(O)2Z 1 In this specification, Z is used to refer to a sulfo-oxo group represented by the formula, where Z 1 This can be hydrogen, the alkyl group, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0106] As used herein, the terms "sulfonylamino" or "sulfonamide" are represented by the formula -S(O)2NH-.

[0107] The term "phosphonyl" is derived from the formula -P(O)(OZ 1 In this specification, Z is used to refer to the phosphooxo group represented by 2 in the formula, where Z 1 This can be hydrogen, the alkyl group, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0108] As used herein, the term "thiol" is represented by the formula -SH.

[0109] The term "thio" as used herein is represented by the formula -S-.

[0110] The term "R" used in this specification 1 "R 2 "R 3 "R n Each of these (where n is some integer) can independently have one or more of the above bases. For example, R 1 If the group is a linear alkyl group, one of its hydrogen atoms can be optionally replaced with a hydroxyl group, alkoxyl group, amine group, alkyl group, halide, etc. Depending on the selected group, the first group may be incorporated into the second group, or alternatively, the first group may pendant (i.e., bond) to the second group. For example, in the phrase "alkyl group containing an amino group," the amino group may be incorporated into the alkyl group's skeleton, or the amino group may bond to the alkyl group's skeleton. The properties of the selected group(s) determine whether the first group is embedded in or bonded to the second group.

[0111] Unless otherwise stated, formulas with chemical bonds shown only by solid lines and without wedges or dashed lines intend to represent their respective possible isomers, e.g., their respective enantiomers, diastereomers, and meso compounds, as well as mixtures of isomers such as racemic or scaremic mixtures.

[0112] Specific aspects of the disclosed materials, compounds, compositions, articles, and methods are shown in detail in the attached examples and drawings.

[0113] compound In one embodiment, the present disclosure relates to a compound of formula A, i.e., [ka] or provide the salt thereof. In the formula, each R 7 is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate; each R 8 is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol; R 9 is hydrogen, alkyl, alkenyl, alkynyl, ester, alkyl ester, or

Chemical formula

[0114] In some embodiments, each R 8 is alkyl. In some embodiments, each R 8 is methyl. In some embodiments, each R 8 is alkyl alcohol.

[0115] In some embodiments, R 9 is hydrogen. In some embodiments, R 9 is

Chemical formula

[0116] In some embodiments, m is an integer from 1 to 3. In some embodiments, X is O. In some embodiments, X is N and R 9 is hydrogen. In some embodiments, X is N and R 9 is

Chemical formula

[0117] In one embodiment, the present disclosure relates to a compound of formula I, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. X is selected from O or N.

[0118] In one embodiment, the present disclosure relates to a compound of formula II, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. X is selected from O or N.

[0119] In one embodiment, the present disclosure relates to a compound of formula III, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate.

[0120] In one embodiment, the present disclosure relates to a compound of formula IV, i.e., [ka] or provide the salt thereof. During the ceremony, Each R 7is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate.

[0121] In one aspect, the present disclosure provides a compound of Formula V, namely,

Chemical formula

[0122] In one aspect, the present disclosure provides a compound of Formula VI, namely,

Chemical formula

[0123] In one aspect, the present disclosure provides a compound of Formula VII, namely,

Chemical formula

[0124] In one aspect, the disclosure provides a compound of Formula VIII, namely,

Chemical formula

[0125] In one aspect, the disclosure provides a compound of Formula IX, namely,

Chemical formula

[0126] In one aspect, the disclosure provides a compound of Formula X, namely,

Chemical formula

[0127] In some embodiments, R 7 is independently

Chemical formula

[0128] In some embodiments, the compound has the formula:

Chemical formula

Chemical formula

[0129] In some embodiments, the compound has the formula:

Chemical formula

Chemical formula

[0130] In some embodiments, the compound has the formula:

Chemical formula

Chemical formula

[0131] Several embodiments, each R 7 R is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate, and in some embodiments, each R 7 R is selected from alkyl, alkenyl, alkynyl, ester, or alkyl ester. In some embodiments, each R 7 R is selected from alkyl or alkenyl. In some embodiments, each R 7 is alkyl. In some embodiments, each R 7 is an alkenyl. In some embodiments, each R 7 is an alkynyl. In some embodiments, each R 7 is an ester. In some embodiments, each R 7 It is an alkyl ester.

[0132] Several embodiments, each R 7 is C 7-17 It is alkyl. In some embodiments, each R 7 is a C7 alkyl group. In some embodiments, each R 7 is a C8 alkyl group. In some embodiments, each R 7 is a C9 alkyl group. In some embodiments, each R 7 is C 10 It is alkyl. In some embodiments, each R 7 is C 11 It is alkyl. In some embodiments, each R 7 is C 12 It is alkyl. In some embodiments, each R 7 is C 13 It is alkyl. In some embodiments, each R 7 is C 14 It is alkyl. In some embodiments, each R 7 is C 15 It is alkyl. In some embodiments, each R 7 is C16 It is alkyl. In some embodiments, each R 7 is C 17 It is alkyl.

[0133] Several embodiments, each R 7 is C 10-21 It is an alkenyl. In some embodiments, each R 7 is C 10 It is an alkenyl. In some embodiments, each R 7 is C 11 It is an alkenyl. In some embodiments, each R 7 is C 12 It is an alkenyl. In some embodiments, each R 7 is C 13 It is an alkenyl. In some embodiments, each R 7 is C 14 It is an alkenyl. In some embodiments, each R 7 is C 15 It is an alkenyl. In some embodiments, each R 7 is C 16 It is an alkenyl. In some embodiments, each R 7 is C 17 It is an alkenyl. In some embodiments, each R 7 is C 18 It is an alkenyl. In some embodiments, each R 7 is C 19 It is an alkenyl. In some embodiments, each R 7 is C 20 It is an alkenyl. In some embodiments, each R 7 is C 21 It is Alkenil.

[0134] Several embodiments, each R 7 teeth [ka] That is the case.

[0135] Several embodiments, each R 7 teeth [ka] That is the case.

[0136] Several embodiments, each R 7 teeth [ka] That is the case.

[0137] Several embodiments, each R 7 teeth [ka] That is the case.

[0138] Several embodiments, each R 7 teeth [ka] That is the case.

[0139] Several embodiments, each R 7 teeth [ka] That is the case.

[0140] Several embodiments, each R 8 R is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol. In some embodiments, each R 8 is alkyl. In some embodiments, each R 8 is an alkenyl. In some embodiments, each R 8 is an alkynyl. In some embodiments, each R 8 is an ester. In some embodiments, each R 8 is an alkyl ester. In some embodiments, each R 8 is an alkyl alcohol. In some embodiments, each R 8 is methyl. In some embodiments, each R8 It is ethyl.

[0141] In some embodiments, at least one R 7 R is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. In some embodiments, at least one R 7 R is selected from alkyl, alkenyl, alkynyl, ester, or alkyl ester. In some embodiments, at least one R 7 is selected from alkyl or alkenyl. In some embodiments, at least one R 7 is alkyl. In some embodiments, at least one R 7 is an alkenyl. In some embodiments, at least one R 7 is an alkynyl. In some embodiments, at least one R 7 is an ester. In some embodiments, at least one R 7 It is an alkyl ester.

[0142] In some embodiments, at least one R 7 teeth [ka] That is the case.

[0143] In some embodiments, at least one R 7 teeth [ka] That is the case.

[0144] In some embodiments, at least one R 7 teeth [ka] That is the case.

[0145] In some embodiments, at least one R 7 teeth [ka] That is the case.

[0146] In some embodiments, at least one R 7 teeth [ka] That is the case.

[0147] In some embodiments, at least one R 7 teeth [ka] That is the case.

[0148] In some embodiments, at least one R 7 is C 7-17 It is alkyl. In some embodiments, at least one R 7 is a C7 alkyl group. In some embodiments, at least one R 7 is a C8 alkyl group. In some embodiments, at least one R 7 is a C9 alkyl group. In some embodiments, at least one R 7 is C 10 It is alkyl. In some embodiments, at least one R 7 is C 11 It is alkyl. In some embodiments, at least one R 7 is C 12 It is alkyl. In some embodiments, at least one R 7 is C 13 It is alkyl. In some embodiments, at least one R 7 is C 14 It is alkyl. In some embodiments, at least one R 7 is C 15 It is alkyl. In some embodiments, at least one R7 is C 16 It is alkyl. In some embodiments, at least one R 7 is C 17 It is alkyl.

[0149] In some embodiments, at least one R 7 is C 10-21 It is an alkenyl. In some embodiments, at least one R 7 is C 10 It is an alkenyl. In some embodiments, at least one R 7 is C 11 It is an alkenyl. In some embodiments, at least one R 7 is C 12 It is an alkenyl. In some embodiments, at least one R 7 is C 13 It is an alkenyl. In some embodiments, at least one R 7 is C 14 It is an alkenyl. In some embodiments, at least one R 7 is C 15 It is an alkenyl. In some embodiments, at least one R 7 is C 16 It is an alkenyl. In some embodiments, at least one R 7 is C 17 It is an alkenyl. In some embodiments, at least one R 7 is C 18 It is an alkenyl. In some embodiments, at least one R 7 is C 19 It is an alkenyl. In some embodiments, at least one R 7 is C 20 It is an alkenyl. In some embodiments, at least one R 7 is C 21 It is Alkenil.

[0150] In some embodiments, at least one R 8R is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol. In some embodiments, at least one R 8 is alkyl. In some embodiments, at least one R 8 is an alkenyl. In some embodiments, at least one R 8 is an alkynyl. In some embodiments, at least one R 8 is an ester. In some embodiments, at least one R 8 is an alkyl ester. In some embodiments, at least one R 8 is an alkyl alcohol. In some embodiments, at least one R 8 is methyl. In some embodiments, at least one R 8 It is ethyl.

[0151] In some embodiments, R 9 is hydrogen, alkyl, alkenyl, alkynyl, ester, alkyl ester, or [ka] Selected from.

[0152] In some embodiments, R 9 is hydrogen. In some embodiments, R 9 is alkyl. In some embodiments, R 9 is an alkenyl. In some embodiments, R 9 is an alkynyl. In some embodiments, R 9 is an ester. In some embodiments, R 9 is an alkyl ester. In some embodiments, R 9 teeth [ka] That is the case.

[0153] In some embodiments, X is selected from O or N. In some embodiments, X is O. In some embodiments, X is N.

[0154] In some embodiments, m is an integer between 0 and 10. In some embodiments, m is an integer between 1 and 3. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10.

[0155] In some embodiments, X is O. In some embodiments, X is N and R 9 is hydrogen. In some embodiments, X is N and R 9 teeth [ka] That is the case.

[0156] In some embodiments, the alkyl group is substituted. In some embodiments, the alkyl group is unsubstituted. In some embodiments, the alkenyl group is substituted. In some embodiments, the alkenyl group is unsubstituted.

[0157] nanoparticles In one aspect, this disclosure is, Compounds of formulas A, I, II, III, IV, V, VI, VII, VIII, IX, or X, Noncationic lipids, Polyethylene glycol lipids, and Sterols The present invention provides nanoparticles containing the following:

[0158] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula A, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0159] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula I, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0160] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula II, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0161] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula III, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0162] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula IV, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0163] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula V, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0164] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula VI, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0165] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula VII, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0166] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula VIII, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0167] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula IX, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0168] In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula X, a noncationic lipid, a polyethylene glycol lipid, and a sterol.

[0169] Various compounds of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X are described in the above selection of compounds. In some embodiments, the nanoparticles contain the compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X in a molar ratio of about 5% to about 80%. In some embodiments, the nanoparticles contain the compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X in a molar ratio of about 20% to about 60%. In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X in molar ratios of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80%. In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X in molar ratios of about 20%. In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X in molar ratios of about 40%. In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X in a molar ratio of about 60%.

[0170] In some embodiments, the nanoparticles contain non-cationic lipids. In some embodiments, the non-cationic lipids interact with the lipids as helper lipids. In some embodiments, the non-cationic lipids include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), and DPPC (1,2-dipalmitoyl-sn-glycero- The noncationic lipid may include, but is not limited to, 1,2-dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho(1'-rac-glycerol) (DOPG), or combinations thereof. In one embodiment, the noncationic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the noncationic lipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). In one embodiment, the noncationic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the noncationic lipid is 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). While several noncationic lipids are described herein, additional noncationic lipids can be used in combination with the compounds disclosed herein.

[0171] In some embodiments, the nanoparticles contain noncationic lipids in a molar ratio of about 10% to about 50%. In some embodiments, the nanoparticles contain noncationic lipids in molar ratios of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%, about 45%, or about 50%. In one embodiment, the nanoparticles contain noncationic lipids in a molar ratio of about 30%.

[0172] In some embodiments, the nanoparticles contain polyethylene glycol lipids (PEG lipids). PEG lipids are incorporated to form a hydrophilic outer layer and stabilize the particles. Non-limiting examples of polyethylene glycol lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol lipids include DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG. In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG). In another embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol,methoxypolyethylene glycol-XXXX, where XXXX represents the molecular weight of the polyethylene glycol portion, for example, DMG-PEG2000 or DMG-PEG5000.

[0173] In some embodiments, the nanoparticles contain polyethylene glycol lipids in a molar ratio of about 0% to about 5%. In some embodiments, the nanoparticles contain polyethylene glycol lipids in molar ratios of about 0%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, or about 5%. In one embodiment, the nanoparticles contain polyethylene glycol lipids in a molar ratio of about 0.75%.

[0174] In some embodiments, the nanoparticles contain sterols. Sterols are well known to those skilled in the art and generally refer to compounds having a perhydrocyclopentanophenanthrene ring system and one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, and sitosterol.

[0175] In some embodiments, the sterol is selected from cholesterol-based lipids. In some embodiments, one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol, DC-Choi(N,N-dimethyl-N-ethylcarboxyamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine, or a combination thereof.

[0176] Sterols can be used to regulate particle permeability and fluidity based on their function in cell membranes. In one embodiment, the sterol is cholesterol.

[0177] In some embodiments, the nanoparticles contain sterols in a molar ratio of about 20% to about 60%. In some embodiments, the nanoparticles contain sterols in molar ratios of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%, about 55%, or about 60%. In one embodiment, the nanoparticles contain sterols in a molar ratio of about 40%.

[0178] In one embodiment, this disclosure, Compounds of formulas A, I, II, III, IV, V, VI, VII, VIII, IX, or X, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dimyristoyl-sn-glycerol, Methoxypolyethylene glycol (DMG-PEG2000), and cholesterol The present invention provides nanoparticles containing the following:

[0179] In one embodiment, this disclosure, Compounds of formulas A, I, II, III, IV, V, VI, VII, VIII, IX, or X, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Dimyristoyl-sn-glycerol, Methoxypolyethylene glycol (DMG-PEG2000), and cholesterol The present invention provides nanoparticles containing the following:

[0180] In one embodiment, this disclosure, Compounds of formulas A, I, II, III, IV, V, VI, VII, VIII, IX, or X, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dimyristoyl-sn-glycerol, Methoxypolyethylene glycol (DMG-PEG2000), and cholesterol The present invention provides nanoparticles containing the following:

[0181] In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X / noncationic lipids / sterols / polyethylene glycol lipids in a ratio of 20:30:40:0.75. In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X / DOPE / cholesterol / PEG lipids in a ratio of 20:30:40:0.75.

[0182] In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X / noncationic lipids / sterols / polyethylene glycol lipids in a ratio of 40:30:40:0.75. In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X / DOPE / cholesterol / PEG lipids in a ratio of 40:30:40:0.75.

[0183] In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X / noncationic lipids / sterols / polyethylene glycol lipids in a ratio of 60:30:40:0.75. In some embodiments, the nanoparticles contain a compound of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X / DOPE / cholesterol / PEG lipids in a ratio of 60:30:40:0.75.

[0184] In one embodiment, the nanoparticles further comprise a drug. In one embodiment, the nanoparticles further comprise a therapeutic agent. In one embodiment, the nanoparticles further comprise a diagnostic agent.

[0185] The drug delivered to the cell may be a polynucleotide. Polynucleotides or oligonucleotides that can be introduced according to the methods herein include all types of DNA, cDNA, and RNA sequences. For example, polynucleotides may be double-stranded DNA, single-stranded DNA, complex DNA, encapsulated DNA, naked RNA, encapsulated RNA, messenger RNA (mRNA), tRNA, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antisense RNA (asRNA), and combinations thereof. Polynucleotides may also be DNA constructs such as expression vectors, expression vectors encoding the desired gene product (e.g., a gene product of the same or different species as the target into which it is introduced). In one embodiment, the drug is mRNA. In one embodiment, the drug is DNA.

[0186] composition As described herein, compositions containing active compounds and certain excipients may be useful in a variety of medical and non-medical applications. For example, pharmaceutical compositions containing active compounds and excipients may be useful for delivering an effective amount of drug to a target that needs it. Functional food compositions containing active compounds and excipients may be useful for delivering an effective amount of functional food, such as a dietary supplement, to a target that needs it. Cosmetic compositions containing active compounds and excipients may be formulated as creams, ointments, balms, pastes, films, or liquids, and may be useful in makeup, hair products, and materials useful for personal hygiene. Compositions containing active compounds and excipients are useful in non-medical applications, such as emulsions or emulsifiers, and may be useful as food ingredients for fire extinguishing, surface disinfection, oil cleaning, etc.

[0187] In certain embodiments, the composition further comprises the agents described herein. For example, in certain embodiments, the agent is a small molecule, organometallic compound, nucleic acid, protein, peptide, polynucleotide, metal, targeted agent, isotope-labeled compound, drug, vaccine, immunological agent, or agent useful for bioprocessing. In certain embodiments, the agent is a polynucleotide. In certain embodiments, the polynucleotide is DNA or RNA. In certain embodiments, the RNA is mRNA, RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. In certain embodiments, the polynucleotide and one or more active compounds are not covalently bonded.

[0188] In one aspect, this disclosure is, Compounds of formulas A, I, II, III, IV, V, VI, VII, VIII, IX, or X, and medication The present invention provides a composition containing the following:

[0189] In one aspect, this disclosure is, Compounds of formulas A, I, II, III, IV, V, VI, VII, VIII, IX, or X, and medication The present invention provides a composition containing nanoparticles containing the following:

[0190] In another embodiment, as described herein, Disclosed are compositions comprising nanoparticles containing compounds of formula A, I, II, III, IV, V, VI, VII, VIII, IX, or X, and a drug. The drug comprises mRNA encoding at least one antigen polypeptide or an immunogenic fragment thereof, which can induce an immune response to the antigen polypeptide.

[0191] In some embodiments, mRNA encoding at least one antigen polypeptide or an immunogenic fragment thereof, which can induce an immune response to the antigen polypeptide, is encapsulated by nanoparticles.

[0192] In some embodiments, the following pharmaceutical compositions are disclosed herein, comprising a pharmaceutically acceptable carrier and nanoparticles containing at least one antigen polypeptide or its immunogenic fragment mRNA capable of inducing an immune response to the antigen polypeptide.

[0193] medication The compounds, compositions, and systems described herein that deliver agents may be therapeutic agents, diagnostic agents, or prophylactic agents. Any compound to be administered to a target may be delivered using the particles or nanoparticles described herein. The agents may be organic molecules (e.g., therapeutic agents, drugs), inorganic molecules, nucleic acids, proteins, amino acids, peptides, polypeptides, polynucleotides, targeting agents, isotope-labeled organic or inorganic molecules, vaccines, immunological agents, etc.

[0194] In certain embodiments, the drug is an organic molecule having pharmaceutical activity, such as a drug. In certain embodiments, the drug is an antibiotic, antiviral agent, anesthetic agent, steroid agent, anti-inflammatory agent, antitumor agent, anticancer agent, antigen, vaccine, antibody, decongestant, antihypertensive agent, sedative, contraceptive, progestin, anticholinergic agent, analgesic, antidepressant, antipsychotic, β-adrenergic blocker, diuretic, cardiovascular activator, vasoactive agent, nonsteroidal anti-inflammatory drug, nutritional supplement, etc.

[0195] In certain embodiments of this disclosure, the drug to be delivered may be a mixture of drugs.

[0196] Diagnostic agents include gases, metals, commercially available imaging agents used in positron emission tomography (PET), computed tomography (CAT), single-photon emission computed tomography, X-ray, fluoroscopy, and magnetic resonance imaging (MRI), as well as contrast agents. Examples of materials suitable for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and X-ray imaging include iodine-based materials.

[0197] Therapeutic and prophylactic agents include, but are not limited to, antibiotics, nutritional supplements, and vaccines. Vaccines may include isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically modified organisms or viruses, cell extracts, and RNA or immunogenic fragments thereof that encode at least one antigen polypeptide (e.g., immunogenic fragments capable of inducing an immune response to an antigen polypeptide). Therapeutic and prophylactic agents may be combined with interleukins, interferons, cytokines, and adjuvants, such as cholera toxin, alum, and Freund's adjuvant. Prophylactic drugs include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, CamphylobacterAntigens of bacterial organisms such as jejuni, as well as antigens of viruses such as smallpox virus, influenza A and B viruses, RSV, parainfluenza virus, measles virus, HIV virus, varicella-zoster virus, herpes simplex virus types 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps virus, rabies virus, rubella virus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A, B, C, D and E viruses, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial This includes antigens of fungi, protozoa, and parasites such as trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni. These antigens may be in the form of whole-organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

[0198] In some embodiments, the drug is a ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one (e.g., at least two, three, four, or five) antigenic polypeptides or immunogenic fragments thereof (e.g., immunogenic fragments capable of inducing an immune response to an antigenic polypeptide).

[0199] In some embodiments, RNA (e.g., mRNA) may be used to induce a balanced immune response to respiratory viruses. The term “respiratory virus” as used herein refers to viruses that cause respiratory illness. Examples include negative-sense single-stranded RNA viruses of the Paramyxoviridae family, such as human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), RSV, and measles virus (MeV). Another example of a respiratory virus is coronavirus. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical symmetric nucleocapsid. Coronaviruses are viral species belonging to the subfamily Coronavirinae of the family Coronaviridae in the order Nidovirales.

[0200] Representative examples of betacoronaviruses include, but are not limited to, Embecovirus 1 (e.g., betacoronavirus 1, human coronavirus OC43, Chinese rat coronavirus HKU24, human coronavirus HKU1, mouse coronavirus), Hibecovirus (e.g., bat Hp-betacoronavirus Zhejiang2013), Merbecovirus (e.g., Hedgehog coronavirus 1, Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV), Japanese house bat coronavirus HKU5, bamboo bat coronavirus HKU4), Novecovirus (e.g., Rousettus bat coronavirus GCCDC1, Rousettus flying fox coronavirus HKU9), and Salvecovirus (e.g., Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2)).

[0201] Representative examples of gamma coronaviruses include, but are not limited to, Segacovirus (e.g., beluga whale coronavirus SQ1) and Igacovirus (e.g., avian coronavirus (IBV)).

[0202] Representative examples of delta coronaviruses include, but are not limited to, anddecoviruses (e.g., duck coronavirus HKU20), brudecoviruses (e.g., bulbul coronavirus HKU11, swine coronavirus HKU15 (PorCoV HKU15), mumppara coronavirus HKU13, and white-eye coronavirus HKU16), herdecoviruses (e.g., night heron coronavirus HKU19), and moor decoviruses (e.g., van coronavirus HKU21).

[0203] In some embodiments, the coronavirus is a human coronavirus. Representative examples of human coronaviruses include, but are not limited to, human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome-associated coronavirus (MERS-CoV).

[0204] In some embodiments, an RNA (e.g., mRNA) polynucleotide has an open reading frame encoding at least one (e.g., at least two, three, four, or five) hMPV, PIV, RSV, MeV, or beta-CoV (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptide, or any combination of two or more antigenic polypeptides. In this specification, the use of the term “antigenic polypeptide” encompasses immunogenic fragments of antigenic polypeptides (immunogenic fragments that induce (or can induce) an immune response to hMPV, PIV, RSV, MeV, or beta-CoV), unless otherwise stated.

[0205] In some embodiments, the agent is an RNA (e.g., mRNA) that can induce a balanced immune response, including both cellular and humoral immunity, against hMPV, PIV, RSV, MeV, and / or beta-CoV (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, and / or HCoV-HKU1), or any combination of two or more of the aforementioned viruses, without exposing the agent to the risk of insertional mutagenesis, for example.

[0206] In some embodiments, the delivered drug is used for gene therapy. In some embodiments, the delivered drug is used for gene editing. In some embodiments, the delivered drug is used for CRISPR-mediated gene editing. In some embodiments, the delivered drug is Cas9 mRNA. In some embodiments, the delivered drug is Cpf1 mRNA. In some embodiments, the delivered drug is guide RNA.

[0207] method In one embodiment, in this specification, Compounds of formulas A, I, II, III, IV, V, VI, VII, VIII, IX, or X, Noncationic lipids, Polyethylene glycol lipids, Sterols, and medication The present invention provides a method for delivering a drug (e.g., a polynucleotide) into a cell, comprising introducing a composition containing nanoparticles into the cell.

[0208] In one embodiment, in this specification, Compound of formula A, [ka] or its salt (In the formula, Each R 7This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate; Each R 8 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol; R 9 is hydrogen, alkyl, alkenyl, alkynyl, ester, alkyl ester, or [ka] Selected from, X is selected from O or N, m is an integer between 0 and 10) and medication A method for delivering a drug into cells is disclosed, which includes introducing a composition containing nanoparticles into cells.

[0209] In one embodiment, in this specification, Compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, or X, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or its salt (In the formula, Each R 7 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, alkyl ketal, alkyl carbonate, or alkyl carbamate. Each R 8 This is independently selected from alkyl, alkenyl, alkynyl, ester, alkyl ester, or alkyl alcohol. X is selected from O or N) Noncationic lipids, Polyethylene glycol lipids, Sterols, and medication A method for delivering a drug into cells is disclosed, which includes introducing a composition containing nanoparticles into cells.

[0210] In some embodiments, nanoparticles containing any of the compounds listed in the above section on compounds are used in the methods herein for the delivery of drugs into cells.

[0211] In some embodiments, the drug is a polynucleotide. In some embodiments, the drug is RNA. In some embodiments, the drug is mRNA. In some embodiments, the drug is DNA. In some embodiments, the drug is a therapeutic agent, a diagnostic agent, or a prophylactic agent.

[0212] In some embodiments, methods for delivering polynucleotides are provided herein. In some embodiments, methods for delivering polynucleotides (e.g., mRNA) to correct mutations in a genome are provided herein. For example, mRNA may be delivered to correct mutations that cause hemophilia (mutations in the genes encoding factor VIII (F8, hemophilia A) or factor IX (F9, hemoglobin B)). In some embodiments, methods for delivering polynucleotides are provided herein. In some embodiments, methods for delivering polynucleotides (e.g., mRNA) to provide mRNA expression (and translation for protein production) in a cell are provided herein. In some embodiments, methods for delivering polynucleotides (e.g., mRNA) to induce an immune response in a subject are provided herein. In some embodiments, an RNA (e.g., mRNA) polynucleotide has an open reading frame encoding at least one (e.g., at least two, three, four, or five) hMPV, PIV, RSV, MeV, and / or beta-CoV (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptide, or any combination of two or more antigenic polypeptides.

[0213] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. [Examples]

[0214] The following examples are provided below to illustrate the compounds, compositions, methods, and results of the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the invention that would be apparent to those skilled in the art.

[0215] Example 1: Bio-inspired lipids Efficient mRNA delivery is a crucial step and challenge in the application of mRNA therapy. Despite promising data from ongoing clinical trials, the discovery and development of more efficient delivery systems are necessary for the clinical use of mRNA.

[0216] Phosphates and phosphoramide lipid-like compounds First, 40 types of ionic lipids were synthesized. These ionic lipids were then combined with DOPE / cholesterol / PEG lipids in three different ratios (20:30:40:0.75, 40:30:40:0.75, and 60:30:40:0.75) to prepare lipid nanoparticles (LNPs), which were then screened in Hep3b cells using mRNA encoding firefly luciferase. ZYB20200113-1 was more effective in mRNA delivery compared to other ionic lipids (Figures 1-3).

[0217] [ka] [ka] [ka] [ka] [ka]

[0218] General procedure for the synthesis of dialkyl-H-phosphonates Alcohol (6.15 mmol) was added to a solution of diphenyl phosphonate 1 (0.7 g, 3.0 mmol) in 3.0 mL of pyridine. The resulting solution was then heated to 75°C and stirred for 3 hours. Pyridine was removed under reduced pressure, and the residue was diluted with 100 mL of DCM and washed with 10 mL of 1N NaOH aqueous solution and 10 mL of water. The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (0%-10% ethyl acetate in hexane) to obtain the desired product.

[0219] General procedure for aminophosphate and phosphamide lipid synthesis To a flame-dried flask containing dialkyl-H-phosphonate (0.35 mmol) and carbon tetrachloride (2.0 mL), a solution of trimethylamine (194.6 μL, 1.4 mmol), DMAP (4.3 mg, 0.035 mmol), and an amino alcohol or amine (2.0 mmol) in 1.0 mL of dry DCM was added dropwise at room temperature with vigorous stirring. The reaction mixture was stirred for 1 hour, diluted with 50 mL of DCM, and washed three times with 50 mL of brine. The organic phase was isolated, dried over anhydrous Na₂SO₄, filtered, and the solvent was removed under vacuum. The residue was purified by silica gel chromatography (0% to 20% [a mixture of 3% NH₄OH and 22% MeOH in dichloromethane]) to obtain the desired product.

[0220] [ka] ZYB20191125-1. 1 H NMR (300 MHz, CDCl3) δ 5.45 - 5.27 (m, 4H), 4.08 (qd, J = 6.8, 1.5 Hz, 4H), 2.77 (d, J = 11.5 Hz, 2H), 2.42 (s, 6H), 2.03 (q, J = 6.7, 5.6 Hz, 8H), 1.69 (p, J = 6.8 Hz, 4H), 1.44 - 1.20 (m, 44H), 0.90 (t, J = 6.9 Hz, 6H). 31P NMR (121 MHz, CDCl3) δ 25.07. MS (m / z): [M+H] + C 39 H 79 NO3P + Calculated value for 640.5792; Measured value: 640.5797.

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[0221] Those skilled in the art will understand that many changes and modifications can be made to preferred embodiments of the present invention, and that such changes and modifications can be made without departing from the spirit of the invention. Accordingly, the appended claims are intended to cover all such equivalent modifications that fall within the true spirit and scope of the invention.

Claims

1. Formula: 【Chemistry 1】 (In the formula, each R ７ However, they became independent, 【Chemistry 2】 (Selected from) A composition for delivering a drug to cells or a target, comprising a compound having [a certain characteristic].

2. Each R ７ but, 【Transformation 3】 The composition according to claim 1.

3. Formula: 【Chemistry 4】 (In the formula, each R ７ but, 【Transformation 5】 (is) A composition for delivering a drug to cells or a target, comprising a compound having [a certain characteristic].

4. A compound as defined in any one of claims 1 to 3, and Drugs, A composition containing the following:

5. The composition according to claim 4, wherein the drug is a polynucleotide.

6. The composition according to claim 4, wherein the drug is RNA.

7. The composition according to claim 4, wherein the drug is mRNA.

8. A compound as defined in any one of claims 1 to 3, Noncationic lipids, Polyethylene glycol lipids, and Sterols Nanoparticles containing these nanoparticles.

9. The nanoparticles according to claim 8, further comprising a drug.

10. The nanoparticle according to claim 9, wherein the drug is a polynucleotide.

11. The nanoparticle according to claim 9, wherein the drug is RNA.

12. The nanoparticle according to claim 9, wherein the drug is mRNA.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound as defined in any one of claims 1 to 3, a composition as defined in any one of claims 4 to 7, or nanoparticles as defined in any one of claims 8 to 12.

14. A composition for use in a method of delivering a drug to cells, comprising a compound specified in any one of claims 1 to 3, wherein the method comprises introducing the composition into cells.

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