Lipids for the delivery of activators into lipid nanoparticles

Abstract

To provide a novel cationic lipid for the delivery of oligonucleotides.SOLUTION: There are provided compounds having the following structure (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein G1 is -N(R3)R4 or -OR5; R1 is C12-C36 alkyl; R2 is C12-C36 alkyl or the like; R3 and R4 are each independently H or C1-C6 alkyl; R5 is H or C1-C6 alkyl; L is -C(=O)-, C6-C12 alkylene or the like; and n is an integer from 1 to 12. There are also provided use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to novel cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-bound lipids, to form lipid nanoparticles that encapsulate nucleic acids and can facilitate intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) both in vivo and in vitro. [Background technology]

[0002] Numerous attempts have been made to influence the delivery of nucleic acids to affect desired responses in biological systems. Nucleic acid-based therapies hold great potential, but realizing this potential requires more effective delivery of nucleic acids to the appropriate sites within cells or organisms. Examples of therapeutic nucleic acids include messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomir, antimir, mimic, supermir, and aptamer. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cell products, for example, to be useful in treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are very broad because they can synthesize components and generate selective protein sequences, whether system-specific or not. Nucleic acid expression products can enhance the levels of existing proteins, replace missing or non-functional proteins, or introduce new proteins and associated functions into cells or organisms.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to induce the expression of specific cell products regulated by miRNAs, for example, to be useful in treating diseases associated with protein or enzyme deficiencies. Because it is possible to inhibit one or more miRNAs that synthesize components and regulate the expression of mRNA products, the therapeutic applications of miRNA inhibition are very broad. Inhibition of endogenous miRNAs can enhance the expression of their downstream target endogenous proteins and restore proper function of cells or organisms as a means of treating diseases associated with specific miRNAs or groups of miRNAs.

[0004] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, thereby downregulating the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. Because oligonucleotide components can be synthesized with nucleotide sequences directed toward target mRNAs, the therapeutic applications of antisense oligonucleotides and RNAi are also very broad. Targets can include mRNAs from normal cells, mRNAs associated with disease conditions such as cancer, and mRNAs from infectious agents such as viruses. Antisense oligonucleotide components have been shown to have the ability to specifically downregulate target proteins through the degradation of similar mRNAs in both in vitro and in vivo models. Furthermore, antisense oligonucleotide components are currently being evaluated in clinical studies.

[0005] However, the use of oligonucleotides in therapeutic applications currently faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has a limited ability to access intracellular regions where the relevant translation mechanisms reside. Lipid nanoparticles formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides are used to inhibit the degradation of RNA in plasma and promote the cellular uptake of oligonucleotides.

[0006] Improvements to cationic lipids and lipid nanoparticles are needed for oligonucleotide delivery. Preferably, these lipid nanoparticles provide an optimal drug-lipid ratio, protect nucleic acids from degradation and clearance in serum, are suitable for systemic or topical delivery, and provide intracellular delivery of nucleic acids. Furthermore, these lipid-nucleic acid particles are highly tolerable and provide appropriate therapeutic indicators, thereby preventing the treatment of patients with effective doses of nucleic acids from being associated with unacceptable toxicity and / or risk to the patient. This disclosure provides these and related advantages. [Overview of the project]

[0007] In short, this disclosure provides lipid compounds, including their stereoisomers, pharmaceutically acceptable salts, and tautomers, which can be used alone or in combination with other lipids, such as neutral lipids, charged lipids, steroids (e.g., all sterols), and / or their analogues, and / or polymer-bound lipids for forming lipid nanoparticles for the delivery of therapeutic agents. In some examples, lipid nanoparticles are used to deliver nucleic acids, such as antisense RNA and / or messenger RNA. Methods are also provided for using lipid nanoparticles to treat a variety of diseases or illnesses, including those caused by infectious agents and / or protein deficiencies.

[0008] In one embodiment, the following structure (I): [ka] [In the formula, G 1 , R 1 , R 2 L and n are as defined herein. A compound represented by, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, is provided.

[0009] Furthermore, a pharmaceutical composition is provided comprising one or more compounds of the above structure (I) and a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-bound lipids. Such compositions are useful for forming lipid nanoparticles for the delivery of therapeutic agents.

[0010] In other embodiments, the Disclosure provides a method for administering a therapeutic agent to a patient in need, comprising manufacturing or preparing a composition of lipid nanoparticles comprising a compound of structure (I) and the therapeutic agent, and delivering or administering the composition to the patient.

[0011] These and other aspects of this disclosure will become clearer by referring to the detailed description below. [Modes for carrying out the invention]

[0012] The following description provides certain specific details to facilitate an overall understanding of the various embodiments of this disclosure. However, those skilled in the art will understand that the disclosure can be carried out without these details.

[0013] This disclosure is based in part on the discovery of novel cationic (amino) lipids that offer advantages when used in lipid nanoparticles for in vivo delivery of activators or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of this disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein that provide enhanced nucleic acid activity and improved tolerance of the composition in vivo, resulting in a significant increase in therapeutic metrics compared to previous nucleic acid-lipid nanoparticle compositions. In other embodiments, the disclosed lipids and lipid nanoparticles comprising them provide improved safety and / or tolerance when used for delivery of activators, such as nucleic acids.

[0014] In certain embodiments, this disclosure provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of mRNA-encoded proteins. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target one specific miRNA, or a group of miRNAs that modulate one target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of a target gene. In some other embodiments, lipid nanoparticles are also useful for the delivery of mRNA and plasmids for the expression of transgenes. In yet another embodiment, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as improved red blood cell production by delivery of appropriate erythropoietin mRNA, or protection against infection by delivery of mRNA encoding an appropriate antigen or antibody.

[0015] The lipid nanoparticles and compositions of the embodiments of this disclosure can be used for a variety of purposes, including the delivery of encapsulated or bound (e.g., complexed) therapeutic agents, such as nucleic acids, to cells, both in vitro and in vivo. Accordingly, the embodiments of this disclosure provide a method for treating or preventing a disease or disorder of a subject requiring such treatment by contacting the subject with lipid nanoparticles encapsulating or bound to a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more of the novel cationic lipids described herein.

[0016] As described herein, embodiments of lipid nanoparticles of this disclosure are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomyl / antimyl), messenger RNA interference complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), and the like. Accordingly, lipid nanoparticles and compositions of specific embodiments of this disclosure can be used to induce the expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or bind to nucleic acids to be expressed in order to produce the desired protein (e.g., messenger RNA or plasmid encoding the desired protein) or to inhibit a process that halts mRNA expression (e.g., a miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of the embodiments of this disclosure can be used to reduce the expression of target genes and proteins both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or bind to nucleic acids (e.g., antisense oligonucleotides or small interfering RNAs (siRNAs)) that reduce the expression of the target genes. The lipid nanoparticles and compositions of the embodiments of this disclosure can also be used for the simultaneous delivery of different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, for example, by providing the desired effect of co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for integration into the host genome).

[0017] Nucleic acids for use in embodiments of this disclosure can be prepared according to any available technique. In the case of mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which is currently the most efficient method for generating specific mRNA of long sequences. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template consisting of upstream bacteriophage promoter sequences (including, but not limited to, those from T7, T3, and SP6 colophages) ligated to downstream sequences encoding the gene of interest. Template DNA can be prepared for in vitro transcription from many sources using appropriate techniques well known in the art, including but not limited to plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0018] RNA transcription is performed in vitro using a linearized DNA template in the presence of adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs), while supporting polymerase activity and minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax Large Scale RNA Production System (Promega) and MegaScript Transcription kits (Life Technologies), as well as commercially available reagents such as RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in this art (e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B.,(2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530). See pp. 101–114; these are all incorporated herein by reference.

[0019] The desired in vitro transcribed mRNA is then purified from undesirable components of the transcription or related reactions (including unintegrated rNTPs, protein enzymes, salts, short RNA oligos, etc.). Techniques for isolating mRNA transcripts are well known in the art. Well-known methods include phenol / chloroform extraction or precipitation with either an alcohol (ethanol, isopropanol) in the presence of a monovalent cation or lithium chloride. Further examples of available purification methods, but not limited to them, include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be carried out using a variety of commercially available kits, including but not limited to the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0020] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous abnormal RNA impurities associated with undesirable polymerase activity that may need to be removed from the full-length mRNA preparation. These include short RNAs resulting from abnormal transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extensions. It has been demonstrated that these contaminants with dsRNA structures can lead to undesirable immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and induce potent immune responses. Additionally, mRNA translation may be dramatically reduced due to decreased protein synthesis during the innate cellular immune response. Therefore, further techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but are not limited to, scalable HPLC purification (see, for example, Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).mRNA purified by HPLC has been reported to be translated at very high levels, particularly in primary cells and in vivo.

[0021] A considerable variety of modifications used to alter specific properties of in vitro transcribed mRNA and improve its utility have been described in the art. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs). This contributes to improved intracellular mRNA stability and the efficiency of mRNA translation. Thus, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5' cap contains a 5'-5'-triphosphate linkage between most 5'-nucleotides and guanine nucleotides. The conjugated guanine nucleotides are methylated at the N7 position. Additional modifications include methylation of the last and second-to-last most 5'-nucleotides with a 2'-hydroxyl group.

[0022] Multiple different cap structures can be used to generate the 5' cap of synthetic mRNA transcribed in vitro. 5' capping of synthetic mRNA can be co-transcribed with chemical cap analogs (i.e., capping during in vitro transcription). For example, anti-reverse cap analog (ARCA) caps contain a 5'-5'-triphosphate guanine-guanine linkage, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription process, up to 20% of the transcript remains uncapped, and synthetic cap analogs are not identical to the 5' cap structure of actual cellular mRNA, potentially leading to reduced translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped after transcription. These can produce a more realistic 5'-cap structure that structurally or functionally more closely mimics the endogenous 5' cap, enhancing the binding of cap-binding proteins, extending half-life, reducing sensitivity to 5' endonucleases, and / or decreasing 5' decapping. Numerous synthetic 5' cap analogs have been developed and are known in this field to enhance mRNA stability and translationability (see, for example, Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0023] At the 3' end, a long chain of adenine nucleotides (poly-A tail) is usually added to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3' hydroxyl group, and poly-A polymerase adds the adenine nucleotide chain to the RNA in a process called polyadenylation. Poly(A) tails have been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. And Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved by various methods, including, but are not limited to, cloning of poly(T) tracts to a DNA template, or post-transcriptional addition using poly(A) polymerase. In the first case, in vitro transcription of mRNA with a poly(A) tail of a predetermined length is possible depending on the size of the poly(T) tract, but additional template operations are required. In the latter case, a poly(A) tail is enzymatically added to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of an adenine residue into the 3' end of the RNA. This eliminates the need for additional DNA template operations, but results in mRNA with poly(A) tails of non-uniform lengths. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including, but not limited to, the Poly(A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit, and Poly(A) Tailing kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerase, etc.

[0025] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcription have been reported to offer advantages in terms of translational efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotes, triggering a potent innate immune response. The ability to distinguish pathogenic from self DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications, since most nucleic acids from natural sources contain modified nucleosides. In contrast, RNA synthesized in vitro lacks these modifications and can therefore be immunostimulant, potentially inhibiting effective mRNA translation as described above.The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby mitigating this undesirable immunostimulatory activity and improving translational capacity (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013; Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, (See D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and techniques known in the art. A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA, either alone or to some extent in combination with other modified nucleosides (see, e.g., US2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to enhance translational ability while reducing its ability to activate immune sensors.

[0026] Other components of mRNA that can be modified to provide benefits in terms of translationability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTR (preferred 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to improve mRNA stability and the translation efficiency of in vitro transcribed mRNA, both or independently (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0027] In addition to mRNA, other nucleic acid payloads may be used for the purposes of this disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic and chemical cleavage of longer precursors, and in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (e.g., Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both are incorporated herein by reference).

[0028] Plasmid DNA, while not limited to the formulations used in embodiments of this disclosure, generally utilizes the in vitro amplification and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of genes in the plasmid of interest that encode resistance to a specific antibiotic (such as penicillin or kanamycin) allows bacteria containing the plasmid of interest to selectively grow in a culture containing the antibiotic. Methods for isolating plasmid DNA are widely used and well-known in this art (see, for example, Heilig, J., Elbing, KL and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstroem, S., Bjoernestedt, R. and Schmidt, SR, (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., 99: 557-566; and US 6,197,553 B1). Plasmid isolation can be performed using various commercially available kits, including, but not limited to, Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as commercially available reagents.

[0029] Various exemplary embodiments of the cationic lipids of this disclosure, and lipid nanoparticles and compositions containing them, as well as their use for delivering active substances (e.g., therapeutic agents) such as nucleic acids for regulating gene and protein expression, will be described in further detail below.

[0030] In this specification, the following terms have the meanings set forth herein unless otherwise specified.

[0031] Unless otherwise specified in the context, throughout this specification and the claims, the word “including” and its variations, such as “including” and “including,” are to be interpreted in an open and comprehensive sense, that is, “including but not limited to.”

[0032] Throughout this specification, any reference to “one embodiment” or “embodiment” means that a particular feature, structure, or characteristic described in relation to the embodiment is included in at least one embodiment of this disclosure. Therefore, the appearance of the phrase “in one embodiment” or “in an embodiment” in various places throughout this specification does not necessarily mean the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0033] Unless otherwise specified, 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 pertains. In the specification and claims, the singular forms "a," "an," and "the" imply the plural meaning unless the context explicitly indicates otherwise.

[0034] The expression "induces the expression of a desired protein" means the ability of nucleic acids to increase the expression of a desired protein. To examine the degree of protein expression, a test sample (e.g., a sample of cultured cells expressing the desired protein) or a model of a test mammal (e.g., a mammal such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey), is brought into contact with nucleic acids (e.g., nucleic acids combined with the lipids of this disclosure). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a sample of cultured cells expressing the desired protein) or a model of a control mammal (e.g., a mammal such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey), which has not been brought into contact with or administered nucleic acids. If the desired protein is present in the control sample or control mammal, a value of 1.0 can be assigned to the expression of the desired protein in the control sample or control mammal. In certain embodiments, induction of the expression of a desired protein is achieved when the ratio of the expression of the desired protein in the test sample or test mammal to the level of expression of the desired protein in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of the expression of the desired protein is achieved when a measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand appropriate assays for determining the expression level of a protein in a sample, such as dot blotting, Northern blotting, in-situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0035] The expression "inhibits target gene expression" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To investigate the degree of gene silencing, a test sample (e.g., a sample of cultured cells expressing the target gene) or a model of a test mammal (e.g., a mammal such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey), is brought into contact with the nucleic acid that silences, reduces, or inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cultured cells expressing the target gene) or a control mammal (e.g., a mammal such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey), that has not been brought into contact with or administered the nucleic acid. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of target gene expression is achieved when the level of target gene expression in the test sample or test mammal is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of target gene expression in the control sample or control mammal. In other words, nucleic acids can silence, reduce, or inhibit the expression of a target gene in a test sample or test mammal by at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the target gene expression level in a control sample or control mammal that has not been in contact with or administered the nucleic acid.Appropriate assays for determining the level of target gene expression include, but are not limited to, testing of protein or mRNA levels using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in-situ hybridization, ELISA, immunoprecipitation, enzyme function testing, and phenotypic assays known to those skilled in the art.

[0036] The “effective dose” or “therapeutic effective dose” of a therapeutic agent, such as an activator or therapeutic nucleic acid, is the amount sufficient to produce the desired effect, for example, an increase or inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. An increase in the expression of a target sequence is achieved when a measurable level is detected, in the case of an expression product that does not exist in the absence of the nucleic acid. If the expression product is present at a certain level before contact with the nucleic acid, an increase in expression is achieved when the multiplier of the increase in value obtained with the nucleic acid, such as mRNA, compared to the control is approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or more. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid, such as an antisense oligonucleotide, is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to a control. Suitable assays for measuring the expression of a target gene or target sequence include, for example, testing of protein or RNA levels using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in-situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays known to those skilled in the art.

[0037] In this specification, the term “nucleic acid” means a polymer comprising at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA can take the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA can take the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified skeletal residues or bindings, which exist synthetically, naturally, and unnaturally, and which have similar binding properties to the reference nucleic acid. Such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, this term encompasses nucleic acids, including known analogues of native nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise noted, a particular nucleic acid sequence also implicitly includes its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" consists of the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups.The “bases” include purines and pyrimidines, and further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural analogues, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifiers that incorporate novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acid prodrugs are included in various embodiments of the present invention.

[0038] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence containing a partial or full-length coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0039] In this specification, “gene product” means a gene product such as an RNA transcript or polypeptide.

[0040] The term "lipids" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids, and are generally characterized by being poorly soluble in water but soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids" (which include fats, oils, and waxes); (2) "complex lipids" (which include phospholipids and glycolipids); and (3) "derived lipids" (e.g., steroids).

[0041] "Steroids" have the following carbon skeleton: [ka] It is a compound containing [this substance]. Steroids are not limited to this, but examples include cholesterol.

[0042] "Cationic lipids" refer to lipids that can be positively charged. Cationic lipids, for example, contain one or more amine groups that carry a positive charge. Preferred cationic lipids are ionizable so that they can exist in a positively charged or neutral form depending on the pH. Ionization of cationic lipids affects the surface charge of lipid nanoparticles under various pH conditions. This charge state can affect the absorption, blood clearance, and tissue distribution of plasma proteins (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), and the ability to form endosomal soluble non-double layer structures important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0043] The term "polymer-bound lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-bound lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in this art, and examples include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0044] The term "neutral lipids" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); steroids such as sphingomyelin (SM), ceramides, and sterols; and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0045] The term "charged lipid" refers to any of several lipid species that exist in either a positively or negatively charged form, regardless of the useful physiological pH range, e.g., pH ~3 to pH ~9. Charged lipids can be synthetic or naturally occurring. Examples of charged lipids include phosphatidylserine, phosphatidylic acid, phosphatidylglycerol, phosphatidylinositol, hemisuccinate sterols, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoyl sterol (e.g., DC-Chol).

[0046] The term “lipid nanoparticles” means particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm), comprising one or more compounds of structure (I) or other specific cationic lipids. In some embodiments, lipid nanoparticles comprising the disclosed cationic lipids (e.g., compounds of structure (I)) are included in formulations that may be used to deliver activators or therapeutic agents, such as nucleic acids (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles comprise the compound of structure (I) and nucleic acids. Such lipid nanoparticles typically comprise the compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-binding lipids. In some embodiments, the activator or therapeutic agent, such as nucleic acids, can be encapsulated in the lipid portion of the lipid nanoparticle, or in an aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses.

[0047] In various embodiments, lipid nanoparticles have an average particle size of approximately 30nm to 150nm, 40nm to 150nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, 70nm to 100nm, 80nm to 100nm, 90nm to 100nm, 70nm to 90nm, 80nm to 90nm, and 70nm to 8nm. The wavelengths are 0 nm, or approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, when nucleic acids are present in lipid nanoparticles, they are resistant to degradation by nucleases in aqueous solutions. Lipid nanoparticles containing nucleic acids and methods for producing the same are disclosed, for example, in U.S. Patent Publications 2004 / 0142025, 2007 / 0042031, and PCT Publications WO2013 / 016058, and WO2013 / 086373 (all of these disclosures are incorporated herein by reference in their entirety for any purpose).

[0048] In this specification, “lipid encapsulation” means lipid nanoparticles that provide an activator or therapeutic agent, such as a nucleic acid (e.g., mRNA), involving complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in lipid nanoparticles.

[0049] In this specification, the term "aqueous solution" means a composition containing water.

[0050] "Serum stability" for nucleic acid-lipid nanoparticles means that nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0051] In this specification, “systemic delivery” means the delivery of a therapeutic product that can expose an active agent to a wide area within a living organism. Some administration techniques can allow certain drugs to be delivered systemically, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the drug is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be done by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0052] In this specification, “local delivery” means the direct delivery of an active agent to a target site within a living organism. For example, a drug can be delivered locally by direct injection to a disease site such as a tumor, another target site such as an inflammatory site, or a target organ such as the liver, heart, pancreas, or kidney. Local delivery can also include topical application or local injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude systemic pharmacological effects.

[0053] "Alkyl" refers to a group of branched or unbranched (i.e., straight-chain) hydrocarbon chains consisting only of carbon and hydrogen atoms, which are saturated or unsaturated (i.e., contain one or more double bonds (alkenyl) and / or triple bonds (alkynyl)), for example, 1 to 24 carbon atoms (C1-C1). 24 Alkyl), 4-20 carbon atoms (C4-C 20 Alkyl), 6-16 carbon atoms (C6-C 16 Alkyl), 6-9 carbon atoms (C6-C9 alkyl), 1-15 carbon atoms (C1-C 15 Alkyl), 1 to 12 carbon atoms (C1-C 12has 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), which is bonded to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless otherwise specified herein, the alkyl group may be optionally substituted.

[0054] "Alkylene" or "alkylene chain" means a branched or unbranched (i.e., straight) divalent hydrocarbon chain consisting only of carbon and hydrogen, which is saturated or unsaturated (i.e., having one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), and which binds the rest of the molecule to other groups, for example, having 1 to 24 carbon atoms (C1-C 24 alkylene), 1 to 15 carbon atoms (C1-C 15 alkylene), 1 to 12 carbon atoms (C1-C 12 alkylene), 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is bonded to the rest of the molecule via a single bond or a double bond, and is bonded to other groups via a single bond or a double bond. The bonding points of the alkylene chain to the rest of the molecule and to other groups can be through one carbon or any two carbons within the chain. Unless otherwise specified herein, the alkylene chain may be optionally substituted.

[0055] "Cycloalkyl" or "carbocyclic" means a stable, non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, including condensed or bridging ring systems, having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, being saturated or unsaturated, and bonded to the rest of the molecule by single bonds. Examples of monocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic groups include adamantyl, norbornyl, dekalinyl, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless otherwise specified herein, cycloalkyl groups may be optionally substituted.

[0056] "Cycloalkylene" refers to a divalent cycloalkyl group. Unless otherwise specified herein, the cycloalkylene group may be optionally substituted.

[0057] A "heterocyclyl" or "heterocyclic" refers to a stable 3- to 18-membered non-aromatic ring group consisting of 2-12 carbon atoms and 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur.

[0058] In this specification, the term “substitution” means, but is not limited to, any of the above groups (e.g., alkyl, alkylene, cycloalkyl, cycloalkylene, or heterocyclyl) in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, but also includes, for example, halogen atoms such as F, Cl, Br, or I; oxo groups (=O); hydroxyl groups (-OH); C1-C 12 Alkyl alkyl group; cycloalkyl group; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O)x R'; and -S(O)xNR'R', where R' is independently H, C1-C each time it appears. 15 It is alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C1-C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group such as an fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR'R').

[0059] "Optional" or "optionally" (e.g., optionally substituted) means that the event of the situation described thereafter may or may not occur, and that description includes both cases in which such event or situation occurs and cases in which it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that the description includes both substituted alkyl groups and alkyl groups without substituents.

[0060] The term "prodrug" refers to a compound that can be converted to a biologically active compound of the Disclosure under physiological conditions or by solvolysis. Therefore, the term "prodrug" means a pharmaceutically acceptable metabolic precursor of a compound of the Disclosure. A prodrug may be inactive when administered to an entity requiring it, but is converted in vivo to the active compound of the Disclosure. Prodrugs are typically readily converted in vivo, for example, by hydrolysis in the blood, to produce the parent compound of the Disclosure. Prodrug compounds often offer advantages in mammalian organisms, such as solubility, histocompatibility, or delayed release (see Bundgard, H., Design of Prodrugs (1985), pp. 7 9, 21 24 (Elsevier, Amsterdam)). The discussion of prodrugs is provided in Higuchi, T., et al., ACS Symposium Series, Vol. 14, and in *Bioreversible Carriers in Drug Design*, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0061] The disclosures disclosed herein also mean that they encompass all pharmaceutically acceptable compounds of the compound of structure (I) that are isotope-labeled by replacing one or more atoms with atoms of different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively. 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S,18 F, 36 Cl, 123 I, and 125 These radiolabeled compounds may be useful in determining or measuring the efficacy of a compound by characterizing, for example, the site of action or mechanism of action, or the binding affinity to a pharmacologically important site of action. Specific isotope-labeled compounds of structure (I), (IA), or (IB), such as compounds incorporating radioisotopes, are useful in studying drug and / or substrate tissue distribution. The radioisotope tritium, i.e. 3 H, and carbon-14, i.e. 14 C is particularly useful for this purpose in terms of ease of integration and detection preparation.

[0062] Deuterium, that is 2 Substitution with heavier isotopes, such as 1H, may result in greater metabolic stability, leading to certain therapeutic benefits such as increased in vivo half-life or reduced required doses, and may therefore be preferable in certain situations.

[0063] 11 C, 18 F, 15 O, 13 Substitution with positron-emitting isotopes such as 16N may be useful in positron emission tomography (PET) studies to investigate substrate-receptor occupancy. The isotope-labeled compound of structure (I) can generally be prepared by using a suitable isotope-labeled reagent in place of the previously used unlabeled reagent, either by conventional techniques known to those skilled in the art or by processes similar to those described in the following manufacturing methods and examples.

[0064] "Stable compound" and "stable structure" refer to a compound that is robust enough to withstand isolation from a reaction mixture to a useful purity and formulation into an effective therapeutic agent.

[0065] "Mammals" include both humans and domesticated animals such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), as well as non-domesticated animals such as wild animals.

[0066] "Pharmacologically acceptable carriers, diluents or excipients" include, but are not limited to, adjuvants, carriers, excipients, flow enhancers, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are approved by the U.S. Food and Drug Administration as acceptable for use in human or livestock animals.

[0067] "Pharmacologically acceptable salts" include both addition salts of acids and bases.

[0068] "Pharmacologically acceptable acid addition salts" means salts formed with inorganic and organic acids that retain the biological efficacy and properties of the free base and are not biological or otherwise undesirable. Examples of inorganic acids, but not limited to, include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids, but not limited to, include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, and 2-hydroxyethanesulfonic acid. Examples include nic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0069] "Pharmacologically acceptable base addition salts" mean salts that retain the biological efficacy and properties of a free acid and are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Preferred inorganic salts are salts of ammonium, sodium, potassium, calcium, and magnesium. Examples of salts derived from organic bases, though not limited to these, include primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and salts of basic ion exchange resins. Examples include salts of ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0070] Crystallization often produces solvates of the compounds of the Disclosure (i.e., compounds of structure (I)). In this specification, the term “solvate” means an aggregate comprising one or more molecules of the compounds of the Disclosure and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the Disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the corresponding solvates. While the solvates of the compounds of the Disclosure may be true solvates, in other cases, the compounds of the Disclosure may simply retain incidental water or be a mixture of water and several incidental solvents.

[0071] "Pharmaceutical composition" means a formulation of the compounds of this disclosure and a medium generally accepted in the art for delivering the bioactive compounds to a mammal (e.g., human). Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients for that purpose.

[0072] In this specification, “to treat” or “to treat” encompasses the treatment of the disease or illness in a mammal (preferably human) having the disease or illness of the interest, and includes: (i) To prevent the occurrence of disease or illness in a mammal, in particular if the mammal is susceptible to the disease but has not yet been diagnosed as having it; (ii) To suppress a disease or illness (i.e., to stop its onset); (iii) to alleviate a disease or illness (i.e., to cause a regression of a disease or illness); or, (iv) Relieving symptoms caused by a disease or illness (i.e., relieving pain without addressing the underlying disease or illness). In this specification, the terms “disease” and “illness” may be used interchangeably, or they may differ in that a particular illness or condition may not have a known causative agent (and therefore its etiology is not yet understood), and thus it may be recognized only as an undesirable illness or syndrome in which some specific symptoms have been identified by a clinician, but which is not yet recognized as a disease.

[0073] The compounds of this disclosure, or any pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined with respect to absolute stereochemistry as (R)- or (S)-, or in the case of amino acids as (D)- or (L)-. This disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or separated using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors, or separation of racemics (or racemics of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). Where a compound described herein contains an olefin double bond or other geometrically asymmetric center, unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, it is also intended to include all tautomer forms.

[0074] A "stereoisomer" refers to a compound that consists of the same atoms bonded together by the same bonds, but has different, incompatible three-dimensional structures. This disclosure intends to cover various stereoisomers and mixtures thereof, and includes "enantiomers," which means two stereoisomers that are mirror images of each other and cannot be superimposed.

[0075] "Tautomerism" refers to a proton shift from one atom in a molecule to another atom in the same molecule. This disclosure includes tautomers of any of the aforementioned compounds.

[0076] compound In one embodiment, the disclosure provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-bound lipids, to form lipid nanoparticles having oligonucleotides. While not constrained by theory, these lipid nanoparticles are thought to protect the oligonucleotides from degradation in serum and provide effective delivery of the oligonucleotides to cells in vitro and in vivo.

[0077] In one embodiment, the compound has the following structure (I): [ka] [In the formula, G 1 is -N(R 3 )R 4 , or -OR 5 and; R 1 This is a branched saturated or unsaturated C which may be optionally substituted. 12 -C 36 It is alkyl; R 2 If L is -C(=O)-, then branched or unbranched saturated or unsaturated C may be optionally substituted. 12 -C 36 Alkyl; or, R 2 L is C6-C 12 Alkylene, C6-C 12 If it is an alkenylene or a C2-C6 alkynylene, branched or unbranched saturated or unsaturated C4-C may optionally be substituted. 36 It is alkyl; R 3 and R 4Each is independently H, or optionally substituted, branched or unbranched saturated or unsaturated C1-C6 alkyl; or R 3 and R 4 L is C6-C 12 Alkylene, C6-C 12 If it is an alkenylene or a C2-C6 alkylene, each is independently a branched or unbranched saturated or unsaturated C1-C6 alkyl which may be optionally substituted; or R 3 and R 4 They, together with the nitrogen to which they are bound, form heterocyclines; R 5 is H, or optionally substituted C1-C6 alkyl; L is -C(=O)-, C6-C 12 Alkylene, C6-C 12 Alkenylene, or C2-C 12 It is an alkynylene (e.g., C2-C6 alkynylene); and, n is an integer between 1 and 12. The compound represented by , or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0078] In some embodiments, G 1 is -N(R 3 )R 4 , or -OR 5 and; R 1 This is a branched saturated or unsaturated C which may be optionally substituted. 12 -C 36 It is alkyl; R 2 This is a branched or unbranched saturated or unsaturated C which may be optionally substituted. 12 -C 36 It is alkyl; R 3 and R 4 Each is independently H, or a branched or unbranched saturated or unsaturated C1-C6 alkyl which may be substituted; or R 3and R 4 together with the nitrogen to which they are attached form a heterocyclyl; R 5 is H or optionally substituted C1-C6 alkyl; L is -C(=O)-; and, n is an integer from 1 to 12, a compound of structure (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0079] In other different embodiments, G 1 is -N(R 3 )R 4 or -OR 5 ; [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​In some more specific embodiments, the following structure (IA): [ka] [In the formula, R 8 and R 9 Each of these is independently H, or optionally substituted branched or unbranched saturated or unsaturated C2-C 12 It is alkyl; however, R 8 and R 9 R 1 However, branched saturated or unsaturated C may be optionally substituted. 12 -C 36 Each element is independently selected to form an alkyl group; and, R 10 and R 11 Each of these is independently H, or optionally substituted branched or unbranched saturated or unsaturated C2-C 12 It is alkyl; however, if L is -C(=O)-, then R 2 However, branched or unbranched saturated or unsaturated C may be optionally substituted. 12 -C 36 It becomes alkyl; and L is C6-C 12 Alkylene, C6-C 12 If it is an alkenylene or a C2-C6 alkynylene, R 2 However, branched or unbranched saturated or unsaturated C4-C may be optionally substituted. 36 R 10 and R 11 These are selected independently. It is a compound represented by [the formula shown].

[0081] In some of the above embodiments, R 8 and R 9 Each of these is independently a branched or unbranched saturated or unsaturated C2-C which may be optionally substituted. 12 It is alkyl. In some embodiments, R 8may be optionally substituted with: C2 alkyl, C4 alkyl, C6 alkyl, C8 alkyl, or C 10 It is alkyl. In some embodiments, R 8 R may be optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl. In some more specific embodiments, R 9 may be optionally substituted with: C4 alkyl, C6 alkyl, C8 alkyl, C 10 Alkyl, or C 12 It is alkyl. In some more specific embodiments, R 9 may be optionally substituted with: C6 alkyl, C8 alkyl, or C 10 It is alkyl. In some more specific embodiments, R 8 R may be optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl, and 9 may be optionally substituted with: C6 alkyl, C8 alkyl, or C 10 It is alkyl.

[0082] In some embodiments, R 10 H is R 11 This is a branched or unbranched saturated or unsaturated C2-C which may be optionally substituted. 12 It is alkyl. In some more specific embodiments, R 11 This is an unbranched C2 alkyl, C6 alkyl, or C which may be optionally substituted. 10 It is alkyl. In some further embodiments, R 11 is a C2 alkyl which may be optionally substituted. In other embodiments, R 11 is a C6 alkyl which may be optionally substituted. In yet another embodiment, R 11 C may be replaced as desired. 10 It is alkyl.

[0083] In a particular embodiment, R 10 and R 11Each of these is independently a branched or unbranched saturated or unsaturated C2-C which may be optionally substituted. 12 It is alkyl. In some embodiments, R 10 may be optionally substituted with: C2 alkyl, C4 alkyl, C6 alkyl, C8 alkyl, or C 10 It is alkyl. In certain embodiments, R 10 R may be optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl. In some embodiments, R 11 may be optionally substituted with: C4 alkyl, C6 alkyl, C8 alkyl, C 10 Alkyl, or C 12 It is alkyl. In some embodiments, R 11 may be optionally substituted with: C6 alkyl, C8 alkyl, or C 10 It is alkyl. In some embodiments, R 10 R may be optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl, and 11 C6 alkyl, C8 alkyl, or C 10 It is alkyl.

[0084] In some embodiments, R 8 , R 9 , R 10 , and R 11 These are, independently, branched or unbranched saturated or unsaturated C6-C, which may be substituted as desired. 12 It is alkyl. In some embodiments, R 8 , R 9 , R 10 , and R 11 These are, independently, branched or unbranched saturated or unsaturated C6-C, which may be substituted as desired. 10 It is alkyl. In some specific embodiments, R 8 , R 9 , R 10 , and R 11These are, independently, branched or unbranched saturated or unsaturated C8-C, which may be optionally substituted. 12 It is alkyl. In some embodiments, R 8 , R 9 , R 10 , and R 11 Each of these is independently a branched or unbranched saturated or unsaturated C2-C6 alkyl group, which may be optionally substituted.

[0085] In some embodiments, R 1 and R 2 Each of these is independently of the branched saturated or unsaturated C, which may be optionally substituted. 12 -C 30 It is alkyl. In some embodiments, R 1 and R 2 Each of these is independently of the branched saturated or unsaturated C, which may be optionally substituted. 12 -C 20 It is alkyl. In some embodiments, R 1 and R 2 Each of these is independently of the branched saturated or unsaturated C, which may be optionally substituted. 15 -C 20 It is alkyl.

[0086] In some of the above embodiments, R 1 and R 2 Each of these is saturated. In a particular embodiment, R 1 and R 2 At least one of them is unsaturated. In some embodiments, R 1 and R 2 At least one of them is independently non-substitutable. In some more specific embodiments, R 1 and R 2 Both are non-substitutions.

[0087] In some embodiments, R 1 and R 2At least one of these is independently substituted (e.g., -OH, -NH2, halo, -SH, -C(=O)-, aminyl, or a combination thereof). In some embodiments, R 1 and R 2 Both are substituted (for example, -OH, -NH2, halo, -SH, -C(=O)-, aminyl, or a combination thereof).

[0088] In some of the above embodiments, R 1 and R 2 The structure is as follows: [ka] That is the case.

[0089] In some embodiments, R 2 The structure is as follows: [ka] It is one of them.

[0090] In some more specific embodiments, R 2 The structure is as follows: [ka] It is one of them.

[0091] In some more specific embodiments, R 1 The structure is as follows: [ka] It is one of them.

[0092] In some embodiments, R 1 and R 2 Both have the following structure: [ka] It is one of them.

[0093] In some of the above embodiments, R 1 The structure is as follows: [ka] That is the case.

[0094] In some embodiments, L is -C(=O)-. In some embodiments, L is an alkylene linker, for example, C6-C 12 It is an alkylene. In some more specific embodiments, L is a C6 alkylene. In other specific embodiments, L is a C7 alkylene. In some embodiments, L is a C8 alkylene. In some embodiments, L is a C9 alkylene. In some embodiments, L is a C 10 It is alkylene. In other specific embodiments, L is C 11 It is alkylene.

[0095] In a particular embodiment, G 1 is -N(R 3 )R 4 In some embodiments, G 1 This is -NH2, -NHCH3, or -N(CH3)2.

[0096] In some embodiments, R 3 and R 4 These, together with the nitrogen to which they are bound, form a heterocycline. In certain embodiments, G 1 The structure is as follows: [ka] It is one of them.

[0097] In other embodiments, G 1 The following structure: [ka] That is the case.

[0098] In a particular embodiment, G 1 is -OR 5 In some embodiments, R 5 It is a C1-C6 alkyl group, and R 5 is substituted with at least one amine. In some specific embodiments, R 5 These are methyl, ethyl, or isopropyl.

[0099] In some specific embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 7, 8, 9, 10, 11, or 12. In some specific embodiments, n is 1. In some specific embodiments, n is 2. In some specific embodiments, n is 3. In some specific embodiments, n is 4. In some specific embodiments, n is 5. In some specific embodiments, n is 6. In some specific embodiments, n is 7. In some specific embodiments, n is 8. In some specific embodiments, n is 9. In some specific embodiments, n is 10. In some specific embodiments, n is 11. In some specific embodiments, n is 12.

[0100] In various different embodiments, the compound is one of the structures shown in Table 1 below. Table 1. Compounds included in the embodiment of the compound of structure (I) [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0101] It is understood that any embodiment of the compound of structure (I) above, and any specific substituents and / or variables in the compound of structure (I) above, can independently be combined with other embodiments and / or substituents and / or variables of the compound of structure (I) to form embodiments of the disclosure not specifically described above. Furthermore, if a list of substituents and / or variables is given for a particular R group, G group, or variable a, b, or n in a particular embodiment and / or claim, it is understood that each individual substituent and / or variable may be removed from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is considered to be within the scope of this disclosure.

[0102] In this specification, it is understood that combinations of substituents and / or variables in the formulas described are permissible only if such contributions result in a stable compound.

[0103] In some embodiments, lipid nanoparticles comprising a compound of structure (I) are provided. The lipid nanoparticles may optionally contain excipients selected from neutral lipids, steroids, and polymer-bound lipids.

[0104] In some embodiments, compositions are provided comprising one or more compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the composition comprises one of the compounds of structure (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-bound lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0105] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:1.

[0106] In various embodiments, the composition further comprises a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol is in the range of about 5:1 to 1:1.

[0107] In various embodiments, the polymer-bound lipid is a PEGylated lipid. For example, in some embodiments, PEGylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEG-S-DAG), e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediole) Examples include PEG-S-DMG, PEG-ceramide, or PEG-dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the PEGylated lipid is in the range of about 100:1 to about 20:1.

[0108] In some embodiments, the composition has the following structure (II): [ka] [In the formula, R 12 and R 13Each of these is independently a branched or unbranched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally have one or more ester bonds interposed, and The average value of w is in the range of 30 to 60. This includes pegylated lipids represented by , or their pharmaceutically acceptable salts, tautomers, or stereoisomers.

[0109] In some embodiments, R 12 and R 13 Each of these is an unbranched saturated alkyl chain containing 12 to 16 carbon atoms independently. In other embodiments, the average value of w is in the range of about 42 to 55, for example, about 49.

[0110] In some embodiments of the above-mentioned composition, the therapeutic agent comprises nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA.

[0111] In other different embodiments, the disclosure relates to a method for administering a therapeutic agent to a patient in need thereof, the method comprising manufacturing or preparing one of the aforementioned compositions and administering the composition to a patient.

[0112] For administration purposes, embodiments of the compounds of this disclosure (typically in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or formulated as pharmaceutical compositions. Pharmaceutical compositions of embodiments of this disclosure comprise the compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the compound of structure (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver, for example, a therapeutic agent for treating a particular disease or illness of interest. Appropriate concentrations and doses can be readily determined by those skilled in the art.

[0113] The administration of the compositions of the embodiments of this disclosure can be carried out through any acceptable method of administering a drug to provide similar utility. The pharmaceutical compositions of the embodiments of this disclosure can be formulated into solid, semi-solid, liquid, or gaseous formulations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes for administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral cavity, rectal, vaginal, and nasal. In this specification, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, and intrasternal injection or infusion techniques. The pharmaceutical compositions of the embodiments of this disclosure are formulated so that the active ingredients contained herein are bioavailable when the composition is administered to a patient. In some embodiments, the composition administered to a subject or patient may take the form of one or more unit dosage forms, for example, a tablet may be one unit dosage form, and a container of the compound of the embodiments of this disclosure in aerosol form may hold multiple unit dosage forms. Methods for actually manufacturing such dosage forms are known or obvious to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In some embodiments, the administered composition in each case comprises a therapeutically effective amount of lipid nanoparticles containing the compound of the Disclosure or a pharmaceutically acceptable salt thereof for the treatment of the disease or illness of interest in accordance with the teachings of the Disclosure.

[0114] The pharmaceutical compositions of the embodiments of this disclosure may be in solid or liquid form. In one embodiment, the carrier is particulate, and the composition is, for example, in the form of a tablet or powder. Alternatively, the carrier may be liquid, and the composition may be, for example, an oral syrup, an injection solution, or an aerosol useful for, for example, inhalation administration.

[0115] When intended for oral administration, the pharmaceutical composition of a particular embodiment is preferably in either solid or liquid form, and this includes semi-solid, semi-liquid, suspension, and gel forms, which are considered as either solid or liquid in this specification.

[0116] As solid compositions for oral administration, pharmaceutical compositions of several embodiments can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, e.g., carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch, lactose, or dextrin; disintegrants, e.g., alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, e.g., magnesium stearate, or Sterotex; flow enhancers, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose, or saccharin; flavorings, e.g., peppermint, methyl salicylate, or orange flavor; and colorants.

[0117] In some embodiments, when the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may include a liquid carrier such as polyethylene glycol or oil, in addition to the raw materials of the types described above.

[0118] The pharmaceutical compositions of some embodiments may be in liquid form, such as elixirs, syrups, solutions, emulsions, or suspensions. The liquids may be, as two examples, for oral administration or for delivery by injection. When intended for oral administration, preferred compositions include, in addition to the compound of structure (I), one or more of the following: sweeteners, preservatives, dyes / colorants, and flavoring agents. Compositions intended for administration by injection may include one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0119] Liquid pharmaceutical compositions of embodiments of this disclosure, whether they are in the form of solutions, suspensions or other methods, may contain one or more of the following adjuvants: sterile diluents, e.g., water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride; non-volatile oils, e.g., synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as a solvent or suspension medium; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates, and tonic modifiers such as sodium chloride or dextrose; agents acting as antifreeze, e.g., sucrose or trehalose. Parenteral formulations may be placed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Physiological saline is a preferred adjuvant. Pharmaceutical compositions for injection are preferably sterile.

[0120] Liquid pharmaceutical compositions of embodiments of the present disclosure intended for either parenteral or oral administration contain an amount of the compound of the present disclosure such that an appropriate dose is obtained.

[0121] The pharmaceutical compositions of embodiments of this disclosure may be intended for topical administration, in which case the carrier may optionally include a solution, emulsion, ointment, or gel base. The base may include, for example, diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as one or more emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. When intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device.

[0122] The pharmaceutical compositions of the embodiments of this disclosure may be intended for rectal administration, for example, in the form of suppositories that dissolve in the rectum and release the drug. Compositions for rectal administration may include an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0123] The pharmaceutical compositions of embodiments of this disclosure may include a variety of materials that alter the physical form of a solid or liquid dosing unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0124] Pharmaceutical compositions of embodiments of the present disclosure, in solid or liquid form, may include agents that bind to the compounds of the present disclosure, thereby assisting in the delivery of the compounds. Suitable agents capable of acting in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0125] The pharmaceutical compositions of embodiments of this disclosure may consist of dosing units that can be administered as aerosols. The term aerosol is used to describe a variety of systems, ranging from colloidal to systems consisting of pressurized packaging. Delivery may be by liquefaction or compressed gas that disperses the active ingredient, or by a suitable pumping system. Aerosols of the compounds of embodiments of this disclosure may be delivered in single-phase, two-phase, or three-phase systems for delivering the active ingredient. Delivery of the aerosol may include necessary containers, activators, valves, sub-containers, etc., which may together form a kit. Those skilled in the art can obtain a preferred aerosol without excessive experimentation.

[0126] Pharmaceutical compositions of embodiments of this disclosure can be prepared by methods well known in the pharmaceutical field. For example, a pharmaceutical composition intended for administration by injection can be prepared by combining liquid lipid nanoparticles of this disclosure with sterile distilled water or other carriers to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the compounds of this disclosure to facilitate the dissolution or homogeneous suspension of the compound in an aqueous delivery system.

[0127] The compositions of embodiments of this disclosure are administered in a therapeutically effective dose, which varies depending on a variety of factors, including the activity of the particular therapeutic agent used: factors such as the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, usual health condition, sex, and diet; the manner and timing of administration; the excretion rate; the combination of drugs; the severity of a particular disorder or disease; and the subject being treated.

[0128] The compositions of the embodiments of this disclosure may also be administered concurrently with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical formulation of the compositions of the embodiments of this disclosure and one or more additional activators, as well as the administration of separate pharmaceutical formulations of the compositions of the embodiments of this disclosure and each activator. For example, the compositions of the embodiments of this disclosure and other activators may be administered together to a patient in a single oral formulation such as a tablet or capsule, or each agent may be administered in a separate oral formulation. When separate formulations are used, the compounds of the embodiments of this disclosure and one or more additional activators may be administered essentially simultaneously, i.e., together, or separately at staggered times, i.e., consecutively, and combination therapy is understood to include all such regimens.

[0129] Methods for producing the above compounds and compositions are described below herein and / or are known in the art.

[0130] It will be understood by those skilled in the art that in the methods described herein, the functional groups of intermediate compounds may need to be protected by appropriate protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, and benzyl. Suitable protecting groups for amino, amidino, and guanidino groups include t-butoxycarbonyl and benzyloxycarbonyl. Suitable protecting groups for mercapto groups include -C(0)-R'' (where R'' is alkyl, aryl, or arylalkyl), p-methoxybenzyl, and trityl. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to the standard techniques known to those skilled in the art and described herein. For the use of protecting groups, see Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd As described in detail in Ed., Wiley, as those skilled in the art will understand, the protecting group may also be a polymer resin such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0131] Such protected derivatives of the compounds of the Disclosure may not possess pharmacological activity themselves, but they may be administered to mammals and subsequently metabolized in the body to form pharmacologically active compounds of the Disclosure. Therefore, such derivatives can be described as “prodrugs.” All prodrugs of the compounds of the Disclosure are included within the scope of the Disclosure.

[0132] Furthermore, compounds of the embodiments of this disclosure, existing in the form of a free base or acid, can be converted to their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid using methods known in the art. Salts of compounds of the embodiments of this disclosure can be converted to their free base or acid form by standard techniques.

[0133] The following general reaction scheme 1 applies to the compound of this disclosure, i.e., structure (I): [ka] [In the formula, G 1 , R 1 , R 2 L and n are as defined herein. This document describes exemplary methods for preparing the compounds shown, or their pharmaceutically acceptable salts, tautomers, or stereoisomers. Those skilled in the art will understand that these compounds can be prepared by similar methods or in combination with other methods known to those skilled in the art. They will also understand that other compounds of structure (I) not specifically shown below can be prepared by using appropriate starting components and, as necessary, modifying the parameters of the synthesis, in a manner similar to that described below. Generally, starting components can be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or prepared as described in this disclosure.

[0134] General reaction scheme 1 [ka]

[0135] General reaction scheme 1 ("Method A") provides an exemplary method for producing the compound of structure (I). 1 , R 2 , L, and n are as defined herein, and X and Y represent interchangeable reactive parts throughout the reaction scheme. Compounds of structures A-1 and A-2 are purchased or prepared according to methods known in the art. Under suitable coupling conditions (e.g., Y is a chloride and the reaction conditions include a suitable base such as triethylamine), the reaction of A-1 and A-2 produces A-3, which then reacts with the reactive group X (e.g., bromide) to form the desired G 1 The compound of structure (I) is produced by a reaction that converts to a group (for example, using a secondary amine). Furthermore, if A-2 is an acid halide (for example, an acid chloride), A-3 may require a reduction step to remove the carbonyl group. In that case, the compound of structure (I) can be produced using appropriate reduction conditions (for example, lithium aluminum hydride).

[0136] General reaction scheme 2 [ka]

[0137] General reaction scheme 2 ("Method B") provides an exemplary method for producing the compound of structure (I). General reaction scheme 2 G 1 , R 1 , R 2L, n, and n are as defined herein, and m is selected such that B-4 is its counterpart (e.g., a compound with one less -CH2- than the compound of structure (I)) and the final compound is as defined in structure (I) herein. Compounds of structure B-1 are purchased or produced according to methods known in the art. Under suitable conditions (e.g., oxalyl chloride), the reaction of B1 produces acyl B-2 chloride, which can then react with B-3 to produce amide B-4. B-4 can be treated with a suitable reducing agent (e.g., lithium aluminum hydride) to produce the compound of structure (I). It should be noted that various alternative strategies for producing the compound of structure (I) are available to those skilled in the art. For example, other compounds of structure (I) can be produced according to similar methods using suitable starting materials. The use of protecting groups as needed and other modifications to the general reaction scheme described above will be readily apparent to those skilled in the art. [Examples]

[0138] The following examples are provided for illustrative purposes only and are not limited thereto.

[0139] Example 1 In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions Lipid nanoparticles were prepared and tested according to the general methods described in PCT publication numbers WO2015 / 199952 and WO2017 / 004143, all of which are incorporated herein by reference. Briefly, cationic lipids, DSPC, cholesterol, and PEG lipids were solubilized in ethanol in a molar ratio of approximately 50:10:38.5:1.5 or approximately 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) were prepared with a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. The mRNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate or acetate buffer (pH 4). Using a syringe pump, the ethanol-soluble lipid solution was mixed with the mRNA aqueous solution in a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate of 15 mL / min or more. Next, the ethanol is removed, and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a sterile filter with a 0.2 μm pore size. The particle size of the lipid nanoparticles is approximately 55–95 nm, and in some cases approximately 70–90 nm, and is determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0140] The experiments will be conducted in 6-8 week old female C57BL / 6 mice (Charles River) or 8-10 week old CD-1 (Harlan) mice (Charles River) in accordance with guidelines established by the Institutional Animal Care Board (ACC) and the Canadian Animal Care Council (CCAC). Various doses of mRNA lipid nanoparticles will be administered systemically by tail vein injection, and the animals will be euthanized at a specific time point after administration (e.g., 4 hours). The liver and spleen will be collected in pre-weighed tubes, weighed, immediately refrozen in liquid nitrogen, and stored at -80°C until analytical processing.

[0141] For liver samples, approximately 50 mg is processed for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon, OH). A 1 / 4-inch ceramic sphere (MP Biomedicals) is added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison, WI), equilibrated to room temperature, is added to the liver tissue. The liver tissue is homogenized twice in a FastPrep24 instrument (MP Biomedicals) at 6.0 m / s for 15 seconds each time. After incubating the homogenate at room temperature for 5 minutes, it is diluted 1:4 with GLB and measured using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μL of the homogenate-diluted tissue is reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then measured using the CentroXS assay system. 3 Quantification is performed using an LB960 luminometer (Berthold Technologies, Germany). The amount of protein being assayed is measured using the BCA protein assay kit (Pierce, Rockford IL). Next, the relative luminescence units (RLU) are normalized to the total protein ug being assayed. A standard curve is created using QuantiL um Recombinant Luciferase (Promega) to convert RLU to luciferase ng.

[0142] Trilink Biotechnologies' FLuc mRNA (L-6107 or L-7202) expresses the luciferase protein first isolated from the firefly (Photinus pyralis). FLuc is commonly used in mammalian cell cultures to measure both gene expression and cell viability. It emits bioluminescence in the presence of its substrate, luciferin. This capped, polyadenylated mRNA is completely substituted with respect to uridine and / or cytidine nucleosides.

[0143] Example 2 PK of formulated lipids A Measurement As explained elsewhere, the pK of formulated cationic lipids a pK correlates with the effectiveness of LNPs in nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). a The preferred range is ~5 to ~7. The pK of each cationic lipid a This is measured using lipid nanoparticles with a fluorescence-based assay for 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipids / DSPC / cholesterol / PEG lipids (50 / 10 / 38.5 / 1.5 mol%) at a total lipid concentration of 0.4 mM are prepared in PBS using the in-line process described in Example 1. TNS is prepared as a 100 mM stock solution in distilled water. The vehicle is diluted to 24 mM lipids with 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH in the range of 2.5 to 11. Aliquots of TNS solution were added to a final concentration of 1 μM, followed by vortex mixing. The fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best-fit analysis was applied to the fluorescence data, and the pKa was measured at the pH that produced half of the maximum fluorescence intensity.

[0144] Table 2. pK of the compound with the selected structure (I) a [Table 2]

[0145] Example 3 Measurement of the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo rodent model expressing luciferase mRNA. The cationic lipids shown in Table 3 have been previously tested with nucleic acids. For comparison, these lipids were used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the in-line mixing method described in Example 1 and PCT / US10 / 22614 (which is incorporated herein by reference in its entirety). The lipid nanoparticles were formulated in the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., 1-(monomethoxy-polyethylene glycol)-2,3-dimiristoylglycerol, mean PEG molecular weight 2000). In alternative embodiments, the cationic lipid, DSPC, cholesterol, and PEG lipid were formulated in molar ratios of approximately 47.5:10:40.8:1.7. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection, as described in Example 1. Activity was compared at mRNA / kg doses of 0.3 mg and 1.0 mg, and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1.

[0146] Table 3. Comparative lipids showing activity in mRNA [Table 3]

[0147] The representative compounds of this disclosure shown in Table 4 were formulated in the following molar ratios: 50% cationic lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMA"), 2-[2-(ω-methoxy(polyethylene glycol)] 2000(ethoxy)-N,N-ditetradecylacetamide, or 47.5% cationic lipid / 10% DSPC / 40.8% cholesterol / 1.7% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection, as described in Example 1. Activity was compared at a dose of 0.5 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1. The compound numbers in Table 4 correspond to the compound numbers in Table 1.

[0148] Table 4. Novel cationic lipids and their activity [Table 4]

[0149] Example 4 Synthesis of compound I-1 [ka] A solution of di-(2-hexyldecanyl)amine (1.4 g) in dichloromethane (20 mL) was treated with a solution of triethylamine (1 mL) and 5-bromopentanoyl chloride (2 g) in dichloromethane (20 mL). The reaction mixture was stirred for 1 hour and then washed with dilute hydrochloric acid aqueous solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was dissolved in a solution of 2 M dimethylamine in tetrahydrofuran (30 mL) and stirred overnight. The solvent was almost completely removed, and the residue was partitioned with hexane and dilute hydrochloric acid aqueous solution. The organic phase was washed with water, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane, followed by 2-12% methanol / dichloromethane, to obtain compound I-1 (0.95 g) as a colorless oil.

[0150] Example 5 Synthesis of compound I-2 [ka] A solution of di-(2-hexyldecanyl)amine (1.5 g) in dichloromethane (10 mL) was treated with triethylamine (20 drops) and 6-bromohexanoyl chloride (1 g) in dichloromethane (10 mL). The reaction mixture was stirred for 30 minutes, and then the solvent was removed under vacuum. The residue was dissolved in dichloromethane, filtered through a silica gel layer, and the solvent was removed. The residue was dissolved in a solution of 2 M dimethylamine in tetrahydrofuran (30 mL) and stirred overnight. The solvent was almost completely removed, and the residue was partitioned with dichloromethane and aqueous sodium bicarbonate. The organic phase was washed with water, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column with a 0-8% methanol / dichloromethane gradient to obtain compound I-2 (0.26 g) as a colorless oil.

[0151] Example 6 Synthesis of compound I-3 [ka] A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (20 mL) was treated with triethylamine (5 mL) and 5-bromopentanoyl chloride (1 g). The reaction mixture was stirred for 10 minutes, filtered, and the solvent was removed. The residue was dissolved in dichloromethane and washed with dilute hydrochloric acid aqueous solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was treated with a solution of diethanolamine (4.9 g) in tetrahydrofuran (20 mL) and heated overnight at 45°C. The reaction mixture was washed twice with water and the solvent was removed. The residue was passed through a silica gel column using a 0-12% methanol / dichloromethane gradient to obtain compound I-3 (0.32 g) as a colorless oil.

[0152] Example 7 Synthesis of compound I-4 [ka] A solution of di-(2-hexyldecyl)amine (1.25 g) in dichloromethane (20 mL) was treated with triethylamine (5 mL) and 5-bromopentanoyl chloride (1 g). The reaction mixture was stirred for 10 minutes, filtered, and the solvent was removed. The residue was dissolved in dichloromethane and washed with dilute aqueous hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was treated with isopropylamine (20 mL) and stirred overnight. The solvent was removed and the residue was washed with hexane and aqueous sodium bicarbonate solution. The solvent was removed and the residue was passed through a silica gel column using a gradient of 0 - 8% methanol / dichloromethane to give Compound I-4 (0.67 g) as a colorless oil.

[0153] Example 8 Synthesis of Compound I-5

Chemical formula

[0154] Example 9 Synthesis of Compound I-6

Chemical formula

[0155] Example 10 Synthesis of compound I-7 [ka] A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (10 mL) was treated overnight with 8-bromooctanoyl chloride (0.52 g) and triethylamine (1 mL). The solvent was removed, and the residue was partitioned three times with hexane and water. The solvent was removed, and the residue was dissolved in 2 M dimethylamine THF (20 mL). The reaction mixture was stirred overnight, filtered, and the solvent was removed under vacuum. The residue was passed through a silica gel column using a 0-6% methanol / dichloromethane gradient. The purified fraction was washed with hexane and aqueous sodium bicarbonate solution. The solvent was removed to obtain compound I-7 (0.65 g) as a colorless oil.

[0156] Example 11 Synthesis of 2-hexyldecanoylamide A solution of 2-hexyldecanoic acid (26 g) in benzene (30 mL) was treated with oxalyl chloride (15 mL). The reaction mixture was stirred until gas generation ceased, then the solvent was removed under vacuum, and the residue was dried under vacuum for 4 hours. Crude 2-hexyldecanoyl chloride was dissolved in dichloromethane (100 mL) and slowly added to a stirred solution of concentrated ammonium hydroxide (150 mL). The reaction mixture was allowed to stand for 2 hours, and the aqueous supernatant was removed. The organic phase was washed twice with water using the same method and filtered. The collected precipitate was dried to obtain crude 2-hexyldecanoylamide (22 g) as a white powder.

[0157] Example 12 Synthesis of 2-hexyldecanylamine A suspension of 2-hexyldecanoylamide (8.2 g) in dry tetrahydrofuran (40 mL) was treated with lithium aluminum hydride (1.1 g, added slowly). The reaction mixture was stirred for 2 hours, and then excess methanol was slowly added. Dichloromethane (150 mL) was added, followed by water (2 mL). The reaction mixture was filtered, and the solvent was removed from the filtrate to obtain crude 2-hexyldecanyamine (4.7 g).

[0158] Example 13 Synthesis of N-(2'-hexyldecanyl)-2-hexyldecanoylamide A solution of 2-hexyldecanylamine (4.7 g) in dichloromethane (100 mL) was treated with 2-hexyldecanoyl chloride (5.5 g, dissolved in 50 mL of dichloromethane), followed by treatment with triethylamine (4 mL). The reaction mixture was stirred for 1 hour and then washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue (~10 g) was combined with the crude product from the second reaction (~7 g) and passed through silica gel (100 g) using a 0-10% methanol / dichloromethane gradient to obtain N-(2'-hexyldecanyl)-2-hexyldecanoylamide (14.4 g).

[0159] Example 14 Synthesis of di-(2-hexyldecanyl)amine A solution of N-(2'-hexyldecanyl)-2-hexyldecanoylamide (14.4 g) in dry tetrahydrofuran (100 mL) was treated with lithium aluminum hydride (2 g) and refluxed overnight. Excess methanol was slowly added to dissolve the excess reducing agent. Dichloromethane (200 mL) was added, followed by water (2 mL). The mixture was then filtered to remove the solvent. The residue was suspended in hexane, filtered, and the solvent was removed. The residue was passed through a silica gel (100 g) column using a gradient of 2% acetic acid / dichloromethane, followed by 2-16% methanol / dichloromethane. The purified fraction was washed with hexane and aqueous sodium bicarbonate. The solvent was removed to obtain di-(2-hexyldecanyl)amine (9.5 g) as a colorless oil.

[0160] Example 15 Synthesis of compound I-8 [ka] A solution of di-(2-hexyldecanyl)amine (1.61 g) in dichloromethane (10 mL) was treated overnight with 9-bromonanoyl chloride (1 g) and N-ethyldiisopropylamine (1 mL). The solvent was removed, and the residue was dissolved in 2 M dimethylamine THF (35 mL). The reaction was stirred overnight, and then the solvent was removed under vacuum. The residue was partitioned with hexane and aqueous sodium bicarbonate. The solvent was removed from the organic fraction, and the residue was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane, and then 2-12% methanol / dichloromethane. The purified fraction was washed with hexane and aqueous sodium bicarbonate. The solvent was removed to obtain compound 1a (0.85 g) as a colorless oil. [ka] A solution of compound 1-8a (0.50 g) in tetrahydrofuran (15 mL) was treated with lithium aluminum hydride (0.28 g, added slowly). The reaction mixture was stirred for 4 hours. Next, excess methanol was slowly added, followed by dichloromethane (50 mL) and water (0.5 mL). The suspension was filtered to remove the solvent. The crude product was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane, and then 2–12% methanol / dichloromethane. The purified fraction was partitioned with hexane and aqueous sodium bicarbonate. After removing the solvent, compound I-8 (0.46 g) was obtained as a colorless oil.

[0161] Example 16 Synthesis of compound I-9 [ka] A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (10 mL) was treated overnight with 8-bromooctanoyl chloride (0.52 g) and triethylamine (1 mL). The solvent was removed, and the residue was partitioned three times with hexane and water. The solvent was removed, and the residue was dissolved in 2 M dimethylamine THF (20 mL). The reaction mixture was stirred overnight, filtered, and the solvent was removed under vacuum. The residue was passed through a silica gel column using a 0-6% methanol / dichloromethane gradient. The purified fraction was washed with hexane and aqueous sodium bicarbonate solution. The solvent was removed to obtain compound I-9a (0.65 g) as a colorless oil. [ka] A solution of compound I-9a (0.55 g) in tetrahydrofuran (20 mL) was treated with lithium aluminum hydride (0.5 g) for 2 hours. Methanol was added to quench the excess reducing agent. Dichloromethane (50 mL) and water (1 mL) were added, and the suspension was filtered. The solvent was removed, and the residue was passed through a silica gel column with a 0-8% methanol / dichloromethane gradient to obtain compound I-9 (0.27 g) as a colorless oil.

[0162] Further embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications (including both U.S. provisional patent applications 62 / 747,557 and 62 / 747,521 filed on 18 October 2018) (if any) referenced herein and / or listed in the application datasheet are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified to provide yet another embodiment using the concepts of various patents, applications, and publications as needed. These and other modifications may be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be construed as limiting the claims to any particular embodiment disclosed in the specification and claims, but rather as including all possible embodiments, along with the entire scope of equivalents to which such claims are granted. Thus, the claims are not limited by this disclosure.

Claims

1. (i) The following structure (I): 【Chemistry 1】 [In the formula, G １ is -N(R ３ ) R 4; R １ C is the branch saturation which may be optionally substituted. １２ -C ３６ It is an alkyl or optionally substituted branched unsaturated C12-C36 alkyl; R ２ is, when L is -C(=O)-, optionally substituted branched or unbranched saturated C １２ -C ３６ alkyl or optionally substituted branched or unbranched unsaturated C12-C36 alkyl; or, R ２ is, when L is C ６ -C １２ alkylene, C ６ -C １２ alkenylene, or C ２ -C ６ alkynylene, optionally substituted branched or unbranched saturated C ４ -C ３６ alkyl or optionally substituted branched or unbranched unsaturated C4-C36 alkyl; R ３ and R ４ Each of these is independently a branched or unbranched saturated or unsaturated C, which may be optionally substituted. １ -C ６ It is alkyl; or, R ３ and R ４ They, together with the nitrogen to which they are bound, form heterocyclines; L is -C (=O)-, C ６ -C １２ Alkylene, C ６ -C １２ Alkenylene, or C ２ -C ６ It is an alkynylene; and n is an integer between 1 and 12. The compound shown, or its pharmaceutically acceptable salt, tautomer, or stereoisomer (ii) Nucleic acids and Lipid nanoparticles (LNPs) for use in methods for inducing protein expression in a target, including those mentioned above.

2. G １ However, -N(R ３ ) R 4; R １ However, the branch saturation C may be arbitrarily substituted. １２ -C ３６ It is an alkyl or optionally substituted branched unsaturated C12-C36 alkyl; R ２ However, branched or unbranched saturated C may be arbitrarily substituted. １２ -C ３６ It is an alkyl or optionally substituted branched or unbranched unsaturated C12-C36 alkyl; R ３ and R ４ However, each of them independently may be arbitrarily substituted for branched or unbranched saturated C １ -C ６ It is an alkyl or optionally substituted branched or unbranched unsaturated C1-C6 alkyl; or R ３ and R ４ However, together with the nitrogen to which they are bound, they form heterocyclines; L is -C (=O)-; and, n is an integer between 1 and 12. The LNP according to claim 1.

3. G １ However, -N(R ３ ) R 4; R １ However, the branch saturation C may be arbitrarily substituted. １２ -C ３６ It is an alkyl or optionally substituted branched unsaturated C12-C36 alkyl; R ２ However, branched or unbranched saturated C may be arbitrarily substituted. ４ -C ３６ It is an alkyl or optionally substituted branched or unbranched unsaturated C4-C36 alkyl; R ３ and R ４ However, each of them independently may be arbitrarily substituted for branched or unbranched saturated C １ -C ６ It is an alkyl or optionally substituted branched or unbranched unsaturated C1-C6 alkyl; or R ３ and R ４ However, together with the nitrogen to which they are bound, they form heterocyclines; L is C ６ -C １２ Alkylene linker, C ６ -C １２ Alkenylene linker, or C ２ -C ６ It is an alkynylene linker; and n is an integer between 1 and 12. The LNP according to claim 1.

4. The compound has the following structure (IA): 【Chemistry 2】 [In the formula, R ８ and R ９ Each of these independently comprises H, or a branched or unbranched saturated C which may be substituted as desired. ２ -C １２ Alkyl or optionally substituted branched or unbranched unsaturated C2-C12 alkyl; however, R ８ and R ９ R １ However, the branch saturation C may be arbitrarily substituted. １２ -C ３６ Each is independently selected to be an alkyl or optionally substituted branched unsaturated C12-C36 alkyl; and, R １０ and R １１ are each independently H, or a branched or unbranched saturated C ２ -C １２ alkyl or a branched or unbranched unsaturated C2-C12 alkyl optionally substituted; provided that when L is -C(=O)-, R ２ is a branched or unbranched saturated C １２ -C ３６ alkyl or a branched or unbranched unsaturated C12-C36 alkyl optionally substituted; and when L is C ６ -C １２ alkylene, C ６ -C １２ alkenylene, or C ２ -C ６ alkynylene, R ２ is a branched or unbranched saturated C ４ -C ３６ alkyl or a branched or unbranched unsaturated C4-C36 alkyl optionally substituted, so that R １０ and R １１ [[ID=3 "6]]are each independently selected. ] The LNP according to any one of claims 1 to 3, which is represented by or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

5. R ８ and R ９ However, each of them independently may be arbitrarily substituted for branched or unbranched saturated C ２ -C １２ The LNP according to claim 4, wherein the LNP is an alkyl or optionally substituted branched or unbranched unsaturated C2-C12 alkyl group.

6. R ８ which may be optionally substituted: C ２ alkyl, C ４ alkyl, C ６ alkyl, C ８ alkyl, or C １０ alkyl, the LNP according to any one of claims 4 or 5.

7. R ８ However, it may be replaced as desired: C ４ Alkyl, C ６ Alkyl, or C ８ The LNP according to any one of claims 4 to 6, wherein it is alkyl.

8. R ９ However, it may be replaced as desired: C ４ Alkyl, C ６ Alkyl, C ８ Alkyl, C １０ Alkyl, or C １２ The LNP according to any one of claims 4 to 7, wherein the LNP is alkyl.

9. R ９ However, it may be replaced as desired: C ６ Alkyl, C ８ Alkyl, or C １０ The LNP according to any one of claims 4 to 8, wherein the LNP is alkyl.

10. R １０ and R １１ However, each of them independently may be arbitrarily substituted for branched or unbranched saturated C ２ -C １２ The LNP according to any one of claims 4 to 9, wherein the LNP is an alkyl or optionally substituted branched or unbranched unsaturated C2-C12 alkyl group.

11. R １０ However, it may be replaced as desired: C ２ Alkyl, C ４ Alkyl, C ６ Alkyl, C ８ Alkyl, or C １０ The LNP according to any one of claims 4 to 10, wherein the LNP is alkyl.

12. R １０ However, it may be replaced as desired: C ４ Alkyl, C ６ Alkyl, or C ８ The LNP according to any one of claims 4 to 11, wherein the LNP is alkyl.

13. R １１ However, the non-branched C may be arbitrarily substituted. ２ Alkyl, C ６ Alkyl, or C １０ The LNP according to any one of claims 4 to 12, wherein the LNP is alkyl.

14. R １１ However, C may be arbitrarily substituted. ２ The LNP according to claim 13, wherein it is alkyl.

15. R １１ However, C may be arbitrarily substituted. ６ The LNP according to claim 13, wherein it is alkyl.

16. R １１ However, C may be arbitrarily substituted. １０ The LNP according to claim 13, wherein it is alkyl.

17. R １１ However, it may be replaced as desired: C ４ Alkyl, C ６ Alkyl, C ８ Alkyl, C １０ Alkyl, or C １２ The LNP according to any one of claims 4 to 12, wherein the LNP is alkyl.

18. R １１ However, it may be replaced as desired: C ６ Alkyl, C ８ Alkyl, or C １０ The LNP according to any one of claims 4 to 12, wherein the LNP is alkyl.

19. R ８ , R ９ , R １０ , and R １１ However, each of them independently may be arbitrarily substituted for branched or unbranched saturated C ６ -C １０ The LNP according to claim 4, wherein the LNP is an alkyl or optionally substituted branched or unbranched unsaturated C6-C10 alkyl group.

20. R ８ , R ９ , R １０ , and R １１ However, each of them independently may be arbitrarily substituted for branched or unbranched saturated C ８ -C １２ The LNP according to claim 4, wherein the LNP is an alkyl or optionally substituted branched or unbranched unsaturated C8-C12 alkyl group.

21. R ８ , R ９ , R １０ , and R １１ However, each independently determines the branched or unbranched saturated C ２ -C ６ The LNP according to claim 4, wherein the LNP is alkyl or branched or unbranched unsaturated C2-C6 alkyl.

22. R ８ , R ９ , R １０ , and R １１ However, each of them independently may be arbitrarily substituted for branched or unbranched saturated C ６ -C １２ The LNP according to claim 4, wherein the LNP is an alkyl or optionally substituted branched or unbranched unsaturated C6-C12 alkyl group.

23. R １ and R ２ However, each branch may be independently and arbitrarily substituted for the saturation C of the branch. １２ -C ３０ The LNP according to claim 1, wherein the LNP is an alkyl or optionally substituted branched unsaturated C12-C30 alkyl.

24. R １ and R ２ However, each branch may be independently and arbitrarily substituted for the saturation C of the branch. １２ -C ２０ The LNP according to claim 1, wherein the LNP is an alkyl or optionally substituted branched unsaturated C12-C20 alkyl.

25. R １ and R ２ However, each branch may be independently and arbitrarily substituted for the saturation C of the branch. １５ -C ２０ The LNP according to claim 1, wherein the LNP is an alkyl or optionally substituted branched unsaturated C15-C20 alkyl.

26. R １ and R ２ The LNP according to any one of claims 1 or 23 to 25, wherein each is saturated.

27. R １ and R ２ The LNP according to claim 1 or any one of claims 23 to 25, wherein at least one of the is unsaturated.

28. R １ and R ２ The LNP according to any one of claims 1 to 27, wherein both are unsubstituted.

29. R １ and R ２ However, each has the following structure: 【Transformation 3】 The LNP according to claim 1.

30. R1 and R ２ However, each has the following structure: 【Chemistry 4】 The LNP described in claim 1, which is one of the LNPs.

31. R ２ However, the structure is as follows: 【Transformation 5】 An LNP according to any one of claims 1 or 3 to 29, which is one of the above.

32. R ２ However, the structure is as follows: 【Transformation 6】 An LNP according to any one of claims 1 or 3 to 29, which is one of the above.

33. R １ However, the structure is as follows: 【Transformation 7】 The LNP according to any one of claims 31 or 32.

34. L is C ６ -C １２ The LNP according to any one of claims 1 or 3 to 33, wherein the LNP is alkylene.

35. G １ However, -N(CH ３ ) ２ The LNP according to claim 1.

36. R ３ and R ４ The LNP according to claim 1, wherein they form a heterocycline together with the nitrogen to which they are bound.

37. G １ However, the structure is as follows: 【Transformation 8】 The LNP described in claim 36 is one of them.

38. G １ However, the structure is as follows: 【Chemistry 9】 The LNP according to claim 1.

39. The LNP according to any one of claims 1 to 38, wherein n is 1, 2, 3, 4, 5, or 6.

40. The LNP according to any one of claims 1 to 38, wherein n is 7, 8, 9, 10, 11, or 12.

41. The compound has the following structure: 【Chemistry 10】 The LNP described in claim 1, which is one of the LNPs.

42. The compound has the following structure: 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 The LNP described in claim 1, which is one of the LNPs.

43. The LNP according to any one of claims 1 to 42, wherein the nucleic acid is messenger RNA.

44. The LNP according to claim 43, wherein the mRNA encodes an antigen or antibody.

45. The LNP according to claim 44, wherein the antigen or antibody protects against infection.

46. The LNP according to claim 43, wherein the messenger RNA encodes a gene modifying enzyme.

47. The LNP according to claim 46, wherein the LNP further comprises a DNA segment for integration into the host genome, or the LNP is used in combination with a DNA segment for integration into the host genome.

Images