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WO2018191719A1 - Administration lipidique d'agents thérapeutiques au tissu adipeux - Google Patents

Administration lipidique d'agents thérapeutiques au tissu adipeux Download PDF

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Publication number
WO2018191719A1
WO2018191719A1 PCT/US2018/027655 US2018027655W WO2018191719A1 WO 2018191719 A1 WO2018191719 A1 WO 2018191719A1 US 2018027655 W US2018027655 W US 2018027655W WO 2018191719 A1 WO2018191719 A1 WO 2018191719A1
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alkyl
independently
occurrence
lipid
carbon
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PCT/US2018/027655
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English (en)
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Paulo Jia Ching LIN
Ying Tam
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Acuitas Therapeutics, Inc.
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Publication of WO2018191719A1 publication Critical patent/WO2018191719A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis

Definitions

  • Embodiments of the present invention generally relate to treating diseases mediated by protein expression in adipose tissue by delivery of therapeutic agents, such as nucleic acids (e.g., oligonucleotides, messenger RNA) to the adipose tissue via intraperitoneal administration.
  • therapeutic agents such as nucleic acids (e.g., oligonucleotides, messenger RNA) to the adipose tissue via intraperitoneal administration.
  • nucleic acid based therapeutics have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within an organism in order to realize this potential.
  • nucleic acid therapeutics are not stabile enough in circulation to provide a therapeutically effective concentration or accumulation in organs after systemic administration other than the liver.
  • free RNA is susceptible to nuclease digestion in plasma and possesses a limited ability to access intracellular compartments where relevant translational machinery resides. Therefore, nucleic acid-based therapies have been unable to effectively treat tissues of the body. Encapsulation in lipid nanoparticles have been shown effective in overcoming some limitations, but systemic administration generally leads to significant accumulation in only a few tissues of the body, e.g., liver. Thus, delivery to other tissues after systemic administration remains a significant challenge.
  • Adipose tissue is a highly active endocrine organ which produces and secretes proteins and adipokines involved in metabolic processes. Obesity, along with a variety of other metabolic disturbances, such as type II diabetes, is at least partly mediated by protein expression in adipocytes.
  • the first line treatment of obesity includes life-style changes and physical exercise, however this approach is often insufficient to normalize body weight and prevent life-threatening complications. Accordingly, alternative approaches are urgently needed.
  • a method of delivery of therapeutic agents to adipose tissue Preferably, such a delivery method would therapeutically target adipose tissue and have a desirable biological effect on the same.
  • a method for administration should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient.
  • embodiments of the present invention provide a method for treating a disease mediated by protein expression in adipose tissue of a subject in need thereof, the method comprising:
  • compositions comprising a lipid nanoparticle, the lipid nanoparticle comprising a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof,
  • lipid nanoparticle encapsulated within or associated with the lipid nanoparticle, thereby delivering the therapeutic agent to adipose tissue of the subject and altering protein expression in the adipose tissue.
  • the invention provides a method for delivering a therapeutic agent to adipose tissue of a subject in need thereof, the method comprising:
  • compositions comprising a therapeutically effective amount of a lipid nanoparticle, the lipid nanoparticle comprising a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof, encapsulated within or associated with the lipid nanoparticle; and intraperitoneally administering the composition to the subject, thereby delivering the therapeutic agent to the adipose tissue of the subject.
  • the presently disclosed methods can be used for treatment of various diseases or conditions, such as those caused by protein expression in adipose tissue.
  • the present invention provides methods for treatment of obesity, type II diabetes, insulin resistance, atherosclerosis or lipid disorders.
  • Fig. 1 shows a comparison of mRNA and luciferase distribution in mice for intravenous and intraperitoneal administration.
  • Fig. 2A illustrates tissue distribution of mRNA and luciferase following intravenous administration of an LNP comprising DLin-MC3-DMA.
  • Fig. 2B shows tissue distribution of mRNA and luciferase following intravenous administration of an LNP comprising Compound 1-6.
  • Fig. 2C depicts tissue distribution of mRNA and luciferase following intraperitoneal administration of an LNP comprising DLin-MC3-DMA.
  • Fig. 2D illustrates tissue distribution of mRNA and luciferase following intraperitoneal administration of an LNP comprising Compound 1-6.
  • Fig. 2E provides a legend identifying the tissue samples for panels shown in each Fig. 2A-D. DETAILED DESCRIPTION
  • Embodiments of the present invention are based, in part, upon the discovery that intraperitoneally administering a therapeutically effective amount of a composition comprising lipid nanoparticles (LNPs) encapsulating or associated with a therapeutic agent (e.g., mRNA) unexpectedly provides advantages previously unknown in the art.
  • a therapeutic agent e.g., mRNA
  • Applicant has unexpectedly discovered that intraperitoneal administration of LNPs encapsulating or associated with a therapeutic agent results in preferential localization of the therapeutic agent in adipose tissue relative to other tissues.
  • the therapeutic agent remains active within the adipose tissue (e.g., induces protein expression in adipose tissue).
  • Altering protein expression in adipose tissue can influence overall metabolism, whole-body energy and adipose conversion and activation. Accordingly, the presently disclosed methods are effective for treatment of various diseases associated with protein expression in adipose tissue, such as obesity, type II diabetes, insulin resistance, atherosclerosis or lipid disorders.
  • the invention provides a method for treating a disease mediated by protein expression in adipose tissue of a subject in need thereof, the method comprising:
  • a composition comprising a lipid nanoparticle, the lipid nanoparticle comprising a therapeutic agent, or a pharmaceutically acceptable salt or prodrugthereof, encapsulated within or associated with the lipid nanoparticle, thereby delivering the therapeutic agent to adipose tissue of the subject and altering protein expression in the adipose tissue.
  • a method for delivering a therapeutic agent to adipose tissue of a subject in need thereof comprises:
  • composition comprising a therapeutically effective amount of a lipid nanoparticle, the lipid nanoparticle comprising a therapeutic agent, or a pharmaceutically acceptable salt or prodrugthereof, encapsulated within or associated with the lipid nanoparticle;
  • compositions intraperitoneally administering the composition to the subject, thereby delivering the therapeutic agent to the adipose tissue of the subject.
  • White adipose tissue stores energy in the form of triacylglycerol.
  • Brown adipose tissue (BAT) has been identified to be an important factor in regulation of energy balance, thereby limiting or controlling weight gain.
  • the conversion of white adipose tissue to brown adipose tissue mediates anti-obesity effects such as resistance to weight gain and improvements in systemic metabolism, including improved glucose tolerance, increased insulin sensitivity and enhanced uptake and metabolism of lipids from the bloodstream.
  • brown adipocytes (BAT cells) surround blood vessels and have been implicated in the protection against development of atherosclerosis.
  • therapeutically targeting protein expression in adipose tissue has applications, both directly in obesity and beyond, to a variety of metabolic disturbances, including type II diabetes, insulin resistance, atherosclerosis and lipid disorders.
  • the presently disclosed methods induce conversion of white adipose tissue to brown adipose tissue. In other embodiments, the disclosed methods induce activation of brown adipose tissue. In some embodiments of the foregoing, the adipose tissue comprises white adipocytes or brown adipocytes.
  • the present invention provides a method for administering a composition comprising lipid nanoparticles for and in vivo delivery of mRNA and/or other oligonucleotides to adipose tissue.
  • the methods are useful for mediating expression of protein encoded by mRNA. In other embodiments, the methods are useful for affecting upregulation of endogenous protein expression in adipose tissue by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA. In other embodiments, this method is useful for down-regulating ⁇ e.g., silencing) the protein levels and/or mRNA levels of target genes. In some other embodiments, the methods are also useful for delivery of mRNA and plasmids for expression of transgenes.
  • the methods are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., conversion of white adipose tissue to brown adipose tissue, or activation of brown adipose tissue.
  • the therapeutic agent may be a non-nucleic acid based agent, such as a small molecule or peptide-based drug.
  • the small molecule or peptide based drug may include drugs used for treating a variety of metabolic disturbances, including obesity, type II diabetes, insulin resistance, atherosclerosis and lipid disorders.
  • therapeutic agents include, but are not limited to acarbose, miglitol, metformin (e.g., metformin-alogliptin, metformin-canagliflozin, metformin-dapagliflozin (Xigduo XR), metformin-empagliflozin (Synjardy), metformin-glipizide, metformin-glyburide (Glucovance), metformin-linagliptin (Jentadueto), metformin-pioglitazone (Actoplus), metformin-repaglinide (PrandiMet), metformin-rosiglitazone (Avandamet), metformin-saxagliptin (Kombiglyze XR), metformin-sitagliptin (Janumet)), bromocriptine (Parlodel), alogliptin (Nesina), alogliptin (N
  • Glucovance chlorpropamide
  • Tolinase tolazamide
  • Tolbutamide Orinase, Tol-Tab
  • rosiglitazone Avandia
  • rosiglitazone-glimepiride Avandaryl
  • rosiglitizone-metformin Amaryl M
  • pioglitazone Actos
  • statins e.g., Fluvastatin, Atorvastatin, Lovastatin, Pravastatin, Simvastatin, Rosuvastatin, Pitavastatin
  • fibrates Gemfibrozil, Fenofibrate, niacin, ezetimibe, cholestyramine, colestipol, or colesevelam.
  • embodiments of methods of the present invention are particularly useful for the delivery of nucleic acids to adipose tissue, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, the methods of the present invention may be used to induce expression of a desired protein both in vitro and in vivo by contacting adipose tissue with a lipid nanoparticle.
  • the method of the present invention may be used to decrease the expression of target genes and/or proteins both in vitro and in vivo by contacting adipose tissue with a lipid nanoparticle.
  • the methods of the present invention may also be used for co-delivery of different nucleic acids ⁇ e.g., mRNA and plasmid DNA) separately or in combination, such as may be useful to provide an effect requiring colocalization of different nucleic acids ⁇ e.g., mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome).
  • Nucleic acids for use with embodiments of this invention may be prepared according to any available technique.
  • the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequence-specific mRNA.
  • In vitro transcription describes a process of template- directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence ⁇ e.g. including but not limited to that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest.
  • Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification ⁇ see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012).
  • RNA polymerase adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts.
  • rNTPs ribonucleoside triphosphates
  • In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies) as well as with commercially available reagents including RNA polymerases and rNTPs.
  • the methodology for in vitro transcription of mRNA is well known in the art. (see, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46;
  • the desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including
  • RNA transcripts unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.).
  • Techniques for the isolation of the mRNA transcripts are well known in the art.
  • Well known procedures include phenol/chloroform extraction or precipitation with either alcohol ⁇ e.g., ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.
  • Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P.J.
  • RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012 ). Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro
  • RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation.
  • RNA impurities include short RNAs that result from abortive 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' extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses.
  • dsRNA double-stranded RNA
  • HPLC purification eliminates immune activation and improves translation of nucleoside- modified, protein-encoding mRNA, Nucl Acid Res, v.
  • Endogenous eukaryotic mRNA typically contain a cap structure on the 5'- end of a mature molecule which plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts.
  • CBP mRNA Cap Binding Protein
  • the 5 '-cap contains a 5 '-5 '-triphosphate linkage between the 5 '-most nucleotide and guanine nucleotide.
  • the conjugated guanine nucleotide is methylated at the N7 position.
  • modifications include methylation of the ultimate and penultimate most 5 '-nucleotides on the 2'-hydroxyl group.
  • 5 '-capping of synthetic mRNA can be performed co- transcriptionally with chemical cap analogs ⁇ i.e., capping during in vitro transcription).
  • the Anti -Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 3'-0-methyl group.
  • ARC A Anti -Reverse Cap Analog
  • the synthetic cap analog is not identical to the 5 '-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability.
  • synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5 '-cap structure that more closely mimics, either structurally or functionally, the endogenous 5 '-cap which have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5' decapping.
  • poly-A tail On the 3 '-terminus, a long chain of adenine nucleotides (poly-A tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl to which poly- A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation.
  • the poly-A tail has been extensively shown to enhance both translational 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.
  • Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase.
  • the first case allows in vitro transcription of mRNA with poly (A) tails of defined length, depending on the size of the poly (T) tract, but requires additional manipulation of the template.
  • poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3 'termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length.
  • 5'- capping and 3 '-poly (A) tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A)
  • modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity (see e.g., Kariko, K. And Weissman, D.
  • modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art.
  • nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA (see, e.g., US Pub. No.2012/0251618). In vitro synthesis of nucleoside-modified mRNA have been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.
  • mRNA which can be modified to provide benefit in terms of translatability and stability
  • 5' and 3' untranslated regions include the 5' and 3' untranslated regions (UTR). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA (see e.g. Pardi, N.,
  • oligonucleotides In addition to mRNA, other nucleic acid payloads may be used for this invention.
  • methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor, in vitro transcription as described above, etc. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.:
  • plasmid DNA preparation for use with this invention commonly utilizes but is not limited to expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest.
  • a gene in the plasmid of interest that encodes resistance to a particular antibiotic penicillin, kanamycin, etc.
  • Methods of isolating plasmid DNA are widely used and well known in the art ⁇ see, e.g., Heilig, J., Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology.
  • Plasmid isolation can be performed using a variety of commercially available kits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and Pure Yield MaxiPrep (Promega) kits as well as with commercially available reagents.
  • a test sample e.g. a sample of cells in culture expressing the desired protein
  • a test mammal e.g. a mammal such as a human or an animal model such as a rodent (e.g. mouse) or a non-human primate (e.g., monkey) model
  • a nucleic acid e.g. nucleic acid in combination with a lipid of the present invention.
  • expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g.
  • a sample of cells in culture expressing the desired protein or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model) that is not contacted with or administered the nucleic acid.
  • a control mammal e.g., a mammal such as a human or an animal model such as a rodent (e.g. mouse) or non-human primate (e.g. monkey) model
  • the expression of a desired protein in a control sample or a control mammal may be assigned a value of 1.0.
  • inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or the control mammal is greater than 1, for example, about 1.1, 1.5, 2.0. 5.0 or 10.0.
  • inducing expression of a desired protein is achieved when any measurable level of the desired protein in the test sample or the test mammal is detected.
  • ⁇ assays to determine the level of protein expression in a sample, for example dot blots, northern blots, 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.
  • the phrase "inhibiting expression of a target gene” refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene.
  • test sample e.g., a sample of cells in culture expressing the target gene
  • test mammal e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model
  • a nucleic acid that silences, reduces, or inhibits expression of the target gene.
  • Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that is not contacted with or administered the nucleic acid.
  • a control sample e.g., a sample of cells in culture expressing the target gene
  • a control mammal e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model
  • the expression of the target gene in a control sample or a control mammal may be assigned a value of 100%.
  • silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relative to the level of target gene expression in a control sample or a control mammal not contacted with or administered the nucleic acid.
  • Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • the subject is a mammal. In some more specific embodiments, the subject is a human.
  • an “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g. an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid.
  • An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid.
  • an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 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 greater.
  • Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%), 15%), 10%), 5%), or 0%.
  • Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors.
  • RNA may be in 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 backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl
  • nucleic acids Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol.
  • Nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • gene refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.
  • Gene product refers to a product of a gene such as an RNA transcript or a polypeptide.
  • lipid 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 divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • a “steroid” is a compound comprising the following carbon skeleton:
  • Non-limiting examples of steroids include cholesterol, and the like.
  • a "cationic lipid” refers to a lipid capable of being positively charged.
  • Exemplary cationic lipids include one or more amine group(s) which bear the positive charge.
  • Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S.C., et al., Adv.
  • polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion.
  • An example of a polymer conjugated lipid is a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-s «-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-5 «-glycero-3- phosphocholine (DPPC), l,2-Dimyristoyl-s «-glycero-3-phosphocholine (DMPC), 1- Palmitoyl-2-oleoyl-s «-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), phophatidylethanolamines such as l,2-Dioleoyl-s «-glycero-3- phosphoethanolamine (DOPE), sphin
  • charged lipid refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g. pH ⁇ 3 to pH ⁇ 9.
  • Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates, dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Choi).
  • lipid nanoparticle refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the compounds of Formula I, II, III, or other specified cationic lipids.
  • lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like).
  • an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA)
  • a target site of interest e.g., cell, tissue, organ, tumor, and the like.
  • the lipid nanoparticles of the invention comprise a nucleic acid.
  • Such lipid nanoparticles typically comprise a compound of Formula I, II, III or other specified cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids.
  • the active agent or therapeutic agent such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.
  • the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 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 n
  • nucleic acids when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.
  • Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos.
  • lipid encapsulated refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both.
  • an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA)
  • nucleic acid e.g., mRNA
  • lipid nanoparticle (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.
  • aqueous solution refers to a composition comprising water.
  • “Serum-stable” in relation to nucleic acid-lipid nanoparticles means that the nucleotide is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.
  • Systemic delivery refers to delivery of a therapeutic product that can result in a broad exposure of an active agent within an organism.
  • Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body.
  • Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.
  • Local delivery refers to delivery of an active agent directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site such as a tumor, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like.
  • Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.
  • Adipose tissue refers to loose connective tissue comprising adipocytes.
  • Adipose tissue includes two types: white adipose tissue (WAT; which stores energy) and brown adipose tissue (BAT; which generates body heat).
  • WAT white adipose tissue
  • BAT brown adipose tissue
  • brown adipose tissue includes beige adipose tissue.
  • Adipose tissue can be found beneath the skin and around internal organs to provide a protective padding.
  • Adipose tissue can also include a stromal vascular fraction of cells including preadipocytes, fibroblasts, vascular endothelial cells and a variety of immune cells such as adipose tissue macrophages.
  • amino acid refers to naturally-occurring and non-naturally occurring amino acids.
  • An amino acid lipid can be made from a genetically encoded amino acid, a naturally occurring non-genetically encoded amino acid, or a synthetic amino acid.
  • amino acids include Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • amino acids also include azetidine, 2-aminooctadecanoic acid, 2-aminoadipic acid, 3-aminoadipic acid, 2,3- diaminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 2,3-diaminobutyric acid, 2,4-diaminobutyric acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid, 2- aminopimelic acid, 2,2'-diaminopimelic acid, 6-aminohexanoic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, desmosine, ornithine, citrulline, N-methylisoleucine, norleucine, tert-leucine, phenylglycine, t-butylglycine, N-methylglycine, sacrosine, N- ethylglycine, cyclohexylglycine
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double (alkenyl) and/or triple bonds (alkynyl)), having, for example, from one to twenty-four carbon atoms (Ci-C 2 4 alkyl), four to twenty carbon atoms (C 4 -C 2 o alkyl), six to sixteen carbon atoms (C 6 -Ci 6 alkyl), six to nine carbon atoms (C 6 -C9 alkyl), one to fifteen carbon atoms (C 1 -C 15 alkyl),one to twelve carbon atoms (C 1 -C 12 alkyl), one to eight carbon atoms (C 1 -C 8 alkyl) or one to six carbon atoms (C 1 -C 6 alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl
  • Alkylene or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e., contains one or more double (alkenylene) and/or triple bonds (alkynylene)), and having, for example, from one to twenty-four carbon atoms (Ci-C 2 4 alkylene), one to fifteen carbon atoms (C 1 -C 15 alkylene),one to twelve carbon atoms (C 1 -C 12 alkylene), one to eight carbon atoms (Ci- C 8 alkylene), one to six carbon atoms (C 1 -C 6 alkylene), two to four carbon atoms (C 2 -C 4 alkylene), one to two carbon atoms (Ci-C 2 alkylene), e.g., methylene, ethylene, propylene, «-butylene, ethenylene, propenylene,
  • the alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond.
  • the points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.
  • an alkylene chain may be optionally substituted.
  • alkenyl refers to an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include, but are not limited to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1 -pentenyl, 2- pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • Alkoxy refers to an alkyl, cycloalkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom.
  • Alkylamino refers to the group -NRR, where R and R' are each either hydrogen or alkyl, and at least one of R and R is alkyl. Alkylamino includes groups such as piped dino wherein R and R form a ring. The term “alkylaminoalkyl” refers to - alkyl- RR.
  • alkynyl includes any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons.
  • Representative straight chain and branched alkynyls include, without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3 -methyl- 1 butynyl, and the like.
  • acyl refers to any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below.
  • Aryl refers to any stable monocyclic, bicyclic, or poly cyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one ring is aromatic. Some examples of an aryl include phenyl, naphthyl, tetrahydro-naphthyl, indanyl, and biphenyl. Where an aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is to the aromatic ring. An aryl may be substituted or unsubstituted.
  • Cyano refers to a functional group of the formula -CN.
  • Cycloalkyl or “carbocyclic ring” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond.
  • Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkyl group may be optionally substituted.
  • Cycloalkylene is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.
  • diacylglycerol or “DAG” includes a compound having 2 fatty acyl chains, both of which have independently between 2 and 30 carbons bonded to the 1- and 2-position of glycerol by ester linkages.
  • the acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (C16), stearoyl (C18), and icosoyl (C20).
  • the fatty acid acyl chains of one compound are the same, i.e., both myristoyl ⁇ i.e., dimyristoyl), both stearoyl ⁇ i.e., distearoyl), etc.
  • heterocycle refers to an aromatic or nonaromatic ring system of from five to twenty -two atoms, wherein from 1 to 4 of the ring atoms are heteroatoms selected from oxygen, nitrogen, and sulfur.
  • a heterocycle may be a heteroaryl or a dihydro or tetrathydro version thereof.
  • Heterocycles include, but are not limited to, pyrrolidine, tetryhydrofuran, thiolane, azetidine, oxetane, thietane, diazetidine, dioxetane, dithietane, piperidine,
  • Heteroaryl refers to any stable monocyclic, bicyclic, or poly cyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur.
  • a heteroaryl examples include acridinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinolinyl.
  • a heteroaryl includes the N-oxide derivative of a nitrogen-containing heteroaryl.
  • alkylamine and “dialkylamine” refer to— NH(alkyl) and — N(alkyl) 2 radicals respectively.
  • alkylphosphate refers to— O— P(Q')(Q")-0— R, wherein Q' and Q" are each independently O, S, N(R) 2 , optionally substituted alkyl or alkoxy; and R is optionally substituted alkyl, co-aminoalkyl or ro-(substituted)aminoalkyl.
  • alkylphosphorothioate refers to an alkylphosphate wherein at least one of Q' or Q" is S.
  • alkylphosphonate refers to an alkylphosphate wherein at least one of Q' or Q" is alkyl.
  • Hydrox alkyl refers to an -O-alkyl radical.
  • alkylheterocycle refers to an alkyl where at least one methylene has been replaced by a heterocycle.
  • co-aminoalkyl refers to -alkyl-NH 2 radical.
  • co- (substituted)aminoalkyl refers to an ⁇ -aminoalkyl wherein at least one of the H on N has been replaced with alkyl.
  • co-phosphoalkyl refers to -alkyl-0— P(Q')(Q")-0— R, wherein Q' and Q" are each independently O or S and R optionally substituted alkyl.
  • co-thiophosphoalkyl refers to co-phosphoalkyl wherein at least one of Q' or Q" is S.
  • R ' is, at each occurrence, independently H, C 1 -C 15 alkyl or cycloalkyl, and x is 0, 1 or 2.
  • the substituent is a C 1 -C 12 alkyl group.
  • the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. 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(- R R ).
  • Optional or “optionally substituted” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
  • optionally substituted alkyl means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.
  • Prodrug is meant to indicate a compound, such as a therapeutic agent, that may be converted under physiological conditions or by solvolysis to a biologically active compound of the invention.
  • prodrug refers to a metabolic precursor of a compound of the invention that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention.
  • Prodrugs are typically rapidly transformed in vivo to yield the parent compound of the invention, for example, by hydrolysis in blood.
  • the prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp.
  • prodrugs are provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
  • prodrug is also meant to include any covalently bonded carriers, which release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs ⁇ e.g., a prodrug of a therapeutic agent) may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine
  • Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group such that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the therapeutic agents of the invention and the like.
  • the invention disclosed herein is also meant to encompass administration of all pharmaceutically acceptable lipid nanoparticles and components thereof ⁇ e.g., cationic lipid, therapeutic agent, etc.) being isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number.
  • isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2 H, 3 H, U C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 31 P, 32 P, 35 S, 18 F, 36 C1, 123 I, and 125 I, respectively.
  • These radiolabeled L Ps could be useful to help determine or measure the effectiveness of the administration of compounds to adipose tissue, by characterizing, for example, the site or mode of action, or binding affinity to
  • LNPs for example, those incorporating a radioactive isotope
  • the radioactive isotopes tritium, i.e., 3 H, and carbon-14, that is, C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
  • substitution with heavier isotopes such as deuterium, that is, 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
  • Isotopically-labeled compounds of Formula I, II or III can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
  • Solid compound and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • “Mammal” includes humans and both domestic animals such as laboratory animals and household pets ⁇ e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.
  • “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, 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, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol,
  • 2-diethylaminoethanol dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, tri ethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine,
  • a "pharmaceutical composition” refers to a formulation of an L P of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all
  • Treating refers to the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes:
  • disease and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.
  • the disease is a metabolic disturbance, for example, obesity, type II diabetes, insulin resistance, atherosclerosis, or lipid disorders.
  • the present invention provides a use of a therapeutically effective amount of a composition for intraperitoneally administration, the composition comprising a lipid nanoparticle and a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof, encapsulated within or associated with the lipid nanoparticle, in the manufacture of a medicament for treating a disease mediated by protein expression in adipose tissue of a subject in need thereof, wherein the administering delivers the therapeutic agent to adipose tissue of the subject and alters protein expression in the adipose tissue.
  • the lipid nanoparticle and therapeutic agent can be prepared according to any of the
  • Certain embodiments provide a method for treating a disease mediated by protein expression in adipose tissue of a subject in need thereof, the method comprising:
  • compositions comprising a lipid nanoparticle, the lipid nanoparticle comprising a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof,
  • lipid nanoparticle encapsulated within or associated with the lipid nanoparticle, thereby delivering the therapeutic agent to adipose tissue of the subject and altering protein expression in the adipose tissue.
  • the present invention provides a method for delivering a therapeutic agent to adipose tissue of a subject in need thereof, the method comprising:
  • composition comprising a therapeutically effective amount of a lipid nanoparticle, the lipid nanoparticle comprising a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof, encapsulated within or associated with the lipid nanoparticle;
  • lipid nanoparticle components can be chosen to effectuate desirable physical characteristics.
  • Some common components of lipid nanoparticles include, but are not limited to cationic lipids, neutral lipids, steroids, and polymer conjugated lipids.
  • the L Ps disclosed herein comprise a cationic lipid.
  • the cationic lipid can be any of a number of lipid species which carry a net positive charge at a selected pH, such as physiological pH.
  • lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl- ⁇ , ⁇ -dimethylammonium bromide (DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP); 3-(N— (N',N'dimethylaminoethane)- carbamoyl)cholesterol (DC-Choi), N-(l-(2,3-dioleoyloxy)propyl)N-2- (
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.
  • LIPOFECTIN® commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3phosphoethanolamine (DOPE)
  • DOPE dioleoyl-sn-3phosphoethanolamine
  • LIPOFECTAMINE® commercially available cationic liposomes comprising N- (l-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl
  • DOGS carboxyspermine
  • the cationic lipid is an amino lipid.
  • Suitable amino lipids useful in the invention include those described in PCT Pub. No. WO
  • Representative amino lipids include, but are not limited to, l,2-dilinoleyoxy-3-
  • Ri and R 2 are either the same or different and independently optionally substituted Cio-C 24 alkyl, optionally substituted C 10 -C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted C10-C24 acyl;
  • R 3 and R4 are either the same or different and independently optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, or optionally substituted C 2 -C 6 alkynyl or R 3 and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
  • R 5 is either absent or present and when present is hydrogen or C 1 -C 6 alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and
  • Ri and R 2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.
  • cationic lipids include those having the following structure:
  • Ri and R 2 are independently selected from the group consisting of H, and C1-C3 alkyls;
  • R 3 and R4 are independently selected from the group consisting of alkyl groups having from about 10 to about 20 carbon atoms, wherein at least one of R 3 and R4 comprises at least two sites of unsaturation.
  • R 3 and R4 may be, for example, dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl.
  • R 3 and R are both linoleyl.
  • R 3 and R4 may comprise at least three sites of unsaturation (e.g., R3 and R4 may be, for example, dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl).
  • the LNPs comprise lipids having the following structure:
  • Ri and R2 are independently selected and are H or C1-C3 alkyls.
  • R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, wherein at least one of R4 and R4 comprises at least two sites of unsaturation.
  • R 3 and R4 are both the same, for example, in some embodiments R3 and R4 are both linoleyl (i.e. CI 8), etc.
  • R3 and R4 are different, for example, in some embodiments R3 is tetradectrienyl (C14) and R4 is linoleyl (CI 8).
  • the cationic lipids of the present invention are symmetrical, i.e., R3 and R4 are the same.
  • both R3 and R4 comprise at least two sites of unsaturation.
  • R 3 and R4 are independently selected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl.
  • R 3 and R4 are both linoleyl.
  • R4 and R4 comprise at least three sites of unsaturation and are
  • the cationic lipid has the formula:
  • n 2 to 20;
  • R 1 is independently, for each occurrence, a non-hydrogen, substituted or unsubstituted side chain of an amino acid
  • R 2 and R N are independently, for each occurrence, hydrogen, an organic group consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, or any combination of the foregoing, and having from 1 to 20 carbon atoms, C ( i- 5) alkyl,
  • Z is NH, O, S,— CH2S— ,— CH2S(0)— , or an organic linker consisting of 1-40 atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfur atoms (preferably, Z is NH or O);
  • R x and R y are, independently, (i) a lipophilic tail derived from a lipid (which can be naturally-occurring or synthetic), phospholipid, glycolipid,
  • the tail optionally includes a steroid; (ii) an amino acid terminal group selected from hydrogen, hydroxyl, amino, and an organic protecting group; or (iii) a substituted or unsubstituted
  • R x and R y are lipophilic tails as defined above and the other is an amino acid terminal group, or both R x and R y are lipophilic tails;
  • R x and R y are interrupted by one or more biodegradable
  • R 11 is a C 2 -C 8 alkyl or alkenyl and each occurrence of R 5 is, independently, H or alkyl; and each occurrence of R 3 and R 4 are, independently H, halogen, OH, alkyl, alkoxy,— NHfe, alkylamino, or dialkylamino; or R 3 and R 4 , together with the carbon atom to which they are directly attached, form a cycloalkyl group (in one preferred embodiment, each occurrence ofR 3 and R 4 are, independently H or C 1 -C 4 alkyl)); and R x and R y each, independently, optionally have one or more carbon-carbon double bonds.
  • the cationic lipid is one of the following:
  • Ri and R 2 are independently alkyl, alkenyl or alkynyl, and each can be optionally substituted;
  • R 3 and R4 are independently a Ci-Ce alkyl, or R 3 and R4 can be taken together to form an optionally substituted heterocyclic ring.
  • a representative useful dilinoleyl amino lipid has the formula:
  • n 0, 1, 2, 3, or 4.
  • the cationic lipid is a DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein 2).
  • the cationic lipid is DLin-MC3-DMA.
  • the cationic lipid has the following structure:
  • Ri and R 2 are each independently for each occurrence optionally substituted C10-C30 alkyl, optionally substituted C10-C30 alkenyl, optionally substituted C1.0-C30 alkynyl or optionally substituted C10-C30 acyl;
  • Cio alkenyl optionally substituted C 2 -C 10 alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonate, alkylamine, hydroxyalkyl, ⁇ -aminoalkyl, ro-(substituted)aminoalkyl, ⁇ -phosphoalkyl, ⁇ - thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl, or heterocycle, or linker- ligand, for example in some embodiments R3 is ( ⁇ 3 ⁇ 4)2 ⁇ (0 ⁇ 2) ⁇ -, wherein n is 1, 2, 3 or 4;
  • E is O, S, N(Q), C(0), OC(O), C(0)0, N(Q)C(0), C(0)N(Q),
  • Q is H, alkyl, ⁇ -aminoalkyl, ro-(substituted)aminoalkyl, ⁇ -phosphoalkyl or ⁇ -thiophosphoalkyl.
  • the cationic lipid has the following structure:
  • Q is H, alkyl, ⁇ -amninoalkyl, ro-(substituted)amninoalky, ⁇ - phosphoalkyl or ⁇ -thiophosphoalkyl;
  • Ri and R2 and R x are each independently for each occurrence H, optionally substituted C 1 -C 10 alkyl, optionally substituted C 10 -C 30 alkyl, optionally substituted C 10 -C 30 alkenyl, optionally substituted C 10 -C 30 alkynyl, optionally substituted C10-C30 acyl, or linker-ligand, provided that at least one of Ri, R2 and R x is not H;
  • R3 is H, optionally substituted C1-C10 alkyl, optionally substituted C 2 -C 10 alkenyl, optionally substituted C 2 -C 10 alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonate, alkylamine, hydroxyalkyl, ⁇ -aminoalkyl, ro-(substituted)aminoalkyl, ⁇ -phosphoalkyl, ⁇ - thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl, or heterocycle, or linker- ligand; and
  • n 0, 1, 2, or 3.
  • the canonic lipid has one of the following structures:
  • the cationic lipid has the structure of Formula I:
  • R a is H or C 1 -C 12 alkyl
  • R la and R lb are, at each occurrence, independently either (a) H or C 1 -C 12 alkyl, or (b) R la is H or C 1 -C 12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either (a) H or C 1 -C 12 alkyl, or (b) R 2a is H or C 1 -C 12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either (a) H or C 1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either (a) H or C 1 -C 12 alkyl, or (b) R 4a is H or C 1 -C 12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R 5 and R 6 are each independently methyl or cycloalkyl;
  • R 7 is, at each occurrence, independently H or C 1 -C 12 alkyl;
  • R 8 and R 9 are each independently unsubstituted C 1 -C 12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7- membered heterocyclic ring comprising one nitrogen atom;
  • a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; e is 1 or 2; and
  • x 0, 1 or 2.
  • L 1 and L 2 are independently -
  • R la and R lb are not isopropyl when a is 6 or n-butyl when a is 8.
  • R la and R lb are not isopropyl when a is 6 or n-butyl when a is 8.
  • R 8 and R 9 are each independently unsubstituted C 1 -C 12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;
  • R a and R b are, at each occurrence, independently H or a substituent.
  • R a and R b are, at each occurrence, independently H, Ci- Ci 2 alkyl or cycloalkyl, for example H or C 1 -C 12 alkyl.
  • the lipid compounds of Formula I have the following structure (la):
  • the lipid compounds of Formula I have the following structure (lb):
  • the lipid compounds of Formula I have the following structure (Ic):
  • a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.
  • b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.
  • c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16. In some certain other embodiments of Formula I, d is 0. In some embodiments, d is 1. In other embodiments, d is 2.
  • d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.
  • a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.
  • a and b and the sum of c and d in Formula I are factors which may be varied to obtain a lipid of formula I having the desired properties.
  • a and b are chosen such that their sum is an integer ranging from 14 to 24.
  • c and d are chosen such that their sum is an integer ranging from 14 to 24.
  • the sum of a and b and the sum of c and d are the same.
  • the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24.
  • a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.
  • e is 1. In other embodiments, e is 2.
  • the substituents at R la , R 2a , R 3a and R 4a of Formula I are not particularly limited. In certain embodiments R la , R 2a , R 3a and R 4a are H at each occurrence. In certain other embodiments at least one of R la , R 2a , R 3a and R 4a is C 1 -C 12 alkyl. In certain other embodiments at least one of R la , R 2a , R 3a and R 4a is C 1 -C 8 alkyl. In certain other embodiments at least one of R la , R 2a , R 3a and R 4a is C 1 -C 6 alkyl.
  • the C 1 -C 8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R la , R lb , R 4a and R 4b are C 1 -C 12 alkyl at each occurrence.
  • R ib R 2b R 3b and R 4b is H or R lb , R 2b , R 3b and R 4b are H at each occurrence.
  • R together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 5 and R 6 of Formula I are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R 5 or R 6 is methyl.
  • R 5 or R 6 is cycloalkyl for example cyclohexyl.
  • the cycloalkyl may be substituted or not substituted.
  • the cycloalkyl is substituted with C 1 -C 12 alkyl, for example tert-butyl.
  • R 7 are not particularly limited in the foregoing embodiments of Formula I. In certain embodiments at least one R 7 is H. In some other embodiments, R 7 is H at each occurrence. In certain other embodiments R 7 is C 1 -C 12 alkyl.
  • one of R 8 or R 9 is methyl. In other embodiments, both R 8 and R 9 are methyl.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.
  • the lipid of Formula I has one of the structures set forth in Table 1 below. TABLE 1: REPRESENTATIVE LIPIDS OF FORMULA I
  • the lipid of Formula I is compound 1-5. In some embodiments the lipid of Formula I is compound 1-6.
  • the cationic lipid has a structure of Formula II:
  • G 3 is C 1 -C 6 alkylene;
  • R a is H or C 1 -C 12 alkyl
  • R la and R lb are, at each occurrence, independently either: (a) H or C 1 -C 12 alkyl; or (b) R la is H or C 1 -C 12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either: (a) H or C 1 -C 12 alkyli or (b)R ⁇ is H or C 1 -C 12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either (a): H or C 1 -C 12 alkyl; or (b) R 3a is H or C 1 -C 12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either: (a) H or C 1 -C 12 alkyl; or (b) R 4a is H or C 1 -C 12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 5 and R 6 are each independently H or methyl
  • R 7 is C4-C20 alkyl
  • R 8 and R 9 are each independently C 1 -C 12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring;
  • a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.
  • the lipid compound has one of the following structures (II A) or (IIB):
  • the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).
  • one of L 1 or L 2 is a direct bond.
  • a "direct bond” means the group (e.g., L 1 or L 2 ) is absent.
  • each of L 1 and L 2 is a direct bond.
  • R la is H or C 1 -C 12 alkyl
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4a is H or C 1 -C 12 alkyl
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 2a is H or C 1 -C 12 alkyl
  • R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 3a is H or C 1 -C 12 alkyl
  • R 3b together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • the lipid compound has one of the following structures (IIC) or (IID):
  • e, f, g and h are each independently an integer from 1 to 12.
  • the lipid compound has structure
  • the lipid compound has structure (IID).
  • structures (IIC) or (IID) are each independently an integer from 4 to 10.
  • a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12.
  • a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.
  • b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some
  • b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.
  • c is 1. In other embodiments, c is
  • c is 3. In yet other embodiments, c is 4. In some
  • c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.
  • d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.
  • e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some
  • e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.
  • f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.
  • g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some
  • g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.
  • h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some
  • e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.
  • a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.
  • the sum of a and b and the sum of c and d of Formula II are factors which may be varied to obtain a lipid having the desired properties.
  • a and b are chosen such that their sum is an integer ranging from 14 to 24.
  • c and d are chosen such that their sum is an integer ranging from 14 to 24.
  • the sum of a and b and the sum of c and d are the same.
  • the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24.
  • a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.
  • R la , R 2a , R 3a and R 4a of Formula II are not particularly limited.
  • at least one of R la , R 2a , R 3a and R 4a is H.
  • R la , R 2a , R 3a and R 4a are H at each occurrence.
  • at least one of R la , R 2a , R 3a and R 4a is C 1 -C 12 alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is C 1 -C 8 alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is C 1 -C 6 alkyl.
  • the C 1 -C 8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R la , R lb , R 4a and R 4b are C 1 -C 12 alkyl at each occurrence.
  • R 4b is H or R lb , R 2b , R 3b and R 4b are H at each occurrence.
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 5 and R 6 of Formula II are not particularly limited in the foregoing embodiments.
  • one of R 5 or R 6 is methyl.
  • each of R 5 or R 6 is methyl.
  • R a is H or C 1 -C 12 alkyl
  • R b is C1-C15 alkyl
  • x is 0, 1 or 2.
  • R b is branched C1-C16 alkyl.
  • R b has one of the following structures:
  • one of R or R 9 is methyl. In other embodiments, both R 8 and R 9 are methyl.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.
  • G 3 is C 2 -C4 alkylene, for example C 3 alkylene.
  • the lipid compound has one of the structures set forth in Table 2 below
  • the lipid of Formula II is compound II-9. In some embodiments the lipid of Formula II is compound 11-10. In some embodiments the lipid of Formula II is compound 11-11. In some embodiments the lipid of Formula II is compound 11-12. In some embodiments the lipid of Formula II is compound 11-14. In some embodiments the lipid of Formula II is compound 11-15.
  • the cationic lipid has a structure of Formula
  • G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or Ci-
  • G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene;
  • R a is H or C 1 -C 12 alkyl
  • R* and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl
  • R 4 is C 1 -C 12 alkyl
  • R 5 is H or C 1 -C 6 alkyl
  • x 0, 1 or 2.
  • the lipid has one of the following structures (IIIA) or (IIIB):
  • A is a 3 to 8-membered cycloalkyl or cycloalkylene ring
  • R 6 is, at each occurrence, independently H, OH or C 1 -C 24 alkyl; n is an integer ranging from 1 to 15.
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure ( ⁇ ).
  • the lipid has one of the following structures (IIIC or (HID):
  • y and z are each independently integers ranging from 1 to 12.
  • L 1 and L 2 are each
  • the lipid has one of the following structures (HIE) or (IIIF):
  • the lipid has one of the followin structures (IIIG), (IIIH), (IIII), or (III J):
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is 4.
  • n is 5.
  • n is 6.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H. In other of the foregoing embodiments, R 6 is C 1 -C24 alkyl. In other embodiments, R 6 is OH.
  • G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C 1 -C24 alkylene or linear C 1 -C 24 alkenylene.
  • R 1 or R 2 is C6-C24 alkenyl.
  • R 1 and R 2 each, independently have the following structure:
  • R 7a and R 7b are, at each occurrence, independently H or C 1 -C 12 alkyl; and a is an integer from 2 to 12,
  • R 7a , R ⁇ and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • at least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is C 1 -C 8 alkyl.
  • C 1 -C 8 alkyl is methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the followin structures:
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula III has one of the structures set forth in Table 3 below.
  • the lipid of Formula III is compound III-3. In some embodiments the lipid of Formula III is compound 111-25. In some embodiments the lipid of Formula III is compound 111-45.
  • the cationic lipid has a structure of formula (IV):
  • X is CR a ;
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
  • R a is, at each occurrence, independently H, C 1 -C 12 alkyl, C 1 -C 12 hydroxylalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminylalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;
  • R is, at each occurrence, independently either: (a) H or C 1 -C 12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 1 and R 2 have, at each occurrence, the following structure, respectively:
  • a 1 and a 2 are, at each occurrence, independently an integer from 3 to 12; b 1 and b 2 are, at each occurrence, independently 0 or 1;
  • c 1 and c 2 are, at each occurrence, independently an integer from 5 to 10; d 1 and d 2 are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
  • alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
  • G 1 and G 2 are each independently
  • X is CH.
  • the sum of a ⁇ ⁇ + c 1 or the sum of a 2 + b 2 + c 2 is an integer from 12 to 26.
  • a 1 and a 2 are independently an integer from 3 to 10.
  • a 1 and a 2 are independently an integer from 3 to 10.
  • b 1 and b 2 are 0. In different embodiments, b 1 and b 2 are 1.
  • c 1 , c 2 , d 1 and d 2 are independently an integer from 6 to 8.
  • c 1 and c 2 are, at each occurrence, independently an integer from 6 to 10
  • d 1 and d 2 are, at each occurrence, independently an integer from 6 to 10.
  • c 1 and c 2 are, at each occurrence, independently an integer from 5 to 9, and d 1 and d 2 are, at each occurrence,
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1. In other embodiments, Z is alkyl.
  • R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • each R is H.
  • at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 1 and R 2 independently have one of the following structures:
  • the compound has one of the following structures:
  • the cationic lipid is a compound having the structure of formula (V):
  • X is CR a ;
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
  • R a is, at each occurrence, independently H, C 1 -C 12 alkyl, C 1 -C 12 hydroxylalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminylalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;
  • R is, at each occurrence, independently either: (a) H or C 1 -C 12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 1 and R 2 have, at each occurrence, the following structure, respectively:
  • R' is, at each occurrence, independently H or C 1 -C 12 alkyl; a 1 and a 2 are, at each occurrence, independently an integer from 3 to 12; b 1 and b 2 are, at each occurrence, independently 0 or 1;
  • c 1 and c 2 are, at each occurrence, independently an integer from 2 to 12; d 1 and d 2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6,
  • G 1 and G 2 are each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
  • G 1 and G 2 are each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
  • G 1 and G 2 are each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbon
  • X is CH.
  • the sum of a ⁇ +d 1 is an integer from 20 to 30, and the sum of a 2 +c 2 +d 2 is an integer from 18 to 30.
  • the sum of a ⁇ +d 1 is an integer from 20 to 30, and the sum of a 2 +c 2 +d 2 is an integer from 20 to 30.
  • a , a , c 1 , c 2 , d 1 and d 2 are selected such that the sum of a ⁇ +d 1 is an integer from 18 to 28, and the sum of a 2 +c 2 +d 2 is an integer from 18 to 28,
  • a 1 and a 2 are independently an integer from 3 to 10, for example an integer from 4 to 9.
  • b 1 and b 2 are 0. In different embodiments b 1 and b 2 are 1.
  • c 1 , c 2 , d 1 and d 2 are independently an integer from 6 to 8.
  • Z is alkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1.
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1. In other embodiments, Z is alkyl.
  • R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • each R is H.
  • at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • each R' is H.
  • the sum of a ⁇ +d 1 is an integer from 20 to 25, and the sum of a 2 +c 2 +d 2 is an integer from 20 to 25.
  • R 1 and R 2 independently have one of the following structures:
  • the compound has one of the following structures:
  • n is 1. In other of the foregoing embodiments of formula (IV) or (V), n is greater than 1.
  • Z is a mono- or polyvalent moiety comprising at least one polar functional group. In some embodiments, Z is a monovalent moiety comprising at least one polar functional group. In other embodiments, Z is a polyvalent moiety comprising at least one polar functional group.
  • the polar functional group is a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylaminyl, heterocyclyl or heteroaryl functional group.
  • Z is hydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl,
  • alkylaminylalkyl heterocyclyl or heterocyclylalkyl.
  • Z has the following structure:
  • R 5 and R 6 are independently H or C 1 -C 6 alkyl
  • R 7 and R 8 are independently H or C 1 -C 6 alkyl or R 7 and R 8 , together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring;
  • x is an integer from 0 to 6.
  • Z has the following structure:
  • R 5 and R 6 are independently H or C 1 -C 6 alkyl
  • R 7 and R 8 are independently H or C 1 -C 6 alkyl or R 7 and R 8 , together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring;
  • x is an integer from 0 to 6.
  • Z has the following structure:
  • R 5 and R 6 are independently H or C 1 -C 6 alkyl
  • R 7 and R 8 are independently H or C 1 -C 6 alkyl or R 7 and R 8 , together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring;
  • x is an integer from 0 to 6.
  • Z is hydroxylalkyl, cyanoalkyl or an alkyl substituted with one or more ester or amide groups.
  • Z has one of the following structures:
  • Z-L has one of the following structures:
  • Z-L has one of the following structures:
  • X is CH and Z-L has one of the following structures:
  • the compound has one of the structures set forth in Table 1 below.
  • the compounds have the following structure of
  • R is, at each occurrence, independently H or OH
  • R 1 and R 2 are each independently branched, saturated or unsaturated Ci 2
  • R 3 and R 4 are each independently H or straight or branched, saturated or unsaturated C 1 -C 6 alkyl; R 5 is straight or branched, saturated or unsaturated C 1 -C 6 alkyl; and n is an integer from 2 to 6.
  • R 1 and R 2 are each independently branched, saturated or unsaturated C12-C30 alkyl, C12-C20 alkyl, or C 15 - C20 alkyl. In some specific embodiments, R 1 and R 2 are each saturated. In certain embodiments, at least one of R 1 and R 2 is unsaturated.
  • R 1 and R 2 have the following structure:
  • the compound has the following structure (VIA):
  • R 6 and R 7 are, at each occurrence, independently H or straight or branched, saturated or unsaturated C1-C14 alkyl;
  • a and b are each independently an integer ranging from 1 to 15, provided that R 6 and a, and R 7 and b, are each independently selected such that R 1 and R 2 , respectively, are each independently branched, saturated or unsaturated C12-C36 alkyl.
  • the compound has the following structure (VIB):
  • R 8 , R 9 , R 10 and R 11 are each independently straight or branched, saturated or unsaturated C4-C12 alkyl, provided that R 8 and R 9 , and R 10 and R 11 , are each independently selected such that R 1 and R 2 , respectively, are each independently branched, saturated or unsaturated Ci 2 -C 36 alkyl.
  • R 8 , R 9 , R 10 and R 11 are each independently straight or branched, saturated or unsaturated C 6 -Cio alkyl.
  • at least one of R 8 , R 9 , R 10 and R 11 is unsaturated.
  • each of R 8 , R 9 , R 10 and R 11 is saturated.
  • the compound has structure (IA), and in other embodiments, the compound has structure (VIB).
  • G 1 is -OH, and in some embodiments G 1 is - R 3 R 4 .
  • G 1 is -NH 2 , - HCH 3 or -N(CH 3 ) 2 .
  • G 1 is
  • n is an integer ranging from 2 to 6, for example, in some embodiments n is 2, 3, 4, 5 or 6. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.
  • R , R , R , R and R is unsubstituted.
  • R , R , R , R 4 and R 5 are each unsubstituted.
  • R 3 is substituted.
  • R 4 is substituted.
  • R5 is substituted.
  • each of R 3 and R 4 are substituted.
  • a substituent on R 3 , R 4 or R 5 is hydroxyl.
  • R 3 and R 4 are each substituted with hydroxyl.
  • At least one R is
  • each R is H.
  • the compound has one of the structures set forth in Table 5 below.
  • the compounds have the following structure of
  • G 1 and G 2 are each independently C 2 -C 12 alkylene or C 2 -C 12 alkenylene;
  • G 3 is C 1 -C 24 alkylene, C2-C24 alkenylene, C 3 -C 8 cycloalkylene or C 3 -C 8 cycloalkenylene;
  • R a , R b , R d and R e are each independently H or C 1 -C 12 alkyl or C 2 -C 12 alkenyl;
  • R c and R f are each independently C 1 -C 12 alkyl or C 2 -C 12 alkenyl
  • R 1 and R 2 are each independently branched C6-C 24 alkyl or branched C 6 -
  • R 4 is C 1 -C 12 alkyl
  • R 5 is H or Ci-Cs alkyl or C 2 -C 8 alkenyl
  • R 6 is H, aryl or aralkyl
  • x 0, 1 or 2
  • each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted.
  • G 3 is unsubstituted.
  • G 3 is C 2 -C 12 alkylene, for example, in some embodiments G 3 is C3-C7 alkylene or in other embodiments G is C3-C12 alkylene. In some embodiments, G 3 is C 2 or C 3 alkylene.
  • the compound has the following structure (VIIA):
  • y and z are each independently integers ranging from 2 to 12, for example an integer from 2 to 6, for example 4.
  • the compound has one of the following structures (VIIB), ( VIIC), (VIID) or ( VIE) :
  • the compound has structure (VIIB), in other embodiments, the compound has structure (VIIC) and in still other embodiments the compound has the structure (VIID). In other embodiments, the compound has structure (VIIE). In some different embodiments of the foregoing, the compound has one of the following structures (VIIF), (VUG), (VIIH) or (VIIJ)
  • y and z are each independently integers ranging from 2 to 12, for example an integer from 2 to 6, for example 4.
  • y and z are each independently an integer ranging from 2 to 10, 2 to 8, from 4 to 10 or from 4 to 7.
  • y is 4, 5, 6, 7, 8, 9, 10, 11 or 12.
  • z is 4, 5, 6, 7, 8, 9, 10, 11 or 12.
  • y and z are the same, while in other embodiments y and z are different.
  • R 1 or R 2 is branched C6-C24 alkyl.
  • R 1 and R 2 each, independently have the following structure:
  • R 7a and R 7b are, at each occurrence, independently H or C 1 -C 12 alkyl; and a is an integer from 2 to 12,
  • R 7a , R ⁇ and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • at least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is C 1 -C 8 alkyl.
  • C 1 -C 8 alkyl is methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the following structures:
  • R b , R c , R e and R f are each independently C3-C12 alkyl.
  • R b , R c , R e and R f are n-hexyl and in other embodiments R b , R c , R e and R f are n-octyl.
  • R 4 is ethyl, propyl, n-butyl, n-hexyl, n- octyl or n-nonyl.
  • R 5 is H, methyl, ethyl, propyl, n-butyl, n- hexyl or n-octyl.
  • R 4 and/or R 5 is optionally substituted with a substituent, for example hydroxyl.
  • R 6 is benzyl and in other embodiments R 6 is H.
  • R 4 , R 5 and R 6 are independently optionally substituted with one or more substituents selected from the group consisting of
  • R g is, at each occurrence independently H or C 1 -C 6 alkyl
  • R h is at each occurrence independently C 1 -C 6 alkyl
  • R 1 is, at each occurrence independently C 1 -C 6 alkylene.
  • R has one of the following structures:
  • the compound has one of the structures set forth in Table 6 below.
  • the compounds have the following structure of Formula (VIII):
  • G 1 and G 2 are each independently C 1 -C 12 alkylene or C2-C12 alkenylene;
  • G 3 is C 1 -C 24 alkylene, C2-C24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene;
  • R a , R b , R d and R e are each independently H or C 1 -C 12 alkyl or C2-C12 alkenyl;
  • R c and R f are each independently C 1 -C 12 alkyl or C2-C12 alkenyl
  • R 1 and R 2 are each independently branched C 6 -C 2 4 alkyl or branched C 6 - C24 alkenyl;
  • R 4 is H, C 1 -C 12 alkyl or C 2 -Ci 2 alkenyl
  • R 5 is C2-C12 alkyl or C2-C12 alkenyl when R 4 is H; or R 5 is C 1 -C 12 alkyl or C2-C12 alkenyl when R 4 is C 1 -C 12 alkyl or C 2 -Ci 2 alkenyl; and
  • x 0, 1 or 2
  • each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene and cycloalkenylene is independently substituted or unsubstituted.
  • G 3 is unsubstituted.
  • G 3 is C 1 -C 12 alkylene, for example, G 3 is C3-C5 alkylene or G 3 is C3-C12 alkylene.
  • the compound has the following structure (VIIIA):
  • the compound has one of the following structures (VIIIB) or (VIIIC):
  • the compound has structure (VIIIB), in other embodiments, the compound has structure (VIIIC).
  • the compound has one of the following structures
  • y and z are each independently integers ranging from 1 to 12.
  • y and z are each independently an integer ranging from 2 to 12, for example from 2 to 10, from 2 to 8, from 4 to 7 or from 4 to 10.
  • y is 4, 5, 6, 7, 8, 9, 10, 11 or 12.
  • z is 4, 5, 6, 7, 8, 9, 10, 11 or 12.
  • y and z are the same, while in other embodiments y and z are different.
  • R 1 or R 2 is branched C 6 -C 24 alkyl.
  • R 1 and R 2 each, independently have the following structure:
  • R a and R are, at each occurrence, independently H or C 1 -C 12 alkyl; and a is an integer from 2 to 12,
  • R 7a , R ⁇ and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • At least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is C 1 -C 8 alkyl.
  • C 1 -C 8 alkyl is methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the following structures:
  • R 4 is H, methyl, ethyl, propyl or octyl.
  • R 5 is methyl, ethyl, propyl, heptyl or octyl, for example n-heptyl or n-octyl.
  • R g is, at each occurrence independently H or C 1 -C 6 alkyl
  • R h is at each occurrence independently C 1 -C 6 alkyl
  • R 1 is, at each occurrence independently C 1 -C 6 alkylene.
  • R has one of the following structures:
  • the compound has one of the structures set forth in Table 7 below.
  • the lipid nanoparticle comprises a neutral lipid.
  • Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monom ethyl PE, 16-0- dimethyl PE, 18-1 -trans PE, l-stearioyl-2-oleo
  • 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 cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.
  • the lipid nanoparticle further comprises a steroid or steroid analogue.
  • the steroid or steroid analogue is cholesterol.
  • the molar ratio of the cationic lipid to cholesterol ranges from about 5 : 1 to 1 : 1.
  • the lipid nanoparticle comprises a polymer conjugated lipid, for example a pegylated lipid.
  • the lipid nanoparticle includes a pegylated diacylglycerol (PEG-DAG) such as
  • PEG-DMG l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
  • PEG-PE pegylated phosphatidylethanoloamine
  • PEG succinate diacylglycerol PEGS-DAG
  • PEG-S-DMG PEG succinate diacylglycerol
  • PEG-S-DMG PEG succinate diacylglycerol
  • PEG-S-DMG a pegylated ceramide
  • PEG- cer PEG dialkoxypropylcarbamate
  • the pegylated lipid has the following structure
  • R 12 and R 13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;
  • w has a mean value ranging from 30 to 60.
  • R 12 and R 13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • the average w ranges from 45 to 55. In other embodiments, the average w ranges from 42 to 55. In some specific embodiments, w is about 49.
  • the pegylated lipid has the following structure
  • the therapeutic agent comprises a nucleic acid.
  • the nucleic acid is selected from antisense and messenger RNA.
  • the lipid nanoparticles of the present invention may be administered alone or may be formulated as pharmaceutical compositions.
  • Pharmaceutical compositions of the present invention comprise a lipid nanoparticle according to any of the foregoing embodiments and one or more pharmaceutically acceptable carrier, diluent or excipient.
  • the lipid nanoparticle is present in an amount which is effective to deliver the therapeutic agent, e.g., for treating a particular disease or condition of interest.
  • the disease or condition of interest is a disease mediated by to protein expression in adipose tissue or adipocytes.
  • the disease or condition is related indirectly to protein expression in adipose tissue or adipocytes.
  • Diseases and conditions include metabolic disturbances, for example, obesity, type II diabetes, insulin resistance, atherosclerosis and lipid disorders. Appropriate
  • concentrations and dosages can be readily determined by one skilled in the art.
  • Intraperitoneal administration of the compositions of the invention can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
  • the pharmaceutical compositions of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Pharmaceutical compositions according to certain embodiments of the methods described herein are formulated into injections. In certain embodiments, administering the composition comprises intraperitoneal injection.
  • compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon intraperitoneal administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient take the form of one or more dosage units, for example, as measured by mg/kg denoting mg of the composition and kg of the subject. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • the composition to be administered will, in any event, contain a therapeutically effective amount of a composition comprising a lipid nanoparticle and a therapeutic agent, or a
  • a pharmaceutical composition of the invention may be in the form of a solid or liquid.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup or injectable liquid.
  • the pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions of the invention may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose.
  • An intraperitoneal preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of
  • a liquid pharmaceutical composition of the invention intended for intraperitoneal administration should contain an amount of a lipid nanoparticle of the invention such that a suitable dosage will be obtained.
  • the pharmaceutical composition of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby assists in the delivery of the compound.
  • Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, or a protein.
  • compositions of the invention may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the invention with sterile, distilled water or other carrier so as to form a solution.
  • a surfactant may be added to facilitate the formation of a
  • Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or
  • compositions of the invention are administered or delivered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • Compositions of the invention may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents.
  • Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the invention and one or more additional active agents, as well as administration of the composition of the invention and each active agent in its own separate pharmaceutical dosage formulation.
  • a composition of the invention and the other active agent can be administered to the patient together in a single intraperitoneal dosage composition such as an injection, or each agent administered in separate intraperitoneal dosage formulations.
  • the compounds of the invention and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.
  • Suitable protecting groups include hydroxy, amino, mercapto and carboxylic acid.
  • Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like.
  • Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like.
  • Suitable protecting groups for mercapto include -C(0)-R" (where R" is alkyl, aryl or arylalkyl), /?-methoxybenzyl, trityl and the like.
  • Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters.
  • Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley.
  • the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.
  • lipids which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the lipids can be converted to their free base or acid form by standard techniques.
  • General Reaction Scheme 1 provides an exemplary method ("Method A") for preparation of Lipids of Formula III.
  • G 1 , G 3 , R 1 and R 3 in General Reaction Scheme A are as defined herein for Formula III, and Gl ' refers to a one-carbon shorter homologue of Gl .
  • Compounds of structure A-1 are purchased or prepared according to methods known in the art. Reaction of A-1 with diol A-2 under appropriate condensation conditions (e.g., DCC) yields ester/alcohol A-3, which can then be oxidized (e.g., PCC) to aldehyde A-4. Reaction of A-4 with amine A-5 under reductive amination conditions yields a lipid of Formula III.
  • DCC condensation conditions
  • PCC oxidized
  • Method B provides a method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "B-8"), wherein R, R 1 , a 1 , a 2 and Z are as defined herein, and PG is an alcohol protecting group such as tetrahydropyran.
  • Compounds of structure B-1 are purchased or prepared according to methods known in the art. Reaction of B-1 with ethyl formate B-2 under Grignard conditions yields alcohol B-3, which can then be coupled with acid B-4 under standard conditions to yield B-5. Removal of the protecting group followed by coupling with acid B-6 yields B-7.
  • Method C provides an alternative method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "C-9"), wherein R, R 1 , a 1 , a 2 and Z are as defined herein and PG is an alcohol protecting group such as tetrahydropyran.
  • Compounds of structure C-l are purchased or prepared according to methods known in the art. The hydroxyl group of Compound C-l is protected using methods known in the art (e.g. pyridinium p-toluenesulfonate, dihydropyran) to yield I.
  • Reaction of C-2 with ethyl formate C-3 under Grignard conditions e.g.
  • Method D provides another alternative method for preparation of exemplary compounds of structure (I) or (II) (i.e., compound "D-6"), wherein R, R 1 , a 1 , a 2 and Z are as defined herein.
  • Compounds of structure D-l are purchased or prepared according to methods known in the art.
  • Compound D-l is used to form D-2 under appropriate conditions (e.g. diethyl acetone dicarboxylate, EtONa).
  • Alcohol D-3 is then coupled to D-2 using standard conditions (e.g. DMAP, EDC HCl) to yield D-4.
  • the carbonyl of D-4 is reduced (e.g. with NaBH 4 ) followed by coupling with acid D-5 (e.g. with DMAP, EDC HCl) to yield the desired product D-6.
  • Method E provides an exemplary method for preparation of compounds of structure (VI).
  • G 1 and n in General reaction Scheme 5 are as defined herein for Formula (VI), and R 1 ' refers to a one-carbon shorter homologue of R 1 .
  • Compounds of structure E-1 are purchased or prepared according to methods known in the art. Reaction of E-1 under appropriate oxidation conditions (e.g., TEMPO) yields aldehyde E-2, which can then undergo a reductive amination with E-3 using an appropriate reagent (e.g., sodium triacetoxyborohydride) to yield a compound of structure of Formula (VI).
  • an appropriate reagent e.g., sodium triacetoxyborohydride
  • Method F provides an exemplary method for preparation of compounds of Formula (VII).
  • R 1 , R 2 , R 4 , R 5 , R 6 , y and z in General Reaction Scheme 6 are as defined herein for Formula (VII).
  • R', X, m and n refer to variables selected such that F-5, F-6, F-8, and F-10 are compounds having a structure of Formula (VII).
  • R' is R 1 or R 2
  • X is Br
  • m is y or z
  • n is an integer ranging from 0 to 23.
  • Compounds of structure F-1 are purchased or prepared according to methods known in the art.
  • Amine/acid F-1 is protected with alcohol F-2 (e.g., benzyl alcohol) using suitable conditions and reagents (e.g., p-TSA) to obtain ester/amine F-3.
  • Ester/amine F-3 is coupled with ester F-4 (e.g., using DIPEA) to afford benzyl ester F-5.
  • Compound F-5 is optionally deprotected using appropriate conditions (e.g., Pd/C, H 2 ) to obtain acid F-6.
  • the acid F-6 can be reacted with amine F-7 (e.g., using oxalyl chloride/DMF) to obtain amide F-8, or alternatively, reacted with alcohol F-9 (e.g., using DCC/DMAP) to yield ester F-10.
  • amine F-7 e.g., using oxalyl chloride/DMF
  • alcohol F-9 e.g., using DCC/DMAP
  • Method G provides an exemplary method for preparation of compounds of Formula (VII).
  • R 1 , R 2 , R 4 , R 5 , y and z in General reaction Scheme 7 are as defined herein for Formula (VII).
  • R', X, m and n refer to variables selected such that G-6 is a compound having a structure of Formula (VII).
  • R' is R 1 or R 2
  • X is Br
  • m is y or z
  • n is an integer ranging from 0 to 23.
  • Compounds of structure G-1 are purchased or prepared according to methods known in the art.
  • Reaction of protected amine/acid G-1 with amine G-2 is carried out under appropriate coupling conditions (e.g., NHS, DCC) to yield amide G-3.
  • appropriate coupling conditions e.g., NHS, DCC
  • acidic conditions e.g., TFA
  • amine G-4 is coupled with ester G-5 under suitable conditions (e.g., DIPEA) to yield G-6, a compound of Formula (VII).
  • Method H provides an exemplary method for preparation of compounds of Formula (VII).
  • R 1 , R 4 , R 5 , R e , R f , y and z in General reaction Scheme 8 are as defined herein for Formula (VII).
  • R', X, m and n refer to variables selected such that H-7 is a compound having a structure of Formula (VII).
  • R' is R 1 or R 2
  • X is Br
  • m is y or z
  • n is an integer ranging from 0 to 23.
  • compounds of structure H-1, H-2, H-4 and H-5 are purchased or prepared according to methods known in the art.
  • amide H-6 is prepared by coupling acid H-4 with amine H-5 under suitable conditions (e.g., oxalyl chloride/DMF). H-3 and H-6 are combined under basic conditions (e.g., DIPEA) to afford H-7, a compound of Formula (VII).
  • suitable conditions e.g., oxalyl chloride/DMF
  • H-3 and H-6 are combined under basic conditions (e.g., DIPEA) to afford H-7, a compound of Formula (VII).
  • General Reaction Scheme 9 (“Method I") provides an exemplary method for preparation of compounds of Formula (VIII).
  • R 1 , R 2 , R 4 , R 5 , y and z in General Reaction Scheme 1 are as defined herein for Formula VIII.
  • R', X, m and n refer to variables selected such that BF is a compound having a structure (VIII).
  • R' is R 1 or R 2
  • X is Br
  • m is y or z
  • n is an integer ranging from 0 to 23.
  • Method J provides an exemplary method for preparation of compounds of Formula (VIII).
  • R 1 , R 2 , R 5 , y and z in General Reaction Scheme 10 are as defined herein for Formula (VIII).
  • R', X, m and n refer to variables selected such that J-6 is a compound having a structure of Formula (VIII).
  • R' is R 1 or R 2
  • X is Br
  • m is y or z
  • n is an integer ranging from 0 to 23.
  • Compounds of structure J-l are purchased or prepared according to methods known in the art. Reaction of J-l with methanol to afford ester J-2 is carried out under appropriate conditions (e.g., acetyl chloride).
  • Ester J-2 can be prepared by adding diamine J-3 using, for example, methanol and heat.
  • the resulting amide J-4 can be coupled to J-5 using basic conditions (e.g., DIPEA) to afford J-6, a compound of Formula (VIII).
  • Method K provides an exemplary method for preparation of compounds of Formula (VIII).
  • R 1 , R 2 , R 4 , R 5 , y and z in General Reaction Scheme 11 are as defined herein for Formula (VIII).
  • R', X, m and n refer to variables selected such that K-8 is a compound having a structure (VIII).
  • R' is R 1 or R 2
  • X is Br
  • m is y or z
  • n is an integer ranging from 0 to 23.
  • Compounds of structure K-1 are purchased or prepared according to methods known in the art.
  • Reaction of K-1 alcohol/amine K-2 proceeds under appropriate conditions (e.g., KC0 3 , Ce 2 C0 3 and Nal) to yield di-ester/alcohol K-3.
  • Mesylate K-4 is obtained (e.g., using MsCl, triethylamine and DMAP) and coupled with amine K-5 (e.g., using heat) to afford di-ester K-6.
  • the final coupling between K-6 and acid chloride K-7 proceeds using basic coupling conditions (e.g., triethylamine, DMAP) to afford K-8, a compound of Formula (VIII).
  • Method L provides an exemplary method for preparation of compounds of Formula (VIII).
  • R 1 , R 2 , R 4 , R 5 , y and z in General Reaction Scheme 12 are as defined herein.
  • R', X, m and n refer to variables selected such that L-8 is a compound having a structure of Formula (VIII).
  • R' is R 1 or R 2
  • X is Br
  • m is y or z
  • n is an integer ranging from 0 to 23.
  • Compounds of structure L-1 are purchased or prepared according to methods known in the art.
  • Reaction of mesylate L-1 is coupled to amine L-2 (e.g., using heat to 75 °C) to yield protected amine L-3, which is reacted with acid chloride L-4 (e.g., using triethylamine/DMAP) to afford amide L-5.
  • the protecting group is removed under acidic conditions (e.g., TFA) and amine L-6 is then coupled with L-7 under basic conditions (e.g., DIPEA) to afford L-8, a compound of Formula (VIII).
  • Lipid nanoparticles, cationic lipids and polymer conjugated lipids were prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2015/199952 and WO 2017/004143, the full disclosures of which are incorporated herein by reference, or were prepared as described herein. Briefly, cationic lipid, DSPC, cholesterol and PEG-lipid were solubilized in ethanol at a molar ratio of about 50: 10:38.5: 1.5 or about 47.5: 10:40.8: 1.7. Lipid nanoparticles (L P) were prepared at 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 buffer, pH 4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1 :5 to 1 :3 (vol/vol) with total flow rates above 15 mL/min. The ethanol was then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 ⁇ pore sterile filter. Lipid nanoparticle particle size was approximately 55-95 nm diameter, and in some instances
  • a comparison of administration methods was performed as follows. A dose of 1.0 mg/kg of lipid nanoparticle/FLuc mRNA was administered either intravenously or intraperitoneally. Lipid nanoparticles were formulated according to Example 1, using Dlin-MC3-DMA or Compound 1-6 as a cationic lipid.
  • the FLuc mRNA (L-6107) from Trilink Biotechnologies will express a luciferase protein, originally isolated from the firefly, photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and polyadenylated mRNA is fully substituted with 5-methylcytidine and pseudouridine.
  • Protein expression in tissue was assessed from animals at 4, 24, and 48 hours post administration or from tissue harvested at a specific time point ⁇ e.g., 4 hours) post-administration. Protein expression in tissue was characterized using IVIS live imaging. As Fig. 1 and 2 indicate, both formulations show significant expression of luciferase in adipose tissue. Fig. 2 shows accumulation and expression in mostly liver and spleen tissue for intravenous treatment. In contrast, the intraperitoneal
  • luciferase in adipose tissue unexpectedly shows significant accumulation of mRNA and luciferase expression in adipose tissue ⁇ e.g., fat pads).
  • the expression of luciferase in adipose tissue is significant because: (1) intraperitoneal administration of the LNP localizes the therapeutic agent at a location other than the liver and spleen ⁇ e.g., in the adipose tissue) and (2) the therapeutic agent remains viable in the adipose tissue.
  • mRNA viability was evidenced by the expression of the luciferase detected in the harvested samples.
  • Figs. 2A-D provide a heat map of fluorescent intensity (i.e., more intense shading indicates greater expression).
  • Fig. 2E is a legend showing the types of tissue samples in each panel of Figs. 2A-D.
  • lipid nanoparticles were formulated according to Example 1, using the following cationic lipids: 1-5, II-9, or 111-45 as a cationic lipid.
  • the CleanCap FLuc mRNA (L-7202) from Trilink Biotechnologies will express a luciferase protein, originally isolated from the firefly, photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and polyadenylated mRNA is fully substituted with 5-methoxyuridine.
  • Protein expression in tissue was assessed from animals at 4 or 16 hours post administration by harvesting fat pads pertaining to different regions
  • Compound 1-5 was prepared according to method B as follows:
  • the purified product (7.4 g) was dissolved in methylene chloride (50 mL) and treated with pyridinum chlorochr ornate (5.2 g) for two hours. Diethyl ether (200 mL) as added and the supernatant filtered through a silica gel bed. The solvent was removed from the filtrate and resultant oil passed down a silica gel (50 g) column using a ethyl acetate/hexane (0-5%) gradient. 6-(2'-hexyldecanoyloxy)dodecanal (5.4 g) was recovered as an oil.
  • Compound 1-6 was prepared according to method B as follows: A solution of nonan-l,9-diol (12.6 g) in methylene chloride (80 mL) was treated with 2- hexyldecanoic acid (10.0 g), DCC (8.7 g) and DMAP (5.7 g). The solution was stirred for two hours. The reaction mixture was filtered and the solvent removed. The residue was dissolved in warmed hexane (250 mL) and allowed to crystallize. The solution was filtered and the solvent removed. The residue was dissolved in methylene chloride and washed with dilute hydrochloric acid. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent removed.
  • Compound II-9 was prepared according to method D as follows:
  • reaction mixture was diluted with hexanes-EtOAc (9: 1) and quenched by adding 0.1 N NaOH (20 mL).
  • the organic phase was separated, washed with sat NaHC0 3 , brine, dried over sodium sulfate, decanted and concentrated to give the desired product II-9b as a slightly yellow cloudy oil (1.07 g, 1.398 mmol).
  • the crude product was dissolved in methylene chloride (150 mL) and treated with pyridinium chlorochromate (6 g) for one hour. Diethyl ether (450 mL) was added and the supernatant filtered through a silica gel bed. The solvent was removed from the filtrate and resultant oil dissolved in hexane. The suspension was filtered through a silica gel bed and the solvent removed, yielding 4-(2'- hexyldecanoyloxy)butan-l-al (1 lg) was obtained as a colorless oil.
  • the crude material was added to 5% sodium hydroxide in a 1 : 10 water/methanol solution (150 mL) and heated at 45°C for one hour. The solution was cooled, diluted with water and extracted with hexane. The organic fractions were dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed down a silica gel (200 g) column using 0-4% methanol/dichloromethane to afford the desired product (15 g). l,19-Di(tetrahydropyranyloxy)nonadecan-10-one.
  • the purified product was dissolved in hexane and washed with sodium hydrogen carbonate solution. The solvent was removed from the organic fraction and the residue dissolved in ⁇ 5 mL hexane. The solution was passed through a silica gel plug, and dried under a nitrogen stream, yielding 1.20 g of the desired product.
  • the purified product was dissolved in hexane and washed with sodium hydrogen carbonate solution. The solvent was removed from the organic fraction and the residue dissolved in ⁇ 2 mL hexane. The solution was passed through a silica gel plug, and dried under a nitrogen stream, yielding 1.75 g of compound VI-4.
  • the mixture was filtered through a pad of silica gel, and washed with a hexane/ethyl acetate gradient (1 :0 to 49: 1) until all unreacted 2-hexyldecyl 6-bromohexanoate was removed. Then the pad was washed with a mixture of hexane/ethyl acetate/triethylamine (400 mL, 4: 1 :0.1) and concentrated to yield the crude product as yellow oil (870 mg). The crude product was purified by flash column chromatography on silica gel (230-400 mesh silica gel, 40 g, gradient from 4 to 5% methanol in dichloromethane). The desired product was afforded as slightly yellow oil (704, 0.8 mmol, 25%).
  • Compound VII-6 was prepared according to method F to yield 84 mg of colorless oil.
  • Compound VII-7 was prepared according to method G as follows: Synthesis of fert-Butyl (4-(dibutylamino)-4-oxobutyl)carbamate. To a solution of 4-((tert-butoxycarbonyl)amino)butyric acid (1.0 eq, 2 mmol, 406 mg), N- hydroxysuccinimide (1.0 eq, 2 mmol, 230 mg) and DMAP (20 mg) in 20 mL of dichloromethane was added ⁇ , ⁇ '-Dicyclohexylcarbodiimide (DCC, 1.2 eq, 2.4 mmol, 494 mg) and the mixture stirred at room temperature for 1 hour. The reaction mixture was filtered.
  • DCC ⁇ , ⁇ '-Dicyclohexylcarbodiimide
  • the extract was concentrated (0.8 g oil/solid) and the crude product was purified by flash column chromatography on silica gel (gradient from 10 to 35% ethyl acetate in hexane). The desired product was afforded as colorless oil (630 mg, 2 mmol, 100%).
  • the mixture was diluted with dichloromethane and saturated sodium bicarbonate was slowly added with stirring. Two phases were separated and the aqueous phase was extracted with dichloromethane (4 ⁇ ). Anhydrous sodium carbonate and brine were added to the aqueous phase. The aqueous phase was extracted further with dichloromethane (2 x). The combined extracts were dried over anhydrous sodium carbonate and sodium sulfate, filtered and concentrated. The resultant residue was taken up in dichloromethane (4 mL), filtered through a pad of silica gel and washed with a mixture of chloroform/ethanol/water/ammonium hydroxide (40:24: 1.5: 1).
  • the crude was taken up in a mixture of hexane/ethyl acetate/triethylamine (20 mL, -85: 15: 1), filtered through a pad of silica gel, and washed with a mixture of hexane/ethyl acetate/triethylamine (100 mL, 4: 1 :0.1). The filtrate was concentrated and the crude product was afforded as yellow oil/solid, 400 mg. The crude product was purified again by flash dry column chromatography on silica gel (gradient from 0 to 4% methanol in chloroform, 0 to 4%) to afford the desired product as slightly yellow oil (213 mg, 0.27 mmol, 44%).
  • Compound VII-8 was prepared according to method G (in a similar manner to compound VII-7) to yield 207 mg (0.25 mmol, 40%) of slightly yellow oil.
  • Compound VII- 10 was prepared according to method H as follows:
  • the residual liquid/solid (yellow) was dissolved in 20 mL of dichloromethane and added to a solution of dioctylamine (1.1 eq, 16.5 mmol, 3.98 g, 4.98 mL), triethylamine (90 mmol, 9.09 g, 12.5 mL) and DMAP (10 mg) in dichloromethane (20 mL) at room temperature.
  • the resulting mixture was concentrated after stirring for 2.5 hours.
  • the concentrated yellow oil/solid was taken up in a mixture of hexane and ethyl acetate (-75:25) and 1M hydrochloric acid was added. The mixture was filtered and two layers were separated.
  • the aqueous layer was extracted with dichloromethane (x 3) and the combined extracts were dried over anhydrous sodium sulfate and concentrated to afford the crude product as yellow oil.
  • the crude oil was purified by flash dry column chromatography on silica gel (gradient from 1 :0 to 4: 1, hexane/ethyl acetate). The desired was afforded as slightly yellow oil (5.09 g, 12.2 mmol, 81%).
  • Compound VII-12 was prepared as follows:
  • Compound VIII-3 was prepared according to method I as follows:
  • the reaction mixture was concentrated, and the residue was taken up in a mixture of hexane/ethyl acetate (ca 5: 1, 100 mL), washed with water, brine, dried over sodium sulfate, filtered and concentrated.
  • the crude product was afforded as slightly yellow oil (-0.6 g).
  • the crude product was diluted in hexane (10 mL) and filtered through a pad of silica gel. The pad was washed with a mixture of hexane/ethyl acetate/triethylamine (200 mL, 4: 1 :0.1). Concentration of the filtrate afforded the crude product as slightly yellow oil.
  • the crude product was purified by flash column chromatography on silica gel (40 g of silica gel; gradient from 0 to 6% methanol in chloroform) to afford the desired product as colorless oil (216 mg, 0.24 mmol, 34 %).
  • Compound VIII-4 was prepared according to method J as follows:
  • the reaction mixture was concentrated and the residue was taken up in a mixture of hexane/ethyl acetate (-5: 1, 100 mL), washed with water, brine, dried over sodium sulfate, filtered and concentrated.
  • the crude product was afforded as slightly yellow oil (-1.2 g).
  • the crude product was diluted in hexane (10 mL) filtered through a pad of silica gel and washed with a mixture of hexane/ethyl acetate (1 :0 to 49: 1) until all unreacted 2-hexyldecyl 6-bromohexanoate was removed.
  • Compound VIII-5 was prepared according to method K as follows: Synthesis of bis(2-Hexyldecyl) 8,8'-((4-hvdroxybutyl)azanediyl)dioctanoate. To a solution of 2-hexyldecyl 8-bromooctanoate (2 eq, 3.09 g, 6.9 mmol) in 30 mL of anhydrous tetrahydrofuran, were added 4-amino-l-butanol (1 eq, 3.45 mmol, 308 mg, 318 ⁇ .), potassium carbonate (2 eq, 6.9 mmol, 954 mg), cesium carbonate (0.3 eq, 1.04 mmol, 337 mg) and sodium iodide (10 mg).
  • the mixture was heated to 64°C using an oil bath in a pressure round-bottom flask under argon atmosphere for 6 days.
  • the resultant crude product was dissolved in hexane (50 mL) and loaded on a short column of silica gel (1 cm height x 6.5 cm width).
  • the column was eluted with hexane (50 mL, fraction 1), a mixture of ethyl acetate/hexane (0 to 3% ethyl acetate).
  • the unreacted 2- hexyldecyl 8-bromooctanoate (1.27 g, 2.84 mmol, 41%, colorless oil) was recovered.
  • the column was eluted with a mixture of hexane/ethyl acetate/triethylamine (-4: 1 :0.1) to afford the crude product as slightly yellow oil (1.2 g).
  • the crude product was further purified by flash dry column chromatography on silica gel (gradient from 0 to 4.2% methanol in chloroform) to afford the desired product (1.28 g, 1.56 mmol, 45%).

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Abstract

L'invention concerne une méthode de traitement d'une maladie médiée par l'expression de protéines dans un tissu adipeux par administration intrapéritonéale d'une composition comprenant une nanoparticule lipidique encapsulant ou associée à un agent thérapeutique (par exemple, un acide nucléique), ce qui permet d'administrer l'agent thérapeutique au tissu adipeux du sujet et de modifier l'expression de la protéine dans le tissu adipeux. L'invention concerne également une méthode d'administration d'un agent thérapeutique au tissu adipeux d'un sujet en ayant besoin.
PCT/US2018/027655 2017-04-13 2018-04-13 Administration lipidique d'agents thérapeutiques au tissu adipeux WO2018191719A1 (fr)

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WO2025106806A1 (fr) 2023-11-17 2025-05-22 Acuitas Therapeutics, Inc. Lipides pégylés
WO2025128696A1 (fr) 2023-12-12 2025-06-19 Acuitas Therapeutics, Inc. Composés lipidiques cationiques destinés à être utilisés dans des nanoparticules lipidiques

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