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WO2025217110A1 - Lipid nanoparticles for drug delivery and methods for making and using the same - Google Patents

Lipid nanoparticles for drug delivery and methods for making and using the same

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Publication number
WO2025217110A1
WO2025217110A1 PCT/US2025/023578 US2025023578W WO2025217110A1 WO 2025217110 A1 WO2025217110 A1 WO 2025217110A1 US 2025023578 W US2025023578 W US 2025023578W WO 2025217110 A1 WO2025217110 A1 WO 2025217110A1
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WIPO (PCT)
Prior art keywords
cooh
peptide
terminal
lipid nanoparticle
lipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2025/023578
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French (fr)
Inventor
Ying Liu
Gang Cheng
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University of Illinois at Urbana Champaign
University of Illinois System
Original Assignee
University of Illinois at Urbana Champaign
University of Illinois System
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Publication of WO2025217110A1 publication Critical patent/WO2025217110A1/en
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Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • Lipid nanoparticles have revolutionized nucleic acid therapeutics, exemplified by their pivotal role in mRNA vaccine during the COVID-19 pandemic. While current LNP systems excel in nucleic acid encapsulation and delivery, their efficacy varies significantly when applied to distinct nucleic acids, such as small interfering RNA (siRNA), messenger RNA (mRNA) or plasmid DNA (pDNA). Ionizable lipids serve as the cornerstone of LNP technology, enabling pH-responsive nucleic acid delivery through dynamic charge modulation during cellular uptake and endosomal escape.
  • siRNA small interfering RNA
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • ionizable lipids such as SM-102 and DLin-MC3-DMA
  • SM-102 and DLin-MC3-DMA are synthetic compounds with non-natural, hydrocarbon tails and tertiary amines, which lack a clear degradation pathway and raise concerns about long-term toxicity.
  • structurally diverse nucleic acids e.g., siRNA, ASOs, mRNA, pDNA
  • siRNA structural duplex structure
  • pDNA large-supercoiled conformation require tailored lipid geometries to prevent aggregation or premature payload leakage, a challenge unmet by mRNA-optimized compositions.
  • a critical barrier in ionizable lipid development lies in the synthesis of complex lipid architectures that balance high transfection efficiency with low immunogenicity.
  • Conventional approaches rely on combinatorial chemistry in early time and more recently Al-guided design, which struggle to generate large, diverse ionizable lipid libraries at a low cost while maintaining synthetic feasibility.
  • asymmetric or biodegradable lipid designs often demand multi-step organic synthesis, limiting scalability and structural diversity.
  • Even advanced strategies, such as directed chemical evolution face trade-offs between transfection efficiency and biocompatibility.
  • Lipids optimized for nucleic acid delivery e.g., C12-200
  • nucleic acid delivery often incorporate non-degradable tertiary amines or rigid hydrocarbon tails, which enhance transfection but trigger immune activation through interactions with receptors like TLR4 and CD1d .
  • This persistent cationic character can induce pro- inflammatory cytokines and type I interferons, compromising safety.
  • the disclosure in one aspect, relates to lipid nanoparticles for use as drug delivery agents.
  • the lipid nanoparticles are composed of lipopeptide conjugates, where a peptide is bonded to a lipid.
  • the lipid nanoparticles described herein represent a new approach in drug delivery, addressing critical challenges in balancing biodegradability, biocompatibility, and organ-specific targeting.
  • the lipid nanoparticles can selectively target and deliver bioactive agents to specific tissues in a subject by modifying specific amino acids and ratios thereof in the peptide of the lipopeptide conjugate.
  • FIGS. 1A-1 C show the design of RmHnC-DOPE lipopeptide library and four-component RmHnC-DOPE LNPs.
  • RmHnC Representative structures of two helper lipids, p-sitosterol, and DMPE PEG-2000.
  • C Formulation showing the combination of these four components with nucleic acids to prepare multiple RmHn-DOPE LNPs for screening.
  • FIGS. 2A-C show the characterization of 13 RmHn-DOPE LNPs and SM102 LNP control loaded with PCSK9 siRNA: particle size, polydispersity index (PDI), zeta potential (measured via Malvern dynamic light scattering), and particle size stability during long-term storage at 4°C.
  • FIG. 2D shows the relative PCSK9 mRNA expression levels in HEPA1-6 cells treated with RH-DOPE LNPs or SM102 control loaded with PCSK9 siRNA (24-well plate, 24-hour incubation, 50 pmol per well, n > 3 replicates). Data are normalized to GAPDH (housekeeping gene) and presented as mean ⁇ SD.
  • FIG. 2E shows the particle size and PDI of R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 control loaded with eGFP pDNA (measured via Malvern DLS). These lipopeptides were selected based on superior siRNA transfection efficiency in vitro.
  • FIG. 2F shows the pKa determination of R3H7C-DOPE, R4H6C-DOPE, and R5H5C-DOPE via pH titration.
  • FIG. 2I shows the quantitative analysis of flow cytometry data: percentage of eGFP-positive cells and mean fluorescence intensity (MFI).
  • 2J shows the expression of firefly luciferase in HepG2 cells treated with flue pDNA loaded R3H7C-DOPE, R4H6C-DOPE, R5H5C- DOPE, S 102, or Lipofectamine 2000 LNP. Data are shown as mean ⁇ SD.
  • FIGS. 3A-3C show the in vitro transfection mechanism for RmHn-DOPE LNPs.
  • A Cellular uptake of Cy5-labeled siRNA in HepG2 cells. Confocal microscopy images of HepG2 cells transfected for 4 h with Cy5-labeled Negative control siRNA (20 pmol/well) encapsulated in R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, or SM-102. The blank control (untreated cells) shows no Cy5 fluorescence. Red fluorescence corresponds to internalized Cy5-siRNA. Scale bar: 10 pm.
  • FIGS. 4A-B show the biodistribution of RmHn-DOPE LNPs in mice.
  • A R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 LNPs loaded with Cy5-labeled negative control siRNA were intravenously administered to C57BL/6 mice via the tail vein. Organs were harvested 2 h postinjection and imaged using a PE Spectrum small animal in vivo imaging system to evaluate biodistribution.
  • flue pDNA-loaded R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 LNPs were intravenously injected into C57BL/6 mice. Substrate was administered 24 h post-injection, and bioluminescence signals were captured using the PE Spectrum imaging system.
  • FIGS. 5A-5C shows sustained PCSK9 silencing and LDL receptor upregulation mediated by PCSK9 siRNA RmHn-DOPE LNPs in C57BL/6 mice.
  • FIGS. 6I-6J show the biocompatibility assessment of lipopeptide LNPs in mice over a 28-day period following tail vein administration.
  • A TBL (Total bilirubin, testing with a blood biochemical analyzer).
  • B ALB (Albumin, testing with a blood biochemical analyzer).
  • C TP (Total protein, testing with a blood biochemical analyzer).
  • D OR (Serum creatinine, testing with a blood biochemical analyzer).
  • E BUN (Blood urea nitrogen, testing with a blood biochemical analyzer).
  • F ALT (Alanine aminotransferase, testing with a blood biochemical analyzer).
  • AST Aspartate aminotransferase, testing with a blood biochemical analyzer.
  • H UA (Uric acid, testing with a blood biochemical analyzer).
  • I Body weight changes in this batch of mice over 28 days.
  • J HE staining of major organs from mice 28 days after tail vein injection of lipopeptide LNP, Scale bar: 100pm.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible subranges) within the indicated range.
  • a component is in an amount of about 1 %, 2%, 3%, 4%, or 5%, where any value can be a lower and upper endpoint of a range, then any range is contemplated between 1% and 5% (e.g., 1% to 3%, 2% to 4%, etc.).
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined.
  • a residue of a chemical species refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species.
  • an ethylene glycol residue in a polyester refers to one or more - OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester.
  • a sebacic acid residue in a polyester refers to one or more -CO(CH2)sCO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.
  • the term “substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described below.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms, such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • substitution or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (/.e., further substituted or unsubstituted).
  • alkyl as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, f-butyl, n- pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
  • the alkyl group can be cyclic or acyclic.
  • the alkyl group can be branched or unbranched.
  • the alkyl group can also be substituted or unsubstituted.
  • the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.
  • a “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms.
  • alkyl group can also be a C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.
  • alkenyl as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond.
  • the alkenyl group is a vinyl group or an allyl group.
  • the alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.
  • groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described here
  • Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like.
  • heterocycloalkenyl is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.
  • the cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted.
  • the cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.
  • alkynyl as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond.
  • the alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.
  • amine or “amino” as used herein are represented by the formula — NA 1 A 2 , where A 1 and A 2 can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • a specific example of amino is — NH 2 .
  • alkylamino as used herein is represented by the formula — NH(-alkyl) and — N(- alkyl) 2 , where alkyl is a described herein.
  • Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino
  • acetyloxy as used herein is represented by the formula — OC(O)CH 3 .
  • amide or “amido” as used herein is represented by the formula — NHC(O)R, where can be hydrogen, an alkyl group, an aryl group, a heteroaryl group, or a cycloalkyl group as defined herein.
  • acetyl as used herein is represented by the formula — C(O)CH3.
  • acetyl amino as used herein is represented by the formula — NHC(O)CH 3 .
  • esters as used herein is represented by the formula — OC(O)A 1 or — C(O)OA 1 , where A 1 can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • ether as used herein is represented by the formula A 1 OA 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein.
  • halo halogen
  • halide halogen or halide
  • hydroxyl or “hydroxy” as used herein is represented by the formula — OH.
  • ketone as used herein is represented by the formula A 1 C(O)A 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • nitro as used herein is represented by the formula — NO 2 .
  • nitrile or “cyano” as used herein is represented by the formula — CN.
  • sil as used herein is represented by the formula — SiA 1 A 2 A 3 , where A 1 , A 2 , and A 3 can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • sulfo-oxo is represented by the formulas — S(O)A 1 , — S(O) 2 A 1 , — OS(O) 2 A 1 , or — OS(O) 2 OA 1 , where A 1 can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • sulfonyl is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2A 1 , where A 1 can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • a 1 S(O)2A 2 is represented by the formula A 1 S(O)2A 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • sulfoxide as used herein is represented by the formula A 1 S(O)A 2 , where A 1 and A 2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
  • R 1 ,” “R 2 ,” “R 3 ,” “R n ,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above.
  • R 1 is a straight chain alkyl group
  • one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like.
  • a first group can be incorporated within second group or, alternatively, the first group can be pendant (/.e. , attached) to the second group.
  • an alkyl group comprising an amino group the amino group can be incorporated within the backbone of the alkyl group.
  • the amino group can be attached to the backbone of the alkyl group.
  • the nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.
  • random copolymeric peptide when referencing a structure of a peptide is defined as peptide having the amino acids as provided in the structure in any random order.
  • the cysteine, arginine, and histidine groups are present in any order and not the specific order as provided in the structure.
  • sequence-specific peptide when referencing a structure of a peptide is defined as peptide having the amino acids as provided in the structure in the order as provided in the structure.
  • the cysteine, arginine, and histidine groups are present in the order as provided in the structure.
  • compounds of the invention may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture.
  • Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers.
  • the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.
  • a specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture.
  • Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula.
  • one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane).
  • the Cahn-lngold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.
  • Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance.
  • the disclosed compounds can be isotopically-labeled or isotopically- substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 0, 35 S, 18 F, and 36 CI, respectively.
  • Compounds further comprise prodrugs thereof and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention.
  • Certain isotopically-labeled compounds of the present invention for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, /.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability.
  • isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.
  • the compounds described in the invention can be present as a solvate.
  • the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate.
  • the compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution.
  • one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates.
  • the invention includes all such possible solvates.
  • ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form.
  • amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, the invention includes all such possible tautomers.
  • polymorphic forms or modifications It is known that chemical substances form solids which are present in different states of order which are termed polymorphic forms or modifications.
  • the different modifications of a polymorphic substance can differ greatly in their physical properties.
  • the compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.
  • administering can refer to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g.
  • a composition the perivascular space and adventitia can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells.
  • parenteral can include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
  • subject can refer to a vertebrate organism, such as a mammal (e.g. human).
  • Subject can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.
  • pharmaceutically acceptable describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
  • pharmaceutically acceptable salts means salts of the active principal agents which are prepared with acids or bases that are tolerated by a biological system or tolerated by a subject or tolerated by a biological system and tolerated by a subject when administered in a therapeutically effective amount.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include, but are not limited to; sodium, potassium, calcium, ammonium, organic amino, magnesium salt, lithium salt, strontium salt or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include, but are not limited to; those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate
  • prodrug represents those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.
  • Prodrugs of the present disclosure can be rapidly transformed in vivo to a parent compound having a structure of a disclosed compound, for example, by hydrolysis in blood.
  • a thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).
  • dose can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.
  • Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art.
  • the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St.
  • compositions ofthe invention Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary.
  • compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.
  • temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).
  • lipid nanoparticles for use as drug delivery agents.
  • the lipid nanoparticles are composed of lipopeptide conjugates, where a peptide is bonded to a lipid.
  • the lipopeptide conjugate has the formula X-L-Y, wherein X is a peptide, L is a linker, and Y is a lipid.
  • the lipid nanoparticles described herein represent a new approach in drug delivery, addressing critical challenges in balancing biodegradability, biocompatibility, and organ-specific targeting.
  • the lipid nanoparticles can selectively target and deliver bioactive agents to specific tissues in a subject by modifying specific amino acids and ratios thereof in the peptide of the lipopeptide conjugate.
  • the peptide of the lipopeptide conjugate includes a plurality of arginine and histidine groups. In one aspect, the peptide includes from about 1 to about 10,000 arginine residues, from about 1 to about 10,000 histidine residues and at least one cysteine residue.
  • the peptide includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, 500, 1 ,000, 1 ,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,500, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500 or about 10,000 arginine residues, where any value can be a lower and upper endpoint of a range (e.g., 50 to 500).
  • the peptide includes 1 , 50, 100, 500, 1 ,000, 1 ,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,500, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500 or about 10,000 histidine residues, where any value can be a lower and upper endpoint of a range (e.g., 50 to 500).
  • the properties of the lipid nanoparticle can be fined tuned for the delivery of bioactive agents to specific tissues and organs in a subject.
  • the arginine-to-histidine (R/H) ratio is from 4:1 to 1 :4.
  • arginine-to-histidine (R/H) ratio is 4:1 , 3.5:1 , 3.0:1 , 2.5:1 , 2:1 , 1.5:1 , 1 :1 , 1 :1.5, 1 :2, 1 :2.5, 1 :3, 1 :3.5, or 1 :4, where any value can be a lower and upper endpoint of a range (e.g., 2:1 to 1 :1.5).
  • the arginine-to- histidine (R/H) ratio is from 2:1 to 1 :2, 1.5:1 to 1 :1.5, or about 1 :1.
  • the arginine-to-histidine (R/H) ratio when it is desirable to deliver a bioactive agent to the liver of a subject, is from 1 :1 to 1 :3, 1 :1 to 1 :2.5, or about 3:7. In another aspect, when it is desirable to deliver a bioactive agent to the spleen of a subject, the arginine-to-histidine (R/H) ratio is from 1 :1 to 1 :2, 1 :1 to 1 :1.75, or 1 :1 to 1 :1.5.
  • the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH 2 -C W R 3 H 3 -COOH, NH2-CWR5H5-COOH, NH2-CWR3H3-COOH, NH 2 -C W R 3 H4- COOH, NH 2 -C W R 3 H 5 -COOH, NH 2 -C W R 3 H 6 -COOH, NH 2 -C W R 3 H7-COOH, NH 2 -CWR 3 H 8 -COOH, NH 2 - CWR 3 H 9 -COOH, NH 2 -C W K 5 H 5 -COOH, NH2-C W K 3 H 3 -COOH , NH 2 -C w Rm-COOH, NH2-C w K n -COOH, NH 2 -C w R 0 Hp-COOH, NH 2 -C w RuK v
  • the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH 2 -R 3 H 3 -COOH, NH2-R5H5-COOH, NH 2 -R 3 H 3 -COOH, NH 2 -R 3 H 4 -COOH, NH 2 -R 3 H 5 -COOH, NH2-R3H6-COOH, NH2-R3H7-COOH, NH2-R3H8-COOH, NH2-R3H9-COOH, NH 2 - K 5 H 5 -COOH, NH 2 -K 3 H 3 -COOH, NH 2 -R 0 H P -COOH, NH 2 -R U K V -COOH, NH 2 -K p H q -COOH, and NH 2 - RxKyHz-COOH; wherein R is arginine, K is lysine, H is histidine,
  • the peptide has a formula selected from the group consisting of NH 2 -Rm-COOH, NH 2 -Kn-COOH, NH2-H0-COOH; wherein R is arginine, K is lysine, H is histidine, NH 2 - is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m, n, and 0 are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
  • the peptide is a sequence-specific peptide having a formula selected from the group consisting of NH 2 -CR-COOH, NH 2 -CR 2 -COOH, NH 2 -CR 3 -COOH, NH2-CR4-COOH, NH 2 -CR 5 - COOH, NH 2 -CR 6 -COOH, NH 2 -CR m -COOH, NH 2 -CHR-COOH, NH 2 -CH 2 R-COOH, NH2-CH3R-COOH, NH 2 -CH 4 R-COOH, NH 2 -CH 5 R-COOH NH 2 -CH m R 2 -COOH, NH 2 -CHR 2 -COOH, NH 2 -CH 2 R 2 -COOH, NH 2 -CH 3 R 2 -COOH, NH 2 -CH 4 R 2 -COOH, NH 2 -CH 5 R 2 -COOH, NH 2 -CH 5 R 2
  • the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH 2 -CK-COOH, NH 2 -CK 2 -COOH, NH 2 -CK 3 -COOH, NH 2 -CK 4 - COOH, NH 2 -CK 5 -COOH, NH 2 -CK 6 -COOH, NH 2 -CK m -COOH, NH 2 -CHK-COOH, NH 2 -CH 2 K-COOH, NH 2 -CH 3 K-COOH, NH 2 -CH 4 K-COOH, NH 2 -CH 5 K-COOH NH 2 -CH m K 2 -COOH, NH 2 -CHK 2 -COOH, NH 2 -CH 2 K 2 -COOH, NH 2 -CH 3 K 2 -COOH, NH 2 -CH 4 K 2 -COOH, NH 2 -CH 5 K-COOH NH 2
  • the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH 2 -RC-COOH, NH 2 -R 2 C-COOH, NH 2 -R 3 C-COOH, NH 2 -R 4 C- COOH, NH 2 -R 5 C-COOH, NH 2 -R 6 C-COOH, NH 2 -R m C-COOH, NH 2 -RHC-COOH, NH 2 -RH 2 C-COOH, NH 2 -RH 3 C-COOH, NH 2 -RH 4 C-COOH, NH 2 -RH 5 C-COOH, NH 2 -RH m C-COOH, NH 2 -R 2 HC-COOH, NH 2 -R 2 H 2 C-COOH, NH 2 -R 2 H 3 C-COOH, NH 2 -R 2 H 4 C-COOH, NH 2 -RC-COOH,
  • the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH 2 -KC-COOH, NH 2 -K 2 C-COOH, NH 2 -K 3 C-COOH, NH 2 -K 4 C- COOH, NH 2 -K 5 C-COOH, NH 2 -K 6 C-COOH, NH 2 -K m C-COOH, NH 2 -KHC-COOH, NH 2 -KH 2 C-COOH, NH 2 -KH 3 C-COOH, NH 2 -KH 4 C-COOH, NH 2 -KH 5 C-COOH, NH 2 -KH m C-COOH, NH 2 -K 2 HC-COOH, NH 2 -K 2 H 2 C-COOH, NH 2 -K 2 H 3 C-COOH, NH 2 -K 2 H 4 C-COOH, NH 2 -KC-COOH,
  • the peptide has a formula selected from the group consisting of NH 2 -CR n - COOH, NH 2 -CK n -COOH, NH 2 -CH n -COOH, NH 2 -R n C-COOH, NH 2 -K n C-COOH, NH 2 -H n C-COOH, NH 2 -R 0 H P -COOH, H 2 -H P R 0 -COOH, NH 2 -CR 0 H P -COOH, NH 2 -CH P R 0 -COOH, NH 2 -R o CH p -COOH, NH 2 - HpCRo-COOH, NH 2 -R O H P C-COOH, NH 2 -H p R o C-COOH, NH 2 -C W R 0 H P -COOH, NH 2 -C W H P R 0 -COOH, NH 2 -COOH,
  • the peptide is a random or block copolymeric peptide having a formula selected from the group consisting of NH 2 -X1 x X2 y X3 z X4 w -COOH, wherein X1 , X2, X3, and X4 is cysteine, arginine, lysine or histidine, NH 2 - is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
  • the peptide has a sequence selected from the group consisting of NH 2 - CRRRRR-COOH, NH 2 -CRRRHHH-COOH, NH 2 -CHHHRRR-COOH, NH 2 -CKKKKK-COOH, NH 2 -CKKK-COOH, NH 2 -CHHHHHRRRR-COOH, NH 2 -CRRRRRHHHHH-COOH, NH 2 -CRRRHHH- COOH, NH 2 -CHHHRRRCOOH, NH 2 -CKKKKKHHHHH-COOH, NH 2 -CHHHHHKKKKK-COOH, NH 2 - CHHHKKK-COOH, NH 2 -CKKKHHH-COOH, NH 2 -RRRRRC-COOH, NH 2 -RRRHHHC-COOH, NH 2 - KKKKKC-COOH, NH 2 -KKKC-COOH, NH 2 -RRRRRHHHHHH
  • RRRHHHHHHHC-COOH NH 2 -RRRHHHHHHC-COOH, NH 2 -RRRHHHHHC-COOH, NH 2 - RRRRHHHHHHC-COOH, NH 2 -RRRRHHHHHHHC-COOH, NH 2 -RRRRHHHHHHHHC-COOH, wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH 2 - is the N-terminal of the peptide, - COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups.
  • the peptide is a block copolymer having a sequence selected from the group consisting of NH 2 -CR m -COOH, NH 2 -CK n -COOH, NH 2 -CR o H p -COOH, NH 2 -CR x K y H z -COOH, NH 2 - RmC-COOH, NH 2 -K n C-COOH, NH 2 -R o H p C-COOH, NH 2 - R x K y H z C-COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, and m, n, o, p, q, x, y, and z are integers from 1 to 1 ,000, NH 2 - is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C
  • the peptide is a random copolymeric peptide having a formula NH 2 -R m H n C- COOH, wherein C is cysteine, R is arginine, H is histidine, COOH is the N-terminal of the peptide, and m and n are integers from 1 to 15.
  • the lipopeptide conjugate includes a linker that covalently bonds the peptide to the lipid.
  • the linker is a residue of a group selected from the group consisting of amine, carboxylate, hydroxyl, thiol, alkene, alkyne, azide, disulfide, maleimide, ester, peptide, acetal, anhydride, halide, vinyl sulfone, methyl acrylate, acrylate, acrylamide, methyl acrylamide, fluoride, chloride, bromide, aldehyde, and ketone.
  • the linker can be present on the peptide or the lipid.
  • the linker is a maleimide bonded to the lipid.
  • the lipid used to produce the lipopeptide conjugate can be selected from a variety of materials used to produce lipid nanoparticles.
  • the lipid is selected from the group consisting of a phosphatidylcholine, a lysophosphatidylcholine, a plasmenylphosphatidylcholine, a phosphatidylethanolamin, a lysophosphatidylethanolamine, a plasmenylphosphatidylethanolamine, a phosphatidylserine, a sphingomyeline, a phosphatidic acid, a lysophosphatidic acid, a phosphatidylinositol, a phosphatidylglycerol, a cardiolipin, a ceramide-1-phosphate, an N-acylsphingosine, a sulfatide, a ganglioside, a sulfoquinovosyl
  • the lipid is a phospholipid.
  • the lipid is a phosphatidylcholine, a phosphatidic acid, a phosphatidylglycerol, a phosphatidylethanolamine, a phosphatidylserine, or a PEG phospholipid.
  • the lipid is 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or any combination thereof.
  • DMPE 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine
  • DPPE dipalmitoyl-sn-glycero-3-phosphoethanolamine
  • DSPE 1,2-distearoyl-sn-glycero-3- phosphoethanolamine
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • the linker is a residue of a maleimide and the lipid is phosphatidylethanolamine.
  • the lipopeptide conjugate has the structure I wherein Ri and R 2 are independently a C8 to C25 alkyl group or a C8 to C25 alkenyl group, o and p are independently integers from 1 to 4, and
  • X is a peptide
  • o and p in structure are each 2.
  • o and p in structure are each 2, and Ri and R 2 are independently an oleyl group or a palmitoyl group.
  • o and p in structure are each 2, and Ri and R 2 are both an oleyl group or a palmitoyl group.
  • the lipopeptide conjugate has the structure depicted in FIG. 1 A.
  • the lipopeptide conjugate can be synthesized by reacting a peptide (X) with a lipid having a linked covalently bonded to the lipid (L-Y).
  • the lipid can be a phospholipid that is covalently bonded to a linker.
  • phosphatidylethanolamine e.g., DOPE
  • a linker group e.g., a maleimide
  • the selection of the linker group can vary depending upon the peptide to be bonded to L-Y.
  • the linker when the peptide has a terminal cysteine group, the linker can be selected so that it reacts with the thiol group of cysteine.
  • the linker is an alkene or a compound with a degree of unsaturation so that the thiol group of cysteine reaction with the alkene via a thiol-ene coupling reaction.
  • the linker possesses a maleimide group.
  • FIG. 1A provides an exemplary reaction scheme for producing the lipopeptide conjugates. Exemplary methods for producing the lipid nanoparticles described herein, as well as characterization information, are provided in the Examples.
  • the lipid nanoparticles are produced by admixing the lipopeptide conjugates described herein with a bioactive agent in a suitable solvent.
  • one or more helper lipids can be added to mixture to produce the lipid nanoparticles.
  • the helper lipid includes a phospholipid (e.g., DOPE), a PEGylated lipid, a sterol (e.g., cholesterol, beta-sitosterol), an any combination thereof.
  • the inclusion of one or more helper lipids can improve the properties of the lipid nanoparticle including, but not limited to, particle stability, delivery efficacy, tolerability and biodistribution.
  • the lipid peptide conjugate to helper lipid is at a molar ratio of 1 :100 to 100:1 . In another aspect, the lipid peptide conjugate to helper lipid is at a molar ratio of 100:1 , 90:1 , 80:1 , 70:1 , 60:1 , 50:1 , 40:1 , 30:1 , 20:1 , 10:1 , 4:1 , 3.5:1 , 3.0:1 , 2.5:1 , 2:1 , 1.5:1 , 1 :1 , 1 :1.5, 1 :2, 1 :2.5, 1 :3, 1 :3.5, or 1 :4, 1 :10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 :100, where any value can be a lower and upper endpoint of a range (e.g., 2:1 to 1 : 1.5).
  • the bioactive agent is selected from the group consisting of Abiraterone Acetate, Brentuximab vedotin, Trastuzumab emtansine, Afatinib, Afinitor® (Everolimus), Aldara® (Imiquimod), Alimta® (Pemetrexed Disodium), Pemetrexed, Palonosetron, Chlorambucil, Nelarabine, Axitinib, Belinostat, Bleomycin, Bortezomib, Cabozantinib- S-Malate, Camptothecin, Capecitabine, Ceritinib, Cerubidine® (Daunorubicin), Crizotinib, Dabrafenib, Dasatinib, Degarelix, Docetaxel, Doxorubic
  • the bioactive agent is selected from the group consisting of antibodies, peptides, therapeutic enzymes, cytokines, interferons, and interleukins.
  • the bioactive agent is a nucleic acid such as, for example, a plasmid DNA, an oligonucleotide, an aptamers, a DNAzyme, a RNA aptamers, a RNA Decoy, an antisense RNA, a ribozymes, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA, an antagomirs, or any combination thereof.
  • a nucleic acid such as, for example, a plasmid DNA, an oligonucleotide, an aptamers, a DNAzyme, a RNA aptamers, a RNA Decoy, an antisense RNA, a ribozymes, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA, an antagomirs, or any combination thereof.
  • the surface charge of the lipid nanoparticles can be modified by modifying the peptide.
  • the number of arginine groups can be modified to alter the surface charge.
  • the lipid nanoparticle has a surface charge of between about -100 mV and about +100 mV at pH 3 to 11 , or -100 mv, -75 mv, - 50 mv, -25 mv, 0 mv, 25 mv, 50 mv, 75 mv, or 100 mv, where any value can be a lower and upper endpoint of a range (e.g., -50 mv to 25 mv, etc.).
  • the size of the lipid nanoparticles can be modified by modified.
  • the lipid nanoparticle is between about 2 nm and about 2,000 m in diameter.
  • the lipid nanoparticle about 2 nm, 10 nm, 25 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, or 200 nm in diameter, where any value can be a lower and upper endpoint of a range (75 nm to 125 nm, etc.).
  • the lipid nanoparticles possess monodisperse hydrodynamic diameters with narrow size distributions (e.g., polydispersity index, PDI ⁇ 0.2).
  • the lipid nanoparticles described herein represent a new approach in drug delivery, addressing critical challenges in balancing biodegradability, biocompatibility, and organ-specific targeting.
  • the lipid nanoparticles can selectively target and deliver bioactive agents to specific tissues in a subject by modifying specific amino acids and ratios thereof in the peptide of the lipopeptide conjugate.
  • the properties of the lipid nanoparticle can be fined tuned for the delivery of bioactive agents to specific tissues and organs in a subject.
  • modifying the arginine-to-histidine (R/H) ratio of the peptide in the lipopeptide conjugate can affect the ability of the lipid nanoparticles to deliver the bioactive agent to specific organs.
  • the lipid nanoparticles described herein can enhance transfection efficiency.
  • the lipid nanoparticles described herein possess a dual-functional architecture.
  • the cationic arginine residues in lipid nanoparticles promote robust electrostatic interactions with the nucleic acid, enabling the formation of stable nanocomplexes that protect genetic payloads from nuclease degradation.
  • histidine residues contribute pH-buffering capacity, which facilitates endosomal escape through the proton sponge effect.
  • the lipid nanoparticles described herein demonstrate significant advantages over commercial transfection reagents. Their biocompatible design minimizes cytotoxicity, maintaining over 98% cell viability compared to Lipofectamine 2000, which causes substantial toxicity (e.g., 73.8 % viability in HepG2 cell) due to its high charge density and structure. Furthermore, lipid nanoparticles described herein exhibit exceptional colloidal stability, remaining intact for over 30 days. The unique attributes of the lipid nanoparticles described herein provide a versatile platform capable of delivering diverse bioactive agents (e.g., nucleic acid) payloads with viral-like efficiency while retaining the safety profile of biodegradable lipids.
  • bioactive agents e.g., nucleic acid
  • lipid nanoparticles described herein can be administered to a subject as a pharmaceutical formulation by a variety of techniques as described further below.
  • the present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one disclosed lipid nanoparticle, at least one product of a disclosed method, or a pharmaceutically acceptable salt thereof.
  • pharmaceutically- acceptable carriers means one or more of a pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, and adjuvants.
  • the disclosed pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy and pharmaceutical sciences.
  • the disclosed pharmaceutical compositions comprise a therapeutically effective amount of at least one disclosed compound, at least one product of a disclosed method, or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutically acceptable carrier, optionally one or more other therapeutic agent, and optionally one or more adjuvant.
  • the disclosed pharmaceutical compositions include those suitable for oral, rectal, topical, pulmonary, nasal, and parenteral administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered.
  • the disclosed pharmaceutical composition can be formulated to allow administration orally, nasally, via inhalation, parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially and intratumorally.
  • parenteral administration includes administration by bolus injection or infusion, as well as administration by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, a therapeutically effective amount of a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof.
  • a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof, or any subgroup or combination thereof may be formulated into various pharmaceutical forms for administration purposes.
  • the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, of the present disclosure can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
  • the pharmaceutical compositions of the present disclosure can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient.
  • compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion.
  • the compounds of the present disclosure, and/or pharmaceutically acceptable salt(s) thereof can also be administered by controlled release means and/or delivery devices.
  • the compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.
  • unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. That is, a “unit dosage form” is taken to mean a single dose wherein all active and inactive ingredients are combined in a suitable system, such that the patient or person administering the drug to the patient can open a single container or package with the entire dose contained therein, and does not have to mix any components together from two or more containers or packages.
  • unit dosage forms are tablets (including scored or coated tablets), capsules or pills for oral administration; single dose vials for injectable solutions or suspension; suppositories for rectal administration; powder packets; wafers; and segregated multiples thereof.
  • This list of unit dosage forms is not intended to be limiting in any way, but merely to represent typical examples of unit dosage forms.
  • compositions disclosed herein comprise a compound of the present disclosure (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents.
  • the disclosed pharmaceutical compositions can include a pharmaceutically acceptable carrier and a disclosed compound, or a pharmaceutically acceptable salt thereof.
  • a disclosed compound, or pharmaceutically acceptable salt thereof can also be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds.
  • the instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered.
  • the pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methodswell known in the art of pharmacy.
  • the compounds described herein are typically to be administered in admixture with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • suitable pharmaceutical diluents, excipients, extenders, or carriers suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the deliverable compound will be in a form suitable for oral, rectal, topical, intravenous injection or parenteral administration.
  • Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used.
  • the compounds may be administered as a dosage that has a known quantity of the compound.
  • oral administration can be a preferred dosage form, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed.
  • other dosage forms may be suitable depending upon clinical population (e.g., age and severity of clinical condition), solubility properties of the specific disclosed compound used, and the like.
  • the disclosed compounds can be used in oral dosage forms such as pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.
  • any convenient pharmaceutical media can be employed.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like
  • oral solid preparations such as powders, capsules and tablets.
  • tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • compositions in an oral dosage form can comprise one or more pharmaceutical excipient and/or additive.
  • suitable excipients and additives include gelatin, natural sugars such as raw sugar or lactose, lecithin, pectin, starches (for example corn starch or amylose), dextran, polyvinyl pyrrolidone, polyvinyl acetate, gum arabic, alginic acid, tylose, talcum, lycopodium, silica gel (for example colloidal), cellulose, cellulose derivatives (for example cellulose ethers in which the cellulose hydroxy groups are partially etherified with lower saturated aliphatic alcohols and/or lower saturated, aliphatic oxyalcohols, for example methyl oxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate), fatty acids as well as magnesium, calcium or aluminum salts of fatty acids with 12 to 22 carbon
  • auxiliary substances useful in preparing an oral dosage form are those which cause disintegration (so-called disintegrants), such as: cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethyl cellulose or microcrystalline cellulose.
  • Conventional coating substances may also be used to produce the oral dosage form.
  • Plasticizing agents that may be considered as coating substances in the disclosed oral dosage forms are: citric and tartaric acid esters (acetyl-triethyl citrate, acetyl tributyl-, tributyl-, triethyl-citrate); glycerol and glycerol esters (glycerol diacetate, -triacetate, acetylated monoglycerides, castor oil); phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-, dipropyl-phthalate), di-(2-methoxy- or 2- ethoxyethyl)-phthalate, ethylphthalyl glycolate, butylphthalylethyl glycolate and butylglycolate; alcohols (propylene glycol, polyethylene glycol of various chain lengths), adipates (diethyladipate, di- (2-methoxy- or 2-ethoxye
  • suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers.
  • the pharmaceutical carrier employed can be, for example, a solid, liquid, or gas.
  • solid carriers include, but are not limited to, lactose, terra alba, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol talc, starch, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • liquid carriers are sugar syrup, peanut oil, olive oil, and water.
  • gaseous carriers include carbon dioxide and nitrogen.
  • a binder can include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • a disintegrator can include, for example, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • an oral dosage form such as a solid dosage form, can comprise a disclosed compound that is attached to polymers as targetable drug carriers or as a prodrug.
  • Suitable biodegradable polymers useful in achieving controlled release of a drug include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, caprolactones, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and hydrogels, preferably covalently crosslinked hydrogels.
  • Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a tablet containing a disclosed compound can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants.
  • Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.
  • Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • a solid oral dosage form such as a tablet
  • enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate.
  • enteric coating materials may be selected on the basis of testing to achieve an enteric coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crushing strength (e.g., see S. C. Porter et al. “The Properties of Enteric Tablet Coatings Made From Polyvinyl Acetate-phthalate and Cellulose acetate Phthalate”, J. Pharm. Pharmacol. 22:42p (1970)).
  • the enteric coating may comprise hydroxypropyl-methylcellulose phthalate, methacrylic acidmethacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate.
  • an oral dosage form can be a solid dispersion with a water soluble or a water insoluble carrier.
  • water soluble or water insoluble carrier include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, hydroxypropylmethyl-cellulose, phosphatidylcholine, polyoxyethylene hydrogenated castor oil, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, or hydroxypropylmethylcellulose, ethyl cellulose, or stearic acid.
  • an oral dosage form can be in a liquid dosage form, including those that are ingested, or alternatively, administered as a mouth wash or gargle.
  • a liquid dosage form can include aqueous suspensions, which contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may also contain various excipients.
  • the pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions, which may also contain excipients such as sweetening and flavoring agents.
  • water particularly sterile water, or physiologically acceptable organic solvents, such as alcohols (ethanol, propanol, isopropanol, 1 ,2-propylene glycol, polyglycols and their derivatives, fatty alcohols, partial esters of glycerol), oils (for example peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soya bean oil, castor oil, bovine hoof oil), paraffins, dimethyl sulfoxide, triglycerides and the like.
  • alcohols ethanol, propanol, isopropanol, 1 ,2-propylene glycol, polyglycols and their derivatives, fatty alcohols, partial esters of glycerol
  • oils for example peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soya bean oil, castor oil, bovine hoof oil
  • paraffins dimethyl sulfoxide, triglycerides and the like.
  • a liquid dosage form such as a drinkable solutions
  • the following substances may be used as stabilizers or solubilizers: lower aliphatic mono- and multivalent alcohols with 2-4 carbon atoms, such as ethanol, n-propanol, glycerol, polyethylene glycols with molecular weights between 200-600 (for example 1 to 40% aqueous solution), diethylene glycol monoethyl ether, 1 ,2- propylene glycol, organic amides, for example amides of aliphatic C1-C6-carboxylic acids with ammonia or primary, secondary or tertiary C1-C4-amines or C1-C4-hydroxy amines such as urea, urethane, acetamide, N-methyl acetamide, N,N-diethyl acetamide, N,N-dimethyl acetamide, lower aliphatic amines and diamines with 2-6 carbon atoms, such
  • solubilizers and emulsifiers such as the following non-limiting examples can be used: polyvinyl pyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phosphatides such as lecithin, acacia, tragacanth, polyoxyethylated sorbitan monooleate and other ethoxylated fatty acid esters of sorbitan, polyoxyethylated fats, polyoxyethylated oleotriglycerides, linolizated oleotriglycerides, polyethylene oxide condensation products of fatty alcohols, alkylphenols or fatty acids or also 1-methyl-3-(2- hydroxyethyl)imidazolidone-(2).
  • solubilizers and emulsifiers such as the following non-limiting examples can be used: polyvinyl pyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phosphatides
  • polyoxyethylated means that the substances in question contain polyoxyethylene chains, the degree of polymerization of which generally lies between 2 and 40 and in particular between 10 and 20.
  • Polyoxyethylated substances of this kind may for example be obtained by reaction of hydroxyl group-containing compounds (for example mono- or diglycerides or unsaturated compounds such as those containing oleic acid radicals) with ethylene oxide (for example 40 Mol ethylene oxide per 1 Mol glyceride).
  • hydroxyl group-containing compounds for example mono- or diglycerides or unsaturated compounds such as those containing oleic acid radicals
  • ethylene oxide for example 40 Mol ethylene oxide per 1 Mol glyceride
  • oleotriglycerides are olive oil, peanut oil, castor oil, sesame oil, cottonseed oil, corn oil. See also Dr. H. P. Fiedler “Lexikon der Hillsstoffe fur Pharmazie, Kostnetik und angrenzende füre” 1971 , pages 191-195.
  • a liquid dosage form can further comprise preservatives, stabilizers, buffer substances, flavor correcting agents, sweeteners, colorants, antioxidants and complex formers and the like.
  • Complex formers which may be for example be considered are: chelate formers such as ethylene diamine retrascetic acid, nitrilotriacetic acid, diethylene triamine pentacetic acid and their salts.
  • a liquid dosage form with physiologically acceptable bases or buffers may optionally be necessary to stabilize a liquid dosage form with physiologically acceptable bases or buffers to a pH range of approximately 6 to 9. Preference may be given to as neutral or weakly basic a pH value as possible (up to pH 8).
  • a parenteral injection form or an intravenous injectable form
  • cosolvents such as alcohols may improve the solubility and/or the stability of the compounds according to the present disclosure in pharmaceutical compositions.
  • a disclosed liquid dosage form, a parenteral injection form, or an intravenous injectable form can further comprise liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
  • compositions of the present disclosure suitable injection, such as parenteral administration, such as intravenous, intramuscular, or subcutaneous administration.
  • Pharmaceutical compositions for injection can be prepared as solutions or suspensions of the active compounds in water.
  • a suitable surfactant can be included such as, for example, hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
  • compositions of the present disclosure suitable for parenteral administration can include sterile aqueous or oleaginous solutions, suspensions, or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some aspects, the final injectable form is sterile and must be effectively fluid for use in a syringe.
  • the pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
  • Injectable solutions for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • a disclosed parenteral formulation can comprise about 0.01-0.1 M, e.g. about 0.05 M, phosphate buffer. In a further aspect, a disclosed parenteral formulation can comprise about 0.9% saline.
  • a disclosed parenteral pharmaceutical composition can comprise pharmaceutically acceptable carriers such as aqueous or non-aqueous solutions, suspensions, and emulsions.
  • pharmaceutically acceptable carriers such as aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include but not limited to water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.
  • a disclosed parenteral pharmaceutical composition can comprise may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • Also contemplated for injectable pharmaceutical compositions are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the subject or patient.
  • the disclosed compounds can also be formulated as a depot preparation.
  • Such long acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.
  • compositions of the present disclosure can be in a form suitable for topical administration.
  • topical application means administration onto a biological surface, whereby the biological surface includes, for example, a skin area (e.g., hands, forearms, elbows, legs, face, nails, anus and genital areas) or a mucosal membrane.
  • a skin area e.g., hands, forearms, elbows, legs, face, nails, anus and genital areas
  • a mucosal membrane e.g., a skin area (e.g., hands, forearms, elbows, legs, face, nails, anus and genital areas) or a mucosal membrane.
  • a topical pharmaceutical composition can be in a form of a cream, an ointment, a paste, a gel, a lotion, milk, a suspension, an aerosol, a spray, foam, a dusting powder, a pad, and a patch. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the present disclosure, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.
  • the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
  • These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on, as an ointment.
  • Ointments are semisolid preparations, typically based on petrolatum or petroleum derivatives.
  • the specific ointment base to be used is one that provides for optimum delivery for the active agent chosen for a given formulation, and, preferably, provides for other desired characteristics as well (e.g., emollience).
  • an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed., Easton, Pa.: Mack Publishing Co. (1995), pp.
  • ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases.
  • Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum.
  • Emulsifiable ointment bases also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.
  • Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.
  • W/O water-in-oil
  • O/W oil-in-water
  • Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight.
  • Lotions are preparations that are to be applied to the skin surface without friction. Lotions are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are typically preferred for treating large body areas, due to the ease of applying a more fluid composition. Lotions are typically suspensions of solids, and oftentimes comprise a liquid oily emulsion of the oil-in-water type. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, such as methylcellulose, sodium carboxymethyl-cellulose, and the like.
  • Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil.
  • Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase.
  • the oil phase also called the “internal” phase, is generally comprised of petrolatum and/or a fatty alcohol such as cetyl or stearyl alcohol.
  • the aqueous phase typically, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. Reference may be made to Remington: The Science and Practice of Pharmacy, supra, for further information.
  • Pastes are semisolid dosage forms in which the bioactive agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from a single-phase aqueous gel.
  • the base in a fatty paste is generally petrolatum, hydrophilic petrolatum and the like.
  • the pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base. Additional reference may be made to Remington: The Science and Practice of Pharmacy, for further information.
  • Gel formulations are semisolid, suspension-type systems.
  • Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.
  • Preferred organic macromolecules, i.e., gelling agents are crosslinked acrylic acid polymers such as the family of carbomer polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the trademark CarbopolTM.
  • hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; modified cellulose, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin.
  • dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.
  • Sprays generally provide the active agent in an aqueous and/or alcoholic solution which can be misted onto the skin for delivery.
  • Such sprays include those formulated to provide for concentration of the active agent solution at the site of administration following delivery, e.g., the spray solution can be primarily composed of alcohol or other like volatile liquid in which the active agent can be dissolved.
  • the carrier evaporates, leaving concentrated active agent at the site of administration.
  • Foam compositions are typically formulated in a single or multiple phase liquid form and housed in a suitable container, optionally together with a propellant which facilitates the expulsion of the composition from the container, thus transforming it into a foam upon application.
  • Other foam forming techniques include, for example the “Bag-in-a-can” formulation technique.
  • Compositions thus formulated typically contain a low-boiling hydrocarbon, e.g., isopropane. Application and agitation of such a composition at the body temperature cause the isopropane to vaporize and generate the foam, in a manner similar to a pressurized aerosol foaming system.
  • Foams can be water-based or aqueous alkanolic, but are typically formulated with high alcohol content which, upon application to the skin of a user, quickly evaporates, driving the active ingredient through the upper skin layers to the site of treatment.
  • Skin patches typically comprise a backing, to which a reservoir containing the active agent is attached.
  • the reservoir can be, for example, a pad in which the active agent or composition is dispersed or soaked, or a liquid reservoir.
  • Patches typically further include a frontal water permeable adhesive, which adheres and secures the device to the treated region. Silicone rubbers with selfadhesiveness can alternatively be used. In both cases, a protective permeable layer can be used to protect the adhesive side of the patch prior to its use.
  • Skin patches may further comprise a removable cover, which serves for protecting it upon storage.
  • Examples of patch configuration which can be utilized with the present invention include a single-layer or multi-layer drug-in-adhesive systems which are characterized by the inclusion of the drug directly within the skin-contacting adhesive.
  • the adhesive not only serves to affix the patch to the skin, but also serves as the formulation foundation, containing the drug and all the excipients under a single backing film.
  • a membrane is disposed between two distinct drug-in-adhesive layers or multiple drug-in-adhesive layers are incorporated under a single backing film.
  • Examples of pharmaceutically acceptable carriers that are suitable for pharmaceutical compositions for topical applications include carrier materials that are well-known for use in the cosmetic and medical arts as bases for e.g., emulsions, creams, aqueous solutions, oils, ointments, pastes, gels, lotions, milks, foams, suspensions, aerosols and the like, depending on the final form of the composition.
  • suitable carriers according to the present invention therefore include, without limitation, water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated protein hydrolysates, liquid lanolin and lanolin derivatives, and like materials commonly employed in cosmetic and medicinal compositions.
  • suitable carriers include, without limitation, alcohols, such as, for example, monohydric and polyhydric alcohols, e.g., ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol, hexyleneglycol, mannitol, and propylene glycol; ethers such as diethyl or dipropyl ether; polyethylene glycols and methoxypolyoxyethylenes (carbowaxes having molecular weight ranging from 200 to 20,000); polyoxyethylene glycerols, polyoxyethylene sorbitols, stearoyl diacetin, and the like.
  • alcohols such as, for example, monohydric and polyhydric alcohols, e.g., ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol, hexyleneglycol, mannito
  • Topical compositions of the present disclosure can, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the dispenser device may, for example, comprise a tube.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.
  • Such notice for example, may include labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising the topical composition of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Another patch system configuration which can be used by the present invention is a reservoir transdermal system design which is characterized by the inclusion of a liquid compartment containing a drug solution or suspension separated from the release liner by a semi-permeable membrane and adhesive.
  • the adhesive component of this patch system can either be incorporated as a continuous layer between the membrane and the release liner or in a concentric configuration around the membrane.
  • Yet another patch system configuration which can be utilized by the present invention is a matrix system design which is characterized by the inclusion of a semisolid matrix containing a drug solution or suspension which is in direct contact with the release liner.
  • the component responsible for skin adhesion is incorporated in an overlay and forms a concentric configuration around the semisolid matrix.
  • compositions of the present disclosure can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
  • compositions containing a compound of the present disclosure, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.
  • the pharmaceutical composition may be packaged in a variety of ways.
  • an article for distribution includes a container that contains the pharmaceutical composition in an appropriate form.
  • Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, foil blister packs, and the like.
  • the container may also include a tamper proof assemblage to prevent indiscreet access to the contents of the package.
  • the container typically has deposited thereon a label that describes the contents of the container and any appropriate warnings or instructions.
  • the disclosed pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • Pharmaceutical compositions comprising a disclosed compound formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the exact dosage and frequency of administration depends on the particular disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the present disclosure.
  • the pharmaceutical composition will comprise from 0.05 to 99 % by weight, preferably from 0.1 to 70 % by weight, more preferably from 0.1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.
  • an appropriate dosage level will generally be about 0.01 to 1000 mg of a compound described herein per kg patient body weight per day and can be administered in single or multiple doses.
  • the dosage level will be about 0.1 to about 500 mg/kg per day, about 0.1 to 250 mg/kg per day, or about 0.5 to 100 mg/kg per day.
  • a suitable dosage level can be about 0.01 to 1000 mg/kg per day, about 0.01 to 500 mg/kg per day, about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg/kg per day.
  • compositions are preferably provided in the form of tablets containing 1.0 to 1000 mg of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated.
  • the compound can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response.
  • Such unit doses as described hereinabove and hereinafter can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day.
  • such unit doses can be administered 1 or 2 times per day, so that the total dosage for a 70 kg adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration.
  • dosage is 0.01 to about 1 .5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years.
  • the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.
  • a typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient.
  • the time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
  • compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological or clinical conditions.
  • compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure relates to a process for preparing such a pharmaceutical composition, characterized in that a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound according to the present disclosure.
  • Classical opioid analgesics including morphine, mediate all of their desired and undesired effects by specific activation of the -opioid receptor ( receptor).
  • the use of morphine for treating chronic pain is limited by the development of constipation, respiratory depression, tolerance and dependence.
  • Analgesic effects can also be mediated through other members of the opioid receptor family such as the K-opioid receptor (K receptor), 6-opioid receptor (6 receptor) and the nociceptin/orphanin FQ peptide receptor (NOP receptor).
  • K receptor K-opioid receptor
  • 6 receptor 6-opioid receptor
  • NOP receptor nociceptin/orphanin FQ peptide receptor
  • the compounds described herein can bind to the kappa opioid receptor (KOR) and behave as an agonist. As shown in the Examples, compounds described herein demonstrate the ability of the compounds described herein to bind to KOR. The ability of the compounds to bind to opioid receptors make them effective in the treatment or prevention of pain. In one aspect, the pain can be chronic or acute pain.
  • KOR kappa opioid receptor
  • a lipid nanoparticle comprising a lipopeptide conjugate and a bioactive agent, wherein the lipopeptide conjugate has the formula X-L-Y, wherein
  • X is a peptide
  • L is a linker
  • Y is a lipid
  • Aspect 2 The lipid nanoparticle of Aspect 1 , wherein the peptide comprises from about 1 to about 10000 arginine residues, from about 1 to about 10000 histidine residues and at least one cysteine residue.
  • Aspect 3 The lipid nanoparticle of Aspect 1 or 2, wherein the peptide comprises arginine residues and histidine residues, wherein the arginine-to-histidine (R/H) ratio in the peptide is from 4:1 to 1 :4.
  • Aspect 4 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH 2 -C W R 3 H 3 -COOH, NH 2 - C W 5 H 5 -COOH, NH 2 -C W 3 H 3 -COOH, NH 2 -C W 3 H 4 -COOH, NH 2 -C W 3 H 5 -COOH, NH 2 -C W 3 H S -COOH, NH 2 -CW 3 H 7 -COOH, NH 2 -CW 3 H 8 -COOH, NH 2 -CW 3 H 9 -COOH, NH 2 -CWK 5 H 5 -COOH, NH 2 -C W K 3 H 3 - COOH, NH 2 -CwRm-COOH, NH 2 -C w K n -COOH, NH 2 -C RuleR o H
  • Aspect 5 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH 2 -R 3 H 3 -COOH, NH 2 - R5H5-COOH, NH 2 -R 3 H 3 -COOH, NH 2 -R 3 H 4 -COOH, NH 2 -R 3 H 5 -COOH, NH 2 -R 3 H S -COOH, NH 2 -R 3 H 7 - COOH, NH 2 -R 3 H 8 -COOH, NH 2 -R 3 H 9 -COOH, NH 2 -K 5 H 5 -COOH, NH 2 -K 3 H 3 -COOH, NH 2 -R 0 H P -COOH, NH 2 -RUK V -COOH, NH 2 -K p H q -COOH, and NH 2 -R
  • Aspect 6 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide has a formula selected from the group consisting of NH 2 -R m -COOH, NH 2 -K n -COOH, NH 2 -H 0 -COOH; wherein R is arginine, K is lysine, H is histidine, NH 2 - is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m, n, and 0 are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
  • Aspect 7 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequencespecific peptide having a formula selected from the group consisting of NH 2 -CR-COOH, NH 2 -CR 2 - COOH, NH 2 -CR 3 -COOH, NH 2 -CR 4 -COOH, NH 2 -CR 5 -COOH, NH 2 -CR 6 -COOH, NH 2 -CR m -COOH, NH 2 -CHR-COOH, NH 2 -CH 2 R-COOH, NH 2 -CH 3 R-COOH, NH 2 -CH 4 R-COOH, NH 2 -CH 5 R-COOH NH 2 - CHmR-COOH, NH 2 -CHR 2 -COOH, NH 2 -CH 2 R 2 -COOH, NH 2 -CH 3 R 2 -COOH, NH 2 -CH 4 R-COOH, NH 2
  • Aspect 8 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequencespecific peptide or polypeptide having a formula selected from the group consisting of NH 2 -CK- COOH, NH 2 -CK 2 -COOH, NH 2 -CK 3 -COOH, NH 2 -CK 4 -COOH, NH 2 -CK 5 -COOH, NH 2 -CK 6 -COOH, NH 2 - CK m -COOH, NH 2 -CHK-COOH, NH 2 -CH 2 K-COOH, NH 2 -CH 3 K-COOH, NH 2 -CH 4 K-COOH, NH 2 -CH 5 K- COOH NH 2 -CH m K-COOH, NH 2 -CHK 2 -COOH, NH 2 -CH 2 K 2 -COOH, NH 2 -CH 3 K 2 -COOH, NH 2 -CH 4 K-CO
  • Aspect 9 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequencespecific peptide or polypeptide having a formula selected from the group consisting of NH 2 -RC- COOH, NH 2 -R 2 C-COOH, NH 2 -R 3 C-COOH, NH 2 -R 4 C-COOH, NH 2 -R 5 C-COOH, NH 2 -R 6 C-COOH, NH 2 -R m C-COOH, NH 2 -RHC-COOH, NH 2 -RH 2 C-COOH, NH 2 -RH 3 C-COOH, NH 2 -RH 4 C-COOH, NH 2 - RHsC-COOH, NH 2 -RH m C-COOH, NH 2 -R 2 HC-COOH, NH 2 -R 2 H 2 C-COOH, NH 2 -R 2 H 3 C-COOH, NH 2 -
  • Aspect 10 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2- KC-COOH, NH2-K2C-COOH, NH2-K3C-COOH, NH2-K4C-COOH, NH2-K5C-COOH, NH 2 -K 6 C-COOH, NH 2 -K m C-COOH, NH2-KHC-COOH, NH2-KH2C-COOH, NH2-KH3C-COOH, NH2-KH4C-COOH, NH 2 - KH5C-COOH, NH 2 -KH m C-COOH, NH 2 -K 2 HC-COOH, NH 2 -K 2 H 2 C-COOH, NH2-K2H3C-COOH, NH 2 - K2H4C-COOH, NH2-K2H
  • Aspect 1 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide has a formula selected from the group consisting of NH 2 -CR n -COOH, NH 2 -CK n -COOH, NH 2 -CH n -COOH, NH 2 -R n C- COOH, NH 2 -K n C-COOH, NH 2 -H n C-COOH, NH 2 -R 0 H P -COOH, H 2 -H p R o -COOH, NH 2 -CR 0 H P -COOH, NH 2 -CH P R 0 -COOH, NH 2 -R 0 CH P -COOH, NH 2 -H P CR 0 -COOH, NH 2 -R 0 H P C-COOH, NH 2 -R 0 H P C-COOH, NH 2 -HpRoC-COOH, NH 2 -
  • Aspect 12 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a random or block copolymeric peptide having a formula selected from the group consisting of NH 2 - X1 x X2yX3 z X4w-COOH, wherein X1 , X2, X3, and X4 is cysteine, arginine, lysine or histidine, NH 2 - is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
  • the peptide is a random or block copolymeric peptide having a formula selected from the group consisting of NH 2 - X1 x X2yX3 z X4w-COOH, wherein
  • Aspect 13 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide has a sequence selected from the group consisting of NH2-CRRRRR-COOH, NH2-CRRRHHH-COOH, NH2- CHHHRRR-COOH, NH2-CKKKKK-COOH, NH2-CKKK-COOH, NH2-CHHHHHRRRR-COOH, NH 2 - CRRRRRHHHHH-COOH, NH2-CRRRHHH-COOH, NH2-CHHHRRRCOOH, NH2-CKKKKKHHHHH- COOH, NH2-CHHHHHKKKKK-COOH, NH2-CHHHKKK-COOH, NH2-CKKKHHH-COOH, NH 2 - RRRRRC-COOH, NH2-RRRHHHC-COOH, NH2-KKKKKC-COOH, NH2-KKKC-COOH, NH2-KKKC-COOH, NH2-KKKC-COOH, NH 2 -
  • Aspect 14 The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a block copolymer having a sequence selected from the group consisting of NH 2 -CR m -COOH, NH 2 -CK n - COOH, NH 2 -CR 0 H P -COOH, NH 2 -CR x KyH z -COOH, NH 2 -R m C-COOH, NH 2 -K n C-COOH, NH 2 -R O H P C- COOH, NH 2 - RxKyHzC-COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, and m, n, o, p, q, x, y, and z are integers from 1 to 1 ,000, NH 2 - is the N-terminal of the peptide, -COOH is the C-terminal of the a -
  • Aspect 15 The lipid nanoparticle of any one of Aspects 1-14, wherein the linker is a residue of a group selected from the group consisting of amine, carboxylate, hydroxyl, thiol, alkene, alkyne, azide, disulfide, maleimide, ester, peptide, acetal, anhydride, halide, vinyl sulfone, methyl acrylate, acrylate, acrylamide, methyl acrylamide, fluoride, chloride, bromide, aldehyde, and ketone.
  • the linker is a residue of a group selected from the group consisting of amine, carboxylate, hydroxyl, thiol, alkene, alkyne, azide, disulfide, maleimide, ester, peptide, acetal, anhydride, halide, vinyl sulfone, methyl acrylate, acrylate, acrylamide, methyl acrylamide, fluoride, chloride,
  • Aspect 16 The lipid nanoparticle of any one of Aspects 1-15, wherein the lipid is selected from the group consisting of a phosphatidylcholine, a lysophosphatidylcholine, a plasmenylphosphatidylcholin, a phosphatidylethanolamin, a lysophosphatidylethanolamine, a plasmenylphosphatidylethanolamin, a phosphatidylserine, a sphingomyeline, a phosphatidic acid
  • a lysophosphatidic acid a phosphatidylinositol, a phosphatidylglycerol, a cardiolipin, a ceramide-1 -phosphate, an N-acylsphingosine, a sulfatide, a ganglioside, a sulfoquinovosyldiacylglycero, and a diphosphorylated hexaacyl Lipid A.
  • Aspect 17 The lipid nanoparticle of any one of Aspects 1-15, wherein the lipid is a phospholipid.
  • Aspect 18 The lipid nanoparticle of any one of Aspects 1-15, wherein the lipid is a phosphatidylcholine, a phosphatidic acid, a phosphatidylglycerol, a phosphatidylethanolamine, a phosphatidylserine, or a PEG phospholipid.
  • Aspect 19 The lipid nanoparticle of Aspect 1 , wherein the linker has a residue of a maleimide and the lipid is phosphatidylethanolamine.
  • Aspect 20 The lipid nanoparticle of Aspect 19, wherein the lipid is 1 ,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine (DMPE), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), or any combination thereof.
  • DMPE 1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine
  • DPPE 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
  • DSPE distearoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • Aspect 21 The lipid nanoparticle of Aspect 19 or 20, wherein the lipid is 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE).
  • DOPE 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine
  • Aspect 22 The lipid nanoparticle of any one of Aspects 19-21 , wherein the peptide is a random copolymeric peptide having a formula NH 2 -RmH n C-COOH, wherein C is cysteine, R is arginine, H is histidine, COOH is the N-terminal of the peptide, and m and n are integers from 1 to 15.
  • Aspect 23 The lipid nanoparticle of any one of Aspects 1-14, wherein the lipopeptide conjugate has the structure I wherein Ri and R 2 are independently a C8 to C25 alkyl group or a C8 to C25 alkenyl group, o and p are independently integers from 1 to 4, and
  • X is a peptide
  • Aspect 24 The lipid nanoparticle of Aspect 23, wherein (a) o and p are each 2 or (b) o and p are each 2 and Ri and R 2 are independently an oleyl group or a palmitoyl group.
  • Aspect 25 The lipid nanoparticle of any one of Aspects 1-24, wherein the lipid nanoparticle further comprises one or more helper lipids.
  • Aspect 26 The lipid nanoparticle of Aspect 25, wherein the helper lipid comprises a phospholipid, PEGylated lipid, a sterol, an any combination thereof.
  • Aspect 27 The lipid nanoparticle of Aspect 25 and 26, wherein the lipid peptide conjugate to helper lipid is at a molar ratio of 1 :100 to 100:1 .
  • Aspect 28 The lipid nanoparticle of any one of Aspects 1-27, wherein the bioactive agent is selected from the group consisting of Abiraterone Acetate, Brentuximab vedotin, Trastuzumab emtansine, Afatinib, Afinitor® (Everolimus), Aldara® (Imiquimod), Alimta® (Pemetrexed Disodium), Pemetrexed, Palonosetron, Chlorambucil, Nelarabine, Axitinib, Belinostat, Bleomycin, Bortezomib, Cabozantinib-S-Malate, Camptothecin, Capecitabine, Ceritinib, Cerubidine® (Daunorubicin), Crizotinib, Dabrafenib, Dasatinib, Degarelix, Docetaxel, Doxorubicin, Epirubicin, Eribulin, Etopo
  • Aspect 29 The lipid nanoparticle of any one of Aspects 1-27, wherein the bioactive agent is selected from the group consisting of antibodies, peptides, therapeutic enzymes, cytokines, interferons, and interleukins.
  • Aspect 30 The lipid nanoparticle of any one of Aspects 1-27, wherein the bioactive agent is a nucleic acid.
  • Aspect 31 The lipid nanoparticle of Aspect 30, wherein the nucleic acid is selected from the group consisting of plasmid DNA, an oligonucleotide, an aptamers, a DNAzyme, a RNA aptamers, a RNA Decoy, an antisense RNA, a ribozymes, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA, an antagomirs, or any combination thereof.
  • the nucleic acid is selected from the group consisting of plasmid DNA, an oligonucleotide, an aptamers, a DNAzyme, a RNA aptamers, a RNA Decoy, an antisense RNA, a ribozymes, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA, an antagomirs, or any combination thereof.
  • Aspect 32 The lipid nanoparticle of Aspect 30 or 31 , wherein the molar ratio of amine or guanidine groups on the side chain of peptide (N) to the phosphate group in the nucleic acid (P) is from about 0.5: 1 to about 100: 1 .
  • Aspect 33 The lipid nanoparticle of any one of Aspects 1 -32, wherein the lipid nanoparticle is between about 2 nm and about 2000 m in diameter.
  • Aspect 34 The lipid nanoparticle of any one of Aspects 1-33, wherein the lipid nanoparticle has a surface charge of between about -100 mV and about +100 mV at pH 3 to 1 1 .
  • Aspect 35 A method for delivering a bioactive agent to a subject, the method comprising administering to the subject the lipid nanoparticle of any one of Aspects 1-34.
  • Aspect 36 The method of Aspect 35, wherein the bioactive agent is a nucleic acid.
  • Aspect 37 The method of Aspect 35, wherein the bioactive agent is a siRNA.
  • Aspect 38 The method of any one of Aspects 35-37, wherein when the bioactive agent is delivered to the lungs, the arginine-to-histidine (R/H) ratio in the peptide is from 2:1 to 1 :2.
  • Aspect 39 The method of any one of Aspects 35-37, wherein when the bioactive agent is delivered to the liver, the arginine-to-histidine (R/H) ratio in the peptide is from 1 :1 to 1 :3.
  • Aspect 40 The method of any one of Aspects 35-37, wherein when the bioactive agent is delivered to the liver, the arginine-to-histidine (R/H) ratio in the peptide is from 1 :1 to 1 :2.
  • the lipopeptide conjugate was synthesized via an Al BN-initiated free radical addition reaction. Briefly, DOPE-maleimide (1.0 equiv) and RmHnC (40 mg, 2.0 equiv) were separately dissolved in 1 mL anhydrous dimethyl sulfoxide (DMSO) under sonication at 25°C for 12 h. The solutions were combined in a 25 mL glass reactor, followed by addition of azobisisobutyronitrile (AIBN, 20 mg, 2.0 equiv relative to DOPE-mal). The reaction mixture was heated to 70°C in a pre-equilibrated metal bath (initial preheating at 50°C) and stirred under inert atmosphere for 16 h.
  • DMSO dimethyl sulfoxide
  • the crude product was diluted with 30 mL deionized (DI) water, agitated for 3 h, and centrifuged at 10,000 xg for 30 min to remove insoluble residues.
  • DI deionized
  • the supernatant was dialyzed against DI water using a 2 kDa molecular weight cut-off (MWCO) membrane for 96 h, with water replaced every 12 h.
  • MWCO molecular weight cut-off
  • the resultant powder was dissolved in a 4:1 (v/v) ethanol/DMSO mixture containing 0.1% trifluoroacetic acid (TFA) to prepare a 10 mg/mL stock solution, which was characterized by MALDI-TOF mass spectrometry and LC-MS.
  • TFA trifluoroacetic acid
  • LNPs were formulated by mixing the synthesized lipopeptide, SM-102, sitosterol/cholesterol (38.5% molar ratio), DOPE/DSPC (10%), and DMPE-PEG2000 (1.5%) at molar ratios of 50% lipopeptide, 38.5% sterol blend, 10% phospholipids, and 1.5% PEGylated lipid.
  • the lipid components were dissolved in ethanol and combined with nucleic acids at an N/P ratio of 6 (1 :3 v/v aqueous buffer) under gentle agitation for 30 min at 25°C.
  • Freshly prepared LNPs were diluted with nuclease- free water and purified via 30 kDa molecular weight cut-off (MWCO) centrifugal filters to achieve >99% buffer exchange efficiency.
  • MWCO molecular weight cut-off
  • Pre-Ultrafiltration EE (Total nucleic acid (post-lysis)-Free nucleic acid (unlysed)) I Total nucleic acid (pre-lysis)x100%
  • HepG2 cells were obtained from the Chinese Academy of Sciences Cell Bank, and HEPA1-6 cells were purchased from Pricella Biotechnology Co., Ltd. (Wuhan, China). Cells were cultured in RPMI 1640, MEM, or DMEM supplemented with 10% FBS and 0.5% penicillin/streptomycin at 37°C under 5% CO 2 . Mycoplasma contamination was routinely monitored.
  • Cytotoxicity of RH-LP LNPs was evaluated using the CCK-8 assay. Briefly, HEPA1-6/HepG2 cells (5.0*10 3 cells/well) were seeded in 96-well plates, cultured for 24 h, and treated with RH-DOPE LNPs (10 pmol nucleic acid/well) in complete medium for 48 h. After adding 10 L CCK-8 solution per well, cells were incubated for 2 h, and absorbance at 450 nm was measured using an EnSpire microplate reader at 25°C. Triplicate measurements were performed for each sample.
  • RNA-loaded RH-DOPE LNPs 50 pmol RNA/well
  • Opti-MEM Opti-MEM
  • RNA-loaded RH-DOPE LNPs 50 pmol RNA/well
  • Opti-MEM Opti-MEM
  • cells were incubated for 24-48 h.
  • Total RNA was extracted using the EASYspin Plus RNA Extraction Kit (Aidlab, China, #RN28) and quantified via NanoDrop One (Thermo Fisher Scientific).
  • Target gene (PCSK9) expression relative to GAPDH was analyzed using the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme Biotech, #0221-01).
  • the qRT-PCR protocol included: reverse transcription (50°C, 3 min), pre-denaturation (95°C, 15 min), 40 cycles of denaturation (95°C, 10 s) and annealing/extension (60°C, 30 s), followed by a melt curve analysis (95°C to 60°C). Naked siRNA, RNAiMAX, and SM-102 LNPs served as controls.
  • eGFP plasmid delivery fluorescence images were captured using an inverted fluorescence microscope. eGFP-positive cells were quantified via flow cytometry (FlowJo software). Lipofectamine 2000 and SM-102 LNPs were used as positive controls.
  • Unbound LNPs were removed by washing three times with ice-cold PBS, and cells were trypsinized, resuspended in PBS, and analyzed using flow cytometry (Cy5 excitation/emission: 649/670 nm; >10,000 events per sample). Untreated cells and cells without LNP exposure served as background controls, and inhibitor cytotoxicity was pre-validated by CCK-8 assay (cell viability >90%).
  • mice received PCSK9 siRNA-loaded LNPs (0.5 mg kg -1 ). Tissues (15 mg) were homogenized using a high-speed tissue homogenizer (TIANGEN, #OSE-Y30), and total RNA was extracted for qRT-PCR analysis of PCSK9 expression relative to GAPDH.
  • the qRT-PCR protocol included reverse transcription (50°C, 3 min), pre-denaturation (95°C, 15 min), 40 cycles of amplification (95°C, 10 s; 60°C, 30 s), and melt curve analysis. PBS and SM-102 LNPs were used as controls.
  • RmHnC-DOPE RmHnC-based lipopeptides
  • the peptides were conjugated to DOPE-Mal via thiol-ene chemistry (FIG. 1C), and LNPs were formulated with helper lipids, p-sitosterol for membrane stability and DMPE PEG-2000 for stabilization and stealth properties (FIG. 1 D) to create four-component RmHn-DOPE LNPs encapsulating nucleic acids (FIG. 1 E).
  • RmHnC-DOPE conjugates were synthesized through an azobisisobutyronitrile (AIBN)- initiated free radical thiol-ene reaction between the cysteine thiol group of the peptides and the maleimide moiety of DOPE-Mal. It should be noted that thiol and maleimide can also undergo the Michael-type reaction 7 , however, the thiol-ene 8 reaction was used to reduce the reaction time.
  • AIBN azobisisobutyronitrile
  • RmHn- DOPE LNPs were prepared by combining the lipopeptide conjugates with p-sitosterol (membrane stabilizer), DMPE PEG-2000, and nucleic acids (e.g., siRNA or pDNA) at a fixed molar ratio. Selfassembly was achieved via microfluidic mixing, yielding monodisperse nanoparticles with diameters tailored for cellular uptake (e.g., 80-120 nm). The inclusion of histidine residues enabled pH- dependent endosomal escape, while arginine provided cationic charge for nucleic acid complexation.
  • p-sitosterol membrane stabilizer
  • nucleic acids e.g., siRNA or pDNA
  • This modular synthesis strategy enabled systematic variation of peptide sequences and lipid compositions, accelerating high-throughput screening of LNP formulations to optimize delivery efficiency.
  • Our approach leverages well-established solid-state peptide synthesis, which ensures scalable production of high-purity peptides and well-characterized lipid chemistry, allowing precise tuning of lipopeptide-LNP structure and functionality.
  • the integration of these established methodologies provides a robust framework for designing biodegradable, high-performance delivery systems.
  • FIG. 2G Cryo-TEM imaging (FIG. 2G) confirmed spherical nanostructures with electron-dense cores, consistent with tightly condensed nucleic acid payloads and uniform lipid bilayer organization.
  • the pH-responsive behavior of top-performing variants was further validated by their pKa values (R3H7C- DOPE: 6.2; R4H6C-DOPE: 6.5; R5H5C-DOPE: 6.0), which aligned with the endosomal pH gradient (4.0-6.5) to enable protonation-dependent membrane fusion and payload release (FIG. 2F).
  • Encapsulation efficiency (EE) for both siRNA and pDNA exceeded 90% across all formulations, with R4H6C-DOPE achieving 95.2 ⁇ 1.8% EE for siRNA and 92.4 ⁇ 2.1% for pDNA, superior to SM-102 LNPs (85.0 ⁇ 3.5% siRNA EE).
  • R3H7C-, R4H6C-, and R5H5C-DOPE LNPs encapsulating PCSK9 siRNA demonstrated dose-dependent gene silencing efficacy in HEPA1-6 cells (FIG. 2E).
  • Quantitative RT-PCR analysis revealed PCSK9 mRNA level was knocked down to 16.9 ⁇ 12.2%, 14.9 ⁇ 1 .0%, and 10.3 ⁇ 1.9% for R3H7C-, R4H6C-, and R5H5C-DOPE LNPs, respectively, compared to 32.6 ⁇ 8.5% observed with SM-102 LNP (p ⁇ 0.05).
  • pDNA plasmid DNA
  • LNPs lipid nanoparticles
  • Fluorescence microscopy demonstrated robust eGFP expression across all three lipopeptide formulations, with R3H7C-DOPE and R4H6C-DOPE LNPs exhibiting fluorescence intensities comparable to or exceeding Lipofectamine 2000, a commercial transfection reagent.
  • SM- 102 LNPs displayed markedly weaker signal intensity, aligning with their suboptimal pDNA delivery efficiency observed in prior studies.
  • FIG. 2I Flow cytometric quantification (FIG. 2I) revealed distinct transfection profiles among LNP variants in HEPG2 cells.
  • MFI mean fluorescence intensity
  • R5H5C-DOPE The exceptional efficacy of R5H5C-DOPE likely arises from its optimized arginine-to-histidine (R/H) ratio (5:5), which balances cationic charge density for stable pDNA complexation and pH-responsive endosomal escape via histidine's buffering capacity 5. This mechanism is further supported by the pKa range (6.0-6.5) of RmHn-DOPE variants, aligning with the acidic endosomal environment to enhance payload release.
  • R/H arginine-to-histidine
  • R5H5C-DOPE LNPs achieved the highest luminescence intensity at 6.3 ⁇ 0.5X10 4 relative light units (RLU), significantly surpassing both the commercial transfection reagent Lipofectamine 2000 (5.3 ⁇ 0.6X 10 4 RLU) and SM-102 LNPs (1 .7 ⁇ 0.8X10 2 RLU).
  • SM-102 LNPs showed limited transfection efficiency, with luminescence levels comparable to the blank control ( ⁇ 500 RLU), highlighting its suboptimal pDNA delivery capability.
  • R5H5C-DOPE’s superior performance correlated with its optimized arginine-to-histidine (5:5) ratio, which balances cationic charge density for stable pDNA complexation and pH-responsive endosomal escape.
  • the cytotoxicity study also showed nearly 100% cell viability for flue pDNA-loaded RmHn-DOPE- and SM102 LNP-treated cells versus 73.8% viability for flue pDNA Lipofectamine 2000-treated cells.
  • RmHn-DOPE LNPs particularly the R3H7C- DOPE variant
  • the superior plasmid transfection efficiency of RmHn-DOPE LNPs arises from their dual-functional architecture.
  • the cationic arginine residues in these LNPs promote robust electrostatic interactions with plasmid DNA, enabling the formation of stable nanocomplexes that protect genetic payloads from nuclease degradation.
  • histidine residues contribute pH-buffering capacity, which facilitates endosomal escape through the proton sponge effect. This mechanism is supported by the optimal pKa range (6.0-6.5) of R3H7C-DOPE, aligning with the acidic environment of endosomes to enhance transfection efficiency.
  • SM-102-based LNPs with tertiary amines limits endosomal buffering and escape — a key bottleneck in non-viral gene delivery.
  • RmHn-DOPE LNPs demonstrate significant advantages over commercial transfection reagents. Their biocompatible design minimizes cytotoxicity, maintaining over 98% cell viability compared to Lipofectamine 2000, which causes substantial toxicity (73.8 % viability in HepG2 cell) due to its high charge density and structure. Furthermore, RmHn-DOPE formulations exhibit exceptional colloidal stability, remaining intact for over 30 days. These attributes position RmHn- DOPE LNPs as a versatile platform capable of delivering diverse nucleic acid payloads with viral-like efficiency while retaining the safety profile of biodegradable lipids.
  • SM-102 LNPs displayed elevated signals in the spleen and liver.
  • R5H5C-DOPE demonstrated intermediate liver targeting (1 .3- fold vs. SM-102), suggesting that lower histidine content (5:5 R/H ratio) may reduce hepatic specificity by altering surface charge dynamics, as observed in lipid designs where imbalanced cationic-to-pH- responsive ratios compromise organotropism.
  • R3H7C-DOPE LNPs The superior performance of R3H7C-DOPE LNPs can be attributed to their optimized design: the arginine-rich domain enhances siRNA complexation and serum stability, while histidine-mediated pH buffering promotes endosomal escape in hepatocytes.
  • SM-102 reliance on tertiary amines and non-degradable hydrocarbon tails likely contributes to its non-specific biodistribution and reduced hepatic transfection.
  • RmHn-DOPE LNPs as a modular platform for precision nucleic acid delivery.
  • the ability to tune R/H ratios for payload- and tissue-specific optimization addresses a critical gap in non-viral gene therapy, particularly for hepatotropic disorders or lung diseases requiring extrahepatic targeting.
  • Future studies should explore correlations between protein corona profiles (e.g., apolipoprotein E for liver targeting or Vtn for lung specificity) and in vivo targeting efficiency, building on emerging insights into LNP-host interactions.
  • ester- linked lipopeptides in RmHn-DOPE LNPs reduces long-term toxicity risks compared to non- degradable lipids like SM-102, positioning them as clinically translatable candidates for systemic gene delivery.
  • RmHn-DOPE LNPs The therapeutic potential of RmHn-DOPE LNPs was evaluated in C57BL/6 mice following intravenous administration of PCSK9 siRNA-loaded formulations. As shown in FIG. 5A, R3H7C- DOPE LNPs achieved a 82.7 ⁇ 15.1 % reduction in hepatic PCSK9 mRNA levels 7 days post-injection, significantly outperforming SM-102 LNPs (46.2%; P ⁇ 0.0001) and untreated controls (no suppression). R4H6C-DOPE and R5H5C-DOPE LNPs showed intermediate efficacy, suppressing PCSK9 expression by 79.1 ⁇ 17.7 % and 29.7 ⁇ 29.5 %, respectively (P ⁇ 0.0001 vs. SM-102), which correlated with their reduced in vitro performance and biodistribution profiles.
  • R3H7C-DOPE LNPs Long-term gene silencing was assessed 28 days post-administration using R3H7C-DOPE LNPs, selected for their superior short-term efficacy (FIG. 5B).
  • R3H7C-DOPE maintained a 85.9 ⁇ 7.3 % reduction in PCSK9 mRNA levels. This sustained silencing aligns with the biodegradability and rapid metabolic clearance of RmHn-DOPE LNPs, which minimize hepatic accumulation and associated toxicity.
  • SM-102’s non-degradable hydrocarbon tails likely contribute to prolonged tissue retention and reduced long-term efficacy, as reported in prior studies.
  • R3H7C-DOPE LNPs The enhanced performance of R3H7C-DOPE LNPs underscores the advantages of their peptide-lipid hybrid design.
  • the arginine-rich domain facilitates efficient siRNA complexation and hepatocyte targeting, while histidine-mediated pH buffering ensures endosomal escape and cytosolic payload release.
  • RmHn-DOPE LNPs represent a new approach in nucleic acid delivery, addressing critical challenges in balancing transfection efficiency, biodegradability, and organspecific targeting.
  • these LNPs achieve >83% PCSK9 mRNA silencing in hepatocytes with sustained efficacy (85.9% suppression at 28 days post-injection), outperforming synthetic SM-102 LNPs (46.2% silencing at 7 days) while maintaining >99% cell viability 16.
  • RmHn-DOPE LNPs are further validated by cryo-TEM, which revealed tightly condensed nucleic acid cores and uniform lipid bilayers, and mechanistic studies confirming histidine’s proton sponge effect in endosomal escape 36. These properties contrast sharply with SM- 102’s reliance on non-degradable hydrocarbon tails and tertiary amines, which contribute to nonspecific biodistribution and long-term toxicity risks.
  • Biocompatibility assessments in murine models revealed no hepatotoxicity or renal impairment, with blood biomarkers (ALT, AST, BUN) and histopathology confirming safety — a critical advantage over Lipofectamine 2000 (73.8% viability) and other synthetic systems.
  • RmHn-DOPE LNPs hold promise for treating hypercholesterolemia via sustained PCSK9 suppression and pulmonary disorders requiring extrahepatic delivery, bridging the gap between biodegradable nanocarriers and precision medicine.

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Abstract

In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to lipid nanoparticles for use as drug delivery agents. The lipid nanoparticles are composed of lipopeptide conjugates, where a peptide is bonded to a lipid. The lipid nanoparticles described herein represent a new approach in drug delivery, addressing critical challenges in balancing biodegradability, biocompatibility, and organspecific targeting. The lipid nanoparticles can selectively target and deliver bioactive agents to specific tissues in a subject by modifying specific amino acids and ratios thereof in the peptide of the lipopeptide conjugate.

Description

LIPID NANOPARTICLES FOR DRUG DELIVERY AND METHODS FOR MAKING AND USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S. provisional application nos. 63/631 ,609 and 63/631 ,620, both filed on April 9, 2024, the contents of which are incorporated by reference herein in their entireties.
BACKGROUND
[0002] Lipid nanoparticles (LNPs) have revolutionized nucleic acid therapeutics, exemplified by their pivotal role in mRNA vaccine during the COVID-19 pandemic. While current LNP systems excel in nucleic acid encapsulation and delivery, their efficacy varies significantly when applied to distinct nucleic acids, such as small interfering RNA (siRNA), messenger RNA (mRNA) or plasmid DNA (pDNA). Ionizable lipids serve as the cornerstone of LNP technology, enabling pH-responsive nucleic acid delivery through dynamic charge modulation during cellular uptake and endosomal escape. However, conventional ionizable lipids, such as SM-102 and DLin-MC3-DMA, are synthetic compounds with non-natural, hydrocarbon tails and tertiary amines, which lack a clear degradation pathway and raise concerns about long-term toxicity. These limitations are exacerbated when delivering structurally diverse nucleic acids (e.g., siRNA, ASOs, mRNA, pDNA), as their distinct charge densities and molecular weights demand adaptable lipid architectures for stable encapsulation and intracellular release. For instance, siRNA’s rigid duplex structure and pDNA’s large-supercoiled conformation require tailored lipid geometries to prevent aggregation or premature payload leakage, a challenge unmet by mRNA-optimized compositions.
[0003] A critical barrier in ionizable lipid development lies in the synthesis of complex lipid architectures that balance high transfection efficiency with low immunogenicity. Conventional approaches rely on combinatorial chemistry in early time and more recently Al-guided design, which struggle to generate large, diverse ionizable lipid libraries at a low cost while maintaining synthetic feasibility. For example, asymmetric or biodegradable lipid designs often demand multi-step organic synthesis, limiting scalability and structural diversity. Even advanced strategies, such as directed chemical evolution, face trade-offs between transfection efficiency and biocompatibility. Lipids optimized for nucleic acid delivery (e.g., C12-200) often incorporate non-degradable tertiary amines or rigid hydrocarbon tails, which enhance transfection but trigger immune activation through interactions with receptors like TLR4 and CD1d . This persistent cationic character can induce pro- inflammatory cytokines and type I interferons, compromising safety.
SUMMARY [0004] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to lipid nanoparticles for use as drug delivery agents. The lipid nanoparticles are composed of lipopeptide conjugates, where a peptide is bonded to a lipid. The lipid nanoparticles described herein represent a new approach in drug delivery, addressing critical challenges in balancing biodegradability, biocompatibility, and organ-specific targeting. The lipid nanoparticles can selectively target and deliver bioactive agents to specific tissues in a subject by modifying specific amino acids and ratios thereof in the peptide of the lipopeptide conjugate.
[0005] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.
[0007] FIGS. 1A-1 C show the design of RmHnC-DOPE lipopeptide library and four-component RmHnC-DOPE LNPs. (A) Synthesis of lipopeptides through the thiol-ene reaction between DOPE- Mal and cysteine residue at the C-terminal of RmHnC peptides (RmHnC, where m represents the number of the arginine residue, n indicates the number of the histidine residue, with n=1-5 and m=1- 9). (B) Representative structures of two helper lipids, p-sitosterol, and DMPE PEG-2000. (C) Formulation showing the combination of these four components with nucleic acids to prepare multiple RmHn-DOPE LNPs for screening.
[0008] FIGS. 2A-C show the characterization of 13 RmHn-DOPE LNPs and SM102 LNP control loaded with PCSK9 siRNA: particle size, polydispersity index (PDI), zeta potential (measured via Malvern dynamic light scattering), and particle size stability during long-term storage at 4°C. FIG. 2D shows the relative PCSK9 mRNA expression levels in HEPA1-6 cells treated with RH-DOPE LNPs or SM102 control loaded with PCSK9 siRNA (24-well plate, 24-hour incubation, 50 pmol per well, n > 3 replicates). Data are normalized to GAPDH (housekeeping gene) and presented as mean ± SD. FIG. 2E shows the particle size and PDI of R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 control loaded with eGFP pDNA (measured via Malvern DLS). These lipopeptides were selected based on superior siRNA transfection efficiency in vitro. FIG. 2F shows the pKa determination of R3H7C-DOPE, R4H6C-DOPE, and R5H5C-DOPE via pH titration. FIG. 2G shows the cryo-TEM images of R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 control (arranged left to right, top to bottom, scale bar = 50 nm). FIG. 2H shows the inverted fluorescence microscopy images and flow cytometry analysis of eGFP expression in HepG2 cells treated with R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, SM102, or Lipofectamine 2000 (loaded with eGFP pDNA, scale bar = 1 mm ). FIG. 2I shows the quantitative analysis of flow cytometry data: percentage of eGFP-positive cells and mean fluorescence intensity (MFI). FIG. 2J shows the expression of firefly luciferase in HepG2 cells treated with flue pDNA loaded R3H7C-DOPE, R4H6C-DOPE, R5H5C- DOPE, S 102, or Lipofectamine 2000 LNP. Data are shown as mean ± SD.
[0009] FIGS. 3A-3C show the in vitro transfection mechanism for RmHn-DOPE LNPs. (A) Cellular uptake of Cy5-labeled siRNA in HepG2 cells. Confocal microscopy images of HepG2 cells transfected for 4 h with Cy5-labeled Negative control siRNA (20 pmol/well) encapsulated in R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, or SM-102. The blank control (untreated cells) shows no Cy5 fluorescence. Red fluorescence corresponds to internalized Cy5-siRNA. Scale bar: 10 pm. (B) HepG2 cells pre-treated with inhibitors (4°C, chlorpromazine [CPZ], methyl-p-cyclodextrin [MpCD], or 5-(N- ethyl-N-isopropyl)amiloride [EIPA]) were transfected with R3H7C-DOPE, R4H6C-DOPE, R5H5C- DOPE, or SM102 LNPs loaded with Cy5-labeled negative control (NC) siRNA. Cellular uptake was analyzed by flow cytometry after 4 h. (C) Relative PCSK9 mRNA expression levels in HEPA1-6 cells pre-incubated with bafilomycin A1 (1 h) and treated with R3H7C-DOPE, R4H6C-DOPE, R5H5C- DOPE, or SM102 LNP loaded with PCSK9 siRNA. Data are normalized to GAPDH and presented as mean ± SD (n > 3).
[0010] FIGS. 4A-B show the biodistribution of RmHn-DOPE LNPs in mice. (A) R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 LNPs loaded with Cy5-labeled negative control siRNA were intravenously administered to C57BL/6 mice via the tail vein. Organs were harvested 2 h postinjection and imaged using a PE Spectrum small animal in vivo imaging system to evaluate biodistribution. (B) flue pDNA-loaded R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 LNPs were intravenously injected into C57BL/6 mice. Substrate was administered 24 h post-injection, and bioluminescence signals were captured using the PE Spectrum imaging system.
[0011] FIGS. 5A-5C shows sustained PCSK9 silencing and LDL receptor upregulation mediated by PCSK9 siRNA RmHn-DOPE LNPs in C57BL/6 mice. (A) Relative PCSK9 mRNA levels in mouse livers 7 days post-IV administration of PCSK9 siRNA-loaded R3H7C-DOPE, R4H6C-DOPE, R5H5C- DOPE, or SM102 LNPs. Data normalized to GAPDH (mean ± SD, n > 4). (B) Longitudinal PCSK9 mRNA suppression 28 days post-injection of PCSK9 siRNA R3H7C-DOPE LNPs (selected for superior 7-day efficacy in panel a) compared to SM102 LNPs. Data normalized to GAPDH (mean ± SD, n > 4). (C) Immunofluorescence staining of LDL receptor (green) in liver sections from PCSK9 siRNA R3H7C-DOPE-treated (28 days, left image) and untreated mice (right image), demonstrating sustained receptor upregulation due to PCSK9 silencing. Nuclei counterstained with DAPI (blue), Scale bar: 50pm.
[0012] FIGS. 6I-6J show the biocompatibility assessment of lipopeptide LNPs in mice over a 28-day period following tail vein administration. (A) TBL (Total bilirubin, testing with a blood biochemical analyzer). (B) ALB (Albumin, testing with a blood biochemical analyzer). (C) TP (Total protein, testing with a blood biochemical analyzer). (D) OR (Serum creatinine, testing with a blood biochemical analyzer). (E) BUN (Blood urea nitrogen, testing with a blood biochemical analyzer). (F) ALT (Alanine aminotransferase, testing with a blood biochemical analyzer). (G) AST (Aspartate aminotransferase, testing with a blood biochemical analyzer). (H) UA (Uric acid, testing with a blood biochemical analyzer). (I) Body weight changes in this batch of mice over 28 days. (J) HE staining of major organs from mice 28 days after tail vein injection of lipopeptide LNP, Scale bar: 100pm.
[0013] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
DETAILED DESCRIPTION
[0014] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.
[0015] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
[0016] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.
[0017] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
[0018] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.
[0019] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.
[0020] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.
[0021] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.
Definitions
[0022] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.
[0023] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an excipient” include, but are not limited to, mixtures or combinations of two or more such excipients, and the like.
[0024] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
[0025] When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
[0026] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible subranges) within the indicated range. Thus, for example, if a component is in an amount of about 1 %, 2%, 3%, 4%, or 5%, where any value can be a lower and upper endpoint of a range, then any range is contemplated between 1% and 5% (e.g., 1% to 3%, 2% to 4%, etc.).
[0027] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
[0028] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more - OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH2)sCO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.
[0029] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (/.e., further substituted or unsubstituted).
[0030] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, f-butyl, n- pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.
[0031] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. In one aspect, the alkenyl group is a vinyl group or an allyl group. In another aspect, the alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.
[0032] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.
[0033] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.
[0034] The term “aldehyde” as used herein is represented by the formula -C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C=O.
[0035] The terms “amine” or “amino” as used herein are represented by the formula — NA1 A2, where A1 and A2 can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is — NH2.
[0036] The term “alkylamino” as used herein is represented by the formula — NH(-alkyl) and — N(- alkyl)2, where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.
[0037] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.
[0038] The term “acetyloxy” as used herein is represented by the formula — OC(O)CH3.
[0039] The term “amide” or “amido” as used herein is represented by the formula — NHC(O)R, where can be hydrogen, an alkyl group, an aryl group, a heteroaryl group, or a cycloalkyl group as defined herein.
[0040] The term “acetyl” as used herein is represented by the formula — C(O)CH3.
[0041] The term “acetyl amino” as used herein is represented by the formula — NHC(O)CH3.
[0042] The term “ester” as used herein is represented by the formula — OC(O)A1 or — C(O)OA1, where A1 can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0043] The term “ether” as used herein is represented by the formula A1OA2, where A1 and A2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein.
[0044] The terms “halo,” “halogen” or “halide,” as used herein can be used interchangeably and refer to F, Cl, Br, or I.
[0045] The term “hydroxyl” or “hydroxy” as used herein is represented by the formula — OH.
[0046] The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1 and A2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0047] The term “azide” or “azido” as used herein is represented by the formula — N3.
[0048] The term “nitro” as used herein is represented by the formula — NO2.
[0049] The term “nitrile” or “cyano” as used herein is represented by the formula — CN.
[0050] The term “silyl” as used herein is represented by the formula — SiA1A2A3, where A1, A2, and A3 can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0051] The term “sulfo-oxo” as used herein is represented by the formulas — S(O)A1 , — S(O)2A1, — OS(O)2A1 , or — OS(O)2OA1, where A1 can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S=O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2A1, where A1 can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfone” as used herein is represented by the formula A1S(O)2A2, where A1 and A2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1 and A2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0052] The term “thiol” as used herein is represented by the formula -SH.
[0053] “R1,” “R2,” “R3,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1 is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (/.e. , attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.
[0054] The term “random copolymeric peptide” when referencing a structure of a peptide is defined as peptide having the amino acids as provided in the structure in any random order. As an example, with the peptide NH2-CWR3H3-COOH, the cysteine, arginine, and histidine groups are present in any order and not the specific order as provided in the structure.
[0055] The term “sequence-specific peptide” when referencing a structure of a peptide is defined as peptide having the amino acids as provided in the structure in the order as provided in the structure. As an example, with the peptide NH2-CHmR2-COOH, the cysteine, arginine, and histidine groups are present in the order as provided in the structure.
[0056] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (/.e., further substituted or unsubstituted). [0057] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis/trans (E/Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.
[0058] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.
[0059] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and I or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or I meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-lngold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.
[0060] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically-labeled or isotopically- substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 170, 35S, 18F, and 36CI, respectively. Compounds further comprise prodrugs thereof and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, /.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can 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 the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.
[0061] The compounds described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates. Unless stated to the contrary, the invention includes all such possible solvates.
[0062] It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form. keto form enol form amide form imidic acid form
Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, the invention includes all such possible tautomers.
[0063] It is known that chemical substances form solids which are present in different states of order which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.
[0064] As used herein, “administering” can refer to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g. by diffusion) a composition the perivascular space and adventitia. For example a medical device such as a stent can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells. The term “parenteral” can include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
[0065] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.
[0066] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
[0067] The term “pharmaceutically acceptable salts”, as used herein, means salts of the active principal agents which are prepared with acids or bases that are tolerated by a biological system or tolerated by a subject or tolerated by a biological system and tolerated by a subject when administered in a therapeutically effective amount. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to; sodium, potassium, calcium, ammonium, organic amino, magnesium salt, lithium salt, strontium salt or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to; those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like.
[0068] The term “pharmaceutically acceptable prodrug” or “prodrug” represents those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. Prodrugs of the present disclosure can be rapidly transformed in vivo to a parent compound having a structure of a disclosed compound, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).
[0069] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.
[0070] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
[0071] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including, but not limited to matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.
[0072] Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A- F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.
[0073] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.
[0074] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
[0075] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).
Lipid Nanoparticles and Methods of Making and Using the Same
[0076] Described herein are lipid nanoparticles for use as drug delivery agents. The lipid nanoparticles are composed of lipopeptide conjugates, where a peptide is bonded to a lipid. In one aspect, the lipopeptide conjugate has the formula X-L-Y, wherein X is a peptide, L is a linker, and Y is a lipid. Each component of the lipopeptide conjugate and methods for making the same are described below.
[0077] Lipopeptide Conjugates
[0078] The lipid nanoparticles described herein represent a new approach in drug delivery, addressing critical challenges in balancing biodegradability, biocompatibility, and organ-specific targeting. The lipid nanoparticles can selectively target and deliver bioactive agents to specific tissues in a subject by modifying specific amino acids and ratios thereof in the peptide of the lipopeptide conjugate.
[0079] In one aspect, the peptide of the lipopeptide conjugate includes a plurality of arginine and histidine groups. In one aspect, the peptide includes from about 1 to about 10,000 arginine residues, from about 1 to about 10,000 histidine residues and at least one cysteine residue. In another aspect, the peptide includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, 500, 1 ,000, 1 ,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,500, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500 or about 10,000 arginine residues, where any value can be a lower and upper endpoint of a range (e.g., 50 to 500). In another aspect, the peptide includes 1 , 50, 100, 500, 1 ,000, 1 ,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,500, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500 or about 10,000 histidine residues, where any value can be a lower and upper endpoint of a range (e.g., 50 to 500).
[0080] In one aspect, by modifying the arginine-to-histidine (R/H) ratio in the peptide, the properties of the lipid nanoparticle can be fined tuned for the delivery of bioactive agents to specific tissues and organs in a subject. In one aspect, the arginine-to-histidine (R/H) ratio is from 4:1 to 1 :4. In another aspect, arginine-to-histidine (R/H) ratio is 4:1 , 3.5:1 , 3.0:1 , 2.5:1 , 2:1 , 1.5:1 , 1 :1 , 1 :1.5, 1 :2, 1 :2.5, 1 :3, 1 :3.5, or 1 :4, where any value can be a lower and upper endpoint of a range (e.g., 2:1 to 1 :1.5). In one aspect, when it is desirable to deliver a bioactive agent to the lungs of a subject, the arginine-to- histidine (R/H) ratio is from 2:1 to 1 :2, 1.5:1 to 1 :1.5, or about 1 :1. In another aspect, when it is desirable to deliver a bioactive agent to the liver of a subject, the arginine-to-histidine (R/H) ratio is from 1 :1 to 1 :3, 1 :1 to 1 :2.5, or about 3:7. In another aspect, when it is desirable to deliver a bioactive agent to the spleen of a subject, the arginine-to-histidine (R/H) ratio is from 1 :1 to 1 :2, 1 :1 to 1 :1.75, or 1 :1 to 1 :1.5.
[0081] In one aspect, the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH2-CWR3H3-COOH, NH2-CWR5H5-COOH, NH2-CWR3H3-COOH, NH2-CWR3H4- COOH, NH2-CWR3H5-COOH, NH2-CWR3H6-COOH, NH2-CWR3H7-COOH, NH2-CWR3H8-COOH, NH2- CWR3H9-COOH, NH2-CWK5H5-COOH, NH2-CWK3H3-COOH , NH2-CwRm-COOH, NH2-CwKn-COOH, NH2-CwR0Hp-COOH, NH2-CwRuKv-COOH, NH2-CwKpHq-COOH, and NH2-CwRxKyHz-COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and m, n, 0, p, q, u, v, x, y, z, and w are integers from 1 to 1 ,000.
[0082] In one aspect, the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH2-R3H3-COOH, NH2-R5H5-COOH, NH2-R3H3-COOH, NH2-R3H4-COOH, NH2-R3H5-COOH, NH2-R3H6-COOH, NH2-R3H7-COOH, NH2-R3H8-COOH, NH2-R3H9-COOH, NH2- K5H5-COOH, NH2-K3H3-COOH, NH2-R0HP-COOH, NH2-RUKV-COOH, NH2-KpHq-COOH, and NH2- RxKyHz-COOH; wherein R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and m, n, 0, p, q, u, v, x, y, and z are integers from 1 to 1 ,000.
[0083] In one aspect, the peptide has a formula selected from the group consisting of NH2-Rm-COOH, NH2-Kn-COOH, NH2-H0-COOH; wherein R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m, n, and 0 are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
[0084] In one aspect, the peptide is a sequence-specific peptide having a formula selected from the group consisting of NH2-CR-COOH, NH2-CR2-COOH, NH2-CR3-COOH, NH2-CR4-COOH, NH2-CR5- COOH, NH2-CR6-COOH, NH2-CRm-COOH, NH2-CHR-COOH, NH2-CH2R-COOH, NH2-CH3R-COOH, NH2-CH4R-COOH, NH2-CH5R-COOH NH2-CHmR2-COOH, NH2-CHR2-COOH, NH2-CH2R2-COOH, NH2-CH3R2-COOH, NH2-CH4R2-COOH, NH2-CH5R2-COOH, NH2-CHmR2-COOH, NH2-CHR3-COOH, NH2-CH2R3-COOH, NH2-CH3R3-COOH, NH2-CH4R3-COOH, NH2-CH5R3-COOH, NH2-CH6R3-COOH, NH2-CH7R3-COOH, NH2-CH8R3-COOH, NH2-CH9R3-COOH, NH2-CHmR3-COOH, NH2-CHR4-COOH, NH2-CH2R4-COOH, NH2-CH3R4-COOH, NH2-CH4R4-COOH, NH2-CH5R4-COOH, NH2-CH6R4-COOH, NH2-CH7R4-COOH, NH2-CH8R4-COOH, NH2-CH9R4-COOH, NH2-CHmR4-COOH, NH2-CHR5-COOH, NH2-CH2R5-COOH, NH2-CH3R5-COOH, NH2-CH4R5-COOH, NH2-CH5R5-COOH, NH2-CH6R5-COOH, NH2-CH7R5-COOH, NH2-CH8R5-COOH, NH2-CH9R5-COOH, NH2-CHmR5-COOH, and NH2-CHmRn- COOH, wherein C is cysteine, R is arginine, H is histidine, NH2- is the N-terminal of the peptide, - COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C- terminal are either modified or unmodified with functional groups.
[0085] In one aspect, the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2-CK-COOH, NH2-CK2-COOH, NH2-CK3-COOH, NH2-CK4- COOH, NH2-CK5-COOH, NH2-CK6-COOH, NH2-CKm-COOH, NH2-CHK-COOH, NH2-CH2K-COOH, NH2-CH3K-COOH, NH2-CH4K-COOH, NH2-CH5K-COOH NH2-CHmK2-COOH, NH2-CHK2-COOH, NH2-CH2K2-COOH, NH2-CH3K2-COOH, NH2-CH4K2-COOH, NH2-CH5K2-COOH, NH2-CHmK2-COOH, NH2-CHK3-COOH, NH2-CH2K3-COOH, NH2-CH3K3-COOH, NH2-CH4K3-COOH, NH2-CH5K3-COOH, NH2-CH6K3-COOH, NH2-CH7K3-COOH, NH2-CH8K3-COOH, NH2-CH9K3-COOH, NH2-CHmK3-COOH, NH2-CHK4-COOH, NH2-CH2K4-COOH, NH2-CH3K4-COOH, NH2-CH4K4-COOH, NH2-CH5K4-COOH, NH2-CH6K4-COOH, NH2-CH7K4-COOH, NH2-CH8K4-COOH, NH2-CH9K4-COOH, NH2-CHmK4-COOH, NH2-CHK5-COOH, NH2-CH2K5-COOH, NH2-CH3K5-COOH, NH2-CH4K5-COOH, NH2-CH5K5-COOH, NH2-CH6K5-COOH, NH2-CH7K5-COOH, NH2-CH8K5-COOH, NH2-CH9K5-COOH, NH2-CHmK5-COOH, and NH2-CHmKn-COOH, wherein C is cysteine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
[0086] In one aspect, the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2-RC-COOH, NH2-R2C-COOH, NH2-R3C-COOH, NH2-R4C- COOH, NH2-R5C-COOH, NH2-R6C-COOH, NH2-RmC-COOH, NH2-RHC-COOH, NH2-RH2C-COOH, NH2-RH3C-COOH, NH2-RH4C-COOH, NH2-RH5C-COOH, NH2-RHmC-COOH, NH2-R2HC-COOH, NH2-R2H2C-COOH, NH2-R2H3C-COOH, NH2-R2H4C-COOH, NH2-R2H5C-COOH, NH2-R2HmC-COOH, NH2-R3HC-COOH, NH2-R3H2C-COOH, NH2-R3H3C-COOH, NH2-R3H4C-COOH, NH2-R3H5C-COOH, NH2-R3H6C-COOH, NH2-R3H7C-COOH, NH2-R3H8C-COOH, NH2-R3H9C-COOH, NH2-R3HmC-COOH, NH2-R4H-COOH, NH2-R4H2C-COOH, NH2-R4H3C-COOH, NH2-R4H4C-COOH, NH2-R4H5C-COOH, NH2-R4 HeC-COOH, NH2-R4H7C-COOH, NH2-R4H8C-COOH, NH2-R4H9C-COOH, NH2-R4HmC- COOH, NH2-R5HCOOH, NH2-R5H2C-COOH, NH2-R5H3C-COOH, NH2-R5 H4C-COOH, NH2-R5H5C- COOH, NH2-R5H6C-COOH, NH2-R5H7C-COOH, NH2-R5 HBC-COOH, NH2-R5H9C-COOH, NH2- R5HmC-COOH, and NH2-RnHmC-COOH, wherein C is cysteine, R is arginine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
[0087] In one aspect, the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2-KC-COOH, NH2-K2C-COOH, NH2-K3C-COOH, NH2-K4C- COOH, NH2-K5C-COOH, NH2-K6C-COOH, NH2-KmC-COOH, NH2-KHC-COOH, NH2-KH2C-COOH, NH2-KH3C-COOH, NH2-KH4C-COOH, NH2-KH5C-COOH, NH2-KHmC-COOH, NH2-K2HC-COOH, NH2-K2H2C-COOH, NH2-K2H3C-COOH, NH2-K2H4C-COOH, NH2-K2H5C-COOH, NH2-K2HmC-COOH, NH2-K3HC-COOH, NH2-K3H2C-COOH, NH2-K3H3C-COOH, NH2-K3H4C-COOH, NH2-K3H5C-COOH, NH2-K3H6C-COOH, NH2-K3H7C-COOH, NH2-K3H8C-COOH, NH2-K3H9C-COOH, NH2-K3HmC-COOH, NH2-K4H-COOH, NH2-K4H2C-COOH, NH2-K4H3C-COOH, NH2-K4H4C-COOH, NH2-K4H5C-COOH, NH2-K4H6C-COOH, NH2-K4H7C-COOH, NH2-K4H8C-COOH, NH2-K4H9C-COOH, NH2-K4HmC-COOH, NH2-K5HCOOH, NH2-K5H2C-COOH, NH2-K5H3C-COOH, NH2-K5H4C-COOH, NH2-K5H5C-COOH, NH2-K5HSC-COOH, NH2-K5H7C-COOH, NH2-K5H8C-COOH, NH2-K5H9C-COOH, NH2-K5HmC-COOH, and NH2-KnHmC-COOH, wherein C is cysteine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N- terminal and C-terminal are either modified or unmodified with functional groups.
[0088] In one aspect, the peptide has a formula selected from the group consisting of NH2-CRn- COOH, NH2-CKn-COOH, NH2-CHn-COOH, NH2-RnC-COOH, NH2-KnC-COOH, NH2-HnC-COOH, NH2-R0HP-COOH, H2-HPR0-COOH, NH2-CR0HP-COOH, NH2-CHPR0-COOH, NH2-RoCHp-COOH, NH2- HpCRo-COOH, NH2-ROHPC-COOH, NH2-HpRoC-COOH, NH2-CWR0HP-COOH, NH2-CWHPR0-COOH, NH2-R0CwHp-COOH, NH2-HPCWR0-COOH, NH2-RoHpCw-COOH, NH2-HPR0CW-COOH, wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, m and n are integers from 0 to 1 ,000, n, o, p, and w are integers from 0 to 1 ,000 but cannot be zero at the same time.
[0089] In one aspect, the peptide is a random or block copolymeric peptide having a formula selected from the group consisting of NH2-X1xX2yX3zX4w-COOH, wherein X1 , X2, X3, and X4 is cysteine, arginine, lysine or histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
[0090] In one aspect, the peptide has a sequence selected from the group consisting of NH2- CRRRRR-COOH, NH2-CRRRHHH-COOH, NH2-CHHHRRR-COOH, NH2-CKKKKK-COOH, NH2- CKKK-COOH, NH2-CHHHHHRRRR-COOH, NH2-CRRRRRHHHHH-COOH, NH2-CRRRHHH- COOH, NH2-CHHHRRRCOOH, NH2-CKKKKKHHHHH-COOH, NH2-CHHHHHKKKKK-COOH, NH2- CHHHKKK-COOH, NH2-CKKKHHH-COOH, NH2-RRRRRC-COOH, NH2-RRRHHHC-COOH, NH2- KKKKKC-COOH, NH2-KKKC-COOH, NH2-RRRRRHHHHHC-COOH, NH2-RRRHHHC-COOH, NH2- KKKKKHHHHHC-COOH, NH2-KKKHHHC-COOH, NH2-RRRHHHHHHHHC-COOH, NH2-
RRRHHHHHHHC-COOH, NH2-RRRHHHHHHC-COOH, NH2-RRRHHHHHC-COOH, NH2- RRRRHHHHHHC-COOH, NH2-RRRRHHHHHHHC-COOH, NH2-RRRRHHHHHHHHC-COOH, wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, - COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups.
[0091] In one aspect, the peptide is a block copolymer having a sequence selected from the group consisting of NH2-CRm-COOH, NH2-CKn-COOH, NH2-CRoHp-COOH, NH2-CRxKyHz-COOH, NH2- RmC-COOH, NH2-KnC-COOH, NH2-RoHpC-COOH, NH2- RxKyHzC-COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, and m, n, o, p, q, x, y, and z are integers from 1 to 1 ,000, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
[0092] In one aspect, the peptide is a random copolymeric peptide having a formula NH2-RmHnC- COOH, wherein C is cysteine, R is arginine, H is histidine, COOH is the N-terminal of the peptide, and m and n are integers from 1 to 15.
[0093] The lipopeptide conjugate includes a linker that covalently bonds the peptide to the lipid. In one aspect, the linker is a residue of a group selected from the group consisting of amine, carboxylate, hydroxyl, thiol, alkene, alkyne, azide, disulfide, maleimide, ester, peptide, acetal, anhydride, halide, vinyl sulfone, methyl acrylate, acrylate, acrylamide, methyl acrylamide, fluoride, chloride, bromide, aldehyde, and ketone. In one aspect, during the synthesis of the lipopeptide conjugate, the linker can be present on the peptide or the lipid. In one aspect, the linker is a maleimide bonded to the lipid.
[0094] The lipid used to produce the lipopeptide conjugate can be selected from a variety of materials used to produce lipid nanoparticles. In one aspect, the lipid is selected from the group consisting of a phosphatidylcholine, a lysophosphatidylcholine, a plasmenylphosphatidylcholine, a phosphatidylethanolamin, a lysophosphatidylethanolamine, a plasmenylphosphatidylethanolamine, a phosphatidylserine, a sphingomyeline, a phosphatidic acid, a lysophosphatidic acid, a phosphatidylinositol, a phosphatidylglycerol, a cardiolipin, a ceramide-1-phosphate, an N-acylsphingosine, a sulfatide, a ganglioside, a sulfoquinovosyldiacylglycero, and a diphosphorylated hexaacyl Lipid A.
[0095] In one aspect, the lipid is a phospholipid. In one aspect, the lipid is a phosphatidylcholine, a phosphatidic acid, a phosphatidylglycerol, a phosphatidylethanolamine, a phosphatidylserine, or a PEG phospholipid. In another aspect, the lipid is 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or any combination thereof. In another aspect, the lipid is one more compounds provided in the table below.
1 ,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1 -
DMPG-NA Phosphatidylglycerol glycerol) (sodium salt)
[0096] In one aspect, the linker is a residue of a maleimide and the lipid is phosphatidylethanolamine.
In one aspect, the lipopeptide conjugate has the structure I wherein Ri and R2 are independently a C8 to C25 alkyl group or a C8 to C25 alkenyl group, o and p are independently integers from 1 to 4, and
X is a peptide.
[0097] In one aspect, o and p in structure are each 2. In another aspect, o and p in structure are each 2, and Ri and R2 are independently an oleyl group or a palmitoyl group. In another aspect, o and p in structure are each 2, and Ri and R2 are both an oleyl group or a palmitoyl group. In another aspect, the lipopeptide conjugate has the structure depicted in FIG. 1 A.
[0098] In one aspect, the lipopeptide conjugate can be synthesized by reacting a peptide (X) with a lipid having a linked covalently bonded to the lipid (L-Y). In one aspect, the lipid can be a phospholipid that is covalently bonded to a linker. For example, phosphatidylethanolamine (e.g., DOPE) can be reacted with a compound possessing a linker group (e.g., a maleimide) to produce L-Y.
[0099] The selection of the linker group can vary depending upon the peptide to be bonded to L-Y. In one aspect, when the peptide has a terminal cysteine group, the linker can be selected so that it reacts with the thiol group of cysteine. In one aspect, the linker is an alkene or a compound with a degree of unsaturation so that the thiol group of cysteine reaction with the alkene via a thiol-ene coupling reaction. In one aspect, the linker possesses a maleimide group. FIG. 1A provides an exemplary reaction scheme for producing the lipopeptide conjugates. Exemplary methods for producing the lipid nanoparticles described herein, as well as characterization information, are provided in the Examples.
[0100] Lipid Nanoparticles
[0101] In one aspect, the lipid nanoparticles are produced by admixing the lipopeptide conjugates described herein with a bioactive agent in a suitable solvent. In one aspect, one or more helper lipids can be added to mixture to produce the lipid nanoparticles. In one aspect, the helper lipid includes a phospholipid (e.g., DOPE), a PEGylated lipid, a sterol (e.g., cholesterol, beta-sitosterol), an any combination thereof. The inclusion of one or more helper lipids can improve the properties of the lipid nanoparticle including, but not limited to, particle stability, delivery efficacy, tolerability and biodistribution. In one aspect, the lipid peptide conjugate to helper lipid is at a molar ratio of 1 :100 to 100:1 . In another aspect, the lipid peptide conjugate to helper lipid is at a molar ratio of 100:1 , 90:1 , 80:1 , 70:1 , 60:1 , 50:1 , 40:1 , 30:1 , 20:1 , 10:1 , 4:1 , 3.5:1 , 3.0:1 , 2.5:1 , 2:1 , 1.5:1 , 1 :1 , 1 :1.5, 1 :2, 1 :2.5, 1 :3, 1 :3.5, or 1 :4, 1 :10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 :100, where any value can be a lower and upper endpoint of a range (e.g., 2:1 to 1 : 1.5). [0102] Depending upon the application of the lipid nanoparticle, a variety of different bioactive agents can be incorporated into the lipid nanoparticle. In one aspect, the bioactive agent is selected from the group consisting of Abiraterone Acetate, Brentuximab vedotin, Trastuzumab emtansine, Afatinib, Afinitor® (Everolimus), Aldara® (Imiquimod), Alimta® (Pemetrexed Disodium), Pemetrexed, Palonosetron, Chlorambucil, Nelarabine, Axitinib, Belinostat, Bleomycin, Bortezomib, Cabozantinib- S-Malate, Camptothecin, Capecitabine, Ceritinib, Cerubidine® (Daunorubicin), Crizotinib, Dabrafenib, Dasatinib, Degarelix, Docetaxel, Doxorubicin, Epirubicin, Eribulin, Etoposide, Raloxifene, Fulvestrant, Folex® (Methotrexate), Pralatrexate, Eribulin Mesylate, Topotecan, Ibritumomab tiuxetan, Ibrutinib, Irinotecan, Ixempra® (Ixabepilone), Jevtana® (Cabazitaxel), Kadcyla® (Ado- Trastuzumab Emtansine), Lenalidomide, Leuprolide Acetate, Vincristine, Methotrexate, Mitomycin C, Mitoxantrone, Nelarabine, Paclitaxel, Prednisone, Promacta® (Eltrombopag Olamine), Raloxifene Hydrochloride, Lenalidomide, Methotrexate, Synribo® (Omacetaxine Mepesuccinate), Targretin® (Bexarotene), Temsirolimus, Treanda® (Bendamustine Hydrochloride), Velban® (Vinblastine Sulfate), Velsar® (Vinblastine Sulfate), Vincasar PFS® (Vincristine Sulfate), Vinorelbine Tartrate, Vorinostat, Capecitabine, Ipilimumab, and Goserelin Acetate.
[0103] In one aspect, the bioactive agent is selected from the group consisting of antibodies, peptides, therapeutic enzymes, cytokines, interferons, and interleukins.
[0104] In one aspect, the bioactive agent is a nucleic acid such as, for example, a plasmid DNA, an oligonucleotide, an aptamers, a DNAzyme, a RNA aptamers, a RNA Decoy, an antisense RNA, a ribozymes, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA, an antagomirs, or any combination thereof.
[0105] Depending upon the selection of the bioactive agent, the surface charge of the lipid nanoparticles can be modified by modifying the peptide. Foe example, the number of arginine groups can be modified to alter the surface charge. In one aspect, wherein the lipid nanoparticle has a surface charge of between about -100 mV and about +100 mV at pH 3 to 11 , or -100 mv, -75 mv, - 50 mv, -25 mv, 0 mv, 25 mv, 50 mv, 75 mv, or 100 mv, where any value can be a lower and upper endpoint of a range (e.g., -50 mv to 25 mv, etc.).
[0106] Depending upon the selection of the bioactive agent and application, the size of the lipid nanoparticles can be modified by modified. In one aspect, the lipid nanoparticle is between about 2 nm and about 2,000 m in diameter. In another aspect, the lipid nanoparticle about 2 nm, 10 nm, 25 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, or 200 nm in diameter, where any value can be a lower and upper endpoint of a range (75 nm to 125 nm, etc.). In another aspect, the lipid nanoparticles possess monodisperse hydrodynamic diameters with narrow size distributions (e.g., polydispersity index, PDI < 0.2).
[0107] Delivery of Bioactive Agents
[0108] The lipid nanoparticles described herein represent a new approach in drug delivery, addressing critical challenges in balancing biodegradability, biocompatibility, and organ-specific targeting. The lipid nanoparticles can selectively target and deliver bioactive agents to specific tissues in a subject by modifying specific amino acids and ratios thereof in the peptide of the lipopeptide conjugate.
[0109] In one aspect, by modifying the arginine-to-histidine (R/H) ratio in the peptide, the properties of the lipid nanoparticle can be fined tuned for the delivery of bioactive agents to specific tissues and organs in a subject. As demonstrated in the Examples, modifying the arginine-to-histidine (R/H) ratio of the peptide in the lipopeptide conjugate can affect the ability of the lipid nanoparticles to deliver the bioactive agent to specific organs.
[0110] In one aspect, when the bioactive agent is a nucleic acid, the lipid nanoparticles described herein can enhance transfection efficiency. Not wishing to be bound by theory, the lipid nanoparticles described herein possess a dual-functional architecture. The cationic arginine residues in lipid nanoparticles promote robust electrostatic interactions with the nucleic acid, enabling the formation of stable nanocomplexes that protect genetic payloads from nuclease degradation. Concurrently, histidine residues contribute pH-buffering capacity, which facilitates endosomal escape through the proton sponge effect.
[0111] As demonstrated in the Examples, the lipid nanoparticles described herein demonstrate significant advantages over commercial transfection reagents. Their biocompatible design minimizes cytotoxicity, maintaining over 98% cell viability compared to Lipofectamine 2000, which causes substantial toxicity (e.g., 73.8 % viability in HepG2 cell) due to its high charge density and structure. Furthermore, lipid nanoparticles described herein exhibit exceptional colloidal stability, remaining intact for over 30 days. The unique attributes of the lipid nanoparticles described herein provide a versatile platform capable of delivering diverse bioactive agents (e.g., nucleic acid) payloads with viral-like efficiency while retaining the safety profile of biodegradable lipids.
[0112] The lipid nanoparticles described herein can be administered to a subject as a pharmaceutical formulation by a variety of techniques as described further below.
[0113] Pharmaceutical Compositions
[0114] In various aspects, the present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one disclosed lipid nanoparticle, at least one product of a disclosed method, or a pharmaceutically acceptable salt thereof. As used herein, “pharmaceutically- acceptable carriers” means one or more of a pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, and adjuvants. The disclosed pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy and pharmaceutical sciences.
[0115] In a further aspect, the disclosed pharmaceutical compositions comprise a therapeutically effective amount of at least one disclosed compound, at least one product of a disclosed method, or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutically acceptable carrier, optionally one or more other therapeutic agent, and optionally one or more adjuvant. The disclosed pharmaceutical compositions include those suitable for oral, rectal, topical, pulmonary, nasal, and parenteral administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. In a further aspect, the disclosed pharmaceutical composition can be formulated to allow administration orally, nasally, via inhalation, parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially and intratumorally.
[0116] As used herein, “parenteral administration” includes administration by bolus injection or infusion, as well as administration by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
[0117] In various aspects, the present disclosure also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, a therapeutically effective amount of a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof. In a further aspect, a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof, or any subgroup or combination thereof may be formulated into various pharmaceutical forms for administration purposes.
[0118] In practice, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, of the present disclosure can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present disclosure can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the present disclosure, and/or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and/or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.
[0119] It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. That is, a “unit dosage form” is taken to mean a single dose wherein all active and inactive ingredients are combined in a suitable system, such that the patient or person administering the drug to the patient can open a single container or package with the entire dose contained therein, and does not have to mix any components together from two or more containers or packages. Typical examples of unit dosage forms are tablets (including scored or coated tablets), capsules or pills for oral administration; single dose vials for injectable solutions or suspension; suppositories for rectal administration; powder packets; wafers; and segregated multiples thereof. This list of unit dosage forms is not intended to be limiting in any way, but merely to represent typical examples of unit dosage forms.
[0120] The pharmaceutical compositions disclosed herein comprise a compound of the present disclosure (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents. In various aspects, the disclosed pharmaceutical compositions can include a pharmaceutically acceptable carrier and a disclosed compound, or a pharmaceutically acceptable salt thereof. In a further aspect, a disclosed compound, or pharmaceutically acceptable salt thereof, can also be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methodswell known in the art of pharmacy.
[0121] Techniques and compositions for making dosage forms useful for materials and methods described herein are described, for example, in the following references: Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.).
[0122] The compounds described herein are typically to be administered in admixture with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The deliverable compound will be in a form suitable for oral, rectal, topical, intravenous injection or parenteral administration. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. The compounds may be administered as a dosage that has a known quantity of the compound.
[0123] Because of the ease in administration, oral administration can be a preferred dosage form, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. However, other dosage forms may be suitable depending upon clinical population (e.g., age and severity of clinical condition), solubility properties of the specific disclosed compound used, and the like. Accordingly, the disclosed compounds can be used in oral dosage forms such as pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.
[0124] The disclosed pharmaceutical compositions in an oral dosage form can comprise one or more pharmaceutical excipient and/or additive. Non-limiting examples of suitable excipients and additives include gelatin, natural sugars such as raw sugar or lactose, lecithin, pectin, starches (for example corn starch or amylose), dextran, polyvinyl pyrrolidone, polyvinyl acetate, gum arabic, alginic acid, tylose, talcum, lycopodium, silica gel (for example colloidal), cellulose, cellulose derivatives (for example cellulose ethers in which the cellulose hydroxy groups are partially etherified with lower saturated aliphatic alcohols and/or lower saturated, aliphatic oxyalcohols, for example methyl oxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate), fatty acids as well as magnesium, calcium or aluminum salts of fatty acids with 12 to 22 carbon atoms, in particular saturated (for example stearates), emulsifiers, oils and fats, in particular vegetable (for example, peanut oil, castor oil, olive oil, sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflower seed oil, cod liver oil, in each case also optionally hydrated); glycerol esters and polyglycerol esters of saturated fatty acids C12H24O2 to C HseCh and their mixtures, it being possible for the glycerol hydroxy groups to be totally or also only partly esterified (for example mono-, di- and triglycerides); pharmaceutically acceptable mono- or multivalent alcohols and polyglycols such as polyethylene glycol and derivatives thereof, esters of aliphatic saturated or unsaturated fatty acids (2 to 22 carbon atoms, in particular 10-18 carbon atoms) with monovalent aliphatic alcohols (1 to 20 carbon atoms) or multivalent alcohols such as glycols, glycerol, diethylene glycol, pentacrythritol, sorbitol, mannitol and the like, which may optionally also be etherified, esters of citric acid with primary alcohols, acetic acid, urea, benzyl benzoate, dioxolanes, glyceroformals, tetrahydrofurfuryl alcohol, polyglycol ethers with C1-C12-alcohols, dimethylacetamide, lactamides, lactates, ethyl carbonates, silicones (in particular medium-viscous polydimethyl siloxanes), calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, magnesium carbonate and the like.
[0125] Other auxiliary substances useful in preparing an oral dosage form are those which cause disintegration (so-called disintegrants), such as: cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethyl cellulose or microcrystalline cellulose. Conventional coating substances may also be used to produce the oral dosage form. Those that may for example be considered are: polymerizates as well as copolymerizates of acrylic acid and/or methacrylic acid and/or their esters; copolymerizates of acrylic and methacrylic acid esters with a lower ammonium group content (for example EudragitR RS), copolymerizates of acrylic and methacrylic acid esters and trimethyl ammonium methacrylate (for example EudragitR RL); polyvinyl acetate; fats, oils, waxes, fatty alcohols; hydroxypropyl methyl cellulose phthalate or acetate succinate; cellulose acetate phthalate, starch acetate phthalate as well as polyvinyl acetate phthalate, carboxy methyl cellulose; methyl cellulose phthalate, methyl cellulose succinate, -phthalate succinate as well as methyl cellulose phthalic acid half ester; zein; ethyl cellulose as well as ethyl cellulose succinate; shellac, gluten; ethylcarboxyethyl cellulose; ethacrylate-maleic acid anhydride copolymer; maleic acid anhydride-vinyl methyl ether copolymer; styrol-maleic acid copolymerizate; 2-ethyl-hexyl-acrylate maleic acid anhydride; cratonic acid-vinyl acetate copolymer; glutaminic acid/glutamic acid ester copolymer; carboxymethylethylcellulose glycerol monooctanoate; cellulose acetate succinate; polyarginine.
[0126] Plasticizing agents that may be considered as coating substances in the disclosed oral dosage forms are: citric and tartaric acid esters (acetyl-triethyl citrate, acetyl tributyl-, tributyl-, triethyl-citrate); glycerol and glycerol esters (glycerol diacetate, -triacetate, acetylated monoglycerides, castor oil); phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-, dipropyl-phthalate), di-(2-methoxy- or 2- ethoxyethyl)-phthalate, ethylphthalyl glycolate, butylphthalylethyl glycolate and butylglycolate; alcohols (propylene glycol, polyethylene glycol of various chain lengths), adipates (diethyladipate, di- (2-methoxy- or 2-ethoxyethyl)-adipate; benzophenone; diethyl- and diburylsebacate, dibutylsuccinate, dibutyltartrate; diethylene glycol dipropionate; ethyleneglycol diacetate, -dibutyrate, -dipropionate; tributyl phosphate, tributyrin; polyethylene glycol sorbitan monooleate (polysorbates such as Polysorbar 50); sorbitan monooleate.
[0127] Moreover, suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers. The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include, but are not limited to, lactose, terra alba, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol talc, starch, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.
[0128] In various aspects, a binder can include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. In a further aspect, a disintegrator can include, for example, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
[0129] In various aspects, an oral dosage form, such as a solid dosage form, can comprise a disclosed compound that is attached to polymers as targetable drug carriers or as a prodrug. Suitable biodegradable polymers useful in achieving controlled release of a drug include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, caprolactones, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and hydrogels, preferably covalently crosslinked hydrogels.
[0130] Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
[0131] A tablet containing a disclosed compound can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
[0132] In various aspects, a solid oral dosage form, such as a tablet, can be coated with an enteric coating to prevent ready decomposition in the stomach. In various aspects, enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate. Akihiko Hasegawa “Application of solid dispersions of Nifedipine with enteric coating agent to prepare a sustained- release dosage form” Chem. Pharm. Bull. 33:1615-1619 (1985). Various enteric coating materials may be selected on the basis of testing to achieve an enteric coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crushing strength (e.g., see S. C. Porter et al. “The Properties of Enteric Tablet Coatings Made From Polyvinyl Acetate-phthalate and Cellulose acetate Phthalate”, J. Pharm. Pharmacol. 22:42p (1970)). In a further aspect, the enteric coating may comprise hydroxypropyl-methylcellulose phthalate, methacrylic acidmethacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate.
[0133] In various aspects, an oral dosage form can be a solid dispersion with a water soluble or a water insoluble carrier. Examples of water soluble or water insoluble carrier include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, hydroxypropylmethyl-cellulose, phosphatidylcholine, polyoxyethylene hydrogenated castor oil, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, or hydroxypropylmethylcellulose, ethyl cellulose, or stearic acid.
[0134] In various aspects, an oral dosage form can be in a liquid dosage form, including those that are ingested, or alternatively, administered as a mouth wash or gargle. For example, a liquid dosage form can include aqueous suspensions, which contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. In addition, oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may also contain various excipients. The pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions, which may also contain excipients such as sweetening and flavoring agents.
[0135] For the preparation of solutions or suspensions it is, for example, possible to use water, particularly sterile water, or physiologically acceptable organic solvents, such as alcohols (ethanol, propanol, isopropanol, 1 ,2-propylene glycol, polyglycols and their derivatives, fatty alcohols, partial esters of glycerol), oils (for example peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soya bean oil, castor oil, bovine hoof oil), paraffins, dimethyl sulfoxide, triglycerides and the like.
[0136] In the case of a liquid dosage form such as a drinkable solutions, the following substances may be used as stabilizers or solubilizers: lower aliphatic mono- and multivalent alcohols with 2-4 carbon atoms, such as ethanol, n-propanol, glycerol, polyethylene glycols with molecular weights between 200-600 (for example 1 to 40% aqueous solution), diethylene glycol monoethyl ether, 1 ,2- propylene glycol, organic amides, for example amides of aliphatic C1-C6-carboxylic acids with ammonia or primary, secondary or tertiary C1-C4-amines or C1-C4-hydroxy amines such as urea, urethane, acetamide, N-methyl acetamide, N,N-diethyl acetamide, N,N-dimethyl acetamide, lower aliphatic amines and diamines with 2-6 carbon atoms, such as ethylene diamine, hydroxyethyl theophylline, tromethamine (for example as 0.1 to 20% aqueous solution), aliphatic amino acids.
[0137] In preparing the disclosed liquid dosage form can comprise solubilizers and emulsifiers such as the following non-limiting examples can be used: polyvinyl pyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phosphatides such as lecithin, acacia, tragacanth, polyoxyethylated sorbitan monooleate and other ethoxylated fatty acid esters of sorbitan, polyoxyethylated fats, polyoxyethylated oleotriglycerides, linolizated oleotriglycerides, polyethylene oxide condensation products of fatty alcohols, alkylphenols or fatty acids or also 1-methyl-3-(2- hydroxyethyl)imidazolidone-(2). In this context, polyoxyethylated means that the substances in question contain polyoxyethylene chains, the degree of polymerization of which generally lies between 2 and 40 and in particular between 10 and 20. Polyoxyethylated substances of this kind may for example be obtained by reaction of hydroxyl group-containing compounds (for example mono- or diglycerides or unsaturated compounds such as those containing oleic acid radicals) with ethylene oxide (for example 40 Mol ethylene oxide per 1 Mol glyceride). Examples of oleotriglycerides are olive oil, peanut oil, castor oil, sesame oil, cottonseed oil, corn oil. See also Dr. H. P. Fiedler “Lexikon der Hillsstoffe fur Pharmazie, Kostnetik und angrenzende Gebiete” 1971 , pages 191-195.
[0138] In various aspects, a liquid dosage form can further comprise preservatives, stabilizers, buffer substances, flavor correcting agents, sweeteners, colorants, antioxidants and complex formers and the like. Complex formers which may be for example be considered are: chelate formers such as ethylene diamine retrascetic acid, nitrilotriacetic acid, diethylene triamine pentacetic acid and their salts.
[0139] It may optionally be necessary to stabilize a liquid dosage form with physiologically acceptable bases or buffers to a pH range of approximately 6 to 9. Preference may be given to as neutral or weakly basic a pH value as possible (up to pH 8).
[0140] In order to enhance the solubility and/or the stability of a disclosed compound in a disclosed liquid dosage form, a parenteral injection form, or an intravenous injectable form, it can be advantageous to employ a-, [3- or y-cyclodextrins or their derivatives, in particular hydroxyalkyl substituted cyclodextrins, e.g. 2-hydroxypropyl-p-cyclodextrin or sulfobutyl-p-cyclodextrin. Also cosolvents such as alcohols may improve the solubility and/or the stability of the compounds according to the present disclosure in pharmaceutical compositions.
[0141] In various aspects, a disclosed liquid dosage form, a parenteral injection form, or an intravenous injectable form can further comprise liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
[0142] Pharmaceutical compositions of the present disclosure suitable injection, such as parenteral administration, such as intravenous, intramuscular, or subcutaneous administration. Pharmaceutical compositions for injection can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
[0143] Pharmaceutical compositions of the present disclosure suitable for parenteral administration can include sterile aqueous or oleaginous solutions, suspensions, or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some aspects, the final injectable form is sterile and must be effectively fluid for use in a syringe. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
[0144] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In some aspects, a disclosed parenteral formulation can comprise about 0.01-0.1 M, e.g. about 0.05 M, phosphate buffer. In a further aspect, a disclosed parenteral formulation can comprise about 0.9% saline.
[0145] In various aspects, a disclosed parenteral pharmaceutical composition can comprise pharmaceutically acceptable carriers such as aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include but not limited to water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like. In a further aspect, a disclosed parenteral pharmaceutical composition can comprise may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. Also contemplated for injectable pharmaceutical compositions are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the subject or patient.
[0146] In addition to the pharmaceutical compositions described herein above, the disclosed compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.
[0147] Pharmaceutical compositions of the present disclosure can be in a form suitable for topical administration. As used herein, the phrase “topical application” means administration onto a biological surface, whereby the biological surface includes, for example, a skin area (e.g., hands, forearms, elbows, legs, face, nails, anus and genital areas) or a mucosal membrane. By selecting the appropriate carrier and optionally other ingredients that can be included in the composition, as is detailed herein below, the compositions of the present invention may be formulated into any form typically employed for topical application. A topical pharmaceutical composition can be in a form of a cream, an ointment, a paste, a gel, a lotion, milk, a suspension, an aerosol, a spray, foam, a dusting powder, a pad, and a patch. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the present disclosure, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.
[0148] In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on, as an ointment.
[0149] Ointments are semisolid preparations, typically based on petrolatum or petroleum derivatives. The specific ointment base to be used is one that provides for optimum delivery for the active agent chosen for a given formulation, and, preferably, provides for other desired characteristics as well (e.g., emollience). As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed., Easton, Pa.: Mack Publishing Co. (1995), pp. 1399-1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight.
[0150] Lotions are preparations that are to be applied to the skin surface without friction. Lotions are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are typically preferred for treating large body areas, due to the ease of applying a more fluid composition. Lotions are typically suspensions of solids, and oftentimes comprise a liquid oily emulsion of the oil-in-water type. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, such as methylcellulose, sodium carboxymethyl-cellulose, and the like.
[0151] Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also called the “internal” phase, is generally comprised of petrolatum and/or a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase typically, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. Reference may be made to Remington: The Science and Practice of Pharmacy, supra, for further information.
[0152] Pastes are semisolid dosage forms in which the bioactive agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from a single-phase aqueous gel. The base in a fatty paste is generally petrolatum, hydrophilic petrolatum and the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base. Additional reference may be made to Remington: The Science and Practice of Pharmacy, for further information.
[0153] Gel formulations are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil. Preferred organic macromolecules, i.e., gelling agents, are crosslinked acrylic acid polymers such as the family of carbomer polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the trademark Carbopol™. Other types of preferred polymers in this context are hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; modified cellulose, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.
[0154] Sprays generally provide the active agent in an aqueous and/or alcoholic solution which can be misted onto the skin for delivery. Such sprays include those formulated to provide for concentration of the active agent solution at the site of administration following delivery, e.g., the spray solution can be primarily composed of alcohol or other like volatile liquid in which the active agent can be dissolved. Upon delivery to the skin, the carrier evaporates, leaving concentrated active agent at the site of administration.
[0155] Foam compositions are typically formulated in a single or multiple phase liquid form and housed in a suitable container, optionally together with a propellant which facilitates the expulsion of the composition from the container, thus transforming it into a foam upon application. Other foam forming techniques include, for example the “Bag-in-a-can” formulation technique. Compositions thus formulated typically contain a low-boiling hydrocarbon, e.g., isopropane. Application and agitation of such a composition at the body temperature cause the isopropane to vaporize and generate the foam, in a manner similar to a pressurized aerosol foaming system. Foams can be water-based or aqueous alkanolic, but are typically formulated with high alcohol content which, upon application to the skin of a user, quickly evaporates, driving the active ingredient through the upper skin layers to the site of treatment. [0156] Skin patches typically comprise a backing, to which a reservoir containing the active agent is attached. The reservoir can be, for example, a pad in which the active agent or composition is dispersed or soaked, or a liquid reservoir. Patches typically further include a frontal water permeable adhesive, which adheres and secures the device to the treated region. Silicone rubbers with selfadhesiveness can alternatively be used. In both cases, a protective permeable layer can be used to protect the adhesive side of the patch prior to its use. Skin patches may further comprise a removable cover, which serves for protecting it upon storage.
[0157] Examples of patch configuration which can be utilized with the present invention include a single-layer or multi-layer drug-in-adhesive systems which are characterized by the inclusion of the drug directly within the skin-contacting adhesive. In such a transdermal patch design, the adhesive not only serves to affix the patch to the skin, but also serves as the formulation foundation, containing the drug and all the excipients under a single backing film. In the multi-layer drug-in-adhesive patch a membrane is disposed between two distinct drug-in-adhesive layers or multiple drug-in-adhesive layers are incorporated under a single backing film.
[0158] Examples of pharmaceutically acceptable carriers that are suitable for pharmaceutical compositions for topical applications include carrier materials that are well-known for use in the cosmetic and medical arts as bases for e.g., emulsions, creams, aqueous solutions, oils, ointments, pastes, gels, lotions, milks, foams, suspensions, aerosols and the like, depending on the final form of the composition. Representative examples of suitable carriers according to the present invention therefore include, without limitation, water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated protein hydrolysates, liquid lanolin and lanolin derivatives, and like materials commonly employed in cosmetic and medicinal compositions. Other suitable carriers according to the present invention include, without limitation, alcohols, such as, for example, monohydric and polyhydric alcohols, e.g., ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol, hexyleneglycol, mannitol, and propylene glycol; ethers such as diethyl or dipropyl ether; polyethylene glycols and methoxypolyoxyethylenes (carbowaxes having molecular weight ranging from 200 to 20,000); polyoxyethylene glycerols, polyoxyethylene sorbitols, stearoyl diacetin, and the like.
[0159] Topical compositions of the present disclosure can, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The dispenser device may, for example, comprise a tube. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may include labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising the topical composition of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
[0160] Another patch system configuration which can be used by the present invention is a reservoir transdermal system design which is characterized by the inclusion of a liquid compartment containing a drug solution or suspension separated from the release liner by a semi-permeable membrane and adhesive. The adhesive component of this patch system can either be incorporated as a continuous layer between the membrane and the release liner or in a concentric configuration around the membrane. Yet another patch system configuration which can be utilized by the present invention is a matrix system design which is characterized by the inclusion of a semisolid matrix containing a drug solution or suspension which is in direct contact with the release liner. The component responsible for skin adhesion is incorporated in an overlay and forms a concentric configuration around the semisolid matrix.
[0161] Pharmaceutical compositions of the present disclosure can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
[0162] Pharmaceutical compositions containing a compound of the present disclosure, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.
[0163] The pharmaceutical composition (or formulation) may be packaged in a variety of ways. Generally, an article for distribution includes a container that contains the pharmaceutical composition in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, foil blister packs, and the like. The container may also include a tamper proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container typically has deposited thereon a label that describes the contents of the container and any appropriate warnings or instructions.
[0164] The disclosed pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Pharmaceutical compositions comprising a disclosed compound formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
[0165] The exact dosage and frequency of administration depends on the particular disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the present disclosure.
[0166] Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05 to 99 % by weight, preferably from 0.1 to 70 % by weight, more preferably from 0.1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.
[0167] In one aspect, an appropriate dosage level will generally be about 0.01 to 1000 mg of a compound described herein per kg patient body weight per day and can be administered in single or multiple doses. In various aspects, the dosage level will be about 0.1 to about 500 mg/kg per day, about 0.1 to 250 mg/kg per day, or about 0.5 to 100 mg/kg per day. A suitable dosage level can be about 0.01 to 1000 mg/kg per day, about 0.01 to 500 mg/kg per day, about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 mg of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response.
[0168] Such unit doses as described hereinabove and hereinafter can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day. In various aspects, such unit doses can be administered 1 or 2 times per day, so that the total dosage for a 70 kg adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration. In a further aspect, dosage is 0.01 to about 1 .5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.
[0169] A typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
[0170] It can be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art. Further, it is noted that the clinician or treating physician will know how and when to start, interrupt, adjust, or terminate therapy in conjunction with individual patient response.
[0171] The disclosed pharmaceutical compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological or clinical conditions.
[0172] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.
[0173] As already mentioned, the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and a pharmaceutically acceptable carrier. Additionally, the present disclosure relates to a process for preparing such a pharmaceutical composition, characterized in that a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound according to the present disclosure.
[0174] Methods of Use
[0175] Classical opioid analgesics, including morphine, mediate all of their desired and undesired effects by specific activation of the -opioid receptor ( receptor). The use of morphine for treating chronic pain, however, is limited by the development of constipation, respiratory depression, tolerance and dependence. Analgesic effects can also be mediated through other members of the opioid receptor family such as the K-opioid receptor (K receptor), 6-opioid receptor (6 receptor) and the nociceptin/orphanin FQ peptide receptor (NOP receptor).
[0176] In one aspect, the compounds described herein can bind to the kappa opioid receptor (KOR) and behave as an agonist. As shown in the Examples, compounds described herein demonstrate the ability of the compounds described herein to bind to KOR. The ability of the compounds to bind to opioid receptors make them effective in the treatment or prevention of pain. In one aspect, the pain can be chronic or acute pain.
[0177] Aspects
[0178] Aspect 1. A lipid nanoparticle comprising a lipopeptide conjugate and a bioactive agent, wherein the lipopeptide conjugate has the formula X-L-Y, wherein
X is a peptide, L is a linker, and Y is a lipid.
[0179] Aspect 2. The lipid nanoparticle of Aspect 1 , wherein the peptide comprises from about 1 to about 10000 arginine residues, from about 1 to about 10000 histidine residues and at least one cysteine residue.
[0180] Aspect 3. The lipid nanoparticle of Aspect 1 or 2, wherein the peptide comprises arginine residues and histidine residues, wherein the arginine-to-histidine (R/H) ratio in the peptide is from 4:1 to 1 :4.
[0181] Aspect 4. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH2-CWR3H3-COOH, NH2- CW 5H5-COOH, NH2-CW 3H3-COOH, NH2-CW 3H4-COOH, NH2-CW 3H5-COOH, NH2-CW 3HS-COOH, NH2-CW 3H7-COOH, NH2-CW 3H8-COOH, NH2-CW 3H9-COOH, NH2-CWK5H5-COOH, NH2-CWK3H3- COOH, NH2-CwRm-COOH, NH2-CwKn-COOH, NH2-C„RoHp-COOH, NH2-CWRUKV-COOH, NH2- CwKpHq-COOH, and NH2-CwRxKyHz-COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and m, n, o, p, q, u, v, x, y, z, and w are integers from 1 to 1 ,000.
[0182] Aspect 5. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH2-R3H3-COOH, NH2- R5H5-COOH, NH2-R3H3-COOH, NH2-R3H4-COOH, NH2-R3H5-COOH, NH2-R3HS-COOH, NH2-R3H7- COOH, NH2-R3H8-COOH, NH2-R3H9-COOH, NH2-K5H5-COOH, NH2-K3H3-COOH, NH2-R0HP-COOH, NH2-RUKV-COOH, NH2-KpHq-COOH, and NH2-RxKyHz-COOH; wherein R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and m, n, 0, p, q, u, v, x, y, and z are integers from 1 to 1 ,000.
[0183] Aspect 6. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide has a formula selected from the group consisting of NH2-Rm-COOH, NH2-Kn-COOH, NH2-H0-COOH; wherein R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m, n, and 0 are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
[0184] Aspect 7. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequencespecific peptide having a formula selected from the group consisting of NH2-CR-COOH, NH2-CR2- COOH, NH2-CR3-COOH, NH2-CR4-COOH, NH2-CR5-COOH, NH2-CR6-COOH, NH2-CRm-COOH, NH2-CHR-COOH, NH2-CH2R-COOH, NH2-CH3R-COOH, NH2-CH4R-COOH, NH2-CH5R-COOH NH2- CHmR-COOH, NH2-CHR2-COOH, NH2-CH2R2-COOH, NH2-CH3R2-COOH, NH2-CH4R2-COOH, NH2- CH5R2-COOH, NH2-CHmR2-COOH, NH2-CHR3-COOH, NH2-CH2R3-COOH, NH2-CH3R3-COOH, NH2- CH4R3-COOH, NH2-CH5R3-COOH, NH2-CH6R3-COOH, NH2-CH7R3-COOH, NH2-CH8R3-COOH, NH2- CH9R3-COOH, NH2-CHmR3-COOH, NH2-CHR4-COOH, NH2-CH2R4-COOH, NH2-CH3R4-COOH, NH2- CH4R4-COOH, NH2-CH5R4-COOH, NH2-CH6R4-COOH, NH2-CH7R4-COOH, NH2-CH8R4-COOH, NH2- CH9R4-COOH, NH2-CHmR4-COOH, NH2-CHR5-COOH, NH2-CH2R5-COOH, NH2-CH3R5-COOH, NH2- CH4R5-COOH, NH2-CH5R5-COOH, NH2-CH6R5-COOH, NH2-CH7R5-COOH, NH2-CH8R5-COOH, NH2- CH9R5-COOH, NH2-CHmRs-COOH, and NH2-CHmRn-COOH, wherein C is cysteine, R is arginine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
[0185] Aspect 8. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequencespecific peptide or polypeptide having a formula selected from the group consisting of NH2-CK- COOH, NH2-CK2-COOH, NH2-CK3-COOH, NH2-CK4-COOH, NH2-CK5-COOH, NH2-CK6-COOH, NH2- CKm-COOH, NH2-CHK-COOH, NH2-CH2K-COOH, NH2-CH3K-COOH, NH2-CH4K-COOH, NH2-CH5K- COOH NH2-CHmK-COOH, NH2-CHK2-COOH, NH2-CH2K2-COOH, NH2-CH3K2-COOH, NH2-CH4K2- COOH, NH2-CH5K2-COOH, NH2-CHmK2-COOH, NH2-CHK3-COOH, NH2-CH2K3-COOH, NH2-CH3K3- COOH, NH2-CH4K3-COOH, NH2-CH5K3-COOH, NH2-CH6K3-COOH, NH2-CH7K3-COOH, NH2-CH8K3- COOH, NH2-CH9K3-COOH, NH2-CHmK3-COOH, NH2-CHK4-COOH, NH2-CH2K4-COOH, NH2-CH3K4- COOH, NH2-CH4K4-COOH, NH2-CH5K4-COOH, NH2-CH6K4-COOH, NH2-CH7K4-COOH, NH2-CH8K4- COOH, NH2-CH9K4-COOH, NH2-CHmK4-COOH, NH2-CHK5-COOH, NH2-CH2K5-COOH, NH2-CH3K5- COOH, NH2-CH4K5-COOH, NH2-CH5K5-COOH, NH2-CH6K5-COOH, NH2-CH7K5-COOH, NH2-CH8K5- COOH, NH2-CH9K5-COOH, NH2-CHmK5-COOH, and NH2-CHmKn-COOH, wherein C is cysteine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
[0186] Aspect 9. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequencespecific peptide or polypeptide having a formula selected from the group consisting of NH2-RC- COOH, NH2-R2C-COOH, NH2-R3C-COOH, NH2-R4C-COOH, NH2-R5C-COOH, NH2-R6C-COOH, NH2-RmC-COOH, NH2-RHC-COOH, NH2-RH2C-COOH, NH2-RH3C-COOH, NH2-RH4C-COOH, NH2- RHsC-COOH, NH2-RHmC-COOH, NH2-R2HC-COOH, NH2-R2H2C-COOH, NH2-R2H3C-COOH, NH2- R2H4C-COOH, NH2-R2H5C-COOH, NH2-R2HmC-COOH, NH2-R3HC-COOH, NH2-R3H2C-COOH, NH2- R3H3C-COOH, NH2-R3H4C-COOH, NH2-R3H5C-COOH, NH2-R3H6C-COOH, NH2-R3H7C-COOH, NH2- R3H8C-COOH, NH2-R3H9C-COOH, NH2-R3HmC-COOH, NH2-R4H-COOH, NH2-R4H2C-COOH, NH2- R4H3C-COOH, NH2-R4H4C-COOH, NH2-R4H5C-COOH, NH2-R4H6C-COOH, NH2-R4H7C-COOH, NH2- R4H8C-COOH, NH2-R4H9C-COOH, NH2-R4HmC-COOH, NH2-R5HCOOH, NH2-R5H2C-COOH, NH2- R5H3C-COOH, NH2-R5H4C-COOH, NH2-R5H5C-COOH, NH2-R5H6C-COOH, NH2-R5H7C-COOH, NH2- RSHSC-COOH, NH2-R5H9C-COOH, NH2-R5HmC-COOH, and NH2-RnHmC-COOH, wherein C is cysteine, R is arginine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups. [0187] Aspect 10. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2- KC-COOH, NH2-K2C-COOH, NH2-K3C-COOH, NH2-K4C-COOH, NH2-K5C-COOH, NH2-K6C-COOH, NH2-KmC-COOH, NH2-KHC-COOH, NH2-KH2C-COOH, NH2-KH3C-COOH, NH2-KH4C-COOH, NH2- KH5C-COOH, NH2-KHmC-COOH, NH2-K2HC-COOH, NH2-K2H2C-COOH, NH2-K2H3C-COOH, NH2- K2H4C-COOH, NH2-K2H5C-COOH, NH2-K2HmC-COOH, NH2-K3HC-COOH, NH2-K3H2C-COOH, NH2- K3H3C-COOH, NH2-K3H4C-COOH, NH2-K3H5C-COOH, NH2-K3H6C-COOH, NH2-K3H7C-COOH, NH2- K3H8C-COOH, NH2-K3H9C-COOH, NH2-K3HmC-COOH, NH2-K4H-COOH, NH2-K4H2C-COOH, NH2- K4H3C-COOH, NH2-K4H4C-COOH, NH2-K4H5C-COOH, NH2-K4H6C-COOH, NH2-K4H7C-COOH, NH2- K4H8C-COOH, NH2-K4H9C-COOH, NH2-K4HmC-COOH, NH2-K5HCOOH, NH2-K5H2C-COOH, NH2- K5H3C-COOH, NH2-K5H4C-COOH, NH2-K5H5C-COOH, NH2-K5H6C-COOH, NH2-K5H7C-COOH, NH2- K5H8C-COOH, NH2-K5H9C-COOH, NH2-K5HmC-COOH, and NH2-KnHmC-COOH, wherein C is cysteine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
[0188] Aspect 1 1. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide has a formula selected from the group consisting of NH2-CRn-COOH, NH2-CKn-COOH, NH2-CHn-COOH, NH2-RnC- COOH, NH2-KnC-COOH, NH2-HnC-COOH, NH2-R0HP-COOH, H2-HpRo-COOH, NH2-CR0HP-COOH, NH2-CHPR0-COOH, NH2-R0CHP-COOH, NH2-HPCR0-COOH, NH2-R0HPC-COOH, NH2-HpRoC-COOH, NH2-CWROHP-COOH, NH2-CWHPRO-COOH, NH2-ROCWHP-COOH, NH2-HPCWRO-COOH, NH2-R0HPCW- COOH, NH2-HPROCW-COOH, wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, m and n are integers from 0 to 1 ,000, n, 0, p, and w are integers from 0 to 1 ,000 but cannot be zero at the same time.
[0189] Aspect 12. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a random or block copolymeric peptide having a formula selected from the group consisting of NH2- X1xX2yX3zX4w-COOH, wherein X1 , X2, X3, and X4 is cysteine, arginine, lysine or histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
[0190] Aspect 13. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide has a sequence selected from the group consisting of NH2-CRRRRR-COOH, NH2-CRRRHHH-COOH, NH2- CHHHRRR-COOH, NH2-CKKKKK-COOH, NH2-CKKK-COOH, NH2-CHHHHHRRRR-COOH, NH2- CRRRRRHHHHH-COOH, NH2-CRRRHHH-COOH, NH2-CHHHRRRCOOH, NH2-CKKKKKHHHHH- COOH, NH2-CHHHHHKKKKK-COOH, NH2-CHHHKKK-COOH, NH2-CKKKHHH-COOH, NH2- RRRRRC-COOH, NH2-RRRHHHC-COOH, NH2-KKKKKC-COOH, NH2-KKKC-COOH, NH2-
RRRRRHHHHHC-COOH, NH2-RRRHHHC-COOH, NH2-KKKKKHHHHHC-COOH, NH2-KKKHHHC-
COOH, NH2-RRRHHHHHHHHC-COOH, NH2-RRRHHHHHHHC-COOH, NH2-RRRHHHHHHC- COOH, NH2-RRRHHHHHC-COOH, NH2-RRRRHHHHHHC-COOH, NH2-RRRRHHHHHHHC- COOH, NH2-RRRRHHHHHHHHC-COOH, wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups.
[0191] Aspect 14. The lipid nanoparticle of any one of Aspects 1-3, wherein the peptide is a block copolymer having a sequence selected from the group consisting of NH2-CRm-COOH, NH2-CKn- COOH, NH2-CR0HP-COOH, NH2-CRxKyHz-COOH, NH2-RmC-COOH, NH2-KnC-COOH, NH2-ROHPC- COOH, NH2- RxKyHzC-COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, and m, n, o, p, q, x, y, and z are integers from 1 to 1 ,000, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
[0192] Aspect 15. The lipid nanoparticle of any one of Aspects 1-14, wherein the linker is a residue of a group selected from the group consisting of amine, carboxylate, hydroxyl, thiol, alkene, alkyne, azide, disulfide, maleimide, ester, peptide, acetal, anhydride, halide, vinyl sulfone, methyl acrylate, acrylate, acrylamide, methyl acrylamide, fluoride, chloride, bromide, aldehyde, and ketone.
[0193] Aspect 16. The lipid nanoparticle of any one of Aspects 1-15, wherein the lipid is selected from the group consisting of a phosphatidylcholine, a lysophosphatidylcholine, a plasmenylphosphatidylcholin, a phosphatidylethanolamin, a lysophosphatidylethanolamine, a plasmenylphosphatidylethanolamin, a phosphatidylserine, a sphingomyeline, a phosphatidic acid
, a lysophosphatidic acid, a phosphatidylinositol, a phosphatidylglycerol, a cardiolipin, a ceramide-1 -phosphate, an N-acylsphingosine, a sulfatide, a ganglioside, a sulfoquinovosyldiacylglycero, and a diphosphorylated hexaacyl Lipid A.
[0194] Aspect 17. The lipid nanoparticle of any one of Aspects 1-15, wherein the lipid is a phospholipid.
[0195] Aspect 18. The lipid nanoparticle of any one of Aspects 1-15, wherein the lipid is a phosphatidylcholine, a phosphatidic acid, a phosphatidylglycerol, a phosphatidylethanolamine, a phosphatidylserine, or a PEG phospholipid.
[0196] Aspect 19. The lipid nanoparticle of Aspect 1 , wherein the linker has a residue of a maleimide and the lipid is phosphatidylethanolamine.
[0197] Aspect 20. The lipid nanoparticle of Aspect 19, wherein the lipid is 1 ,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine (DMPE), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), or any combination thereof.
[0198] Aspect 21. The lipid nanoparticle of Aspect 19 or 20, wherein the lipid is 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE). [0199] Aspect 22. The lipid nanoparticle of any one of Aspects 19-21 , wherein the peptide is a random copolymeric peptide having a formula NH2-RmHnC-COOH, wherein C is cysteine, R is arginine, H is histidine, COOH is the N-terminal of the peptide, and m and n are integers from 1 to 15.
[0200] Aspect 23. The lipid nanoparticle of any one of Aspects 1-14, wherein the lipopeptide conjugate has the structure I wherein Ri and R2 are independently a C8 to C25 alkyl group or a C8 to C25 alkenyl group, o and p are independently integers from 1 to 4, and
X is a peptide.
[0201] Aspect 24. The lipid nanoparticle of Aspect 23, wherein (a) o and p are each 2 or (b) o and p are each 2 and Ri and R2 are independently an oleyl group or a palmitoyl group.
[0202] Aspect 25. The lipid nanoparticle of any one of Aspects 1-24, wherein the lipid nanoparticle further comprises one or more helper lipids.
[0203] Aspect 26. The lipid nanoparticle of Aspect 25, wherein the helper lipid comprises a phospholipid, PEGylated lipid, a sterol, an any combination thereof.
[0204] Aspect 27. The lipid nanoparticle of Aspect 25 and 26, wherein the lipid peptide conjugate to helper lipid is at a molar ratio of 1 :100 to 100:1 .
[0205] Aspect 28. The lipid nanoparticle of any one of Aspects 1-27, wherein the bioactive agent is selected from the group consisting of Abiraterone Acetate, Brentuximab vedotin, Trastuzumab emtansine, Afatinib, Afinitor® (Everolimus), Aldara® (Imiquimod), Alimta® (Pemetrexed Disodium), Pemetrexed, Palonosetron, Chlorambucil, Nelarabine, Axitinib, Belinostat, Bleomycin, Bortezomib, Cabozantinib-S-Malate, Camptothecin, Capecitabine, Ceritinib, Cerubidine® (Daunorubicin), Crizotinib, Dabrafenib, Dasatinib, Degarelix, Docetaxel, Doxorubicin, Epirubicin, Eribulin, Etoposide, Raloxifene, Fulvestrant, Folex® (Methotrexate), Pralatrexate, Eribulin Mesylate, Topotecan, Ibritumomab tiuxetan, Ibrutinib, Irinotecan, Ixempra® (Ixabepilone), Jevtana® (Cabazitaxel), Kadcyla® (Ado-Trastuzumab Emtansine), Lenalidomide, Leuprolide Acetate, Vincristine, Methotrexate, Mitomycin C, Mitoxantrone, Nelarabine, Paclitaxel, Prednisone, Promacta® (Eltrombopag Olamine), Raloxifene Hydrochloride, Lenalidomide, Methotrexate, Synribo® (Omacetaxine Mepesuccinate), Targretin® (Bexarotene), Temsirolimus, Treanda® (Bendamustine Hydrochloride), Velban® (Vinblastine Sulfate), Velsar® (Vinblastine Sulfate), Vincasar PFS® (Vincristine Sulfate), Vinorelbine Tartrate, Vorinostat, Capecitabine, Ipilimumab, and Goserelin Acetate.
[0206] Aspect 29. The lipid nanoparticle of any one of Aspects 1-27, wherein the bioactive agent is selected from the group consisting of antibodies, peptides, therapeutic enzymes, cytokines, interferons, and interleukins.
[0207] Aspect 30. The lipid nanoparticle of any one of Aspects 1-27, wherein the bioactive agent is a nucleic acid.
[0208] Aspect 31. The lipid nanoparticle of Aspect 30, wherein the nucleic acid is selected from the group consisting of plasmid DNA, an oligonucleotide, an aptamers, a DNAzyme, a RNA aptamers, a RNA Decoy, an antisense RNA, a ribozymes, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA, an antagomirs, or any combination thereof.
[0209] Aspect 32. The lipid nanoparticle of Aspect 30 or 31 , wherein the molar ratio of amine or guanidine groups on the side chain of peptide (N) to the phosphate group in the nucleic acid (P) is from about 0.5: 1 to about 100: 1 .
[0210] Aspect 33. The lipid nanoparticle of any one of Aspects 1 -32, wherein the lipid nanoparticle is between about 2 nm and about 2000 m in diameter.
[0211] Aspect 34. The lipid nanoparticle of any one of Aspects 1-33, wherein the lipid nanoparticle has a surface charge of between about -100 mV and about +100 mV at pH 3 to 1 1 .
[0212] Aspect 35. A method for delivering a bioactive agent to a subject, the method comprising administering to the subject the lipid nanoparticle of any one of Aspects 1-34.
[0213] Aspect 36. The method of Aspect 35, wherein the bioactive agent is a nucleic acid.
[0214] Aspect 37. The method of Aspect 35, wherein the bioactive agent is a siRNA.
[0215] Aspect 38. The method of any one of Aspects 35-37, wherein when the bioactive agent is delivered to the lungs, the arginine-to-histidine (R/H) ratio in the peptide is from 2:1 to 1 :2.
[0216] Aspect 39. The method of any one of Aspects 35-37, wherein when the bioactive agent is delivered to the liver, the arginine-to-histidine (R/H) ratio in the peptide is from 1 :1 to 1 :3.
[0217] Aspect 40. The method of any one of Aspects 35-37, wherein when the bioactive agent is delivered to the liver, the arginine-to-histidine (R/H) ratio in the peptide is from 1 :1 to 1 :2.
[0218] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure. EXAMPLES
[0219] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure.
MATERIALS AND METHODS
[0220] Synthesis of RmHnC-DOPE Peptide-Lipid Conjugate
[0221] The lipopeptide conjugate was synthesized via an Al BN-initiated free radical addition reaction. Briefly, DOPE-maleimide (1.0 equiv) and RmHnC (40 mg, 2.0 equiv) were separately dissolved in 1 mL anhydrous dimethyl sulfoxide (DMSO) under sonication at 25°C for 12 h. The solutions were combined in a 25 mL glass reactor, followed by addition of azobisisobutyronitrile (AIBN, 20 mg, 2.0 equiv relative to DOPE-mal). The reaction mixture was heated to 70°C in a pre-equilibrated metal bath (initial preheating at 50°C) and stirred under inert atmosphere for 16 h. Post-reaction, the crude product was diluted with 30 mL deionized (DI) water, agitated for 3 h, and centrifuged at 10,000 xg for 30 min to remove insoluble residues. The supernatant was dialyzed against DI water using a 2 kDa molecular weight cut-off (MWCO) membrane for 96 h, with water replaced every 12 h. The dialyzed solution was centrifuged again (10,000 xg, 30 min), and the purified product was lyophilized at -80°C for 48 h. The resultant powder was dissolved in a 4:1 (v/v) ethanol/DMSO mixture containing 0.1% trifluoroacetic acid (TFA) to prepare a 10 mg/mL stock solution, which was characterized by MALDI-TOF mass spectrometry and LC-MS.
[0222] LNP Preparation and Characterization
[0223] LNPs were formulated by mixing the synthesized lipopeptide, SM-102, sitosterol/cholesterol (38.5% molar ratio), DOPE/DSPC (10%), and DMPE-PEG2000 (1.5%) at molar ratios of 50% lipopeptide, 38.5% sterol blend, 10% phospholipids, and 1.5% PEGylated lipid. The lipid components were dissolved in ethanol and combined with nucleic acids at an N/P ratio of 6 (1 :3 v/v aqueous buffer) under gentle agitation for 30 min at 25°C. Freshly prepared LNPs were diluted with nuclease- free water and purified via 30 kDa molecular weight cut-off (MWCO) centrifugal filters to achieve >99% buffer exchange efficiency.
[0224] Encapsulation Efficiency Analysis
[0225] Pre- and post-ultrafiltration samples, including retentate and filtrate aliquots collected during purification, were treated with 1% Triton X-100 to lyse LNPs. Free and total nucleic acid concentrations were quantified using the Ribogreen™ assay (Thermo Fisher Scientific) with a siRNA standard curve. Encapsulation efficiency (EE) was calculated as follows: [0226] Pre-Ultrafiltration EE= (Total nucleic acid (pre-lysis)-Free nucleic acid (pre-lysis)) I Total nucleic acid (pre-lysis)x100%
[0227] Pre-Ultrafiltration EE= (Total nucleic acid (post-lysis)-Free nucleic acid (unlysed)) I Total nucleic acid (pre-lysis)x100%
[0228] Cell Culture and Animal Studies
[0229] HepG2 cells were obtained from the Chinese Academy of Sciences Cell Bank, and HEPA1-6 cells were purchased from Pricella Biotechnology Co., Ltd. (Wuhan, China). Cells were cultured in RPMI 1640, MEM, or DMEM supplemented with 10% FBS and 0.5% penicillin/streptomycin at 37°C under 5% CO2. Mycoplasma contamination was routinely monitored.
[0230] Animal experiments were conducted at Westlake University Laboratory Animal Center and approved by the Institutional Animal Ethics Committee of Westlake University (Protocol LLSC- 2024022001). Female C57BL/6 mice (6-8 weeks, 18-20 g) were purchased from the Westlake University Animal Center and housed in a barrier facility under a 12 h light/dark cycle at 20°C and 40% humidity.
[0231] In vitro Cytotoxicity Assay
[0232] Cytotoxicity of RH-LP LNPs was evaluated using the CCK-8 assay. Briefly, HEPA1-6/HepG2 cells (5.0*103 cells/well) were seeded in 96-well plates, cultured for 24 h, and treated with RH-DOPE LNPs (10 pmol nucleic acid/well) in complete medium for 48 h. After adding 10 L CCK-8 solution per well, cells were incubated for 2 h, and absorbance at 450 nm was measured using an EnSpire microplate reader at 25°C. Triplicate measurements were performed for each sample.
[0233] In vitro siRNA and eGFP Plasmid Delivery
[0234] For siRNA delivery, HEPA1-6/HepG2 cells (5*104 cells/well) in 24-well plates were treated with RNA-loaded RH-DOPE LNPs (50 pmol RNA/well) diluted in Opti-MEM for 4 h. After replacing with fresh medium, cells were incubated for 24-48 h. Total RNA was extracted using the EASYspin Plus RNA Extraction Kit (Aidlab, China, #RN28) and quantified via NanoDrop One (Thermo Fisher Scientific). Target gene (PCSK9) expression relative to GAPDH was analyzed using the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme Biotech, #0221-01). The qRT-PCR protocol included: reverse transcription (50°C, 3 min), pre-denaturation (95°C, 15 min), 40 cycles of denaturation (95°C, 10 s) and annealing/extension (60°C, 30 s), followed by a melt curve analysis (95°C to 60°C). Naked siRNA, RNAiMAX, and SM-102 LNPs served as controls.
[0235] For eGFP plasmid delivery, fluorescence images were captured using an inverted fluorescence microscope. eGFP-positive cells were quantified via flow cytometry (FlowJo software). Lipofectamine 2000 and SM-102 LNPs were used as positive controls.
[0236] Investigation of Cellular Uptake Mechanisms of RH-DOPE LNPs [0237] To investigate the endocytic pathways of RH-DOPE LNPs in HepG2 cells, cells were seeded in 24-well plates at a density of 5x104 cells/well and cultured in DMEM supplemented with 10% FBS and 0.5% penicillin/streptomycin at 37°C under 5% CO2 for 24 h. Prior to LNP treatment, cells were pretreated with pathway-specific inhibitors: 20 pM chlorpromazine (CPZ, clathrin-mediated endocytosis inhibitor) for 30 min, 5 mM methyl-p-cyclodextrin (MpCD, lipid raft disruptor) for 1 h, 50 pM EIPA (macropinocytosis inhibitor) for 30 min, or incubated at 4°C (energy-dependent endocytosis inhibition) for 1 h. Cells were then exposed to Cy5-labeled RH-DOPE LNPs (20pmol siRNA /well) in serum-free medium for 4 h at 37°C (4°C group maintained at low temperature). Unbound LNPs were removed by washing three times with ice-cold PBS, and cells were trypsinized, resuspended in PBS, and analyzed using flow cytometry (Cy5 excitation/emission: 649/670 nm; >10,000 events per sample). Untreated cells and cells without LNP exposure served as background controls, and inhibitor cytotoxicity was pre-validated by CCK-8 assay (cell viability >90%).
[0238] In vivo PCSK9 siRNA Delivery
[0239] Mice received PCSK9 siRNA-loaded LNPs (0.5 mg kg-1). Tissues (15 mg) were homogenized using a high-speed tissue homogenizer (TIANGEN, #OSE-Y30), and total RNA was extracted for qRT-PCR analysis of PCSK9 expression relative to GAPDH. The qRT-PCR protocol included reverse transcription (50°C, 3 min), pre-denaturation (95°C, 15 min), 40 cycles of amplification (95°C, 10 s; 60°C, 30 s), and melt curve analysis. PBS and SM-102 LNPs were used as controls.
[0240] RESULTS
[0241] Lipopeptide Synthesis and Characterization
[0242] A library of RmHnC-based lipopeptides (RmHnC-DOPE) was designed to enable systematic investigation of its structure-property relationship for nucleic acid delivery. As illustrated in FIG. 1A, the lipopeptides incorporate arginine (R) for nucleic acid binding, histidine for pH-responsive behavior, and a terminal cysteine for conjugation to the maleimide-functionalized phospholipid DOPE. Thirteen distinct peptide sequences (RmHn, where m = 1-9 arginines and n = 1-5 histidines) were synthesized (FIG. 1 B) to explore the impact of its sequence on LNP properties. The peptides were conjugated to DOPE-Mal via thiol-ene chemistry (FIG. 1C), and LNPs were formulated with helper lipids, p-sitosterol for membrane stability and DMPE PEG-2000 for stabilization and stealth properties (FIG. 1 D) to create four-component RmHn-DOPE LNPs encapsulating nucleic acids (FIG. 1 E).
[0243] The RmHnC-DOPE conjugates were synthesized through an azobisisobutyronitrile (AIBN)- initiated free radical thiol-ene reaction between the cysteine thiol group of the peptides and the maleimide moiety of DOPE-Mal. It should be noted that thiol and maleimide can also undergo the Michael-type reaction7, however, the thiol-ene8 reaction was used to reduce the reaction time. RmHn- DOPE LNPs were prepared by combining the lipopeptide conjugates with p-sitosterol (membrane stabilizer), DMPE PEG-2000, and nucleic acids (e.g., siRNA or pDNA) at a fixed molar ratio. Selfassembly was achieved via microfluidic mixing, yielding monodisperse nanoparticles with diameters tailored for cellular uptake (e.g., 80-120 nm). The inclusion of histidine residues enabled pH- dependent endosomal escape, while arginine provided cationic charge for nucleic acid complexation.
[0244] This modular synthesis strategy enabled systematic variation of peptide sequences and lipid compositions, accelerating high-throughput screening of LNP formulations to optimize delivery efficiency. Our approach leverages well-established solid-state peptide synthesis, which ensures scalable production of high-purity peptides and well-characterized lipid chemistry, allowing precise tuning of lipopeptide-LNP structure and functionality. The integration of these established methodologies provides a robust framework for designing biodegradable, high-performance delivery systems.
[0245] LNP Physicochemical Characterization
[0246] The 13 RmHn-DOPE LNP variants exhibited well-defined physicochemical properties essential for nucleic acid delivery (FIGS. 2A-C). All formulations demonstrated monodisperse hydrodynamic diameters (80-120 nm) with narrow size distributions (polydispersity index, PDI < 0.2), comparable to the SM-102 benchmark formulation (110 nm, PDI = 0.15). Surface charge analysis revealed moderate cationic zeta potentials (+5 to +15 mV), which balanced efficient siRNA/pDNA complexation through electrostatic interactions while minimizing nonspecific binding to serum proteins or cell membranes. Stability assessments under refrigerated storage (4°C) showed that R3H7C-DOPE, R4H6C-DOPE, and R5H5C-DOPE maintained <5% size variation over 30 days, whereas SM-102 LNPs aggregated beyond 150 nm after 14 days, highlighting the superior colloidal stability of the lipopeptide designs.
[0247] Cryo-TEM imaging (FIG. 2G) confirmed spherical nanostructures with electron-dense cores, consistent with tightly condensed nucleic acid payloads and uniform lipid bilayer organization. The pH-responsive behavior of top-performing variants was further validated by their pKa values (R3H7C- DOPE: 6.2; R4H6C-DOPE: 6.5; R5H5C-DOPE: 6.0), which aligned with the endosomal pH gradient (4.0-6.5) to enable protonation-dependent membrane fusion and payload release (FIG. 2F). Encapsulation efficiency (EE) for both siRNA and pDNA exceeded 90% across all formulations, with R4H6C-DOPE achieving 95.2 ± 1.8% EE for siRNA and 92.4 ± 2.1% for pDNA, superior to SM-102 LNPs (85.0 ± 3.5% siRNA EE).
[0248] In vitro siRNA Delivery and Gene Silencing
[0249] R3H7C-, R4H6C-, and R5H5C-DOPE LNPs encapsulating PCSK9 siRNA demonstrated dose-dependent gene silencing efficacy in HEPA1-6 cells (FIG. 2E). Quantitative RT-PCR analysis revealed PCSK9 mRNA level was knocked down to 16.9±12.2%, 14.9±1 .0%, and 10.3±1.9% for R3H7C-, R4H6C-, and R5H5C-DOPE LNPs, respectively, compared to 32.6±8.5% observed with SM-102 LNP (p<0.05). This improved silencing performance correlated with optimized arginine-to- histidine (R/H) ratios in the lipopeptide architecture (3:7, 4:6, and 5:5 for respective formulations), which facilitated efficient siRNA complexation through balanced cationic charge density and pH- responsive endosomal membrane disruption. Cytotoxicity assessment via CCK-8 assays showed exceptional biocompatibility profiles for RmHn-DOPE variants, maintaining >99% cell viability at therapeutic siRNA doses (50 pmol/well), while SM-102 LNPs demonstrated comparable viability (99%) under identical conditions.
[0250] Plasmid DNA Transfection Efficiency
[0251] The plasmid DNA (pDNA) transfection performance of RmHn-DOPE lipid nanoparticles (LNPs) was systematically evaluated eGFP-encoded pDNA in HepG2 and HEPA1-6 cells (FIGS. 2H- I). Fluorescence microscopy demonstrated robust eGFP expression across all three lipopeptide formulations, with R3H7C-DOPE and R4H6C-DOPE LNPs exhibiting fluorescence intensities comparable to or exceeding Lipofectamine 2000, a commercial transfection reagent. In contrast, SM- 102 LNPs displayed markedly weaker signal intensity, aligning with their suboptimal pDNA delivery efficiency observed in prior studies.
[0252] Flow cytometric quantification (FIG. 2I) revealed distinct transfection profiles among LNP variants in HEPG2 cells. R3H7C-DOPE LNPs achieved 19.1 ± 0.8% eGFP-positive cells, modestly exceeding SM-102 LNPs (16.7 ± 0.6%, P = 0.0014), but demonstrated a striking 11-fold increase in mean fluorescence intensity (MFI) (2.0 ± 0.2 x 1O5 vs. SM-102's 1.8 ± 0.3 x 104), indicating superior transgene expression per transfected cell. While Lipofectamine 2000 demonstrated higher transfection efficiency (55.4±7.8%), its MFI (2.6±0.3xl05) was statistically comparable to R3H7C- DOPE (P=0.07). Strikingly, R4H6C-DOPE and R5H5C-DOPE LNPs exhibited progressive enhancement in performance, with R4H6C-DOPE yielding 43.8 ± 2.9% eGFP-positive cells (MFI=3.0±0.8 105) and R5H5C-DOPE achieving 94.0 ± 1.0% positivity (MFI=7.1±0.6x105). The exceptional efficacy of R5H5C-DOPE likely arises from its optimized arginine-to-histidine (R/H) ratio (5:5), which balances cationic charge density for stable pDNA complexation and pH-responsive endosomal escape via histidine's buffering capacity 5. This mechanism is further supported by the pKa range (6.0-6.5) of RmHn-DOPE variants, aligning with the acidic endosomal environment to enhance payload release.
[0253] The transfection efficiency of flue pDNA delivered by RmHn-DOPE LNPs was quantitatively evaluated using bioluminescence imaging (FIG. 2J). R5H5C-DOPE LNPs achieved the highest luminescence intensity at 6.3±0.5X104 relative light units (RLU), significantly surpassing both the commercial transfection reagent Lipofectamine 2000 (5.3±0.6X 104RLU) and SM-102 LNPs (1 .7±0.8X102 RLU). The lipopeptide variants exhibited a stepwise enhancement in efficacy: R3H7C- DOPE (5O 4±0.9x103 RLU) and R4H6C-DOPE (3.5±0.4x104 RLU) demonstrated intermediate performance, while R5H5C-DOPE’s luminescence intensity was 365-fold higher than SM-102 (P < 0.0001) and 1.2-fold higher than Lipofectamine 2000 (P = 0.1).
[0254] In consistent with eGFP pDNA, SM-102 LNPs showed limited transfection efficiency, with luminescence levels comparable to the blank control (<500 RLU), highlighting its suboptimal pDNA delivery capability. In contrast, R5H5C-DOPE’s superior performance correlated with its optimized arginine-to-histidine (5:5) ratio, which balances cationic charge density for stable pDNA complexation and pH-responsive endosomal escape. The cytotoxicity study also showed nearly 100% cell viability for flue pDNA-loaded RmHn-DOPE- and SM102 LNP-treated cells versus 73.8% viability for flue pDNA Lipofectamine 2000-treated cells.
[0255] The observed disparity in transfection efficiency between siRNA and pDNA mediated by R3H7C-DOPE LNPs likely stems from payload-specific intracellular trafficking kinetics. While siRNA requires rapid endosomal escape (2-4 hours post-internalization) for immediate RNA-induced silencing complex (RISC) engagement, pDNA necessitates prolonged endosomal retention to facilitate nuclear translocation and transcription. R3H7C-DOPE’s higher histidine content (7:3 R/H ratio) may prematurely disrupt endosomes before pDNA reaches the nucleus, highlighting the need for stoichiometric tuning to match payload requirements.
[0256] The superior plasmid transfection efficiency of RmHn-DOPE LNPs, particularly the R3H7C- DOPE variant, arises from their dual-functional architecture. The cationic arginine residues in these LNPs promote robust electrostatic interactions with plasmid DNA, enabling the formation of stable nanocomplexes that protect genetic payloads from nuclease degradation. Concurrently, histidine residues contribute pH-buffering capacity, which facilitates endosomal escape through the proton sponge effect. This mechanism is supported by the optimal pKa range (6.0-6.5) of R3H7C-DOPE, aligning with the acidic environment of endosomes to enhance transfection efficiency. In contrast, the diminished performance of R5H5C-DOPE underscores the critical balance required between arginine and histidine content. Excess histidine (e.g., n=5) reduces cationic charge density, weakening DNA condensation, while insufficient histidine, as seen in SM-102-based LNPs with tertiary amines, limits endosomal buffering and escape — a key bottleneck in non-viral gene delivery.
[0257] RmHn-DOPE LNPs also demonstrate significant advantages over commercial transfection reagents. Their biocompatible design minimizes cytotoxicity, maintaining over 98% cell viability compared to Lipofectamine 2000, which causes substantial toxicity (73.8 % viability in HepG2 cell) due to its high charge density and structure. Furthermore, RmHn-DOPE formulations exhibit exceptional colloidal stability, remaining intact for over 30 days. These attributes position RmHn- DOPE LNPs as a versatile platform capable of delivering diverse nucleic acid payloads with viral-like efficiency while retaining the safety profile of biodegradable lipids.
[0258] RmHn-DOPE LNP in vitro transfection mechanism study
[0259] Cellular uptake and intracellular trafficking mechanisms of RmHn-DOPE LNPs were systematically investigated using Cy5-labeled siRNA and pathway-specific inhibitors. Confocal microscopy revealed robust internalization of R3H7C-DOPE and R5H5C-DOPE LNPs in HepG2 cells, with mean fluorescence intensity (MFI) values exceeding SM-102 LNPs by 1.7 to 2.3 fold (P < 0.0001). Energy-dependent endocytosis was confirmed by a > 40 % reduction in MFI at 4°C across all formulations (P < 0.0001). Inhibitor studies further delineated entry pathways: ElPA-mediated macropinocytosis dominated for RmHnC-DOPE (50% uptake inhibition with chlorpromazine; P = 0.0007), meanwhile RmHnC-DOPE showed partial reliance on lipid raft/caveolae pathways (50% inhibition with methyl-p-cyclodextrin; P < 0.0001). clathrin-mediated endocytosis played a negligible role (< 20% inhibition with CPZ).
[0260] The pH-responsive endosomal escape mechanism was validated through bafilomycin A1 pretreatment, which abolished PCSK9 siRNA-mediated gene silencing by 51.3-60.1% for R3H7C- DOPE and R5H5C-DOPE LNPs (P < 0.05), contrasting with SM-102’s moderate 39.1 % reduction (P > 0.1). This disparity underscores the critical role of histidine’s buffering capacity in RmHn-DOPE formulations, which facilitates endosomal membrane disruption through proton sponge effects.
[0261] These findings collectively demonstrate that RmHn-DOPE LNPs leverage clathrin-mediated uptake and histidine-driven pH sensitivity to achieve efficient cytosolic siRNA delivery. The superior performance of R3H7C-DOPE, particularly its balance of arginine-mediated cellular entry and histidine-enabled endosomal escape, positions it as a promising alternative to synthetic lipids like SM-102. The data further highlight the importance of molecular tailoring in lipid design to optimize nucleic acid delivery while minimizing reliance on non-degradable components.
[0262] Biodistribution of RmHn-DOPE LNPs
[0263] The biodistribution profiles and therapeutic efficacy of RmHn-DOPE LNPs were evaluated in C57BL/6 mice following intravenous administration. Cy5-labeled siRNA-loaded LNPs exhibited distinct organ accumulation patterns 2 hours post-injection (FIG. 4A). R3H7C-DOPE and R4H6C- DOPE LNPs showed preferential accumulation in the liver, with fluorescence intensities 2.1- and 1 .8- fold higher than SM-102 LNPs, respectively (P < 0.0001). This aligns with studies demonstrating that modular lipopeptide architectures enhance colloidal stability and minimize opsonization, promoting passive liver targeting via the reticuloendothelial system . In contrast, SM-102 LNPs displayed elevated signals in the spleen and liver. R5H5C-DOPE demonstrated intermediate liver targeting (1 .3- fold vs. SM-102), suggesting that lower histidine content (5:5 R/H ratio) may reduce hepatic specificity by altering surface charge dynamics, as observed in lipid designs where imbalanced cationic-to-pH- responsive ratios compromise organotropism.
[0264] Bioluminescence imaging of flue pDNA-loaded LNPs (FIG. 4B) further validated organspecific delivery. At 24 h post-injection, R5H5C-DOPE demonstrates lung-specific expression while exhibiting no expression in the liver. This finding is partially consistent with biodistribution experimental results, maintaining high pulmonary specificity, though the absence of pDNA expression in the liver may be attributed to differences in the types of nucleic acids delivered. In contrast, flue pDNA delivered via SM-102 predominantly (>80%) expresses in the liver. Comparatively, our lipopeptide LNP demonstrates pronounced active extrahepatic targeting capability, representing a significant advantage for non-liver targeted delivery applications. The sustained luciferase activity underscores the ability of RmHn-DOPE LNPs to protect plasmid DNA during systemic circulation and facilitate efficient transfection in hepatocytes.
[0265] The superior performance of R3H7C-DOPE LNPs can be attributed to their optimized design: the arginine-rich domain enhances siRNA complexation and serum stability, while histidine-mediated pH buffering promotes endosomal escape in hepatocytes. In contrast, SM-102’s reliance on tertiary amines and non-degradable hydrocarbon tails likely contributes to its non-specific biodistribution and reduced hepatic transfection. These findings underscore the advantages of natural, degradable lipopeptides over synthetic lipids, offering enhanced targeting precision and therapeutic efficacy with reduced off-target risks.
[0266] These findings establish RmHn-DOPE LNPs as a modular platform for precision nucleic acid delivery. The ability to tune R/H ratios for payload- and tissue-specific optimization addresses a critical gap in non-viral gene therapy, particularly for hepatotropic disorders or lung diseases requiring extrahepatic targeting. Future studies should explore correlations between protein corona profiles (e.g., apolipoprotein E for liver targeting or Vtn for lung specificity) and in vivo targeting efficiency, building on emerging insights into LNP-host interactions. Additionally, the biodegradability of ester- linked lipopeptides in RmHn-DOPE LNPs reduces long-term toxicity risks compared to non- degradable lipids like SM-102, positioning them as clinically translatable candidates for systemic gene delivery.
[0267] In vivo PCSK9 siRNA transfection efficacy of RmHn-DOPE LNPs
[0268] The therapeutic potential of RmHn-DOPE LNPs was evaluated in C57BL/6 mice following intravenous administration of PCSK9 siRNA-loaded formulations. As shown in FIG. 5A, R3H7C- DOPE LNPs achieved a 82.7 ± 15.1 % reduction in hepatic PCSK9 mRNA levels 7 days post-injection, significantly outperforming SM-102 LNPs (46.2%; P < 0.0001) and untreated controls (no suppression). R4H6C-DOPE and R5H5C-DOPE LNPs showed intermediate efficacy, suppressing PCSK9 expression by 79.1 ± 17.7 % and 29.7 ± 29.5 %, respectively (P < 0.0001 vs. SM-102), which correlated with their reduced in vitro performance and biodistribution profiles.
[0269] Long-term gene silencing was assessed 28 days post-administration using R3H7C-DOPE LNPs, selected for their superior short-term efficacy (FIG. 5B). R3H7C-DOPE maintained a 85.9 ± 7.3 % reduction in PCSK9 mRNA levels. This sustained silencing aligns with the biodegradability and rapid metabolic clearance of RmHn-DOPE LNPs, which minimize hepatic accumulation and associated toxicity. In contrast, SM-102’s non-degradable hydrocarbon tails likely contribute to prolonged tissue retention and reduced long-term efficacy, as reported in prior studies.
[0270] As FIG. 5C, the immunofluorescence staining results shown in the figure also demonstrate that R3H7C-DOPE lipopeptide LNP could still significantly upregulate LDL receptor expression on the hepatocyte surface 28 days post-administration, which is further mediated by the downstream regulatory effects of reduced PCSK9 mRNA levels
[0271] The enhanced performance of R3H7C-DOPE LNPs underscores the advantages of their peptide-lipid hybrid design. The arginine-rich domain facilitates efficient siRNA complexation and hepatocyte targeting, while histidine-mediated pH buffering ensures endosomal escape and cytosolic payload release. These results highlight the potential of RmHn-DOPE LNPs as a durable and liver- specific platform for RNAi therapeutics, addressing critical limitations of synthetic lipid formulations in achieving sustained gene silencing with minimal off-target effects.
[0272] Biocompatibility Test Results
[0273] Based on the blood biochemical analysis of mice administered with lipopeptide LNP and SM- 102 via tail vein injection, our lipopeptide LNP did not induce abnormal elevations in biochemical parameters, indicating well-preserved hepatic and renal functions. Additionally, histopathological examination through HE staining of major organs demonstrated that lipopeptide LNP, similar to SM- 102, caused no significant morphological damage to the major organs of the experimental subjects. These findings collectively suggest the favorable safety profile of lipopeptide LNP in murine models. These findings also demonstrate that our lipopeptide exhibits excellent biocompatibility in vivo, further supporting its favorable biological safety profile in animal models.
[0274] CONCLUSION
[0275] The development of RmHn-DOPE LNPs represents a new approach in nucleic acid delivery, addressing critical challenges in balancing transfection efficiency, biodegradability, and organspecific targeting. By integrating cationic arginine domains for nucleic acid condensation and pH- responsive histidine residues for endosomal escape, these LNPs achieve >83% PCSK9 mRNA silencing in hepatocytes with sustained efficacy (85.9% suppression at 28 days post-injection), outperforming synthetic SM-102 LNPs (46.2% silencing at 7 days) while maintaining >99% cell viability 16. This performance is attributed to the tunable arginine-to-histidine (R/H) ratio, which optimizes colloidal stability and minimizes opsonization, enabling liver-specific accumulation (2.1 -fold higher fluorescence intensity vs. SM-102) through passive targeting via the reticuloendothelial system 15. Remarkably, R5H5C-DOPE LNPs demonstrated lung-specific plasmid DNA expression, underscoring the modularity of this platform for payload- and tissue-specific adaptation — a feature aligned with emerging strategies in organ-targeted LNP design.
[0276] The structure of RmHn-DOPE LNPs are further validated by cryo-TEM, which revealed tightly condensed nucleic acid cores and uniform lipid bilayers, and mechanistic studies confirming histidine’s proton sponge effect in endosomal escape 36. These properties contrast sharply with SM- 102’s reliance on non-degradable hydrocarbon tails and tertiary amines, which contribute to nonspecific biodistribution and long-term toxicity risks. Biocompatibility assessments in murine models revealed no hepatotoxicity or renal impairment, with blood biomarkers (ALT, AST, BUN) and histopathology confirming safety — a critical advantage over Lipofectamine 2000 (73.8% viability) and other synthetic systems.
[0277] This work redefines ionizable lipid engineering by harmonizing natural chemistry with therapeutic versatility. The adaptive molecular geometry of RmHn-DOPE decouples lipid design from payload-specific optimization, enabling applications ranging from transient gene regulation to CRISPR-based epigenome editing. Clinically, RmHn-DOPE LNPs hold promise for treating hypercholesterolemia via sustained PCSK9 suppression and pulmonary disorders requiring extrahepatic delivery, bridging the gap between biodegradable nanocarriers and precision medicine.
[0278] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.
REFERENCES
(1) Tang, Q.; Cao, B.; Lei, X.; Sun, B. B.; Zhang, Y. Q.; Cheng, G. Dextran-Peptide Hybrid for Efficient Gene Delivery. Langmuir 2014, 30 (18), 5202-5208. DOI: Doi 10.1021/La500905z.
(2) Hu, Y.; Wang, H. F.; Song, H. Q.; Young, M.; Fan, Y. Q.; Xu, F. J.; Qu, X. J.; Lei, X.; Liu, Y.; Cheng, G. Peptide-grafted dextran vectors for efficient and high-loading gene delivery. Biomater Sci- t/ 2019, 7 (4), 1543-1553. DOI: 10.1039/c8bm01341 a.
(3) Tang, Q.; Lei, X.; Cao, B.; Sun, B. B.; Zhang, Y. Q.; Cheng, G. A naturally derived dextran-peptide vector for microRNA antagomir delivery. Rsc Adv 2015, 5 (35), 28019-28022. DOI: 10.1039/c4ra12878h.
(4) Qu, X.; Hu, Y.; Wang, H.; Song, H.; Young, M.; Xu, F.; Liu, Y.; Cheng, G. Biomimetic Dextran- Peptide Vectors for Efficient and Safe siRNA Delivery. ACS Appl. Bio Mater. 2019, 2 (4), 1456-1463.
(5) Tang, Q.; Cao, B.; Wu, H.; Cheng, G. Cholesterol-peptide hybrids to form liposome-like vesicles for gene delivery. Pios One 2013, 8 (1), e54460. DOI: 10.1371/journal. pone.0054460
PONE-D- 12-36530 [pii],
(6) Tang, Q.; Cao, B.; Cheng, G. Co-delivery of small interfering RNA using a camptothecin prodrug as the carrier. Chemical Communications 2014, 50 (1 1), 1323-1325. DOI: 10.1039/c3cc47970f.
(7) Mather, B. D.; Viswanathan, K.; Miller, K. M.; Long, T. E. Michael addition reactions in macromolecular design for emerging technologies. Progress in Polymer Science 2006, 31 (5), 487- 531. DOI: DOI 10.1016/j.progpolymsci.2006.03.001 .
(8) Dondoni, A. The emergence of thiol-ene coupling as a click process for materials and bioorganic chemistry. Angew Chem Int Ed Engl 2008, 47 (47), 8995-8997. DOI: 10.1002/anie.200802516.

Claims

1. A lipid nanoparticle comprising a lipopeptide conjugate and a bioactive agent, wherein the lipopeptide conjugate has the formula X-L-Y, wherein
X is a peptide,
L is a linker, and
Y is a lipid.
2. The lipid nanoparticle of claim 1 , wherein the peptide comprises from about 1 to about 10000 arginine residues, from about 1 to about 10000 histidine residues and at least one cysteine residue.
3. The lipid nanoparticle of claim 1 or 2, wherein the peptide comprises arginine residues and histidine residues, wherein the arginine-to-histidine (R/H) ratio in the peptide is from 4:1 to 1:4.
4. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH2-CWR3H3- COOH, NH2-CW 5H5-COOH, NH2-CWR3H3-COOH , NH2-CW 3H4-COOH, NH2-C„ 3H5- COOH, NH2-CWR3H6-COOH, NH2-CWR3H7-COOH, NH2-CW 3H8-COOH, NH2-CWR3H9- COOH, NH2-CWK5H5-COOH, NH2-CWK3H3-COOH, NH2-C„Rm-COOH, NH2-C„Kn-COOH, NH2-CwRoHp-COOH, NH2-CWRUKV-COOH, NH2-CwKpHq-COOH, and NH2-CwRxKyHz- COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N- terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and m, n, 0, p, q, u, v, x, y, z, and w are integers from 1 to 1 ,000.
5. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a random copolymeric peptide having a formula selected from the group consisting of NH2-R3H3- COOH, NH2-R5H5-COOH, NH2-R3H3-COOH, NH2-R3H4-COOH, NH2-R3H5-COOH, NH2- R3H6-COOH, NH2-R3H7-COOH, NH2-R3H8-COOH, NH2-R3H9-COOH, NH2-K5H5-COOH, NH2-K3H3-COOH, NH2-ROHP-COOH, NH2-RUKV-COOH, NH2-KpHq-COOH, and NH2- RxKyHz-COOH; wherein R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and m, n, 0, p, q, u, v, x, y, and z are integers from 1 to 1 ,000.
6. The lipid nanoparticle of any one of claims 1-3, wherein the peptide has a formula selected from the group consisting of NH2-Rm-COOH, NH2-Kn-COOH, NH2-Ho-COOH; wherein R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m, n, and o are integers from 1 to 1 ,000, and N-terminal and C- terminal are either modified or unmodified with functional groups.
7. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a sequence-specific peptide having a formula selected from the group consisting of NH2-CR-COOH, NH2-CR2- COOH, NH2-CR3-COOH, NH2-CR4-COOH, NH2-CR5-COOH, NH2-CR6-COOH, NH2-CRm- COOH, NH2-CHR-COOH, NH2-CH2R-COOH, NH2-CH3R-COOH, NH2-CH4R-COOH, NH2- CH5R-COOH NH2-CHmR-COOH, NH2-CHR2-COOH, NH2-CH2R2-COOH, NH2-CH3R2- COOH, NH2-CH4R2-COOH, NH2-CH5R2-COOH, NH2-CHmR2-COOH, NH2-CHR3-COOH, NH2-CH2R3-COOH, NH2-CH3R3-COOH, NH2-CH4R3-COOH, NH2-CH5R3-COOH, NH2- CH6R3-COOH, NH2-CH7R3-COOH, NH2-CH8R3-COOH, NH2-CH9R3-COOH, NH2-CHmR3- COOH, NH2-CHR4-COOH, NH2-CH2R4-COOH, NH2-CH3R4-COOH, NH2-CH4R4-COOH, NH2-CH5R4-COOH, NH2-CH6R4-COOH, NH2-CH7R4-COOH, NH2-CH8R4-COOH, NH2- CH9R4-COOH, NH2-CHmR4-COOH, NH2-CHR5-COOH, NH2-CH2R5-COOH, NH2-CH3R5- COOH, NH2-CH4R5-COOH, NH2-CH5R5-COOH, NH2-CH6R5-COOH, NH2-CH7R5-COOH, NH2-CH8R5-COOH, NH2-CH9R5-COOH, NH2-CHmR5-COOH, and NH2-CHmRn-COOH, wherein C is cysteine, R is arginine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N- terminal and C-terminal are either modified or unmodified with functional groups.
8. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2-CK- COOH, NH2-CK2-COOH, NH2-CK3-COOH, NH2-CK4-COOH, NH2-CKS-COOH, NH2-CKe- COOH, NH2-CKm-COOH, NH2-CHK-COOH, NH2-CH2K-COOH, NH2-CH3K-COOH, NH2- CH4K-COOH, NH2-CH5K-COOH NH2-CHmK-COOH, NH2-CHK2-COOH, NH2-CH2K2- COOH, NH2-CH3K2-COOH, NH2-CH4K2-COOH, NH2-CH5K2-COOH, NH2-CHmK2-COOH, NH CHKs-COOH, NH2-CH2K3-COOH, NH2-CH3K3-COOH, NH2-CH4K3-COOH, NH2- CH5K3-COOH, NH2-CH6K3-COOH, NH2-CH7K3-COOH, NH2-CH8K3-COOH, NH2-CH9K3- COOH, NH^CHmKs-COOH, NH2-CHK4-COOH, NH2-CH2K4-COOH, NH2-CH3K4-COOH, NH2-CH4K4-COOH, NH2-CH5K4-COOH, NH2-CH6K4-COOH, NH2-CH7K4-COOH, NH2- CHSK4-COOH, NH2-CH9K4-COOH, NH2-CHmK4-COOH, NH2-CHKs-COOH, NH2-CH2Ks- COOH, NH2-CH3K5-COOH, NH2-CH4KS-COOH, NH2-CH5K5-COOH, NH2-CH6K5-COOH, NH2-CH7K5-COOH, NH2-CH8K5-COOH, NH2-CH9K5-COOH, NH2-CHmK5-COOH, and NH2- CHmKn-COOH, wherein C is cysteine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
9. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2-RC- COOH, NH2-R2C-COOH, NH2-R3C-COOH, NH2-R4C-COOH, NH2-R5C-COOH, NH2-R6C- COOH, NH2-RmC-COOH, NH2-RHC-COOH, NH2-RH2C-COOH, NH2-RH3C-COOH, NH2- RH4C-COOH, NH2-RH5C-COOH, NH2-RHmC-COOH, NH2-R2HC-COOH, NH2-R2H2C- COOH, NH2-R2H3C-COOH, NH2-R2H4C-COOH, NH2-R2H5C-COOH, NH2-R2HmC-COOH, NH2-R3HC-COOH, NH2-R3H2C-COOH, NH2-R3H3C-COOH, NH2-R3H4C-COOH, NH2- R3H5C-COOH, NH2-R3H6C-COOH, NH2-R3H7C-COOH, NH2-R3H8C-COOH, NH2-R3H9C- COOH, NH2-R3HmC-COOH, NH2-R4H-COOH, NH2-R4H2C-COOH, NH2-R4H3C-COOH, NH2-R4H4C-COOH, NH2-R4H5C-COOH, NH2-R4H6C-COOH, NH2-R4H7C-COOH, NH2- R4H8C-COOH, NH2-R4H9C-COOH, NH2-R4HmC-COOH, NH2-R5HCOOH, NH2-R5H2C- COOH, NH2-R5H3C-COOH, NH2-R5H4C-COOH, NH2-R5H5C-COOH, NH2-R5H6C-COOH, NH2-R5H7C-COOH, NH2-R5H8C-COOH, NH2-R5H9C-COOH, NH2-R5HmC-COOH, and NH2-RnHmC-COOH, wherein C is cysteine, R is arginine, H is histidine, NH2- is the N- terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
10. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a sequence-specific peptide or polypeptide having a formula selected from the group consisting of NH2-KC- COOH, NH2-K2C-COOH, NH2-K3C-COOH, NH2-K4C-COOH, NH2-KSC-COOH, NH2-KeC- COOH, NH2-KmC-COOH, NH2-KHC-COOH, NH2-KH2C-COOH, NH2-KH3C-COOH, NH2- KH4C-COOH, NH2-KH5C-COOH, NH2-KHmC-COOH, NH2-K2HC-COOH, NH2-K2H2C- COOH, NH2-K2H3C-COOH, NH2-K2H4C-COOH, NH2-K2H5C-COOH, NH2-K2HmC-COOH, NH2-K3HC-COOH, NH2-K3H2C-COOH, NH2-K3H3C-COOH, NH2-K3H4C-COOH, NH2- K3H5C-COOH, NH2-K3H6C-COOH, NH2-K3H7C-COOH, NH2-K3H8C-COOH, NH2-K3H9C- COOH, NH2-K3HmC-COOH, NH2-K4H-COOH, NH2-K4H2C-COOH, NH2-K4H3C-COOH, NH2-K4H4C-COOH, NH2-K4H5C-COOH, NH2-K4H6C-COOH, NH2-K4H7C-COOH, NH2- K4H8C-COOH, NH2-K4H9C-COOH, NH2-K4HmC-COOH, NH2-K5HCOOH, NH2-K5H2C- COOH, NH2-K5H3C-COOH, NH2-K5H4C-COOH, NH2-K5H5C-COOH, NH2-K5H6C-COOH, NH2-K5H7C-COOH, NH2-KSH8C-COOH, NH2-K5H9C-COOH, NH2-KsHmC-COOH, and NH2- KnHmC-COOH, wherein C is cysteine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, m and n are integers from 1 to 1 ,000, and N-terminal and C-terminal are either modified or unmodified with functional groups.
11. The lipid nanoparticle of any one of claims 1-3, wherein the peptide has a formula selected from the group consisting of NH2-CRn-COOH, NH2-CKn-COOH, NH2-CHn-COOH, NH2- RnC-COOH, NH2-KnC-COOH, NH2-HnC-COOH, NH2-RoHp-COOH, H2-HpRo-COOH, NH2- CRoHp-COOH, NH2-CHPRO-COOH, NH2-ROCHP-COOH, NH2-HPCRO-COOH, NH2-R0HPC- COOH, NH2-HPROC-COOH, NH2-CWROHP-COOH, NH2-CWHPRO-COOH, NH2-ROCWHP- COOH, NH2-HpCwRo-COOH, NH2-ROHPCW-COOH, NH2-HPROCW-COOH, wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, - COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, m and n are integers from 0 to 1 ,000, n, o, p, and w are integers from 0 to 1 ,000 but cannot be zero at the same time.
12. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a random or block copolymeric peptide having a formula selected from the group consisting of NH2- X1xX2yX3zX4w-COOH, wherein X1 , X2, X3, and X4 is cysteine, arginine, lysine or histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
13. The lipid nanoparticle of any one of claims 1-3, wherein the peptide has a sequence selected from the group consisting of NH2-CRRRRR-COOH, NH2-CRRRHHH-COOH, NH2-CHHHRRR-COOH, NH2-CKKKKK-COOH, NH2-CKKK-COOH, NH2- CHHHHHRRRR-COOH, NH2-CRRRRRHHHHH-COOH, NH2-CRRRHHH-COOH, NH2- CHHHRRRCOOH, NH2-CKKKKKHHHHH-COOH, NH2-CHHHHHKKKKK-COOH, NH2- CHHHKKK-COOH, NH2-CKKKHHH-COOH, NH2-RRRRRC-COOH, NH2-RRRHHHC- COOH, NH2-KKKKKC-COOH, NH2-KKKC-COOH, NH2-RRRRRHHHHHC-COOH, NH2- RRRHHHC-COOH, NH2-KKKKKHHHHHC-COOH, NH2-KKKHHHC-COOH, NH2- RRRHHHHHHHHC-COOH, NH2-RRRHHHHHHHC-COOH, NH2-RRRHHHHHHC- COOH, NH2-RRRHHHHHC-COOH, NH2-RRRRHHHHHHC-COOH, NH2- RRRRHHHHHHHC-COOH, NH2-RRRRHHHHHHHHC-COOH, wherein C is cysteine, R is arginine, K is lysine, H is histidine, NH2- is the N-terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups.
14. The lipid nanoparticle of any one of claims 1-3, wherein the peptide is a block copolymer having a sequence selected from the group consisting of NH2-CRm-COOH, NH2-CKn- COOH, NH2-CRoHP-COOH, NH2-CRxKyHz-COOH, NH2-RmC-COOH, NH2-KnC-COOH, NH2-ROHPC-COOH, NH2- RxKyHzC-COOH; wherein C is cysteine, R is arginine, K is lysine, H is histidine, and m, n, o, p, q, x, y, and z are integers from 1 to 1 ,000, NH2- is the N- terminal of the peptide, -COOH is the C-terminal of the peptide, N-terminal and C-terminal are either modified or unmodified with functional groups, and w, x, y, and z are integers from 1 to 1 ,000 but cannot be zero at the same time.
15. The lipid nanoparticle of any one of claims 1-14, wherein the linker is a residue of a group selected from the group consisting of amine, carboxylate, hydroxyl, thiol, alkene, alkyne, azide, disulfide, maleimide, ester, peptide, acetal, anhydride, halide, vinyl sulfone, methyl acrylate, acrylate, acrylamide, methyl acrylamide, fluoride, chloride, bromide, aldehyde, and ketone.
16. The lipid nanoparticle of any one of claims 1-15, wherein the lipid is selected from the group consisting of a phosphatidylcholine, a lysophosphatidylcholine, a plasmenylphosphatidylcholine, a phosphatidylethanolamin, a lysophosphatidylethanolamine, a plasmenylphosphatidylethanolamine, a phosphatidylserine, a sphingomyeline, a phosphatidic acid, a lysophosphatidic acid, a phosphatidylinositol, a phosphatidylglycerol, a cardiolipin, a ceramide-1- phosphate, an N-acylsphingosine, a sulfatide, a ganglioside, a sulfoquinovosyldiacylglycero, and a diphosphorylated hexaacyl Lipid A.
17. The lipid nanoparticle of any one of claims 1-15, wherein the lipid is a phospholipid.
18. The lipid nanoparticle of any one of claims 1-15, wherein the lipid is a phosphatidylcholine, a phosphatidic acid, a phosphatidylglycerol, a phosphatidylethanolamine, a phosphatidylserine, or a PEG phospholipid.
19. The lipid nanoparticle of claim 1 , wherein the linker has a residue of a maleimide and the lipid is phosphatidylethanolamine.
20. The lipid nanoparticle of claim 19, wherein the lipid is 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), or any combination thereof.
21. The lipid nanoparticle of claim 19 or 20, wherein the lipid is 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).
22. The lipid nanoparticle of any one of claims 19-21 , wherein the peptide is a random copolymeric peptide having a formula NH2-RmHnC-COOH, wherein C is cysteine, R is arginine, H is histidine, COOH is the N-terminal of the peptide, and m and n are integers from 1 to 15.
23. The lipid nanoparticle of any one of claims 1-14, wherein the lipopeptide conjugate has the structure I wherein Ri and R2 are independently a C8 to C25 alkyl group or a C8 to C25 alkenyl group, o and p are independently integers from 1 to 4, and
X is a peptide.
24. The lipid nanoparticle of claim 23, wherein (a) o and p are each 2 or (b) o and p are each 2 and Ri and R2 are independently an oleyl group or a palmitoyl group.
25. The lipid nanoparticle of any one of claims 1-24, wherein the lipid nanoparticle further comprises one or more helper lipids.
26. The lipid nanoparticle of claim 25, wherein the helper lipid comprises a phospholipid, PEGylated lipid, a sterol, an any combination thereof.
27. The lipid nanoparticle of claim 25 and 26, wherein the lipid peptide conjugate to helper lipid is at a molar ratio of 1 : 100 to 100: 1.
28. The lipid nanoparticle of any one of claims 1-27, wherein the bioactive agent is selected from the group consisting of Abiraterone Acetate, Brentuximab vedotin, Trastuzumab emtansine, Afatinib, Afinitor® (Everolimus), Aldara® (Imiquimod), Alimta® (Pemetrexed Disodium), Pemetrexed, Palonosetron, Chlorambucil, Nelarabine, Axitinib, Belinostat, Bleomycin, Bortezomib, Cabozantinib-S-Malate, Camptothecin, Capecitabine, Ceritinib, Cerubidine® (Daunorubicin), Crizotinib, Dabrafenib, Dasatinib, Degarelix, Docetaxel, Doxorubicin, Epirubicin, Eribulin, Etoposide, Raloxifene, Fulvestrant, Folex® (Methotrexate), Pralatrexate, Eribulin Mesylate, Topotecan, Ibritumomab tiuxetan, Ibrutinib, Irinotecan, Ixempra® (Ixabepilone), Jevtana® (Cabazitaxel), Kadcyla® (Ado- Trastuzumab Emtansine), Lenalidomide, Leuprolide Acetate, Vincristine, Methotrexate, Mitomycin C, Mitoxantrone, Nelarabine, Paclitaxel, Prednisone, Promacta® (Eltrombopag Olamine), Raloxifene Hydrochloride, Lenalidomide, Methotrexate, Synribo® (Omacetaxine Mepesuccinate), Targretin® (Bexarotene), Temsirolimus, Treanda® (Bendamustine Hydrochloride), Velban® (Vinblastine Sulfate), Velsar® (Vinblastine Sulfate), Vincasar PFS® (Vincristine Sulfate), Vinorelbine Tartrate, Vorinostat, Capecitabine, Ipilimumab, and Goserelin Acetate.
29. The lipid nanoparticle of any one of claims 1-27, wherein the bioactive agent is selected from the group consisting of antibodies, peptides, therapeutic enzymes, cytokines, interferons, and interleukins.
30. The lipid nanoparticle of any one of claims 1-27, wherein the bioactive agent is a nucleic acid.
31. The lipid nanoparticle of claim 30, wherein the nucleic acid is selected from the group consisting of plasmid DNA, an oligonucleotide, an aptamers, a DNAzyme, a RNA aptamers, a RNA Decoy, an antisense RNA, a ribozymes, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA, an antagomirs, or any combination thereof.
32. The lipid nanoparticle of claim 30 or 31 , wherein the molar ratio of amine or guanidine groups on the side chain of peptide (N) to the phosphate group in the nucleic acid (P) is from about 0.5:1 to about 100:1.
33. The lipid nanoparticle of any one of claims 1-32, wherein the lipid nanoparticle is between about 2 nm and about 2000 pm in diameter.
34. The lipid nanoparticle of any one of claims 1-33, wherein the lipid nanoparticle has a surface charge of between about -100 mV and about +100 mV at pH 3 to 11.
35. A method for delivering a bioactive agent to a subject, the method comprising administering to the subject the lipid nanoparticle of any one of claims 1-34.
36. The method of claim 35, wherein the bioactive agent is a nucleic acid.
37. The method of claim 35, wherein the bioactive agent is a siRNA.
38. The method of any one of claims 35-37, wherein when the bioactive agent is delivered to the lungs, the arginine-to-histidine (R/H) ratio in the peptide is from 2:1 to 1 :2.
39. The method of any one of claims 35-37, wherein when the bioactive agent is delivered to the liver, the arginine-to-histidine (R/H) ratio in the peptide is from 1 :1 to 1 :3.
40. The method of any one of claims 35-37, wherein when the bioactive agent is delivered to the liver, the arginine-to-histidine (R/H) ratio in the peptide is from 1 :1 to 1 :2.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100240731A1 (en) * 2007-10-02 2010-09-23 Mdrna, Inc. Lipopeptides for delivery of nucleic acids
WO2013032643A2 (en) * 2011-08-31 2013-03-07 Dicerna Pharmaceuticals, Inc. Lipids capable of conformational change and their use in formulations to deliver therapeutic agents to cells
WO2018227231A1 (en) * 2017-06-13 2018-12-20 Monash University Non-viral gene delivery agent comprising lipopeptide (lp) compounds
WO2022260480A1 (en) * 2021-06-11 2022-12-15 주식회사 나이벡 Nanoparticle comprising peptide-lipid conjugate for delivering oligonucleotide into target cell and pharmaceutical composition comprising same
CN116983268A (en) * 2023-08-04 2023-11-03 清华大学 A polypeptide-modified liposome for targeted drug delivery and its application

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100240731A1 (en) * 2007-10-02 2010-09-23 Mdrna, Inc. Lipopeptides for delivery of nucleic acids
WO2013032643A2 (en) * 2011-08-31 2013-03-07 Dicerna Pharmaceuticals, Inc. Lipids capable of conformational change and their use in formulations to deliver therapeutic agents to cells
WO2018227231A1 (en) * 2017-06-13 2018-12-20 Monash University Non-viral gene delivery agent comprising lipopeptide (lp) compounds
WO2022260480A1 (en) * 2021-06-11 2022-12-15 주식회사 나이벡 Nanoparticle comprising peptide-lipid conjugate for delivering oligonucleotide into target cell and pharmaceutical composition comprising same
EP4353268A1 (en) * 2021-06-11 2024-04-17 Nibec Co., Ltd. Nanoparticle comprising peptide-lipid conjugate for delivering oligonucleotide into target cell and pharmaceutical composition comprising same
CN116983268A (en) * 2023-08-04 2023-11-03 清华大学 A polypeptide-modified liposome for targeted drug delivery and its application

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
"Bioreversible Carriers in Drug Design", 1987, AMERICAN PHARMACEUTICAL ASSOCIATION AND PERGAMON PRESS
"Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology", article "Drug Delivery to the Gastrointestinal Tract"
"Larock's Comprehensive Organic Transformations", vol. 1-5, 1989, ELSEVIER SCIENCE PUBLISHERS, article "Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms"
"March's Advanced Organic Chemistry", 1979, JOHN WILEY AND SONS
"Pharmaceutical Dosage Forms", 1981
"Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences", vol. 61, 1993
"Remington: The Science and Practice of Pharmacy", vol. 7, 1995, MACK PUBLISHING CO., pages: 1399 - 1404
AKIHIKO HASEGAWA: "Application of solid dispersions of Nifedipine with enteric coating agent to prepare a sustained-release dosage form", CHEM. PHARM. BULL., vol. 33, 1985, pages 1615 - 1619
ANSEL: "Introduction to Pharmaceutical Dosage Forms", 1976
ASAI TOMOHIRO ET AL: "Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM NL, vol. 444, no. 4, 31 January 2014 (2014-01-31), pages 599 - 604, XP028618788, ISSN: 0006-291X, DOI: 10.1016/J.BBRC.2014.01.107 *
CHUANMEI TANG ET AL: "Modular Design of Lipopeptide-Based Organ-Specific Targeting (POST) Lipid Nanoparticles for Highly Efficient RNA Delivery", ADVANCED MATERIALS, VCH PUBLISHERS, DE, vol. 37, no. 11, 9 February 2025 (2025-02-09), pages n/a, XP072850284, ISSN: 0935-9648, DOI: 10.1002/ADMA.202415643 *
DONDONI, A.: "The emergence of thiol-ene coupling as a click process for materials and bioorganic chemistry", ANGEW CHEM INTED ENGL, vol. 47, no. 47, 2008, pages 8995 - 8997, XP072081529, DOI: 10.1002/anie.200802516
DR. H. P. FIEDLER, LEXIKON DER HILLSSTOFFE FÜR PHARMAZIE, KOSTNETIK UND ANGRENZENDE GEBIETE, 1971, pages 191 - 195
FIESERFIESER'S: "Reagents for Organic Synthesis", vol. 1-40, 1991, JOHN WILEY AND SONS
HU, Y.WANG, H. F.SONG, H. Q.YOUNG, M.FAN, Y. Q.XU, F. J.QU, X. J.LEI, X.LIU, Y.CHENG, G.: "Peptide-grafted dextran vectors for efficient and high-loading gene delivery", BIOMATER SCI-UK, vol. 7, no. 4, 2019, pages 1543 - 1553
KANG JI HEE ET AL: "Self-Assembling Lipid-Peptide Hybrid Nanoparticles of Phospholipid-Nonaarginine Conjugates for Enhanced Delivery of Nucleic Acid Therapeutics", BIOMACROMOLECULES, vol. 18, no. 11, 9 October 2017 (2017-10-09), US, pages 3733 - 3741, XP055794024, ISSN: 1525-7797, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acs.biomac.7b01084> DOI: 10.1021/acs.biomac.7b01084 *
MATHER, B. D.VISWANATHAN, K.MILLER, K. M.LONG, T. E.: "Michael addition reactions in macromolecular design for emerging technologies", PROGRESS IN POLYMER SCIENCE, vol. 31, no. 5, 2006, pages 487 - 531, XP025078607, DOI: 10.1016/j.progpolymsci.2006.03.001
QIONG TANG ET AL: "Cholesterol-Peptide Hybrids to Form Liposome-Like Vesicles for Gene Delivery", PLOS ONE, vol. 8, no. 1, 30 January 2013 (2013-01-30), pages e54460, XP055758931, DOI: 10.1371/journal.pone.0054460 *
QU, XHU, Y.WANG, H.SONG, H.YOUNG, M.XU, F.LIU, Y.CHENG, G.: "Biomimetic Dextran-Peptide Vectors for Efficient and Safe siRNA Delivery", ACS APPL. BIO MATER., vol. 2, no. 4, 2019, pages 1456 - 1463
S. C. PORTER ET AL.: "The Properties of Enteric Tablet Coatings Made From Polyvinyl Acetate-phthalate and Cellulose acetate Phthalate", J. PHARM. PHARMACOL., vol. 22, 1970, pages 42
T. HIGUCHIV. STELLA: "A.C.S. Symposium Series", vol. 14, article "Pro-drugs as Novel Delivery Systems"
TANG, Q.CAO, B.CHENG, G.: "Co-delivery of small interfering RNA using a camptothecin prodrug as the carrier", CHEMICAL COMMUNICATIONS, vol. 50, no. 11, 2014, pages 1323 - 1325
TANG, Q.CAO, B.LEI, X.SUN, B. B.ZHANG, Y. Q.CHENG, G.: "Dextran-Peptide Hybrid for Efficient Gene Delivery", LANGMUIR, vol. 30, no. 18, 2014, pages 5202 - 5208, XP093076236, DOI: 10.1021/la500905z
TANG, Q.CAO, B.WU, H.CHENG, G.: "Cholesterol-peptide hybrids to form liposome-like vesicles for gene delivery", PLOS ONE, vol. 8, no. 1, 2013, pages e54460, XP055758931, DOI: 10.1371/journal.pone.0054460
TANG, Q.LEI, X.CAO, B.SUN, B. B.ZHANG, Y. Q.CHENG, G.: "A naturally derived dextran-peptide vector for microRNA antagomir delivery", RSC ADV, vol. 5, no. 35, 2015, pages 28019 - 28022, XP055470223, DOI: 10.1039/C4RA12878H

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