EP4313937A1 - Lipides ionisables et nanoparticules lipidiques comprenant lesdits lipides ionisables pour l'administration d'agents thérapeutiques - Google Patents

Lipides ionisables et nanoparticules lipidiques comprenant lesdits lipides ionisables pour l'administration d'agents thérapeutiques

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Publication number
EP4313937A1
EP4313937A1 EP22720433.6A EP22720433A EP4313937A1 EP 4313937 A1 EP4313937 A1 EP 4313937A1 EP 22720433 A EP22720433 A EP 22720433A EP 4313937 A1 EP4313937 A1 EP 4313937A1
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EP
European Patent Office
Prior art keywords
chch2ch
lipid
independently
formula
alkyl
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
Application number
EP22720433.6A
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German (de)
English (en)
Inventor
Bruno De Geest
Emily DE LOMBAERDE
Niek Sanders
Yong Chen
Zifu ZHONG
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Universiteit Gent
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Universiteit Gent
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Publication of EP4313937A1 publication Critical patent/EP4313937A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/04—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/02—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D223/04—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings with only hydrogen atoms, halogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms

Definitions

  • lipid nanoparticles suitable for use in lipid nanoparticles for delivery of a therapeutic agent, such as a nucleic acid, to a subject in need thereof.
  • a therapeutic agent such as a nucleic acid
  • the recent success of mRNA vaccines may in part be attributed to the development of lipid nanoparticle (LNP) delivery systems.
  • the nanostructural properties of the mRNA LNP bear a resemblance to viral systems in terms of their size, lipid envelope and the internal genomic material that contributes to their application as delivery vehicles for vaccines and other therapeutics.
  • lipid nanoparticles also protects the mRNA from enzymatic attack and enhances cell uptake.
  • the mRNA is bound by an ionizable lipid that occupies the central core of the LNP.
  • Polyethylene glycol (PEG) lipid forms the surface of the LNP, along with distearoylphosphatidylcholine (DSPC) and/or 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), which is bilayer forming.
  • DSPC distearoylphosphatidylcholine
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • the hydrolysis of an internal ester bond in the lipid structure results in the formation of a zwitterionic lipid structure, which is no longer capable of complexing the nucleic acid, in particular mRNA, and, hence, the nucleic acid will be released and able to perform its in vivo therapeutic function.
  • the ionizable lipids according to the invention comprise two linear or branched hydrocarbon chains, each connected to the ionizable amine via a C 2-20 alkyl chain and a degradable ester bond. Upon hydrolysis of the degradable ester bonds, the linear or branched hydrocarbon chains are released from the zwitterionic lipid structure without impacting its charge shifting capacity.
  • the ionizable lipids according to the invention allow degradation of the linear or branched hydrocarbon chains under in vivo conditions, thereby reducing or even avoiding accumulation of the linear or branched hydrocarbon chains.
  • a first aspect the present application provides an ionizable lipid having a structure of Formula (I), or in particular of Formula (IA) - wherein R 1 is hydrogen, C 1-12 alkyl, - wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-8 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S; - wherein R 2 and R 3 are each independently C 2-20 alkyl, preferably wherein R 2 and R 3 are each independently
  • the invention provides an ionizable lipid having a structure of Formula (IA) ( ) - wherein R 1 is hydrogen, C 1-12 alkyl, , whe 1 2 rein Y , Y , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-8 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S; - wherein R 2 and R 3 are each independently C2-20alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; more preferably wherein R 2 and R 3 are each independently C2-3alkyl; most preferably wherein R 2 and R 3 are ethyl; and - wherein R 4 and R 5 are each independently selected from
  • the invention provides an ionizable lipid having a structure of Formula (IA), - wherein R 1 is hydrogen, C 1-12 alkyl, , , or , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-8 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); - wherein R 2 and R 3 are each independently C 2-20 alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; more preferably wherein R 2 and R 3 are each independently C 2-3 alkyl; most preferably wherein R 2 and R 3 are ethyl; and - wherein R 1 is hydrogen, C 1-12
  • R 2 is equal to R 3 and R 2 and R 3 are each a -CH 2 CH 2 - or - CH 2 CH 2 CH 2 - spacer.
  • the ionizable lipid thus has a structure according to Formula (II) or (III), , (II) (III) wherein R 1 , R 4 , and R 5 have the same meaning as defined herein.
  • the ionizable lipid thus has a structure according to Formula (II), wherein R 1 , R 4 , and R 5 have the same meaning as defined herein.
  • the ionizable lipid has a structure according to formula (I), (IA), (II) or (III), wherein R 1 is hydrogen, C 1-6 alkyl, ; wherein Y 1 , Y 2 , and Y 3 are each independently C 1-6 alkyl, preferably wherein Y 1 , Y 2 , and Y 3 are each independently C 2-4 alkyl; and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-6 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, and wherein R 2 , R 3 , R 4 , and R 5 have the same meaning as defined herein.
  • R 1 is hydrogen, methyl, ethyl, n-propyl, ; wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-3alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 - N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, more preferably, wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a piperazine, azepane (hexahydroazepine), pyrrolidine, imidazolidine,
  • R 1 is H, CH 3 , ethyl, n-propyl, , , or . In embodiments, R 1 is or .
  • the ionizable lipid has a structure of Formula (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), or (XIII),
  • the ionizable lipid has a structure of Formula (IV), (V), (VI), (VII), or (VIII), wherein R 4 and R 5 have the same meaning as defined herein.
  • R 4 and R 5 are the same or different; preferably R 4 and R 5 are the same.
  • the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (I.1) or Formula (IA.1) in an alkaline medium or in a mixture of THF/H 2 O/NaOH; and - purifying an ionizable lipid having a structure of Formula (I.2) or Formula (IA.2), respectively, wherein R 2 and R 3 are each independently C 2-20 alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; and wherein R 4 and R 5 are each independently selected from C 3-27 alkyl or C 3-27 alkenyl.
  • the method further comprises: - reacting the ionizable lipid having a structure of Formula (I.2) or Formula (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XV) or Formula (XVA), respectively; and (XVA) - reacting the acid chloride lipid intermediate of Formula (XV) or Formula (XVA) with , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-8alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S , thereby obtaining an ionizable lipid having a structure of Formula (I.3), (I
  • R 2 and R 3 are each independently C 2-20 alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; and wherein R 4 and R 5 are each independently selected from C 3-27 alkyl or C 3-27 alkenyl.
  • the methods as taught herein may further comprise: - reacting the ionizable lipid having a structure of Formula (IA.1) in an alkaline medium or in a mixture of THF/H 2 O/NaOH; and - purifying an ionizable lipid having a structure of Formula (IA.2) (IA.2).
  • the methods as taught herein may further comprise: - reacting the ionizable lipid having a structure of Formula (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XVA); and - reacting the acid chloride lipid intermediate of formula (XVA) with , , or , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-8alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s), thereby obtaining an ionizable lipid having a structure of Formula ((IA.3), (IA.4) or (
  • lipid nanoparticle comprising an ionizable lipid according to Formula (I) or Formula (IA) as specified herein.
  • the invention provides a lipid nanoparticle (LNP) comprising an ionizable lipid, wherein the ionizable lipid has a structure of Formula (IA), wherein R 1 is hydrogen, C 1-12 alkyl, , , or , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-8 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R 2 and R 3
  • the LNP further comprises: - a PEGylated lipid; preferably wherein the PEGylated lipid is selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPE)-PEG, distearoyl-rac-glycerol (DSG)-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; - a helper lipid; preferably wherein the helper lipid is selected from the group consisting of a sterol, particularly cholesterol, dioleoylphosphatidylethanolamine (DOPE), and 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC); and/or - a therapeutic agent
  • Another aspect of the present application provides for the use of an ionizable lipid as envisaged herein for producing a lipid nanoparticle (LNP).
  • Another aspect of the present application provides a method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent, the method comprising: - mixing an aqueous solution comprising a therapeutic agent with an organic solvent, such as an ethanolic solution, comprising an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, a PEGylated lipid, and a helper lipid, particularly cholesterol; and - removing the organic solvent such as ethanol, thereby obtaining LNP comprising the therapeutic agent.
  • an organic solvent such as an ethanolic solution
  • the invention relates to method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent, the method comprising: - mixing an aqueous solution comprising a therapeutic agent with an organic solvent comprising an ionizable lipid, a PEGylated lipid, and a helper lipid, wherein the ionizable lipid has a structure of Formula (IA), wherein R 1 is hydrogen, C 1-12 alkyl, , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-8alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R 2 and R 3 are each
  • the PEGylated lipid is selected from the group consisting of DMG- PEG, DSPE-PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; and/or the helper lipid is selected from the group consisting of a sterol, particularly cholesterol, DOPE, and DSPC.
  • the step of removing the organic solvent such as ethanol is performed by dialysis, spin filtration, or evaporation.
  • the therapeutic agent is a nucleic acid, such as DNA or RNA, preferably wherein the therapeutic agent is mRNA; and/or the therapeutic agent is a protein.
  • a further aspect provides a lipid nanoparticle comprising a therapeutic agent, for use in human or veterinary medicine, in particular for use in a method of delivering a therapeutic agent to a subject, particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein.
  • the invention relates to a lipid nanoparticle comprising a therapeutic agent, for use in a method of delivering a therapeutic agent to a subject, particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid having a structure of Formula (IA), wherein R 1 is hydrogen, , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-8alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R 2 and R 3 are each independently C2-20alkyl; wherein R 4 and R 5 are each independently selected from C
  • R 1 is hydrogen,
  • the present application also provides a method for delivering a therapeutic agent to a subject, wherein the method comprises administering a composition comprising a lipid nanoparticle comprising a therapeutically effective amount of the therapeutic agent to the subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein.
  • R 1 and R 6 are as further specified herein.
  • R 2 and R 3 are carbon spacers, as further specified herein.
  • R 4 and R 5 are linear or branched, and/or saturated or unsaturated alkyl chains, as further specified herein.
  • Figure 2 represents the chemical structure (FIG.2A) and 1 H-NMR spectrum (FIG.2B) of SME.
  • FIG. 2A shows the chemical structure with numbered atom positions.
  • peaks are annotated with the corresponding atom position and integrated.
  • X-axis shows chemical shift in ppm
  • Y-axis shows peak intensity in arbitrary units.
  • the term “one or more”, such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6 or ⁇ 7 etc. of said members, and up to all said members.
  • Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may.
  • alkyl refers to a hydrocarbyl group of Formula CnH 2 n+1 wherein n is a number of at least 1. Alkyl groups may be linear, or branched. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain.
  • C3-27alkyl refers to a hydrocarbyl group of Formula CnH 2 n+1 wherein n is a number ranging from 3 to 27, i.e. 3 ⁇ n ⁇ 27.
  • C 3-27 alkyl groups include all linear or branched alkyl groups having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 carbon atoms.
  • the term “lower alkyl groups” as used herein refers to an alkyl group which typically comprises from 1 to 10 carbon atoms (i.e. C1-10alkyl), preferably from 1 to 6 carbon atoms (i.e.
  • C1-6alkyl more preferably 1, 2, 3, 4, 5 or 6 carbon atoms, which may be linear or branched.
  • a lower alkyl group thus includes for example methyl, ethyl, n- propyl, i-propyl, 2-methyl-ethyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, and hexyl and its isomers, and the like.
  • alkenyl refers to an unsaturated hydrocarbyl group, which may be linear or branched, comprising one or more carbon-carbon double bonds, such as 1, 2, 3 or 4 carbon-carbon double bonds.
  • a subscript refers to the number of carbon atoms that the named group may contain.
  • a C3-27alkenyl group includes all linear or branched alkyl groups having 3 to 27 carbon atoms, i.e.
  • fatty acid or “fatty acid moiety” as used herein generally refers to carboxylic acid with a saturated or unsaturated aliphatic chain of carbon atoms.
  • fatty acid includes saturated and unsaturated fatty acids.
  • the fatty acids or fatty acid moieties may be naturally occurring or synthetic fatty acids or fatty acid moieties.
  • saturated fatty acid refers to a carboxylic acid with an aliphatic chain of carbon atoms having the Formula CH 3 (CH 2 )nCOOH, wherein n is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • the term “unsaturated fatty acid” refers to a carboxylic acid with an aliphatic chain of carbon atoms having one or more double bonds between carbon atoms.
  • branched fatty acids or “branched chain fatty acids” refer to fatty acids comprising a main saturated or unsaturated aliphatic chain of carbon atoms, which is substituted with one or more lower alkyl groups, particularly substituted with a C 1-6 alkyl group, such as a methyl, ethyl, n-propyl, butyl, pentyl or hexyl.
  • branched fatty acids refer to carboxylic acids having a saturated or unsaturated aliphatic chain of carbon atoms, wherein said aliphatic chain of carbon atoms comprises at least one tertiary carbon atom or possibly a quaternary carbon atom, i.e.
  • substituted as used in the present application is meant to indicate that one or more hydrogens on the atom indicated in the expression using “substituted” is replaced with a selection from the indicated group, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a chemically stable compound.
  • substituents include alkyl groups, more in particular lower alkyl groups.
  • saturated heterocyclic ring or “saturated heterocyclic structure” generally refers to a ring structure, wherein the ring atoms comprise carbon atoms and one or more non-carbon or heteroatoms, preferably nitrogen, oxygen or sulphur.
  • unsaturated heterocyclic ring or “unsaturated heterocyclic structure” generally refers to a ring structure, wherein the ring atoms comprise carbon atoms and one or more non-carbon or heteroatoms, preferably nitrogen, oxygen or sulphur, wherein the ring structure comprises at least one double bond, particularly a carbon-carbon double bond.
  • An unsaturated heterocyclic ring may be an aromatic ring, also referred to as a heteroaryl ring.
  • the terms “molecular weight” or “molecular mass”, as used herein, refer to the mass of a molecule.
  • the molecular mass can be measured directly using mass spectrometry.
  • the present disclosure generally relates to a novel class of ionizable lipids and their use in the manufacture of LNP as a delivery system for a therapeutic agent, particularly a nucleic acid, such as mRNA.
  • the ionizable lipids according to the present invention have a charge-shifting feature.
  • the ionizable lipid comprises an ionizable amine (Structure A).
  • the cationic charge of the ionizable amine is compensated by an anionic charge of a carboxylic acid, resulting in a zwitterionic structure (Structure B), for instance at pH conditions between pH 1 and pH 14, particularly between pH 5 and pH 7.4.
  • Structure B a zwitterionic structure
  • the latter is no longer capable of complexing a nucleic acid, such as mRNA, and hence, the nucleic acid will be released from the lipid nanoparticle and is able to perform its therapeutic function.
  • the ionizable lipids according to the invention comprise two linear or branched hydrocarbon chains each connected to the ionizable amine via a C2-20alkyl chain and a degradable ester bond.
  • the linear or branched hydrocarbon chains are released from the structure without impacting the charge shifting capacity (Structure C).
  • the ionizable lipids according to the invention allow satisfactory release of the therapeutic agent such as mRNA, while at the same time reducing or even avoiding accumulation of the linear or branched hydrocarbon chains under in vivo conditions.
  • a first aspect the present application provides an ionizable lipid having a general structure of Formula (I) (I) , - wherein R 1 is hydrogen, C 1-12 alkyl, , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-8 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure comprising N as a heteroatom, and optionally further comprising O or S as heteroatom(s); - wherein R 2 and R 3 are each independently C 2-20 alkyl; - wherein R 4 and R 5 are each independently selected from C 3-27 alkyl or C 3-27 alkenyl; and - wherein R 6 is C 2-10 alkyl, preferably wherein R 6 is C 2-6 alkyl.
  • the invention provides an ionizable lipid having a structure of Formula (IA) - wherein R 1 is hydrogen, C 1-12 alkyl, , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-8alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure comprising N as a heteroatom, and optionally further comprising O or S as heteroatom(s); - wherein R 2 and R 3 are each independently C2-20alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; more preferably wherein R 2 and R 3 are each independently C2-3alkyl; most preferably wherein R 2 and R 3 are ethyl; and - wherein R 1 is hydrogen, C
  • m is an integer ranging from 2 to 10, from 2 to 8, or from 2 to 6, more in particular m is 2, 3, 4 or 5.
  • an ionizable liquid having a structure of Formula (IA) wherein R 1 is hydrogen, C 1-12 alkyl, , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-8alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S as heteroatom(s); wherein R 2 and R 3 are each independently C2-20alkyl; and wherein R 4 and R 5 are each independently selected from C3-27alkyl or C3-27alkenyl.
  • R 1 is hydrogen, C 1-12 alkyl, , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, and wherein Z 1 , Z 2 , Z
  • R 2 and R 3 may be the same or different.
  • R 2 and R 3 are the same.
  • R 2 and R 3 are each independently a lower alkyl. More in particular, R 2 and R 3 are each independently C 2-6 alkyl. In embodiments, R 2 and R 3 are each independently C2-3alkyl. Even more in particular, R 2 and R 3 are each independently -CH 2 CH 2 - (i.e.
  • R 1 is generally a C 1-12 alkyl or a residue comprising at least one nitrogen.
  • R 1 is a lower alkyl, more in particular R 1 is a C1-6alkyl.
  • R 1 is methyl, ethyl or n-propyl.
  • R 1 is a C 1-12 alkyl group substituted with an amine, i.e.
  • R 1 is a residue of formula , wherein Y 1 is a C 1-12 alkyl, and Z 1 and Z 2 are each independently C 1-8 alkyl or hydrogen; more in particular wherein Y 1 is a C 2-4 alkyl and Z 1 and Z 2 are each independently C 1-6 alkyl or C 1-3 alkyl.
  • R 1 is -CH 2 CH 2 N(CH 3 ) 2 , -CH 2 CH 2 CH 2 N(CH 3 ) 2 , -CH 2 CH 2 N(CH 2 CH 3 ) 2 or -CH 2 CH 2 CH 2 N(CH 2 CH 3 ) 2 .
  • R 1 comprises a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom, linked to a carbon spacer Y 2 or Y 3 .
  • R 1 is a residue of formula or , wherein Y 2 and Y 3 are a C 1-12 alkyl, and Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-8alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s), more in particular wherein Y 2 and Y 3 are a C2-4alkyl and Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-6alkyl or C1-3alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N- Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s).
  • Y 2 and Y 3 are -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -.
  • the N-Z 3 -Z 4 or the N-Z 5 - N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, from 2 to 8 carbon atoms and optionally an oxygen or sulphur atom.
  • the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a heteroaryl structure comprising one or two nitrogen atoms and, optionally an oxygen or sulphur atom, as the heteroatom(s).
  • Non limiting examples of such saturated or unsaturated heterocyclic structures N-Z 3 -Z 4 or N-Z 5 -N-Z 6 include aziridine, azirine, azetidine, azete, diazetidine, diazete, pyrrolidine, pyrroline, pyrrole, imidazolidine, imidazole, pyrazolidine, pyrazole, thiazolidine, thiazole, isothiazolidine, isothiazole, piperidine, pyridine, diazinane (e.g. piperazine, hexahydropyrimidine, hexahydropyridazine), diazine (e.g.
  • the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated heterocyclic structure comprising one or two nitrogen atoms, from 2 to 8 carbon atoms and optionally an oxygen or sulphur atom.
  • the N-Z 3 -Z 4 cyclic structure is aziridine, azirine, azetidine, azete, pyrrolidine, pyrroline, pyrrole, piperidine, pyridine, azepane (hexahydroazepine), azepine, azocane, azocine, azonane, or azonine ring structure.
  • the N-Z 3 -Z 4 cyclic structure is azepane (hexahydroazepine).
  • the N-Z 5 -N-Z 6 cyclic structure is diazetidine, diazete, imidazolidine, imidazole, pyrazolidine, pyrazole, diazinane (e.g. piperazine, hexahydropyrimidine, hexahydropyridazine), or diazine (e.g. pyrazine, pyrimidine, pyridazine) ring structure.
  • the N-Z 5 -N-Z 6 cyclic structure is piperazine.
  • Z 7 is methyl or ethyl.
  • R 1 may be or ; wherein Z 1 , Z 2 , Z 3 , and Z 4 are each independently C 1-3 alkyl and/or wherein the N- Z 3 -Z 4 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S.
  • R 1 may be or ; wherein Z 1 , Z 2 , Z 3 , and Z 4 are each independently C1-3alkyl and/or wherein the N-Z 3 -Z 4 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one nitrogen atom and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, preferably wherein the N-Z 3 -Z 4 cyclic structure is an azepane (hexahydroazepine), pyrrolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure.
  • Z 1 , Z 2 , Z 3 , and Z 4 are each independently C1-3alkyl and/or wherein the N-Z 3 -Z 4 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one nitrogen atom and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, preferably wherein the N-Z
  • Some particularly preferred ionizable lipids include the lipids according to Formula (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), or (XIII) as shown elsewhere herein, and wherein R 4 and R 5 are each independently selected from C3-27alkyl or C3-27alkenyl.
  • R 4 and R 5 correspond to the hydrophobic hydrocarbon chains of the lipid, wherein the hydrocarbon chain can be saturated or unsaturated.
  • R 4 and R 5 may be the same or different.
  • such ionizable lipids advantageously allow the formation of lipid nanoparticles comprising therapeutic agents such as mRNA which provide for efficient delivery of the therapeutic agent such as the mRNA both in vitro and in vivo, while at the same time also having low cytotoxicity.
  • said fatty acid moiety is a saturated fatty acid moiety or an unsaturated fatty acid moiety.
  • said fatty acid moiety is a linear or branched fatty acid moiety.
  • a preferred saturated fatty acid moiety typically comprises from 8 to 26 carbon atoms, more preferably from 12 to 24 carbon atoms, still more preferably from 12 to 18 carbon atoms.
  • Non-limiting examples of suitable saturated fatty acids are caprylic acid (i.e., octanoic acid), capric acid (i.e., decanoic acid), lauric acid (i.e., dodecanoic acid), myristic acid (i.e., tetradecanoic acid), palmitic acid (i.e., hexadecanoic acid), stearic acid (i.e., octadecanoic acid), arachidic acid (i.e., eicosanoic acid), behenic acid (i.e., docosanoic acid), lignoceric acid (i.e., tetracosanoic acid), and cerotic acid (i.e., hexacosanoic acid).
  • caprylic acid i.e., octanoic acid
  • capric acid i.e., decanoic acid
  • lauric acid i.e., do
  • Preferred unsaturated fatty acid moieties comprises from 12 to 22 carbon atoms, more preferably from 14 to 22 carbon atoms.
  • suitable unsaturated fatty acids are myristoleic acid (i.e., (Z)- Tetradec-9-enoic acid or 9-cis-tetradecenoic acid), palmitoleic acid (i.e., hexadec-9-enoic acid or 9-cis-hexadecenoic acid), sapienic acid (i.e., (Z)-6-Hexadecenoic acid or cis-6- hexadecenoic acid), oleic acid (i.e., (9Z)-Octadec-9-enoic acid or cis-9-Octadecenoic acid), elaidic acid (i.e., (E)-octadec-9-enoic acid), vaccenic acid (i.e., (E)-Octadec acid),
  • R 4 and R 5 are each independently selected from the compounds listed in Table 1.
  • the ionizable lipid has a structure according to Formula (IA) or (II), wherein: - R 1 is hydrogen, methyl, ethyl, n-propyl, ; wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-3 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsatur
  • - R 1 is H, CH 3 , ethyl, n-propyl, ; - R 2 and R 3 are each independently C 2-6 alkyl; and -
  • the ionizable lipid has a structure according to Formula (II), wherein: - R 1 is or ; wherein Z 1 , Z 2 , Z 3 , and Z 4 are each independently C1-3alkyl and/or wherein the N-Z 3 -Z 4 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z 3 -Z 4 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one nitrogen atom and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, more preferably wherein the N-Z 3 -Z 4 cyclic structure is an azepane (hexahydroazepine), pyrrolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure; and - R 4 and R 5 are the same and are selected from [CH 3
  • - R 1 is H, CH 3 , ethyl, n-propyl, , , or ; and - R 4 and R 5 are the same and are selected from [CH 3 (CH 2 ) 7 ][
  • the ionizable lipid has a structure according to Formula (II), wherein: - R 1 is or ; wherein Z 1 , Z 2 , Z 3 , and Z 4 are each independently C1-3alkyl and/or wherein the N-Z 3 -Z 4 cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z 3 -Z 4 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one nitrogen atom and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, more preferably wherein the N-Z 3 -Z 4 cyclic structure is an azepane (hexahydroazepine), pyrrolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure; and - R 4 and R 5 are the same and are selected from [CH 3
  • Such ionizable lipids advantageously allow the formation of lipid nanoparticles comprising therapeutic agents such as mRNA which provide for efficient delivery of the therapeutic agent such as the mRNA both in vitro and in vivo, while at the same time having low cytotoxicity to human cells.
  • the ionizable lipid has a structure according to Formula (II) or Formula (III), wherein: - R 1 is hydrogen, methyl, ethyl, n-propyl, -CH 2 CH 2 N(CH 3 )2, -CH 2 CH 2 CH 2 N(CH 3 )2, - CH 2 CH 2 N(CH 2 CH 3 ) 2 , -CH 2 CH 2 CH 2 N(CH 2 CH 3 ) 2 , or a residue of formula , wherein Y 2 and Y 3 are -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -, Z 7 is methyl or ethyl, and wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom(s).
  • - R 1 is hydrogen, methyl, ethyl, n
  • the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a pyrrolidine, imidazolidine, piperidine, piperazine, morpholine, thiomorpholine, hexahydroazepine, azocane, or azonane ring structure.
  • the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (I.1) in an alkaline medium or in a mixture of THF/H 2 O/NaOH,; and - purifying an ionizable lipid having a structure of Formula (I.2), (I.2) wherein R 2 and R 3 are each independently C2-20alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; and wherein R 4 and R 5 are each independently selected from C3- 27alkyl or C3-27alkenyl, as specified elsewhere herein.
  • the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (I.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XV); and - reacting the acid chloride lipid intermediate of formula (XV) with in wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom(s), thereby obtaining an ionizable lipid having a structure of Formula (I.3), (I.4) or (I.5)
  • R 2 and R 3 are each independently C2-20alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; and wherein R 4 and R 5 are each independently selected from C3- 27alkyl or C3-27alkenyl, as specified elsewhere herein.
  • the method for producing an ionizable lipid as envisaged herein comprises: - reacting a diC2-20alcoholamine, preferably a diC 2-6 alcoholamine, more preferably diethanolamine, in a solution of methylacrylate, thereby obtaining an intermediate an intermediate with Formula (XIVA), (XIVA), wherein R 2 and R 3 are each independently C 2-20 alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl, as specified elsewhere herein; - purifying the intermediate of Formula (XIVA); and - reacting the intermediate of Formula (XIVA) with (i) a C 3-27 alkyl acid chloride or C 3-27 alkenyl acid chloride or with (ii) a C 3-27 alkyl acid or C 3-27 alkenyl acid in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (IA.1), (I.1A), wherein R 2 and R 3 are each
  • R 4 and R 5 may be each independently selected from C 7-25 alkyl or C 13-21 alkenyl.
  • the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (IA.1) in an alkaline medium or in a mixture of THF/H 2 O/NaOH, and - purifying an ionizable lipid having a structure of Formula (IA.2), (I.2A), wherein R 2 and R 3 are each independently C 2-20 alkyl, preferably wherein R 2 and R 3 are each independently C 2-6 alkyl; and wherein R 4 and R 5 are each independently selected from C 3-27 alkyl or C 3-27 alkenyl, as specified elsewhere herein, or from C 7-25 alkyl or C 13-21 alkenyl, or from CH 3 (CH 2 ) 6 -, CH 3 (CH 2 ) 8 -, CH 3 (CH 2 ) 10 -, CH 3 (CH 2 ) 12 -, CH 3 (CH 2 ) 14 -, CH 3 (CH 2 ) 16 -, CH 3 (CH 2 )
  • the method further comprises: - reacting the ionizable lipid having a structure of Formula (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XVA), (XVA); and - reacting the acid chloride lipid intermediate of formula (XVA) with , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C 1-8 alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom(s)., as specified elsewhere herein, thereby obtaining an ionizable lipid having a structure of Formula (IA.
  • lipid nanoparticle comprising an ionizable lipid according to the present application.
  • the lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the different lipid components, such as the ionizable lipid as envisaged herein, the PEGylated lipid and the helper lipids, the degree of lipid saturation, the nature of the PEGylation, and the like.
  • the LNP as envisaged herein comprises: - an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as further specified above, in particular wherein R 1 , Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , N-Z 3 -Z 4 , N-Z 5 -N-Z 6 , R 2 , R 3 , R 4 , and R 5 , and R 6 are as specified above; - at least one PEGylated lipid; and - at least one helper lipid, particularly a sterol and, optionally, one other helper lipid.
  • the LNP further comprises a therapeutic agent.
  • the ionizable lipid as envisaged herein is particularly an ionizable cationic lipid. Such lipids may become positively charged lipids that are able to associate with nucleic acids in lipid/LNP- based delivery systems. A positive charge on the LNP also promotes association with the negatively charged cell membrane to enhance cellular uptake.
  • the lipid component of the LNP as envisaged herein may include one or more PEGylated lipids.
  • a PEGylated lipid is a lipid modified with polyethylene glycol. This may improve the water-solubility and stability of the LNP.
  • a PEGylated lipid may be selected from the non-limiting group consisting of PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated dialkylamines, PEGylated diacylglycerols, PEGylated dialkylglycerols, and mixtures thereof.
  • the PEGylated lipid is selected from the group consisting of DMG-PEG, DSPE- PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa.
  • the PEGylated lipid is a PEG-OH lipid.
  • a "PEG-OH lipid”, also referred to herein as "hydroxy-PEGylated lipid”, is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid and/or on the PEG chain.
  • a PEG-OH or hydroxy-PEGylated lipid comprises a hydroxyl group at the terminus of the PEG chain.
  • Suitable helper lipids are generally known in the art.
  • a preferred helper lipid is a steroid or a sterol, more preferably cholesterol.
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • a particularly preferred sterol is cholesterol or an analogue thereof.
  • Other examples include ergosterol and phytosterols.
  • Other possible helper lipids include dioleoylphosphatidylethanolamine (DOPE), and 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • DOPE dioleoylphosphatidylethanolamine
  • DSPC 1,2- distearoyl-sn-glycero-3-phosphocholine
  • a therapeutic agent as envisaged herein refers to any compound, which when administered to a subject, particularly a cell of a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • the therapeutic agent may be a small molecule, a nucleic acid or a protein or polypeptide.
  • the terms “protein” or “polypeptide” generally encompass proteins encoded by any open reading frame (ORF) of a genome. Where a single ORF encodes a pre-protein which is processed into one, two or more mature proteins, the term may encompass both the pre-protein and the processed mature proteins.
  • nucleic acid as used herein means a polymer of any length composed essentially of nucleotides, e.g., deoxyribonucleotides and/or ribonucleotides.
  • nucleic acid further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g. chemically synthesised) DNA, RNA or DNA/RNA hybrids.
  • a nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised.
  • a “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
  • nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides.
  • the therapeutic agent is a protein or polypeptide, or a nucleic acid, such as DNA or RNA, particularly mRNA.
  • the nucleic acid is a mRNA encoding a therapeutic protein or polypeptide.
  • the mRNA encodes multiple proteins or polypeptides.
  • the mRNA may be designed to encode proteins or polypeptides of interest selected from any of several target categories including, but not limited to, antibodies, vaccines, therapeutic proteins or peptides, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.
  • “Therapeutic protein” refers to a protein that, when administered to a cell has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • the nucleic acid is a mRNA encoding a vaccine antigen.
  • the term “vaccine” refers to a biological preparation that improves immunity to a particular disease.
  • the LNP as envisaged herein has a mean diameter of 10-500 nm, 20- 400 nm, 30-300 nm, or 40-200 nm, such as a mean diameter of 50-150 nm, 50-200 nm, 80- 100 nm or 80-200 nm.
  • Particularly preferred lipid nanoparticles comprise - an ionizable lipid as envisaged herein, particularly an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as further specified above, in particular wherein R 1 , Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , N-Z 3 -Z 4 , N-Z 5 -N-Z 6 , R 2 , R 3 , R 4 , R 5 , and R 6 are as specified above; - at least one PEGylated lipid; - cholesterol, and, optionally, one other helper lipid, and - a therapeutic agent, particularly a nucleic acid, such as mRNA, encoding for a therapeutic protein or a vaccine antigen.
  • a therapeutic agent particularly a nucleic acid, such as mRNA, encoding for a therapeutic protein or a
  • lipid nanoparticles comprising a therapeutic agent.
  • the formation of lipid nanoparticles may be accomplished by methods known in the art.
  • the method for preparing a lipid nanoparticle comprises the steps of: - mixing an aqueous solution comprising a therapeutic agent with an organic solvent, such as an ethanolic solution, comprising an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as specified herein, a PEGylated lipid, and one or more helper lipids, particularly cholesterol; and - removing the organic solvent such as ethanol, thereby obtaining LNP comprising the therapeutic agent.
  • an organic solvent such as an ethanolic solution
  • the PEGylated lipid is selected from the group consisting of DMG- PEG, DSPE-PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; and/or the helper lipid is selected from the group consisting of a sterol, particularly cholesterol, DOPE, and DSPC.
  • the step of removing the organic solvent such as ethanol is performed by dialysis, spin filtration, or evaporation.
  • the organic solvent may be ethanol, propanol, isopropanol, or chloroform.
  • the therapeutic agent is a nucleic acid, such as DNA or RNA, preferably wherein the therapeutic agent is mRNA; and/or the therapeutic agent is a protein.
  • the ionizable lipid as taught herein may be mixed with the therapeutic agent in a ratio of 50:1 to 1:1.
  • the ionizable lipid as taught herein may be mixed with the therapeutic agent in a ratio of 40:1 to 1:1, 30:1 to 2:1, 25:1 to 2:1, 20:1 to 2:1, 10:1 to 2:1, or 5:1 to 2:1.
  • the ratio when the therapeutic agent is a nucleic acid such as mRNA, the ratio may be expressed as the N:P ratio.
  • N:P ratio refers to the proportion of the number of ionisable lipid molecules (expressed as “nitrogen” or “N”) to the number of nucleotides (expressed as “phosphor” or “P”).
  • the ionizable lipid as taught herein may be mixed with the nucleic acid such as mRNA in a N:P ratio of 50:1 to 1:1.
  • the ionizable lipid as taught herein may be mixed with the nucleic acid such as mRNA in a N:P ratio of 40:1 to 1:1, 30:1 to 2:1, 25:1 to 2:1, 20:1 to 2:1, 10:1 to 2:1, or 5:1 to 2:1.
  • the present application further provides pharmaceutical compositions comprising the lipid nanoparticles as envisaged herein, particularly further comprising one or more other pharmaceutically acceptable ingredients, as known to the skilled person, including, but are not limited to polymeric excipients, for instance chitosan derivatives or salts thereof; lipid excipients, such as phospholipids; surfactants; solvents; buffering agents, and the like.
  • the nanoparticles are provided in a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a subject contemplated by the present application.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the lipid nanoparticles and any other optional agent(s) are combined to facilitate administration.
  • the components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency.
  • parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form.
  • Pharmaceutical parenteral formulations include aqueous solutions of the ingredients.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • a further aspect provides a lipid nanoparticle comprising a therapeutic agent, for use in veterinary or human medicine, in particular in a method of delivering the therapeutic agent to a subject, and more particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, in particular wherein R 1 , Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , N-Z 3 -Z 4 , N-Z 5 -N-Z 6 , R 2 , R 3 , R 4 , R 5 , and R 6 are as specified herein.
  • a further embodiment provides a lipid nanoparticle or composition as defined herein comprising a therapeutic agent, in particular a nucleic acid encoding an antigen, for use as a vaccine.
  • the vaccine may be used for inducing an immune response, in particular an immune response against a disease associated antigen or cells expressing a disease associated antigen, such as an immune response against e.g. an infectious agent or cancer. Accordingly, the vaccine may be used for prophylactic and/or therapeutic treatment of a disease involving a disease associated antigen or cells expressing a disease associated antigen.
  • the present application also provides a method for delivering a therapeutic agent, particularly a protein or nucleic acid, wherein the method comprises administering a composition comprising a lipid nanoparticle comprising a therapeutically effective amount of the therapeutic agent to the subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as specified herein.
  • the therapeutic agent is a protein and the method for delivering a therapeutic level of a protein of interest to a subject, comprises administering a composition to the subject in need thereof, said composition comprising a lipid nanoparticle comprising a nucleic acid encoding for the protein of interest, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein.
  • the present application further provides a method for treating a subject comprising administering to a subject in need thereof a composition comprising a lipid nanoparticle comprising a therapeutically effective amount of a therapeutic agent, particularly a protein or nucleic acid, to the subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, in particular wherein R 1 , Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , R 2 , R 3 , R 4 , R 5 , and R 6 are as specified herein.
  • the therapeutic agent is a protein or polypeptide and the method for treating a subject comprises administering a composition comprising a lipid nanoparticle comprising a nucleic acid encoding for the protein or polypeptide of interest, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, in particular wherein R 1 , Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , R 2 , R 3 , R 4 , R 5 , and R 6 are as specified herein.
  • the terms “subject” or “patient” are generally used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non- human mammals and primates.
  • Preferred subjects or patients are human subjects.
  • a phrase such as “a subject in need of treatment” includes subjects that would benefit from treatment of a given condition, particularly proliferative diseases. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in whom said condition is to be prevented.
  • treat or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed proliferative disease, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of proliferative diseases.
  • the present application also provides aspects and embodiments as set forth in the following Statements: 1.
  • R 1 is hydrogen, C 1-12 alkyl, , wherein Y 1 , Y 2 , and Y 3 are each independently C 1-12 alkyl,
  • R 1 is hydrogen, C 1-6 alkyl, ; wherein Y 1 , Y 2 , and Y 3 are each independently C1-6alkyl, preferably wherein Y 1 , Y 2 , and Y 3 are each independently C2-4alkyl, and wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-6alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); preferably wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 and Z 7 are each independently C1-3alkyl and/or wherein the N-Z 3 -Z 4 or the N-Z 5 -N-Z 6 cyclic structure is a saturated or unsaturated heterocycl
  • An ionizable lipid according to any one of statements 1 to 6, wherein R 4 and R 5 are the same or different; preferably wherein R 4 and R 5 are the same.
  • a lipid nanoparticle comprising an ionizable lipid as defined in any one of statements 1 to 7.
  • the LNP further comprises: - a PEGylated lipid; preferably wherein the PEGylated lipid is selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPE)-PEG, distearoyl-rac-glycerol (DSG)-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; - a helper lipid; preferably wherein the helper lipid is selected from the group consisting of cholesterol, dioleoylphosphatidylethanolamine (DOPE)
  • DOPE 1,2-dimyristoyl-rac-g
  • a method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent comprising: - mixing an aqueous solution comprising a therapeutic agent with an ethanolic solution comprising an ionizable lipid as defined in any one of statements 1 to 7, a PEGylated lipid, and a helper lipid; and - removing ethanol, thereby obtaining LNP comprising the therapeutic agent. 14.
  • - the PEGylated lipid is selected from the group consisting of DMG-PEG, DSPE-PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; and/or - the helper lipid is selected from the group consisting of cholesterol, DOPE, and DSPC; and/or - ethanol is removed by dialysis, spin filtration, or evaporation; and/or - the therapeutic agent is a nucleic acid, such as DNA or RNA, preferably wherein the therapeutic agent is mRNA; and/or - the therapeutic agent is a protein.
  • a lipid nanoparticle comprising a therapeutic agent, for use in a method of delivering a therapeutic agent to a subject, particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid as defined in any one of statements 1 to 7.
  • R 1 amine or nitrogen containing moiety
  • Compound 7 or compound 9 is dissolved in a mixture of THF/H 2 O/NaOH and reacted overnight followed by purification by silica column chromatography, yielding compounds 7a and 9a.. Subsequently, these compounds are reacted with oxalylchloride in presence of a catalytic amount of dimethylformamide.
  • Example 7 Synthesis of a comparative ionizable lipid, referred to herein as “SME”
  • SME comparative ionizable lipid
  • BME ionizable lipid according to an embodiment of the invention
  • R 1 Me
  • Example 8 provides the synthesis of the ionizable lipid BME, i.e. the compound having a structure of Formula (IA), wherein R 1 is Me; R 2 and R 3 are each -CH 2 CH 2 -; R 4 and R 5 are each [CH3(CH2)7][CH3(CH2)5]CH- (compound 7 herein).
  • Hexyldecanoic acid (2.4 Eq) was dissolved in 5 mL anhydrous DCM.
  • OME ionizable lipid according to an embodiment of the invention
  • Example 11 provides the synthesis of the ionizable lipid BC7, i.e. the compound having a structure of Formula (IA), wherein R 1 is -CH2CH2-N(CH2)6; R 2 and R 3 are each -CH2CH2-; R 4 and R 5 are each [CH3(CH2)7][CH3(CH2)5]CH- (compound 8.2 herein).
  • BDMA ionizable lipid according to an embodiment of the invention
  • Example 14 Synthesis of lipid nanoparticles (LNP) according to embodiments of the invention
  • LNP comprising mRNA were prepared using the comparative ionizable lipid SME (prepared according to Example 7) and the ionizable lipids illustrating the invention BME, OME, ODMA, BC7, OC7, or BDMA (prepared according to Examples 8, 9, 10, 11, 12, or 13 respectively).
  • Luciferase-coding self-amplifying (sa)RNAs derived from Venezuelan Equine Encephalitis Virus (VEEV) were synthesized by in vitro transcription (IVT) from a I-SceI linearized plasmid (pV01) using the MEGAscript® kit (Thermo Fisher Scientific, Massachusetts, US). Subsequently, the saRNA was purified using silica-based columns (RNeasy Mini Kit, Qiagen, Hilden, Germany) and capped using the ScriptCapTM Cap 1 Capping System Kit (Cellscript, Wisconsin, US) according to the manufacturer’s instructions.
  • RNA was purified again using silica-based columns and the concentration and quality was determined spectrophotometrically (Nanodrop, Thermo Fisher Scientific, Massachusetts, US) and by gel electrophoresis, respectively.
  • Formulation of mRNA in LNP Lipids were dissolved in absolute ethanol and mixed with DMG-PEG 2K (Avanti Polar Lipid), cholesterol and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) in a ratio of 50: 1.5: 38.5: 10 (Lipid: DMG-PEG 2K: Cholesterol: DOPE).
  • Sa-RNA was dissolved in RNase free NaOAC buffer (7,5mM, pH 4,5).
  • Lipid nanoparticles were produced by mixing ethanolic solutions comprising all lipids with aqueous solutions comprising mRNA in acetate buffer (5 mM, pH 4).
  • Example 15 In vitro transfection using lipid nanoparticles (LNP) according to embodiments of the invention saRNA-LNP comprising the comparative ionizable lipid SME or the ionizable lipids illustrating the invention BME, OME, ODMA, BC7, OC7, or BDMA, prepared according to Example 14 were used to transfect different cell types in vitro: HeLa cells ( Figure 3), MC38 cells ( Figure 4), and DC2.4 cells ( Figure 5) Different N:P ratios were tested namely N:P 5:1 (panel A), 10:1 (panel B) and 20:1 (panel C).
  • ionizable lipids and saRNA to be mixed was calculated based on the average molecular mass of nucleotides and the molecular weight of the lipids.
  • In vitro mRNA transfection Human HeLa cells, mouse MC38 cells and mouse DC2.4 cells were seeded in a 24-well plate at a concentration of 50.000 cells/well (500 ⁇ L cell suspension/well). Cells were transfected with 10 ⁇ L saRNA-LNP (0.5 ⁇ g) per well and the plate was incubated for 24 hours in the incubator (37°C, 5% CO2, 95% humidity).
  • Example 16 Cytotoxicity of lipid nanoparticles (LNP) according to embodiments of the invention
  • LNP lipid nanoparticles
  • MTT assays were performed using the best performing LNP illustrating the invention of Example 15, namely LNP comprising OC7, ODMA, or BDMA, and using the comparative LNP comprising SME.
  • LNP lipid nanoparticles
  • LNPs (N:P 5:1) were made as described previously and were added to each well in following concentration range: 9 ⁇ g/mL, 3 ⁇ g/mL, 1 ⁇ g/mL, 0.33 ⁇ g/mL, 0.11 ⁇ g/mL, 0.037 ⁇ g/mL.
  • the 96-well plate was incubated for 24 hours at 37 °C.
  • 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) stock-solution was prepared by dissolving the yellow tetrazolium salt in phosphate-buffered saline (PBS) (50 mg in 10 mL).
  • PBS phosphate-buffered saline
  • Luciferase-coding self-amplifying (sa)RNAs derived from Venezuelan Equine Encephalitis Virus (VEEV) were synthesized by in vitro transcription (IVT) from a I-SceI linearized plasmid (pV01) using the MEGAscript® kit (Thermo Fisher Scientific, Massachusetts, US).
  • IVTT in vitro transcription
  • pV01 I-SceI linearized plasmid
  • MEGAscript® kit Thermo Fisher Scientific, Massachusetts, US.
  • the saRNA was prepared using cleancap reagent AU (TriLink Biotechnologies) and MEGAscript kit (Life Technologies, Waltham) to do transcript and capping in one step.
  • mice Female 6 weeks BALB/cJRj mice were purchased from Janvier labs (Le Genest-Saint-Isle, France) and housed in individual ventilated cages under 14 hours light and 10 hours dark cycle. Mice were accommodated for two weeks prior to the experiments. mRNA formulation and in vivo evaluation The luciferase encoding saRNA was formulated with the LNPs containing respectively OC7, ODMA and BDMA as ionizable lipids at a N:P ratio of 5:1.
  • mice were anesthetized by isoflurane and injected intramuscularly in hindlimb muscle with 1 ⁇ g saRNA formulated in the different LNPs that were dispersed in 50 ⁇ L PBS.
  • the luciferase expression as a function of time was monitored by in vivo bioluminescence imaging.
  • ROI region of interest
  • Results are provided in Figure 7A for OC7-LNPs, Figure 7B for ODMA-LNPs, and Figure 7C for BDMA-LNPs.
  • the area under curve (AUC) was calculated by Prism 8.0.2 and provided in Figure 7D.
  • the corresponding data in Figure 7 indicate that mRNA formulated in LNP containing OC7 exhibited the highest in vivo mRNA delivery.

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Abstract

L'invention concerne une nouvelle classe de composés lipidiques ionisables ainsi que des nanoparticules lipidiques comprenant lesdits composés lipidiques ionisables. Avantageusement, lors de l'hydrolyse d'une liaison ester interne, qui est susceptible de se produire dans des conditions typiques in vivo, une structure lipidique zwittérionique est obtenue, qui n'est plus capable de complexer un acide nucléique, qui sera ainsi libérée de la nanoparticule lipidique et est ensuite capable d'effectuer sa fonction thérapeutique.
EP22720433.6A 2021-04-02 2022-04-04 Lipides ionisables et nanoparticules lipidiques comprenant lesdits lipides ionisables pour l'administration d'agents thérapeutiques Pending EP4313937A1 (fr)

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