WO2020044349A1 - Composés, conjugués et compositions destinés à être utilisés dans les méthodes d'administration trans-membranaire de molécules - Google Patents

Composés, conjugués et compositions destinés à être utilisés dans les méthodes d'administration trans-membranaire de molécules Download PDF

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WO2020044349A1
WO2020044349A1 PCT/IL2019/050976 IL2019050976W WO2020044349A1 WO 2020044349 A1 WO2020044349 A1 WO 2020044349A1 IL 2019050976 W IL2019050976 W IL 2019050976W WO 2020044349 A1 WO2020044349 A1 WO 2020044349A1
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formula
conjugate
group
precursor
set forth
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PCT/IL2019/050976
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Ilan Ziv
Hagit Grimberg
Joseph Dubrovsky
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Aposense Ltd.
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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
    • A61K47/51—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
    • A61K47/559—Redox delivery systems, e.g. dihydropyridine pyridinium salt redox systems
    • 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
    • A61K47/554—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 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0072—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the A ring of the steroid being aromatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00

Definitions

  • the invention relates to compounds, conjugates and compositions that that are capable of trans-membrane delivery of molecules and macromolecules into cells in vitro and/or in vivo, destined for utilization for biological purposes e.g., treatment of medical diseases, disorders, conditions and symptoms of a subject.
  • OD Oligonucleotide drugs
  • OD are macromolecule drugs that comprise sequences of nucleosides or nucleotides. OD may hold the promise for revolutionary medical treatments for numerous medical disorders. OD are single-stranded or double- stranded, natural or modified, RNA or DNA molecules, or combinations thereof, as known in the art.
  • Examples for OD are, among others, siRNA (small interfering RNA), being substrates for the RNA-induced silencing complex (RISC); siRNA sequences that are substrates for the Dicer endonuclease [Dicer substrates); dsiRNA], microRNA (miRNA), messenger RNA (mRNA) drugs, or DNA sequences designed to serve as antisense oligonucleotides (ASO); all of which are active in down-regulation of expression of target genes.
  • siRNA small interfering RNA
  • RISC RNA-induced silencing complex
  • dsiRNA] microRNA
  • mRNA messenger RNA
  • ASO antisense oligonucleotides
  • the invention is based on a novel molecular delivery system (MDS), being chemical moieties that have the structure as will be detailed herein below, and which, upon conjugation to a cargo drug, such as, for example, a macromolecular drug, entail delivery of the OD across phospholipid membranes into cells, where it exerts its respective biological activity, for example, gene silencing.
  • MDS molecular delivery system
  • a cargo drug such as, for example, a macromolecular drug
  • the present invention provides conjugates, having the structure as set forth in
  • D is a pharmaceutically active agent; in some embodiments D is a drug, to be delivered across biological membranes ⁇ i.e., a cargo drug), selected from the group consisting of a small-molecule drug, an antibiotic drug, a peptidic drug, a protein, and an OD ⁇ i.e., a drug being a native or modified, single- stranded or double-stranded, DNA or RNA, siRNA, dsiRNA, ASO, or a combination thereof);
  • y, z and w are each independently an integer, selected from the group consisting of 0, 1 , 2, 3 or 4; at least one of y, z or w is different from 0; In some embodiments wherein any of y, z or w is 0 the respective E, E’, E” moiety(s) is/are null;
  • E, E’, or E connected to said D with one or more bonds, can be the same or different, each having independently a structure as set forth in general Formula (II), including pharmaceutically acceptable salts, hydrates, solvates and metal chelates thereof:
  • Ri R 2 and R S are each selected from null, Ci, C 2 , C 3 , CT.CT or C 6 linear, branched or cyclic, alkylene, alkenylene, alkynylene, each optionally interrupted by at least one heteroatom; arylene or heteroarylene moiety, comprising 5, 6, 7, 8, 9 or 10 carbon atoms; each optionally substituted by at least one Ci, C 2 , C 3 , CT.CT or C 6 linear, branched or cyclic, alkyl, alkenyl, alkynyl, each optionally interrupted by at least one heteroatom; each optionally substituted by at least one group selected from amine, amide, halide, CN, guanidine;
  • Z groups are each selected from:
  • A, and B are each independently a straight or branched Ci, C 2 , C3 or C4 alkyl, alkenyl, alkynyl, aryl, heteroaryl, or the corresponding alkylene, alkenylene, alkynylene, arylene or heteroarylene;
  • Z groups can be the same or different;
  • n is an integer, selected independently from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • k and p each are integers selected from 0, 1 , 2, 3, or 4.
  • Z groups can be spaced by 0, 1, 2, 3, 4, 5 or 6 R 4 groups (i.e. in some embodiments one or more R4 groups may be positioned between two or more Z groups), wherein each R 4 group is selected from group consisting of null, and a straight or branched Ci, CF ,C 3 , C 4 , C5, C 6 alkylene chain;
  • Y is selected from null, -O- or -S-;
  • R5 and Re are each independently selected from null, or a Ci, C2, C3, Cd.O or Ce, linear, branched or cyclic, alkylene alkenylene, alkynylene; optionally interrupted by at least one heteroatom;
  • a is an integer selected from 0, 1 , 2, 3, 4, 5, or 6;
  • Wi and W2 are each independently a functional group, capable of connecting to said drug D; said Wi and W2 are each selected from the group consisting of null, hydroxyl, di-hydroxyl, ether, amide, natural or modified nucleoside, and a moiety of Formula (I 1 ):
  • Q is selected from the group consisting of null, a secondary or tertiary amine, and oxygen; wherein each of the open valencies indicated by a ' /N/ ' represent the connection to the drug D; and wherein each of E, E’, or E” can be further linked to at least one of the following: a protecting group PG, phosphate group, sulfate group, carboxyl group, or a solid support and any combinations thereof.
  • Z is guanidine, having the structure as set forth in Formula (lib):
  • Z is a tertiary amine of the Formula (lid):
  • each of a and c are integers, each being selected independently from 0, 1, 2, or
  • Z is a substituted phenyl of general Formula (lie), wherein k and p are integers independently selected from 0, 1, 2, 3, 4, 5, or 6:
  • each of E, E' or E" is independently a moiety having the structure as set forth in Formula (III):
  • a are integers, each being selected independently from 0, 1,2, or 3; b is an integer of 0, 1 or 2; c are integers, each being selected independently from 0, 1, 2, or 3; d are integers, each being selected independently from 0, 1 , 2, 3, 4, 5, or 6; n and m are integers, each being selected independently from 0, 1, 2, 3, 4, 5, or 6.
  • E, E' or E" has the structure as set forth in Formula (nia):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the Invention also provides a Precursor Conjugate, being any E, E' or E" moiety of the Invention, linked to protecting group (s) for alcohol and / or protecting group (s) for amine, e.g., a protecting group for guanidine; wherein said protecting group(s) is / are destined to be removed or modified during chemical processing of the molecule, e.g., during conjugation to a cargo drug.
  • a Precursor Conjugate being any E, E' or E" moiety of the Invention, linked to protecting group (s) for alcohol and / or protecting group (s) for amine, e.g., a protecting group for guanidine; wherein said protecting group(s) is / are destined to be removed or modified during chemical processing of the molecule, e.g., during conjugation to a cargo drug.
  • the Precursor Conjugate comprises a Protecting group (PG) for alcohol, being Dimethoxytrityl [bis-(4-methoxyphenyl) phenylmethyl] (DMT) and / or phosphoramidite and / or a PG for amine, such as a protecting group for guanidine [e.g. tert- butyloxycarbonyl] (BOC group).
  • PG Protecting group
  • PG Protecting group for alcohol
  • DMT Dimethoxytrityl [bis-(4-methoxyphenyl) phenylmethyl]
  • BOC group a protecting group for guanidine
  • E, E' or E" is a precursor molecule, having the structure as set forth in Formula (nib) and Formula (Illb’):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the Invention provides a Conjugate, having the structure as set forth in the following Formula [Cn-I]:
  • each hydrocarbon linker chain comprises 2-6 carbon atoms, and wherein Z in Formula [Cn-I] is hydrogen or a phosphate group.
  • Z in Formula [Cn-I] is hydrogen or a phosphate group.
  • the Invention provides a Precursor Conjugate, having the structure as set forth in the Formula [Cn-I], but wherein the guanidine groups and the tertiary amine group are protected by protecting groups (PG) for amines, as known in the art.
  • This Conjugate is designated [Cn-I-Precursor] and Formula [Cn-I-Precursor’], including pharmaceutically-acceptable salts, hydrates, solvates and metal chelates thereof; wherein each hydrocarbon linker chain comprises 2, 3, 4, 5, or 6 carbon atoms, and wherein Z is hydrogen or a phosphate group.
  • the Invention provides a Conjugate wherein E, E' or E" has the structure as set forth in the following Formula (IV):
  • each of m, n, p, r, t, q, is independently selected from an integer of 0, 1, 2, 3, 4, 5, or 6.
  • E, E' or E" has the structure as set forth in Formula (IVa):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • E, E' or E" is a precursor molecule, having the structure as set forth in Formula (IVb) and Formula (IVb’):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the Invention provides a Conjugate, having the structure as set forth in the following Formula [Cn-II] :
  • each hydrocarbon linker chain comprises 1, 2, 3, 4, 5 or 6 carbon atoms, and wherein Z is hydrogen or a phosphate group.
  • the Invention provides a Precursor Conjugate, having the structure as set forth in the Formula [Cn-P], but wherein the guanidine groups and the tertiary amine group are protected by protecting groups (PG) for amines, as known in the art.
  • This Conjugate is designated [Cn-P- Precursor],
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • Some embodiments of the invention relate to a method for delivery of a drug across a biological membrane into cells, either in vitro or in vivo ⁇ , said method comprising contacting the cells with a Conjugate as described herein.
  • Another embodiment of the Invention relates to a method for treating a medical disorder in a patient in need; the method comprising administering to the patient a therapeutically-effective amount of a pharmaceutical composition, that comprises a Conjugate of the Invention, that includes a drug D, that is useful for the treatment of the disease of said patient, e.g., an antibiotic drug for the treatment of bacterial infection, and a pharmaceutically-acceptable salt or carrier.
  • a pharmaceutical composition comprising a conjugate or precursor of the preceding claims.
  • the invention further provides a Conjugate as disclosed herein above and below for use in the delivery of a drug into biological cells, wherein said cells are in culture, or in a living animal or a human subject.
  • the invention further provides a Conjugate as disclosed herein above and below for use in the delivery of a drug across a phospholipid membrane.
  • the invention further provides a pharmaceutical composition comprising a Conjugate disclosed herein above and below for use in the treatment of a medical disease, disorder, condition or symptom (said disease, disorder, condition or symptom corresponds to the indication of drug D of said Conjugate).
  • the invention further provides a Conjugate as disclosed herein above and below for use in the induction of endocytosis and/or flip-flop in a phospholipid membrane.
  • the Invention also discloses Examples describing methods for chemical synthesis of E moieties of the Invention, and their assembly into useful Conjugates.
  • Figure 1 exemplifies the mode of linkage of an E moiety of the Invention, according to Formula [Cn-P] to an oligonucleotide chain, and respective red-ox- mediated cleavage of the E moiety.
  • Fig. la shows a Conjugate of the Invention, comprising an RNA strand, wherein an E moiety according to Formula (IV) is linked at the 5'-end of the oliginucleotide chain;
  • Fig. lb exemplifies red-ox-mediated cleavage of the disulfide group of this E moiety according to Formula (IV) in reductive conditions, such as those that prevail within the cytoplasm, with consequent release of the RNA drug.
  • Figure 2 exemplifies the Mechanism of Action (MOA) of a Conjugate of the Invention, wherein the Conjugate is according to Formula [Cn-(II)].
  • the RNA Duplex is a Dicer substrate of 25/27 -nucleotide long, with a phosphate group linked at the 5'-end of each strand.
  • Fig. 2(a) demonstrates the intact Conjugate;
  • Fig. 2(b) demonstrates the cleavage and removal of the E and E' moieties in the reductive conditions that prevail in the cytoplasm, leaving for each E or E' moiety a short residual stump;
  • RNA Duplex demonstrates interaction of the RNA Duplex with the Dicer endonuclease, that results in a double-strand break, leaving a 21/21 RNA Duplex, and removal of the stump of the E' moiety, leaving one remaining stump of E, linked at the 5'-end and at an position internal to the phosphate group of the passenger strand;
  • Fig. 2(d) demonstrates the removal of the sense (passenger) strand by the enzyme helicase (i.e. , a cytoplasmatic enzyme, capable of separating RNA strands), with concomitant removal of the remaining stump of E, since it is linked to the passenger strand. Consequently, there is release of the intact antisense strand, free to enter the RNA-induced silencing complex (RISC), in order to induce the desired gene silencing [Fig. 2(e)].
  • helicase i.e. , a cytoplasmatic enzyme, capable of separating RNA strands
  • the invention relates to Conjugates and Precursors thereof, comprising macromolecule drugs such as OD, linked to a novel molecular delivery system (MDS), that can deliver a cargo drug across phospholipid biological membranes into cells, to exert biological activity, such as silencing the expression of a target gene.
  • MDS novel molecular delivery system
  • This delivery system is useful, among others, to enable the trans-membrane delivery of macromolecule drugs, such as genetic drugs, for example, siRNA or dsiRNA, antisense oligonucleotides (ASO), or therapeutic proteins.
  • the invention is based on the discovery and development of novel compounds by the Inventors, that manifest good performance in gene silencing, by combining efficacious trans-membrane delivery across phospholipid membranes, with subsequent robust reduction-based liberation of the cargo OD into the cytoplasm, to exert its biological effect on the cytoplasmatic gene-silencing molecular apparatus.
  • D is a drug to be delivered across biological membranes (i.e., a cargo drug), selected from the group consisting of a small-molecule drug, an antibiotic drug, a peptidic drug, a protein drug, and an OD (i.e., a native or modified, single-stranded or double- stranded, DNA or RNA, siRNA, dsiRNA, ASO, and a combination thereof);
  • a cargo drug selected from the group consisting of a small-molecule drug, an antibiotic drug, a peptidic drug, a protein drug, and an OD (i.e., a native or modified, single-stranded or double- stranded, DNA or RNA, siRNA, dsiRNA, ASO, and a combination thereof);
  • y, z and w are each an integer, independently selected from the group consisting of 0, 1, 2, 3 or 4, wherein if any of y, z or w is 0, it means that the respective E moiety (or moieties) is (are) null; at least one of y, z or w is different from 0;
  • E, E’, or E” connected to said D with one or more bonds can be the same or different, each having independently a structure as set forth in general Formula (II):
  • Ri R 2 and R S are each selected from null, Ci, C 2 , C 3 , CdX ’ s or C 6 linear, branched or cyclic, alkylene, alkenylene, alkynylene, each optionally interrupted by at least one heteroatom; arylene or heteroarylene moiety, comprising 5, 6, 7, 8, 9 or 10 carbon atoms; each optionally substituted by at least one Ci, C 2 , C 3 , C 4 ,C 5 or C 6 linear, branched or cyclic, alkyl, alkenyl, alkynyl, each optionally interrupted by at least one heteroatom; each optionally substituted by at least one group selected from amine, amide, halide, CN, guanidine;
  • Z groups are each selected from:
  • A, and B are each independently a straight or branched Ci, C 2 , C 3 or C 4 alkyl, alkenyl, alkynyl, aryl, heteroaryl, or the corresponding alkylene, alkenylene, alkynylene, arylene or heteroarylene;
  • Z groups can be the same or different;
  • n is an integer, selected independently from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • k and p each are integers selected from 0, 1, 2, 3, or 4;
  • n is an integer, selected independently from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein 0 means null;
  • Z groups can be spaced by 0, 1, 2, 3, 4, 5 or 6 R4 groups, wherein each R4 group is selected from group consisting of null, and a straight or branched Ci, C 2 ,C 3 , C 4 , C 5 , C 6 alkyl chain; R4 that is 0 means null;
  • Y is selected from the group consisting of null, -O- or -S-;
  • Rs and R 6 are each independently selected from null, or a Ci, C2, C3, C 4 ,C 5 or Ce , linear, branched or cyclic, alkylene alkenylene, alkynylene ; optionally interrupted by at least one heteroatom
  • a is an integer of 0, 1, 2, 3, 4, 5, or 6;
  • Wi and W2 are each independently a functional group, capable of connecting to said drug D; said Wi and W2 are each selected from the group consisting of null, hydroxyl, di-hydroxyl, ether, amide, natural or modified nucleoside, and a moiety of Formula (I 1 ):
  • Q is selected from the group consisting of null, -CH2-, a secondary or tertiary amine, and oxygen; wherein each of the open valencies indicated by a ' /x/ ' represent the connection to the drug D; and wherein each of E, E’, or E” can be further linked to at least one of the following: a protecting group PG, phosphate group, sulfate group, carboxyl group, or a solid support and any combinations thereof. [0059] E, E’, or E” can be further linked to at least one of the following: D as defined above; a protecting group, as defined herein (e.g.
  • an E, E' or E" moiety may be linked to one D moiety via one or more points.
  • E, E' or E" may be linked to D via one or more W moieties.
  • Z is a tertiary amine of the formula (lid):
  • each of a and c are integers, each being selected independently from 0, 1,2, or 3.
  • the Invention also provides a Precursor Conjugate, being any E, E' or E" moiety of the Invention, linked to protecting group (s) for alcohol and / or protecting group (s) for amine, wherein said protecting group(s) is / are destined to be removed or modified during chemical processing of the molecule, e.g., during conjugation to a cargo drug.
  • a Precursor Conjugate being any E, E' or E" moiety of the Invention, linked to protecting group (s) for alcohol and / or protecting group (s) for amine, wherein said protecting group(s) is / are destined to be removed or modified during chemical processing of the molecule, e.g., during conjugation to a cargo drug.
  • the Precursor Conjugate comprises a PG for alcohol, being Dimethoxytrityl [bis-(4-methoxyphenyl) phenylmethyl] (DMT) and / or phosphoramidite.
  • E, E' or E" has the structure as set forth in Formula (III):
  • E, E' or E" has the structure as set forth in Formula (nia):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • the Invention provides a Precursor of E, E' or E" according to Formula (Illb) and Formula (Illb’):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the Invention provides a Conjugate, having the structure as set forth in the following Formula [Cn-I]:
  • each hydrocarbon linker chain comprises 1 , 2, 3, 4, 5 or 6 carbon atoms, and wherein Z is hydrogen or a phosphate group.
  • the Invention provides a Precursor Conjugate, having the structure as set forth in the Formula [Cn-I], but wherein the guanidine groups and the tertiary amine group are protected by protecting groups (PG) for amines, as known in the art.
  • This Conjugate is designated [Cn-I-Precursor] and Formula [Cn-I-Precursor’], including pharmaceutically-acceptable salts, hydrates, solvates and metal chelates thereof wherein each hydrocarbon linker chain comprises 1, 2, 3, 4, 5, or 6 carbon atoms, and wherein Z is hydrogen or a phosphate group.
  • E, E' or E has the structure as set forth in Formula (IV):
  • each of m, n, p, r, t, q, is independently selected from an integer of 0, 1, 2, 3, 4, 5, or 6.
  • E, E' or E" has the structure as set forth in Formula (IVa):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • E, E' or E" is a precursor molecule, having the structure as set forth in Formula (IVb) and Formula (IVb’):
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the Invention provides a Conjugate, having the structure as set forth in the following Formula [Cn-II] :
  • each hydrocarbon linker chain comprises 1, 2, 3, 4, 5 or 6 carbon atoms, and wherein Z is hydrogen or a phosphate group.
  • the Invention provides a Precursor Conjugate, having the structure as set forth in the Formula [Cn-P], but wherein the guanidine groups and the tertiary amine group are protected by protecting groups (PG) for amines, as known in the art.
  • PG protecting groups
  • any hydrocarbon chain presented in the above formula may be straight or branched and may include 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • D drug or "Cargo Drug” (i.e . , moiety D) in the context of the present Invention, refers to a molecule that is aimed to be delivered across phospholipid membranes into cells, by the Conjugates of the Invention.
  • D can be either a small-molecule drug, (e.g. antibiotic drug), or a macromolecular drug, such as oligonucleotide drug (OD), peptidic drug, or a protein drug such as a monoclonal antibody.
  • the term“drug” or“medicament” in the context of the present invention relates to a chemical substance, that when administered to a patient suffering from a disease, is capable of exerting beneficial effects on the patient.
  • the beneficial effects can range from amelioration of symptoms, to counteracting the effects of an endogenous or exogenous agent or substance (e.g., proteins), that play(s) a role in etiology and / or pathogenesis of the disease process.
  • the drug may comprise a small molecule or be a macromolecule such as single- or double-stranded RNA or DNA, or a protein, administered to inhibit gene expression or activity.
  • the drug may comprise siRNA, dsiRNA or ASO.
  • the drug is aimed at treating degenerative disorders, cancer, ischemic, infectious, toxic or traumatic insults, an inherited or acquired metabolic disease, or immune-mediated disorders.
  • Oligonucleotide drug refers in the context of the Invention, to a drug that comprises nucleosides or nucleotides.
  • Oligonucleotide drugs are single-stranded or double- stranded, natural or modified RNA or DNA.
  • OD can be siRNA (small interfering RNA), a substrate for the Dicer enzyme (dsiRNA), microRNA (miRNA), messenger RNA (mRNA), or DNA sequences, which are designed to serve as antisense oligonucleotides (ASO).
  • Linkage between the nucleotide building blocks of the OD can be, among others, via phosphate- triester bridges, via phosphorothioate bonds, or via any other method known in the art.
  • the nucleotides which serve as the building blocks of the OD can be either natural or modified nucleotides, such as locked nucleic acids (LNA).
  • OD which are embodiments of the Invention are:
  • siRNA being an RNA duplex, wherein each RNA strand is 19-21 -nucleotide long, aimed at silencing gene expression via the RISC (RNA-induced silencing complex) cytoplasmatic protein complex;
  • siRNA substrate for Dicer being an RNA duplex, wherein each RNA strand is 24-30-nucleotide long.
  • the dsiRNA Duplex consists of one strand of 25 nucleotides, while the second strand consists of 27 nucleotides, with an overhang of 2 nucleotides at the 3'-end of the Guide ( antisense ) strand.
  • the dsiRNA Duplex consists of one strand of 24 nucleotides, while the second strand consists of 27 nucleotides.
  • the dsiRNA Duplex comprises RNA strands of equal length, each consisting of 27 nucleotides.
  • ASO Antisense Oligonucleotide
  • ASO being a synthetic, single stranded, natural or modified DNA or RNA oligonucleotide, usually 15-20 nucleotide long.
  • the sequence of the ASO is antisense, i.e., it is complementary to the sense sequence of a specific mRNA that encodes for a protein, which synthesis is sought to be inhibited. Binding of the ASO to said complementary sequence blocks the ability of ribosomes to move along the mRNA, thus preventing synthesis of the protein, or alternatively, hastens the rate of degradation of the mRNA.
  • antibiotic drugs Another class of drugs which can be potentially useful upon conjugation to E, E', or E" according to Formulae I, II, III, IV, Cn-III, or Cn-IV, is antibiotic drugs.
  • a major current problem in medicine is the prevalent emergence of strains of bacteria that are resistant to antibiotic drugs. Consequently, antibiotic renitence is a major cause of death worldwide.
  • a major mechanism of antibiotic resistance is development of bacterial strains which manifest limited passage of the antibiotic drug through the bacterial cell membranes.
  • the molecular delivery system of the invention may help combat antibiotic resistance via enhancing the delivery of antibiotic drugs through the membranes of the bacterial cells.
  • a "nucleoside” in the context of the present invention is defined as a chemical moiety, that comprises a nitrogenous base (nucleobase), and a sugar of five- or six-carbon atoms (e.g. , ribose or deoxyribose).
  • the nucleobases are selected from natural or modified purines (e.g., adenine, guanine) and natural or modified pyrimidines (e.g., thymine, cytosine, uracil).
  • the nucleobase can be modified by various modifications, as known in the art (e.g., methylation, acetylation).
  • the sugar moiety of the nucleoside can also be modified, as known in the art [e.g. , 2'-deoxy derivative, methylation at the 2’ position of the ribose, installment of a 2'-fluoro atom or 2'-0-methoxyethyl, or having a bridge connecting the 2' oxygen and 4' carbon atoms, thus generating locked nucleic acid (LNA)].
  • LNA locked nucleic acid
  • the nucleoside comprises a pyrimidine derivative, selected from natural or modified cytosine, thymine and uracil, and the sugar moiety is either ribose or deoxyribose.
  • a "nucleotide”, in the context of the Invention, is a nucleoside as defined above, linked to a natural or modified phosphate group. Nucleotides are the building blocks of the oligonucleotides.
  • a "Precursor Conjugate” in the context of the invention is defined as an E, E' or E” moiety, having the structure as set forth in any of II, PI, IV, Cn-PI, or Cn-IV, that is attached to protecting groups, for example, PG for alcohol or amine, as defined below.
  • a "protecting group” in the context of the invention is defined as a chemical group that is destined to be removed or modified during the synthesis of the Conjugate of the Invention. Such removal or modification may occur at various stages of the synthesis; for example, without limitation, during the attachment of E, E' or E" moieties to D, in the case that D is a macromolecule drug, such as an oligonucleotide drug (OD).
  • the protecting group is a protecting group for alcohol, as defined below.
  • a "protecting group for alcohol” in the context of the Invention refers to a chemical group attached to a hydroxyl group, in order to "mask” it during certain chemical reactions, and which is potentially removed thereafter, as known in the art.
  • protecting groups are Acetyl (Ac), Benzoyl (Bz), Benzyl (Bn), b- Methoxyethoxymethyl ether (MEM), Dimethoxytrityl [bis-(4-methoxyphenyl) phenylmethyl] (DMT), Methoxymethyl ether (MOM), Methoxytrityl [(4- methoxyphenyl)diphenylmethyl] (MMT), p-Methoxy- benzyl ether (PMB), Pivaloyl (Piv), Tetrahydropyranyl (THP), Tetrahydrofuran (THF), Trityl (triphenylmethyl, Tr), Silyl ether [e.g., trimethylsilyl (Ac), Benz
  • a "protecting group for amine” in the context of the Invention refers to a chemical group attached to an amine group, in order to "mask” it during certain chemical reactions, and which is potentially removed thereafter, as known in the art.
  • Examples for protecting groups for amine within the scope of the Invention, which are provided in a non-limiting manner are: Carbobenzyloxy (Cbz) group; p-Methoxybenzyl carbonyl (Moz or MeOZ) group; terf-Butyloxycarbonyl (BOC) group; 9-Fluorenylmethyloxycarbonyl (FMOC) group; Phenoxyacetyl (PAC) group; 4-tertbutylphenoxyacetyl (t-PAC) group; Acetyl (Ac) group; Benzoyl (Bz) group, Benzyl (Bn) group; Carbamate group; p- Methoxybenzyl (PMB); 3,4-Dimethoxybenzyl (DMPM); p-methoxyphenyl (PMP) group; Tosyl (Ts) group; Troc (trichloroethyl chloroformate ) group, 2-(trimethylsilyl) ethyl carbamate
  • linkage point to a solid support in the context of the Invention, means a point of attachment of an E, E' or E" moiety to a solid support during chemical synthesis.
  • CPG Controlled Pore Glass
  • CPG may be used as a solid support, for attachment of a 3'-end of the oligonucleotide during the synthesis of the oligonucleotide chains of the invention.
  • alkyl/akylene , alkenyl/alkenylen / akynyl/alkynylene optionally interrupted by at least one heteroatom should be understood to encompass any of the hydrocarbons listed wherein in some embodiments a heteroatom is connected between any two carbon atoms in the hydrocarbon chain. The connection with said heteroatom may be formed by any type of bond including a single bond, a double bond and so forth.
  • biological membrane refers to any phospholipid membrane that is related to a biological system.
  • phospholipid membranes are plasma membranes of cells, intracellular membranes, or phospholipid membranes associated with biological barriers, such as the blood-brain- barrier (BBB), the blood-ocular-barrier (BOB), or the blood-placenta barrier (BPB).
  • BBB blood-brain- barrier
  • BOB blood-ocular-barrier
  • BBP blood-placenta barrier
  • the term "fUp-flop" refers to movement of an amphipatic compound, (namely, a molecule that possesses both hydrophobic and hydrophilic elements) from one leaflet of a phospholipid membrane bilayer into the other leaflet.
  • endocytosis refers to the process by which a living cell takes up molecules bound to its surface; said process comprising folding of the plasma membrane inward, thus bringing said molecules into the cell, into vesicles designated endosomes. Subsequently, the molecules may advantageously be released into to the cytoplasm, to reach and exert activity on cytoplasmatic protein complexes such as RISC, culminating in gene silencing.
  • Embodiments of the invention further relate to the use of
  • Conjugates according to the invention comprising therapeutically-useful drugs, such as protein drugs or OD (e.g., siRNA, dsiRNA or ASO), for the treatment of medical disorders, in a subject in need thereof.
  • the medical disorders may be, without limitation, degenerative disorders, cancer, vascular disorders, metabolic disorders, traumatic, toxic or ischemic insults, infections (e.g., viral or bacterial) or immune-mediated disorders, in which specific protein(s) play(s) a role in either disease etiology or pathogenesis.
  • modulation of expression of the gene(s) encoding for these disease- related proteins, through siRNA or antisense mechanisms, or modulation of the activity of the respective disease-related protein by a therapeutic protein may have beneficial effects in inhibiting disease-related processes, or in treating an underlying cause of the disease.
  • Conjugates according to embodiments of the invention may be used as antisense, siRNA or dsiRNA therapy, which is a form of medical treatment, that comprises the administration of a single-stranded or a double- stranded nucleic acid sequences (DNA, RNA or chemical analogues thereof), that bind either to a DNA sequence that encodes for a specific protein, or to a messenger RNA (mRNA) that translates into a protein.
  • mRNA messenger RNA
  • This treatment may act to inhibit the expression of disease-related genes, thereby preventing the production of disease-related proteins, which may play a role in the etiology or pathogenesis of disease.
  • the Conjugates of the invention may comprise therapeutic proteins, or protein / nucleic acid complexes, such as a Cas9-RNA complex, capable of performing gene editing.
  • Embodiments of the invention also provide pharmaceutical compositions, comprising the conjugates described herein, and pharmaceutically-acceptable carrier(s) or salt(s).
  • the conjugates and pharmaceutical compositions of the invention may be used in vitro (e.g., in cell culture), ex vivo, or in vivo, in a living subject, including in the clinical setting.
  • conjugates of the invention or pharmaceutical compositions comprising conjugates of the invention, for use in the treatment of medical disorders, in a patient in need thereof.
  • Further embodiments of the invention include the use of Conjugates of the invention, in the preparation of pharmaceutical compositions for the treatment of medical disorders, in a patient in need thereof.
  • the medical disorder is cancer, metabolic disease, infectious disease, degenerative disease, vascular disease, trauma, or an immune- mediated disease.
  • Said pharmaceutical compositions can comprise pharmaceutically- acceptable ingredients as known in the art, included to enable beneficial properties to said composition, in aspects such as slow-release, prolongation of residence time, dispersion or safety.
  • a conjugate according to embodiments of the invention may be advantageous in improving the efficacy of the delivery of siRNA, dsiRNA, ASO, or a therapeutic protein such as an antibody, through cell membranes, or through additional biological barriers, such as the Blood-Brain-Barrier (BBB), in comparison to the efficacy of delivery of the same therapeutic agents, which are devoid of conjugation of E, E' or E" moieties of the Invention.
  • BBB Blood-Brain-Barrier
  • conjugates of the Invention may improve the performance of a macromolecule drug in one or more aspects, in addition to efficacy, for example, safety or pharmacokinetics.
  • the conjugates of the Invention may be administered via any mode of administration known in the art, including, among others, per os, intravenous, intramuscular, subcutaneous, intra-tracheal, intra-bronchial, intra-peritoneal or intra thecal routes.
  • Conjugates of the Invention wherein D is an OD
  • D is an OD
  • D can be synthesized, in a non-limiting manner, according to the following method: initially, a gene to be silenced is chosen, based on its role in disease etiology or pathogenesis. Then, based on bio- inform atic methodologies, as known in the art, the nucleotide sequences to be incorporated in the conjugate are designed and determined [typically 19-21 base-pairs double-stranded siRNA for a RISC substrate; or 24-29 base-pairs double-stranded RNA for a Dicer substrate (dsiRNA)]. Synthesis is carried-out in the 3' to 5' direction of the oligo nucleotide.
  • Solid phase synthesis is applied, using protected building blocks, such as protected 2'- deoxynucleosides (dA, dC, dG, and dT), ribonucleotides (A, C, G, and U), or chemically modified nucleosides, e.g. [LNA (locked nucleic acids), or BNA (bridged-nucleic-acids)].
  • protected building blocks such as protected 2'- deoxynucleosides (dA, dC, dG, and dT), ribonucleotides (A, C, G, and U), or chemically modified nucleosides, e.g. [LNA (locked nucleic acids), or BNA (bridged-nucleic-acids)].
  • the building blocks are provided as nucleotide Precursors, wherein the 5'- and the 3'- hydroxyl groups are protected by the alcohol protecting groups DMT and phosphoramidite, respectively. These groups are sequential
  • the E groups are provided as Precursor Conjugates, wherein each being an E, E' or E" moiety of the Invention, linked to protecting group(s), as described above.
  • protecting group can be any protecting group for hydroxyl known in the art, phosphoramidite and DMT [Dimethoxytrityl bis-(4-methoxyphenyl) phenyl methyl] are customarily often used in oligonucleotide synthesis.
  • conjugates of the current Invention provide, as exemplified for conjugates (Cn-I) and (Cn-II) above, an option to link E, E', or E" moieties to either the 5' -end of an oligonucleotide strand, the 3' -end of an oligonucleotide strand, or also at internal positions) along the oligonucleotide chain.
  • the E moieties of the Invention can become integrated within the oligonucleotide chain, similar to any inherent, natural oligonucleotide building block.
  • the linkage between the nucleotides can be performed via standard phosphotriester bonds, or through synthetic phosphorothioate bonds, which may provide advantages such as stability in the blood or favorable binding to blood proteins, or via any other nucleotide linkage strategy, as known in the art.
  • the product Upon completion of the assembly of the chain, the product is released from the solid support into solution, de-protected, and collected.
  • the desired Conjugate is then isolated by high-performance liquid chromatography (HPLC), in order to obtain the desired conjugate of the Invention in high purity.
  • HPLC high-performance liquid chromatography
  • each of the complementary RNA strands is synthesized separately.
  • annealing of the two strands is performed as known in the art, to yield the desired double-stranded siRNA or dsiRNA, which is then subjected to purification and aliquoting.
  • phospholipid biological membranes selected from a group consisting of cell membranes and biological barriers, wherein said biological barriers are selected, for example, from the blood-brain-barrier, the blood-ocular-barrier or the blood-fetal-barrier; the method comprising contacting the cells or respective biological barriers with a Conjugate of the invention.
  • the invention provides a method for delivery a drug into biological cehs, wherein said cells are in culture, or in a hving animal, or in a human subject; the method comprising contacting the cehs with a conjugate, or with a pharmaceutical composition that comprises the conjugate of the Invention.
  • the contact with the cell can be achieved through any route of drug administration known in the art, such as per-os, intravenous, subcutaneous, or intramuscular route.
  • each of E, E’ or E has independently the structure as set forth in any of Formulae III, Ilia, mb, IV, IVa and IVb and Cn-I, Cn-II, or a respective precursor.
  • the invention also comprises methods for specific inhibition of gene expression, in vitro, ex vivo or in vivo.
  • the method may include utilization of a conjugate according to any of any of Formulae I, PI, Ilia, mb, IV, IVa, IVb, Cn-I, Cn-II or a precursor thereof, or a pharmaceutical composition, that includes said conjugate, wherein D is .s iRNA, dsiRNA or an ASO, designed to silence the expression of a specific target gene.
  • the target gene encodes for a pathogenic protein that has a role in the etiology or pathogenesis of a disease.
  • D is a therapeutic protein.
  • the Invention provides, in a non- limiting manner, a method for induction of endocytosis and / or flip-flop within a biological membrane; said method comprising contacting the biological membrane with a conjugate of the Invention, or with a pharmaceutical composition that includes said conjugate, that comprises an OD and E, E' or E" moiety(ies), each having the structure as set forth in any of Formulae III, Ilia, nib, IV, IVa or IVb or Cn-I, Cn-P, or precursor thereof, thus achieving endocytosis and / or flip-flop of said Conjugate, at the biological membrane.
  • the basis for said induction of endocytosis or flip-flop may relate to the structure of the conjugate of the Invention.
  • the conjugate approaches the outer leaflet of the membrane, with its cylindrical RNA duplex being parallel to the membrane surface, and its E moieties being are oriented towards the membrane core, perpendicular to the membrane surface. This orientation thus anchors the RNA Duplex to the membrane's outer leaflet.
  • endosomal escape Subsequent release of the conjugate from the endosomal membrane (often designated “endosomal escape ”) may enable the Conjugate to reach the cytoplasm, where, in the case of an OD, it can exert gene silencing effects, mediated by RISC or DICER protein complexes. Therefore, both endocytosis and flip-flop are processes that are supportive of initiation and / or propagation of trans membrane delivery of the conjugate of the Invention, harboring its macromolecule cargo drug, into cells.
  • a method for induction of endocytosis and / or flip-flop in a phospholipid membrane, such as the plasma membrane is within the scope of the Invention; said method comprising contacting the membrane with the conjugates of the Invention, thereby triggering/? ip-flop or endocytosis.
  • Conjugates according to embodiments of the invention may be used for the treatment of medical disorders.
  • Embodiments of the invention include methods for medical treatment, comprising administering to a patient in need, therapeutically effective amounts of a pharmaceutical composition, comprising a conjugate according to any of Formulae III, Ilia, Illb, IV, IVa and IVb and Cn-I, Cn-II, or a precursor thereof, wherein D is a drug useful for treatment of the respective medical disorder.
  • the method is for genetic medical treatment with siRNA, dsiRNA or ASO as therapeutic agents.
  • Said method comprises the administering to a patient in need, therapeutically effective amounts of a pharmaceutical composition, comprising a conjugate of the invention, according to any of Formulae III, Ilia, Illb, IV, IVa and IVb and Cn-I, Cn-II, or a precursor thereof, wherein D is siRNA, dsiRNA, mRNA, miRNA, ASO or a therapeutic protein, useful in inhibition of expression of a gene, or in blocking activity of a respective protein, that plays a role in the etiology or pathogenesis of a disease in the specific patient.
  • Said treatment may involve delivery of the drug into cells in either culture in vitro, ex vivo (i.e., cells taken out of a living tissue, to optionally be returned to the patient after a therapeutic manipulation), or into cells in a living animal or a human subject in vivo.
  • the cell is a neoplastic cell.
  • the neoplastic cell is a cell of a primary or metastatic tumor.
  • the cell may be an eukaryotic cell, an eukaryotic cell transfected by an oncogenic agent, a human cell, a cell-line, a cell that is a pre-cancerous cell, or any combination thereof.
  • D is a protein, administered as a replacement therapy, i.e., to replace a mutated, malfunctioning protein, thus addressing a physiological need.
  • D is a protein that has as role in gene regulation, including, among others, proteins that have a role in DNA or RNA editing (adding, disrupting or changing the sequence of specific genes).
  • said protein may be a member of the CRISPRs (clustered regularly interspaced short palindromic repeats)-related proteins.
  • said protein can be the Cas9 protein (CRISPR associated protein 9), an RNA-guided DNA nuclease enzyme, or an analogue thereof, potentially loaded with its guide oligonucleotide sequence.
  • a method for genetic treatment of a medical disorder comprises administration to a patient in need, therapeutically effective amounts of a pharmaceutical composition, comprising a Conjugate according to any of Formulae III, Ilia, nib, IV, IVa and IVb and Cn-I, Cn-P, or a precursor thereof, wherein D is a CRISPR protein, such as Cas9, administered together with an appropriate guide oligonucleotide, thus achieving delivery of the protein, loaded with a respective guide oligonucleotide into the cells, where the CRISPR protein can exert its genome editing activity.
  • a pharmaceutical composition comprising a Conjugate according to any of Formulae III, Ilia, nib, IV, IVa and IVb and Cn-I, Cn-P, or a precursor thereof, wherein D is a CRISPR protein, such as Cas9, administered together with an appropriate guide oligonucleotide, thus achieving delivery of the protein, loaded with a respective guide oligonucleotide into the cells
  • a guide oligonucleotide in this context is a sequence of RNA or DNA that guides the Cas9 protein to a specific locus (place) on the genomic DNA, in order to induce DNA cleavage at that site, thus enabling repair of the local defect in the genome.
  • the guide oligonucleotide is a short segment of RNA, the sequence of which is complementary to the sequence of the target DNA locus.
  • conjugates according to embodiments of the invention, and the respective pharmaceutical compositions, as well as the respective methods may be beneficial, among others, in the treatment of medical disorders, selected, among others, from the group consisting of cancer, toxic insults, metabolic disease, ischemic disease, infectious disease, vascular disorders, protein storage disease, trauma, immune-mediated disease, degenerative diseases, inherited or acquired medical disorders.
  • the Invention provides a method for treatment of a medical disorder, said method comprising administering to a patient in need, therapeutically effective amounts of a pharmaceutical composition, that comprises a conjugate according to any of any of Formulae III, Ilia, Illb, IV, IVa and IVb and Cn- I, Cn-II, or a precursor thereof, wherein D is drug useful for the treatment of this medical disorder.
  • the medical disorder is cancer.
  • cancer refers to the presence of cells that manifest characteristics that are typical of cancer-causing cells, such as uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti- apoptotic activity, rapid growth and proliferation rate, or certain characteristic morphology and cellular markers known to be associated with cancer.
  • cancer cells are in the form of a solid tumor, existing either locally within an animal, or circulating in the bloodstream as independent cells, as are, for example, leukemic cells.
  • Conjugates according to embodiments of the invention may be useful, among others, in the treatment of neurodegenerative disorders, such as Alzheimer’s disease, Motor Neuron Disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis and Creutzfeldt-Jacob disease.
  • Conjugates according to embodiments of the invention may be useful, among others, for the delivery of antibiotics to combat bacterial, fungal, or other parasitic infections; or delivery of antiviral agents to combat viral infractions. Accordingly, D of the Conjugates of the invention may have anti- infective properties, thus being useful for the treatment of infectious diseases, such as bacterial or viral infections.
  • Examples of viral infections are, without limitation, respiratory syncytial vims (RSV), human immunodeficiency vims (HIV); hepatotropic viruses, such as hepatitis C vims (HCV) or hepatitis B vims (HBV); infection by orthomyxoviridae, such as influenza vims A, influenza vims B, influenza vims C, or infections by parainfluenza viruses.
  • RSV respiratory syncytial vims
  • HCV hepatitis C vims
  • HBV hepatitis B vims
  • infection by orthomyxoviridae such as influenza vims A, influenza vims B, influenza vims C, or infections by parainfluenza viruses.
  • an embodiment of the Invention is a Conjugate of E, E' or E" moiety (or moieties), linked to an antiviral or antibacterial drug.
  • Such drug can be, among others, an OD, which sequence is aimed at interacting with the genetic material of the infective agent, thus interfering with genetic processes that have a role in the replication, metabolism, infectiveness, or survival of said pathogen.
  • Such genetic sequences can be siRNA or dsiRNA, specifically-designed to silence the expression of gene(s) of the infective agent (e.g ., a virus).
  • the utility of the conjugates of the Invention in combating infection can be in at least one of the following utilizations: either in the delivery of therapeutically- useful agents across biological membranes into cells of the host (e.g., a human patient); or across biological membranes into cells of the pathogen (e.g., bacteria or virus).
  • the host e.g., a human patient
  • the pathogen e.g., bacteria or virus
  • conjugates according to embodiments of the invention may be useful, among others, in the delivery of genetic treatments, aimed at down-regulating the expression of a gene or genes responsible for said metabolic disorder, or for administration of a protein, to replace a defective mutated protein, that has a role in the disease etiology or pathogenesis.
  • the Invention relates to the potential utilization of the conjugates of the Invention to enhance delivery of chemical agents across phospholipid membranes into cells of plants, thus being potentially beneficial for utilizations in agriculture.
  • delivery can have various useful utilizations in agriculture.
  • such delivery in plants can assist in improving crop quality and quantity, among others, by improving plant's genetics, or by eradication of various pathogens: insects, bacteria or fungi.
  • Example 1 General procedure for the synthesis of a conjugate of the invention wherein D moieties are oligonucleotides:
  • a gene to be silenced is chosen, based on its role in disease etiology or pathogenesis. Then, based on bioinformatic methodologies known in the art, the nucleotide sequences to be incorporated in the conjugate are designed and determined [typically 19-21 base-pairs double-stranded siRNA for a RISC substrate, or 24-29 base- pairs double- stranded RNA for a Dicer substrate (dsiRNA)]. [00129] Synthesis is carried-out in the 3' to 5' direction of the oligonucleotide.
  • Solid phase synthesis is applied, using protected building blocks, derived from protected 2'- deoxynucleosides (dA, dC, dG, and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g. [LNA (locked nucleic acids), or BNA (bridged-nucleic-acids)].
  • the building blocks are provided as nucleotide Precursors, wherein the 5'- and the 3'- hydroxyl groups are protected by DMT and phosphoramidite, respectively. These groups are sequentially removed during the reactions of coupling the nucleotide to the growing oligonucleotide chain, in an order as determined by the desired nucleotide sequence.
  • the E groups are provided as Precursoe conjugates, each being an E, E' or E" moiety of the Invention, linked to protecting group, as described above.
  • the protecting group can be any protecting group for hydroxyl known in the art, phosphoramidite and DMT [Dimethoxytrityl bis-(4-methoxyphenyl) phenyl methyl] are customarily often used in oligonucleotide synthesis.
  • a major advantage of conjugates of the current Invention is that they provide the option of linking E, E', or E" moieties to either the 5 '-end of an oligonucleotide strand, the 3' -end of an oligonucleotide strand, or also at an internal position along the oligonucleotide chain.
  • the E moieties of the Invention can become integrated within the oligonucleotide chain, similar to any inherent, natural oligonucleotide building block.
  • the linkage between the nucleotides can be via standard phosphotriester bonds, or through synthetic phosphorothioate bonds, which may provide advantages such as stability in the blood, or in binding to blood proteins, or via any other nucleotide linkage methodology, as known in the art.
  • the product Upon completion of the assembly of the chain, the product is released from the solid support into solution, de-protected, and collected. The desired conjugate is then isolated by high-performance liquid chromatography (HPLC), to obtain the conjugate of the Invention in high purity.
  • HPLC high-performance liquid chromatography
  • each complementary RNA strand is synthesized separately, and then annealing of the two strands is performed in standard conditions, as known in the art, yielding the desired double-stranded siRNA or dsiRNA, which is then subjected to purification and aliquoting.
  • Example 2 Procedure for the synthesis of Formula (IV) - Precursor
  • a potential Mechanism of Action of the Conjugates of the Invention comprising OD is described herein in a non-limiting manner.
  • Said MOA comprises four steps:
  • the Conjugate of the Invention is siRNA or dsiRNA, linked to 2-3 E moieties, each having the structure as set forth in Formulae I, II, PI, or IV, Cn-III, or Cn-IV, or a precursor thereof
  • the Conjugate approaches the outer leaflet of the membrane in a position wherein the cylindrical RNA Duplex is parallel to the membrane surface, and the E, E' or E" moieties are oriented towards the membrane core, perpendicular to the membrane surface. This approach acts to anchor the OD to the membrane surface.
  • Relaxation of the hendins energy may take place through either endocytosis, flip-flop or both. Both processes are supportive of initiation and / or propagation of trans-membrane delivery of a conjugate of the Invention, comprising the macromolecular drug into the cytoplasm, either directly, or through the endosomal compartment.
  • E moieties within the cvtonlasm in a red-ox-mediated process, with release of the carso drus. While one or more E moieties, as described above, is required for the trans-membrane passage of siRNA or dsiRNA conjugates, it is desirable to remove these delivery moieties, once the conjugate reaches the cytoplasm, and excrete them from the body, thus liberating the cargo drug to approach its cytoplasmatic sites of action. In the case that the cargo drug is siRNA or dsiRNA, this cleavage enables to avoid steric hindrance in the interaction of the siRNA or dsiRNA with the gene silencing protein complexes (Dicer and RISC).
  • the E moieties of the Invention comprise a disulfide moiety.
  • the disulfide Under oxidative conditions, such as those that prevail in the extracellular milieu, the disulfide normally manifests high stability, and therefore enables the conjugate of the Invention, upon its systemic administration in vivo, to distribute widely in the body, and contact the huge cell membrane pool.
  • the cytoplasm is a highly reductive environment, mainly due to its high concentrations of reduced glutathione (GSH), being continuously generated within the cytoplasm of any living cell, reaching about three to four-orders of magnitude higher concentrations in the cytoplasm, in comparison to the concentrations of glutathione in the extracellular space: 1-5 mM versus 3-5 mM, respectively. Due to these remarkable reductive conditions within the cytoplasm, disulfide groups of E moieties undergo robust reduction in the cytoplasmatic milieu (demonstrated in Figure 1). Consequently, there is release of the Cargo drug (e.g., dsiRNA), to exert its pharmacological actions at its target sites in the cytoplasm (e.g.
  • GSH reduced glutathione
  • the E moieties of the Invention are excreted from the body via the bile and / or the urine, similar to other sterol- based molecules (e.g., estrogens), either directly, or following metabolism (e.g., cytochrome-P-450-mediated hydroxylation or glucoronidation in the liver).
  • sterol- based molecules e.g., estrogens
  • metabolism e.g., cytochrome-P-450-mediated hydroxylation or glucoronidation in the liver.
  • RNA Duplex Interaction of the liberated OD with the cvtoDlasmatic sites for sene silencins Following the reduction- induced cleavage and removal of the E moieties, a short stump is left per each E moiety, still linked to the RNA Duplex.
  • Each stump comprises a short hydrocarbon chain, linked to a thiol group (exemplified in Figure lb and Figure 2b).
  • the RNA Duplex can then interact with the Dicer endonuclease.
  • This interaction is initiated by binding of the 3'-end of the Guide (Antisense) strand Duplex, which has a 2- nucleotide overhang, to a hydrophobic pocket of the Dicer protein; and interaction of the phosphate group of the Passenger (Sense) strand, with a respective positively-charged pocket on the protein surface.
  • This anchoring process positions the RNA Duplex on the protein surface and enables the enzyme to perform an accurate double-strand break of the RNA Duplex, leaving a 21/21 -nucleotide double-helix, with one E stump remaining on the Passenger (Sense) strand.
  • the enzyme helicase a cytoplasmatic enzyme, capable of separating RNA strands. This action removes the residual stump of the E moiety, thus releasing an intact antisense strand, to enter the RNA-induced silencing complex (RISC), in order to induce the desired gene silencing.
  • RISC RNA-induced silencing complex

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Abstract

L'invention concerne un système d'administration de dmgs à travers des membranes biologiques, notamment des conjugués associés, des procédés pour leur préparation et des méthodes pour l'utilisation.
PCT/IL2019/050976 2018-08-30 2019-08-29 Composés, conjugués et compositions destinés à être utilisés dans les méthodes d'administration trans-membranaire de molécules WO2020044349A1 (fr)

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WO2018127927A1 (fr) * 2017-01-09 2018-07-12 Aposense Ltd. Composés et procédés pour l'administration transmembranaire de molécules
WO2019008574A1 (fr) * 2017-07-04 2019-01-10 Aposense Ltd. Composés et procédés pour l'administration transmembranaire de molécules
WO2019130319A1 (fr) * 2018-01-01 2019-07-04 Aposense Ltd. Composés et procédés pour l'administration transmembranaire de molécules

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024062489A1 (fr) * 2022-09-22 2024-03-28 Aposense Ltd. Systèmes d'administration transmembranaire et utilisations associées

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