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JP-7854977-B2 - Compounds and methods for transmembrane delivery of molecules

JP7854977B2JP 7854977 B2JP7854977 B2JP 7854977B2JP-7854977-B2

Inventors

  • ジヴ、イーラン
  • グリムベルグ、ハギット
  • ドュブロフスキー、ヨセフ

Assignees

  • アポセンス リミテッド

Dates

Publication Date
20260507
Application Date
20231129
Priority Date
20180101

Claims (10)

  1. A conjugate having the structure shown in formula (I) below, or a pharmaceutically acceptable salt thereof. During the ceremony, D is RNA, DNA, or any combination thereof, selected from single-stranded or double-stranded, natural or modified RNA or DNA ; y, z, and w are integers independently selected from the group consisting of 0, 1, 2, 3, and 4, and at least two of y, z, and w are non-zero; E, E', or E'' may be identical or different from each other, and independently have the structure shown in formula (II) below. During the ceremony, M1 , M2 , M3 , and M4 are independently selected from the group consisting of N', N'', null, and ether; N' and N'' are independently selected from the group consisting of -N( CH3 )- and -NH-; M1 , M2 , M3 , and M4 may be the same or different from each other; N' and N'' may be the same or different from each other; The L portion is a linker selected from the group consisting of null, C1 , C2 , C3 , C4 , C5 , and C6 alkylenes; G1 , G2 , G3 , and G4 independently represent a hydrogen atom or a methyl group; at least two of the G1 , G2 , G3 , or G4 groups are hydrogen atoms; a, b, c, d, and e are integers independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6, where 0 = null; a, b, c, d, and e may be identical or different from each other; g represents an integer selected from 0, 1, 2, 3, 4, or 5; W is the structure shown in the following equation (II 1 ). During the ceremony, J is selected from the group consisting of null, -CH2- , and oxygen. (i) W is bonded to D via one -O- group , and another -O- group is further bonded to an alcohol protecting group, hydrogen, or phosphoric acid, or (ii) W is bonded to D via two -O- groups, The aforementioned alcohol protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether, ethoxyethyl ether (EE), phosphoramidite, and N-hydroxysuccinimide (NHS).
  2. g is 1, c and d independently represent integers 1, 2, or 3. c and d may be the same or different from each other. The conjugate according to claim 1.
  3. In the above formula (II 1 ) representing W, The aforementioned J is null or -CH2- , The aforementioned W is bonded to the aforementioned D, and further bonded to hydrogen or phosphoric acid, All of the aforementioned G1 , G2 , G3 , or G4 groups are hydrogen atoms, The conjugate according to claim 1, wherein D is coupled to at least two of E, E', or E''.
  4. Each of the above N' and N'' is -N( CH3 )-, The conjugate according to claim 1, wherein D is an oligonucleotide, and E, E', or E'' have the same structure.
  5. The conjugate according to claim 1, wherein each of y, z, and w is 1.
  6. The aforementioned parts E, E', or E'' are Is it a compound having the structure shown in formula (V) below? Is it a compound having the structure shown in formula (VI) below? Is it a compound having the structure shown in formula (VII) below? Is it a compound having the structure shown in formula (XIIa) below? or containing a pharmaceutically acceptable salt thereof, The conjugate according to claim 1, wherein q is an integer of 1, 2, 3, 4, 5, or 6.
  7. A precursor molecule of the conjugate according to claim 1, It consists of a compound having the structure of E in formula (II), In formula (II) above, W is a precursor molecule which is a structure shown in the following formula. In the formula, J is null, and the -O- group of W is bonded to a protecting group of (i) a phosphoramidite or (ii) an alcohol. The aforementioned alcohol protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether, ethoxyethyl ether (EE), phosphoramidite, and N-hydroxysuccinimide (NHS). When one of M1 , M2 , M3 , or M4 is a secondary amine, the secondary amine is bonded to an amine protecting group, and the amine protecting group is a carbobenzyloxy (Cbz) group, a p-methoxybenzylcarbonyl (Moz or MeOZ) group, a tert-butyloxycarbonyl (BOC) group, a 9-fluorenylmethyloxycarbonyl (FMOC) group, a phenoxyacetyl (PAC) group, a 4-tert-butylphenoxyacetyl (t-PAC) group, an acetyl (Ac) group, a benzoyl (Bz) group, a benzyl (Bn) group, a carbamate group, a p-methoxybenzyl (PMB), a 3,4-dimethoxybenzyl (DMPM), a p-methoxyphenyl (PMP) group, a tosyl (Ts) group, a Troc (trichloroethyl chloroformate) group, or a 2-(trimethylsilyl)ethyl carbamate (TEOC).
  8. A pharmaceutical composition, The conjugate described in claim 1, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  9. Use of the pharmaceutical composition according to claim 8 for manufacturing a medicine for the treatment of a patient's illness.
  10. The use according to claim 9, wherein the treatment is a treatment that involves downregulating the expression of genes related to the disease.

Description

This invention relates to delivery systems, cargo compounds, and polymers, as well as compounds and conjugates, and methods for delivering molecules and polymers across biological membranes to cells, for use in biological purposes in vitro and in vivo. Oligonucleotide drugs (ODs) are macromolecular drugs containing sequences of nucleosides or nucleotides. ODs hold the potential for innovative treatments for many medical disorders. As is known in the art, ODs are single-stranded or double-stranded, native or modified RNA or DNA molecules, or combinations thereof. Examples of ODs include, in particular, siRNA (small interfering RNA) that is a substrate for RNA-induced silencing complexes (RISCs); siRNA sequences that are substrates for Dicer endonucleases (dsiRNA); microRNA (miRNA), messenger RNA (mRNA) drugs; or DNA sequences designed to function as antisense oligonucleotides (ASOs), all of which are active in the downstream regulation of target gene expression. Due to the large, heavily charged structure of orbital diuretics (OD), there is an unmet need for delivery systems that can deliver OD across hydrophobic phospholipid membranes to cells. For the purpose of OD utilization in clinical settings, some features of OD conjugates bound to delivery systems may include redox-sensitive cleavable groups that are advantageous, such as activity in the presence or absence of plasma proteins, or that are stable in the extracellular compartment but undergo efficient cleavage under reducing conditions where cytoplasmic reduction is dominant, thus enabling cargo drugs such as OD to exert their activity against cytoplasmic targets such as Dicer or RISC. The mode of binding of the E portion of the present invention to the oligonucleotide chain according to formula (III), and the respective redox-mediated cleavage of the E portion are illustrated. Figure 1A shows an RNA chain to which the E portion according to formula (III) is bound at an internal position.Figure 1B illustrates the redox-mediated cleavage of the disulfide group in this E portion by formula (III) under reducing conditions, such as when RNA drugs are dominant in the cytoplasm, along with the resulting release of RNA drugs.The mechanism of action (MOA) of the conjugate of the present invention is illustrated, and the conjugate is given by formula [Cn-2-(III)]. The RNA double strand is a 25/27 nucleotide length Dicer substrate, and each strand has a phosphate group attached at the 5' end. The W group of the internally located E portion follows formula (II 1 ), and J is -CH2- . Figure 2A shows the intact conjugate.Figure 2B shows the cutting and removal of the E, E', and E'' portions under reducing conditions, which are dominant in the cytoplasm, leaving short residual edges on each of the E, E', or E'' portions.Figure 2C illustrates the interaction between the RNA double strand and Dicer endonuclease, resulting in a double-strand break that leaves the 21/21 RNA double strand, as well as the removal of the E'' portion, leaving residual E and E' fragments joined at the 5' end and within the passenger strand.Figure 2D shows the removal of the sense (passenger) strand by an enzymatic helicase (i.e., a cytoplasmic enzyme capable of separating RNA strands), along with the simultaneous removal of residual fragments of the E and E' portions bound to the passenger strand.As a result, intact antisense strands are released and enter the RNA-induced silencing complex (RISC) to induce the desired gene silencing [Figure 2E].The performance of the present invention's formula [Cn-1-(IX)], i.e., the conjugate having E and E' portions according to formula (IX), in silencing the expression of the ApoC3 gene after intravenous administration in a mouse model in vivo is described (Figure 3A).Gene silencing in the liver (Figure 3B). The experimental groups were: (i) Vehicle: 5% glucose in water for injection, (ii) ApoC3 "naked" dsiRNA (without binding of the molecular nanomotor portion), (iii) [Cn-1-(IX)]-Kras dsiRNA conjugate (unrelated RNA sequence conjugated to the Apo-Si molecular nanomotor delivery system), and (iv) Target [Cn-1-(IX)]-ApoC3 dsiRNA conjugate.In vitro silencing of EGFP gene expression involves [Cn-2-(VIII)], which has E, E', and E'' portions according to formula (VIII), respectively. Figure 4A shows cultured HeLa cells.Figure 4B shows the dose-response curves for cultured 3T3 cells in the concentration range of 0–300 nM.The biological performance of two conjugates of the present invention, conjugate [Cn-1-(VIII-M)] and conjugate [Cn-2-(VIII-M)], in silencing EGFP gene expression in vitro in 3T3 cells is described. A clear dose/response was observed with a very significant logarithmic decay and curve fitting of R² 0.97 for both cell lines. For conjugate [Cn-1-(VIII-M)] (dotted line), which has two E-molets, the IC50 was found to be 2.2 nM, while for conjugate [Cn-2-(VIII-M)] (solid line), which has three E-molets, the IC50 was found to be 0.8 nM. This invention