Carrier peptide conjugate, composition and application thereof

Abstract

A carrier peptide conjugate comprising one or more carrier peptide groups and one or more functional groups; each carrier peptide group is linked to at least one of the functional groups or at least one further carrier peptide group via at least one linking group, and each functional group is linked to at least one of the carrier peptide groups or at least one further functional group via at least one linking group; the length of each carrier peptide group is 5-40 amino acid residues, and each carrier peptide group contains one or more amino acid sequence segments which are connected in series and are shown as a formula (I); each functional group is independently one of a functional oligonucleotide group and a delivery aid group, and at least one functional group is a functional oligonucleotide group. The carrier peptide conjugate provided by the invention can be effectively targeted to the kidney or kidney tissue.

Description

Technical Field

[0001] The present disclosure relates to a carrier peptide conjugate, a composition containing such conjugates, and uses thereof. Background Art

[0002] The anatomical structure of the kidney consists of glomeruli and renal tubules. Many chronic diseases, such as hypertension, diabetes, etc., involve the renal tubules, and ultimately lead to the occurrence of kidney diseases and progress to end-stage renal failure, which requires expensive renal replacement therapy.

[0003] Early diagnosis or targeted early treatment is the key to preventing and treating kidney diseases. In particular, regardless of primary kidney diseases or secondary renal damage such as hypertension and diabetes, tubulointerstitial fibrosis is their common pathological feature and the main factor determining the disease progression and prognosis. Among them, the pathophysiological changes of renal tubular epithelial cells are crucial in the occurrence and progression of tubulointerstitial fibrosis.

[0004] A major challenge in the development of drugs for the diagnosis and treatment of kidney diseases is the lack of effective means for targeted drug delivery to target tissues in the kidney, especially the renal tubules. Many oligonucleotide drugs that show excellent pharmaceutical activity in preclinical pharmaceutical studies are difficult to effectively reach specific target organs or target tissues due to the lack of effective delivery carriers. Therefore, there is a significant practical need in the art for targeted drugs that can effectively target and enter target tissues in the kidney, especially the renal tubules, and release functional oligonucleotides in kidney tissue cells to achieve regulation of the mRNA level. Summary of the Invention

[0005] The present invention provides a carrier peptide conjugate that can effectively target and enter target tissues in the kidney, such as the renal tubules, and release functional oligonucleotides in kidney tissue cells to achieve regulation of the mRNA level.

[0006] In one aspect, the present disclosure provides a carrier peptide conjugate, the carrier peptide conjugate containing one or more carrier peptide groups and one or more functional groups, wherein each of the carrier peptide groups is connected to at least one of the functional groups or at least one additional carrier peptide group through a linking group, and each functional group is connected to at least one of the carrier peptide groups or at least one additional functional group through a linking group; the length of each carrier peptide group is 5 to 40 amino acid residues, each carrier peptide group contains 1 amino acid sequence segment represented by formula (I) or multiple tandemly linked amino acid sequence segments represented by formula (I), and the total number of amino acid residues (X A ) and (X B ) in all amino acid sequence segments represented by formula (I) accounts for more than 50% of the total number of amino acid residues of the carrier peptide group.

[0007] -(X B ) n2 -(X A ) n1 -Z n0 - Formula (I)

[0008] wherein each (X A ) independently represents an amino acid residue having an acidic side chain group, each (X B ) independently represents an amino acid residue having a basic side chain group, each Z independently represents any amino acid residue, each n1 and n2 independently represent an integer from 1 to 10, and n0 represents an integer from 0 to 2; each of said functional groups is independently one of a functional oligonucleotide group and a delivery adjuvant group, and at least one functional group is a functional oligonucleotide group.

[0009] In yet another aspect, the present disclosure also provides a pharmaceutical composition comprising one or more of the conjugate of the present disclosure, its pharmaceutically acceptable salt, metabolite or prodrug, and a pharmaceutically acceptable excipient.

[0010] In yet another aspect, the present disclosure also provides the use of one or more of the conjugate of the present disclosure, its pharmaceutically acceptable salt, metabolite or prodrug, and the pharmaceutical composition in the preparation of a drug for treating and / or preventing kidney-related diseases.

[0011] In yet another aspect, the present disclosure also provides a method for treating and / or preventing kidney-related diseases, the method comprising administering to a subject an effective amount of one or more of the conjugate of the present disclosure, its pharmaceutically acceptable salt, metabolite or prodrug, and the pharmaceutical composition.

[0012] In addition, the present disclosure also provides a kit comprising one or more of the conjugate of the present disclosure, its pharmaceutically acceptable salt, metabolite or prodrug, and the pharmaceutical composition.

[0013] Incorporated by reference

[0014] All publications mentioned in this specification, including patents, patent applications or non-patent literature, are incorporated herein by reference to the same extent as if each individual publication was specifically and individually incorporated herein by reference.

[0015] Beneficial effects

[0016] The carrier peptide conjugate of the present disclosure can efficiently and specifically target kidney tissues and enter the cytoplasm of renal tubular epithelial cells. The carrier peptide conjugate of the present disclosure has excellent ability to target and deliver functional oligonucleotides and delivery adjuvants into renal tubular cells.

[0017] For example, the carrier peptide conjugate containing a fluorescent group and a functional oligonucleotide group provided by the present disclosure can efficiently target and enter kidney tissues when administered intravenously. In addition, the conjugate shows rapid, long-lasting, and strong fluorescence signals in renal tubules, especially in the proximal renal tubules. These results indicate that the carrier peptide conjugate of the present disclosure has the ability to deliver the functional oligonucleotide group to the kidney. Therefore, the carrier peptide conjugate provided by the present disclosure can efficiently target the kidney, release the functional oligonucleotide in kidney tissues and cells, and provide effective assistance for the treatment of related diseases.

[0018] For another example, in the experiment of mouse renal tubular cells, on the 2nd day after administration of the carrier peptide conjugate containing the Cy5 fluorescent group and the functional oligonucleotide group of the present disclosure, the distribution range of the Cy5 fluorescence imaging signal coincides with the green fluorescence signal of part of the anti-AQP1 antibody, and mostly coincides with the cell nucleus and is near the blue signal showing the cell nucleus, and clearly shows the cell boundary. On the 5th day after administration, obvious red Cy5 fluorescence imaging is still visible, and the imaging distribution range coincides with the green fluorescence signal of part of the anti-AQP1 antibody, and clearly shows the cell boundary, and does not enter glomerular cells that do not express the AQP1 protein. It shows that the carrier peptide conjugate of the present disclosure can enter glomerular cells efficiently for a long time and exhibits good specific targeting effect.

[0019] For another example, in the in-vivo experiment of mice, compared with the blank control group, after administration of the carrier peptide conjugate containing the siRNA group as the functional oligonucleotide group of the present disclosure, the relative expression level of SOD1 mRNA in the kidneys of mice is significantly inhibited at different time points, and the highest inhibition rate can reach 47.49%, showing a high inhibitory activity; moreover, the inhibition rate is still on a downward trend on the 29th day, indicating that the carrier peptide conjugate of the present disclosure is likely to stably and efficiently inhibit the target mRNA for a longer time; on the other hand, compared with the inhibition rate in the medulla, the inhibition rate of SOD1 mRNA in the cortex is significantly higher, indicating that the carrier peptide conjugate of the present disclosure can effectively target and enter the proximal renal tubular tissue with a higher content in the cortex and has excellent specific delivery effect.

[0020] For another example, in in-vivo mouse experiments, compared with the blank control group, after administering the conjugate of the active carrier peptide containing D-configured and L-configured amino acid residues and the siRNA group of the present disclosure, the relative expression levels of SOD1 mRNA in the mouse kidneys were significantly inhibited. The average inhibition rate of the siRNA conjugate of the active carrier peptide containing D-configured amino acid residues reached 51.56%, and the average inhibition rate of the siRNA conjugate of the carrier peptide group containing L-configured amino acid residues also reached 49.42%. This indicates that various carrier peptide conjugates of the present disclosure can effectively target and enter the cortex and have a good effect on inhibiting the target mRNA. Description of the Drawings

[0021] Figure 1A-1E Graphs showing fluorescence imaging of the heart, liver, spleen, lung, and kidney in mice at D2, D3, D5, D8, and D14 after injection of PBS, reference conjugate 1, or carrier peptide conjugate 1 of the present disclosure, respectively.

[0022] Figure 2A-2C Graphs showing confocal microscopy imaging of kidney tissue sections in control and experimental groups of mice at day 8 or day 14 after administration of PBS or carrier peptide conjugate 1 of the present disclosure, respectively.

[0023] Figure 3A-3C Graphs showing confocal microscopy imaging of kidney tissue sections in each group of mice at day 14 after administration of PBS, reference conjugate 1, or carrier peptide conjugate 1 of the present disclosure, respectively.

[0024] Figure 4A-4C Graphs showing fluorescence imaging of different organs in rats at day 3, day 6, and day 10 after administration of PBS, carrier peptide conjugate 1 of the present disclosure, or reference conjugate 1, respectively.

[0025] Figure 5A-5D Graph showing confocal microscopy imaging of a kidney tissue section in a mouse at day 2 after administration of carrier peptide conjugate 1 of the present disclosure (40X field of view); Figure 5E Graph showing an overlay of different fluorescence imaging graphs of a kidney tissue section in a mouse at day 5 after administration of carrier peptide conjugate 1 of the present disclosure (20X field of view).

[0026] Figure 6A and Figure 6B Graphs showing the relative expression levels of SOD1 mRNA in the renal cortex and renal medulla of mice at different time points after administration of the carrier peptide conjugate of the present disclosure or PBS, respectively.

[0027] Figure 7 Graph showing the relative expression level of SOD1 mRNA in the renal cortex of a mouse at day 8 after administration of the carrier peptide conjugate of the present disclosure or PBS. Detailed implementation manners

[0028] The following provides a detailed description of the specific implementation manners of the present disclosure. It should be understood that the specific implementation manners described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0029] In the present disclosure, SOD1 mRNA refers to the mRNA with Genbank accession number NM_011434.2 or NM_000454.5.

[0030] Definition

[0031] In the foregoing and following of the present disclosure, a polypeptide sequence refers to a polypeptide sequence formed by multiple amino acid monomers connected through dehydration condensation reactions at the carboxyl and amino positions to form peptide bonds. Unless otherwise specified, the amino terminus of the polypeptide sequence refers to the end of the polypeptide formed by condensation that contains an amino group that has not undergone the above dehydration condensation reaction, and the carboxyl terminus of the polypeptide sequence refers to the end of the polypeptide formed by condensation that contains a carboxyl group that has not undergone the above dehydration condensation reaction. Unless otherwise stated, in the polypeptide sequences mentioned in the present disclosure, each amino acid residue is connected in sequence from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) from left to right via peptide bonds (i.e., amide bonds). Unless otherwise stated, in a peptide sequence represented by a sequence of capital letters of letter abbreviations, the connection between the amino acid residue represented by the left letter and the amino acid residue represented by the right letter is the formation of a peptide bond between the carboxyl group of the amino acid represented by the left letter and the amino group of the amino acid represented by the right letter. For example, in the polypeptide sequence KKEEE, the connection relationship between the second letter K and the third letter E is that the carboxyl group of lysine represented by K forms a peptide bond with the amino group of glutamate represented by E.

[0032] In the above and below of this article, unless otherwise specified, in a polypeptide sequence, each letter represents an amino acid monomer or an amino acid residue. An amino acid monomer refers to an amino acid in which the reactive amino group or reactive carboxyl group is protected by a protecting group, and an amino acid residue refers to a group formed by an amino acid losing a hydrogen from its amino group and / or a hydroxyl group from its carboxyl group. For the sake of simplicity, according to the context, when describing a preparation method, an amino acid or the corresponding capital letter refers to an amino acid monomer, and when describing an amino acid sequence, an amino acid or the corresponding capital letter represents an amino acid residue. Among them, according to the context, G represents a glycine monomer or a glycine residue in a polypeptide sequence, A represents an alanine monomer or an alanine residue in a polypeptide sequence, V represents a valine monomer or a valine residue in a polypeptide sequence, L represents a leucine monomer or a leucine residue in a polypeptide sequence, I represents an isoleucine monomer or an isoleucine residue in a polypeptide sequence, P represents a proline monomer or a proline residue in a polypeptide sequence, F represents a phenylalanine monomer or a phenylalanine residue in a polypeptide sequence, Y represents a tyrosine monomer or a tyrosine residue in a polypeptide sequence, W represents a tryptophan monomer or a tryptophan residue in a polypeptide sequence, S represents a serine monomer or a serine residue in a polypeptide sequence, T represents a threonine monomer or a threonine residue in a polypeptide sequence, C represents a cysteine monomer or a cysteine residue in a polypeptide sequence, M represents a methionine monomer or a methionine residue in a polypeptide sequence, N represents an asparagine monomer or an asparagine residue in a polypeptide sequence, Q represents a glutamine monomer or a glutamine residue in a polypeptide sequence, D represents an aspartic acid monomer or an aspartic acid residue in a polypeptide sequence, E represents a glutamic acid monomer or a glutamic acid residue in a polypeptide sequence, K represents a lysine monomer or a lysine residue in a polypeptide sequence, R represents an arginine monomer or an arginine residue in a polypeptide sequence, and H represents a histidine monomer or a histidine residue in a polypeptide sequence.

[0033] A variety of protecting groups can be used in the present disclosure. Generally, a protecting group renders a chemical functional group insensitive to specific reaction conditions and can be added to and removed from the functional group in the molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, which are hereby incorporated by reference in their entireties. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, examples of protecting groups for the amino group in the carrier peptide, active carrier peptide, or carrier peptide group herein include, but are not limited to, C1-C6 alkyl acyl, tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). In some embodiments, examples of protecting groups for the carboxyl group in the carrier peptide, active carrier peptide, or carrier peptide group herein include, but are not limited to, amino, C1-C6 alkylamino, or C1-C6 alkoxy. In some embodiments, non-exclusive examples of hydroxyl protecting groups for the nucleotide or oligonucleotide group herein include dimethoxytrityl (DMTr), monomethoxytrityl, 9-phenyloxanthren-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthren-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups for the nucleotide or oligonucleotide group herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4′-dimethoxytrityl), and TMTr (4,4′,4″-trimethoxytrityl).

[0034] In the above and below, unless otherwise specified, in a nucleotide sequence, the capital letters C, G, U, and A represent the types of nucleotides defined by the corresponding bases; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluoro group; the lowercase letter combination moe represents that the nucleotide adjacent to the left of the letter combination moe is a nucleotide modified with a methoxyethyl group; the lowercase letter d represents that the nucleotide adjacent to the right of the letter d is a 2'-deoxyribonucleotide; the lowercase letter s represents that there is a phosphorothioate subunit linkage between the two nucleotides adjacent to the left and right of the letter s; P1 represents that the nucleotide adjacent to the right of the P1 is a nucleotide modified with a 5'-phosphate nucleotide or a 5'-phosphate analog. In some embodiments, P1 is VP, Ps, or P representing specific modifications. Among them, the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP); the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a nucleotide modified with a phosphorothioate; the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0035] In the above and below, the "fluoro-modified nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2'-position of the ribose group of the nucleotide with a fluoro group, and the "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2'-position of the ribose group of the nucleotide with a non-fluoro group. A "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide. Such as an isonucleotide, a bridged nucleic acid (abbreviated as BNA), or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.

[0036] In the context of this disclosure, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at corresponding positions do not pair in a complementary form.

[0037] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between two nucleotide sequences involved; "essentially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "fully reverse complementary" means that there are no base mismatches between two nucleotide sequences.

[0038] In the above and below, when a nucleotide sequence has a "nucleotide difference" from another nucleotide sequence, it means that compared with the latter, the base type of the nucleotide at the same position has changed. For example, when a nucleobase in the latter is A, and the corresponding nucleobase at the same position in the former is U, C, G, or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at this position. In some embodiments, when a nucleotide at the original position is replaced by a non-nucleoside nucleotide or its equivalent, a nucleotide difference is also considered to have occurred at this position.

[0039] In the above and below, especially when describing the preparation method of a double-stranded oligonucleotide or a vector peptide conjugate containing a double-stranded oligonucleotide, unless otherwise specified, the "nucleoside monomer" refers to the modified or unmodified RNA phosphoramidite monomer (sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites) used in nucleic acid phosphoramidite solid-phase synthesis according to the type and order of nucleotides in the double-stranded oligonucleotide or vector peptide conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method well-known to those skilled in the art for RNA synthesis. The nucleoside monomers used in this disclosure are all commercially available.

[0040] It should be noted that, in the above and below texts of this article, A appearing in the polypeptide sequence represents alanine, and A appearing in the nucleic acid molecule represents the purine base adenine; T appearing in the polypeptide sequence represents threonine, and T appearing in the nucleic acid molecule represents thymine; G appearing in the polypeptide sequence represents glycine, and G appearing in the nucleic acid molecule represents guanine; C appearing in the polypeptide sequence represents cysteine, and C appearing in the nucleic acid molecule represents cytosine. Those skilled in the art are familiar with the different meanings represented by A, T, G, and C in the polypeptide sequence and the nucleic acid molecule respectively, and there will be no confusion.

[0041] The "carrier peptide group" used in the present disclosure is a polypeptide compound formed by connecting a certain number of amino acids through peptide bonds, and a group formed after removing one or more atoms. The carrier peptide group can be connected to a functional group through a covalent bond or a linking group to form a conjugate, thereby realizing the delivery of the functional group.

[0042] In the context of the present disclosure, unless otherwise specified, "conjugation" means that two or more chemical moieties each having a specific function are connected to each other in a covalent connection manner; correspondingly, "conjugate" means a compound formed by covalent connection between these individual chemical moieties. Further, "carrier peptide conjugate" means a compound formed by covalently connecting one or more carrier peptide groups and a chemical moiety having a specific function. The carrier peptide conjugate should be understood, according to the context, as the general term for multiple carrier peptide conjugates or a specific one or more carrier peptide conjugates represented by a certain chemical formula. As used in the present disclosure, the "carrier peptide conjugate" includes a carrier peptide group and a functional group moiety, wherein the functional group refers to a chemical moiety formed after removing one or more atoms from a drug molecule. Within the scope of the present disclosure, each of the said functional groups is independently selected from a functional oligonucleotide group formed by a functional oligonucleotide and a delivery aid group formed by a delivery aid. Those skilled in the art will understand that the removal of the above one or more atoms will not destroy the activity or stability of the drug molecule. For example, when the functional group is a functional oligonucleotide group, the oligonucleotide group can be a chemical moiety formed after removing the hydrogen atom in the phosphate ester bond in the functional oligonucleotide molecule, or a chemical moiety formed after removing the hydrogen atom in the 5'-hydroxyl group of the 5'-terminal nucleotide of the sense strand or antisense strand in the functional oligonucleotide molecule, or a chemical moiety formed after removing the hydrogen atom in the 3'-hydroxyl group of the 3'-terminal nucleotide of the sense strand or antisense strand in the functional oligonucleotide molecule.

[0043] As used above or below, the term "substituted" or "replaced" group includes, but is not limited to, substituted alkyl, substituted alkoxy, and substituted amino groups. Among them, unless otherwise specified, the "substituted" or "replaced" group refers to a group formed by replacing one or more hydrogen atoms in the group with substituents. For example, "substituted alkoxy" refers to a group formed by replacing one or more hydrogen atoms in the alkoxy group with substituents. Those skilled in the art will understand that various substituents can be included in the compounds applicable to the present disclosure, as long as the introduction of the substituents does not affect the function of the present disclosure and can achieve the purpose of the present disclosure, they can be used in the present disclosure. In some embodiments, the substituents are selected from the group consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituents, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), -CN, -NO2, -CO2H, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 Haloalkyl). In some embodiments, the substituent is one of C1-C3 alkyl, C6-C8 aryl, -OC1-C3 alkyl, -OC1-C3 alkylphenyl, halogen, -OH, -NH2, -CN or -NO2. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitutions or substitution patterns that are spatially unrealistic, synthetically infeasible and / or inherently unstable.

[0044] As used herein, the term "subject" refers to any animal, such as a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and any kind of poultry.

[0045] As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradicating or ameliorating the underlying disorder being treated. In addition, a therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0046] As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit", the conjugates or pharmaceutical compositions of the present disclosure can be administered to a subject at risk of developing a kidney-related disease, or to a subject reporting one or more physiological symptoms of a kidney-related disease, even if a diagnosis of the disease has not yet been made. In some embodiments, the physiological symptoms include, but are not limited to, one or more of edema, abnormal urine, hypertension, low back pain, soreness of the waist, anemia, and fatigue.

[0047] As used herein, the "functional oligonucleotide" refers to an oligonucleotide having the function of upregulating or downregulating the level of target mRNA capable of regulating the expression of a target gene, including single-stranded or double-stranded oligonucleotides. Functional single-stranded oligonucleotides such as single-stranded ribonucleic acid (ssRNA), single-stranded antisense oligonucleotides (ASO), or microRNA (miRNA); functional double-stranded oligonucleotides such as small activating RNAs (saRNA) capable of upregulating the level of target mRNA for target gene expression or small interfering RNAs (siRNA) capable of downregulating the level of target mRNA for target gene expression.

[0048] In the context of the present disclosure, the "modified amino acid" includes constructing a carrier peptide group in the present disclosure by replacing natural L-amino acids with D-amino acids, or substituting natural L-amino acids with amino acid analogs, which include structural analogs of amino acids, such as salts or esters of natural amino acids. In addition, the carboxyl terminus of a polypeptide can be a carboxyl group, an amide group obtained by amidating the carboxyl group, or a substance generated by incorporating one of the above amino acid analogs. In addition, one or more natural peptide bonds in the above carrier peptide group can be replaced by any one of the following groups, which include but are not limited to: sulfonamide, retroamide, aminooxy-containing bond, ester, alkyl ketone, α,α-difluoroketone, α-fluoroketone, peptidomimetic bond (N-alkylated glycinamide bond). In addition, the modified amino acid can be an amino acid with a modified side chain, such as one of 4-fluorophenylalanine, 4-isolysine, 3-aminoproline, 2-nitrotyrosine, or N-alkylhistidine; or a β-branched amino acid or β-branched amino acid analog having a chirality opposite to that of the natural chirality at the β-side chain carbon atom, such as allothreonine, alloisoleucine, and their derivatives. Representative modified amino acids are disclosed in Baran et al., Biochemistry, 2017, 56(30): 3863-3873 and Mehta et al., Tetrahedron Letters, 2017, 58(14): 1357-1372, which are incorporated herein by reference in their entireties. In some embodiments, at least one modified amino acid in the carrier peptide group is a D-amino acid. In some embodiments, all modified amino acids are D-amino acids. In some embodiments, the carrier peptide group further encompasses a carrier peptide group in which all amino acids are D-amino acids and the connection direction of each amino acid is opposite, that is, the amino acid sequence of the carrier peptide group remains unchanged, but each amino acid is a D-amino acid and the connection direction is changed to the carboxyl terminus - amino acid (C-terminal - N-terminal direction). At this time, the peptide bond formed by the carboxyl group of one amino acid and the amino group of another amino acid is changed to the peptide bond formed by the amino group of this amino acid and the carboxyl group of the other amino acid, and the formed carrier peptide group is substantially in a mirror-symmetric conformation with the corresponding carrier peptide group formed by all L-amino acids.

[0049] In some embodiments, the carrier peptide conjugates of the present disclosure contain free carboxyl or amino groups. These carboxyl or amino groups can form salts by reacting with corresponding bases or acids. In some embodiments, the carrier peptide conjugates of the present disclosure further contain oligonucleotide groups. In some embodiments, the oligonucleotide group is an siRNA group. In some embodiments, a pharmaceutically acceptable salt of the carrier peptide conjugate containing the siRNA group is the sodium salt of the carrier peptide conjugate in which the hydrogen ions of the hydroxyl groups in all the phosphate groups in the conjugate are replaced by sodium ions. In these carrier peptide conjugates containing oligonucleotide groups, each adjacent nucleotide is linked by a phosphodiester bond or a phosphorothioate bond, and the non-bridging oxygen or sulfur atom in the phosphodiester bond or phosphorothioate bond bears a negative charge, which can exist in the form of a hydroxyl or mercapto group, and the hydrogen ions in the hydroxyl or mercapto group can also be partially or completely replaced by cations. Therefore, in some embodiments, the present disclosure also provides pharmaceutically acceptable salts of the carrier peptide conjugates. The pharmaceutically acceptable salts can be salts formed by carboxylate ions formed after the carboxyl group loses hydrogen ions and cations, or salts formed by organic ammonium cations formed after the amino group gains hydrogen ions and anions, and / or can be salts formed by phosphate groups in the phosphate groups or phosphorothioate groups in the oligonucleotide groups and cations. The cations can be any cations, such as metal cations, ammonium ion NH4 + , and one of the organic ammonium cations. For the consideration of improving solubility, in some embodiments, the cations are selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal ions can be K + and / or Na +, the cation formed by the tertiary amine can be the ammonium ion formed by triethylamine and / or the ammonium ion formed by N,N - diisopropylethylamine. The anion can be any anion, such as one or more of chloride ions and organic acid ions. In some embodiments, the anion can be, for example, chloride ions or acetate anions. Therefore, the carrier peptide conjugate described in the present disclosure can exist at least partially in the form of a salt. In some embodiments, in the carrier peptide conjugate containing an oligonucleotide group described in the present disclosure, the carrier peptide group does not contain a free carboxyl group or amino group, and the non - bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond in the oligonucleotide group is at least partially bound to a sodium ion. The pharmaceutically acceptable salt of the carrier peptide conjugate containing an oligonucleotide group described in the present disclosure refers to the water - soluble salt of the conjugate, such as one of alkali metal salts or ammonium salts. In some embodiments, the pharmaceutically acceptable salt is the partial or total water - soluble salt of the conjugate. In some embodiments, the water - soluble salt can be an amine salt, an alkali metal salt or an alkaline earth metal salt. In some embodiments, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts and quaternary ammonium salts. The alkali metal salt is selected from potassium salts or sodium salts, and the alkaline earth metal salt is selected from calcium salts or magnesium salts. In some embodiments, the tertiary amine salt is triethylamine salt, triisopropylamine salt or N,N - diisopropylethylamine salt. In some embodiments, the pharmaceutically acceptable salt of the conjugate is a mixture of the methylamine salt and ammonium salt of the conjugate. In some embodiments, the pharmaceutically acceptable salt of the carrier peptide conjugate refers to the sodium salt or partial sodium salt of the conjugate.

[0050] Unless otherwise specified, in the context, when separately referring to the carrier peptide conjugate containing an oligonucleotide described in the application or method provided by the present disclosure, including but not limited to the carrier peptide conjugate represented by any structural formula described in the application or method provided by the present disclosure, it also refers to the pharmaceutically acceptable salt of the conjugate according to the context.

[0051] The carrier peptide conjugate of the present disclosure

[0052] In one aspect, the present disclosure provides a carrier peptide conjugate, which contains one or more carrier peptide groups and one or more functional groups. Each of the carrier peptide groups is connected to at least one of the functional groups or at least one other carrier peptide group through a linking group, and each functional group is connected to at least one of the carrier peptide groups or at least one other functional group through a linking group; the length of each carrier peptide group is 5 to 40 amino acid residues, and each carrier peptide group contains 1 amino acid sequence segment represented by formula (I) or multiple tandemly - linked amino acid sequence segments represented by formula (I). The amino acid residues (X A ) and (X B)The proportion of the total number in the total number of amino acid residues of the carrier peptide group is greater than 50%,

[0053] -(X B ) n2 -(X A ) n1 -Z n0 - Formula (I)

[0054] Wherein, each (X A ) independently represents an amino acid residue having an acidic side chain group, each (X B ) independently represents an amino acid residue having a basic side chain group, each Z independently represents any amino acid residue, each n1 and n2 independently represent an integer from 1 to 10, and n0 represents an integer from 0 to 2; each of the said functional groups is independently one of a functional oligonucleotide group and a delivery aid group, and at least one functional group is a functional oligonucleotide group.

[0055] In some embodiments, the number of carrier peptide groups in the conjugate is n, and the number of functional groups is m. n is an integer selected from 1 to 8, and m is an integer selected from 1 to 4. In some embodiments, n is an integer selected from 1 to 4, and m is an integer selected from 1 to 3.

[0056] In some embodiments, each linking group linking the carrier peptide is independently linked to the carboxyl group at the carboxyl terminus of the carrier peptide group, or to the amino group at the amino terminus of the carrier peptide group.

[0057] In some embodiments, each of the said carrier peptide groups or additional functional groups is independently linked to the functional group through a linking group. In some embodiments, each of the said carrier peptide groups or additional functional groups is separately linked to the functional group through one of the said linking groups. To simplify the synthesis process, in some embodiments, all carrier peptide groups and all additional functional groups are linked to the functional group through the linking group. In some embodiments, all carrier peptide groups and all additional functional groups are linked to the functional group through the same linking group. In the context, "additional functional group" refers to other functional groups of the present disclosure that are different from the functional group covalently linked to all linking groups; "additional carrier peptide group" refers to other carrier peptide groups of the present disclosure that are different from the described carrier peptide group. In some embodiments, the conjugate contains multiple linking groups, wherein any 1, 2, 3, 4, 5, or 6 of the said carrier peptide groups or functional groups are linked to the functional group through one of the said linking groups, and the remaining carrier peptide groups are linked to the functional group through one or more other linking groups.

[0058] In some embodiments, in the carrier peptide conjugate of the present disclosure, the linking group comprises a branching group, 1 to 6 side chain moieties, and 1 conjugating linking group. The branching group is connected to the conjugating linking group and each of the side chain moieties respectively. The side chain moieties are connected to the branching group and the functional group or the carrier peptide group respectively. The conjugating linking group is connected to the branching group and the functional group or the carrier peptide group respectively. In some embodiments, the conjugate has the structure shown in formula (101):

[0059]

[0060] wherein each of the linking groups is composed of k L A , L B and L C . L A is a side chain moiety, L B is a conjugating linking group connecting L C and N u . L C is a branching group, N u and each PP independently represents the functional group or the carrier peptide group, and at least one of N u and all PPs is the functional group, and at least one is the carrier peptide group; each k is independently an integer from 1 to 6, n 101 is an integer from 1 to 3; and the sum of all k is an integer from 1 to 8;

[0061] The branching group L C is a 2- to 7-valent, straight-chain or branched-chain C1-C 25 or C3-C 15 saturated hydrocarbon group, or one or more carbon atoms in the straight-chain or branched-chain saturated hydrocarbon group are replaced by one or more linking combinations selected from the group consisting of: C(O), NH, O, S, CH═N, S(O)2, OP(O)2, C5-C8 sub-glycosyl group, C2-C5 sub-alkenyl group, C2-C5 sub-alkynyl group, C6-C 10 sub-aryl group, C3-C8 sub-heterocyclic group, and C5-C 10 sub-heteroaryl group; and / or

[0062] If there is a methylene group in the branched saturated hydrocarbon group, then one or more of the methylene groups are optionally replaced by one or more selected from the group consisting of: a nitrogen atom, a trivalent C3-C 10 cycloalkyl group, a trivalent C3-C 10 heterocyclic group, a trivalent C6-C 10 aryl group, and a trivalent C5-C 10 heteroaryl group, and

[0063] Wherein, the saturated hydrocarbon group optionally has a substituent selected from any one or more of the following groups: C1-C5 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl);

[0064] The side chain portion L A is a covalent bond, or C1-C 20 alkylene, or one or more carbon atoms in the alkylene are replaced by one or more selected from the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 subglycosyl, C2-C5 alkenylene, C2-C5 alkynylene, C6-C 10 arylene, C3-C8 heterocyclene and C5-C 10 heteroarylene; wherein, the alkylene optionally has a substituent selected from any one or more of the following groups: C1-C5 alkyl, C6-C 10 aryl, C5-C 10heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH-SC1-C5 alkyl, -SC1-C5 alkylphenyl, -C1-C5 alkyl-SH, -OH, -SH, -NH2, -C1-C5 alkyl-NH2, -N(C1-C5 alkyl)(C1-C5 alkyl), -NH(C1-C5 alkyl), -N(C1-C5 alkyl)(C1-C5 alkylphenyl), -NH(C1-C5 alkylphenyl), nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl).

[0065] In the foregoing and hereinafter, the term "optionally" means that the event cited thereafter occurs or does not occur, or the substance described exists or does not exist. For example, "L C is a 2-7 valent straight-chain or branched C1-C 25 saturated hydrocarbon group, and one or more methylenes in the straight-chain or branched saturated hydrocarbon group are optionally replaced by one or more selected from the group consisting of the following groups" means "L C is a 2-7 valent straight-chain or branched C1-C 25 alkylene group, or L C is a group formed by replacing one or more methylenes in a 2-7 valent straight-chain or branched C1-C 25 saturated hydrocarbon group with one or more selected from the group consisting of the following groups".

[0066] The conjugating linking group L B serves to link Nu to the branching group L C . Therefore, any linking group that can achieve the above linking and does not damage the corresponding therapeutic or prophylactic function of the functional group can achieve the object of the present invention. In some embodiments, the L B is one of the following linking groups: phosphodiester subunit, phosphorothioate subunit, amide group, ester group, oxygen atom, disulfide group. The phosphodiester subunit, phosphorothioate subunit, ester group, and disulfide group have the structures shown in formulas (L46)-(L49) respectively:

[0067]

[0068] Among them, represents the site of covalent attachment of the group. In some embodiments, L B is a phosphodiester subunit, a phosphorothioate subunit or a disulfide subunit. In some embodiments, when L B is a phosphodiester subunit or a phosphorothioate subunit, Nu is a functional oligonucleotide group, and L B is attached to the phosphate in the non-terminal nucleotide of the functional oligonucleotide group, or to the 3'-position of the 3'-terminal nucleotide of the nucleotide sequence, the phosphodiester subunit or the phosphorothioate subunit can be attributed to the phosphodiester subunit or the phosphorothioate subunit in the functional oligonucleotide group, that is, the branched group L C directly forms a phosphoester bond with the phosphodiester subunit or the phosphorothioate subunit attributed to the functional oligonucleotide group.

[0069] In some embodiments, in the carrier peptide conjugate of the present disclosure, Nu represents the functional group, each PP is the same or different and independently represents one of the carrier peptide groups, and each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4)-(L23) and their linking combinations:

[0070]

[0071] wherein each j1 is an integer from 1 to 10;

[0072] each R' is a C1-C 10 alkyl;

[0073] each Ra is a hydrogen atom, a C1-C 10 alkyl, or is selected from the group consisting of radical groups (L24)-(L37):

[0074]

[0075] represents the site where the group is attached to the rest of the molecule.

[0076] In some embodiments, the length of LA is 3-35 atoms, where the length of LA refers to the number of chain-forming atoms in the longest atomic chain formed by the atom directly connected to L in LA to the atom directly connected to PP in LA. In some embodiments, the length of LA is 3-20 atoms. In some embodiments, the length of LA is 4-15 atoms. In some embodiments, each L C is directly connected to the atom forming the longest atomic chain of the chain-forming atoms from the atom directly connected to L in LA to the atom directly connected to PP in LA. In some embodiments, each L Ais a linking combination of at least two of the groups (L4)-(L9), (L13), (L14), (L17), (L18). In some embodiments, each L A is a linking combination of at least two of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L17), (L18).

[0077] The branched group L C functions to covalently link the conjugate linking group L B with one or more side chain moieties L A so as to achieve the conjugate linking of one or more carrier peptide groups and functional groups, and thus, through the combined action of one or more carrier peptide groups, the functional groups can be delivered more efficiently. In some embodiments, each L C has a structure represented by the formula -H1-H2-, where H1 is selected from the group consisting of the groups (L4)-(L23) and their linking combinations; H2 is selected from any one of the groups represented by the formula (B1)-(B5):

[0078]

[0079] wherein, the carbon atoms in the formula (B1)-(B3) are directly connected to one or more L A and the nitrogen atom is used to connect with H1 via an imino or amide bond.

[0080] Each nitrogen atom in the formula (B4) is used to connect with one L A via an imino or amide bond; one oxygen atom is used to connect with H1 via an ether bond, an ester bond or a phosphate ester bond, and the other oxygen atom forms a hydroxyl group with a hydrogen atom;

[0081] One of the nitrogen atoms in the formula (B5) is used to connect with H1 via an imino or amide bond, and each of the remaining nitrogen atoms is respectively used to connect with one L A via an imino or amide bond, represents the site of covalent connection of the group.

[0082] Considering the suitability of synthesis convenience, structural stability and / or cost, in some embodiments, H1 is selected from the group consisting of linking combinations of at least two of the groups (L4)-(L9), (L13), (L14), (L17) and (L18). In some embodiments, H1 is selected from linking combinations of at least two of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L17) and (L18). In some embodiments, the length of H1 is 5-20 atoms, and the length refers to the length of H1 from the atom connected to L BThe number of atoms in the longest atomic chain between the site of attachment to the site attached to H2.

[0083] In some embodiments, L C has the structure shown in formula (201a):

[0084]

[0085] wherein, n 201 and m 201 are each independently an integer from 1 to 10, and the sum of n 201 and m 201 is not greater than 10 or not greater than 5.

[0086] At this time, the linking group has the structure shown in formula (201L):

[0087]

[0088] The conjugate has the structure shown in formula (201):

[0089]

[0090] In some embodiments, each k and n 101 are independently an integer from 1 to 3, and the sum of all k is not greater than 6.

[0091] In some embodiments, in formula (201a), (201L) and (201), each L A is a linking combination of at least 2 of the groups (L4)-(L9), (L13), (L14), (L17) and (L18). In some embodiments, L A has a length of 3 to 35 atoms, 3 to 20 atoms, or 4 to 15 atoms, wherein the length of the L A refers to the number of chain-forming atoms in the longest atomic chain formed by the atoms directly connected to L A in L C to the atoms directly connected to PP in L A and any cycloalkylidene, arylidene, heterocycloalkylidene or heteroarylidene is counted as one atom.

[0092] In some embodiments, m is 1 in the carrier peptide conjugate, and n carrier peptide groups are linked to the functional group through an amino-terminal (N-terminal) or carboxyl-terminal (C-terminal) amino acid.

[0093] In some embodiments, L A has the structure shown in formula (202):

[0094]

[0095] Among them, n 202 , m 202 , p 202 , q 202 are each independently an integer from 1 to 5, and i 202 is an integer from 0 to 5, R 202 is H or an amino protecting group, and the L A is connected to PP in formula (201) through an imino group and is connected to the L C .

[0096] In some embodiments, n 202 , m 202 , p 202 and q 202 are each independently 2 or 3, i 202 is 3 or 4, and R 202 is H or a C2-C7 acyl group. In some embodiments, n 202 , all m 202 , q 202 and i 202 sum to no more than 26, or no more than 11, or no more than 6. In some embodiments, R 202 is an acetyl group.

[0097] In some embodiments, the conjugate has a structure as shown in formula (103):

[0098]

[0099] Among them, R 107 is amino or hydroxyl; n 103 and m 103 are each independently an integer from 1 to 10; n 101 is an integer from 1 to 3.

[0100] At this time, the linking group has a structure as shown in formula (103L):

[0101]

[0102] In some embodiments, n 103 is an integer from 1 to 3, and m 103 is an integer from 3 to 6.

[0103] In some embodiments, the conjugate has a structure as shown in formula (104A) or (104B), where m 104 , n 104 , p 104 , q 104 are each independently an integer from 1 to 8, and R 114is H or C 1-10 hydrocarbyl acyl, such as acetyl. In some embodiments, m 104 , n 104 , p 104 , q 104 are each independently an integer from 4 to 6. In some embodiments, m 104 and n 104 are 6, p 104 is 5, q 104 is 4. In some embodiments, n 101 is an integer from 1 to 3, and R 114 is H or C acetyl.

[0104]

[0105] At this time, the linking group has a structure shown in formula (104LA) or (104LB):

[0106]

[0107] In some embodiments, n 104 is an integer from 1 to 3, m 104 is an integer from 3 to 6, p 104 and q 104 are each independently an integer from 2 to 6.

[0108] For the sake of simplicity in synthesis, in some embodiments, the conjugate has a structure shown in formula (104A) or (104B), and n 104 is 4 or 6, m 104 is 4 or 6, p 104 is 4 or 6, q 104 are each independently 2, 4 or 5. In some embodiments, n 104 , m 104 , p 104 are 6, and q 104 is 5. At this time, the carrier peptide conjugate of the present disclosure containing the above linking group can be synthesized from commercially available raw materials using the well-known solid-phase phosphoramidite method and diazo-alkyne click chemistry synthesis method in the art.

[0109] In some embodiments, the conjugate has a structure shown in formula (801):

[0110]

[0111] wherein, p 801 and q 801 are integers from 1 to 6, and R 801 is H or C 2-10An acyl group, such as an acetyl group. At this time, the solid-phase phosphoramidite method and the diazo-alkyne click chemistry synthesis method well-known in the art can be used to synthesize the carrier peptide conjugate of the present disclosure containing the above linking group from commercially available raw materials. At this time, the linking group has the structure shown in formula (801L):

[0112]

[0113] In some embodiments, the conjugate has the structure shown in formula (901):

[0114]

[0115] wherein, p 901 and q 901 are integers from 1 to 6. At this time, the solid-phase phosphoramidite method and the Michael addition method well-known in the art can be used to synthesize the carrier peptide conjugate of the present disclosure containing the above linking group from commercially available raw materials. At this time, the linking group has the structure shown in formula (901L):

[0116]

[0117] In some embodiments, the linking group can also be the linking group disclosed in WO2019128611A1, and the above-mentioned document is incorporated herein by reference in its entirety.

[0118] In some embodiments, the length of the carrier peptide group is 10 to 30 amino acid residues; the total number of amino acid residues (X A ) and (X B ) in all the sequence segments shown in formula (I) accounts for more than 70%, preferably at least 90%, of the total number of amino acid residues of the carrier peptide.

[0119] In some embodiments, each of n1 and n2 is independently an integer from 1 to 5. In some embodiments, each of n1 and n2 is independently 2 or 3. In some embodiments, the ratio of n1:n2 in at least one or each of formula (I) is from 1:3 to 3:1.

[0120] In some embodiments, the sequence segment of formula (I) is selected from one of the sequences shown in the following structural formulas: -(X B )1-(X A )3-Z n0 -, -(X B )1-(X A )2-Z n0 -, -(X B )1-(X A )1-Z n0 -, -(X B)2-(X A )6-Z n0 -、-(X B )2-(X A )5-Z n0 -、-(X B )2-(X A )4-Z n0 -、-(X B )2-(X A )3-Z n0 -、-(X B )2-(X A )2-Z n0 -、-(X B )2-(X A )1-Z n0 -、-(X B )3-(X A )9-Z n0 -、-(X B )3-(X A )8-Z n0 -、-(X B )3-(X A )7-Z n0 -、-(X B )3-(X A )6-Z n0 -、-(X B )3-(X A )5-Z n0 -、-(X B )3-(X A )4-Z n0 -、-(X B )3-(X A )3-Z n0 -、-(X B )3-(X A )2-Z n0 -、-(X B )3-(X A )1-Z n0 -、-(X B )4-(X A ) 10 -Z n0 -、-(X B )4-(X A )9-Z n0 -、-(X B )4-(X A )8-Z n0 -、-(X B )4-(X A )7-Z n0-, -(X B )4-(X A )6-Z n0 -, -(X B )4-(X A )5-Z n0 -, -(X B )4-(X A )4-Z n0 -, -(X B )4-(X A )3-Z n0 -, -(X B )4-(X A )2-Z n0 -, -(X B )5-(X A ) 10 -Z n0 -, -(X B )5-(X A )9-Z n0 -, -(X B )5-(X A )8-Z n0 -, -(X B )5-(X A )7-Z n0 -, -(X B )5-(X A )6-Z n0 -, -(X B )5-(X A )5-Z n0 -, -(X B )5-(X A )4-Z n0 -, -(X B )5-(X A )3-Z n0 -, -(X B )5-(X A )2-Z n0 -, -(X B )6-(X A ) 10 -Z n0 -, -(X B )6-(X A )9-Z n0 -, -(X B )6-(X A )8-Z n0 -, -(X B )6-(X A )7-Z n0 -, -(X B )6-(XA )6-Z n0 -、-(X B )6-(X A )5-Z n0 -、-(X B )6-(X A )5-Z n0 -、-(X B )6-(X A )3-Z n0 -、-(X B )6-(X A )2-Z n0 -、-(X B )7-(X A ) 10 -Z n0 -、-(X B )7-(X A )9-Z n0 -、-(X B )7-(X A )8-Z n0 -、-(X B )7-(X A )7-Z n0 -、-(X B )7-(X A )6-Z n0 -、-(X B )7-(X A )5-Z n0 -、-(X B )7-(X A )4-Z n0 -、-(X B )7-(X A )3-Z n0 -、-(X B )8-(X A ) 10 -Z n0 -、-(X B )8-(X A )9-Z n0 -、-(X B )8-(X A )8-Z n0 -、-(X B )8-(X A )7-Z n0 -、-(X B )8-(X A )6-Z n0 -、-(X B )8-(X A )5-Zn0 -,-(X B )8-(X A )4-Z n0 -,-(X B )8-(X A )3-Z n0 -,-(X B )9-(X A ) 10 -Z n0 -,-(X B )9-(X A )9-Z n0 -,-(X B )9-(X A )8-Z n0 -,-(X B )9-(X A )7-Z n0 -,-(X B )9-(X A )6-Z n0 -,-(X B )9-(X A )5-Z n0 -,-(X B )9-(X A )4-Z n0 -,-(X B )9-(X A )3-Z n0 -,-(X B ) 10 -(X A ) 10 -Z n0 -,-(X B ) 10 -(X A )9-Z n0 -,-(X B ) 10 -(X A )8-Z n0 -,-(X B ) 10 -(X A )7-Z n0 -,-(X B ) 10 -(X A )6-Z n0 -,-(X B ) 10 -(X A )5-Z n0 - or -(X B ) 10 -(X A)4-Z n0 -,

[0121] In some embodiments, each (X A ) is an amino acid residue having a carboxyl functional group and independently represents aspartic acid, glutamic acid, argininosuccinic acid, or cysteine. In some embodiments, each (X A ) in the carrier peptide group is the same. In some embodiments, each (X A ) represents an amino acid residue formed by glutamic acid.

[0122] In some embodiments, each (X B ) is an amino acid residue having a basic side chain group and independently represents an amino acid residue formed by lysine, arginine, histidine, or ornithine. In some embodiments, each (X B ) in the carrier peptide group is the same. In some embodiments, each (X B ) represents an amino acid residue formed by lysine or arginine.

[0123] n0 is an integer from 0 to 2. In some embodiments, n0 is 0 or 1. In some embodiments, n0 is 0, that is, the amino acid sequence (I) does not contain the amino acid residue Z.

[0124] In some embodiments, the sequence of formula (I) is selected from the following sequences: -(KKEEE)-, -(RREEE)-, -(KKEE)-, -(KKKEEE)-, and -(KKKEE)-. In some embodiments, at least one or each sequence of formula (I) is -(KKEEE)- and has the structure shown in formula (900):

[0125]

[0126] wherein, represents the site of covalent attachment of the group.

[0127] In some embodiments, each carrier peptide group is an amino acid sequence segment represented by formula (II):

[0128] E N -[(X B ) n2 -(X A ) n1 -Z n0 n3 -Z′ n4 -EC Formula (II)

[0129] wherein, -[(X B ) n2 -(X A )​n1 -Z n0 n3 - represents n3 amino acid sequence segments shown in formula (I) in series, where n3 represents the number of amino acid sequence segments shown in formula (I) in series, n3 is an integer from 1 to 10 or from 2 to 6, Z' represents any amino acid residue, n4 represents the number of Z', and n4 is an integer from 1 to 2;

[0130] E N and E C respectively represent an amino-terminal linker and a carboxyl-terminal linker. In some embodiments, E N is selected from one of H, an amino protecting group, and a covalent attachment site, E C is selected from one of H, a carboxyl protecting group, and a covalent attachment site, and at least one of E N and E C is a covalent attachment site. In some embodiments, E N is an amino protecting group, and E C is a covalent attachment site; in some embodiments, E N is a covalent attachment site, and E c is a carboxyl protecting group tube. In some embodiments, the amino protecting group is an acyl group, such as acetyl; or the carboxyl protecting group is an amino group.

[0131] In the above and below, n3 "serial" amino acid sequence segments mean that these n3 amino acid sequence segments are connected end to end one by one, and between two adjacent amino acid sequence segments, the amino terminus of one amino acid sequence segment is connected to the carboxyl terminus of another amino acid sequence segment via a peptide bond, and the length of the formed amino acid sequence segment is correspondingly the sum of the n3 amino acid sequence segments shown in formula (I) in series.

[0132] In some embodiments, each Z and Z' in the amino acid sequence segment shown in formula (II) is the same or different and independently represents any amino acid residue. Here, it can be, for example, an amino acid residue formed by alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and / or citrulline.

[0133] In some embodiments, each amino acid sequence segment shown in formula (II) independently has a sequence shown in one of SEQ ID NOs: 13 - 17:

[0134]

[0135] ​In some embodiments, each amino acid sequence segment represented by formula (II) independently has the sequence shown in SEQ ID NO: 13, and an amide group is formed at the carboxyl terminus of the sequence, and the amino terminus is the site of covalent attachment.

[0136] In the carrier peptide conjugate of the present disclosure, each carrier peptide group can be linked to any position in the functional group, and each functional group can be linked to any amino acid in the carrier peptide group. In some embodiments, each functional group is linked to the amino acid at the carboxyl terminus or the amino terminus of the carrier peptide.

[0137] As described above, the target cells of the carrier peptide conjugate of the present disclosure are cells that have a receptor capable of binding to the carrier peptide group on the cell surface. Therefore, the carrier peptide conjugate of the present disclosure can target any target tissue or target organ in a subject in which the above target cells are present. By using double-stranded oligonucleotide groups of different target mRNAs, the function of regulating the target mRNA level in these target tissues or target organs can be exerted, so as to achieve the treatment and / or prevention of diseases or symptoms in the kidney.

[0138] In some embodiments, the target organ is the kidney. In some embodiments, the target tissue is renal tubular epithelial tissue, particularly proximal renal tubular epithelial tissue. In some embodiments, the target cells are renal tubular epithelial cells, particularly proximal renal tubular epithelial cells. In some embodiments, the target mRNA is SOD1 mRNA. The carrier peptide conjugate containing these siRNA groups exhibits significantly improved stability in plasma, low off-target effects, and also exhibits high target mRNA silencing activity.

[0139] In some embodiments, at least one of the functional groups is a delivery aid group, and the delivery aid group is selected from one or more of C8-C 30 alkyl or alkenyl groups, cholesterol groups, lipid groups, palmitic acid groups, and cholic acid groups. The conjugate of the present disclosure with a delivery aid group can better affinity with cell membranes, has better bioavailability, and / or enables the conjugate of the present disclosure to be more effectively delivered to relevant targeted cells.

[0140] In some embodiments, at least one of the functional groups is a functional oligonucleotide group that has a therapeutic effect on kidney-related diseases or symptoms. The oligonucleotide group can modulate the level of target mRNA in cells in the kidney. By including this oligonucleotide group, the conjugate of the present disclosure can specifically reach and enter cells in the kidney, and then, through the action of this oligonucleotide group, such as the RNA interference effect, modulate the level of the relevant target mRNA in the cell, such as inhibiting the level of renal urate transporter mRNA, thereby treating and / or alleviating the relevant disease process or symptoms. In some embodiments, the functional oligonucleotide group is a single-stranded oligonucleotide group. In some embodiments, the functional oligonucleotide group is an siRNA group. The siRNA group refers to a chemical moiety formed by an siRNA molecule losing one or more atoms or functional groups. In some embodiments, the functional oligonucleotide group is a saRNA group. The saRNA group refers to a chemical moiety formed by a saRNA molecule losing one or more atoms or functional groups. In some embodiments, the siRNA or saRNA contains a sense strand and an antisense strand, and the sense strand and the antisense strand each contain 15-25 nucleotides; each nucleotide is a modified or unmodified nucleotide.

[0141] In some embodiments, the functional oligonucleotide group is a single-stranded oligonucleotide group, for example, it can be a group formed by removing one or more atoms from single-stranded ribonucleic acid (ssRNA) or a group formed by removing one or more atoms from single-stranded antisense oligonucleotide (ASO). At this time, the carrier peptide group can be connected to any nucleotide in the single-stranded oligonucleotide through the above connection method. In some embodiments, the carrier peptide group is connected to the first nucleotide at the 3′ end or the first nucleotide at the 5′ end of the single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide group is a group formed by one strand of a functional double-stranded oligonucleotide group, such as a group formed by the sense strand or the antisense strand of siRNA.

[0142] In some embodiments, the double-stranded oligonucleotide group is an siRNA group. The sense strand and the antisense strand of the siRNA each comprise 15-25 modified or unmodified nucleotides. The siRNA contains a sense strand and an antisense strand. The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. The nucleotide sequence II is at least partially reverse complementary to the first nucleotide sequence. The first nucleotide sequence is the first nucleotide sequence in the mRNA expressed by the target gene in the target cells in the kidney or tissues in the kidney. The length of the first nucleotide sequence is the same as that of the nucleotide sequence I. The target cells are cells having receptors on the surface that can bind to the vector peptide group.

[0143] In some embodiments, both the nucleotide sequence I and the nucleotide sequence II consist of 19 nucleotides. The nucleotide sequence II is substantially reverse complementary, essentially reverse complementary, or completely reverse complementary to the nucleotide sequence I. Substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences. Essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences. Completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0144] In some embodiments, in the direction from the 5′ end to the 3′ end, at least the nucleotides at positions 2-19 of the nucleotide sequence II are completely reverse complementary to the nucleotides at positions 1-18 of the nucleotide sequence I. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or there is a base mismatch between the second nucleotide in the nucleotide sequence II in the 5′-to-3′ direction and the second nucleotide in the nucleotide sequence I in the 3′-to-5′ direction. By including this base mismatch, the target gene expression inhibitory activity of the functional double-stranded oligonucleotide can be further enhanced while maintaining a low off-target effect.

[0145] In some embodiments, the sense strand and the antisense strand may have the same or different lengths. The length of the sense strand is 19 - 23 nucleotides, and the length of the antisense strand is 19 - 26 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the functional double-stranded oligonucleotide can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand in the functional double-stranded oligonucleotide is 19 / 21, 21 / 23 or 23 / 25.

[0146] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. Nucleotide sequence III and nucleotide sequence IV have the same length, both being 1 - 4 nucleotides. Nucleotide sequence III is linked to the 5′ end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3′ end of nucleotide sequence II. Nucleotide sequence IV is at least partially reverse complementary to a second nucleotide sequence, which is a segment of the mRNA expressed by the target gene in the target cells in the kidney or tissues in the kidney, adjacent to the above-mentioned first nucleotide sequence and having the same length as nucleotide sequence IV. In some embodiments, nucleotide sequence III and nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary. In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide sequence IV is determined.

[0147] In some embodiments, the sense strand and the antisense strand have different lengths. The antisense strand further contains nucleotide sequence V, and the length of nucleotide sequence V is 1 to 3 nucleotides, which is linked to the 3′ end of the antisense strand to form a 3′ overhang of the antisense strand. In some embodiments, the sense strand further contains nucleotide sequence VI, and the length of nucleotide sequence VI is 1 to 3 nucleotides, which is linked to the 3′ end of the sense strand to form a 3′ overhang of the sense strand.

[0148] In some embodiments, the functional double-stranded oligonucleotide comprises nucleotide sequence V but does not comprise nucleotide sequence VI. Thus, the length ratio of the sense strand to the antisense strand can be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the functional double-stranded oligonucleotide comprises nucleotide sequences V and VI. In some embodiments, the length of nucleotide sequence V is the same as or different from the length of nucleotide sequence VI. Thus, the length ratio of the sense strand to the antisense strand can be (19-26):(19-26). In some embodiments, the length of nucleotide sequence V and / or VI is 2 nucleotides, and thus, the length ratio of the sense strand to the antisense strand can be 19 / 21, 21 / 21, 21 / 23, 23 / 23, 23 / 25 or 25 / 25.

[0149] Each nucleotide in nucleotide sequence V can be any nucleotide. For the convenience of synthesis and cost saving, in some embodiments, nucleotide sequence V is two consecutive thymidine deoxyribonucleotides (dTdT) or two consecutive uridine ribonucleotides (UU); or, in order to improve the affinity between the antisense strand and the target mRNA, nucleotide sequence V is completely reverse complementary to a third nucleotide sequence, and the third nucleotide sequence is a segment of the mRNA expressed by the target gene in the target cells in the kidney or tissues in the kidney, which is adjacent to the above-mentioned first nucleotide sequence or second nucleotide sequence and has the same length as nucleotide sequence V. Therefore, in some embodiments, the length ratio of the sense strand to the antisense strand is 19 / 21 or 21 / 23, and at this time, the functional double-stranded oligonucleotide has better mRNA silencing activity.

[0150] Each nucleotide in nucleotide sequence VI can be any nucleotide. For the convenience of synthesis and cost saving in synthesis, in some embodiments, nucleotide sequence VI is two consecutive thymidine deoxyribonucleotides (dTdT) or two consecutive uridine ribonucleotides (UU); or, in order to improve the affinity between the sense strand and the antisense strand in the functional double-stranded oligonucleotide, nucleotide sequence VI is the same as the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the functional double-stranded oligonucleotide comprises nucleotide sequences V and VI, and the length ratio of the sense strand to the antisense strand is 21 / 21 or 23 / 23, and at this time, the functional double-stranded oligonucleotide has better mRNA silencing activity.

[0151] As described above, the nucleotides in the functional double-stranded oligonucleotides are modified or unmodified nucleotides. In some embodiments, the nucleotides in the functional double-stranded oligonucleotides are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the functional double-stranded oligonucleotides are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the regulatory function of the functional double-stranded oligonucleotides on the target mRNA of target gene expression.

[0152] In some embodiments, at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by substituting the hydroxyl group at the 2'-position of the ribose group of a nucleotide with another group, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide does not significantly weaken or lose the function of the siRNA in inhibiting gene expression. For example, the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 can be selected.

[0153] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the functional double-stranded oligonucleotide is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modifying group; that is, at least a part of the phosphate groups and / or ribose groups in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand are phosphate groups with modifying groups and / or ribose groups with modifying groups.

[0154] In some embodiments, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide. The vector peptide conjugate provided by the present disclosure has achieved a high balance between plasma stability and gene silencing efficiency in animal experiments.

[0155] In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II. The number of fluorinated modified nucleotides in nucleotide sequence I is no more than 5. In the direction from the 5'-end to the 3'-end, the 7th, 8th, and 9th nucleotides of nucleotide sequence I are fluorinated modified nucleotides; the number of fluorinated modified nucleotides in nucleotide sequence II is no more than 7, and the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II are fluorinated modified nucleotides.

[0156] In some embodiments, in the sense strand in the 5′-to-3′ direction, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorine-modified nucleotides; in the antisense strand in the 5′-to-3′ direction, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides.

[0157] In some embodiments, the nucleotide sequence II includes deoxynucleotides, 2′-fluorine-modified nucleotides, and stabilized-modified nucleotides; the number of deoxynucleotides and the number of stabilized-modified nucleotides are each independently 1-3, and the number of 2′-fluorine-modified nucleotides is 2-5; in the 5′-to-3′ direction, the 2′-fluorine-modified nucleotides include the 2nd nucleotide and the 6th nucleotide in the nucleotide sequence II; the deoxynucleotide includes the 14th nucleotide in the nucleotide sequence II; the stabilized-modified nucleotide includes the 13th nucleotide in the nucleotide sequence II; the stabilized-modified nucleotide refers to a nucleotide in which the 2′-hydroxyl group of the ribose of the nucleotide is replaced by a stabilized-modifying group, and compared with a double-stranded oligonucleotide in which the nucleotide at the corresponding position is an unmodified nucleotide, the thermal stability of the double-stranded oligonucleotide formed by the antisense strand containing the stabilized-modified nucleotide is increased, and the steric hindrance of the stabilized-modifying group is greater than that of 2′-O-methyl.

[0158] In the context of the present disclosure, a "fluorine-modified nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2′ position of the ribosyl group of the nucleotide with fluorine, and has the structure shown in the following formula (7). A "non-fluorine-modified nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2′ position of the ribosyl group of the nucleotide with a non-fluorine group, or a nucleotide analogue. In some embodiments, each non-fluorine-modified nucleotide is selected from one of a nucleotide formed by replacing the hydroxyl group at the 2′ position of the ribosyl group of the nucleotide with a non-fluorine group or a nucleotide analogue.

[0159] These nucleotides formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with a non-fluorine group are well known to those skilled in the art, and these nucleotides may be selected from one of 2′-alkoxy-modified nucleotides, 2′-substituted alkoxy-modified nucleotides, 2′-alkyl-modified nucleotides, 2′-substituted alkyl-modified nucleotides, 2′-amino-modified nucleotides, 2′-substituted amino-modified nucleotides, and 2′-deoxynucleotides.

[0160] In some embodiments, the 2′-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2′-OMe), as shown in formula (8). In some embodiments, the 2′-substituted alkoxy-modified nucleotide can be, for example, a 2′-O-methoxyethyl-modified nucleotide (2′-MOE), as shown in formula (9). In some embodiments, the 2′-amino-modified nucleotide (2′-NH2) is as shown in formula (10). In some embodiments, the 2′-deoxynucleotide (DNA) is as shown in formula (11), where Base represents a nucleic acid base in formulas (7)-(11):

[0161]

[0162] A nucleotide analogue refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide. In some embodiments, the nucleotide analogue can be a non-natural nucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide.

[0163] BNA refers to a constrained or inaccessible nucleotide. BNA can contain a bridged structure with a "fixed" C3′-endo sugar puckering in a five-membered, six-membered, or seven-membered ring. Usually, this bridge is incorporated into the 2′-, 4′-positions of the ribose to provide a 2′,4′-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cET BNA, etc., where LNA is as shown in formula (12), ENA is as shown in formula (13), and cET BNA is as shown in formula (14), where Base represents a nucleic acid base:

[0164]

[0165] An acyclic nucleotide is a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), where UNA is as shown in formula (15) and GNA is as shown in formula (16), where Base represents a nucleic acid base:

[0166]

[0167]

[0168] In the above formulas (15) and (16), R is selected from H, OH, or alkoxy (-O-alkyl).

[0169] An isonucleotide refers to a compound formed by a change in the position of the base in the ribose ring of a nucleotide. In some embodiments, the isonucleotide may be a compound formed by the movement of the base from the 1′-position to the 2′-position or 3′-position of the ribose ring, as shown in Formula (17) or (18).

[0170]

[0171] In the compounds of Formula (17) - Formula (18) above, Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or the non-fluorine group as described above.

[0172] In some embodiments, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide. In the above and below, the methoxy-modified nucleotide refers to a nucleotide formed by substituting the 2′-hydroxy group of the ribosyl group with a methoxy group.

[0173] In the above and below, "fluorinated modified nucleotide", "2′-fluorinated modified nucleotide", "nucleotide in which the 2′-hydroxy group of the ribose group is substituted with fluorine" and "nucleotide having a 2′-fluorinated ribosyl group" have the same meaning, and all refer to a compound having the structure shown in Formula (7) formed by substituting the 2′-hydroxy group of the nucleotide with fluorine; "methoxy-modified nucleotide", "2′-methoxy-modified nucleotide", "nucleotide in which the 2′-hydroxy group of the ribose group is substituted with a methoxy group" and "nucleotide having a 2′-methoxy ribosyl group" have the same meaning, and all refer to a compound having the structure shown in Formula (8) formed by substituting the 2′-hydroxy group of the ribose group of the nucleotide with a methoxy group.

[0174] In some embodiments, the functional double-stranded oligonucleotide is a double-stranded oligonucleotide having the following modifications: in the sense strand, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides.

[0175] In some embodiments, the functional double-stranded oligonucleotide is a double-stranded oligonucleotide having the following modifications: in the direction from the 5'-end to the 3'-end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the functional double-stranded oligonucleotide are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand are methoxylated nucleotides; in the direction from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the double-stranded oligonucleotide are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides;

[0176] Alternatively, in the direction from the 5'-end to the 3'-end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand are methoxylated nucleotides; in the direction from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides;

[0177] Alternatively, in the direction from the 5'-end to the 3'-end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I in the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand are methoxylated nucleotides; in the direction from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand are methoxylated nucleotides.

[0178] The double-stranded oligonucleotide having the above modifications can make ribonuclease in the blood not easily cleave the nucleic acid, thereby increasing the stability of the nucleic acid and enabling the nucleic acid to have stronger performance in resisting nuclease hydrolysis. At the same time, the functional double-stranded oligonucleotide having the above modifications still has a high regulatory function on the target mRNA.

[0179] In some embodiments, at least a part of the phosphate ester groups in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand of the functional double-stranded oligonucleotide is a phosphate ester group having a modifying group. In some embodiments, the phosphate ester group having a modifying group is a phosphorothioate subunit formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the phosphate ester group having a modifying group is a phosphorothioate subunit having the structure shown in formula (1):

[0180]

[0181] This modification can stabilize the double-stranded structure of the double-stranded oligonucleotide and maintain high specificity and high affinity of base pairing.

[0182] In some embodiments, in the functional double-stranded oligonucleotide, phosphorothioate subunit linkages are present at least at one position selected from the group consisting of: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above. In some embodiments, phosphorothioate subunit linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, phosphorothioate subunit linkages are present at all of the above positions except the 3' end of the sense strand. In some embodiments, phosphorothioate subunit linkages are present at least at one of the following positions:

[0183] between the first and second nucleotides at the 5' end of the sense strand;

[0184] between the second and third nucleotides at the 5' end of the sense strand;

[0185] between the first and second nucleotides at the 3' end of the sense strand;

[0186] between the second and third nucleotides at the 3' end of the sense strand;

[0187] between the first and second nucleotides at the 5' end of the antisense strand;

[0188] between the second and third nucleotides at the 5' end of the antisense strand;

[0189] between the first and second nucleotides at the 3' end of the antisense strand; and

[0190] between the second and third nucleotides at the 3' end of the antisense strand.

[0191] In some embodiments, phosphorothioate subunit linkages are present between any unmodified nucleotide and the nucleotide adjacent thereto in the sense strand and / or the antisense strand.

[0192] In some embodiments, the 5' terminal nucleotide of the antisense strand in the functional double-stranded oligonucleotide is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog.

[0193] Commonly used 5'-phosphate nucleotides or nucleotides modified with 5'-phosphate analogs are well known to those skilled in the art. For example, a 5'-phosphate nucleotide may have the following structure:

[0194]

[0195] For another example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following 4 kinds of nucleotides modified with 5'-phosphate analogs:

[0196]

[0197]

[0198] Among them, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base and is selected from A, U, C, G, or T.

[0199] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), and the nucleotide modified with a 5'-phosphate analog is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification, as shown in formula (3), or a phosphorothioate-modified nucleotide, as shown in formula (5).

[0200] In some embodiments, in the carrier peptide conjugate of the present disclosure, the functional double-stranded oligonucleotide group is formed by removing one or more atoms from siRNA.

[0201] In some embodiments, the carrier peptide conjugate of the present disclosure is composed of 1 carrier peptide group, 1 linking group, and 1 functional group. The carrier peptide group has the structure shown in the following formula (19):

[0202]

[0203] The linking group has the structure shown in formula (104LA), (104LB), (801L), or (901L), and the functional group is a siRNA group.

[0204] As described above, in the carrier peptide conjugate of the present disclosure, at least one functional group is a functional oligonucleotide group. In the carrier peptide conjugate of the present disclosure, when there are multiple carrier peptide groups, the multiple carrier peptide groups can be linked to the same nucleotide in the functional oligonucleotide group or separately linked to different nucleotides. In order to reduce the influence of steric hindrance on the activity of the functional oligonucleotide, in some embodiments, each covalent bond and / or linking group linked to the carrier peptide group is linked to nucleotides at different positions in the functional oligonucleotide respectively. In some embodiments, each covalent bond and / or linking group linked to the carrier peptide group is linked to adjacent nucleotides. In some embodiments, there is at least 1 nucleotide interval between the nucleotides to which it is linked. In some embodiments, there is at least 3 nucleotide intervals between the nucleotides to which it is linked. In some embodiments, there is at least 5 nucleotide intervals between the nucleotides to which it is linked.

[0205] In some embodiments, the functional oligonucleotide group is a double-stranded oligonucleotide group, and the double-stranded oligonucleotide involved herein contains a sense strand and an antisense strand. Each of the carrier peptide groups is independently linked to the sense strand or the antisense strand. In some embodiments, some of the carrier peptide groups are linked to the sense strand, and some of the carrier peptide groups are linked to the antisense strand. In some embodiments, all of the carrier peptide groups are linked to the antisense strand. In some embodiments, all of the carrier peptide groups are linked to the sense strand.

[0206] In some embodiments, at least one of the carrier peptide groups is linked to the first nucleotide at the 3′ end or 5′ end of the sense strand or the antisense strand. In some embodiments, in the carrier peptide conjugate, the number n of the carrier peptide groups is an integer from 1 to 3. The structure of the carrier peptide conjugate is as described above.

[0207] In some embodiments, n = 1, and the carrier peptide group is linked to the first nucleotide at the 3′ end or 5′ end of the sense strand or the antisense strand. At this time, the carrier peptide conjugate of the present disclosure has a structure shown in any one of formulas (301)-(304):

[0208]

[0209] where 3′ and 5′ respectively represent the first nucleotides at the 3′ end or 5′ end in the sense strand or the antisense strand;

[0210] LK represents a linking group or a covalent bond;

[0211] PP represents a carrier peptide group, and the meanings of "3′", "5′", "LK" and "PP" used in the structural formulas in the following text of the present disclosure are the same as those here.

[0212] In some embodiments, n = 2, one of the carrier peptide groups is linked to the first nucleotide at the 3'-end or 5'-end of the sense strand, and the other carrier peptide group is linked to any one of the nucleotides at other positions in the sense strand. The nucleotides at other positions refer to the nucleotides in the sense strand other than the first nucleotide at the 3'-end or 5'-end. At this time, the carrier peptide conjugate of the present disclosure has the structure shown in formula (305) or formula (306):

[0213]

[0214] Alternatively, one carrier peptide group is linked to the first nucleotide at the 3'-end of the sense strand, and the other carrier peptide group is linked to the first nucleotide at the 5'-end of the sense strand. At this time, the carrier peptide conjugate of the present disclosure has the structure shown in formula (307):

[0215]

[0216] In some embodiments, n = 3, at least one of the carrier peptide groups is linked to the first nucleotide at the 3'-end or 5'-end of the sense strand. In some embodiments, two of the carrier peptide groups are respectively linked to the first nucleotides at the 3'-end and 5'-end of the sense strand, and the other carrier peptide group is linked to any one of the nucleotides at other positions in the sense strand. At this time, the carrier peptide conjugate of the present disclosure has the structure shown in formula (308):

[0217]

[0218] Alternatively, in some embodiments, all of the carrier peptide groups are linked to the first nucleotide at the 3'-end or 5'-end of the sense strand. At this time, the carrier peptide conjugate of the present disclosure has the structure shown in formula (309):

[0219]

[0220] In the formula, R I is defined and has the same selection range as the definition and selection range of the linking group in the above text. The carrier peptide group can be linked to any technically feasible position on the nucleotide. For example, it can be linked to the ribose ring of the nucleotide, and the linking position can be the 2', 3' or 5' position of the ribose ring; or, each carrier peptide group is linked to the base of the nucleotide. To simplify the synthesis process, in some embodiments, each carrier peptide group is linked to the ribose ring of the nucleotide, and the linking position is the 3' or 5' position of the ribose ring.

[0221] In some embodiments, the carrier peptide conjugate of the present disclosure comprises a carboxyl group or an amino group and a functional oligonucleotide group, and the carboxyl group or the amino group can form a salt with a corresponding base or acid. In some embodiments, the functional oligonucleotide group is an siRNA group. In some embodiments, the pharmaceutically acceptable salt of the carrier peptide conjugate comprising an siRNA group is the sodium salt of the carrier peptide conjugate in which the hydrogen ions of the hydroxyl groups in all the phosphate groups in the conjugate are replaced by sodium ions. In these carrier peptide conjugates comprising oligonucleotide groups, each adjacent nucleotide is linked by a phosphodiester bond or a phosphorothioate bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond bears a negative charge, which can exist in the form of a hydroxyl group or a mercapto group, and the hydrogen ions in the hydroxyl group or mercapto group can also be partially or completely replaced by cations. Therefore, in some embodiments, the present disclosure also provides pharmaceutically acceptable salts of the carrier peptide conjugate. The pharmaceutically acceptable salt can be a salt formed by a carboxylate ion formed after the carboxyl group loses a hydrogen ion and a cation, or an organic ammonium cation formed after the amino group obtains a hydrogen ion and an anion, and / or can be a salt formed by a phosphate group in a phosphate group or a phosphorothioate group in the oligonucleotide group and a cation. The cation can be any cation, such as a metal cation, an ammonium ion NH4 + , or one of the organic ammonium cations. For the consideration of improving solubility, in some embodiments, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal ion can be K + and / or Na +, the cation formed by the tertiary amine can be the ammonium ion formed by triethylamine and / or the ammonium ion formed by N,N-diisopropylethylamine. The anion can be any anion, such as one or more of chloride ions and organic acid ions. In some embodiments, the anion can be, for example, a chloride ion or an acetate anion. Thus, the carrier peptide conjugate described in the present disclosure can exist at least partially in the form of a salt. In some embodiments, in the carrier peptide conjugate containing an oligonucleotide group described in the present disclosure, one end of the carrier peptide group is connected to a linking group, and the other end is a protected amino group or carboxyl group. In some embodiments, the amino terminus of the carrier peptide group is connected to the linking group, and the carboxyl terminus is an amide group. In some embodiments, one or all of the carboxyl groups and amino groups in the amino acid side chains of the carrier peptide group exist in a free form. In some embodiments, the carboxyl group in the amino acid side chain combines with a cation to form a salt, such as an alkali metal salt or an ammonium salt. In some embodiments, the amino group in the amino acid side chain combines with an anion to form a salt, such as a hydrochloride or an acetate. In some embodiments, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond in the oligonucleotide group binds at least partially to an alkali metal ion. The pharmaceutically acceptable salt of the carrier peptide conjugate containing an oligonucleotide group described in the present disclosure refers to the water-soluble salt of the conjugate, such as one of an alkali metal salt or an ammonium salt. In some embodiments, the pharmaceutically acceptable salt of the carrier peptide conjugate refers to the sodium salt or a partial sodium salt of the conjugate.

[0222] Unless otherwise specified, in the context, when the carrier peptide conjugate containing an oligonucleotide described in the application or method provided by the present disclosure is mentioned alone, including but not limited to the carrier peptide conjugate represented by any structural formula described in the application or method provided by the present disclosure, it also refers to the pharmaceutically acceptable salt of the conjugate according to the context.

[0223] In context, a "metabolite" refers to a derivative of a compound that is formed when the compound is metabolized in a subject. The metabolite can also be produced by sophisticated chemical synthesis methods after the structure of the derivative is determined using spectroscopy and other analytical means. The term "metabolism" refers to the sum total of processes by which a particular substance is altered by an organism (including, but not limited to, hydrolysis reactions and enzyme-catalyzed reactions). Thus, enzymes may effect specific structural changes to a compound. For example, cytochrome P450 catalyzes a variety of oxidation and reduction reactions, while uridine diphosphate glucuronosyltransferase catalyzes the conversion of activated glucuronic acid molecules to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines, and free sulfhydryls. More information about metabolism can be found in The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). In some embodiments, metabolites of the carrier peptide conjugates of the present disclosure include, but are not limited to: derivatives formed when the carrier peptide group is metabolized, derivatives formed when the linking group is metabolized, and derivatives formed when the functional group is metabolized. In some embodiments, a metabolite of the carrier peptide conjugate of the present disclosure is a compound formed by removing one or more nucleotides from a functional oligonucleotide group.

[0224] In context, a "prodrug" refers to any compound that, when administered to a biological system, yields a biologically active compound due to spontaneous chemical reactions, enzyme-catalyzed chemical reactions, and / or metabolic chemical reactions, or a combination of each. In some embodiments, a prodrug is formed using a group linked to a drug-related functional group such as HO-, HS-, HOOC-, -NHR, which dissociates in vivo. In some embodiments, prodrugs include, but are not limited to, carboxylic acid esters in which the group is alkyl, aryl, aralkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl, and esters of hydroxyl, thiol, and amine in which the linked group is acyl, alkoxycarbonyl, aminocarbonyl, phosphate esters, or sulfate esters. The groups illustrated are merely exemplary, and those skilled in the art are able to prepare various other known prodrugs. In some embodiments, a prodrug of the carrier peptide conjugate of the present disclosure is a compound formed by adding one or more nucleotides that are readily cleaved and removed by nucleases in vivo to a functional oligonucleotide group.

[0225] Synthesis of the carrier peptide conjugate of the present disclosure

[0226] Those skilled in the art can prepare the carrier peptide conjugates of the present disclosure by any suitable method. In some embodiments, the synthesis method of the carrier peptide conjugates of the present disclosure includes contacting a functional molecule and an active carrier peptide under the conditions of a conjugation reaction, and separating to obtain the carrier peptide conjugates of the present disclosure, wherein the active carrier peptide includes the above-mentioned carrier peptide group and one or more reactive groups, and the functional molecule includes the above-mentioned functional group and one or more reactive groups. The conjugation reaction refers to a reaction in which two reactant molecules each remove one or more atoms and then are connected to each other through the formed covalent bond to form a new molecule.

[0227] In some embodiments, the synthesis method of the carrier peptide conjugates of the present disclosure includes contacting an active carrier peptide with one or more functional molecules containing conjugation reaction active groups under the conditions of a conjugation reaction, so that each carrier peptide group is connected to one or more of the functional molecules or another one or more carrier peptide groups, and each functional molecule is connected to one or more of the carrier peptide groups or another one or more functional molecules through a covalent bond and / or a linking group.

[0228] In some embodiments, an active carrier peptide is contacted with the same or different functional molecules containing conjugation reaction active groups multiple times, so that the active carrier peptide is connected to one or more functional molecules through a covalent bond and / or a linking group. Optionally, the method may further include contacting the conjugate molecule of the carrier peptide and the functional molecule obtained in the above step with one or more other active carrier peptides under the conditions where a conjugation reaction can occur.

[0229] In some embodiments, the conjugation reaction is one of an addition reaction, a coupling reaction, and a substitution reaction. In some embodiments, the conditions for the conjugation reaction are the conditions for a coupling reaction. Specifically, in a solvent, under the conditions of a coupling reaction, a functional molecule containing a conjugation-reactive group and an active carrier peptide are contacted. The coupling reaction conditions may be condensation reaction conditions or thiol-disulfide exchange reaction conditions. In some embodiments, the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions, or click chemistry conditions. Correspondingly, the groups with conjugation-reactive activity contained in the active carrier peptide and the functional molecule are active groups capable of undergoing acylation condensation, dehydration condensation, or click chemistry coupling. For example, one of them is a group containing an acyl halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group, or one of them is a group containing an alkynyl group, and the other is a group containing an azide group. In some embodiments, the method for synthesizing the carrier peptide conjugate of the present disclosure includes separately synthesizing a functional molecule and a carrier peptide molecule having a coupling-reactive group, and contacting them with each other under the conditions of a coupling reaction to obtain the carrier peptide conjugate of the present disclosure. Those skilled in the art can understand that for the carrier peptide conjugates of the present disclosure containing different types of functional groups, different active groups and different coupling reaction conditions can be selected according to needs and conventional technical means in the art. Common methods for obtaining carrier peptide conjugates, that is, polypeptide conjugates, are known to those skilled in the art. For example, the methods described in Y. Zhou et al., Carbohydrate Polymers (2021), https: / / doi.org / 10.1016 / j.carbpo1.2021.118662; Ollivier, N. et al., Nat Protoc 10, 269-292 (2015), and Stanley Stein et al., Bioconjugate Chem. 1991, 2, 6, 464-465 are incorporated herein by reference in their entireties respectively.

[0230] Each of the functional molecules is independently selected from one of a functional oligonucleotide and a delivery aid. The functional oligonucleotide or the delivery aid is a compound capable of forming the oligonucleotide group or the delivery aid group as described above. In some embodiments, at least one of the functional molecules is a delivery aid molecule, and the delivery aid molecule is selected from one or more of C8-C 30 alkanes or alkenes, cholesterol molecules, lipid molecules, palmitic acid molecules, and cholic acid molecules. In some embodiments, at least one of the functional groups is an oligonucleotide molecule. The oligonucleotide molecule is capable of adjusting the level of target mRNA in cells in the kidney.

[0231] In some embodiments, the functional group in the carrier peptide conjugate of the present disclosure is a double-stranded oligonucleotide group. At this time, the synthesis method of the carrier peptide conjugate of the present disclosure includes providing a sense strand and an antisense strand of the double-stranded oligonucleotide, annealing the sense strand and the antisense strand to obtain the double-stranded oligonucleotide, wherein the sense strand and the reaction strand each contain 15-25 nucleotides, and each nucleotide is a modified or unmodified nucleotide. At least one of the sense strand or the antisense strand is a sense strand or an antisense strand connected with the carrier peptide group, and the total number of the carrier peptide groups connected to the sense strand or the antisense strand is n. The carrier peptide group is formed by removing one or more atoms from the carrier peptide of the present disclosure. Each carrier peptide group is connected to the sense strand and / or the antisense strand by a covalent bond or through a linking group. Each carrier peptide group is composed of 5-40 amino acids, wherein each amino acid is a modified or unmodified amino acid, and each carrier peptide group is connected to the double-stranded oligonucleotide through the carboxyl terminus or the amino terminus. In some embodiments, the number of the carrier peptide groups, the positions and manners of connecting to the sense strand and / or the antisense strand are as described above.

[0232] In some embodiments, the synthesis method further includes separating and purifying the double-stranded oligonucleotide.

[0233] In some embodiments, the nucleotide single strand connected with n carrier peptide groups can be prepared by the following method: in a solvent, under the conditions of a coupling reaction, contacting the oligonucleotide single strand with an active group R x1 and a compound with an active group R x2 and a carrier peptide group, and reacting to obtain an oligonucleotide single strand with n carrier peptide groups. The molar ratio of the oligonucleotide single strand with an active group R x1 and the compound with an active group R x2 and a carrier peptide group is 1:1 - 1:n. In some embodiments, the carrier peptide group is connected to the oligonucleotide single strand through a linking group, and the active group R x1 and the active group R x2 are groups capable of generating a linking group through a coupling reaction. In some embodiments, the compound with an active group R x2 and a carrier peptide group is the above-mentioned active carrier peptide. In some embodiments, the active group R x2 is the aforementioned reactive group. In some embodiments, the compound with an active group R x2 and a carrier peptide group is obtained by a deprotection reaction of the carrier peptide containing a protected reactive group of the present disclosure.

[0234] The active carrier peptide comprises one or more of the above-mentioned carrier peptide groups and one or more reactive groups, each of the reactive groups being covalently linked to an amino acid residue in the carrier peptide group, the reactive group comprising one or more functional groups or protected functional groups, the functional group being capable of undergoing a conjugation reaction. In some embodiments, the reactive group is capable of reacting with a functional molecule to form a linking group in the carrier peptide conjugate of the present disclosure, thereby achieving the conjugate linkage between the carrier peptide group and the functional group.

[0235] In some embodiments, the number of the reactive groups is 1-3. In some embodiments, in some embodiments, the active carrier peptide comprises only 1 reactive group. In some embodiments, the active carrier peptide is composed of 1-3 carrier peptide groups and 1-3 reactive groups. In some embodiments, the active carrier peptide is composed of 1 carrier peptide group and 1 reactive group. The reactive group may comprise one or more functional groups capable of undergoing a conjugation reaction. In some embodiments, each reactive group independently comprises 1-3 functional groups capable of undergoing a conjugation reaction. In some embodiments, in some embodiments, the active carrier peptide is composed of 1 carrier peptide group and 1 reactive group. In some embodiments, the reactive group has the structure shown in formula (501):

[0236]

[0237] wherein, L R represents any linking group capable of linking the functional group to the carrier peptide group, G F represents the functional group, n501 is an integer from 1 to 3, represents the linking site of the covalent linkage of the groups; or, L R is a straight-chain or branched-chain C1-C 25 or C3-C 15 saturated hydrocarbon group with a valence of 2-7, one or more methylenes (-CH2-) in the straight-chain or branched-chain saturated hydrocarbon group being optionally replaced by one or more linking combinations selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 sub-glycosyl group, C2-C5 sub-alkenyl group, C2-C5 sub-alkynyl group, C6-C 10 sub-aryl group, C3-C8 sub-heterocyclic group and C5-C 10 sub-heteroaryl group; and / or

[0238] if there is a methine group in the branched-chain saturated hydrocarbon group ( methanetriyl), wherein one or more of the methine groups are optionally replaced by one or more selected from the group consisting of: nitrogen atoms, trivalent C3-C 10 Cycloalkyl, trivalent C3-C 10 Heterocyclic group, trivalent C6-C 10 Aryl and trivalent C5-C 10 heteroaryl; and / or

[0239] The saturated hydrocarbon group may optionally have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O) (C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl). In some embodiments, the reactive group is the above-mentioned reactive group R x2 .

[0240] In the above and below, the word "optionally" means that the event cited thereafter occurs or does not occur, or the substance described exists or does not exist, for example, "L R A 2-7 valent straight or branched C1-C 25 Saturated hydrocarbon group, wherein one or more methylene groups in the straight or branched saturated hydrocarbon group are optionally replaced by one or more selected from the group consisting of the following groups" means "L R A 2-7 valent straight or branched C1-C 25 Alkylene, or L R A 2-7 valent straight or branched C1-C 25 A group formed by replacing one or more methylene groups in a saturated hydrocarbon group with one or more selected from the group consisting of the following groups.

[0241] Those skilled in the art are aware of various reactive groups suitable for conjugating an active carrier peptide with a functional molecule through a conjugation reaction. The conjugation reaction refers to a reaction in which two reactant molecules each remove one or more atoms and then are connected to each other through the formed covalent bond to form a new molecule. In some embodiments, the conjugation reaction is one of an addition reaction, a coupling reaction, or a substitution reaction. The addition reaction can be, for example, oxidative addition of an unsaturated bond, Michael addition, alkyne-diazo cycloaddition, Diels-Alder cycloaddition, or ring-opening addition. The coupling reaction can be, for example, acylation coupling, phosphoramidite coupling, or condensation coupling. The substitution reaction can be, for example, transesterification or disulfide exchange reaction. Those skilled in the art can understand that the conjugation reaction described in the present disclosure can also be other types of reactions as long as the connection between the carrier peptide group and the functional group can be achieved.

[0242] Those skilled in the art will understand that in some embodiments, the reactive group can be the active amino group of the first amino acid residue at the amino terminus in the amino acid sequence of the carrier peptide, or the active carboxyl group of the first amino acid residue at the carboxyl terminus, or the side chain of an amino acid residue with a functional group capable of undergoing a conjugation reaction. The amino group, carboxyl group, or side chain is conjugated to the functional molecule through a conjugation reaction. In some embodiments, in order to minimize interference between the carrier peptide group and the functional group, and to fully exert the delivery function and the therapeutic or prophylactic function respectively, and / or for efficient and convenient synthesis, the carrier peptide group and the reactive group are respectively connected to a linker group. After forming an active carrier peptide, the reactive group reacts with the functional molecule to form a carrier peptide conjugate capable of delivering the functional group to the kidney.

[0243] In some embodiments, the functional group capable of undergoing a conjugation reaction is selected from one or more of an azide group, an alkyne group, a dibenzocyclooctyne group, a thiol group, an amino group, a hydroxyl group, a carboxyl group, an acyl halide group, an aldehyde group, a carbonate group, an aminooxy group, an active ester group, a disulfide group, an o-pyridyl disulfide group, a maleimide group, a tosylate group, a tetrazine group, a trans-cyclooctene group, a hydrazide group, and a phosphoramidite group. In some embodiments, for convenient synthesis, each of the functional groups is independently selected from one of an azide group, a thiol group, a maleimide group, a disulfide group, or an o-pyridyl disulfide group.

[0244] Those skilled in the art can understand that when the active carrier peptide contains multiple reactive groups, if the functional groups capable of undergoing conjugation reactions among the multiple reactive groups are functional groups that can react with the same functional molecule to form a covalent bond, for example, when it contains two functional groups that can react with acylating reactive functional groups (such as carboxyl group, activated ester group or acyl chloride group), such as amino group and hydroxyl group, in order to achieve selective reaction and obtain a carrier peptide conjugate with a specific structure, it is necessary to first protect the first functional group and allow the second functional group to undergo conjugation reaction to form a covalent bond. After the reaction is completed, the first functional group is deprotected. The conditions for protection and deprotection are all conventional reaction conditions in the art. For the convenience of storage and transportation, in some embodiments, all reactive groups are protected reactive groups. In some embodiments, when needed, specific protecting groups or all protecting groups are deprotected and then used to form a carrier peptide conjugate through conjugation reaction.

[0245] The preparation of the active carrier peptide can be achieved by selecting appropriate reactive groups and connecting them to the sequence of the carrier peptide group, and this method is known to those skilled in the art. In some embodiments, the active carrier peptide described in the present disclosure can be prepared by the following method: under the conditions that can make the carrier peptide and the reactive molecule undergo conjugation reaction, the carrier peptide is contacted with the reactive molecule, and the active carrier peptide is separated. Among them, the carrier peptide is a carrier peptide containing the amino acid sequence shown in the above formula (I), and the reactive molecule refers to a precursor molecule that can obtain the above reactive group through conjugation reaction. In some embodiments, the carrier peptide is the amino acid sequence shown in formula (II). Those skilled in the art know various reactive groups suitable for conjugating the carrier peptide with the functional molecule through conjugation reaction.

[0246] In some embodiments, in order to ensure the delivery activity of the carrier peptide group, the reactive group is an amino acid residue, one end of the residue is connected to the first amino acid at the carboxyl terminus or amino terminus in the carrier peptide sequence through a peptide bond, and the other end of the residue contains the above functional group. At this time, these amino acid residues are not regarded as part of the carrier peptide sequence. And among them, the functional group capable of undergoing conjugation reaction is the amino group, carboxyl group or functional group on the side chain of the amino acid residue, and the linking group L Ris the remainder after removing the functional group from the amino acid residue. In some embodiments, at least one of the reactive groups is a linking combination of one or more of a cysteine residue, an alanine residue, or an azidohomoalanine residue. In some embodiments, the cysteine residue is a group represented by formula (L38) or (L39). In some embodiments, the azidohomoalanine residue is a group represented by formula (L40) or (L41). In some embodiments, the alanine residue is a group represented by formula (L42) or (L43). In some embodiments, the reactive group has a structure represented by formula (L401), (L402), or (L403):

[0247]

[0248] wherein, represents the site where the group is linked to the rest of the molecule;

[0249] n 38 、n 39 、n 40 、n 41 and n 401 are each independently an integer from 1 to 10;

[0250] R 38 、R 40 and R 42 are each independently a hydroxyl group, a protected hydroxyl group, an amino group, or a site of covalent attachment of a group;

[0251] R 39 、R 41 and R 43 are each independently hydrogen, an amino protecting group, or a site of covalent attachment of a group.

[0252] In some embodiments, for simplicity of structure, n 38 、n 39 、n 40 、n 41 and n 401 are independently integers from 1 to 5. In some embodiments, n 38 、n 39 、n 40 are all 1, n 41 and n 401 are both 4. In some embodiments, R 38 and R 40 are each independently a hydroxyl group or an amino group, and R 39 and R 41 are each independently hydrogen or an amino protecting group. In some embodiments, R 42 and R 43All are sites for covalent attachment of groups. In some embodiments, R 42 forms an amide bond linkage with the imino group in the linking group represented by formula (L38) or (L40); in some embodiments, R 43 forms an amide bond linkage with the carbonyl group in the linking group represented by formula (L39), (L41), (L401) or (L402).

[0253] In some embodiments, the reactive group is attached to the amino-terminal amino acid of the carrier peptide via an amide bond, and the reactive group has a structure represented by formula (L404) or (L405):

[0254]

[0255] In some embodiments, the carrier peptide group is formed by removing one or more atoms from the amino acid residues of the carrier peptide, and the reactive group can be attached to any amino acid in the carrier peptide. In some embodiments, for the convenience of synthesis, the carrier peptide group is formed by removing one or more atoms from the amino-terminal and / or carboxyl-terminal amino acid residues of the carrier peptide, and the reactive group is attached to the amino-terminal amino acid and / or carboxyl-terminal amino acid of the carrier peptide group. In some embodiments, all reactive groups are attached to the amino-terminal amino acid of the carrier peptide. In some embodiments, the active carrier peptide comprises 1 carrier peptide group and 1 reactive group attached to the amino terminus or carboxyl terminus of the carrier peptide.

[0256] In some embodiments, the reactive group is an azidohomoalanine residue represented by formula (L41), and the active carrier peptide is a compound represented by formula (903):

[0257]

[0258] In some embodiments, the reactive group in the active carrier peptide is obtained by subjecting one or more functional monomers containing functional groups capable of undergoing conjugation reactions and carrier peptides to the conditions of the solid-phase peptide synthesis method. The functional monomers contain functional groups capable of undergoing conjugation reactions and functional groups capable of forming peptide bond linkages with carrier peptides by the solid-phase peptide synthesis method. In some embodiments, the functional monomers are known to those skilled in the art and can be synthesized or commercially obtained by those skilled in the art by known methods. In some embodiments, the functional monomers include, but are not limited to, compounds represented by formula (110A), (110B) or (110C):

[0259]

[0260] Those skilled in the art can prepare the carrier peptide molecules corresponding to the carrier peptide groups of the present invention by any suitable technical means. Those skilled in the art know that the carrier peptide can be obtained by successively connecting amino acid monomers starting from a solid-phase carrier under solid-phase synthesis conditions according to the disclosed amino acid sequence of the carrier peptide. The reaction conditions for solid-phase peptide synthesis are known to those skilled in the art, and when the sequence is known and the amino acid monomers used are commercially available, it can be easily obtained by commercial custom synthesis. In some embodiments, the reaction conditions for solid-phase peptide synthesis can be found in Atherton, E.; Sheppard, R.C. Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press. 1989., and the entire content thereof is incorporated herein by reference.

[0261] Those skilled in the art can obtain single-stranded oligonucleotides with the active group R x1 by various methods. In some embodiments, the single-stranded oligonucleotides with the active group R x1 can be obtained by nucleic acid synthesis methods well-known to those skilled in the art, such as phosphoramidite solid-phase synthesis or phosphodiester / phosphotriester liquid-phase synthesis. In some embodiments, the single-stranded oligonucleotides with the active group R x1 are obtained by the phosphoramidite solid-phase synthesis method, which includes successively connecting nucleoside monomers in the order of nucleotides in the single-stranded oligonucleotide under phosphoramidite solid-phase synthesis conditions, wherein at least one nucleoside monomer is a nucleoside monomer with the active group R x1 . The phosphoramidite solid-phase synthesis method is well-known to those skilled in the art, and its process and conditions are disclosed in detail in Methods in Molecular Biology, vol. 288: Oligonucleotide Synthesis: Methods and Applications, P17-P31, and the entire content thereof is incorporated herein by reference.

[0262] The solvent can be any solvent that can achieve the coupling reaction. In some embodiments, the solvent is an organic solvent, such as at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol, and tetrahydrofuran. In some embodiments, the amount of the solvent is 2-6 L / mol relative to the single-stranded oligonucleotide with the active group R x1 , for example, it can be 2.5-4.5 L / mol.

[0263] In some embodiments, the coupling reaction conditions are condensation reaction conditions or thiol-disulfide exchange reaction conditions.

[0264] In some embodiments, the coupling reaction conditions are condensation reaction conditions, and the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions, or click chemistry reaction conditions. The active group R x1 and the active group R x2 are groups capable of undergoing the aforementioned condensation reaction. In some embodiments, the condensation reaction conditions are acylation condensation reaction conditions, and the active groups R x1 and R x2 are groups capable of undergoing acylation condensation reaction to form R I In some embodiments, the condensation reaction conditions are dehydration condensation reaction conditions, and one of the active groups R x1 and R x2 is a group containing an acyl halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. In some embodiments, the condensation reaction conditions are click chemistry conditions, and one of the active groups R x1 and R x2 is a group containing an alkynyl group, and the other is a group containing an azide group.

[0265] In some embodiments, the single-stranded oligonucleotide having the active group R x1 is prepared by contacting a single-stranded oligonucleotide having the active group R x0 with a crosslinking agent under the conditions of a coupling reaction. The crosslinking agent contains a click chemistry active group and an acylation group. The active group and R x0 form a covalent bond through a coupling reaction with the acylation group, connecting the click chemistry active group to the single-stranded oligonucleotide.

[0266] In some embodiments, the active group R x1 is an active group having 1-3 click chemistry active groups at the end, and the click chemistry active group contains a terminal alkynyl group. In some embodiments, the acylation group is an active ester group, such as one of an NHS ester group, an iminoester group, and a pentafluorophenyl ester group. Those skilled in the art can obtain the crosslinking agent through various methods. For example, when the acylation group is a pentafluorophenyl ester group and the click chemistry group contains a terminal alkynyl group, the crosslinking agent can be prepared as described in Prepared by the method described in Scheme 1a(A) of Michael E., et al. "Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides." Bioconjugate chemistry 26.8(2015): 1451-1455. In some embodiments, the active group R x0 is an amino group. In some embodiments, the coupling conditions are basic conditions. In some embodiments, the basic conditions are conditions in the presence of a weak base aqueous solution, such as conditions in the presence of an aqueous sodium bicarbonate solution. Those skilled in the art can obtain the single-stranded oligonucleotide with the active group R x0 in various ways. In some embodiments, the single-stranded oligonucleotide with the active group R x0 is prepared by using a phosphoramidite monomer containing an active group at the corresponding position during the synthesis of the single-stranded oligonucleotide. Those skilled in the art can obtain the phosphoramidite monomer containing an active group in various ways. In some embodiments, the active group R x0 is an amino group, and the phosphoramidite monomer containing R x0 can be commercially obtained or prepared by methods well-known to those skilled in the art. For example, the phosphoramidite monomer containing R x0 can be the readily commercially available 6-(trifluoroacetamido)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer, wherein the active group R x0 is an amino group, and this active group R x0 can be obtained by removing the trifluoroacetyl protecting group through a deprotection reaction (such as ammoniaolysis with concentrated ammonia water) that is easily achievable by those skilled in the art after connecting the phosphoramidite monomer to the single-stranded oligonucleotide by the phosphoramidite solid-phase synthesis method.

[0267] In some embodiments, the coupling reaction conditions are one of the thiol-disulfide exchange reactions, and one of the active groups R x1 and R x2 is a group containing a thiol, and the other contains a leaving group connected by a disulfide bond. To avoid side reactions, in some embodiments, R x1 in the above-mentioned phosphoramidite monomer containing the active group R x1 exists in the form of protected R x1 ′, and the preparation method further includes separating to obtain the oligonucleotide single-stranded containing R x1 ′ by contacting the prepared oligonucleotide single-stranded containing Rx1 Steps of the single-stranded oligonucleotide. In some embodiments, the R x1 ′ contains a disulfide leaving group, the deprotection reaction conditions are thiol-disulfide exchange reaction conditions, and the deprotection reagent is a disulfide activator. In some embodiments, the disulfide activator is 2,2′-dipyridyldisulfide. Those skilled in the art can obtain the above-mentioned phosphoramidite monomer containing the active group R x1 or R x1 ′ through various methods. In some embodiments, the phosphoramidite monomer containing the active group R x1 or R x1 ′ is commercially available. For example, the phosphoramidite monomers shown in formulas (105A), (105B), or (105C) can be obtained through commercial purchase.

[0268]

[0269] where n 105 is an integer from 1 to 10, and m 105 = m 103 .

[0270] In some embodiments, n 105 is an integer from 2 to 6, such as 4 or 5.

[0271] Those skilled in the art can obtain the compound having the active group R x2 and the carrier peptide group through various means. In some embodiments, the coupling reaction conditions are the conditions of dehydration condensation reaction for the condensation reaction. One of the active groups R x1 and R x2 is a group containing an acyl halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. The compound having the active group R x2 and the carrier peptide group can be obtained through commercial customization. In some embodiments, the coupling reaction conditions are click chemistry reaction conditions. The active group R x2 is a compound containing an azide group and a carrier peptide group. The compound having the active group R x2 and the carrier peptide group can be obtained through commercial customization.

[0272] In some embodiments, the linking group is the linking group shown in formula (103L). The carrier peptide conjugate of the present disclosure can be prepared by the following method: In a solvent, at normal temperature and pressure, under thiol-disulfide exchange reaction conditions, the single-stranded oligonucleotide having a thiol exchange group, namely R x1 , shown in formula (106) and n phosphoramidites containing R x2Contact with a compound of a carrier peptide group, and isolate a carrier peptide group-oligonucleotide single-strand conjugate linked by a linking group; anneal the carrier peptide group-oligonucleotide single-strand conjugate with another single strand of the carrier peptide conjugate of the present disclosure to form a double-stranded oligonucleotide, and isolate the carrier peptide conjugate of the present disclosure.

[0273]

[0274] where n 107 = n 103 , pp represents the carrier peptide group;

[0275]

[0276] wherein, Nu′ represents a single-stranded oligonucleotide, m 106 = m 103 ; R 106 is a thiol-exchange agent residue. In some embodiments, R 106 is C7-C 12 aryl or heteroaryl. In some embodiments, R 106 is 2-pyridyl.

[0277] The solvent and the thiol-disulfide exchange reaction conditions are the solvents and reaction conditions commonly used in the art for thiol-disulfide exchange reactions. For example, in the presence of an aqueous ammonium acetate solution with a concentration of 0.05-1 M, the reaction is carried out at normal temperature and pressure for 2-10 h, for example, 4-8 h. The dosage ratio of the solvent to the compound of formula (106) can be 100:1-2000:1 L / mol; the molar ratio of the compound of formula (107) to the compound of formula (106) can be 1:1-15:1, for example, 4:1-10:1.

[0278] Any suitable separation method can be used to separate the carrier peptide-oligonucleotide single-strand conjugate from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the carrier peptide-oligonucleotide single-strand conjugate can be separated by a chromatographic method. For example, the following chromatographic conditions can be used for separation: a C18 reverse-phase chromatographic column as the stationary phase, and a solvent with a ratio of 100 mM TEAA (PH = 7.0-7.3): acetonitrile of 5%-75% (V / V) as the mobile phase for gradient elution. In some embodiments, the solvent can be directly removed to obtain the crude product of the carrier peptide-oligonucleotide single-strand conjugate, and this crude product can be directly used in subsequent reactions.

[0279] The carrier peptide shown in formula (107) can be obtained by the method described in the preparation section of the carrier peptide above.

[0280] Those skilled in the art can obtain the single-stranded oligonucleotide with a thiol exchange group shown in formula (106) by various methods. In some embodiments, the compound shown in formula (106) can be prepared by the following method: in a solvent, under the conditions of a thiol-disulfide exchange reaction, contacting the compound shown in formula (108) with the thiol-disulfide exchange agent shown in formula (111), and separating to obtain the compound shown in formula (106).

[0281]

[0282] wherein, m 106 , Nu′, R 106 are defined and selected within the same ranges as described above; the selection of the solvent and the thiol-disulfide exchange reaction conditions is the same as described above. The molar ratio of the compound of formula (111) to the compound of formula (108) is in a large excess, for example, it can be 10:1 - 1000:1, and in some embodiments, it is 50:1 - 200:1.

[0283] Any suitable separation method can be used to separate the compound of formula (106) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the compound of formula (106) can be separated by a chromatographic method. For example, the following conditions can be used for separation: using an aqueous ethanol solution of 20% (V / V) as the mobile phase, performing gel desalting purification in a purifier, and collecting the product eluate at a wavelength of 280 nm. In some embodiments, the solvent can be directly removed by filtration to obtain the crude product of the compound of formula (106), and this crude product can be directly used in subsequent reactions.

[0284] The thiol-disulfide exchange reagent shown in formula (111) can be prepared by methods well-known to those skilled in the art. In some embodiments, the compound shown in formula (111) is 2,2′-dithiopyridine which can be obtained commercially.

[0285] Those skilled in the art can obtain the single-stranded oligonucleotide with a thiol group shown in formula (108) by various methods. In some embodiments, the single-stranded oligonucleotide shown in formula (108) can be prepared by the following method: in an aqueous solution of a reducing agent, under the conditions of a reduction reaction, contacting the single-stranded oligonucleotide shown in formula (109) with the reducing agent, and separating to obtain the single-stranded oligonucleotide shown in formula (108). In some embodiments, the reducing agent is a reducing agent capable of reducing a disulfide bond to a thiol group. In some embodiments, the reducing agent is TCEP (tris(2-carboxyethyl)phosphine), and the conditions of the reduction reaction are to react in an aqueous solution of TCEP under normal temperature and pressure.

[0286]

[0287] Among them, the definitions and selection ranges of m106 and Nu′ are the same as those described above; the reducing agent and reduction reaction conditions are the commonly used reducing agent and reaction conditions in the field for reduction reactions. For example, in some embodiments, the reducing agent is TCEP. The reaction conditions are to react at normal temperature and pressure for 1 - 5 hours. In some embodiments, the reaction duration is 2 - 3 hours, and the reducing agent is in a large excess. In some embodiments, the "large excess" means that the mass ratio of the reducing agent to the single-stranded oligonucleotide can be 3:1 - 1:1; in some embodiments, the mass ratio of the reducing agent to the single-stranded oligonucleotide can be 1.5:1.

[0288] Any suitable separation method can be used to separate the compound of formula (108) from the reaction mixture. In some embodiments, the reaction solution can be diluted with purified water and filtered, and then ultrafiltration and centrifugation are repeated until the conductivity of the ultrafiltered liquid is measured to be below 100 s, and the product in the filter membrane is collected to obtain the compound of formula (108).

[0289] Those skilled in the art can obtain the single-stranded oligonucleotide shown in formula (109) through various methods. In some embodiments, the single-stranded oligonucleotide shown in formula (109) is prepared by using the phosphoramidite monomer shown in formula (105A) during the solid-phase synthesis of the single-stranded oligonucleotide. The phosphoramidite monomer shown in formula (105A) is readily available to those skilled in the art. In some embodiments, the phosphoramidite monomer shown in formula (105A) is commercially available.

[0290]

[0291] where n 105 is an integer from 1 to 10, m 105 = m 103 ;

[0292] Meanwhile, modified nucleotide groups can also be introduced into the functional double-stranded oligonucleotides described in the present disclosure by using nucleoside monomers with corresponding modifications. The methods for preparing nucleoside monomers with corresponding modifications and the methods for introducing modified nucleotide groups into double-stranded oligonucleotides are also well-known to those skilled in the art.

[0293]

[0294] where, R 107 is -NH2 or -OH, n 107 = n 103 .

[0295] In some embodiments, in the carrier peptide conjugate of the present disclosure, each carrier peptide group is linked to one single strand of the double-stranded oligonucleotide through a linking group represented by formula (201L), and L in formula (201L) AIt has the structure shown in formula (202). At this time, the carrier peptide conjugate of the present disclosure can be prepared by the following method: in a solvent, under the conditions of click chemistry reaction, an oligonucleotide single strand containing a carbon-carbon triple bond and a click peptide (ClickPeptide) containing an -N3 group are subjected to click chemistry reaction to introduce a carrier peptide group into the oligonucleotide single strand containing an active group, and the carrier peptide-oligonucleotide single strand conjugate connected by a linking group is obtained by separation, wherein commercial customization services for click peptides are already available; the carrier peptide group-oligonucleotide single strand conjugate is annealed with another single strand of the carrier peptide conjugate of the present disclosure to form a double-stranded oligonucleotide, and the carrier peptide conjugate of the present disclosure is obtained by separation. The click chemistry reaction conditions are the commonly used click chemistry reaction conditions in the art. In some embodiments, the reaction conditions of the click chemistry are that under heating conditions and in the presence of a catalyst, the click peptide is contacted with the oligonucleotide single strand. In some embodiments, the catalyst is an aqueous solution containing a copper catalyst. In some embodiments, the catalyst is an aqueous solution of cuprous ion (Cu(I)), and the molar ratio of the cuprous ion to the click peptide is 1:6 - 1:1. In some embodiments, the catalyst is an aqueous solution of cuprous ion formed by the mixed reaction of copper sulfate, TBTA, and sodium ascorbate, wherein the molar ratio of copper sulfate to TBTA is 3:1 - 1:3, and the molar ratio of copper sulfate to sodium ascorbate is 1:5 - 1:10. Preferably, the molar ratio of copper sulfate to TBTA is 1:1, and the molar ratio of copper sulfate to sodium ascorbate is 1:7.5. In some embodiments, the carbon-carbon triple bond is present in cyclooctyne, and the click chemistry reaction conditions do not include a catalyst. In some embodiments, the heating conditions refer to reacting in a water bath at 30 - 60 °C. In some embodiments, the heating conditions refer to reacting in a water bath at 30 - 50 °C. In some embodiments, the molar ratio of the click peptide to the oligonucleotide single strand is 10:1 - 3:1. In some embodiments, the molar ratio of the click peptide to the oligonucleotide single strand is 5:1 - 4:1. In some embodiments, the reaction end point is determined by monitoring the content of reactants and / or products in the reaction mixture by HPLC. In some embodiments, the click chemistry reaction conditions are the conditions described in Zengmin Li; Tae Seok Seo; Jingyue Ju (2004). 1,3-Dipolar cycloaddition of azides with electron-deficient alkynes under mild condition in water., 45(15), 3143 - 3146, and its entire content is incorporated herein by reference in its entirety.

[0296] Any suitable separation method can be used to separate the carrier peptide-oligonucleotide single-strand conjugate from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the carrier peptide-oligonucleotide single-strand conjugate can be separated by chromatography. For example, the separation can be carried out under the following chromatography conditions: a C18 reverse-phase chromatography column as the stationary phase, and a solvent with a ratio of 100 mM TEAA (pH = 7.0 - 7.3) and a mixed solvent of acetonitrile / isopropanol (V / V = 1:1) of 5% - 75% (V / V) as the mobile phase for gradient elution. In some embodiments, the solvent can be directly removed to obtain the crude product of the carrier peptide-oligonucleotide single-strand conjugate, and this crude product can be directly used for subsequent reactions.

[0297] In some embodiments, the carrier peptide-oligonucleotide single-strand conjugate is a conjugate represented by formula (203). At this time, the single-stranded oligonucleotide linked with the carrier peptide group can be prepared by the following method:

[0298]

[0299] wherein the definitions of Nu′ and PP are the same as those described above.

[0300] Under the conditions of click chemistry, a click polypeptide containing a reactive group R as shown in formula (204A) x2 is contacted with a single-stranded oligonucleotide containing a reactive group R as shown in formula (205) x1 , and the carrier peptide-oligonucleotide single-strand conjugate shown in formula (203) is separated and obtained, wherein R x1 and R x2 are azide groups. The conditions of the click chemistry are the same as those described above.

[0301]

[0302] Those skilled in the art can obtain the compound containing an azide group and a carrier peptide group as shown in formula (204A) by various methods. In some embodiments, the compound shown in formula (204A) is obtained by commercial order.

[0303] Those skilled in the art can obtain the single-stranded oligonucleotide shown in formula (205) through various methods. For example, under normal pressure and room temperature, and under alkaline conditions, the single-stranded oligonucleotide shown in formula (206) can be contacted with the active ester shown in formula (207), and the single-stranded oligonucleotide shown in formula (205) can be separated and obtained. In some embodiments, the alkaline condition refers to the condition in the presence of an aqueous sodium bicarbonate solution. In some embodiments, the active ester shown in formula (207) is in a large excess compared to the single-stranded oligonucleotide shown in formula (205); in some embodiments, the molar ratio of the active ester shown in formula (207) to the single-stranded oligonucleotide shown in formula (206) is 200:1 - 50:1; in some embodiments, the molar ratio of the active ester shown in formula (207) to the single-stranded oligonucleotide shown in formula (206) is 150:1 - 80:1.

[0304] Those skilled in the art can use any suitable separation method to separate the single-stranded oligonucleotide shown in formula (205) from the reaction mixture. In some embodiments, the single-stranded oligonucleotide shown in formula (205) can be obtained by adding a mixed solvent of acetonitrile and PBS to the reaction solution, centrifuging, adding a nucleic acid precipitation solution (for example, it can be an easily commercially available 10% aqueous trichloroacetic acid solution) to the obtained supernatant, and separating the precipitate.

[0305]

[0306] Those skilled in the art can obtain the active ester shown in formula (207) through various methods. In some embodiments, the active ester shown in formula (207) is obtained by using the preparation method disclosed in Scheme 1a(A) in Stergaard, Michael E., et al. "Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides." Bioconjugate chemistry 26.8 (2015): 1451 - 1455, with the only difference being that the crosslinking agent compound shown in formula (208) is used to replace the initial reactant RCO(CH2)3COOH in Scheme 1a(A), and the entire content thereof is incorporated herein by reference in its entirety. stergaard, Michael E., et al. "Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides." Bioconjugate chemistry 26.8 (2015): 1451 - 1455, Scheme 1a(A), incorporated herein by reference in its entirety, except that the crosslinking agent compound shown in formula (208) is used to replace the initial reactant RCO(CH2)3COOH in Scheme 1a(A).

[0307]

[0308] Those skilled in the art can obtain the compound represented by formula (208) in various ways. In some embodiments, the compound represented by formula (208) is obtained by the following method: in a solvent, under the conditions of ester hydrolysis, hydrolyze the ester represented by formula (209), and separate to obtain the compound represented by formula (208). In some embodiments, the conditions for ester hydrolysis refer to the conditions in an alkaline aqueous solution in the presence of a catalyst. In some embodiments, the alkaline aqueous solution is an aqueous solution of sodium hydroxide, and the catalyst is ethanol, where the molar ratio of sodium hydroxide to the compound represented by formula (209) is 7:1 - 3:1, the concentration of the sodium hydroxide aqueous solution is 2M - 5M, and the volume ratio of ethanol to the sodium hydroxide aqueous solution is 7:1 - 3:1. Those skilled in the art can use various methods to separate the compound represented by formula (208). In some embodiments, the pH value of the reaction solution can be adjusted to 5 - 6 by using an acidic solution, and then the compound represented by formula (208) can be separated by column chromatography.

[0309]

[0310] Those skilled in the art can obtain the compound represented by formula (209) by various methods. In some embodiments, it can be prepared by the following method: under the conditions of a condensation reaction, contact the compound represented by formula (210) with the compound represented by formula (211), and separate to obtain the compound represented by formula (209):

[0311]

[0312]

[0313] In some embodiments, the condensation reaction conditions are amidation reaction conditions. For example, it can be in an organic solvent in the presence of 1-hydroxybenzotriazole (HOBt), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and N,N-diisopropylethylamine (DIEA). In some embodiments, the organic solvent is DMF. In some embodiments, the molar ratio of the compound represented by formula (210) to the compound represented by formula (211) is 1:2 - 1:5. In some embodiments, the molar ratio of the compound represented by formula (210) to 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine is 1:3 - 1:10, and the dosage ratio of DMF to the compound represented by formula (210) is 7 ml / g - 20 ml / g. Those skilled in the art can isolate the compound represented by formula (209) by various methods. In some embodiments, water and ethyl acetate can be added to the reaction solution for extraction to obtain an organic phase, and the obtained organic phase is successively washed with 10% citric acid, sodium bicarbonate, and saturated brine solution, and the solvent is evaporated to obtain the compound represented by formula (209). Those skilled in the art can obtain the compound represented by formula (210) by various methods. In some embodiments, the compound represented by formula (210) is prepared according to the method of Scheme 3 disclosed in WO2014025805A1. Those skilled in the art can obtain the compound represented by formula (211) by various methods. In some embodiments, the compound represented by formula (211) is obtained by commercial purchase.

[0314] Those skilled in the art can obtain the single-stranded oligonucleotide represented by formula (206) by various methods. For example, during the synthesis of the single-stranded oligonucleotide, phosphoramidite monomers containing reactive groups can be used at corresponding positions. In some embodiments, the phosphoramidite monomer containing a reactive group is 6-(trifluoroacetamido)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer. The phosphoramidite monomer is easily obtained by those skilled in the art. In some embodiments, the phosphoramidite monomer is obtained by commercial purchase.

[0315] In some embodiments, in the carrier peptide conjugate of the present disclosure, each carrier peptide group is connected to one single strand of the double-stranded oligonucleotide through a linking group represented by formula (201L), and k in formula (201L) is 1, L Ais an amino acid residue. At this time, the carrier peptide conjugate of the present disclosure can be prepared by the following method: in a solvent, under the conditions of an acylation reaction, a single-stranded oligonucleotide containing an amino group, an active carrier peptide containing an amino group, and a bis-reaction-site acyl compound are respectively contacted for an acylation reaction. By forming a bis-amide bond, the carrier peptide group is introduced into the single-stranded oligonucleotide containing an active group, and the carrier peptide-single-stranded oligonucleotide conjugate connected by the above linking group is obtained by separation; the carrier peptide group-single-stranded oligonucleotide conjugate is annealed with the other single strand of the carrier peptide conjugate of the present disclosure to form a double-stranded oligonucleotide, and the carrier peptide conjugate of the present disclosure is obtained by separation. The solvent, reaction conditions, etc. of the acylation reaction are as described above. The active carrier peptide containing a carboxyl group can be obtained by commercial customization or by preparation as described above. The bis-reaction-site acyl compound can be, for example, a dianhydride or an active ester-acyl halide compound, and the types of these compounds and the acylation reaction method are well known to those skilled in the art.

[0316] Furthermore, by forming the carrier peptide conjugate of the present disclosure, the carrier peptide conjugate of the present disclosure can rapidly, efficiently, and safely deliver a functional molecule in the form of a functional group to the kidney, and release or not release the corresponding functional molecule after reaching the target tissue or cell, thereby exerting a preventive or therapeutic effect.

[0317] Pharmaceutical composition

[0318] On the other hand, the present disclosure provides a pharmaceutical composition, which contains one or more of the above-mentioned carrier peptide conjugate, its pharmaceutically acceptable salt, metabolite or prodrug as an active ingredient, and a pharmaceutically acceptable excipient.

[0319] The pharmaceutically acceptable excipient is one or more of various components conventionally used in the art, such as a solvent, a protective agent, an osmotic pressure regulator, and one or more of other pharmaceutically acceptable carriers.

[0320] For example, when the pharmaceutical composition is an injection solution, the pharmaceutically acceptable excipient is a solvent, such as deionized water, water for injection, ethanol or a pH buffer solution. The pH buffer solution can be a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH value of 7.5 - 8.5 and / or a phosphate buffer solution with a pH value of 5.5 - 8.5. For example, it can be p a phosphate buffer solution with a pH value of 5.5 - 8.5.

[0321] The dosage of the solvent is adjusted according to the required solution concentration. Based on the functional group in the carrier peptide conjugate, the concentration of the carrier peptide conjugate in the injection solution can be 0.01 mg / mL - 20 mg / mL, 0.1 mg / mL - 10 mg / mL, or 0.5 mg / mL - 5 mg / mL.

[0322] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0323] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmoles per kilogram (mOsm / kg). Those skilled in the art can easily determine the content of the osmotic pressure regulator according to the required osmotic pressure. In some embodiments, the dosage of the preparation made from the pharmaceutical composition will be adjusted depending on the mode of administration during administration.

[0324] In some embodiments, the pharmaceutical composition may be a liquid preparation, such as an injection; or it may be a freeze-dried powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous injection administration, and may also be used, but is not limited to, for delivering the pharmaceutical composition to the lungs by spraying, or to other organ tissues (such as the liver) through the lungs by spraying, or by oral administration, etc. In some embodiments, the pharmaceutical composition is administered by subcutaneous injection.

[0325] The other pharmaceutically acceptable carriers may be carriers commonly used in the field of peptide conjugate administration, such as, but not limited to, magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylenephosphate) (PPEEA), and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and one or more of their derivatives.

[0326] In some embodiments, in the pharmaceutical composition, there is no particular requirement for the contents of the carrier peptide conjugate and the other pharmaceutically acceptable carriers. In some embodiments, the weight ratio of the functional group to the pharmaceutically acceptable carrier in the carrier peptide conjugate may be 1:(1 - 500). In some embodiments, the above weight ratio is 1:(1 - 50).

[0327] In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various components commonly used in the art, such as one or more of a diluent, a binder, a disintegrant, a lubricant, a capsule agent, a protective agent, an osmotic pressure regulator, or a solvent.

[0328] For example, when the pharmaceutical composition is an injection solution, the pharmaceutically acceptable excipients include a solvent such as deionized water, water for injection, ethanol, or a pH buffer solution.

[0329] The pH buffer solution can be a tris (hydroxymethyl) aminomethane hydrochloride buffer solution with a pH value of 7.5 - 8.5 and / or a phosphate buffer solution with a pH value of 5.5 - 8.5. For example, it can be a phosphate buffer solution with a pH value of 5.5 - 8.5. The dosage of the solvent is adjusted according to the required solution concentration. Calculated based on the functional groups, the concentration of the functional groups in the injection solution is a solution of 0.01 mg / mL - 5 mg / mL siRNA, 0.1 mg / mL - 5 mg / mL, or 0.5 mg / mL - 3 mg / mL.

[0330] The cryoprotectant protects the carrier peptide conjugate by weakening crystallization or reducing physical damage. The cryoprotectant can be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the cryoprotectant can be 0.01 - 30% by weight.

[0331] The osmotic pressure regulator can be, for example, sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200 - 700 milliosmoles per kilogram (mOsm / kg). Those skilled in the art can easily determine the content of the osmotic pressure regulator according to the required osmotic pressure. In some embodiments, the dosage of the preparation made from the pharmaceutical composition will be adjusted according to the different administration methods.

[0332] In some embodiments, the pharmaceutical composition can be in the form of a liposomal preparation. At this time, the pharmaceutically acceptable excipients are the pharmaceutically acceptable carriers used in the liposomal preparation. In some embodiments, the pharmaceutically acceptable carriers used in the liposomal preparation include amine-containing transfection compounds (hereinafter also referred to as organic amines), co-lipids, and / or polyethylene glycolylated lipids. Among them, the organic amines, co-lipids, and polyethylene glycolylated lipids can be selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, co-lipids, and polyethylene glycolylated lipids described in Chinese Patent Application CN103380113A (which is incorporated herein by reference in its entirety).

[0333] In some embodiments, the organic amine can be the compound shown in formula (800) described in Chinese Patent Application CN103380113A or its pharmaceutically acceptable salt:

[0334]

[0335] Wherein:

[0336] Each X 101 Or X 102 Each is independently O, S, N-A, or C-A, where A is hydrogen or C1-C20 Hydrocarbon chain;

[0337] Each Y 101 or Z 101 is independently C=O, C=S, S=O, CH-OH or SO2;

[0338] Each R 101 、R 102 、R 103 、R 104 、R 105 、R 106 or R 107 is independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;

[0339] x is an integer from 1 to 10;

[0340] n 800 is an integer from 1 to 3, m 800 is an integer from 0 to 20, and p is 0 or 1; provided that if m 800 =p=0, then R 102 is hydrogen;

[0341] And, if n 800 or m 800 is at least one of 2, then R 103 and the nitrogen in formula (800) form a structure as shown in formula (802) or formula (803):

[0342]

[0343] wherein g, e and f are independently integers from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (800).

[0344] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (800) is independently an optionally substituted or unsubstituted, branched or straight-chain alkyl or alkenyl group having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0345] In some embodiments, if n 800and m 800 each independently has a value of 1 or 3, then R 103 can be any one of the following formulas (804) - (813):

[0346]

[0347]

[0348] wherein, in formulas (804) - (813), g, e, and f are each independently an integer from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates a possible connection point of R 103 to the nitrogen atom in formula (800), where each H at any * position can be replaced to achieve connection to the nitrogen atom in formula (800).

[0349] wherein, the compound shown in formula (800) can be prepared according to the description in Chinese Patent Application CN103380113A.

[0350] In some embodiments, the organic amine is the organic amine shown in formula (814) and / or the organic amine shown in formula (815):

[0351]

[0352] The auxiliary lipid is cholesterol, an analogue of cholesterol, and / or a derivative of cholesterol;

[0353] The polyethylene glycolated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0354] In some embodiments, in the pharmaceutical composition, the molar ratio among the organic amine, the auxiliary lipid, and the polyethylene glycolated lipid is (19.7 - 80):(19.7 - 80):(0.3 - 50), for example, it can be (50 - 70):(20 - 40):(3 - 20).

[0355] In some embodiments, the pharmaceutical composition particles formed by the carrier peptide conjugate described in the application or method provided by the present disclosure and the above amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, more usually, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. For example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0356] In some embodiments, in the pharmaceutical composition formed by the carrier peptide conjugate described in the application or method provided by the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the functional group to all lipids (such as organic amines, co-lipids, and / or polyethylene glycolated lipids) ranges from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10. For example, the weight ratio of the functional group to all lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

[0357] In some embodiments, the components of the pharmaceutical composition may exist independently when sold and may exist in the form of a liquid preparation when in use. In some embodiments, the pharmaceutical composition described in the application or method provided by the present disclosure can be prepared by various known methods, only by replacing the drug active ingredient in the known pharmaceutical composition in the art with the carrier peptide conjugate described in the application or method provided by the present disclosure; in some embodiments, it can be prepared according to the following method:

[0358] Suspend the organic amine, co-lipid, and polyethylene glycolated lipid in alcohol according to the above molar ratio and mix well to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2 - 25 mg / mL, for example, it can be 8 - 18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, and for example, it can be ethanol.

[0359] Dissolve the carrier peptide conjugate described in the application or method provided by the present disclosure in a buffer salt solution to obtain an aqueous solution of the carrier peptide conjugate. The concentration of the buffer salt solution is 0.05 - 0.5 M, for example, it can be 0.1 - 0.2 M, adjust the pH of the buffer salt solution to 4.0 - 5.5, for example, it can be 5.0 - 5.2, and the amount of the buffer salt solution is such that the concentration of the functional group does not exceed 0.6 mg / mL, for example, it can be 0.2 - 0.4 mg / mL. The buffer salt is selected from one or more of soluble acetates and soluble citrates, and for example, it can be sodium acetate and / or potassium acetate.

[0360] Mix the lipid solution and the aqueous solution of the carrier peptide conjugate, and incubate the resulting product at 40 - 60 °C for at least 2 minutes, for example, it can be 5 - 30 minutes, to obtain the incubated liposomal preparation. The volume ratio of the lipid solution to the aqueous solution of the carrier peptide conjugate is 1:(2 - 5), for example, it can be 1:4.

[0361] Concentrate or dilute the incubated liposomal preparation, remove impurities, and sterilize to obtain the pharmaceutical composition provided by the present disclosure, whose physicochemical parameters are as follows: the pH value is 6.5 - 8, the encapsulation efficiency is not less than 80%, the particle size is 40 - 200 nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250 - 400 mOsm / kg; for example, the physicochemical parameters can be that the pH value is 7.2 - 7.6, the encapsulation efficiency is not less than 90%, the particle size is 60 - 100 nm, the polydispersity index is not higher than 0.20, and the osmotic pressure is 300 - 400 mOsm / kg.

[0362] Among them, concentration or dilution can be carried out before, after, or simultaneously with the removal of impurities. The method for removing impurities can adopt various existing methods. For example, a tangential flow system or a hollow fiber column can be used for ultrafiltration under the condition of 100 kDa, and the ultrafiltration exchange solution is phosphate buffer (PBS) with a pH of 7.4. The method for sterilization can adopt various existing methods. For example, it can be filtered and sterilized through a 0.22 μm filter. In some embodiments, the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation can be used for, but is not limited to, subcutaneous, intramuscular, or intravenous injection administration, and can also be delivered by, but is not limited to, eye drops, nasal administration, oropharyngeal inhalation, spray administration, etc. In some embodiments, the pharmaceutical composition is used for intravenous injection or subcutaneous injection administration.

[0363] Applications of the carrier peptide conjugate and pharmaceutical composition of the present disclosure

[0364] In some embodiments, the present disclosure also provides the use of the carrier peptide conjugate and / or the pharmaceutical composition of the present disclosure in the preparation of a drug for treating and / or preventing kidney-related diseases. In some embodiments, the disease is a disease related to the mRNA of the target gene expressed in cells in the kidney.

[0365] In some embodiments, the present disclosure also provides a method for treating and / or preventing kidney-related diseases, which includes administering to a subject an effective amount of one or more of the carrier peptide conjugate of the present disclosure, its pharmaceutically acceptable salt, metabolite, or prodrug, and the pharmaceutical composition of the present disclosure. In some embodiments, the disease is a disease related to the target mRNA in cells in the kidney.

[0366] In some embodiments, the target mRNA in the above-mentioned use or method is SOD1. By administering the carrier peptide conjugate and / or pharmaceutical composition provided by the present disclosure to a subject in need, the functional group can be targeted and delivered to the relevant tissues and / or cells in the kidney, and then the therapeutic and / or prophylactic effects can be exerted through the functional group. For example, when the functional group is an oligonucleotide group, the purpose of preventing and / or treating a pathological condition or disease caused by the expression of a specific gene in cells is achieved through the mechanism of regulating gene expression. Therefore, the carrier peptide composition and / or pharmaceutical composition provided by the present disclosure can be used for preventing and / or treating the pathological condition or disease, or for preparing a drug for preventing and / or treating the pathological condition or disease described herein.

[0367] In some embodiments, the present disclosure also provides the use of one or more of the carrier peptide conjugate of the present disclosure, its pharmaceutically acceptable salts, metabolites or prodrugs, and the pharmaceutical composition in the preparation of a drug for treating kidney-related diseases. In some embodiments, the present disclosure also provides a method for treating kidney-related diseases, the method comprising administering an effective amount of the carrier peptide conjugate and / or pharmaceutical composition of the present disclosure to a subject in need.

[0368] As used herein, the term "administering / giving" refers to placing the carrier peptide conjugate and / or pharmaceutical composition in a subject by a method or route that causes at least partial localization of the carrier peptide conjugate and / or pharmaceutical composition at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. Generally, local administration results in delivery of more of the carrier peptide conjugate and / or pharmaceutical composition to a specific site compared to the entire body of the subject; while systemic administration results in delivery of the carrier peptide conjugate and / or pharmaceutical composition to substantially the entire body of the subject.

[0369] The subject can be administered by any suitable route known in the art, including but not limited to: oral or parenteral routes such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), rectal administration, and local administration (including buccal administration and sublingual administration). The frequency of administration can be once or multiple times per day, per week, every two weeks, every three weeks, per month, every two months, every three months, every six months, or annually.

[0370] The dosage of the carrier peptide conjugate and / or pharmaceutical composition described in the present disclosure can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, weight and gender of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD50 (the lethal dose that causes 50% of the population to die) and ED50 (in a quantal response, it refers to the dose that can cause 50% of the maximum response intensity, and in a qualitative response, it refers to the dose when 50% of the experimental subjects show a positive response). The dosage range for human use can be derived based on the data obtained from cell culture analysis and animal studies.

[0371] When using the method provided by the present disclosure for treating and / or preventing kidney-related diseases, the amounts of the functional carrier peptide and the functional group in the provided carrier peptide conjugate and / or pharmaceutical composition can be easily determined by those skilled in the art according to the desired effect. For example, in some embodiments, the functional group is a double-stranded oligonucleotide, and the amount of the functional double-stranded oligonucleotide in the provided carrier peptide conjugate is an amount sufficient to reduce the expression of the target gene and result in an extracellular concentration of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM or up to about 5 nM at the surface of the target cell. The amount required to achieve this local concentration will vary with various factors, including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, whether the delivery is local or systemic, etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.

[0372] Kit

[0373] The present disclosure provides a kit, which contains an effective dose of one or more of the carrier peptide conjugate provided by the present disclosure, its pharmaceutically acceptable salts, metabolites or prodrugs, and the pharmaceutical composition of the present disclosure.

[0374] In some embodiments, the kit described herein can provide the carrier peptide conjugate and / or pharmaceutical composition in one container. In some embodiments, the kit described herein can contain a container providing a pharmaceutically acceptable excipient. In some embodiments, the kit can further contain other components, such as stabilizers or preservatives, etc. In some embodiments, the kit described herein can contain at least one other therapeutic agent in a container different from the container providing the carrier peptide conjugate and / or pharmaceutical composition described herein. In some embodiments, the kit can contain instructions for mixing the carrier peptide conjugate and / or pharmaceutical composition with a pharmaceutically acceptable carrier and / or excipient or other components (if any).

[0375] In the kit of the present disclosure, the carrier peptide conjugate and the pharmaceutically acceptable carrier and / or excipient, and the pharmaceutical composition and / or the pharmaceutically acceptable carrier and / or excipient can be provided in any form, such as liquid form, dry form, and / or lyophilized form. In some embodiments, the carrier peptide conjugate and the pharmaceutically acceptable carrier and / or excipient, and the pharmaceutical composition and / or the optional pharmaceutically acceptable excipient are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

[0376] The present disclosure will be further illustrated by examples below, but the present disclosure is not limited thereby.

[0377] Examples

[0378] Unless otherwise specified, the reagents and cultures used in the following examples are commercially available products, and the operations such as nucleic acid electrophoresis and real-time PCR are carried out according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory (1989)).

[0379] For the amino acid sequences in the following examples, unless otherwise specified, the amino acids used in preparing the sequences and the amino acid residues in the sequences are natural (unmodified) amino acids and natural amino acid residues. For the conjugates containing siRNA sequences in the following examples, unless otherwise specified, the obtained conjugates are the sodium salts of the conjugates in which the hydrogen ions of the hydroxyl groups in all the phosphate groups in the conjugates are replaced by sodium ions.

[0380] The present method relates to the synthesis of a carrier peptide conjugate, including separately synthesizing a siRNA sense strand with a reactive group and an active carrier peptide molecule. Under the conditions of a coupling reaction, they are brought into contact with each other to obtain a sense strand linked with a carrier peptide group; subsequently, the sense strand is contacted with the siRNA antisense strand to finally obtain the desired carrier peptide conjugate.

[0381] Preparation Example 1 Synthesis of Carrier Peptide Conjugate 1

[0382] (1-1) The active carrier peptide 1 was synthesized according to the solid-phase synthesis method of polypeptides, and the specific steps are as follows:

[0383] (1-1-1) Deprotection: Take 0.5 g of Rink amide MBHA resin (amino-protected resin, purchased from Gil Biochemical Co., Ltd.) with a substitution degree of 0.67 mmol / g, add 10 mL of a DMF solution of 20 v / v% piperidine, and react at room temperature for 25 min to obtain the resin with deprotected amino groups.

[0384] (1-1-2) Coupling: Take 0.67 mmol of the monomer with the structure shown in formula (110A) ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-azidohexanoic acid (Adamas, catalog number 3313309D)) and 1.34 mmol of 1-hydroxybenzotriazole (HOBT), dissolve them in 20 mL of DMF, then add 1.34 mmol of N,N-diisopropylcarbodiimide (DIC), and react at room temperature for 5 minutes to obtain a solution of the activated monomer of formula (110A).

[0385]

[0386] Mix the obtained solution of the activated monomer of formula (110A) with the resin with deprotected amino group obtained in step (1-1), react at room temperature and normal pressure for 120 minutes, remove the solvent in the reaction solution under reduced pressure, and wash successively with DCM, DMF, DCM, and DMF at room temperature and normal pressure, 25 mL each time, to obtain the resin connected with the monomer of formula (110A) with protected amino group.

[0387] Add 20 v / v% acetic anhydride / DCM solution (the addition amount is 10 mL of solution per 0.5 g of resin), react at room temperature under an argon atmosphere for 40 mmin; wash the resin successively with DCM, DMF, DCM, and DMF at room temperature and normal pressure, 25 mL each time.

[0388] (1-1-3) For the resin obtained in step (1-1-2), repeat the deprotection method in step (1-1-1) and the coupling method in step (1-1-2), use the amino-protected amino acid monomer as the raw material, and couple the remaining amino acid monomers corresponding to the amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the direction from the carboxyl terminus to the amino terminus in sequence to obtain a peptide resin connected with 16 amino acids in the amino acid sequence shown in SEQ ID NO: 1 and with the terminal amino group protected:

[0389] KKEEEKKEEEKKEEEK (SEQ ID NO: 1);

[0390] Among them, the amino-protected lysine (K) monomer is shown in formula (112A); the amino-protected glutamic acid (E) monomer is shown in formula (112B):

[0391]

[0392] (1-1-4) Cleavage: Prepare a cleavage solution with a volume ratio of TFA: benzyl methyl sulfide: phenol: TIS: water = 87.5:5:2.5:2.5:2.5; after all monomers are connected, at room temperature and atmospheric pressure, contact the peptide resin with the protected terminal amino group with the cleavage solution. The dosage of the cleavage solution is 5 mL of the cleavage solution per 0.5 g of resin, react for 3 hours, filter, collect the filtrate, add it to MTBE with a volume 4 times that of the filtrate, and centrifuge to obtain a solid.

[0393] (1-1-5) Purify the obtained solid by reverse-phase preparative column chromatography. After collecting the fractions containing the product, concentrate to remove the solvent and lyophilize to obtain the active carrier peptide 1. The column chromatography conditions are as follows: stationary phase: ChromCore 10-120 C18 preparative column, 10 μm, 21.2×250 mm; mobile phase: gradient elution is used, mobile phase A: 0.1% TFA / water, mobile phase B: acetonitrile; gradient conditions: 0-20 min, 10%-40% B; 20-30 min, 40% B; 30-40 min, 40%-50% B.

[0394] The obtained active carrier peptide 1 has the structure shown in formula (903):

[0395]

[0396] After diluting the obtained active carrier peptide 1 to a concentration of 0.2 mg / mL with ultrapure water (resistivity 18.2 MΩ·cm (25 °C)), use a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier) to detect the molecular weight. The theoretical molecular weight is 2272.501, and the measured molecular weight is 2944.56 (including the trifluoroacetamide protecting group). The results show that the measured value is consistent with the theoretical value, indicating that the active carrier peptide 1 is obtained.

[0397] (1-2) Synthesis of the carrier peptide conjugate 1

[0398] (1-2-1) Synthesis of the sense strand of the siRNA sequence

[0399] Through the nucleic acid solid-phase synthesis method (solid-phase phosphoramidite method), connect the nucleoside monomers one by one in the 3′-5′ direction according to the siRNA sequence shown in SEQ ID NO: 2 to obtain the sIRNA sense strand of the carrier peptide conjugate:

[0400] UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmsAmsAm

[0401] (SEQ ID NO: 2)

[0402] Among them, the capital letters C, G, U, and A represent the base composition of nucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluoro group; the lowercase letter s indicates that there is a phosphorothioate linkage between the two nucleotides on the left and right of the letter s.

[0403] By the solid-phase phosphoramidite method, nucleoside monomers are sequentially linked one by one in the 3′-5′ direction according to the nucleotide arrangement order of the sense strand. Each connection of a nucleoside monomer includes four reaction steps: deprotection, coupling, capping, and oxidation or sulfurization. Among them, when a phosphoester linkage is used between two nucleotides, when connecting the latter nucleoside monomer, it includes four reaction steps: deprotection, coupling, capping, and oxidation. When a phosphorothioate linkage is used between two nucleotides, when connecting the latter nucleoside monomer, it includes four reaction steps: deprotection, coupling, capping, and sulfurization. The synthesis conditions are given as follows:

[0404] The nucleoside monomers are provided in an acetonitrile solution with a concentration of 0.1M. The conditions for each deprotection reaction are the same, that is, the temperature is 25°C, the reaction time is 70 seconds, the deprotection reagent is a dichloromethane solution of dichloroacetic acid (3% v / v), and the molar ratio of dichloroacetic acid to the 4,4′-dimethoxytrityl protecting group on the solid support is 5:1.

[0405] The conditions for each coupling reaction are the same, including a temperature of 25°C, a molar ratio of the nucleic acid sequence linked to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence linked to the solid support and the coupling reagent of 1:65, a reaction time of 600 seconds, and the coupling reagent is a 0.5M acetonitrile solution of 5-(Ethylthio)-1H-tetrazole (ETT).

[0406] The conditions for each capping step are the same, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence linked to the solid support is acetic anhydride:N-methylimidazole:nucleic acid sequence linked to the solid support = 1:1:1.

[0407] The conditions for each oxidation reaction are the same, including a temperature of 25°C, a reaction time of 15 seconds, and the oxidation reagent is an iodine water solution with a concentration of 0.05M. The molar ratio of iodine to the nucleic acid sequence linked to the solid support in the coupling step is 30:1. The reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0408] The conditions for each step of the vulcanization reaction are the same, including a temperature of 25 °C, a reaction time of 300 seconds, and the vulcanizing reagent is hydrocarbyl xanthate. The molar ratio of the vulcanizing reagent to the nucleic acid sequence linked to the solid-phase carrier in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:1.

[0409] (1-2-2) In the 3′-5′ direction of solid-phase synthesis, after the last nucleoside monomer linked to the 5′ end of the sense strand, another alkynyl-C5 phosphoramidite monomer shown in formula (902) (purchased from Di Na Xing Ke Co., Ltd.) is linked according to the solid-phase synthesis method:

[0410]

[0411] The linking method is the same as the above four-step reactions of deprotection, coupling, capping, and oxidation to obtain a sense strand of siRNA with an alkynyl end.

[0412] (1-2-3) Synthesis of the sense strand linked with the carrier peptide group:

[0413] (1-2-3-1) Ammonolysis: Add the sense strand with an alkynyl end obtained from the above process (20 mg, 1.0 eq) to ammonia water with a concentration of 25 wt%, the dosage of ammonia water is 0.5 ml / μmol, react at 55 °C for 16 h, remove the liquid, and concentrate to dryness under vacuum to obtain a purified sense strand with an alkynyl end.

[0414] (1-2-3-2) Click chemistry coupling: Dissolve the sense strand with an alkynyl end (20 mg, 1.0 eq) after ammonolysis in 1.2 mL of purified water to obtain an aqueous solution of the sense strand. Dissolve the active carrier peptide 1 (12 mg, 3.0 eq) prepared according to the method described in step (1-1) in 1.2 mL of DMF to obtain a solution of the active carrier peptide 1. Mix the solution of the active carrier peptide 1 with the aqueous solution of the sense strand to obtain a reaction system. Add a mixture of 50 mM aqueous copper sulfate solution (188 μL, 3.0 eq), 200 mM aqueous THPTA solution (48 μL, 3.0 eq), and 1 M aqueous sodium ascorbate solution (48 μL, 15.0 eq) to the above reaction system, and react at a constant temperature of 40 °C for 3 h to obtain a product mixture of the sense strand linked with the carrier peptide group.

[0415] Purify the above product mixture using an Agilent semi-preparative reversed-phase column chromatography, stationary phase: XBridgePrep / C18, 10 μm, 19 * 250 mm; Gradient elution was used for the mobile phase: Eluent component A: 100 mM TEAA (pH = 7.0 - 7.3), Eluent component B: acetonitrile, gradient: B% 5 - 65%, 0 - 40 min. The product eluent was collected, concentrated to remove the solvent, and a sense strand linked with a carrier peptide group was obtained.

[0416] Molecular weight detection was performed on a liquid chromatography - mass spectrometry (LC - MS, purchased from Waters, model: LCT Premier). The theoretical molecular weight of the sense strand linked with the carrier peptide group was 8958.3, measured value: [M + H + = 8957.3. The molecular weight detected by LC - MS was consistent with the theoretical value, indicating that the sense strand linked with the carrier peptide group was successfully obtained. The sense strand linked with the carrier peptide group has the structure shown in the following formula (905):

[0417]

[0418] Among them, represents the siRNA sense strand group, having the nucleotide sequence shown in SEQ ID NO: 2. The carrier peptide group is linked to the 5'-terminal nucleotide of the siRNA sense strand group; the 5'-hydroxyl group of the ribose on the 5'-terminal nucleotide of the sense strand forms a phosphoester bond with the P atom in the structure of formula (905).

[0419] (1 - 2 - 4) Synthesis of siRNA antisense strand

[0420] According to the above solid - phase phosphoramidite method, nucleoside monomers were connected one by one according to the sequence of SEQ ID NO: 3 to prepare an antisense strand having the sequence shown in SEQ ID NO: 3:

[0421]

[0422] In the 3'-5' direction, after connecting the last nucleoside monomer at the 5'-end of the antisense strand, the Cy5 phosphoramidite monomer (purchased from Shanghai Zhaowei Technology Development Co., Ltd., product number OP - 057, having the structure shown in formula (907)) was linked to the 5'-end of the antisense strand through four steps of deprotection, coupling, capping, and oxidation:

[0423]

[0424] In this step, the differences between the deprotection, coupling, capping, and oxidation reaction conditions used and the corresponding reaction conditions for nucleic acid solid-phase synthesis in Preparation Example 2 are as follows: 1) The deprotection reaction time is extended to 300 seconds; 2) The Cy5 coupling reaction time is extended to 900 seconds; 3) The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the carrier is added to the AMA solution (a mixed solution of 40 wt% aqueous methylamine solution and 25 wt% aqueous ammonia solution with a volume ratio of 1:1), and the amount of the AMA solution is 0.5 ml / μmol. The reaction is carried out in a water bath at 25 °C for 2 h. The remaining carrier is removed by filtration, and the supernatant is concentrated to dryness under vacuum. The obtained siRNA antisense strand linked with the Cy5 fluorescent group has the structure shown in formula (908):

[0425]

[0426] Among them, represents the siRNA antisense strand group, having the sequence shown in SEQ ID NO: 3. The Cy5 fluorescent group is linked to the 5'-position of the ribose of the 5'-terminal nucleotide of the siRNA antisense strand group; the hydroxyl group at the 5'-position of the ribose on the 5'-terminal nucleotide of the antisense strand forms a phosphoester bond with the P atom in the structure of formula (908). Molecular weight detection is carried out on a liquid chromatography-mass spectrometry (LC-MS, purchased from Waters, model: LCT Premier). The theoretical molecular weight of the antisense strand linked with the Cy5 fluorescent group is 7630.4, and the measured value: [M+H + = 7628.7. The measured value is consistent with the theoretical value, indicating that the antisense strand linked with the Cy5 fluorescent group is successfully obtained.

[0427] (1-2-5) Synthesis of the carrier peptide conjugate 1

[0428] The siRNA sense strand and the siRNA antisense strand linked with the carrier peptide group are respectively dissolved in water for injection to form a solution of 0.5 nmol / μL and mixed. They are left standing at 90 °C for 10 min, cooled naturally to room temperature, and freeze-dried to obtain the solid of the carrier peptide conjugate 1.

[0429] Preparation Example 2-3 Synthesis of the carrier peptide conjugates 2 and 3 provided by the present disclosure

[0430] In this preparation example, the carrier peptide conjugates 2 and 3 were prepared and obtained according to the following method:

[0431] (2-1) According to the method for synthesizing the active carrier peptide 1 obtained in Preparation Example 1, the active carrier peptide 2 was synthesized according to the amino acid sequence shown in SEQ ID NO: 1; the difference is that both the lysine (K) with protected amino group and the glutamic acid (E) with protected amino group for synthesizing the active carrier peptide 2 are in D configuration.

[0432] (2-2) According to the method for synthesizing the carrier peptide conjugate 1 in Preparation Example 2, the nucleoside monomers were sequentially linked to the active carrier peptide 2 from the 3'-5' direction according to the siRNA sequence shown in SEQ ID NO: 2 to obtain the siRNA sense strand of the carrier peptide conjugate 2 with a linked active carrier peptide group. The nucleoside monomers were sequentially linked according to the sequence of the siRNA sequence shown in SEQ ID NO: 3 from the 3'-5' direction to obtain the siRNA antisense strand. The sense strand with a linked carrier peptide group and the siRNA antisense strand were separately dissolved in water for injection to form a solution of 0.5 nmol / μL and mixed, left standing at 90 °C for 10 min, naturally cooled to room temperature, and freeze-dried to obtain the solid of the carrier peptide conjugate 2. After the synthesis of the carrier peptide conjugate 2 was completed, the obtained carrier peptide conjugate was diluted to a concentration of 0.2 mg / mL with ultrapure water (prepared by a Milli-Q ultrapure water instrument, resistivity 18.2 MΩ·cm (25 °C)), and the molecular weight was detected using a liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier).

[0433] The theoretical molecular weight of the sense strand of the carrier peptide conjugate 2 is 8958.3, measured value: [M+H + = 8957.3, the theoretical molecular weight of the antisense strand is 7096.8, measured value: [M+H + = 7095.9, and the LC-MS measured value is consistent with the theoretical value, indicating that the target-designed carrier peptide conjugate 2 was obtained.

[0434] (3-1) According to the methods described in the above steps (2-1) and (2-2), the carrier peptide conjugate 3 was prepared. The difference is only that the lysine (K) monomer in the L configuration and the glutamic acid (E) monomer in the L configuration were used to replace the lysine monomer and the glutamic acid monomer in step (2-1), respectively. The molecular weight was detected using a liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier).

[0435] The theoretical molecular weight of the sense strand of the carrier peptide conjugate 3 is 8958.3, measured value [M+H +=8957.3, the theoretical molecular weight of the antisense strand is 7096.8, measured value: [M+H + =7095.9, the measured value is consistent with the theoretical value, indicating that the target-designed vector peptide conjugate 3 has been obtained.

[0436] Compare the synthesis of reference conjugate 1 and reference siRNA2 in Preparation Examples 4-5

[0437] In this preparation example, reference conjugate 1 and reference siRNA2 were prepared by the following method:

[0438] Reference conjugate 1 has the same sense strand as vector peptide conjugate 1 (i.e., SEQ ID NO: 2). However, it does not contain the nucleotide sequence of active vector peptide 1.

[0439] (4-1) According to the nucleic acid solid-phase synthesis method (solid-phase phosphoramidite method) described in step (1-2-1) of Preparation Example 1, and the ammonolysis method described in step (1-2-3-1) for synthesis, the difference is that the connection of the alkynyl group described in (1-2-2) is not carried out, and ammonolysis is directly carried out to obtain the sense strand of siRNA of reference conjugate 1;

[0440] (4-2) According to the solid-phase synthesis method described in step (1-2-4) of Preparation Example 1, the antisense strand of siRNA with a Cy5 fluorescent group at the 5′ end was prepared. The obtained antisense strand of siRNA linked with the Cy5 fluorescent group has the structure shown in the above formula (908).

[0441] (4-3) According to the method described in step (1-2-5) of Preparation Example 1, annealing was carried out to obtain reference conjugate 1 provided in the present disclosure. Reference conjugate 1 is a conjugate of an siRNA group and a Cy5 fluorescent group, and the Cy5 fluorescent group is linked to the 5′ end of the siRNA antisense strand. Molecular weight detection was carried out using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The theoretical molecular weight of the sense strand of reference conjugate 1 is 6183.2, measured value [M+H + =6183.4, the theoretical molecular weight of the antisense strand is 7630.4, measured value: [M+H +=7629.9. The measured value is consistent with the theoretical value, indicating that the reference conjugate 2 with the target design is obtained. According to the above method, the reference siRNA2 is prepared. The difference is that after the ligation of the antisense strand nucleoside monomers is completed, no additional Cy5 phosphoramidite monomer is ligated. The molecular weight is detected by a liquid chromatography-mass spectrometry (LC-MS, purchased from Waters Corporation, model: LCT Premier). The theoretical molecular weight of the sense strand of the reference siRNA2 is 6183.2, and the measured value [M+H + =6183.4. The theoretical molecular weight of the antisense strand is 7096.8, and the measured value: [M+H + =7095.9. The measured value is consistent with the theoretical value, indicating that the reference siRNA2 with the target design is obtained.

[0442] Experimental Example 1 Effect of the vector peptide conjugate of the present disclosure on targeting mouse kidney tissue

[0443] Twenty-five female C57BL / 6 mice aged 6-8 weeks (purchased from Beijing Spearf Bio-Technology Co., Ltd.) are selected. Among them, 10 mice are experimental group mice, 10 mice are reference control group mice, and the remaining 5 mice are blank control group mice. The body weight of each mouse is about 25 g.

[0444] (1.1) Fluorescent imaging experiment of organs of experimental group mice

[0445] (1.1.1) Intravenous injection

[0446] The vector peptide conjugate 1 is dissolved in a phosphate buffer solution (PBS) with pH 7.4 to form a vector peptide conjugate 1 solution with a concentration of 1.125 mg / mL. The vector peptide conjugate 1 solution is injected into the tail vein of 10 experimental group mice once, and each mouse is injected with 0.2 mL of the solution. The dosing dose is calculated according to the body weight of the mouse. Calculated by the amount of the vector peptide, the dosing dose is 9 mg / kg. The dosing day is recorded as D1.

[0447] (1.1.2) Anesthesia and perfusion

[0448] On D2, D3, D5, D8, and D14 after dosing, 2 mice are selected from 12 experimental group mice and anesthetized by intraperitoneal injection of anesthetic. When the muscles of the mice are completely relaxed and the eyelid response disappears, it indicates that the mice enter a deep anesthesia state. Among them, the anesthetic is a mixture of Zoletil (tiletamine hydrochloride and zolazepam hydrochloride for injection) and xylazine hydrochloride (purchased from Suzhou Kunchen Biotechnology Co., Ltd.). The mass ratio of Zoletil to xylazine hydrochloride is 4:1. Calculated by the body weight of the mouse, the total dosing dose of the anesthetic is 62.5 mg / kg.

[0449] Place the above 2 deeply anesthetized mice in the supine position. Use surgical scissors to cut open the ribs and diaphragm upward from both sides of the sternum of the mice to expose the heart. Insert a syringe into the left ventricle of the mice, cut open the right auricle, and slowly perfuse about 20 mL of normal saline until the liver turns white. Stop perfusion, take out the lung, liver, and kidney tissues of the mice, and store them in 20 mL of 4% paraformaldehyde fixative (purchased from Beyotime Biotechnology Co., Ltd.).

[0450] (1.1.3) Imaging of organ tissues:

[0451] For the heart, liver, spleen, lung, and kidney of each mouse, fluorescence imaging was performed in an IVIS Lumina III (PE) small animal in vivo imaging system (purchased from PerkinElmer Enterprise Management (Shanghai) Co., Ltd.). The imaging results are shown in Figure 1. Among them, the excitation wavelength of the imaging system was set to 480 nm, the emission wavelength was 520 nm, and the exposure time was 1 s.

[0452] (1.2) Fluorescence imaging experiment of organ tissues of mice in the blank control group:

[0453] According to the above steps (1.1), on D2, D3, D5, D8, and D14, one mouse was selected from 5 mice in the blank control group for the experiment. The only difference was that in step (1.1.1), instead of injecting the PBS solution with pH 7.4 into the tail vein of 5 control group mice, the injection volume of PBS was 0.2 mL / mouse.

[0454] (1.3) Fluorescence imaging experiment of organ tissues of mice in the reference control group

[0455] According to the above steps (1.1), on D2, D3, D5, D8, and D14, one mouse was selected from 10 control group mice for the experiment. The only difference was that in step (1.1.1), instead of injecting the reference conjugate 1 with the same concentration into the tail vein of 10 control group mice, the injection volume for each mouse was 0.2 mL / mouse.

[0456] The results of steps (1.1), (1.2), and (1.3) are as Figure 1A-1E shown.

[0457] (1.4) Imaging of kidney tissue sections:

[0458] Further, for the other 2 experimental group mice, 2 control group mice and 2 reference control groups, the administration was carried out according to the methods described in the above steps (1.1.1), (1.2) and (1.3) respectively. At D8 and D14 after administration to each group of mice, one blank control group mouse, one reference control group and one experimental group mouse were anesthetized and perfused respectively. The specific method of the specific operation was the same as that described in the aforementioned step (1.1.2). The difference was that kidney tissues of each mouse were taken, dehydrated overnight in the presence of sucrose, sectioned coronally, cryosectioned, stained with DAPI, and confocal imaging was performed on the kidney tissue sections. The DAPI laser intensity was 0.2%, the gain was 650V, the Cy5 laser intensity was 0.5%, and the gain was 650V. The results of the experimental group mice, reference control group mice and blank control group mice at D8 are respectively as Figure 2A-2C shown, and the results at D14 are respectively as Figure 3A-3C shown.

[0459] Figure 1A-1E Figures respectively showing the fluorescence imaging of the heart, liver, spleen, lungs and kidneys in the body of mice at D2, D3, D5, D8 and D14 after injection of PBS, reference conjugate 1 or the carrier peptide conjugate 1 of the present disclosure.

[0460] From Figure 1A-1E the imaging results, it can be seen that no fluorescence imaging was observed in the tissues of the mice injected with the PBS solution at each time point; among the 10 mice injected with the carrier peptide conjugate 1, there was no obvious fluorescence signal in the heart, spleen and lung tissues, the fluorescence signal in the liver tissue was significantly lower than that in the kidney, but obvious fluorescence signals were visible in the kidney tissues. Further, at D5, the fluorescence signal of the carrier peptide conjugate 1 of the present disclosure in the kidney tissue was significantly higher than that of the reference conjugate 1; at D8, the carrier peptide conjugate 1 of the present disclosure could still show a strong fluorescence signal in the kidney tissue, while the reference conjugate 1 showed a weak fluorescence signal; at D14, the carrier peptide conjugate 1 of the present disclosure could still show a fluorescence signal in the kidney tissue, while the reference conjugate 1 did not show a fluorescence signal.

[0461] The results show that the carrier peptide conjugate 1 containing a functional oligonucleotide group provided by the present disclosure can efficiently target the kidney tissue of mice and has stronger stability or a longer duration.

[0462] Figure 2A-2C Figures respectively showing the confocal microscopy imaging of the kidney tissue sections in the body of each group of mice after 8 days of PBS, reference conjugate 1 or the carrier peptide conjugate 1 of the present disclosure.

[0463] Figure 3A-3CThey are confocal microscopy images of kidney tissue sections in each group of mice 14 days after PBS, reference conjugate 1, or carrier peptide conjugate 1 of the present disclosure, respectively.

[0464] Figure 2A and 3A They are confocal microscopy images of kidney tissue sections in control group mice at day 8 or day 14 after administration of PBS, respectively; Figure 2B and 3B They are confocal microscopy images of kidney tissue sections in experimental group mice at day 8 or day 14 after administration of reference conjugate 1, respectively. Figure 2C and 3C They are confocal microscopy images of kidney tissue sections in experimental group mice at day 8 or day 14 after administration of carrier peptide conjugate 1 of the present disclosure, respectively.

[0465] Figure 2A-2C In 3A - 3C, the blue DAPI fluorescence shows the position of cell nuclei, and the red Cy5 fluorescence signal represents the position of carrier peptide conjugate 1 or reference conjugate 1 containing the Cy5 fluorophore. From Figure 2A-2B and the imaging results of 3A - 3B, it can be seen that only the fluorescence imaging showing the cell nuclei of the kidney tissue stained by DAPI can be seen in the kidney tissues of 1 mouse injected with PBS solution and the kidney injected with reference conjugate 1.

[0466] In contrast, from Figure 2C the imaging results, it can be seen that obvious red Cy5 fluorescence imaging can be seen in the kidney tissue of the mouse injected with carrier peptide conjugate 1 at day 8. And the imaging signal distribution range mostly coincides with the cell nuclei and is near the blue signal showing the cell nuclei, and clearly shows the boundary of the tubular cross-section, which indicates that the carrier peptide conjugate 1 provided by the present disclosure can effectively deliver functional oligonucleotides specifically into renal tubular epithelial cells.

[0467] From Figure 3C the imaging results, it can be seen that weaker red Cy5 fluorescence imaging can still be seen in the kidney tissue of the mouse injected with carrier peptide conjugate 1 at day 14, which indicates that in the carrier peptide conjugate 1 provided by the present disclosure, the contained functional oligonucleotides can continuously play their roles for a long time.

[0468] In summary, the carrier peptide conjugate 1 containing functional oligonucleotides provided by the present disclosure can efficiently target renal tubular tissues in mouse kidneys, can effectively deliver functional oligonucleotides specifically into renal tubular epithelial cells, and can continuously play their roles for a long time.

[0469] Experimental Example 2 Effects of the carrier peptide conjugate of the present disclosure on targeting rat kidney tissues at different times

[0470] (2.1) According to the method described in step (1.1) of Experimental Example 1, the targeting effect of the carrier peptide conjugate 1 entering the rat kidney tissue at different times was investigated. The differences are as follows:

[0471] i) The experimental animals used were SD rats (purchased from Spf (Beijing) Biotechnology Co., Ltd.), and their weights were all about 150 g;

[0472] ii) In step (1.1.1), the carrier peptide conjugate 1 with a concentration of 0.2 mg / ml was administered. Two rats were in each group, and 0.75 mL of the solution was administered to each. The dosage was calculated according to the body weight of the rats. Calculated by the amount of the carrier peptide, the dosage was 1 mg / kg. The day of administration was recorded as D1 and served as the experimental group.

[0473] (2.2) According to the method described in step (1.2) of Experimental Example 1, 0.75 mL of PBS solution was administered, and the fluorescence results of PBS in the rat kidney tissue at different times were investigated. The difference was only that the experimental animals used were rats; after administering PBS, one rat was anesthetized and perfused on the 3rd, 6th, and 10th days respectively, and the heart, liver, spleen, lung, and kidney tissues were taken and the fluorescence intensity was measured, serving as the blank control group.

[0474] (2.3) According to the method described in step (1.3) of Experimental Example 1, 0.75 mL of the reference conjugate 1 solution with a concentration of 0.2 mg / ml was administered. Five rats were selected in each group. After administering the reference conjugate 1, two rats were anesthetized and perfused on the 3rd and 6th days respectively, and one rat was anesthetized and perfused on the 10th day. The heart, liver, spleen, lung, and kidney tissues were taken and the fluorescence intensity was measured, serving as the reference control group.

[0475] The results of the above steps (2.1), (2.2), and (2.3) are respectively shown in Figure 4A-4C as follows.

[0476] Figure 4A-4C They are the images of fluorescence imaging of different organs in rats on the 3rd, 6th, and 10th days after administering PBS, the carrier peptide conjugate 1 of the present disclosure, or the reference conjugate 1. From Figure 4A-4CAs shown by the imaging results, no fluorescence imaging was observed in any of the tissues of the 1 rat injected with the PBS solution. The rats injected with reference conjugate 1 showed strong fluorescence signals in the kidneys on the 3rd and 6th days after administration, but the fluorescence signals rapidly weakened and disappeared at subsequent times, indicating that without the carrier peptide group, siRNA was metabolized out of the body within a few days. In contrast, for the rats injected with carrier peptide conjugate 1, obvious fluorescence signals were visible in the kidney tissues from the 3rd to the 10th day after administration, indicating that the carrier peptide conjugate 1 of the present disclosure could rapidly target the rat kidney tissues after administration and maintain the targeted delivery effect for a long time.

[0477] Experimental Example 3 Effect of the carrier peptide conjugate of the present disclosure on targeting mouse renal tubular cells

[0478] According to the method of Experimental Example 1, the targeting effect of carrier peptide conjugate 1 on entering mouse renal tubular cells was investigated. The difference was only that the drug administered was carrier peptide conjugate 1, and a fluorescently labeled anti-aquaporin (AQP1) antibody was administered simultaneously. This antibody specifically targets the AQP1 protein mainly expressed in the renal proximal tubular epithelial cells and shows a strong green fluorescence signal. Confocal imaging was performed on kidney tissue sections at the 2nd and 5th days after administration of carrier peptide conjugate, and the results are shown in Figure 5A-5D and Figure 5E as shown.

[0479] Figure 5A-5D is a confocal microscopy image of a mouse kidney tissue section (40X field of view) at the 2nd day after administration of the carrier peptide conjugate 1 of the present disclosure. Among them, Figure 5A-5C are images of Cy5 fluorescence imaging, anti-AQP1 antibody fluorescence imaging, and DAPI fluorescence imaging respectively, Figure 5D is Figure 5A-5C is the superimposed image. As can be seen from the imaging results of Figure 5A-5D , the blue fluorescence imaging stained by DAPI represents the nuclei of the kidney tissues, and the imaging of the renal tubular cells with high AQP1 expression shown by the green fluorescence of the anti-AQP1 antibody together show some cell structures in the kidney tissues; meanwhile, obvious red Cy5 fluorescence imaging was visible in the kidney tissues of the mice injected with carrier peptide conjugate 1 at the 2nd day after administration, and the imaging signal distribution range overlapped with the green fluorescence signal of some anti-AQP1 antibodies, and mostly overlapped with the cell nuclei and was near the blue signal showing the cell nuclei, and clearly showed the cell boundaries, indicating that the carrier peptide conjugate of the present disclosure could efficiently target the renal tubular tissues in the mouse kidneys and could effectively deliver the delivery adjuvant and siRNA specifically into some renal tubular epithelial cells.

[0480] Figure 5EThis is an overlay image (20X field of view) of different fluorescence imaging diagrams obtained by confocal microscopy of kidney tissue sections in mice on the 5th day after administration of the carrier peptide conjugate 1 of the present disclosure. As can be seen from Figure 5E the imaging results, obvious red Cy5 fluorescence imaging was still visible on the 5th day after administration, and the imaging distribution range coincided with the green fluorescence signal of part of the anti-AQP1 antibody, clearly showing the cell boundaries; at the same time, the cluster of cells formed by the blue nuclear signals (circled by a dashed line) had no adjacent green and red signals, indicating that the carrier peptide conjugate of the present disclosure did not enter glomerular cells that do not express the AQP1 protein and still exhibited good specific targeting effects at a relatively long time on the 5th day after administration.

[0481] Experimental Example 4 Activity of the Carrier Peptide Conjugate of the Present Disclosure in Mice

[0482] This experimental example investigated the inhibitory activity of the carrier peptide conjugate 2 on SOD1 mRNA in different tissues of mice, especially in the kidney.

[0483] The mice used in this experiment were purchased from SPF Biotechnology Co., Ltd., with a strain of C57BL / 6, a grade of SPF, all female, and 6 - 8 weeks old.

[0484] The carrier peptide conjugate 2 was dissolved in PBS to a solution of 1.8 mg / mL (calculated as siRNA). C57BL / 6 mice (female, with an average body weight of about 20 g, 6 - 8 weeks old, purchased from SPF Biotechnology Co., Ltd.) were randomly grouped, with 5 mice in each group and numbered respectively. By subcutaneous injection in the nape of the neck, each mouse in one group was given the carrier peptide conjugate 2 solution. Weighed and recorded the body weight before administration, and administered the drug according to the body weight, with a dosing volume of 5 mL / kg for all, as the test group; in addition, each of the 5 mice in another group was given PBS, with a dosing volume of 5 mL / kg for all, as the blank control group; in addition, each of the 5 mice in another group was given the reference siRNA2, with a dosing volume of 5 mL / kg for all, as the reference control group.

[0485] Calculating the dosing time point as the 1st day, on the 8th day, 15th day, and 29th day after administration, 5 mice from the test group and the blank control group were sacrificed, and the left renal cortex tissue and renal medulla tissue were taken and stored in RNAlater. The cutting method is as follows:

[0486] Cut off the outer 1 / 3 part of the kidney along the longitudinal section, cut it into small pieces of 2 - 3 mm with a blade and put them into RNAlater as the renal cortex part; take the remaining inner part of the kidney with a lighter color and harder texture, remove the adhering cortex completely as the renal medulla part. Cut the renal cortex part and the renal medulla part into small pieces respectively and store them in RNA later.

[0487] Total RNA was extracted from the cells in each well using TRI Reagent (Sigma, catalog number T9424) according to the method described in the instruction manual.

[0488] For the cells in each well, 1 μg of total RNA was taken respectively, and the reagents provided by the reverse transcription kit Goldenstar TM RT6cDNASynthesis Kit (purchased from Beijing Tsingke New Industry Biotechnology Co., Ltd., catalog number TSK301M) were used. Among them, Goldenstar TM Oligo(dT) 17 was used as the primer. A 20-μL reverse transcription reaction system was prepared according to the reverse transcription operation steps in the kit instruction manual to perform reverse transcription on the total RNA of the cells in each well. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the reverse transcription reaction system was incubated at 50 °C for 50 min, then incubated at 85 °C for 5 min, and finally incubated at 4 °C for 30 s. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0489] For each reverse transcription reaction system, 5 μL of the above solution containing cDNA was taken as the template respectively, and the reagents provided by the SYBR qPCR SuperMix Plus kit (purchased from Novoprotein Scientific Inc., catalog number E096-01B) were used to prepare a 20-μL qPCR reaction system. Among them, the PCR primer sequences for amplifying the target gene SOD1 and the internal reference gene GAPDH are shown in Table 1, and the final concentration of each primer was 0.25 μM. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using the three-step method. The amplification program was pre-denaturation at 95 °C for 10 min, then denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. After repeating the above denaturation, annealing, and extension processes 40 times, a product W1 containing the amplified target gene SOD1 and internal reference gene GAPDH was obtained. The product W1 was then incubated at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s in sequence. The real-time fluorescence quantitative PCR instrument collected the melting curves of the target gene and the internal reference gene GAPDH in the product W1 respectively to obtain the Ct values of the target gene SOD1 and the internal reference gene GAPDH.

[0490] Table 1 Primer information

[0491]

[0492] The comparative Ct (ΔΔCt) method was used to perform relative quantification calculations on the target gene SOD1 in each test group (8 groups of vector peptide conjugates, 10 groups of reference siRNAs), and the calculation method was as follows:

[0493] ΔCt (test group) = Ct (target gene of the test group) - Ct (reference gene within the test group)

[0494] ΔCt (control group) = Ct (target gene of the control group) - Ct (reference gene within the control group)

[0495] ΔΔCt (test group) = ΔCt (test group) - ΔCt (average of the control group)

[0496] ΔΔCt (control group) = ΔCt (control group) - ΔCt (average of the control group)

[0497] Among them, ΔCt (average of the control group) is the arithmetic mean of the ΔCt (control group) of the two culture wells of the blank control group. Thus, each culture well of the test group and the blank control group corresponds to a ΔΔCt value.

[0498] Based on the average value of the blank control group, the expression level of SOD1 mRNA in the test group was normalized, and the average value of the expression level of SOD1 mRNA in the blank control group was defined as 100%,

[0499] Relative expression level of SOD1 mRNA in the test group = 2 -ΔΔCt(测试组) × 100%

[0500] Inhibition rate of SOD1 mRNA in the test group = (1 - relative expression level of SOD1 mRNA in the test group) × 100%

[0501] Figure 6A and Figure 6B are respectively graphs of the relative expression levels of SOD1 mRNA in the renal cortex and renal medulla of mice at different time points after administration of the vector peptide conjugate or PBS of the present disclosure. According to Figure 6A and 6BThe results showed that, compared with the blank control group at different time points, after administration of the vector peptide conjugate 2, the relative expression level of SOD1 mRNA in the kidneys of mice was significantly inhibited at different time points, and the highest inhibition rate could reach 47.49%; showing relatively high inhibitory activity; moreover, the inhibition rate still had a good inhibitory effect on the 29th day, indicating that the vector peptide conjugate of the present disclosure could still stably and efficiently inhibit the target mRNA for a longer time; in addition, compared with the reference control group at different time points, the relative expression level of SOD1 mRNA in the kidneys of mice by the vector peptide conjugate 2 was lower than that of the reference control group, indicating that the vector peptide conjugate of the present disclosure could more efficiently inhibit the target mRNA. In addition, since SOD1 mRNA is generally expressed in vivo, the difference in the inhibition rate can reflect the specific difference in the targeting and entry of the vector peptide into cells. At D15 and D29, compared with the inhibition rate in the medulla, the inhibition rate of SOD1 mRNA in the cortex was significantly higher, indicating that the vector peptide conjugate of the present disclosure could effectively target and enter the proximal tubular tissue with a higher content in the cortex.

[0502] Experimental Example 5 Activity of the Vector Peptide Conjugate of the Present Disclosure in Mice

[0503] This experimental example investigated the inhibitory activity of vector peptide conjugate 2 and vector peptide conjugate 3 on SOD1 mRNA in different tissues of the kidneys of mice in vivo.

[0504] The mice in this experiment were purchased from Spf Biotechnology Co., Ltd., with a strain of C57BL / 6, a grade of SPF, all female, 6 - 8 weeks old, and a body weight of about 20 g.

[0505] The vector peptide conjugate 2 and the vector peptide conjugate 3 were respectively dissolved in PBS to a solution of 1.8 mg / mL (calculated based on siRNA). The C57BL / 6 mice were randomly grouped, with 5 mice in each group and numbered respectively. By subcutaneous injection in the nape of the neck, each mouse in one group was respectively given the vector peptide conjugate 2 solution. Weighed and recorded the body weight before administration, and administered the drug according to the body weight, with a dosing volume of 5 mL / kg for all, as test group 1; each mouse in a group of 5 mice was respectively given the vector peptide conjugate 3 solution. Weighed and recorded the body weight before administration, and administered the drug according to the body weight, with a dosing volume of 5 mL / kg for all, as test group 2;

[0506] Calculated with the dosing time point as the 1st day, on the 8th day after administration, 5 mice from test group 1, test group 2, and the blank control group were sacrificed, and the left renal cortex tissues were taken and stored with RNAlater. The cutting method is as follows:

[0507] Cut off the outer 1 / 3 part of the kidney along the longitudinal section, cut it into small pieces of 2-3 mm with a blade and put them into RNAlater as the renal cortex part. Cut the renal cortex part into small pieces and store them in RNA later.

[0508] Extract the total RNA and reverse transcription from the tissue cells of each group by the same method as in Experimental Example 5, and perform relative quantitative calculation on the target gene SOD1 in each test group. The primer information used is shown in Table 1. The inhibition rate results are respectively as Figure 7 shown.

[0509] The experimental results are as Figure 7 shown. Figure 7 is a graph showing the relative expression levels of SOD1 mRNA in the renal cortex of mice on the 8th day after administration of the vector peptide conjugate or PBS of the present disclosure. According to Figure 7 the results, compared with the blank control group, the relative expression levels of SOD1 mRNA in the kidneys of mice after administration of vector peptide conjugate 2 and vector peptide conjugate 3 were significantly inhibited. The average inhibition rate of vector peptide conjugate 2 reached 51.56%, and the average inhibition rate of vector peptide conjugate 3 reached 49.42%, showing high inhibitory activity; indicating that the vector peptide conjugate of the present disclosure can effectively target and enter the proximal renal tubular tissue with a higher content in the cortex, and has a good effect of inhibiting the target mRNA.

[0510] Some embodiments of the present disclosure have been described in detail above. However, the present disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

[0511] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above embodiments can be combined in any suitable manner. In order to avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0512] In addition, any combination can be made between the various different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, it should also be regarded as the content disclosed by the present disclosure.

Claims

1. A carrier peptide conjugate comprising one or more carrier peptide groups and one or more functional groups, in, Each of the carrier peptide groups is connected to at least one of the functional groups or at least one other carrier peptide group via a linking group, and each functional group is connected to at least one of the carrier peptide groups or at least one other functional group via a linking group; the length of each carrier peptide group is 5 to 40 amino acid residues, each carrier peptide group contains one amino acid sequence segment as shown in formula (I) or multiple amino acid sequence segments as shown in formula (I) connected in series, and the amino acid residues (X) in all the amino acid sequence segments as shown in formula (I) are A ) and (X B ) accounts for more than 50% of the total number of amino acid residues in the carrier peptide group, -(X B ) n2 -(X A ) n1 -Z n0 - Formula (I) Among them, each (X A ) independently represent amino acid residues having acidic side chain groups, each (X B ) independently represents an amino acid residue having a basic side chain group, each Z independently represents any amino acid residue, each n1 and n2 independently represents an integer from 1 to 10, and n0 represents an integer from 0 to 2; Each of the functional groups is independently one of a functional oligonucleotide group and a delivery aid group, and at least one functional group is a functional oligonucleotide group.

2. The conjugate according to claim 1, wherein The number of the carrier peptide groups in the conjugate is n, and the number of the functional groups is m, where n is an integer selected from 1-8, and m is an integer selected from 1-4; or, n is an integer selected from 1-4, and m is an integer selected from 1-3.

3. The conjugate according to claim 1 or 2, wherein Each linking group connected to the carrier peptide is independently connected to the carboxyl group at the carboxyl terminal of the carrier peptide group, or to the amino group at the amino terminal of the carrier peptide group.

4. The conjugate according to claim 3, wherein The conjugate has a structure as shown in formula (101): Wherein, each of the linking groups is composed of k LA, L B and L C Composition, L A is the side chain part, L B To connect L C and Nu conjugated linker, L C is a branching group, Nu and each PP independently represent the functional group or the carrier peptide group, and at least one of Nu and all PP is the functional group, and at least one is the carrier peptide group; each k is independently an integer of 1-6, n 101 is an integer from 1 to 3; and the sum of all k is an integer from 1 to 8; The branching group L C A 2-7 valent straight or branched C1-C 25 Saturated hydrocarbon group, or One or more carbon atoms in the straight or branched saturated hydrocarbon group are replaced by one or more connected combinations selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidyl, C2-C5 alkenyl, C2-C5 alkynyl, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 Heteroarylene; and / or If there are methine groups in the branched saturated hydrocarbon group, one or more methine groups are optionally replaced by one or more selected from the group consisting of the following groups: nitrogen atoms, trivalent C3-C 10 Cycloalkyl, trivalent C3-C 10 Heterocyclic group, trivalent C6-C 10 Aryl and trivalent C5-C 10 heteroaryl, and Wherein, the saturated hydrocarbon group optionally has any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O) (C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl); The side chain part L A is a covalent bond, or C1-C 20 Alkylene, or one or more carbon atoms in the alkylene are replaced by one or more selected from the group consisting of the following groups: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidylene, C2-C5 alkenylene, C2-C5 alkynylene, C6-C 10 Arylene, C3-C8 heterocyclylene and C5-C 10 wherein the alkylene group may have any one or more substituents selected from the group consisting of: C1-C5 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH-SC1-C5 alkyl, -SC1-C5 alkylphenyl, -C1-C5 alkyl-SH, -OH, -SH, -NH2, -C1-C5 alkyl-NH2, -N(C1-C5 alkyl)(C1-C5 alkyl), -NH(C1-C5 alkyl), -N(C1-C5 alkyl)(C1-C5 alkylphenyl), -NH(C1-C5 alkylphenyl), nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH (C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl) and -NHSO2(phenyl).

5. The conjugate according to claim 4, wherein The L B is one of the following linking groups: a phosphodiester subunit, a phosphorothioate subunit, an amide group, an ester group, an oxygen atom, or a disulfide group; Or, L B It is a phosphodiester subunit, a phosphorothioate subunit or a disulfide subunit.

6. The conjugate according to claim 4 or 5, wherein Nu represents the functional group, each PP is the same or different and independently represents a carrier peptide group, and 1) Each LA is a covalent bond, or each L A Selected from the group consisting of groups (L4) to (L23) and their linked combinations; and / or 2) Each L C -H1-H2-, wherein H1 is selected from the group consisting of groups (L4)-(L23) and their connection combinations: In the formula, each j1 is an integer from 1 to 10; Each R′ is C1-C 10 alkyl; Each Ra is a hydrogen atom, C1-C 10 Alkyl, or selected from the group consisting of radicals (L24)-(L37): H2 is selected from any one of the groups shown in formula (B1)-(B5), Wherein, the carbon atoms in formula (B1)-(B3) are directly connected to one or more L A Connection, the nitrogen atom is used to connect to H1 via an imino or amide bond; Each nitrogen atom in formula (B4) is used to bind to one L via an imino or amide bond. A Connection; one oxygen atom is used to connect to H1 via an ether bond, ester bond or phosphate bond, and the other oxygen atom forms a hydroxyl group with the hydrogen atom; In formula (B5), one of the nitrogen atoms is used to connect to H1 via an imino group or an amide bond, and each of the remaining nitrogen atoms is used to connect to one L via an imino group or an amide bond. A connect, Indicates the site to which a group is covalently attached.

7. The conjugate according to claim 6, wherein Each L C For -H1-H2-, H1 and each L A Independently selected from the group consisting of a linked combination of at least two of the groups (L4)-(L9), (L13), (L14), (L17) and (L18); Alternatively, H1 and each L A Independently selected from a combination of at least two of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L17) and (L18); Alternatively, the length of H1 is 5-20 atoms, and the length refers to the length of H1 from L B The number of atoms in the longest atomic chain from the site of attachment to the site of attachment to H2.

8. The conjugate according to claim 6, wherein Each L A It is a combination of at least two of the groups (L4)-(L9), (L13), (L14), (L17) and (L18).

9. The conjugate according to claim 8, wherein L A The length of L is 3-35 atoms, 3-20 atoms, or 4-15 atoms, wherein L A The length is L A Zhong and L C Directly connected to the atom with L A The number of atoms in the longest chain of atoms formed by the atom directly connected to PP in the alkylene group is the number of atoms in the longest chain of atoms, and any cycloalkylene, arylene, heterocyclylene or heteroarylene group is calculated as one atom.

10. The conjugate according to claim 6, wherein the conjugate has a structure as shown in formula (104A), wherein m 104 、n 104 、p 104 ,q 104 Each independently represents an integer of 1-8, or an integer of 4-6, or m 104 and n 104 6, p 104 5, q 104 is 4; R 114 H or C 1-10 Alkyl acyl, or R 114 is H or acetyl, n 101 An integer from 1 to 3:

11. The conjugate according to any one of claims 1 to 10, wherein The length of the carrier peptide group is 10 to 30 amino acid residues; the amino acid residues (X A ) and (X B ) accounts for no less than 70%, or no less than 90% of the total number of amino acid residues in the carrier peptide group.

12. The conjugate according to claim 1 or 11, wherein Each n1 and n2 is independently an integer of 1-5; or, each n1 and n2 is independently 2 or 3; or, in at least one or each amino acid sequence represented by formula (I), n1:n2=1:3 to 3:

1.

13. The conjugate according to any one of claims 1 to 12, wherein Each (X A ) independently represents an amino acid residue formed by aspartic acid, glutamic acid, argininosuccinic acid or cysteine; or, each (X A ) are the same; or, each (X A ) all represent amino acid residues formed from glutamic acid; Each (X B ) independently represent amino acid residues formed by lysine, arginine, histidine or ornithine; or, each (X B ) are the same; or, each (X B ) represent amino acid residues formed by lysine or arginine.

14. The conjugate according to claim 13, wherein At least one or each n0 is zero.

15. The conjugate according to claim 14, wherein Each sequence of formula (I) is independently one of the sequences shown in SEQ ID NOs: 8-12: -(KKEEE)-(SEQ ID NO: 8) -(RREEE)-(SEQ ID NO:9) -(KKEE)-(SEQ ID NO: 10) -(KKKEEE)-(SEQ ID NO: 11) -(KKKEE)-(SEQ ID NO: 12).

16. The conjugate according to claim 1 or 15, wherein Each carrier peptide group is an amino acid sequence segment represented by formula (II): E N -[(X B ) n2 -(X A ) n1 -Z n0 n3 -Z′ n4 -E C Formula (II)​ Among them, -[(X B ) n2 -(X A ) n1 -Z n0 ] n3 - represents n3 tandem amino acid sequence segments of formula (I), n3 is an integer of 1-10 or 2-6, Z′ represents any amino acid residue, and n4 is an integer of 1-2; E N and E C represents the amino-terminal linker and the carboxyl-terminal linker, respectively; or, E N is selected from one of H, an amino protecting group and a covalently attached site, E C is selected from one of H, a carboxyl protecting group and a site of covalent attachment, and E N and E C At least one of which is a covalent attachment site; Alternatively, each amino acid sequence segment represented by formula (II) independently has a sequence represented by one of SEQ ID NOs: 13-17: (KKEEE)3K (SEQ ID NO: 13) (RREEE)3R (SEQ ID NO: 14) (KKEE)5K (SEQ ID NO: 15) (KKKEEE)3K (SEQ ID NO: 16) (KKKEE)3K (SEQ ID NO: 17); Alternatively, each amino acid sequence segment represented by formula (II) independently has the sequence represented by SEQ ID NO: 13, and the carboxyl terminus of the sequence forms an amide group, and the amino terminus is a covalent bonding site.

17. The conjugate of any one of claims 1 to 16, wherein the target organ is the kidney; Alternatively, the target tissue is renal tubular epithelial tissue, and the target cell is a renal tubular epithelial cell; optionally, the target tissue is renal proximal tubular epithelial tissue, and the target cell is a renal proximal tubular epithelial cell.

18. The conjugate according to claim 17, wherein At least one or each of the functional groups is a functional oligonucleotide group having a therapeutic effect on kidney-related diseases or symptoms.

19. The conjugate according to claim 18, wherein The functional oligonucleotide group is a double-stranded oligonucleotide group, which is formed by removing one or more atoms from siRNA or saRNA, wherein the siRNA or saRNA contains a sense strand and an antisense strand, and the sense strand and the antisense strand each contain 15-25 nucleotides; each of the nucleotides is a modified or unmodified nucleotide.

20. The conjugate of claim 19, wherein The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. The nucleotide sequence II is at least partially reverse complementary to the target mRNA. The target mRNA is a target mRNA in a target cell in a target tissue or target organ. The target cell is a cell having a receptor on its surface that can bind to the carrier peptide.

21. The conjugate according to claim 19 or 20, wherein At least one or all of the carrier peptide groups are linked to the sense strand; Alternatively, at least one of the carrier peptide groups is linked to the first nucleotide at the 3' end or the first nucleotide at the 5' end of the sense strand.

22. The conjugate according to claim 21, wherein the conjugate has a structure shown in formula (104A), wherein m 104 and n 104 6, p 104 5, q 104 is 4; R 114 For acetyl, n 101 is 1; Nu represents a siRNA group, the siRNA comprises a sense strand and an antisense strand, the phosphorus atom shown in formula (104A) forms a phosphate bond with the 5′-hydroxyl group of the ribose of the first nucleotide at the 5′-end of the sense strand; the 5′-end or 3′-end of the antisense strand is optionally connected to a fluorescent group; PP represents a carrier peptide group shown in (SEQ ID NO: 13), and the carbonyl group shown in formula (104A) is connected to the amino group at the amino terminal of the carrier peptide group by forming an amide bond.

23. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 22, or one or more of its pharmaceutically acceptable salts, metabolites or prodrugs, and pharmaceutically acceptable excipients.

24. Use of one or more of the conjugate according to any one of claims 1 to 22, its pharmaceutically acceptable salt, metabolite or prodrug and the pharmaceutical composition according to claim 23 in the preparation of a medicament for treating and / or preventing kidney-related diseases.

25. A kit comprising one or more of the conjugate according to any one of claims 1 to 22, a pharmaceutically acceptable salt, metabolite or prodrug thereof and the pharmaceutical composition according to claim 23.

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