Disclosure of Invention
It is an object of the present invention to provide novel compounds for use in anti-hepatitis b virus/hepatitis delta virus therapy.
In a first aspect of the invention there is provided a compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound is a bis-Gap modified oligonucleotide;
Wherein,
(A) The oligonucleotide has a sequence shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5;
| SEQ ID NO.2 | GCAGAGGTGAAGCGAAGTGC |
| SEQ ID NO.3 | GTGCAGAGGTGAAGCGAAGT |
| SEQ ID NO.4 | AGAGGTGAAGCGAAGTGCAC |
| SEQ ID NO.5 | GTGAAGCGAAGTGCACACGG |
(b) The double-Gap modification refers to that each nucleoside part in the oligonucleotide is modified in a mode shown in a formula I;
P1-S1-P2-S2-P3 (I)
In the formula I, the compound (I),
P1 represents a modification region consisting of at least 1 contiguous nucleotide at the 5' end;
P2 represents a modified region consisting of at least 1 contiguous nucleotide from the 3' -end of the S1 region;
p3 represents a modification region consisting of at least 1 contiguous nucleotide at the 3' end;
S1 represents a spacer region consisting of at least 1 contiguous nucleotide, and
S2 represents a spacer region consisting of at least 1 contiguous nucleotide from the 3' -end of the P2 region;
And
In the modification region, all nucleoside portions have glycosyl modification, all nucleoside portions optionally have base modification, and
In the spacer region, all nucleoside moieties are devoid of glycosyl modification, and all nucleoside moieties are optionally base modified;
And
(C) The internucleoside linkages of the oligonucleotide are unmodified or modified internucleoside linkages, and the modified internucleoside linkages refer to that part or all of the phosphodiester linkage internucleoside linkages in the oligonucleotide are each independently replaced by an internucleoside linkage selected from the group consisting of phosphorothioate linkage internucleoside linkages, phosphorodithioate linkage internucleoside linkages or a combination thereof.
In another preferred embodiment, each of P1, P2 and P3 is a modified nucleoside (preferably, the modified nucleoside comprises a modified sugar and a modified or unmodified base), and each of S1 and S2 is an unmodified nucleoside or a sugar in an unmodified nucleoside.
In another preferred embodiment, the glycosyl modification is selected from the group consisting of a 2' -O-methylated glycosyl modification, a 2' -O-methoxyethylated glycosyl modification, or a combination thereof, preferably a 2' -O-methoxyethylated glycosyl modification (moe).
In another preferred embodiment, the base modification is a 5-methyl modification (5-methyl, 5 Me) of the cytosine (C) base.
In another preferred embodiment, the internucleoside linkages of the oligonucleotide are phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, or a combination thereof.
In another preferred embodiment, the internucleoside linkage of the oligonucleotide is a phosphorothioate linkage.
In another preferred embodiment, P1 represents a modification region consisting of 2 to 6 (preferably 3 to 4) consecutive nucleotides at the 5' end.
In another preferred embodiment, P2 represents a modification region consisting of 1 to 5 (preferably 1,2, 3 or 4) consecutive nucleotides.
In another preferred embodiment, P3 represents a modification region consisting of 2 to 6 (preferably 3 or 4) consecutive nucleotides.
In another preferred embodiment, S1 represents a spacer region consisting of 3 to 7 (preferably 4, 5, 6 or 7, more preferably 4, 5 or 6, most preferably 5 or 6) consecutive nucleotides.
In another preferred embodiment, S2 represents a spacer region consisting of 3 to 7 (preferably 4, 5, 6 or 7, more preferably 4, 5 or 6, most preferably 5 or 6) consecutive nucleotides.
In another preferred embodiment, the oligonucleotide of (a) has a sequence as set forth in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5; the double-Gap modification of (b) means that each nucleoside moiety in the oligonucleotide is modified in a manner as shown in formula I; P1-S1-P2-S2-P3 (I), wherein P1 represents a modification region consisting of 2 to 6 consecutive nucleotides at the 5' end, P2 represents a modification region consisting of 1 to 5 consecutive nucleotides, P3 represents a modification region consisting of 2 to 6 consecutive nucleotides, S1 represents a spacer region consisting of 4, 5 or 6 consecutive nucleotides, S2 represents a spacer region consisting of 4, 5 or 6 consecutive nucleotides, and in said modification region, all nucleoside moieties have a glycosyl modification and all nucleoside moieties optionally have a base modification, and in said spacer region, all nucleoside moieties do not have a glycosyl modification and all nucleoside moieties optionally have a base modification, wherein said glycosyl modification is selected from the group consisting of a 2' -O-methylated glycosyl modification, a 2' -O-methoxyethylated glycosyl modification, or a combination thereof, said base modification is a cytosine (C) methyl at position 5, and (C) said oligonucleotide internucleoside linkage is an internucleoside linkage, and said internucleoside linkage is a phosphoester linkage, independently selected from the group of two-or three-part of-nucleoside linkages, phosphorodithioate internucleoside linkages, or combinations thereof.
In another preferred embodiment, (a) the oligonucleotide has a sequence as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5, (b) the double-Gap modification means that each nucleoside moiety in the oligonucleotide is modified in the manner shown in formula I, P1-S1-P2-S2-P3 (I) wherein P1 represents a modification region consisting of 3 to 4 consecutive nucleotides at the 5' end, P2 represents a modification region consisting of 1,2, 3 or 4 consecutive nucleotides, P3 represents a modification region consisting of 3 or 4 consecutive nucleotides, S1 represents a spacer region consisting of 5 or 6 consecutive nucleotides, S2 represents a spacer region consisting of 5 or 6 consecutive nucleotides, and in the modification region, all nucleoside moieties are sugar-based modified and all nucleoside moieties are optionally present, and in the spacer region, all nucleoside moieties are not sugar-based modified and all nucleoside moieties are present, P2 represents a modification region consisting of 3 to 4 consecutive nucleotides, P2 represents a modification region consisting of 3 or 4 consecutive nucleotides, S1 represents a spacer region consisting of 5 or 6 consecutive nucleotides, S2 represents a spacer region consisting of 5 or 6 consecutive nucleotides, and in the modification region, all nucleoside moieties are sugar-based modifications, O-methyl-modified, O-substituted-amino acid modified, O-methyl modified, and the combination thereof.
In another preferred embodiment, the oligonucleotide has a sequence as shown in SEQ ID NO.2 or SEQ ID NO. 5.
In another preferred embodiment, the oligonucleotide has a sequence as shown in SEQ ID NO. 2.
In another preferred embodiment, the glycosyl modification is a2 '-O-methoxyethylated glycosyl modification when the oligonucleotide has the sequence shown in SEQ ID NO.2, and/or the glycosyl modification is a 2' -O-methylated glycosyl modification when the oligonucleotide has the sequence shown in SEQ ID NO. 5.
In another preferred embodiment, the oligonucleotide has a sequence as set forth in SEQ ID NO.2 and the glycosyl modification is a 2' -O-methoxyethylated glycosyl modification.
In another preferred embodiment, (a) the oligonucleotide has a sequence as shown in SEQ ID NO.2, (b) the double-Gap modification means that each nucleoside moiety in the oligonucleotide is modified in the manner shown in formula I, P1-S1-P2-S2-P3 (I), wherein P1 represents a modification region consisting of 3 to 4 consecutive nucleotides at the 5 'end, P2 represents a modification region consisting of 1, 2,3 or 4 consecutive nucleotides, P3 represents a modification region consisting of 3 or 4 consecutive nucleotides, S1 represents a spacer consisting of 5 or 6 consecutive nucleotides, S2 represents a spacer consisting of 5 or 6 consecutive nucleotides, and in the modification region, all nucleoside moieties are sugar-based modified and all nucleoside moieties are optionally base-modified, and in the spacer, all nucleoside moieties are not sugar-based modified and all nucleoside moieties are optionally base-modified, wherein the sugar-base is a 2' -O-methoxy-base-based nucleoside or a combination of the modifications, and (C) the phosphorothioate bond is a methyl bond between the nucleotides.
In another preferred embodiment, the compounds are selected from tables A and B
Table A
In Table A, A/T/G/C represents a conventional unmodified deoxyribonucleotide residue, A/T/G/C represents a 2' -O-methyl modified nucleobase, and the internucleoside linkages of each oligonucleotide are phosphorothioate internucleoside linkages;
Table B
In Table B, A/T/G represents a conventional unmodified deoxyribonucleotide residue and C represents a 5-methyl-modified deoxyribonucleotide residue, A/T/G/C represents a 2' -O-methoxyethylated modified nucleotide base, and the internucleoside linkages of each oligonucleotide are phosphorothioate internucleoside linkages.
In another preferred embodiment, the compound is selected from the group consisting of PA0088, PA0089, PA0114, PA0090, PA0115, PA0091, PA0116, PA0092, PA0093, PA0094 and PA0095.
In another preferred embodiment, the compound is selected from the group consisting of:
5'-Gmoe*5MeCmoe*Amoe*Gmoe*dA*dG*dG*dT*dG*Amoe*dA*dG*5MedC*dG*dA*dA*Gmoe*Tmoe*Gmoe*5MeCmoe-3'(PA0088); Or 5'-Gmoe*5MeCmoe*Amoe*dG*dA*dG*dG*dT*dG*Amoe*dA*dG*5MedC*dG*dA*dA*dG*Tmoe*Gmoe*5MeCmoe-3'(PA0089).
Wherein, represents phosphorothioate, meo in Amoe, gmoe,5Me Cmoe and Tmoe represents the presence of a 2 '-O-methoxyethylated glycosyl modification of the glycosyl group of the corresponding nucleoside (i.e. Amoe, gmoe, cmoe and Tmoe represent 2' -O-methoxyethyl modified nucleosides), dA, dG,5Me dC, dT represent unmodified nucleosides, and5Me in5Me dC represents the presence of a methyl modification at the 5-position of the base.
In a second aspect of the present invention there is provided a pharmaceutical composition comprising a compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable carrier.
In a third aspect of the present invention there is provided the use of a compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as described in the third aspect, in the manufacture of a medicament for the treatment and/or prophylaxis of a disease associated with hepatitis b virus or hepatitis delta virus.
In another preferred embodiment, the disease comprises one or more of a disease associated with hepatitis B virus infection, a disease associated with co-infection of hepatitis B virus and hepatitis D virus.
In another preferred example, the disease may be an acute disease or a chronic disease.
In another preferred embodiment, the disease comprises viral hepatitis b, viral hepatitis d, liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC), or a combination thereof.
In another preferred embodiment, the disease comprises acute or chronic liver disease.
In a fourth aspect of the present invention there is provided a method of treating and/or preventing a hepatitis b virus or hepatitis d virus associated disease, the method comprising the step of administering to a subject in need thereof a safe and effective amount of a compound according to the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the second aspect.
In another preferred embodiment, the disease is as defined in the third aspect.
In another preferred embodiment, the method is for administering a safe and effective amount of a compound according to the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the second aspect, to a subject in need thereof by intravenous injection and/or subcutaneous injection.
In another preferred embodiment, the subject is a mammal, preferably the subject is selected from the group consisting of a human, a rat, a mouse, or a combination thereof.
In a fifth aspect of the present invention there is provided a method of modulating HBV DNA/RNA, HBsAg and/or HBeAg expression comprising the step of contacting a subject with a compound of claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, thereby modulating HBV DNA/RNA, HBsAg and/or HBeAg expression.
In another preferred embodiment, the method is non-therapeutic in vitro.
In another preferred embodiment, the subject is a cell.
In another preferred embodiment, the modulation is inhibition of HBV DNA/RNA, HBsAg and/or HBeAg expression, or reduction of HBV RNA and/or HBeAg levels in the extracellular space (e.g., in the cell culture medium).
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Detailed Description
The inventors have conducted long and intensive studies and have unexpectedly found that modification of an oligonucleotide having a specific sequence (particularly having a sequence as shown in SEQ ID NO. 2) in a double-Gap modification manner can promote, even significantly promote, the antiviral activity of the antisense oligonucleotide, as well as the concentration of HBsAg at the cellular level. Based on the above findings, the inventors have completed the present invention.
Terminology
The term "oligonucleotide" refers to an oligomer of nucleotides in ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA). The term includes oligonucleotides consisting of modified or unmodified nucleobases, modified or unmodified sugars (ribose or deoxyribose) and modified or unmodified internucleoside linkages (phosphodiester linkages), as well as functionally similar oligonucleotides having non-naturally occurring portions in which one or more bases of the oligonucleotide may optionally be replaced (e.g., T replaced with a modified or unmodified U). Particularly modified or substituted oligonucleotides may be preferred over natural forms due to desirable properties such as, for example, reduced immunoreactivity, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and/or improved stability to nuclease-mediated degradation. In the present invention, oligonucleotides may be single-stranded or double-stranded, including single-stranded molecules such as antisense oligonucleotides (ASOs), and aptamers and mirnas, etc., as well as double-stranded molecules such as small interfering RNAs (sirnas) or small hairpin RNAs (shrnas). The oligonucleotides may include various modifications, such as stable modifications, and thus may be subjected to at least one modification on the phosphodiester bond (partially or wholly) and/or on the sugar and/or base or include at least one modification group. For example, an oligonucleotide may be subjected to one or more modifications including, but not limited to, or may be fully modified so as to contain all of the bonds or sugars or bases having the modifications described (that is, each phosphodiester bond, sugar, and base that make up the oligonucleotide is unmodified, or partially modified or fully modified). In the present invention, the modified internucleoside linkage may be a phosphorothioate linkage and/or a phosphorodithioate linkage. Other modifications useful for the present invention include, but are not limited to, modifications at the 2 'position of the sugar, including 2' -O-alkyl modifications (such as 2 'O-methyl modifications, 2' -O-methoxyethyl modifications (2 'MOE)), 2' -amino modifications, 2 '-halogen modifications (such as 2' -fluoro substitutions), acyclic nucleotide analogs. Other 2' position modifications are also well known in the art and may be used, such as locked nucleic acids. In particular, the oligonucleotides have modified bonds throughout or have each bond modified, e.g., phosphorothioate, have 3 '-caps and/or 5' -caps, including terminal 3'-5' bonds. The base modification may include 5 'methylation of cytosine bases (5' methylcytosine) and/or 4 'sulfation of uracil bases (4' thiouracil). When the synthesis conditions are chemically compatible, then different chemically compatible modified linkages can be combined, for example, oligonucleotides having a phosphorothioate linkage, a 2' ribose modification (e.g., 2' O-methylation), and a modified base (e.g., 5' methylcytosine). All of these various modifications (e.g., each phosphorothioate linkage, each 2' modified ribose, and each modified base) can be used to further fully modify the oligonucleotide.
For brevity, unless specifically stated otherwise, an oligonucleotide as expressed herein in the form of a DNA/RNA sequence or as defined in the manner shown, for example, in SEQ ID NO.2 includes the modified or unmodified case of an oligonucleotide having that sequence.
The term "antisense oligonucleotide" refers to a single stranded oligonucleotide having a nucleobase sequence that allows hybridization to a corresponding segment of a target nucleic acid.
As used herein, the term "nt" refers to a nucleotide.
The term "complementary" refers to the ability of the nucleobase sequence of an antisense oligonucleotide to base pair exactly with the corresponding nucleobase sequence in the target nucleic acid and is mediated by hydrogen bonding between the corresponding nucleobases, e.g., adenine base pairs with thymine (or uracil) and guanine pairs with cytosine.
In the present invention, the terms "comprising," "including," or "containing" mean that the various ingredients may be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.
In the present invention, the term "pharmaceutically acceptable" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Thus, it is not useful to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.
As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention with bases that are suitable for use as medicaments.
Unless otherwise indicated, all compounds present in the present invention are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all the compounds of the invention, each chiral carbon atom may optionally be in the R configuration or in the S configuration, or in a mixture of R and S configurations.
Some of the compounds of the present invention may be crystallized or recrystallized from water or various organic solvents, in which case various solvates may be formed. Solvates of the present invention include stoichiometric solvates such as hydrates and the like, as well as compounds containing variable amounts of water formed when prepared by the low pressure sublimation drying process.
The present invention provides methods, compounds and compositions for modulating HBV DNA/RNA and expression of HBsAg and HBeAg. In embodiments, compounds suitable for modulating HBV DNA/RNA and HBsAg, HBeAg expression are antisense oligonucleotides.
In certain embodiments, modulation may be performed in a cell. In certain embodiments, the modulation is in an animal. In certain embodiments, the animal is a human. In certain embodiments HBV RNA levels are reduced. In certain embodiments HBV-DNA levels are reduced. In certain embodiments HBsAg levels are reduced. In certain embodiments HBeAg levels are reduced. The decrease occurs in a dose and time dependent manner.
Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, the HBV-related disease, disorder, or condition is an acute or chronic liver disease. In certain embodiments, the liver diseases, disorders, and conditions include viral hepatitis b, viral hepatitis d, liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and the like.
In certain embodiments, the method of treatment comprises administering to an individual in need thereof an HBV antisense oligonucleotide by intravenous or subcutaneous injection.
Oligonucleotide (antisense oligonucleotide)
The present invention unexpectedly found oligonucleotides (or referred to as antisense oligonucleotides) capable of inhibiting the expression of viral proteins such as Hepatitis B Virus (HBV) surface antigen (HBsAg) and e antigen (HBeAg). Typically, the antisense oligonucleotide provided by the invention is capable of complementing a segment of a genotype-wide conserved sequence of the hepatitis B virus genome. For example, the antisense oligonucleotide provided by the invention can be complementary to, for example, a segment of a conserved genotype sequence shown as SEQ ID NO.6 in a hepatitis B virus genome (genotype D) shown as SEQ ID NO.1 or a sequence having homology of 96% or more and 98% or more with the sequence.
Preferably, the sequences of the oligonucleotides of the invention are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, more preferably as shown in SEQ ID NO. 2.
In another preferred embodiment, the oligonucleotide is complementary to at least a portion of the fragment of SEQ ID NO. 6.
In another preferred embodiment, the oligonucleotide is complementary to at least a portion of SEQ ID NO.6 and at the same time complementary to at least a portion of SEQ ID NO. 1.
In another preferred embodiment, the oligonucleotide is at least 96% complementary to SEQ ID NO. 6.
Double-Gap modified oligonucleotides
The invention also provides modified antisense oligonucleotides. The preferred antisense oligonucleotide provided by the invention modifies ribose of 5 'and 3' terminal nucleotide of the oligonucleotide through double-Gap design, improves affinity with target RNA, enhances drug effect, can reduce onset dose, increases safety window, and simultaneously, ribose modification at two ends further improves stability of the oligonucleotide. The more preferred modified antisense oligonucleotides provided by the invention are phosphorothioate modified oligonucleotides, wherein the phosphorothioate modification improves the stability of the oligonucleotide in vivo, enhances plasma protein binding and facilitates the distribution of the oligonucleotide to target tissues such as the liver. The most preferred antisense oligonucleotide provided by the invention is a double Gap antisense oligonucleotide, which is modified by a nucleotide ribose part at the 5 'end and the 3' end of the oligonucleotide and between the sequences, and is modified by the base of a cytosine nucleotide in a CpG sequence (namely cytosine (C) -phosphate (p) -guanine (G)), so that the affinity of the oligonucleotide is further improved, and the off-target toxicity caused by the activation of an innate immunity Toll-like receptor is reduced.
In one embodiment, a compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof, is provided, wherein the compound is a modified or unmodified oligonucleotide having a length of 14 to 26nt (e.g., 16 to 24nt, preferably 18 to 22nt, most preferably 20 nt), and the oligonucleotide has the structural features of formula I
P1-S1-P2-S2-P3 formula I
Wherein P1, P2 and P3 are modified 1 or more nucleotides, S1 and S2 are deoxyribonucleosides, the spacer region shown by S1 is Gap1, and the spacer region shown by S2 is Gap2.
Wherein the modification is one or more modifications selected from the group consisting of:
(i) Modification of the nucleoside including 2 '-O-methylated glycosyl modification, 2' -O-methoxyethylated glycosyl modification, and/or methylation modification at position 5 of cytosine;
(ii) Modification of internucleoside linkages, wherein part or all of internucleoside linkages in the oligonucleotide are replaced by phosphorothioate internucleoside linkages and/or phosphorodithioate ester internucleoside linkages.
In another preferred embodiment, each nucleoside of P1, P2 and P3 is a modified nucleoside (preferably, the modified nucleoside comprises a modified sugar and a modified or unmodified base);
Each nucleoside of S1 and S2 is an unmodified nucleoside or a sugar in an unmodified nucleoside;
the individual nucleoside portions of the oligonucleotide are each independently linked via a phosphodiester linkage or a phosphorothioate linkage (preferably, each linked via a phosphorothioate linkage).
In another preferred embodiment, the internucleoside linkages in the modified oligonucleotide are at least partially modified internucleoside linkages (i.e., phosphorothioate internucleoside linkages).
In another preferred embodiment, all internucleoside linkages in the modified oligonucleotide are modified internucleoside linkages (i.e., phosphorothioate internucleoside linkages).
In another preferred embodiment, the P1 corresponds to a region of 2-6 nt where the modified 5' end is present.
In another preferred embodiment, the S1 corresponds to the inclusion of L1 consecutive nucleotides without a nucleotide modification in the spacer region Gap1, wherein L1 is a positive integer from 3 to 7, preferably 4, 5 or 6, more preferably 5 or 6.
In another preferred embodiment, the P2 corresponds to a region of 2-5 nt where modifications are present.
In another preferred embodiment, the S2 corresponds to the inclusion of L2 consecutive nucleotides without a nucleotide modification in the spacer region Gap2, wherein L1 is a positive integer from 3 to 7, preferably 4, 5 or 6, more preferably 5 or 6.
In another preferred embodiment, the P3 corresponds to a region of 2-6 nt from the 3' end.
In another preferred embodiment, P1 is 3, 4 or 5nt in length, S1 is 4, 5 or 6nt in length, P2 is 2,3 or 4nt in length, S2 is 4, 5 or 6nt in length, and/or P3 is 3, 4 or 5nt in length.
In another preferred embodiment, in P1, P2 and P3, the modified nucleosides are each independently glycosyl modified nucleosides, or glycosyl and base modified nucleosides.
In another preferred embodiment, the nucleoside modified with a glycosyl group is a nucleoside having a glycosyl group containing a2 '-O-methyl group or a 2' -O-methoxyethyl group.
In another preferred embodiment, the nucleoside with both the sugar group and the base modified is a nucleoside with the sugar group containing a2 '-O-methyl group or a 2' -O-methoxyethyl group and the base being 5-methylcytosine.
In another preferred embodiment, in P1, P2 and P3, the modified nucleosides are each independently glycosyl modified nucleosides, or modified nucleoside moieties each of which is glycosyl and base when the base is cytosine;
Wherein the nucleoside modified with a sugar group is a nucleoside having a sugar group containing a2 '-O-methyl group or a 2' -O-methoxyethyl group, and the nucleoside modified with both a sugar group and a base is a nucleoside having a sugar group containing a2 '-O-methyl group or a 2' -O-methoxyethyl group and a base of 5-methylcytosine. .
In another embodiment, the invention provides an oligonucleotide, or an optical isomer, pharmaceutically acceptable salt, hydrate, or solvate thereof, the antisense oligonucleotide sequence being selected from SEQ ID NO.2-5, and wherein one or more T's may be optionally substituted with U.
| SEQ ID NO.2 | GCAGAGGTGAAGCGAAGTGC |
| SEQ ID NO.3 | GTGCAGAGGTGAAGCGAAGT |
| SEQ ID NO.4 | AGAGGTGAAGCGAAGTGCAC |
| SEQ ID NO.5 | GTGAAGCGAAGTGCACACGG |
In another preferred example, the modified oligonucleotide is PA0088, the sequence of which is SEQ ID No.2, sugar modifications and base modifications ：5'-Gmoe*5MeCmoe*Amoe*Gmoe*dA*dG*dG*dT*dG*Amoe*dA*dG*5MedC*dG*dA*dA*Gmoe*Tmoe*Gmoe*5MeCmoe-3',, wherein phosphorothioates, amoe, gmoe,5Me Cmoe, tmoe:2' -O-methoxyethyl modified monomers, dA, dG,5Me dC, dT: natural DNA monomers,5Me: base 5-methyl modification.
In another preferred example, the modified oligonucleotide is PA0089, the sequence of which is SEQ ID No.2, sugar modifications and base modifications ：5'-Gmoe*5MeCmoe*Amoe*dG*dA*dG*dG*dT*dG*Amoe*dA*dG*5MedC*dG*dA*dA*dG*Tmoe*Gmoe*5MeCmoe-3', wherein phosphorothioates, amoe, gmoe,5Me Cmoe, tmoe:2' -O-methoxyethyl modified monomers, dA, dG,5Me dC, dT: natural DNA monomers,5Me: base 5-methyl modification.
Preparation of antisense oligonucleotides
The antisense oligonucleotides of the invention can be prepared and synthesized by conventional synthetic methods in the oligonucleotide industry. For example, phosphorothioate linkages can be synthesized on a GE OP 100-like device using standard phosphoramidite chemical synthesis and using 1, 2-benzodithiol-3-keto-1, 1-dioxide as the oxidizing agent instead of iodine.
Pharmaceutical compositions and methods of administration
Since the compound (or the modified or unmodified oligonucleotide) of the present invention has excellent ability to inhibit replication of hepatitis B virus DNA, the compound and isomers (e.g., optical isomers), crystal forms, solvates, pharmaceutically acceptable inorganic substances, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient are useful for treating, preventing and alleviating diseases associated with or caused by infection with hepatitis B virus (i.e., hepatitis B virus) or co-infection with hepatitis B virus and hepatitis D virus. These diseases may be acute or chronic. According to the prior art, the compounds of the present invention are useful for the treatment of hepatitis B, pancreatic cancer, cirrhosis, hepatocellular carcinoma, etc. (such as pancreatic cancer, cirrhosis, and hepatocytes caused by chronic hepatitis B).
The pharmaceutical compositions provided herein comprise a safe and effective amount of a compound of the present invention or other pharmaceutically acceptable forms thereof (e.g., optical isomers, pharmaceutically acceptable salts, hydrates, or solvates thereof), and a pharmaceutically acceptable adjuvant, diluent, or carrier. By "safe and effective amount" is meant an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 10-500mg of the compound of the invention per dose. Preferably, the "one dose" is an ampoules or penicillin bottles.
By "pharmaceutically acceptable carrier" is meant one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.
Application method
The compounds of the present invention and compositions containing them may be administered by any suitable means, for example, oral ingestion, oral inhalation, by subcutaneous, intravenous injection or infusion. The compounds of the present invention or compositions containing them may be administered in the form of a dosage unit formulation containing a non-toxic pharmaceutically acceptable carrier or diluent, or may be in the form of an immediate release or sustained release formulation.
The topical effective dosing regimen for administration of agents directed to antisense oligonucleotides of the invention to humans, following dosing regimens commonly used for other antisense oligonucleotides, the routine use of 100-500mg of compound per week for parenteral administration is well established in the art.
In accordance with the disclosure presented herein, it is useful to treat subjects with HBV infection or HBV/HDV co-infection with a pharmaceutically acceptable antisense oligonucleotide formulation.
The main advantages of the invention include:
(a) The bis-Gap modified compounds or oligonucleotides of the invention have significantly improved antiviral ability compared to unmodified or non-bis-Gap modified oligonucleotides
(B) The compound or the oligonucleotide provided by the invention can effectively inhibit viral gene products at the transcriptional level in vitro, thereby obviously inhibiting hepatitis B virus antigens (such as HBsAg).
(C) The modified oligonucleotide provided by the invention has the capability of inhibiting hepatitis B virus antigen (such as HBsAg) which is equivalent to or even remarkably excellent as that of an unmodified oligonucleotide while improving in vivo stability through modification.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.
It will be appreciated that the oligonucleotides used in the examples can be obtained by the person skilled in the art from the sequences described in the examples and the corresponding modifications, according to techniques conventional in the art (e.g.standard solid phase synthesis methods) and using starting materials (e.g.modified or unmodified nucleosides) which are commercially available or synthesized according to methods of the prior art.
Example 1 antiviral Effect of antisense oligonucleotides of different Gap modification modes in HepG2.2.15 cell lines
The hepg2.2.1.5 cell line stably expresses replication HBV virus and secretes HBV viral particles, HBsAg and HBeAg into the cell supernatant. HepG2.2.1.5 cells were cultured using DMEM @ F12| medium (Hyclone) containing 10% FBS (ExCell Bio) and 400. Mu.g/ml G418 and were used after three generations.
On day 0, the siRNA was diluted in PBS at 2 concentrations (final concentrations of 1nM and 10 nM) in a gradient. HepG2.2.15 cells were washed with DPBS, digested with trypsin, 10% FBSDMEM F12 medium (without G418) was adjusted to the appropriate density, seed cells were simultaneously transfected with siRNA into HepG2.2.15 cells using Lipofectamine RNAiMax, and inoculated into 96-well plates at a density of 225,00 cells per well, with 135. Mu.l per well of culture medium. Cells were incubated in a 5% CO2, 37℃incubator for 3 days. The collected cell supernatants were assayed for HBsAg by ELISA. Meanwhile, cellTiter-Glo detects cell viability.
ELISA was briefly described by adding 50. Mu.l of the sample and the standard to the reaction plate, adding 50. Mu.l of the enzyme conjugate to each well, shaking and mixing, incubating at 37℃for 60 minutes, washing the plate 5 times with a wash solution, adding 50. Mu.l of the luminescent substrate to each well, mixing, reacting at room temperature in the absence of light for 10 minutes, and finally detecting the chemiluminescent intensity with an ELISA.
CellTiter-Glo is briefly described as 100. Mu.l CTG per well, reacted in the dark for 10min. The enzyme-labeled instrument detects the chemiluminescent intensity.
The antisense oligonucleotides used are shown in Table 1.
TABLE 1 description of oligonucleotides used in example 1
A/T/G/C represents a conventional unmodified deoxyribonucleotide residue, A/T/G/C represents a 2' -O-methyl modified nucleobase, and the internucleoside linkages of each oligonucleotide are phosphorothioate internucleoside linkages.
The relative inhibition rate of transfected antisense oligonucleotide to HepG2.2.1.5 expression secretion HBsAg was calculated as:
relative inhibition = 100% [1- (treatment/blank HBsAg concentration) ]
The relative inhibition rates of the antisense oligonucleotide treatment groups are shown in Table 2
Table 2 relative inhibition of HepG2.2.1.5 expression of secreted HBsAg by the oligonucleotides of example 1
As can be seen from Table 2 and FIG. 1, the addition of the double-Gap modification contributes to an increase in the relative inhibition of HBsAg compared to the antisense oligonucleotide PA0087 in the single-Gap modification mode. Combining the results of the two experiments showed that the weighted averages of PA0075, PA0077, PA0079 and PA0081 were significantly better than the antisense oligonucleotides of the single Gap modification mode.
Example 2 antiviral Effect of antisense oligonucleotides of different Gap modification modes in HepG2.2.15 cell lines
The hepg2.2.1.5 cell line stably expresses replication HBV virus and secretes HBV viral particles, HBsAg and HBeAg into the cell supernatant. HepG2.2.1.5 cells were cultured using DMEM @ F12| medium (Hyclone) containing 10% FBS (ExCell Bio) and 400. Mu.g/ml G418 and were used after three generations.
On day 0, the siRNA was diluted in PBS at 2 concentrations (final concentrations of 10nM and 30 nM) in a gradient. HepG2.2.15 cells were washed with DPBS, digested with trypsin, 10% FBSDMEM F12 medium (without G418) was adjusted to the appropriate density, seed cells were simultaneously transfected with siRNA into HepG2.2.15 cells using Lipofectamine RNAiMax, and inoculated into 96-well plates at a density of 225,00 cells per well, with 135. Mu.l per well of culture medium. Cells were incubated in a 5% CO2, 37℃incubator for 3 days. The collected cell supernatants were assayed for HBsAg by ELISA. Meanwhile, cellTiter-Glo detects cell viability.
ELISA was briefly described by adding 50. Mu.l of the sample and the standard to the reaction plate, adding 50. Mu.l of the enzyme conjugate to each well, shaking and mixing, incubating at 37℃for 60 minutes, washing the plate 5 times with a wash solution, adding 50. Mu.l of the luminescent substrate to each well, mixing, reacting at room temperature in the absence of light for 10 minutes, and finally detecting the chemiluminescent intensity with an ELISA.
CellTiter-Glo is briefly described as 100. Mu.l CTG per well, reacted in the dark for 10min. The enzyme-labeled instrument detects the chemiluminescent intensity.
The antisense oligonucleotides used are shown in Table 3.
TABLE 3 description of oligonucleotides used in example 2
A/T/G represents a conventional unmodified deoxyribonucleotide residue and C represents a 5-methyl modified deoxyribonucleotide residue, A/T/G/C represents a 2' -O-methoxyethyl modified nucleotide base, and the internucleoside linkages of each oligonucleotide are phosphorothioate internucleoside linkages.
The relative inhibition rate of transfected antisense oligonucleotide to HepG2.2.1.5 expression secretion HBsAg was calculated as:
relative inhibition = 100% [1- (treatment/blank HBsAg concentration) ]
The relative inhibition rates of the antisense oligonucleotide treatment groups are shown in Table 4
Table 4 relative inhibition of HepG2.2.1.5 expression of secreted HBsAg by the oligonucleotides of example 2
As can be seen from Table 4 and FIG. 2, the addition of the double-Gap modified antisense oligonucleotide helps to increase the relative inhibition of HBsAg compared to the single-Gap modified antisense oligonucleotide Bepirovirsen in clinical phase III. The results of the two experiments were combined to show that the weighted averages of PA0088 and PA0089 were significantly better than the antisense oligonucleotides of the single Gap modification mode.
Example 3 antiviral EC50 values of antisense oligonucleotides of different Gap modification modes in HepG2.2.15 cell lines
The hepg2.2.1.5 cell line stably expresses replication HBV virus and secretes HBV viral particles, HBsAg and HBeAg into the cell supernatant. HepG2.2.1.5 cells were cultured using DMEM @ F12| medium (Hyclone) containing 10% FBS (ExCell Bio) and 400. Mu.g/ml G418 and were used after three generations.
On day 0, the siRNA was gradient diluted with PBS to concentrations of 100nM, 33.33nM, 11.11nM, 3.70nM, 1.23nM, 0.41nM, 0.14nM and 0.05nM (note: GSK-3228836 compound concentrations: 40nM, 13.33nM, 4.44nM, 1.48nM, 0.49nM, 0.16nM, 0.05nM and 0.02 nM). HepG2.2.15 cells were washed with DPBS, digested with trypsin, conditioned with 10% FBSDMEM F12| medium (without G418) to the appropriate density, and siRNA transfected into HepG2.2.15 cells using Lipofectamine RNAiMax simultaneously, seeded into 96-well plates at 225,00 cells per well at 135. Mu.l per well of culture medium. Cells were incubated in a 5% CO2, 37℃incubator for 3 days. The collected cell supernatants were assayed for HBsAg by ELISA. Meanwhile, cellTiter-Glo detects cell viability.
ELISA was briefly described by adding 50. Mu.l of the sample and the standard to the reaction plate, adding 50. Mu.l of the enzyme conjugate to each well, shaking and mixing, incubating at 37℃for 60 minutes, washing the plate 5 times with a wash solution, adding 50. Mu.l of the luminescent substrate to each well, mixing, reacting at room temperature in the absence of light for 10 minutes, and finally detecting the chemiluminescent intensity with an ELISA.
CellTiter-Glo is briefly described as 100. Mu.l CTG per well, reacted in the dark for 10min. The enzyme-labeled instrument detects the chemiluminescent intensity.
The antisense oligonucleotides used are shown in Table 5.
TABLE 5 description of oligonucleotides used in example 3
A/T/G represents a conventional unmodified deoxyribonucleotide residue and C represents a 5-methyl modified deoxyribonucleotide residue, A/T/G/C represents a 2' -O-methoxyethyl modified nucleotide base, and the internucleoside linkages of each oligonucleotide are phosphorothioate internucleoside linkages.
The relative inhibition rate of transfected antisense oligonucleotide to HepG2.2.1.5 expression secretion HBsAg was calculated as:
relative inhibition = 100% [1- (treatment/blank HBsAg concentration) ]
The relative inhibition rates of the antisense oligonucleotide treatment groups are shown in Table 6
TABLE 6 relative inhibition and cell viability of the oligonucleotides of example 3 on HepG2.2.1.5 expression of secreted HBsAg
As can be seen from Table 6 and FIG. 3, the bis-Gap modified compound helps to increase the relative inhibition of HBsAg. Combining the results of the two experiments showed that the weighted averages of PA0088 and PA0089 were significantly better than the antisense oligonucleotides of the single Gap modification pattern, EC50 was 10.5 and 9.6 times that of control Bepirovirsen (i.e., GSK-3228836), respectively.
Example 4 antiviral Effect of double Gap antisense oligonucleotides PA0088, PA0089 and Bepirovirsen in AAV-HBV mouse model
Dose-escalating antisense oligonucleotide PA0020 was evaluated for antiviral activity in c57 mice infected with adeno-associated virus carrying HBV1.3 ploidy (AAV-HBV, acanthopanax) and continuously replicating HBV-DNA and expressing HBV antigen. The oligonucleotide PA0020 used is shown in table 7.
| Oligonucleotides | SEQ ID | Oligonucleotide modification |
| PA0088 | SEQ ID NO.2 | GCAGAGGTGAAGCGAAGTGC |
| PA0089 | SEQ ID NO.2 | GCAGAGGTGAAGCGAAGTGC |
| Bepirovirsen | SEQ ID NO.2 | GCAGAGGTGAAGCGAAGTGC |
A model of persistent hepatitis B infection mice was prepared by tail vein injection of male C57BL/6 mice with 5X1010 rAAV8-1.3HBV (Acanthopanax senticosus). After stable replication of HBV virus was confirmed, the HBV virus was randomly divided into 4 groups (5 groups each) according to body weight, the first group was a control group (Vehicle) which was perfused with physiological saline every day, the second group to the fourth group were intraperitoneally injected Bepirovirsen, PA0088 and PA0089, respectively, at a frequency of 1 time per week, and the doses were 30mg/kg,60mg/kg,60mg/kg,90mg/kg 5 in this order. Blood was taken 1-2 times per week, the qPCR method analyzed the HBV nucleic acid (HBV-DNA) load in serum, the ELISA method analyzed the HBsAg and HBeAg concentrations in serum, and the graph was drawn 4.
As shown in FIG. 4, the serum of animals in the control group showed smooth fluctuation of HBsAg and HBV-DNA, and the serum of animals in the Bepirovirsen, PA0088 and PA0089 groups showed continuous decrease of HBV-DNA, and the end of administration was reduced by >3log10, i.e., by >99.9% from day 0. HBsAg and HBeAg also decreased continuously in serum from animals in Bepirovirsen, PA0088 and PA0089 groups, with the end of dosing decreasing by >2log10 on day 0, and PA0088 decreasing slightly more than control drug Bepirovirsen. Therefore, PA0088 and PA0089 can reduce HBV-DNA and surface antigen, and may be used in clinical treatment of hepatitis B. In addition, as shown in fig. 5, all groups had a constant increase in body weight, and control group Bepirovirsen had no further increase in body weight after 42 days of dosing, while PA0088 and PA0089 had a greater increase in body weight, indicating that PA0088 and PA0089 were safer than Bepirovirsen.
In particular, further methylation of cytosine bases of CpG sequences, i.e., substitution of cytosine with 2' -O-methoxyethylated-5-methylcytosine, reduces the likelihood of activation of Toll-like receptors (TLRs), particularly TLR9, thereby reducing the risk of immune off-target toxicity.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.