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Definitions

• This application relates to STOPSTM antiviral compounds that are S-antigen transport inhibiting oligonucleotide polymers, processes for making them and methods of using them to treat diseases and conditions.
• the STOPSTM compounds described herein are antiviral oligonucleotides that can be at least partially phosphorothioated and exert their antiviral activity by a non sequence dependent mode of action.
• the term“Nucleic Acid Polymer” (NAP) has been used in the literature to refer to such oligonucleotides, although that term does not necessarily connotate antiviral activity.
• NAP Nucleic Acid Polymer
• a number of patent applications filed in the early 2000s disclosed the structures of certain specific compounds and identified various structural options as potential areas for future experimentation.
• Some embodiments described herein relate to a modified oligonucleotide or complex thereof having sequence independent antiviral activity against hepatitis B, that can include an at least partially phosphorothioated sequence of alternating A and C units, wherein:
• the A units comprise one or more selected from:
• the C units comprise one or more selected from
• each terminal is independently hydroxyl, an 0,0-dihydrogen phosphorothioate, a dihydrogen phosphate, an endcap or a linking group;
• each internal is a phosphorus-containing linkage to a neighboring A or C unit, the phosphorus-containing linkage being a phosphorothioate linkage or a modified linkage selected from phosphodiester, phosphorodithioate, methylphosphonate, diphosphorothioate, 5’-phosphoramidate, 3’,5’ -phosphor diamidate, 5’-thiophosphoramidate, 3’,5’-thiophosphordiamidate or diphosphodiester; and
• sequence independent antiviral activity against hepatitis B is greater than that of a reference compound
• sequence of alternating A and C units comprises a Ribo-A unit
• sequence further comprises at least one A unit that is not a Ribo- A unit
• sequence of alternating A and C units comprises a Ribo-C unit
• sequence further comprises at least one C unit that is not a Ribo- C unit.
• Some embodiments described herein relate to a method of treating a HBV and/or HDV infection that can include administering to a subject identified as suffering from the HBV and/or HDV infection an effective amount of a modified oligonucleotide modified oligonucleotide as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide as described herein.
• Some embodiments disclosed herein relate to a method of inhibiting replication of HBV and/or HDV that can include contacting a cell infected with the HBV and/or HDV with an effective amount of a modified oligonucleotide modified oligonucleotide as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide as described herein.
• FIG. 1 illustrates an embodiment of a modified oligonucleotide that comprises a CT-ealkylene linkage.
• FIG. 2 illustrates an embodiment of a modified oligonucleotide that comprises a propylene oxide linkage.
• FIG. 3 A illustrates an embodiment of a modified oligonucleotide having cholesterol attached via a 5’ tetraethylene glycol (TEG) linkage.
• TEG tetraethylene glycol
• FIG. 3B illustrates an embodiment of a modified oligonucleotide having cholesterol attached via a 3’ TEG linkage.
• FIG. 3C illustrates an embodiment of a modified oligonucleotide having a tocopherol (Vitamin E) attached via a 5’ TEG linkage.
• FIG. 3D illustrates an embodiment of a modified oligonucleotide having a tocopherol (Vitamin E) attached via a 3’ TEG linkage.
• FIGS. 4 A and 4B illustrate embodiments of modified oligonucleotides having GalNac attached via a linking group.
• FIG. 5 illustrates an embodiment of a reaction scheme for preparing a 5’- EP building block.
• FIG. 6A illustrates embodiments of modified oligonucleotides and corresponding values of sequence independent antiviral activity against hepatitis B (as determined by HBsAg Secretion Assay) and cytotoxicity.
• FIG. 6B illustrates embodiments of modified oligonucleotides and corresponding values of sequence independent antiviral activity against hepatitis B (as determined by HBsAg Secretion Assay) and cytotoxicity.
• FIG. 7 illustrates an embodiment of a reaction scheme for preparing compound 5’-VP.
• FIG. 8 illustrates an embodiment of a reaction scheme for preparing compounds 8-5 and 8-6.
• FIG. 9A illustrates an embodiment of a reaction scheme for preparing compound 9R.
• FIG. 9B illustrates an embodiment of a reaction scheme for preparing compound 9S.
• FIG. 10 illustrates an embodiment of a reaction scheme for preparing compounds 10-5 and 10-6.
• FIG. 11A illustrates an embodiment of a reaction scheme for preparing compound HR.
• FIG. 11B illustrates an embodiment of a reaction scheme for preparing compound 11S.
• FIG. 12 illustrates liver exposure results following subcutaneous administration to non-human primates of embodiments of modified oligonucleotide compounds.
• FIG. 13 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 14 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 15 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 16 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 17 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 18 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 19 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 20 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 21 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 22 illustrates PBMC assay results illustrating the immune reaction of embodiments of modified oligonucleotide compounds.
• FIG. 23 illustrates a graph that is utilized in connection with the HBsAg Secretion Assays described in Examples B3 and B4. DETAILED DESCRIPTION
• the hepatitis B virus is a DNA virus and a member of the Hepadnaviridae family. HBV infects more than 300 million worldwide and is a causative agent of liver cancer and liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV can be acute and/or chronic. Acute HBV infection can be either asymptomatic or present with symptomatic acute hepatitis. HBV is classified into eight genotypes, A to H.
• HBV is a partially double-stranded circular DNA of about 3.2 kilobase (kb) pairs.
• the HBV replication pathway has been studied in great detail. T.J. Liang, Heptaology (2009) 49(5 Suppl):S13-S21.
• One part of replication includes the formation of the covalently closed circular (cccDNA) form.
• cccDNA covalently closed circular
• HBV carriers can transmit the disease for many years. An estimated 257 million people are living with hepatitis B virus infection, and it is estimated that over 750,000 people worldwide die of hepatitis B each year.
• immunosuppressed individuals or individuals undergoing chemotherapy are especially at risk for reactivation of an HBV infection.
• HBV can be transmitted by blood, semen, and/or another body fluid. This can occur through direct blood-to-blood contact, unprotected sex, sharing of needles, and from an infected mother to her baby during the delivery process.
• the HBV surface antigen (HBsAg) is most frequently used to screen for the presence of this infection.
• Currently available medications do not cure an HBV and/or HDV infection. Rather, the medications suppress replication of the virus.
• the hepatitis D virus is a DNA virus, also in the Hepadnaviridae family of viruses. HDV can propagate only in the presence of HBV. The routes of transmission of HDV are similar to those for HBV. Transmission of HDV can occur either via simultaneous infection with HBV (coinfection) or in addition to chronic hepatitis B or hepatitis B carrier state (superinfection). Both superinfection and coinfection with HDV results in more severe complications compared to infection with HBV alone. These complications include a greater likelihood of experiencing liver failure in acute infections and a rapid progression to liver cirrhosis, with an increased risk of developing liver cancer in chronic infections. In combination with hepatitis B, hepatitis D has the highest fatality rate of all the hepatitis infections, at 20%. There is currently no cure or vaccine for hepatitis D.
• the term “antiviral” has its usual meaning as understood by those skilled in the art and thus includes an effect of the presence of the oligonucleotides or other material that inhibits production of viral particles, typically by reducing the number of infectious viral particles formed in a system otherwise suitable for formation of infectious viral particles for at least one virus.
• the antiviral oligonucleotide has antiviral activity against multiple different virus, e.g., both HBV and HDV.
• oligonucleotide (or“oligo”) has its usual meaning as understood by those skilled in the art and thus refers to a class of compounds that includes oligodeoxynucleotides, oligodeoxyribonucleotides and oligoribonucleotides.
• oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, including reference to oligonucleotides composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) internucleoside (backbone) linkages as well as “modified” or substituted oligonucleotides having non- naturally-occurring portions which function similarly.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• mimetics mimetics thereof, including reference to oligonucleotides composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) internucleoside (backbone) linkages as well as “modified” or substituted oligonucleotides having non- naturally-occurring portions which function similarly.
• modified oligonucleotide has its usual meaning as understood by those skilled in the art and includes oligonucleotides having one or more of various modifications, e.g., stabilizing modifications, and thus can include at least one modification in the internucleoside linkage and/or on the ribose, and/or on the base.
• a modified oligonucleotide can include modifications at the 2'-position of the ribose, acyclic nucleotide analogs, methylation of the base, phosphorothioated (PS) linkages, phosphorodithioate linkages, methylphosphonate linkages, linkages that connect to the sugar ring via sulfur or nitrogen, and/or other modifications as described elsewhere herein.
• a modified oligonucleotide can include one or more phosphorothioated (PS) linkages, instead of or in addition to PO linkages.
• modified oligonucleotides that include such PS linkages are considered to be in the same class of compounds because even though the PS linkage contains a phosphorous-sulfur double bond instead of the phosphorous-oxygen double bond of a PO linkage, both PS and PO linkages connect to the sugar rings through oxygen atoms.
• phosphorothioated oligonucleotide has its usual meaning as understood by those skilled in the art and thus refers to a modified oligonucleotide in which all of the phosphodiester internucleoside linkages have been replaced by phosphorothioate linkages.
• phosphorothioated oligonucleotide is synonymous with “fully phosphorothioated” oligonucleotide.
• a phosphorothioated oligonucleotide (or a sequence of phosphorothioated oligonucleotides within a partially phosphorothioated oligonucleotide) can be modified analogously, including (for example) by replacing one or more phosphorothioated internucleoside linkages by phosphodiester linkages.
• modified phosphorothioated oligonucleotide refers to a phosphorothioated oligonucleotide that has been modified in the manner analogous to that described herein with respect to oligonucleotides, e.g., by replacing a phosphorothioated linkage with a modified linkage such as phosphodiester, phosphorodithioate, methylphosphonate, diphosphorothioate, 5’- phosphoramidate, 3’,5’-phosphordiamidate, 5’-thiophosphoramidate, 3’,5’- thiophosphordiamidate or diphosphodiester.
• a modified linkage such as phosphodiester, phosphorodithioate, methylphosphonate, diphosphorothioate, 5’- phosphoramidate, 3’,5’-phosphordiamidate, 5’-thiophosphoramidate, 3’,5’- thiophosphordiamidate or di
• An at least partially phosphorothioated sequence of a modified oligonucleotide can be modified similarly, and thus, for example, can be modified to contain a non-phosphorothioated linkage such as phosphodiester, phosphorodithioate, methylphosphonate, diphosphorothioate 5’-phosphoramidate, 3’,5’- phosphordiamidate, 5’-thiophosphoramidate, 3’,5’-thiophosphordiamidate or diphosphodiester.
• a non-phosphorothioated linkage such as phosphodiester, phosphorodithioate, methylphosphonate, diphosphorothioate 5’-phosphoramidate, 3’,5’- phosphordiamidate, 5’-thiophosphoramidate, 3’,5’-thiophosphordiamidate or diphosphodiester.
• modification by inclusion of a phosphodiester linkage may be considered to result in a modified phosphorothioated oligonucleotide, or to a modified phosphorothioated sequence, respectively.
• a phosphorothioated linkage may be considered to result in a modified oligonucleotide or a modified phosphodiesterified sequence, respectively.
• the term “stereochemically defined phosphorothioate linkage” has its usual meaning as understood by those skilled in the art and thus refers to a phosphorothioate linkage having a phosphorus stereocenter with a selected chirality (R or S configuration).
• a composition containing such a dinucleotide or oligonucleotide can be enriched in molecules having the selected chirality.
• the stereopurity of such a composition can vary over a broad range, e.g. from about 51% to about 100% stereopure. In various embodiments, the stereopurity is greater than 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or in a range defined as having any two of the foregoing stereopurity values as endpoints.
• sequence independent antiviral activity has its usual meaning as understood by those skilled in the art and thus refers to an antiviral activity of an oligonucleotide (e.g., a modified oligonucleotide) that is independent of the sequence of the oligonucleotide.
• Methods for determining whether the antiviral activity of an oligonucleotide is sequence independent are known to those skilled in the art and include the tests for determining if an oligonucleotide acts predominantly by a non-sequence complementary mode of action as disclosed in Example 10 of U.S. Patent Nos. 7,358,068; 8,008,269; 8,008,270 and 8,067,385, which is hereby incorporated herein by reference and particularly for the purpose of describing such tests.
• a and C units e.g., alternating A and C units, or AC units
• the terms“A” and“C” refer to the modified adenosine-containing (A) units and modified cystosine-containing (C) units set forth in Tables 1 and 2 below, respectively.
• An embodiment provides a STOPSTM modified oligonucleotide compound having sequence independent antiviral activity against hepatitis B, comprising an at least partially phosphorothioated sequence of alternating A and C units, wherein the A units are any one or more selected from those set forth in Table 1 and the C units are any one or more selected from those set forth in Table 2.
• Various combinations of A and C units can be included in the at least partially phosphorothioated AC sequence, including the combinations described in Table 3 below.
• a modified oligonucleotide as described herein comprises an at least partially phosphorothioated sequence of alternating A and C units that has a sequence length of about 8 units, about 10 units, about 12 units, about 14 units, about 16 units, about 18 units, about 20 units, about 24 units, about 30 units, about 34 units, about 36 units, about 38 units, about 40 units, about 44 units, about 50 units, about 60 units, about 76 units, about 100 units, about 122 units, about 124 units, about 150 units, about 172 units, about 200 units, or a sequence length in a range between any two of the aforementioned values.
• the at least partially phosphorothioated sequence of alternating A and C units has a sequence length in the range of 8 units to 200 units.
• the at least partially phosphorothioated sequence of alternating A and C units has a sequence length that is in any one or more (as applicable) of the following ranges: about 8 units to about 36 units; about 16 units to about 36 units; 20 units to 36 units; 16 units to 30 units; 18 units to 60 units; 20 units to 30 units; 30 units to 50 units; 34 units to 46 units, 36 units to 44 units; 44 units to 200 units; 44 units to 150 units; 44 units to 120 units; 50 units to 200 units; 50 units to 150 units; 50 units to 120 units; 60 units to 200 units; 60 units to 150 units; and/or 60 units to 120 units.
• a modified oligonucleotide can comprise a single at least partially phosphorothioated sequence of alternating A and C units in some embodiments, or in other embodiments the modified oligonucleotide can comprise a plurality of at least partially phosphorothioated sequences of alternating A and C units that are linked together.
• a modified oligonucleotide that contains a single at least partially phosphorothioated sequence of alternating A and C units can have the same sequence length as that sequence. Examples of such sequence lengths are described elsewhere herein.
• a modified oligonucleotide that contains a plurality of at least partially phosphorothioated sequences of alternating A and C units can have sequence length that is the result of linking those sequences as described elsewhere herein.
• sequence lengths for a modified oligonucleotide that contains a plurality of at least partially phosphorothioated sequences of alternating A and C units are expressed elsewhere herein in terms of the lengths of the individual sequences, and also taking into account the length of the linking group.
• a modified oligonucleotide as described herein can comprises a plurality of at least partially phosphorothioated sequences of alternating A and C units.
• the sequence of alternating A and C units comprises a Ribo-A unit
• the sequence further comprises at least one A unit that is not a Ribo-A unit.
• the sequence of alternating A and C units comprises a Ribo-C unit
• the sequence further comprises at least one C unit that is not a Ribo-C unit.
• the modified oligonucleotide can contain one or more of various nucleotide units (known to those skilled in the art, e.g., thymine, cytosine, adenine, guanine and modified versions thereof) that are not A or C units, e.g., as an end group(s) and/or as a linking group(s) between two or more at least partially phosphorothioated sequences of alternating A and C units.
• the modified oligonucleotide comprises one or more cytosine units that link together at least two or more of the at least partially phosphorothioated sequences of alternating A and C units.
• the two or more at least partially phosphorothioated sequences of alternating A and C units, which are linked together by a non-A/non-C linking group are identical to one another.
• An example of such a modified oligonucleotide is (AQs-cytosine- (AC)s.
• AC AQs-cytosine-
• Such a modified oligonucleotide that comprises a plurality of identical sequences that are joined together may be referred to herein as a concatemer.
• the two or more at least partially phosphorothioated sequences of alternating A and C units that are linked together can also be different from one another.
• An example of such a modified oligonucleotide is (AC)x-cytosine- (AC)l6.
• the modified oligonucleotide can contain two or more different A groups and/or two or more different C groups.
• an A or C group is replaced by a different A or C group, such a replacement is not ordinarily considered to interrupt the alternating sequence of A and C units.
• at least some of the A units are not 2 ⁇ - methylated on the ribose ring and/or at least some of the C units are not 2’O-methylated on the ribose ring.
• the group linking the two at least partially phosphorothioated sequences of alternating A and C units is itself an A or C unit that interrupts the alternating sequence of A and C units.
• an at least partially phosphorothioated 16-mer of alternating A and C units may be linked by an A unit to another such 16-mer to form (AC)8-A-(AC)8.
• such a 16-mer may be linked by a C unit to another such 16-mer to form (AC)8-C-(AC)8.
• the modified oligonucleotide may be referred to herein as a concatemer.
• the two or more at least partially phosphorothioated sequences of alternating A and C units that are linked together can also be different from one another.
• modified oligonucleotides include (AC)s-A-(AC)ie and (AC)8-C-(AC)i6.
• the modified oligonucleotide comprises a 5’ endcap.
• the 5’ endcap is selected from OH Hd .
• R 1 and R 2 are each individually selected from hydrogen, deuterium, phosphate, thioCi-6alkyl, and cyano.
• R 1 and R 2 are both hydrogen and the modified oligonucleotide comprises a vinyl phosphonate endcap. In other embodiments, R 1 and R 2 are not both hydrogen.
• the 5’ endcap is selected from H0' . " ⁇ HO D ⁇ i
• the modified oligonucleotide comprises a 3’ and/or 5’ linking group.
• modified oligonucleotide compounds comprising A and C units as described herein, such as the A and C units of Tables 1 and 2, wQ
• At least one terminal can be a linking group.
• Various linking groups known to those skilled in the art can be used to link the modified oligonucleotide to another moiety (such as one or more second oligonucleotides and/or targeting ligands).
• the linking group comprises a non-A/non-C linking group or an A or C unit that interrupts the alternating sequence of A and C units as discussed above, or the linking group comprises a C2- 6alkylene linkage (FIG. 1), a Cv-ealkylene oxide linkage, such as a propylene oxide linkage (FIG. 2), or a tetraethylene glycol (TEG) linkage (FIGS. 3A-D).
• two, three, four or more of the modified oligonucleotides can be connected to each other in various ways.
• the modified oligonucleotides can be connected end-to-end via 3’ and/or 5’ linking groups, and/or a linking group can be connected to a one 3’ or 5’ end of multiple modified oligonucleotides, e.g., as illustrated in FIGS. 1 and 2.
• the modified oligonucleotide further comprises a targeting ligand that is attached to the modified oligonucleotide via the linking group.
• the targeting ligand is, or comprises, a N- acetylgalactosamine (GalNac) (e.g., triantennary-GalNAc), a tocopherol or cholesterol.
• FIGS. 3A and 3B illustrate embodiments of modified oligonucleotides having cholesterol attached via a 5’ TEG linking group and a 3’TEG linking group, respectively.
• 3C and 3D illustrate embodiments of modified oligonucleotides having a tocopherol (Vitamin E) attached via a 5’ TEG linking group and a 3’TEG linking group, respectively.
• FIGS. 4A and 4B illustrate embodiments of modified oligonucleotides having GalNac attached via a linking group.
• the GalNac is a triantennary GalNac.
• the at least partially phosphorothioated sequence of alternating A and C units can include modification(s) to one or more phosphorothioated linkages.
• the inclusion of such a modified linkage is not ordinarily considered to interrupt the alternating sequence of A and C units because those skilled in the art understand that such a sequence may be only partially phosphorothioated and thus may comprise one or more modifications to a phosphorothioate linkage.
• the modification to the phosphorothioate linkage is a modified linkage selected from phosphodiester, phosphorodithioate, methylphosphonate, diphosphorothioate and diphosphodi ester.
• the modified linkage is a phosphodiester linkage.
• the at least partially phosphorothioated sequence of alternating A and C units can have various degrees of phosphorothioation.
• the at least partially phosphorothioated sequence of alternating A and C units is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% phosphorothioated.
• the at least partially phosphorothioated sequence of alternating A and C units is at least 85% phosphorothioated.
• the at least partially phosphorothioated sequence of alternating A and C units is fully phosphorothioated.
• the at least partially phosphorothioated sequence of alternating A and C units can include stereochemical modification(s) to one or more phosphorothioated linkages.
• the modified oligonucleotides described herein can comprise at least one stereochemically defined phosphorothioate linkage.
• the stereochemically defined phosphorothioate linkage has an R configuration.
• the stereochemically defined phosphorothioate linkage has an S configuration.
• modified oligonucleotide compounds comprising A and C units as described herein, such as the A and C units of Tables 1 and 2, respectively, contain internal linkages between the A and C units as well as terminal groups at the 3’ and 5’ ends.
• a and C units described herein such as w Q wQ the A and C units of Tables 1 and 2, respectively, each represents an internal or a wQ wQ
• each terminal is independently hydroxyl, an O,O-dihydrogen phosphorothioate, a dihydrogen phosphate, an endcap or a linking group.
• each internal is a phosphorus-containing linkage to a neighboring A or C unit, the phosphorus-containing linkage being a phosphorothioate linkage or a modified linkage selected from phosphodiester, phosphorodithioate, methylphosphonate, diphosphorothioate 5’-phosphoramidate, 3’,5’-phosphordiamidate, 5’-thiophosphoramidate, 3’,5’-thiophosphordiamidate or diphosphodiester.
• the STOPSTM compounds described herein are antiviral oligonucleotides.
• a modified oligonucleotide as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units has sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is greater than that of a reference compound.
• the sequence independent antiviral activity against hepatitis B is at least 2-fold greater than a reference compound.
• the sequence independent antiviral activity against hepatitis B is in the range of from 2-fold greater than a reference compound to 5-fold greater than a reference compound.
• sequence independent antiviral activity against hepatitis B is at least 5 -fold greater than a reference compound. In another embodiment, the sequence independent antiviral activity against hepatitis B is in the range of from 5-fold greater than a reference compound to 10-fold greater than a reference compound. In another embodiment, the sequence independent antiviral activity against hepatitis B is at least 10-fold greater than a reference compound. In another embodiment, the sequence independent antiviral activity against hepatitis B is in the range of from 10-fold greater than a reference compound to 25 -fold greater than a reference compound. In another embodiment, the sequence independent antiviral activity against hepatitis B is at least 25 -fold greater than a reference compound.
• 2-fold, 5-fold, 10-fold and 25- fold refer to the increased potency of a modified oligonucleotide as described herein as compared to another compound in HBsAg Secretion Assay, as indicated by an ECso value that is one-half, one-fifth, one-tenth or one-twenty-fifth that of a reference compound, respectively.
• a modified oligonucleotide having a potency that is two-fold greater than a reference compound has an ECso value in HBsAg Secretion Assay that is one-half that of the ECso value of a reference compound.
• a modified oligonucleotide having a potency that is five-fold greater than a reference compound has an ECso value in HBsAg Secretion Assay that is one-fifth that of a reference compound.
• a modified oligonucleotide having a potency that is ten-fold greater than a reference compound has an ECso value in HBsAg Secretion Assay that is one-tenth that of a reference compound.
• a modified oligonucleotide having a potency that is twentyfive-fold greater than a reference compound has an ECso value in HBsAg Secretion Assay that is one-twenty-fifth that of a reference compound.
• the reference compound can be the phosphorothioated AC 40-mer oligonucleotide known as REP 2139 discussed above.
• the reference compound can be the AC 40-mer oligonucleotide having the structure 5’mApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmCpsmApsmC 3’ (2’-OMe-A, 2’-OMe-C).
• a modified oligonucleotide as described herein comprising an at least partially phosphorothioated sequence of alternating A and C units, has sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nanomolar (nM); in a“B” activity range of 30 nM to less than 100 nM; in a“C” activity range of 100 nM to less than 300 nM; or in a “D” activity range of greater than 300 nM.
• a modified oligonucleotide as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units has sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is less than 50 nM.
• the modified oligonucleotides described herein can be prepared in the form of various complexes.
• an embodiment provides a chelate complex of a modified oligonucleotide as described herein.
• a chelate complex comprises a calcium, magnesium or zinc chelate complex of the modified oligonucleotide.
• the modified oligonucleotides described herein can also be prepared in the form of various monovalent counterion complexes.
• a counterion complex comprises a lithium, sodium or potassium complex of the modified oligonucleotide.
• An embodiment provides a modified oligonucleotide or complex thereof having sequence independent antiviral activity against hepatitis B, comprising an at least partially phosphorothioated sequence of alternating A and C units as described herein, wherein;
• the at least partially phosphorothioated sequence of alternating A and C units is at least 85% phosphorothioated
• the at least partially phosphorothioated sequence of alternating A and C units has a sequence length in the range of 36 units to 44 units;
• the A units comprise at least 12 2’-OMe-A units (e.g., at least 15 2’-OMe- A units) and at least 1 Ribo-A unit (e.g., at least 2 Ribo-A units);
• the C units comprise at least 15 LNA-5mC units.
• the modified oligonucleotide has an ECso value, as determined by HBsAg Secretion Assay, that is less than 100 nM (e.g., less than 50 nM or less than 30 nM).
• An embodiment provides a modified oligonucleotide or complex thereof having sequence independent antiviral activity against hepatitis B, comprising an at least partially phosphorothioated sequence of alternating A and C units as described herein, wherein;
• the at least partially phosphorothioated sequence of alternating A and C units is at least 85% phosphorothioated
• the at least partially phosphorothioated sequence of alternating A and C units has a sequence length in the range of 36 units to 44 units;
• the A units comprise at least 15 2’-OMe-A units
• the C units comprise at least 7 LNA-5mC units.
• the modified oligonucleotide has an ECso value, as determined by HBsAg Secretion Assay, that is less than 100 nM (e.g., less than 50 nM or less than 30 nM).
• An embodiment provides a modified oligonucleotide or complex thereof having sequence independent antiviral activity against hepatitis B, comprising an at least partially phosphorothioated sequence of alternating A and C units as described herein, wherein;
• the at least partially phosphorothioated sequence of alternating A and C units is at least 85% phosphorothioated
• the at least partially phosphorothioated sequence of alternating A and C units has a sequence length in the range of 36 units to 44 units;
• the A units comprise at least 15 2’-OMe-A units
• the C units comprise at least 3 LNA-5mC units.
• the modified oligonucleotide has an ECso value, as determined by HBsAg Secretion Assay, that is less than 100 nM (e.g., less than 50 nM or less than 30 nM).
• An embodiment provides a modified oligonucleotide or complex thereof having sequence independent antiviral activity against hepatitis B, comprising an at least partially phosphorothioated sequence of alternating A and C units as described herein, wherein;
• the at least partially phosphorothioated sequence of alternating A and C units is at least 85% phosphorothioated
• the at least partially phosphorothioated sequence of alternating A and C units has a sequence length in the range of 36 units to 44 units;
• the A units comprise at least 18 2’-OMe-A units
• the C units comprise at least 15 LNA-5mC units; and [0093] the modified oligonucleotide has an ECso value, as determined by HBsAg Secretion Assay, that is less than 100 nM (e.g., less than 50 nM or less than 30 nM).
• the modified oligonucleotides described herein can be prepared in various ways.
• the building block monomers described in Tables 4 and 5 are employed to make the modified oligonucleotides described herein by applying standard phosphoramidite chemistry.
• the building blocks described in Tables 4 and 5 and other building block phosphoramidite monomers can be prepared by known methods or obtained from commercial sources (Thermo Fischer Scientific US, Hongene Biotechnology USA Inc., Chemgenes Corporation). Exemplary procedures for making modified oligonucleotides are set forth in the Examples below.
• the STOPSTM modified oligonucleotides described herein can also be prepared using dinucleotides that comprise or consist of any two of the building block monomers described in Tables 4 and 5. Exemplary procedures for making dinucleotides and the corresponding modified oligonucleotides are set forth in the Examples below.
• An embodiment provides a dinucleotide comprising, or consisting of, an A unit and a C unit connected by a stereochemically defined phosphorothioate linkage, wherein the A unit is selected from any of the building block monomers described in Table 4 and the C unit is selected from any of the building block monomers described in Table 5, and wherein wQ
• each is independently hydroxyl, an O,O-dihydrogen phosphorothioate, an 0,0- dihydrogen phosphate, a phosphoramidite, a dimethoxytrityl ether, or the stereochemically
• the is a phosphoramidite of the following formula (A):
• R 1 and R 2 of formula (A) are each individually a Ci-6alkyl, and R 3 is a Ci-6alkyl or a cyanoCi-6alkyl.
• the phosphoramidite of the formula (A) is a phosphoramidite of the following formula (Al):
• the stereochemically defined phosphorothioate linkage that is a phosphorothioate.
• the stereochemically defined phosphorothioate linkage is a phosphorothioate of the following Formula (Bl) or (B2):
• R 4 of formulae (Bl) and (B2) is a Ci-6 alkyl or a cyanoCi-6 alkyl.
• the phosphorothioates of the formulae (Bl) and (B2) are phosphorothioates of the following Formulae (B3) or (B4), respectively:
• Various embodiments provide methods of making a modified oligonucleotide as described herein, comprising coupling one or more dinucleotides as described herein. Exemplary methods of carrying out such coupling are illustrated in the Examples below.
• Some embodiments described herein relate to a pharmaceutical composition, that can include an effective amount of a compound described herein (e.g., a STOPSTM modified oligonucleotide compound or complex thereof as described herein) and a pharmaceutically acceptable carrier, excipient or combination thereof.
• a pharmaceutical composition described herein is suitable for human and/or veterinary applications.
• a“carrier” refers to a compound that facilitates the incorporation of a compound into cells or tissues.
• DMSO dimethyl sulfoxide
• a“carrier” refers to a compound that facilitates the incorporation of a compound into cells or tissues.
• DMSO dimethyl sulfoxide
• a“diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable.
• a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation.
• a common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.
• an“excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition.
• A“diluent” is a type of excipient.
• Proper formulation is dependent upon the route of administration chosen.
• Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, rectal, topical, aerosol, injection and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections.
• Pharmaceutical compositions will generally be tailored to the specific intended route of administration.
• the liposomes may be targeted to and taken up selectively by the organ.
• compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.
• compounds used in a pharmaceutical composition may be provided as salts with pharmaceutically compatible counterions.
• Some embodiments described herein relate to a method of treating a HBV and/or HDV infection that can include administering to a subject identified as suffering from the HBV and/or HDV infection an effective amount of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein.
• Other embodiments described herein relate to using a modified oligonucleotide or complex thereof as described herein in the manufacture of a medicament for treating a HBV and/or HDV infection.
• Still other embodiments described herein relate to the use of a modified oligonucleotide or complex thereof as described herein or a pharmaceutical composition that includes a modified oligonucleotide as described herein for treating a HBV and/or HDV infection.
• a modified oligonucleotide or complex thereof may be administered to a subject in need thereof as indicated elsewhere herein.
• the modified oligonucleotide or complex thereof is administered to the subject by a parenteral route.
• the modified oligonucleotide or complex thereof is administered to the subject intravenously.
• the modified oligonucleotide or complex thereof is administered to the subject subcutaneously.
• a modified oligonucleotide or complex thereof such as REP 2139, REP 2055 or those described in U.S. Patent Nos. 7,358,068; 8,008,269; 8,008,270 and 8,067,385
• a primate was considered unlikely to be safe and effective because of the relatively high dosages believed required to achieve efficacy and the concomitant increase in the potential risk of safety concerns such as undesirable injection site reactions.
• prior clinical studies involving the administration of REP 2139 to humans are believed to have utilized only intravenous routes. At the dosage levels that were believed to be necessary for efficacy, it is believed that safety concerns such as undesirable injection site reactions would have precluded subcutaneous administration.
• liver exposure following subcutaneous administration to non-human primates is much higher than expected based on liver exposure levels resulting from otherwise comparable intravenous dosing.
• This finding means that embodiments of modified oligonucleotides or complexes thereof as described herein, and particularly embodiments of highly potent STOPSTM compounds or complexes as described herein, can be safely and effectively administered to primates via subcutaneous administration at dosages lower than previously considered likely to be effective. These lower dosages reduce the risk profile (e.g., reduce risk of injection site reactions) and thus provide a clinically acceptable safety profile for human use.
• Some embodiments disclosed herein relate to a method of treating a HBV and/or HDV infection that can include contacting a cell infected with the HBV and/or HDV with an effective amount of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein.
• a method of treating a HBV and/or HDV infection comprises safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• a modified oligonucleotide or complex thereof as described herein in the manufacture of a medicament for treating a HBV and/or HDV infection.
• Still other embodiments described herein relate to the use of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein for treating a HBV and/or HDV infection.
• such uses comprise safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• Some embodiments disclosed herein relate to a method of inhibiting replication of HBV and/or HDV that can include contacting a cell infected with the HBV and/or HDV with an effective amount of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein.
• a method of inhibiting replication of HBV and/or HDV comprises safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• FIG. 1 Another embodiments described herein relate to using a modified oligonucleotide or complex thereof as described herein in the manufacture of a medicament for inhibiting replication of HBV and/or HDV. Still other embodiments described herein relate to the use of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein, for inhibiting replication of HBV and/or HDV.
• such uses for inhibiting replication of HBV and/or HDV comprise safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• the HBV infection can be an acute HBV infection. In some embodiments, the HBV infection can be a chronic HBV infection.
• Some embodiments disclosed herein relate to a method of treating liver cirrhosis that is developed because of a HBV and/or HDV infection that can include administering to a subject suffering from liver cirrhosis and/or contacting a cell infected with the HBV and/or HDV in a subject suffering from liver cirrhosis with an effective amount of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein.
• such a method of treating liver cirrhosis that is developed because of a HBV and/or HDV infection comprises safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• modified oligonucleotide or complex thereof relate to using a modified oligonucleotide or complex thereof as described herein in the manufacture of a medicament for treating liver cirrhosis that is developed because of a HBV and/or HDV infection, with an effective amount of the modified oligonucleotide(s). Still other embodiments described herein relate to the use of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein for treating liver cirrhosis that is developed because of a HBV and/or HDV infection.
• such uses for treating liver cirrhosis comprise safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• liver cancer such as hepatocellular carcinoma
• a method of treating liver cancer that is developed because of a HBV and/or HDV infection that can include administering to a subject suffering from the liver cancer and/or contacting a cell infected with the HBV and/or HDV in a subject suffering from the liver cancer with an effective amount of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• Other embodiments described herein relate to using a modified oligonucleotide or complex thereof as described herein in the manufacture of a medicament for treating liver cancer (such as hepatocellular carcinoma) that is developed because of a HBV and/or HDV infection.
• liver cancer such as hepatocellular carcinoma
• uses for treating liver cancer comprise safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• Some embodiments disclosed herein relate to a method of treating liver failure that is developed because of a HBV and/or HDV infection that can include administering to a subject suffering from liver failure and/or contacting a cell infected with the HBV and/or HDV in a subject suffering from liver failure with an effective amount of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein.
• such a method of treating liver failure that is developed because of a HBV and/or HDV infection comprises safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an“A” activity range of less than 30 nM.
• modified oligonucleotide or complex thereof relate to using a modified oligonucleotide or complex thereof as described herein in the manufacture of a medicament for treating liver failure that is developed because of a HBV and/or HDV infection. Still other embodiments described herein relate to the use of a modified oligonucleotide or complex thereof as described herein, or a pharmaceutical composition that includes an effective amount of a modified oligonucleotide or complex thereof as described herein for treating liver failure that is developed because of a HBV and/or HDV infection.
• such uses for treating liver failure comprise safe and effective subcutaneous administration of the modified oligonucleotide or complex thereof to a human at a dosage lower than otherwise expected based on liver levels observed following otherwise comparable intravenous administration.
• the modified oligonucleotide or complex thereof is REP-2139 or a complex thereof.
• the modified oligonucleotide or complex thereof comprises a highly potent STOPSTM compound or complex thereof as described herein.
• the STOPSTM compound or complex thereof is a modified oligonucleotide or complex thereof as described herein, comprising an at least partially phosphorothioated sequence of alternating A and C units, having sequence independent antiviral activity against hepatitis B, as determined by HBsAg Secretion Assay, that is in an “A” activity range of less than 30 nM.
• Suitable indicators include, but are not limited to, a reduction in viral load indicated by reduction in HBV DNA (or load), HBV surface antigen (HBsAg) and HBV e-antigen (HBeAg), a reduction in plasma viral load, a reduction in viral replication, a reduction in time to seroconversion (virus undetectable in patient serum), an increase in the rate of sustained viral response to therapy, an improvement in hepatic function, and/or a reduction of morbidity or mortality in clinical outcomes.
• HBV DNA or load
• HBV surface antigen HBV surface antigen
• HBV eAg HBV e-antigen
• an effective amount of a modified oligonucleotide or complex thereof as described herein is an amount that is effective to achieve a sustained virologic response, for example, a sustained viral response 12 month after completion of treatment.
• Subjects who are clinically diagnosed with an HBV and/or HDV infection include“naive” subjects (e.g., subjects not previously treated for HBV and/or HDV) and subjects who have failed prior treatment for HBV and/or HDV (“treatment failure” subjects).
• Treatment failure subjects include“non-responders” (subjects who did not achieve sufficient reduction in ALT levels, for example, subject who failed to achieve more than 1 log 10 decrease from base-line within 6 months of starting an anti-HBV and/or anti-HDV therapy) and “relapsers” (subjects who were previously treated for HBV and/or HDV whose ALT levels have increased, for example, ALT > twice the upper normal limit and detectable serum HBV DNA by hybridization assays).
• Further examples of subjects include subjects with a HBV and/or HDV infection who are asymptomatic.
• a modified oligonucleotide or complex thereof as described herein can be provided to a treatment failure subject suffering from HBV and/or HDV. In some embodiments, a modified oligonucleotide or complex thereof as described herein can be provided to a non-responder subject suffering from HBV and/or HDV. In some embodiments, a modified oligonucleotide or complex thereof as described herein can be provided to a relapser subject suffering from HBV and/or HDV. In some embodiments, the subject can have HBeAg positive chronic hepatitis B. In some embodiments, the subject can have HBeAg negative chronic hepatitis B.
• the subject can have liver cirrhosis.
• the subject can be asymptomatic, for example, the subject can be infected with HBV and/or HDV but does not exhibit any symptoms of the viral infection.
• the subject can be immunocompromised.
• the subject can be undergoing chemotherapy.
• agents that have been used to treat HBV and/or HDV include interferons (such as IFN-a and pegylated interferons that include PEG-IFN-a-2a), and nucleosides/nucleotides (such as lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide and tenofovir disoproxil).
• interferons such as IFN-a and pegylated interferons that include PEG-IFN-a-2a
• nucleosides/nucleotides such as lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide and tenofovir disoproxil.
• Resistance can be a cause for treatment failure.
• the term“resistance” as used herein refers to a viral strain displaying a delayed, lessened and/or null response to an anti-viral agent.
• a modified oligonucleotide or complex thereof as described herein can be provided to a subject infected with an HBV and/or HDV strain that is resistant to one or more anti-HBV and/or anti-HDV agents.
• development of resistant HBV and/or HDV strains is delayed when a subject is treated with a modified oligonucleotide as described herein compared to the development of HBV and/or HDV strains resistant to other HBV and/or HDV anti-viral agents, such as those described.
• a modified oligonucleotide or complex thereof as described herein can be used in combination with one or more additional agent(s) for treating and/or inhibiting replication HBV and/or HDV.
• Additional agents include, but are not limited to, an interferon, nucleoside/nucleotide analogs, a capsid assembly modulator, a sequence specific oligonucleotide (such as anti-sense oligonucleotide and/or siRNA), an entry inhibitor and/or a small molecule immunomodulator.
• a modified oligonucleotide or complex thereof as described herein can be used as a first treatment in combination with one or more second treatment(s) for HBV, wherein the second treatment comprises a second oligonucleotide having sequence independent antiviral activity against hepatitis B, an siRNA oligonucleotide (or nucleotides), an anti-sense oligonucleotide, a nucleoside, an interferon, an immunomodulator, a capsid assembly modulator, or a combination thereof.
• the second treatment comprises a second oligonucleotide having sequence independent antiviral activity against hepatitis B, an siRNA oligonucleotide (or nucleotides), an anti-sense oligonucleotide, a nucleoside, an interferon, an immunomodulator, a capsid assembly modulator, or a combination thereof.
• additional agents include recombinant interferon alpha 2b, IFN-a, PEG-IFN-a-2a, lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide. tenofovir disoproxil, JNJ-3989 (ARO-HBV), RG6004, GSK3228836, AB-729, VTR-2218, DCR-HBVS, JNJ-6379, GLS4, ABI-H0731, JNJ-440, NZ-4, RG7907, AB-423, AB-506 and ABI-H2158.
• the additional agent is a capsid assembly modulator (CAM).
• the additional agent is an anti-sense oligonucleotide (ASO).
• a modified oligonucleotide or complex thereof as described herein can be administered with one or more additional agent(s) together in a single pharmaceutical composition.
• a modified oligonucleotide or complex thereof as described herein can be administered with one or more additional agent(s) as two or more separate pharmaceutical compositions. Further, the order of administration of a modified oligonucleotide or complex thereof as described herein with one or more additional agent(s) can vary.
• a series of modified oligonucleotides containing phosphorothioated sequences of alternating A and C units were synthesized on an ABI 394 synthesizer using standard phosphoramidite chemistry.
• the solid support was controlled pore glass (CPG, 1000A, Glen Research, Sterling VA) and the building block monomers are described in Tables 4 and 5.
• the reagent (dimethylamino-methylidene) amino)-3H-l,2,4-dithiazaoline-3-thione (DDTT) was used as the sulfur-transfer agent for the synthesis of oligoribonucleotide phosphorothioates (PS linkages).
• CPG controlled pore glass
• Samples were dissolved in deionized water (1.0 mL) and quantitated as follows: Blanking was first performed with water alone (1 mL). 20 ul of sample and 980 uL of water were mixed well in a microfuge tube, transferred to cuvette and absorbance reading obtained at 260 nm. The crude material is dried down and stored at -20°C.
• the crude oligomers were analyzed and purified by HPLC (Dionex PA 100).
• the purified dry oligomer was then desalted using Sephadex G-25M (Amersham Biosciences).
• the cartridge was conditioned with 10 mL of water.
• the purified oligomer dissolved thoroughly in 2.5 mL RNAse free water was applied to the cartridge with very slow dropwise elution.
• the salt free oligomer was eluted with 3.5 ml water directly into a screw cap vial.
• Table 6 summarizes the sequence length, alternating A and C units and whether the backbone is phosphorothioate (PS) or phosphodiester (PO) for the resulting exemplified modified oligonucleotides.
• the 5’-vinyl phosphonate building block (5’-VP) was prepared as follows:
• Table 7 summarizes the sequence length, alternating A and C units, and 5’ modification for the resulting exemplified modified phosphorothioated oligonucleotides.
• the dinucleotide building blocks 9R and 9S were prepared as follows:
• the modified method also used a longer coupling time (8 min) and a greater number of equivalents of amidites (8 equivalents).
• Table 9 summarizes the sequence length, alternating A and C units, the number and type (R or S) of stereochemically defined phosphorothioate (PS) linkages, and 5’-modification for the resulting exemplified modified phosphorothioated oligonucleotides.
• the dinucleotide building blocks 11R and HS were prepared as follows:
• amido-bridge nucleic acid (AmNA-(N-Me)) modification and spirocyclopropylene-bridged nucleic acid (scp-BNA) modification was evaluated by preparing a series of modified phosphorothioated oligonucleotides.
• the AmNA-N-Me 6 -N- benzoyladenosine (A Bz ), 4-A-benzoyl -5-methyl cytidine were obtained from Luxna Biotech Co, Ltd and scp-BNA phosphoramidite monomers with 6-A-benzoyladenosine (A Bz ), 4 -N- benzoyl -5-methyl cytidine were synthesized by using the procedure described in the references Takao Yamaguchi, Masahiko Horiba and Satoshi Obika; Chem. Commun. 2015, 51, 9737-9740, and Masahiko Horiba, Takao Yamaguchi, and Satoshi Obika; Journal of Organic Chemistry, 2016, 81, 11000-11008.
• the monomers were dried in a vacuum desiccator with desiccant (P2O5, at room temperature for 24 hours).
• the synthesis was carried out on a 1 mM scale in a 3’ to 5’ direction with the phosphoramidite monomers diluted to a concentration of 0.12 M in anhydrous CH3CN in the presence of 0.3 M 5-(benzylthio)-lH-tetrazole activator (coupling time 16-20 min) to a solid bound oligonucleotide followed by modified capping, oxidation and deprotection to afford the modified oligonucleotides.
• the stepwise coupling efficiency of all modified phosphoramidites was more than 97%.
• DDTT dimethylamino-methylidene amino-3H-l, 2, 4- dithiazaoline-3-thione was used as the sulfur- transfer agent for the synthesis of the oligoribonucleotide phosphorothioates.
• Oligonucleotide-bearing solid supports were washed with 20 % DEA solution in acetonitrile for 15 min then the column was washed thoroughly with AcCN. The support was heated at 65 °C with diisopropylamine:water:methanol (1 : 1 :2) for 5 h in a heat block to cleave from the support and deprotect the base labile protecting groups.
• Table 13 summarizes the sequence length, alternating A and C units, and 5’ modification for the resulting exemplified modified phosphorothioated oligonucleotides.
• FIGS. 3A-D and Table 14 illustrate the structures and summarize the sequence length, alternating A and C units, and targeting ligands for the resulting exemplified modified phosphorothioated oligonucleotides.
• GalNAc-3 and GalNAc-5 amidites were purchased from AM Chemicals LLC and Glen Research respectively.
• GalNAc-4 and GalNAc- 6 were obtained from AM Chemicals LLC.
• Table 15 illustrates the structures and summarizes the sequence length, alternating A and C units, and targeting ligands for the resulting exemplified modified phosphorothioated oligonucleotides.
• the effect of attaching a targeting ligand was evaluated by preparing a series of modified phosphorothioated oligonucleotides.
• N-acetylgalactosamine (GalNAc) was attached to phosphorothioated oligonucleotides via a linking group by preparing the starting oligonucleotides, forming a precursor by attaching a C6-NH2 linking group at the 5’-terminal, and then reacting the precursor with a GalNAc ester.
• the sequences were synthesized at 10 pmol scale using universal support (Loading 65 pmol/g).
• the C6-NH2 linker was attached to the 5’-terminal to form the precursor by reacting with 6-(4-monomethoxytritylamino)hexyl- (2-cyanoethyl)-(N, N-diisopropyl)-phosphoramidite in 0.1 M acetonitrile was a coupling time of 10 min.
• the phosphorothioated oligonucleotide-bearing solid supports were heated at room temperature with aqueous ammonia/methylamine (1 : 1) solution for 3 h in a shaker to cleave from the support and deprotect the base labile protecting groups.
• Table 16 illustrates the structures and summarizes the sequence length, alternating A and C units, and targeting ligands for the resulting exemplified modified phosphorothioated oligonucleotides.
• GalNAc- 1 and GalNAc-2 were prepared in accordance with procedures described in J. Med. Chem. 2016 59(6) 2718-2733 and WO 2017/021385A1, respectively
• Table 17 summarizes the sequence length, alternating A and C units, the number and type (R or S) of stereochemically defined phosphorothioate (PS) linkages and LNA modification for the resulting exemplified 5’-EP endcapped modified phosphorothioated oligonucleotides.
• FIG. 6A describes compound nos. 282-295, which were prepared in accordance with the methods described above.
• nmLNA N-methyl LNA
• C incorporation was evaluated by preparing a series of phosphorothioated oligonucleotides in accordance with the methods described above.
• the nmLNA monomers were obtained from commercial sources (Bio- Synthesis Inc., Lewisville, TX). The results are summarized in Table 32.
• HepG2.2. l 5 cells were maintained in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, 1% Glutamine, 1% non-essential amino acids, 1% Sodium Pyruvate and 380 ug/ml G418. Cells were maintained at 37°C in a 5% CO2 atmosphere.
• FBS fetal bovine serum
• penicillin/streptomycin 1%
• Glutamine 1% non-essential amino acids
• 1% Sodium Pyruvate 380 ug/ml G418.
• HepG2.2.15 cells were grown in DMEM medium as described above. Cells were plated at a concentration of 45,000 cells/well in collagen-I coated 96 well plates. Immediately after addition of the cells, test compounds are added.
• Selected compounds may also be tested following Lipofectamine® RNAiMAX transfection.
• Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher) is used following the manufacturer's instructions.
• the 50% inhibitory concentration (EC50) and 50% cytotoxic concentration (CC50; below) were assessed by solubilizing in 1 X PBS to 100 X the desired final testing concentration. Each compound was then serially diluted (1 :3) up to 8 distinct concentrations to 10X the desired final testing concentration in DMEM medium with 10% FBS. A 10 pL sample of the 10X compounds in cell culture media was used to treat the HepG2.2.l5 cells in a 96-well format. Cells were initially incubated with compounds for 3 days at 37°C in a 5% CO2 atmosphere.
• the EC50 the concentration of the drug required for reducing HBsAg secretion by 50% in relation to the untreated cell control value was calculated from the plot of the percentage reduction of the HBsAg level against the drug concentrations using Microsoft Excel (forecast function).
• HepG2.2. l 5 cells were cultured and treated as above. At Day 6, cellular cytotoxicity was assessed using a cell proliferation assay (CellTiter-Glo Luminescent Cell Viability Assay; Promega) according to the manufacturer’s instructions or a suitable alternative.
• a cell proliferation assay CellTiter-Glo Luminescent Cell Viability Assay; Promega
• the CC50 the concentration of the drug required for reducing cell viability by 50% in relation to the untreated cell control value was calculated from the plot of the percentage reduction of viable cells against the drug concentrations using Microsoft Excel (forecast function).
• Potency A: > 5-fold higher than (2’-OMe-A; 2’-OMe-C); B: > 2-fold higher than (2’-OMe-A; 2’-OMe-C) and ⁇ 5-fold higher than (2’-OMe-A; 2’-OMe-C); C: higher than or equal to (2’-OMe-A; 2’-OMe-C) and ⁇ 2-fold higher than (2’-OMe-A; 2’-OMe- C); D: lower than (2’-OMe-A; 2’-OMe-C).
• Potency A: ECso ⁇ 30 nM; B: ECso ⁇ 30 nM and ECso ⁇ 100 nM; C: ECso ⁇ 100 nM and ECso ⁇ 300 nM; D: ECso > 300 nM.
• Cytotoxicity A: CCso > 1000 nM; B: CCso ⁇ 1000 nM
• HepG2-NTCP cells were maintained in DMEM/F12 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, 1% Glutamine, 1% non-essential amino acids, 1% Sodium Pyruvate. Cells were maintained at 37°C in a 5% CO2 atmosphere. [0218] HepG2-NTCP cells were resuspended with above mentioned medium and plated at a concentration of 15,000 cells/well in collagen-I coated 96 well plates.
• FBS fetal bovine serum
• the cells were infected with HBV (purified HBV from Hep D38 cells) at 200 moi (ge) in the presence of 4% PEG8000 and 2% DMSO and incubated at 37°C overnight. The inoculum was vacuumed and cells were washed three times with DMEM/F12 with 2% FBS before replacing with the HepG2-NTCP culture medium.
• HBV purified HBV from Hep D38 cells
• test compounds were diluted 3 -fold with Opti-MEM I media and mixed with Lipofectamine® RNAiMAX transfection reagent following the manufacturer's instructions. After media replacement on Day 8, the test compounds were transfected as described. After incubation for an additional 3 days, the supernatant was harvested and HBsAg was measured by ELISA (Diasino). The cell viability was measured with CellTiter-Glo (Promega).
• the EC50 the concentration of the drug required for reducing HBsAg secretion by 50% in relation to the untreated cell control value, was calculated from the plot of the percent reduction of the HBsAg level against the drug concentrations using the Microsoft Excel forecast function or GraphPad Prism and summarized in Table 36.
• A ECso ⁇ 30 nM
• B ECso ⁇ 30 nM and ECso ⁇ 100 nM
• C ECso ⁇ 100 nM and ECso ⁇ 300 nM
• D ECso > 300 nM.
• Cytotoxicity A: CCso > 1000 nM; B: CCso ⁇ 1000 nM
• HepG2.2. l 5 cells were maintained in DMEM/F12 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, 1% Glutamine, 1% non-essential amino acids, 1% Sodium Pyruvate. Cells were maintained at 37°C in a 5% CO2 atmosphere.
• FBS fetal bovine serum
• HepG2.2. l 5 cells were grown in DMEM/F12 medium as described above. Cells were seeded at a concentration of 35,000 cells/well in collagen-I coated 96-well plates. Immediately after addition of the cells, add test compounds. Do double transfections on day 0 and 3.
• RNAiMAX transfection Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher, cat#: 13778-150) is used following the manufacturer's instructions. [0227] A: mix RNAiMAX (0.3ul/well for 96-well plate) with Opti-MEM I (make 20% extra), incubate for 5 min at RT.
• [0228] B dilute combinations of ASOs and modified oligonucleotides in Opti- MEM I to make 40x of final concentration (8-point, 3-fold dilution, include concentration OnM). The top concentration is about 100 - 200 folds of ECso value. Then mix equal volume dilutions from both compoundl and compound2 at opposite direction as indicated in the graph shown in FIG. 23.
• Synergy volume ⁇ 25 indicates no synergism/antagonism.
• Synergy volume 25-50 indicates minor synergism/antagonism.
• Synergy volume 50-100 indicates moderate synergism/antagonism.
• Percentage of cell viability (well/average of no drug control)* 100. Monitor cytotoxicity as previously described.
• ASO- 1 is an unconjugated HBV ASO SSO-l as disclosed in in Javanbakht, H. et al.
• ASO-2 is an ASO having a structure as described for the ASO referred to as Sequence #9 in U.S. application serial number 62/855,793, which is hereby incorporated herein by reference and particularly for the purpose of describing the structure of the Sequence #9.
• sequence independent antiviral activity against hepatitis B was determined as described below and summarized in Table 38.
• HBV antiviral assay uses HepG2.2.15 cells, which have been transfected with HBV genome, and extracellular HBV DNA quantification as endpoint. Cell viability is assessed in parallel by measuring the intracellular ATP content using the CellTiter-Glo ® reagent from Promega.
• HepG2.2. l 5 cells were grown in DMEM/F12 medium as described above. Cells were seeded at a concentration of 35,000 cells/well in collagen-I coated 96-well plates. Immediately after addition of the cells, add test compounds. Do double transfections on day 0 and 3.
• Extracellular DNA was isolated with QIAamp 96 DNA Blood Kit per the manufacturer’s manual. HBV DNA was then quantified by qPCR with HBV specific primers and probes as specified below using the FastStart Universal MasterMix from Roche on an ABI- 7900HT. The PCR cycle program consisted of 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min.
• Lipofectamine® RNAiMAX transfection Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher, cat#: 13778-150) is used following the manufacturer's instructions.
• B dilute combinations of a CAM, ASO or ETV with modified oligonucleotides in Opti-MEM I to make 40x of final concentration (8-point, 3-fold dilution, include concentration OnM). The top concentration is about 100 - 200 folds of ECso value. Then mix equal volume dilutions from both compoundl and compound2 at opposite direction as indicated in the graph shown in FIG. 23.
• HBsAg Quantification [0246] Secreted HBsAg was measured quantitatively using HBsAg ELISA kit (Autobio-CL0310). Synergy values for combinations of modified oligonucleotides with ASOs are provided in Table 38.
• X CAM compound 1 is a CAM having a structure as described for the CAM compounc referred to as compound 3 in WO2017/181141, which is hereby incorporated herein by reference and particularly for the purpose of describing the structure of the compound 3.
• CAM compound 2 is a CAM having a structure as described for the CAM compound referred to as compound 1 in U.S. serial no. 62/805,725, which is hereby incorporated herein by reference and particularly for the purpose of describing the structure of the compound 1.
• ASO-l is as described above for Table 37.
• Terminal liver exposures in non-human primates were evaluated by dosing exemplified modified oligonucleotide compounds to female cynomolgus monkeys by either the intravenous (IV) or subcutaneous (SC) route.
• IV route the compound was administered in sterile phosphate-buffered saline (PBS) vehicle and infused over a 2-hr period at 1 mL/kg.
• SC subcutaneous
• the vehicle was also sterile PBS and the compound was administered as a single bolus at 1 mL/kg.
• liver exposure following subcutaneous administration to non-human primates is much higher than expected based on liver exposure levels resulting from otherwise comparable intravenous dosing.
• PBMC peripheral blood mononuclear cells
• PBMC (1 milbon/mL) were suspended in complete culture (RPMI supplemented with 10% heat inactivated-low IgG FBS and PSG) and plated at 100 pL/well in a 96- well round bottom plate.
• PBMC were treated with test articles (list on next slide) (concentration range: 10 mM to 0 mM -3 fold dilution) and PHA and Poly IC (concentration range: 10 pg/mL to 0 pg/mL -3 fold dilution). All was set up in triplicates.
• Cytokines GM-CSF, IL-lb, IL-2, IL-6, IL-10, IL-8, IL-l2p70, IFNg, TNFa
• Cytokine IFNa was tested by standard ELISA. Results are expressed as pg/ml calculated based on the standard curve.

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