Compositions and methods for treating transthyretin (TTR)-mediated amyloidosis

Abstract

Disclosed herein are methods for treating hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) in a human patient in need thereof by administering an effective amount of a transthyretin (TTR) inhibitor composition.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 560,667, filed September 19, 2017, U.S. Provisional Patent Application No. 62 / 561,182, filed September 20, 2017, and U.S. Provisional Patent Application No. 62 / 581,005, filed November 2, 2017, which are hereby incorporated by reference for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing submitted via EFS-Web, which is hereby incorporated by reference in its entirety. Said ASCII copy, created in XX / 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0003] Transthyretin (TTR) is a tetrameric protein primarily produced in the liver. When mutations in the TTR gene destabilize this tetrameric protein, the monomers misfold and aggregate to form TTR amyloid fibrils (ATTR). Tissue deposition results in systemic ATTR amyloidosis (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). Over 100 TTR mutations have been reported, resulting in a variety of disease symptoms.

[0004] TTR amyloidosis manifests in various ways. When the peripheral nervous system is more significantly affected, the disease is called familial amyloid polyneuropathy (FAP). When the lesions are primarily in the heart and not the nervous system, the disease is called familial amyloid cardiomyopathy (FAC). A third major type of TTR amyloidosis is called leptomeningeal / CNS (central nervous system) amyloidosis.

[0005] The most common mutations associated with familial amyloidotic polyneuropathy (FAP) and ATTR-associated cardiomyopathy are Val30Met (Non-Patent Document 4) and Val122Ile (Non-Patent Document 5), respectively.

[0006] Current treatment options for FAP focus on stabilizing or reducing the amount of circulating amyloidogenic proteins. Orthotopic liver transplantation reduces mutant TTR levels (Non-Patent Document 6) and has been reported to improve survival in early-stage FAP patients, although wild-type TTR may continue to be deposited (Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12).

[0007] Tafamidis and diflunisal can stabilize circulating TTR tetramers and slow disease progression (Non-Patent Document 13; Non-Patent Document 4; Non-Patent Document 14; Non-Patent Document 15). However, symptoms continue to worsen during treatment in the majority of patients, highlighting the need for novel disease-modifying treatment options for FAP.

[0008] For a description of dsRNAs targeting TTR, see, for example, Patent Documents 1 and 2. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] PCT / US2009 / 061381 specification (International Publication No. 2010 / 048228 pamphlet) [Patent Document 2] PCT / US2010 / 055311 specification (International Publication No. 2011 / 056883 pamphlet) [Non-patent literature]

[0010] [Non-Patent Document 1] Coutinho et al., 「Forty years of experience with type I amyloid neuropathy. Review of 483 cases」. In: Glenner et al., Amyloid and Amyloidosis, Amsterdam: Excerpta Media, 1980 pg.88-93

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non - Patent Document 12

Non - Patent Document 13

Non - Patent Document 14

Non - Patent Document 15

[0011] Described herein are methods of treating hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) (with or without polyneuropathy and / or cardiomyopathy) in a human patient in need thereof, the method comprising administering to the patient a patisiran drug formulation as described in Table 1A, Table 1B, or Table 1C at a dose of 0.3 mg siRNA per kg of body weight, wherein patisiran is administered intravenously once every three weeks, the method resulting in stabilization or improvement of FAP stage, PND score, modified Neuropathy Impairment Score (mNIS+7) or other neuropathy-related clinical endpoint, serum percent TTR concentration, cardiac markers, and / or echocardiographic parameters.

[0012] Also described herein are methods for reducing or inhibiting an increase in the Neuropathy Impairment Score (NIS) or modified NIS (mNIS+7) in a human subject by administering an effective amount of a transthyretin (TTR) inhibitory composition, wherein the effective amount reduces the serum TTR protein concentration in the human subject to less than 50 μg / ml, or by at least 80%. Also described herein are methods for adjusting the dosage of a TTR inhibitory composition in response to an increase in NIS or treatment of familial amyloidotic polyneuropathy (FAP) by administering a TTR inhibitory composition to a subject with an increase in NIS or FAP and determining the TTR protein level in the subject with an increase in NIS or FAP. In some embodiments, if the TTR protein level is higher than 50 μg / ml, the next dose of the TTR inhibitory composition administered to the subject is increased, and if the TTR protein level is less than 50 μg / ml, the next dose of the TTR inhibitory composition administered to the subject is decreased. Also described herein are formulated versions of TTR inhibitor siRNAs. [Brief explanation of the drawings]

[0013] [Figure 1]1 is a graph showing the relationship between the progression of ΔNIS or ΔmNIS+7 and TTR concentration. [Figure 2] 1 is a graph showing the relationship between the progression of ΔNIS or ΔmNIS+7 and TTR concentration. [Figure 3] 1 is a structural formula of the sense and antisense strands of patisiran. [Figure 4] FIG. 1 is a graph showing improvement in neurological impairment compared to baseline. [Figure 5] 1 shows the effect of patisiran on mNIS+7. [Figure 6] The effect of patisiran on other secondary endpoints is shown. [Figure 7] 1 is a graph showing serum TTR concentrations in study participants. [Figure 8] 1 shows the relationship between serum TTR reduction and mNIS+7 score at 18 months. [Figure 9] Shifts in both PND scores and FAP status at 18 months are shown. [Figure 10] 1 is a graph showing the results of study participants in an 18-month double-blind study treated with patisiran for 12 months. [Figure 11] 1 is a graph showing the results of study participants in the 24-month study. DETAILED DESCRIPTION OF THE INVENTION

[0014] As described in further detail below, disclosed herein are methods of treating hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) (with or without polyneuropathy and / or cardiomyopathy) in a human patient in need thereof, the method comprising administering to the patient a patisiran drug formulation as described in Table 1A, Table 1B, or Table 1C at a dose of 0.3 mg siRNA per kg of body weight, wherein patisiran is administered intravenously once every three weeks, the method resulting in stabilization or improvement of FAP stage, PND score, modified Neuropathy Impairment Score (mNIS+7) or other neuropathy-related clinical endpoint, serum percent TTR concentration, cardiac markers, and / or echocardiographic parameters. Also disclosed are methods for reducing or inhibiting an increase in the Neuropathy Impairment Score (NIS) or modified NIS (mNIS+7) in a human subject by administering an effective amount of a transthyretin (TTR) inhibitory composition, such that the effective amount reduces serum TTR protein concentrations to less than 50 μg / ml, or by at least 80%.

[0015] In one embodiment, the TTR inhibitor composition is patisiran, such as, for example, a patisiran drug formulation. Patisiran is a TTR-specific small interfering ribonucleic acid (siRNA) formulated as a liver-targeting lipid nanoparticle (LNP) for intravenous (IV) administration.

[0016] TTR inhibitor composition The methods described herein include administering a TTR inhibitory composition. The TTR inhibitory composition may be any compound that reduces TTR protein levels in the serum of a human subject. Examples include, but are not limited to, RNAi, such as siRNA. Examples of siRNA include siRNAs that target the TTR gene, such as patisirin (described in further detail below) and revusiran. Examples also include antisense RNA. See U.S. Pat. No. 8,697,860 for examples of antisense RNAs that target the TTR gene.

[0017] TTR inhibitor compositions inhibit expression of the TTR gene. As used herein, "transthyretin" ("TTR") refers to a cellular gene. TTR is also known as ATTR, HsT2651, PALB, prealbumin, TBPA, and transthyretin (prealbumin, amyloidosis type I). The sequence of human TTR mRNA transcripts can be found at NM_000371. The sequence of mouse TTR mRNA can be found at NM_013697.2, and the sequence of rat TTR mRNA can be found at NM_012681.1.

[0018] The terms "silence," "inhibit expression," "downregulate expression," "suppress expression," and the like, as they refer to the TTR gene, refer to at least partial suppression of expression of the TTR gene, which suppression is manifested by a reduction in the amount of mRNA that can be isolated from a first cell or group of cells that has been treated to inhibit expression of the TTR gene, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been treated (control cells). The degree of inhibition is usually measured by the following:

number

[0019] Alternatively, the degree of inhibition can be given by the reduction of a parameter functionally related to TTR gene expression, such as the amount of protein encoded by the TTR gene secreted by cells, or the reduction of the number of cells exhibiting a certain phenotype, such as apoptosis.In principle, TTR gene silencing can be determined by any suitable assay in any cell that expresses a target constitutively or by genetic engineering.However, if a reference is needed to determine whether a certain dsRNA inhibits the expression of TTR gene to a certain extent and therefore falls within the scope of the present invention, the assay provided in the following examples will serve as such a reference.

[0020] RNAi In some embodiments, the methods described herein use a TTR inhibitory composition, such as an RNAi, e.g., an siRNA, e.g., a dsRNA, to inhibit expression of the TTR gene. In one embodiment, the siRNA is a dsRNA targeting the TTR gene. The dsRNA comprises an antisense strand having a complementary region complementary to at least a portion of the mRNA formed upon expression of the TTR gene, the complementary region being less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhangs. TTR-inhibiting siRNAs are described in International Patent Application No. PCT / US2009 / 061381 (WO 2010 / 048228) and International Patent Application No. PCT / US2010 / 055311 (WO 2011 / 056883), both of which are incorporated herein by reference in their entireties.

[0021] In one embodiment, the TTR inhibitory composition is patisiran, which is described in more detail below. In another embodiment, the TTR inhibitory composition is revusiran, an siRNA specific for trivalent GalNAc glycocluster-conjugated TTR. For a comprehensive description of revusiran, see International Application No. PCT / US2012 / 065691 and U.S. Patent Application Publication No. 20140315835, the contents of which are incorporated by reference in their entireties.

[0022] dsRNA comprises two RNA strands that are sufficiently complementary to hybridize and form a duplex structure. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary, generally perfectly complementary, to the target sequence derived from the sequence of the mRNA formed during the expression of the TTR gene, and the other strand (the sense strand) contains a region complementary to the antisense strand, so that when combined under suitable conditions, these two strands hybridize to form a duplex structure. The term "antisense strand" refers to the strand of dsRNA that contains a region that is substantially complementary to the target sequence. As used herein, the term "complementary region" refers to the region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence as defined herein. If the complementary region is not perfectly complementary to the target sequence, mismatches are most tolerated in the terminal regions, and if present, are generally located in one or more terminal regions, for example, within 6, 5, 4, 3, or 2 nucleotides from the 5' and / or 3' end. The term "sense strand," as used herein, refers to the strand of a dsRNA that contains a region that is substantially complementary to a region of the antisense strand. Generally, the duplex structure is 15 to 80, or 15 to 60, or 15 to 30, or 25 to 30, or 18 to 25, or 19 to 24, or 19 to 21, or 19, 20, or 21 base pairs in length. In one embodiment, the duplex is 19 base pairs in length. In another embodiment, the duplex is 21 base pairs in length.

[0023] Each strand of the dsRNA is generally 15 to 80, or 18 to 60, or 15 to 30, or 18 to 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In another embodiment, each strand is 25 to 30 nucleotides in length. Each strand of the duplex may have the same length or different lengths. When two different siRNAs are used in combination, the length of each strand of each siRNA may be the same or different.

[0024] The dsRNA may include one or more single-stranded overhangs consisting of one or more nucleotides. In one embodiment, at least one end of the dsRNA may include a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2, nucleotides. In another embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotide overhangs at each of its 3' and 5' ends beyond the sense strand. In a further embodiment, the sense strand of the dsRNA has 1 to 10 nucleotide overhangs at each of its 3' and 5' ends beyond the antisense strand.

[0025] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence relative to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising a second nucleotide sequence under specified conditions to form a duplex structure, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions may also be applied, such as physiologically relevant conditions that may be encountered in an organism. One of skill in the art will be able to determine the optimal set of conditions for determining the complementarity of two sequences depending on the ultimate application of the hybridized nucleotides.

[0026] This includes base pairing over the entire length of the first and second nucleotide sequences, between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, these two sequences may be perfectly complementary, or they may form one or more, but generally no more than four, three, or two mismatched base pairs upon hybridization, while retaining the ability to hybridize under conditions most relevant to the end application. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides long and another oligonucleotide 23 nucleotides long (where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide) may still be referred to as "fully complementary" for purposes of this description.

[0027] As used herein, "complementary" sequences may also include or be formed entirely by non-Watson-Crick base pairs and / or base pairs formed with non-naturally occurring modified nucleotides, so long as the above requirements for hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairing.

[0028] The terms "complementary," "fully complementary," and "substantially complementary" may be used herein with reference to matching bases between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context in which they are used.

[0029] As used herein, a polynucleotide "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding TTR), including the 5' UTR, open reading frame (ORF), or 3' UTR. For example, a polynucleotide is complementary to at least a portion of a TTR mRNA if its sequence is substantially complementary to an uninterrupted portion of an mRNA encoding TTR.

[0030] dsRNA can be synthesized by standard methods known in the art, as discussed further below, for example, by using an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0031] modified dsRNA In some embodiments, the dsRNA used in the methods described herein is chemically modified to enhance stability. Nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Specific examples of dsRNA compounds useful in the present invention include RNAs containing modified backbones or lacking natural internucleoside linkages. As defined herein, dsRNAs with modified backbones include those that retain a phosphorus atom in the backbone and those that lack a phosphorus atom in the backbone. For purposes of this specification, and as sometimes referred to in the art, modified dsRNAs that lack a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides.

[0032] Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0033] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,4 Nos. 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is incorporated herein by reference.

[0034] Modified dsRNA backbones that do not contain internal phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, or mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0035] Representative patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,471,968; 5,472,969; 5,473,969; 5,474,969; 5,475,969; 5,476,969; 5,477,969; 5,478,969; 5,479,969; 5,480,062; 5,481,062; 5,482,063; 5,483,064; 5,484,065; 5,485,066; 5,486,067; 5,487,068; 5,488,069; 5,489,069; 5,490,069; 5,491,069; 5,492,069; 5,493,069; 5,494,069; 5,495,069; 5,496,069; 5,497,069; 5,498,069; 5,499,069; 5,500,069; 5,501,069; 5,502, Nos. 489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is incorporated herein by reference.

[0036] In other suitable dsRNA mimics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimic, that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of dsRNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The acid groups are retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. Representative patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. No. 5,539,082; U.S. Pat. No. 5,714,331; and U.S. Pat. No. 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0037] Another embodiment of the present invention is dsRNA with phosphorothioate backbone, and oligonucleosides with heteroatom backbone of the above-referenced US Patent No. 5,489,677, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- (known as methylene (methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- (wherein natural phosphodiester backbone is represented as -OPO-CH2-) and amide backbone of the above-referenced US Patent No. 5,602,240. Also preferred are dsRNA with morpholino backbone structure of the above-referenced US Patent No. 5,034,506.

[0038] Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) nCH3)]2, where n and m are from 1 to about 10, are particularly preferred. Other preferred dsRNAs contain one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group that improves the pharmacokinetic properties of a dsRNA, or a group that improves the pharmacodynamic properties of a dsRNA, and other substituents with similar properties. Preferred modifications include 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxy-alkoxy group. Further preferred modifications include the group O(CH2)2ON(CH3)2, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as described in the Examples herein below, and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described in the Examples herein below.

[0039] Other preferred modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the dsRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide, or in 2'-5'-linked dsRNAs and at the 5' position of the 5'-terminal nucleotide. DsRNAs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427 Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the present application and each of which is incorporated herein by reference in its entirety.

[0040] dsRNA may also contain modified or substituted nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-aminoadenine, 5-methyl-2-methyl-3-methyl-4-methyl-5-methyl-6-methyl-1-methyl-2-methyl-3-methyl-4-methyl-1-methyl-2-methyl-3 ... These include other synthetic and natural nucleobases such as 8-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-dazaadenine, and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds that characterize the present invention.These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0041] Representative United States patents that teach the routine preparation of the above and other modified nucleobases include, but are not limited to, U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,300; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; and 5,484,908. Nos. 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is incorporated herein by reference, as well as U.S. Pat. No. 5,750,692, which is also incorporated herein by reference.

[0042] Pachisiran In one embodiment, the TTR inhibitory composition is patisiran, a TTR-specific small interfering ribonucleic acid (siRNA) formulated as a liver-targeting lipid nanoparticle (LNP) for intravenous (IV) administration (Akinc A, Zumbuehl A, et al. "A combinatorial library of lipid-like materials for delivery of RNAi therapeutics." Nat Biotechnol. 2008;26(5):561-569). This TTR siRNA has a target region within the 3'UTR region of the TTR gene to ensure and support homology with WT TTR and all reported TTR mutations. After LNP-mediated delivery to the liver, patisiran targets TTR mRNA for degradation, resulting in a potent and sustained reduction of mutant and WT TTR protein via the RNAi mechanism.

[0043] TTR siRNA (also known as ALN-18328) consists of sense and antisense strands containing the following sequences; lowercase letters indicate 2'-O-methyl nucleotides.

[0044] [Table 1]

[0045] Typically, the patisiran drug substance, i.e., siRNA, is in the form of a pharmaceutically acceptable salt. In some embodiments, the patisiran drug substance is patisiran sodium. The molecular formula of patisiran sodium is C 412 H 480 N 148 Na 40 O 290 P 40 The molecular weight is 14,304 Da. The structural formulae of the sense strand and antisense strand are shown in Figure 3.

[0046] The manufacturing process involves synthesizing two single-stranded oligonucleotides of a duplex by conventional solid-phase oligonucleotide synthesis. After purification, the two oligonucleotides are annealed to form a duplex.

[0047] The patisiran drug formulation contains TTR siRNA ALN-18328 and lipid excipients (DLin-MC3-DMA, DSPC, cholesterol, and PEG) in isotonic phosphate-buffered saline. 2000 -C-DMG).

[0048] The formulations of patisiran drug formulations are shown in Table 1A, Table 1B, or Table 1C below; in some embodiments, the concentration or amount of any one component is ±0.01, 0.05, 0.1, 0.5, 1.0, 5.0, or 10.0% of the concentration or amount listed in these tables:

[0049] [Table 2]

[0050] [Table 3]

[0051] [Table 4]

[0052] In some embodiments, the patisiran drug formulation is provided in a container, such as a glass vial, in the following amounts per vial:

[0053] [Table 5]

[0054] The injectable patisiran solution contains 2mg / mL of TTR siRNA drug substance.In some embodiments, the patisiran drug formulation is packaged in a 10mL glass vial with a fill volume of 5.5mL.In some embodiments, the patisiran drug formulation is packaged in a single-use vial with 10mg in 5mL.

[0055] In some embodiments, the container closure system consists of a United States Pharmacopoeia / European Pharmacopoeia (USP / EP) Type I borosilicate glass vial, a Teflon-coated butyl rubber stopper, and an aluminum flip-off cap.

[0056] Tetramer stabilizers In some embodiments, the methods described herein involve co-administering a tetramer stabilizer with another TTR inhibitor composition.

[0057] Tetramer stabilizers are compounds that bind to the TTR protein and stabilize the TTR tetramer. Mutations that destabilize the TTR tetramer lead to TTR misfiling and aggregation.

[0058] Examples of tetramer stabilizers include tafamidis and diflunisal. Both tafamidis and diflunisal can slow disease progression (Berk et al., "Repurposing diflunisal for familial amyloid polyneuropathy: a randomized clinical trial," JAMA 2013, 310:2658-2667; Coelho et al., 2012; Coelho et al., "Long-term effects of tafamidis for the treatment of transthyretin familial amyloid polyneuropathy," J Neurol 2013, 260:2802-2814; Lozeron et al., "Effect of tafamidis on disability and safety in late-onset Met30 transthyretin familial amyloid polyneuropathy"). on disability and safety of Tafamidis in late onset of Met30 transthyretin familial amyloid polyneuropathy).Eur J Neurol 2013,20:1539-1545).

[0059] Subjects and Diagnosis Disclosed herein is a method of treating hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) in a human patient in need thereof, wherein the patient may have polyneuropathy and / or cardiomyopathy, the method comprising administering to the patient a patisiran drug formulation as described in Table 1A, Table 1B, or Table 1C at a dose of 0.3 mg siRNA per kg of body weight, wherein patisiran is administered intravenously once every three weeks, the method resulting in stabilization or improvement of FAP stage, PND score, modified Neuropathy Impairment Score (mNIS+7) or other neuropathy-related clinical endpoint, serum percent TTR concentration, cardiac markers, and / or echocardiographic parameters.

[0060] Also disclosed herein are methods for reducing or inhibiting an increase in the Neuropathy Impairment Score (NIS) or modified NIS (mNIS+7) in a human subject, wherein the human subject has a TTR-related disorder. In some embodiments, the TTR-related disorder is one of the diseases caused by mutations in the transthyretin (TTR) gene. In certain embodiments, the disease is TTR amyloidosis, which manifests in various forms, including familial amyloid polyneuropathy (FAP), transthyretin-mediated amyloidosis (ATTR), and symptomatic polyneuropathy. When the peripheral nervous system is more prominently affected, the disease is referred to as FAP. When lesions are primarily in the heart and not the nervous system, the disease is referred to as familial amyloid cardiomyopathy (FAC). The third major type of TTR amyloidosis is called leptomeningeal / CNS (central nervous system) amyloidosis. ATTR affects the autonomic nervous system.

[0061] In some embodiments, a human subject with a TTR-related disorder has a mutant TTR gene. Over 100 TTR mutations have been reported, resulting in various disease symptoms. The most common mutations associated with FAP and ATTR-related cardiomyopathy are Val30Met and Val122Ile, respectively. TTR mutations cause protein misfolding, accelerating the process of TTR amyloid formation and are the most important risk factor for the development of clinically significant TTR amyloidosis (also known as ATTR amyloidosis-transthyretin type). Over 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.

[0062] In some embodiments, a human subject is selected to receive treatment for any form of TTR amyloidosis if they are an adult (age 18 or older) with a biopsy-confirmed diagnosis of TTR amyloidosis and mild to moderate neuropathy. In further embodiments, the human subject also has one or more of the following: a Karnofsky Performance Status (KPS) of ≥ 60%; a Body Mass Index (BMI) of 17-33 kg / m 2 adequate liver and kidney function (aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 2.5 × upper limit of normal (ULN), total bilirubin within the normal range, albumin > 3 g / dL, and international normalized ratio (INR) ≤ 1.2; serum creatinine ≤ 1.5 ULN); and hepatitis B virus and hepatitis C virus seronegativity.

[0063] In another embodiment, human subjects are excluded from treatment if they have undergone a liver transplant; undergone planned surgery during treatment; are HIV positive; have received an investigational drug other than tafamidis or diflunisal within the last 30 days; have a New York Heart Association heart failure classification of >2; are pregnant or nursing; have known or suspected systemic bacterial, viral, parasitic, or fungal infection; have unstable angina, uncontrolled clinically significant cardiac arrhythmia; or have previously had a severe reaction to a liposomal formulation or have known hypersensitivity to an oligonucleotide.

[0064] Neuropathic Impairment Score (NIS) The methods disclosed herein reduce or prevent the increase of the Neuropathy Impairment Score (NIS) in a human subject by administering a transthyretin (TTR) inhibitor composition. The NIS refers to a scoring system that measures muscle weakness, sensation, and reflexes, particularly in peripheral neuropathy. The NIS score assesses muscle weakness in standard muscle groups (1 = 25% loss, 2 = 50% loss, 3 = 75% loss, 3.25 = movement against gravity, 3.5 = movement with gravity removed, 3.75 = muscle spasm but no movement, and 4 = paralysis), standard muscle stretch reflex groups (0 = normal, 1 = low, 2 = absent), and touch, vibration, joint position and movement, and pins-and-needles sensation (all scored for the index finger and big toe: 0 = normal, 1 = low, 2 = absent). The scores are adjusted for age, sex, and physical strength.

[0065] In one embodiment, the method of reducing the NIS score results in a decrease in the NIS of at least 10%. In other embodiments, the method results in a decrease in the NIS score of at least 5, 10, 15, 20, 25, 30, 40%, or at least 50%. In other embodiments, the method prevents an increase in the NIS score, e.g., the method results in a 0% increase in the NIS score.

[0066] Methods for determining the NIS of a human subject are well known to those skilled in the art and can be found in:

[0067] Dyck, PJ et al., "Longitudinal assessment of diabetic polyneuropathy using a composite score in the Rochester Diabetic Neuropathy Study cohort," Neurology 1997. 49(1):pp.229-239.

[0068] Dyck PJ. "Detection, characterization, and staging of polyneuropathy: assessed in diabetics." Muscle Nerve. 1988 Jan;11(1):21-32.

[0069] Modified Neuropathy Impairment Score (mNIS+7) In some embodiments, the methods disclosed herein reduce or inhibit the increase of a human subject's modified Neuropathy Impairment Score (mNIS+7) upon administration of a transthyretin (TTR) inhibitor composition. As known to those skilled in the art, mNIS+7 refers to an assessment of neurological impairment (NIS) based on clinical examination combined with electrophysiological measures of large and small nerve fiber function (NCS and QST) and measures of autonomic function (postural blood pressure).

[0070] The mNIS+7 score is a modified version of the NIS+7 score (corresponding to the NIS+7 tests). The NIS+7 analyzes muscle weakness and muscle stretch reflexes. Five of the seven tests include nerve conduction attributes: peroneal nerve compound muscle action potential amplitude, motor nerve conduction velocity and motor nerve distal latency (MNDL), tibial MNDL, and sural sensory nerve action potential amplitude. These values ​​are corrected for age, sex, height, and weight variables. The remaining two of the seven tests include vibration detection threshold and heart rate slowing with deep breathing.

[0071] The mNIS+7 score modifies the NIS+7 to take into account the use of Smart Somatotopic Quantitative Sensation Testing, a novel autonomic assessment, and the use of compound muscle action potentials of the ulnar, peroneal, and tibial nerve amplitudes, and sensory nerve action potentials of the ulnar and sural nerves (Suanprasert, N. et al., "Retrospective study of a TTR FAP cohort to modify NIS+7 for therapeutic trials," J. Neurol. Sci., 2014. 344(1-2):ppg. 121-128).

[0072] In some embodiments, the method of reducing the mNIS+7 score results in a decrease in mNIS+7 of at least 10%. In other embodiments, the method results in a decrease in mNIS+7 score of at least 5, 10, 15, 20, 25, 30, 40%, or at least 50%. In other embodiments, the method inhibits an increase in mNIS+7, e.g., the method results in a 0% increase in mNIS+7.

[0073] Quality of life and neuropathy-related clinical endpoints In some embodiments, the methods disclosed herein stabilize or improve quality of life and / or neuropathy-related clinical endpoints. For example, the methods described herein can improve or stabilize quality of life, exercise intensity, disability, gait speed, nutritional status, and / or autonomic symptoms in a human patient with hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis), with or without polyneuropathy and / or cardiomyopathy, comprising administering to the patient a patisiran drug formulation as described in Table 1A, Table 1B, or Table 1C at a dose of 0.3 mg siRNA per kg body weight, wherein patisiran is administered intravenously once every three weeks.

[0074] In some embodiments, the methods described herein can improve or stabilize at least one neuropathy-related clinical endpoint selected from the group consisting of Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (QOL-DN), NIS-W; Rasch-built Composite Disability Scale (R-ODS); 10-Meter Walk Test (10-MWT); modified Body Mass Index (mBMI); and COMPASS-31 score in a human patient with hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis), with or without polyneuropathy and / or cardiomyopathy, the methods comprising administering to the patient a patisiran drug formulation as described in Table 1A, Table 1B, or Table 1C at a dose of 0.3 mg siRNA per kg body weight, wherein patisiran is administered intravenously once every three weeks.

[0075] FAP stage and PND score In some embodiments, the methods described herein stabilize or improve the polyneuropathy disorder (PND) score and familial amyloidotic polyneuropathy (FAP) stage as described herein. The PND score is determined as follows: PND I: maintains walking ability, paresthesia; PND II: reduced walking ability, but able to walk without a cane or crutch; PND IIIA: walks with one cane or crutch; PND IIIB: walks with two canes or crutches; PND IV: wheelchair-bound or bedridden. The FAP stage is as follows: FAP I: walking movement is intact; FAP II: requires assistance with walking movement; FAP III: wheelchair-bound or bedridden.

[0076] Serum TTR protein concentration The methods described herein include administering to a human subject an effective amount of a transthyretin (TTR) inhibitor composition, e.g., patisiran, wherein the effective amount reduces the serum TTR protein concentration of the human subject to less than 50 μg / ml, or by at least 80%. The serum TTR protein concentration can be determined directly using any method known to those skilled in the art, for example, an antibody-based assay, e.g., ELISA. Alternatively, the serum TTR protein concentration may be determined by measuring the amount of TTR mRNA. In a further embodiment, the serum TTR protein concentration is determined by measuring the concentration of a surrogate, e.g., vitamin A or retinol-binding protein (RBP). In one embodiment, the serum TTR protein concentration is determined using an ELISA assay, as described in the Examples below.

[0077] In some embodiments, the serum TTR protein concentration is reduced to less than 50 μg / ml, or less than 40 μg / ml, 25 μg / ml, or 10 μg / ml, hi some embodiments, the serum TTR protein concentration is reduced by 80%, or 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 95%.

[0078] Cardiac markers and echocardiographic parameters In some embodiments, the methods described herein treat a patient in need of treatment for hereditary transthyretin-mediated amyloidosis with cardiomyopathy (hATTR amyloidosis), wherein the methods result in improvement or stabilization of cardiac markers and / or echocardiographic parameters compared to baseline.

[0079] An example of a cardiac marker is serum NT-proBNP concentration. An example of an echocardiographic parameter is left ventricular (LV) strain or LV wall thickness.

[0080] AUC AUC refers to the area under the curve of the concentration of a composition, such as TTR, in the plasma of the bloodstream over time after a dose of a drug, such as a TTR inhibitor composition, is administered to a patient.AUC is affected by the rate of absorption of the composition into the patient's plasma and the rate of its removal from the plasma.As known to those skilled in the art, AUC can be determined by calculating the integral of the plasma composition concentration after drug administration.In another embodiment, AUC can be predicted using the following formula: Prediction AUC = (D × F) / CL (where D is the dosage concentration, F is a measure of bioavailability, and CL is the predicted clearance rate.) Those skilled in the art will appreciate that predicted AUC values ​​include an error range of ±3-4 fold.

[0081] In some embodiments, data for determining AUC are obtained by collecting blood samples from the patient at various time intervals after administration of the drug. In one aspect, the mean AUC in the patient's plasma after administration of the TTR inhibitor composition ranges from about 9,000 to about 18,000.

[0082] It is understood that plasma TTR concentrations may vary widely from subject to subject due to variability in metabolism and / or potential interactions with other therapeutic agents. In one embodiment of the present invention, plasma TTR concentrations may vary from subject to subject. Similarly, peak plasma concentrations (C max ) or time to reach maximum plasma concentration (T max) or the area under the curve from time 0 to the last measurable concentration (AUC last Values ​​such as the plasma concentration (TTR) or total area under the plasma concentration-time curve (AUC) may also vary from subject to subject. Due to this variability, the amount necessary to constitute a "therapeutically effective amount" of a compound, such as a TTR inhibitor composition, may vary from subject to subject.

[0083] Pharmaceutical Composition The methods described herein include administering a TTR inhibitory composition, e.g., an siRNA that targets the TTR gene, e.g., patisiran. In some embodiments, the TTR inhibitory composition is a pharmaceutical composition.

[0084] As used herein, a "pharmaceutical composition" includes a TTR inhibitor composition and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include pharmaceutically acceptable excipients such as, but not limited to, inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while cornstarch and alginic acid are suitable disintegrants. Binders can include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If necessary, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

[0085] The pharmaceutical compositions of the present invention can be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical; pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal; oral; or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular, administration.

[0086] This composition can be delivered in a way that targets specific tissues, such as the liver (for example, the hepatocytes of the liver).The pharmaceutical composition can be delivered directly to the brain by injection.Injection can be by stereotactic injection into specific regions of the brain (for example, the substantia nigra, cortex, hippocampus, striatum, or globus pallidus), or dsRNA can be delivered to multiple regions of the central nervous system (for example, multiple regions of the brain and / or to the spinal cord).dsRNA can also be delivered to diffuse regions of the brain (for example, diffuse delivery to the cortex of the brain).

[0087] In one embodiment, dsRNA targeting TTR can be delivered using a cannula or other delivery device with one end implanted in a tissue, such as the brain, for example, the substantia nigra, cortex, hippocampus, striatum, corpus callosum, or globus pallidus. The cannula can be connected to a reservoir of the dsRNA composition. Flow or delivery can be mediated by a pump, such as an osmotic pump or minipump, such as an Alzet pump (Durect, Cupertino, CA). In one embodiment, the pump and reservoir are implanted in an area remote from the tissue, for example, the abdomen, and delivery is achieved by a conduit leading from the pump or reservoir to the release site. Infusion of the dsRNA composition into the brain can last for several hours or days, for example, 1, 2, 3, 5, or 7 days or more. Brain delivery devices are described, for example, in U.S. Patent Nos. 6,093,180 and 5,814,014.

[0088] Dosage and timing Those skilled in the art will appreciate that certain factors can influence the dosage and timing required for effective treatment of a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimation of effective dosages and in vivo half-lives of TTR inhibitor compositions encompassed by the present invention can be performed using conventional methods or based on in vivo testing using appropriate animal models, as described elsewhere herein.

[0089] Generally, suitable dosages of pharmaceutical compositions of TTR inhibitory compositions can range from 0.01 to 200.0 milligrams per kilogram of recipient body weight per day, generally from 1 to 50 mg per kilogram of body weight per day.

[0090] For example, the TTR inhibitor composition may be an siRNA and may be administered at a dose of 0.01 mg / kg, 0.05 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.628 mg / kg, 2 mg / kg, 3 mg / kg, 5.0 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg per dose. In another embodiment, the dosage is between 0.15 mg / kg and 0.3 mg / kg. For example, the TTR inhibitor composition can be administered at a dose of 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, or 0.3 mg / kg. In one embodiment, the TTR inhibitor composition is administered at a dose of 0.3 mg / kg.

[0091] The pharmaceutical composition (e.g., patisiran) can be administered once daily, or once or twice every 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. The unit dosage can be compounded for delivery over several days, for example, using a conventional sustained release formulation that provides sustained release of the TTR inhibitor composition over several days. Sustained release formulations are well known in the art and are particularly useful for delivering drugs at specific sites, such as may be used with the agents of the present invention.

[0092] In one embodiment, the TTR inhibitor composition is patisiran, e.g., a patisiran drug formulation, and the dosage is 0.3 mg / kg, and this dose is administered once every 21 days or 3 weeks. In some embodiments, the dose, e.g., an effective amount, is administered about every 3 weeks or about every 21 days. In another embodiment, the effective amount is 0.3 mg / kg, and this effective amount is administered by a 70-minute infusion at 1 mL / min for 15 minutes followed by 3 mL / min for 55 minutes once every 21 days or 3 weeks. In another embodiment, the effective amount is 0.3 mg / kg, and this effective amount is administered by a 60-minute infusion at 3.3 mL / min for 15 minutes followed by 3.3 mL / min for 55 minutes in two doses every 21 to 28 days.

[0093] In some embodiments, the method comprises administering patisiran, e.g., a patisiran drug formulation, at a dosage of 0.3 mg siRNA per kg body weight administered by intravenous infusion over approximately 80 minutes once every three weeks. In some embodiments, the method comprises administering patisiran at a dosage of 0.3 mg siRNA per kg body weight administered by intravenous infusion at 3.3 mL / min over 60 minutes, or using a microdosing regimen (1.1 mL / min for 15 minutes, followed by 3.3 mL / min for the remaining doses) over 70 minutes.

[0094] The dosage of the TTR inhibitor composition can be adjusted in response to an increase in NIS or treatment of FAP by administering the TTR inhibitor composition and determining the subject's TTR protein level: if the TTR protein level is greater than 50 μg / ml, the next dose of the TTR inhibitor composition administered to the subject is increased; if the TTR protein level is less than 50 μg / ml, the next dose of the TTR inhibitor composition administered to the subject is decreased.

[0095] The TTR inhibitor composition can be administered in combination with other known agents effective in treating pathological processes mediated by target gene expression. In one embodiment, patisiran is administered with a tetramer stabilizer, such as tafamidis or diflunisal. In either case, the administering physician can adjust the amount and timing of patisiran and / or tetramer stabilizer administration based on observed results using standard efficacy measures known in the art or described herein. [Example]

[0096] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0097] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0098] The terms "patisiran" and "patisiran drug formulation" are used interchangeably in this example and refer to the formulated siRNA as described in Table 1A, Table 1B, and Table 1C.

[0099] Example 1: Safety and Efficacy of Patisiran for TTR Amyloidosis A Phase I clinical trial found that patisiran reduced TTR levels in patients over a 28-day period. The results of this study were published in the New England Journal of Medicine (Coelho et al., N Engl J Med 2013;369:819-29). This publication is incorporated by reference for all purposes. A summary of the study design and results is reproduced below.

[0100] This was a multicenter, randomized, single-blind, placebo-controlled, dose-ranging study to determine the safety and efficacy of a single dose of patisiran in patients with TTR amyloidosis or healthy adults. Men and women aged 18 to 45 years were eligible for the study if they were healthy (as determined based on medical history, physical examination, and 12-lead electrocardiogram), had a BMI of 18.0 to 31.5, had adequate liver function and blood counts, and were not of childbearing potential.

[0101] Participant series (four participants per series) were randomly assigned in a 3:1 ratio to receive patisiran at doses of 0.01 to 0.5 mg / kg or placebo (normal saline). Patisiran was administered intravenously over 15 and 60 minutes, respectively. To reduce the risk of infusion-related reactions, patients received similar premedications the night before and on the day of infusion. These medications included dexamethasone, acetaminophen, diphenhydramine or cetirizine, and ranitidine.

[0102] The pharmacodynamic activity of patisiran, as reflected by serum TTR levels, was measured using a validated enzyme-linked immunosorbent assay (ELISA) for total TTR (Charles River Laboratories, Wilmington, MA). Baseline levels of TTR, retinol-binding protein, and vitamin A for each patient were defined as the mean of four measurements taken before patisiran administration. Adverse events were monitored from the start of drug administration through day 28. Safety monitoring also included hematological evaluations, blood chemistry analysis, and thyroid function tests.

[0103] The plasma pharmacokinetics of the TTR siRNA contained in patisiran was determined using a validated ELISA-based hybridization assay. The ATTO-Probe-HPLC assay (lower limit of quantitation, 1.0 ng per milliliter) (Tandem Laboratories, Salt Lake City, UT) was used for detection and quantification of the siRNA. Pharmacokinetic estimates were determined using WinNonlin (Pharsight, Princeton, NJ).

[0104] Knockdown of TTR, vitamin A, and retinol binding protein was measured compared to baseline levels (data not shown).

[0105] result No significant changes in TTR levels (compared to placebo) were observed at the two lowest patisiran doses. However, substantial TTR knockdown was observed in all participants receiving doses of 0.15 to 0.5 mg / kg (data not shown). TTR knockdown was rapid, potent, and sustained at all three dose levels, and was highly significant compared to placebo by day 28 (P<0.001). Given the robust responses seen at 0.15 and 0.3 mg / kg and the modest, gradual improvement at 0.5 mg / kg, only one participant received the 0.5 mg / kg dose.

[0106] Response kinetics were consistent among participants (data not shown), particularly at doses of at least 0.3 mg / kg, with a >50% reduction by day 3, nadir levels by approximately day 10, continued >50% suppression at day 28, and complete recovery by day 70. Participants receiving 0.15 mg / kg, 0.3 mg / kg, and 0.5 mg / kg had peak TTR knockdown of 85.7%, 87.6%, and 93.8%, respectively. The mean nadirs at the 0.15 mg / kg and 0.3 mg / kg doses were 82.3% (95% confidence interval (CI), 67.7 to 90.3) and 86.8% (95% CI, 83.8 to 89.3), respectively; these nadirs showed little variability between participants when analyzed by either absolute TTR levels or percent TTR knockdown and were highly significant when compared with placebo (P<0.001) (data not shown).

[0107] The degree of knockdown appeared to determine the duration of suppression, with mean reductions at day 28 of 56.6% (95% CI, 11.6–78.7) and 67.1% (95% CI, 45.5–80.1) for participants receiving the 0.15 mg / kg and 0.3 mg / kg doses, respectively, and a 76.8% reduction at day 28 for one patient receiving the 0.5 mg / kg dose. The TTR knockdown observed in humans at the 0.3 mg / kg dose was virtually identical to that seen in nonhuman primates at the same dose level (data not shown). These TTR reductions by patisiran correlated with changes in retinol-binding protein and vitamin A levels (data not shown).

[0108] The use of patisiran did not result in any significant changes in hematology, liver, or kidney measurements or thyroid function, and there were no drug-related serious adverse events or discontinuations of study drug due to adverse events (data not shown).

[0109] The plasma pharmacokinetic profile of patisiran showed that the peak plasma concentrations of TTR siRNA and the area under the curve values ​​up to the final day increased approximately dose-proportionally over the dose range tested (data not shown).

[0110] Specificity of Patisiran To further demonstrate the specificity of patisiran's effects, we also measured TTR in a group of healthy volunteers in a phase 1 trial of ALN-PCS, which contains siRNA targeting PCSK9 (a cholesterol-lowering target) formulated in the same type of lipid nanoparticles used in patisiran. A single dose of 0.4 mg / kg ALN-PCS (the so-called control siRNA) had no effect on TTR (data not shown), indicating that the effect of patisiran on TTR was due to specific targeting by the siRNA and not a nonspecific effect of the lipid nanoparticle formulation.

[0111] Further evidence supporting the specificity and mechanism of action of patisiran's pharmacodynamic effects was provided by detecting predicted TTR mRNA cleavage products in circulating extracellular RNA using a 5' RACE (rapid amplification of complementary DNA ends) assay on blood samples obtained from participants receiving a 0.3 mg / kg dose. To collect blood samples, clotted blood samples (from subjects pre-dose and 24 hours post-dose) were centrifuged at 1200 x g for 20 minutes, followed by serum collection. The serum was centrifuged once more at 1200 x g for 10 minutes to remove suspended cellular material and then frozen. The thawed serum was mixed with lithium chloride (1 M final concentration) and incubated at 4°C for 1 hour. The samples were spun at 120,000 x g for 2 hours at 4°C to pellet the RNA, and total RNA was isolated from the pellet by Trizol extraction (Life Technologies, Grand Island, New York, USA) and isopropanol precipitation.

[0112] To detect TTR siRNA-mediated cleavage products, isolated RNA was used for ligation-mediated RACE PCR using the GeneRacer kit (Life Technologies). RNA was ligated to a GeneRacer adapter and reverse transcribed using a TTR-specific reverse primer (5'-aatcaagttaaagtggaatgaaaagtgcctttcacag-3') (SEQ ID NO: 3). Two rounds of PCR were then performed using the GeneRacer GR5' forward primer, complementary to the adapter, and a TTR-specific reverse primer (5'-gcctttcacaggaatgttttattgtctctg-3') (SEQ ID NO: 4). Nested PCR was performed with the GR5' nested primer and a TTR-specific reverse nested primer (5'-ctctgcctggacttctaacatagcatatgaggtg-3') (SEQ ID NO: 5). PCR products were cloned using a TOPO-Blunt vector (Life Technologies). The cloned inserts were amplified by colony PCR using M13 forward and reverse primers. These amplicons were sequenced with T7 promoter primers at the Macrogen sequencing facility. Sequences from 96 clones were aligned with human TTR using CLC WorkBench.

[0113] TTR mRNA was detected in both pre-dose samples and samples obtained 24 hours after drug administration. Consistent with an RNAi mechanism, the expected mRNA cleavage products were absent in pre-dose samples but present in post-dose samples in all three participants (data not shown).

[0114] Tandem Labs qualified and implemented an LC / MS / MS assay for quantifying wild-type and mutant TTR in human serum. Serum samples were digested using chymotrypsin, then processed by protein precipitation extraction, and then analyzed by LC / MS / MS. The chymotryptic peptides TTRW-1, corresponding to wild-type TTR, and V30M-1, corresponding to mutant V30M, were monitored according to their unique specific mass-to-charge ratio transitions. Standard calibration data obtained using stable isotope-labeled peptides (TTRW-1-D8 and V30M-1-D8) were used to calculate the endogenous peptide fragments (TTRW-1 and V30M-1) in human serum samples. A linear calibration curve was constructed using 1 / x2 weighted least-squares regression analysis using the standard peak area ratios (i.e., TTRW-1-D8 relative to the internal standard TTRW-L1-D16 and V30M-1-D8 relative to V30M-L1-D16). The qualified LC / MS / MS method achieved a lower limit of quantitation (LLOQ) of 5 ng / ml with a standard curve ranging from 5 to 2500 ng / ml.

[0115] Example 2: Multiple-dose study of the safety and efficacy of patisiran therapy for familial amyloidotic polyneuropathy This Phase II clinical trial assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple ascending intravenous doses of patisiran in patients with TTR-mediated FAP. The data were presented at the International Symposium on Familial Amyloidotic Polyneuropathy (ISFAP) in November 2013.

[0116] Eligible patients were adults (≥18 years) with a biopsy-confirmed diagnosis of ATTR amyloidosis and mild to moderate neuropathy; Karnofsky Performance Status (KPS) ≥60%; and body mass index (BMI) 17–33 kg / m 2adequate hepatic and renal function (aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 2.5 × upper limit of normal (ULN), total bilirubin within the normal range, albumin > 3 g / dL, and international normalized ratio (INR) ≤ 1.2; serum creatinine ≤ 1.5 ULN); and hepatitis B and C seronegative. Patients were excluded if they had undergone liver transplantation; underwent planned surgery during the study; were HIV-positive; had received an investigational drug other than tafamidis or diflunisal within 30 days; had New York Heart Association heart failure classification > 2; were pregnant or breastfeeding; had known or suspected systemic bacterial, viral, parasitic, or fungal infection; had unstable angina pectoris, uncontrolled clinically significant cardiac arrhythmias; or had a previous severe reaction to a liposomal drug or known hypersensitivity to oligonucleotides.

[0117] This was a multicenter, international, open-label, multiple-ascending dose phase II study of patisiran in patients with FAP. Three patient cohorts received two doses of patisiran, each administered as an intravenous (IV) infusion. Cohorts 1-3 received two doses of patisiran, 0.01, 0.05, and 0.15 mg / kg, respectively, every four weeks (Q4W); cohorts 4 and 5 both received two doses of patisiran at 0.3 mg / kg Q4W. All patients in cohorts 6-9 received two doses of patisiran at 0.3 mg / kg Q3W. All patients received premedication consisting of dexamethasone, paracetamol (acetaminophen), an H2 blocker (e.g., ranitidine or famotidine), and an H1 blocker (e.g., cetirizine, hydroxyzine, or fexofenadine) before each patisiran infusion to reduce the risk of infusion-related reactions.Patisiran was administered IV at 3.3 mL / min for 60 minutes or over 70 minutes using a microdosing regimen (1.1 mL / min for 15 minutes, followed by 3.3 mL / min for the remaining dose).

[0118] All patients were assessed for serum total TTR protein levels using an enzyme-linked immunosorbent assay (ELISA). In addition, wild-type and mutant TTR protein were separately and specifically measured in the serum of patients with the Val30Met mutation using a proprietary mass spectrometry method (Charles River Laboratories, Quebec, Canada). Serum samples were collected at screening and on days 0, 1, 2, 7, 10, 14, 21, 22, and 23 (Q3W only); 28 and 29 (Q4W only); 30 (Q4W only); 31 (Q3W only); 35 and 38 (Q4W only), as well as follow-up days 42, 49, 56, 112, and 208.

[0119] Plasma concentration-time profiles of TTR siRNA were generated based on blood samples collected on Day 0 and the following time points: predose (within 1 hour of the scheduled start of dosing), end of infusion (EOI), 5, 10, and 30 minutes after infusion, and 1, 2, 4, 6, 24, 48, 168, 336, and 504 hours (Day 21, Q3W regimen only), and 672 hours (Day 28, Q4W regimen only). Additional samples were collected on Days 84 and 180 for the Q4W regimen and Days 35, 91, and 187 for the Q3W regimen. Cohorts 3–9 also analyzed both free and encapsulated TTR siRNA from Day 0, EOI, and 2-hour postinfusion blood samples. Serum TTR siRNA was analyzed using a validated ATTO-Probe high-performance liquid chromatography (HPLC) assay (Tandem Laboratories, Salt Lake City, Utah, USA). PK analysis was performed using noncompartmental and / or compartmental evaluation of TTR siRNA plasma concentration-time data to determine PK parameter estimates using the validated software program WinNonlin®. After administration, urine samples were analyzed for excreted TTR siRNA levels, and renal clearance (CL) was assessed. R ) was measured.

[0120] Serum vitamin A and retinol-binding protein (RBP) levels were measured by HPLC and turbidimetry, respectively, at the same time points as indicated for total TTR (Biomins Specialized Medical Pathology, Lyon, France).

[0121] The mean and variance of TTR knockdown from baseline in the PP population were calculated (baseline was defined as the mean of all pre-dose values). PD data (natural log-transformed versus baseline TTR) were analyzed using analysis of variance (ANOVA) and analysis of covariance (ANCOVA) with Tukey's post-hoc test for individual pairwise comparisons (between dose levels). Nadir TTR levels were defined as the lowest level for each patient in the 28-day period (21-day period for the Q3W group) after each dose administration (first dose period, second dose period: days 1-28, 29-56, and 1-21, 22-42 for the Q4W and Q3W groups, respectively). The relationship between TTR and RBP or vitamin A compared to baseline, and the relationship between wild-type and V30M TTR levels, were examined by linear regression. Power model analysis was used to determine dose proportionality of patisiran components in PK parameters. AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA) coding system, version 15.0, and descriptive statistics provided for AEs, laboratory data, vital sign data, and ECG interval data. All statistical analyses were performed using SAS software, version 9.3 or higher. Efficacy and Pharmacodynamics: Mean (SD) baseline serum TTR protein levels were similar across dose cohorts: 272.9 (98.86), 226.5 (12.67), 276.1 (7.65), 242.6 (38.30), and 235.5 (44.45) μg / mL for the 0.01, 0.05, 0.15, 0.3 mg / kg Q4W, and 0.3 mg / kg Q3W dose groups, respectively.

[0122] Significant reductions in TTR (p<0.001 by post hoc test after ANCOVA) were observed after the first and second patisiran doses in the 0.3 mg / kg Q4W and Q3W cohorts compared with the 0.01 mg / kg dose cohort (data not shown). In patients with the Val30Met mutation, similar levels of knockdown were observed for wild-type and mutant TTR (data not shown). Serum TTR knockdown levels were correlated with RBP (r 2 =0.89, p<10 -15 ) and Vitamin A (r 2 =0.90, p<10 -15 ) was highly correlated with reduced circulating levels (data not shown).

[0123] Patients receiving tafamidis or diflunisal had significantly increased baseline serum TTR levels compared with patients not receiving stabilizer therapy (p<0.001 by ANOVA) (data not shown), but patisiran treatment resulted in similar TTR knockdown in these two patient groups (data not shown).

[0124] Pharmacokinetics: Mean concentrations of the patisiran TTR siRNA component decreased after EOI (data not shown), and there was no accumulation of siRNA after the second dose on Days 21 / 28. Measurement of encapsulated vs. unencapsulated TTR siRNA concentrations after each dose demonstrated the stability of the circulating LNP formulation. For both the first and second doses, mean maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from 0 to the last measurable time point (AUC0-last) increased dose-proportionally across the dose range tested. Cmax and AUC0-last after Dose 1 and Dose 2 were similar, indicating no accumulation. Median terminal half-lives of patisiran on Days 0 and 21 / 28 ranged from 39 to 59 hours at doses >0.01 mg / kg and were relatively stable when comparing Dose 1 and Dose 2 for each dose cohort.

[0125] These Phase II data demonstrate that treatment of FAP patients with patisiran resulted in a robust, dose-dependent, and statistically significant knockdown of serum TTR protein levels. Two consecutive doses of patisiran at 0.3 mg / kg, administered 3–4 weeks apart, achieved a sustained mean TTR reduction of >80%, with a maximum knockdown of 96% in the Q3W group. These knockdown rates are consistent with those observed in a single, ascending-dose, placebo-controlled Phase 1 study of patisiran (Coelho et al. 2013a). Evidence from other systemic amyloid diseases indicates that clinical disease improvement or stabilization can be achieved with as little as a 50% reduction in disease-causing proteins (Lachmann et al. 2003; Lachmann et al. 2007). The extent of TTR knockdown by patisiran was not affected by patients' use of tafamidis or diflunisal, suggesting that these TTR stabilizers do not interfere with patisiran's pharmacological activity. In patients with the Val30Met mutation, patisiran suppressed the production of both mutant and wild-type TTR; the latter remains amyloidogenic in patients with late-onset FAP after liver transplantation (Yazaki et al., 2003; Liepnieks et al., 2010).

[0126] Example 3: Administration of patisiran reduces neurological impairment as measured by NIS and mNIS+7 An open-label extension (OLE) study has been and will be conducted in FAP patients using the protocol described in Example 2. Administration of patisiran reduced both NIS and mNIS+7.

[0127] Eligible FAP patients previously treated in a phase 2 trial were enrolled in a phase 2 OLE study. Clinical endpoints were assessed every 6 months, and treatment with 0.30 mg / kg once every 3 weeks for up to 2 years was ongoing. Study objectives included effects on neurological impairment (mNIS+7 and NIS), quality of life, mBMI, disability, exercise capacity, grip strength, autonomic symptoms, nerve fiber density in skin biopsies, cardiac lesions (in the cardiac subgroup), and serum TTR levels.

[0128] The demographic characteristics of the patients are shown below.

[0129] [Table 6]

[0130] Baseline characteristics included the following:

[0131] [Table 7]

[0132] As shown in the table below, administration of patisiran resulted in a reduction in serum TTR levels. Patisiran achieved a sustained reduction in serum TTR of approximately 80%, with a further nadir of up to 88% between doses.

[0133] [Table 8]

[0134] As shown in the table below, patisiran treatment resulted in a change in mNIS+7 when measured at 6 and 12 months.

[0135] [Table 9]

[0136] Treatment with patisiran resulted in changes in NIS at 6 and 12 months, as shown in the table below.

[0137] [Table 10]

[0138] The relationship between progression of ΔNIS or ΔmNIS+7 and TTR concentrations was examined by linear regression, as shown in Figures 1 and 2. TTR and mean pre-treatment trough values ​​[TTR] correlated with the change in mNIS+7 at 6 months.

[0139] NIS and mNIS+7 were measured at 0, 6, and 12 months. ΔNIS or ΔmNIS+7 from 0 to 6 months and 0 to 12 months were used as response variables. Predictor variables included two different TTR concentration measures: area under the TTR protein concentration curve ("AUC"), and mean percent knockdown compared to baseline at days 84 and 168 (for the 0-6 month comparison) and days 84, 168, 273, and 357 (for the 0-12 month comparison).

[0140] For all TTR measurements, "baseline" was defined as the mean of all pre-dose values. TTR AUC was calculated using the trapezoidal method, starting with raw TTR concentrations (μg / mL) and baseline values ​​(inserted at day 0) through day 182 (for the 0-6 month comparison) or day 357 (for the 0-12 month comparison). Percent knockdown relative to baseline was calculated at each scheduled time point. Linear regression was performed, and P values ​​associated with testing the null hypothesis of no association between predictor and response variables are reported.

[0141] At 12 months, the mean mNIS+7 and NIS change were -2.5 and 0.4 points, respectively, which compare favorably with the sharp increases in estimated mNIS+7 and NIS (e.g., 10-18 point increases) at 12 months from prior FAP studies in patient populations with similar baseline NIS. The favorable effect of patisiran on progression of neuropathy impairment scores correlated with the degree of TTR reduction. This demonstrates that reduction of serum TTR burden with patisiran translates into clinical benefit in FAP patients.

[0142] Example 4: A Single Randomized, Double-Blind, Placebo-Controlled Phase 3 Study of Patisiran in Patients with hATTR Amyloidosis with Polyneuropathy Study design: The efficacy and safety of patisiran were determined in a single randomized, double-blind, placebo-controlled phase 3 study (APOLLO) in patients with hATTR amyloidosis with polyneuropathy. The primary efficacy endpoint was the change from baseline in the mNIS+7 composite neurological impairment score at 18 months. Secondary endpoints included the Norfolk QOL-DN quality of life score and measures of motor strength (NIS-W), physical disability (R-ODS), gait speed (10-meter walk test), nutritional status (mBMI), and autonomic symptoms (COMPASS-31). Exploratory endpoints included cardiac measures in patients with evidence of cardiac involvement at baseline, as well as measures of dermal amyloid burden and nerve fiber density in skin biopsies.

[0143] overview: APOLLO met its primary endpoint (mNIS+7) and also demonstrated highly statistically significant effects on Norfolk QOL-DN and all other secondary endpoints, demonstrating the clinical benefit of patisiran in hATTR amyloidosis with polyneuropathy. More than 50% of patients treated with patisiran showed improvement in neurological impairment compared to baseline at 18 months.

[0144] Protocol Overview: Patients were treated with patisiran as described above. Briefly, patients received patisiran (see Table 1) at a dose of 0.3 mg siRNA per kg of body weight intravenously every three weeks (Q3W). Patisiran was administered intravenously, for example, at 3.3 mL / min over 60 minutes, or over 70 minutes using a microdose regimen (1.1 mL / min for 15 minutes, followed by 3.3 mL / min for the remaining doses).

[0145] In some embodiments, patients received premedication, for example, one hour before patisiran infusion, the night before and / or on the day of the infusion to reduce the risk of infusion-related reactions. This medication included dexamethasone, acetaminophen, diphenhydramine or cetirizine, and ranitidine. In some embodiments, the following premedication regimen can be used: IV dexamethasone 10 mg, or equivalent; and oral paracetamol / acetaminophen 500 mg, or equivalent; and IV histamine H1 receptor antagonist (H1 blocker): diphenhydramine 50 mg, or equivalent other IV H1 blocker, or hydroxyzine 25 mg, or fexofenadine 30 or 60 mg PO, or cetirizine 10 mg PO; and IV histamine H2 receptor antagonist (H2 blocker): ranitidine 50 mg, or famotidine 20 mg, or equivalent dose of other H2 blocker.

[0146] Baseline characteristics: APOLLO enrolled 225 patients (148 in the patisiran group and 77 in the placebo group). Patients were enrolled between December 2013 and January 2016 at 44 centers in 19 countries across North America, Europe, Asia-Pacific, and Central / South America. The majority of patients were Caucasian (72.4%), 74.2% were male, and most were elderly, with a median age of 62 years (range, 24-83 years). Similar proportions of FAP stage I and stage II patients were present, with mean mNIS+7 scores of 80.9 (range, 8-165) and 74.6 (range, 11-153.5) in the patisiran and placebo groups, respectively. 42.7% of patients had a Val30Met mutation, compared with 57.3% with non-Val30Met mutations. Fifty-six percent had echocardiographic evidence of cardiac amyloid pathology, and 52.9% of all patients had a history of TTR tetramer stabilizer use. Treatment groups were balanced with respect to age, sex, disease stage, baseline mNIS+7, and history of TTR tetramer stabilizer use. Patisiran-treated patients were more likely to be Caucasian (76.4% vs. 64.9%), have a higher proportion of non-Val30Met mutations (62.2% vs. 48.1%), have baseline echocardiographic evidence of cardiac disease (cardiac subpopulation, 60.8% vs. 46.8%), and were more likely to be enrolled in North America (25% vs. 13%).

[0147] TTR genotype As noted above, 42.7% of patients had Val30Met mutations, compared with 57.3% with non-Val30Met mutations. The non-Val30Met mutations found in patients are listed below.

[0148] [Table 11]

[0149] Disease Stage The following table provides the stages of FAP:

[0150] [Table 12]

[0151] breakdown: A total of 185 patients completed study treatment, with a higher completer rate in the patisiran group (92.6% vs. 62.3% in the patisiran and placebo groups, respectively).A total of 193 patients completed the study, with a higher completer rate in the patisiran group (93.2% vs. 71.4% in the patisiran and placebo groups, respectively).

[0152] Six (7.8%) placebo patients had rapid disease progression at 9 months (a mNIS+7 increase of ≥ 24 points with FAP stage progression as determined by the Clinical Adjudication Committee) compared with one (0.7%) in the patisiran group.

[0153] In the placebo group, the main reasons for discontinuing study treatment were subject withdrawal of consent (15.6%), adverse events (9.1%), disease progression (5.2%), and death (5.2%), while the main reasons for dropping out of the study were subject withdrawal of consent (14.3%), adverse events (7.8%), and death (5.2%).

[0154] In the patisiran group, the main reasons for discontinuing study treatment were death (3.4%) and adverse events (2%), and the main reasons for dropping out of the study were death (4.1%) and adverse events (1.4%).

[0155] Of 189 patients who completed APOLLO and were potentially eligible for enrollment in the global open-label extension study, 186 (98.4%) enrolled in the ongoing global open-label extension study.

[0156] Summary of effectiveness A summary of the results for both mNIS+7 and secondary endpoints is shown in the table below.

[0157] [Table 13]

[0158] [Table 14]

[0159] [Table 15]

[0160] mNIS+7 The study met its primary efficacy endpoint. In the modified intent-to-treat (mITT) population, the patisiran group demonstrated improvement in neurological impairment compared to baseline at 18 months (mNIS+7 LS mean (SEM) change of -6.0 (1.7) points), whereas the placebo group demonstrated worsening of neurological impairment (mNIS+7 LS mean (SEM) change of +28.0 (2.6) points), corresponding to a highly significant reduction in neuropathy progression with patisiran compared with placebo (LS mean difference of -34.0 points, 95% CI: -39.9, -28.1, p=9.26E-24). Similar results were observed in the PP population. The benefit of patisiran was observed as early as 9 months (LS mean difference of -16.0 points, 95% CI: -20.7, -11.3), with all components of the mNIS+7 showing consistent effects in favor of patisiran.

[0161] As shown in Figure 4, improvement in neurological impairment (mNIS+7 change of <0 points) compared to baseline at 18 months was seen in 56.1% (95% CI: 48.1%, 64.1%) of patients on patisiran compared to only 3.9% (95% CI: 0.0%, 8.2%) on placebo (odds ratio of 40.0, p=1.82E-15).

[0162] As shown in Figure 5, the effect of patisiran on mNIS+7 was observed across patient subgroups defined by age, sex, race, geographic region, TTR genotype, neuropathy severity, disease stage, and prior TTR tetramer stabilizer use.

[0163] Patients experienced sustained efficacy of patisiran over 30 and 36 months of treatment regimens, as measured by mNIS+7 scores.

[0164] Secondary endpoints Six secondary endpoints also achieved statistical significance by hierarchical testing.

[0165] As shown in Figure 6, mITT population, the LS mean (SEM) change from baseline on the Norfolk QOL-DN at 18 months was -6.7 (1.8) points for patisiran, corresponding to an improvement in quality of life, compared with +14.4 (2.7) points for placebo, indicating a worsening of quality of life. The LS mean difference between treatment groups was -21.1 points (95% CI: -27.2, -15.0, p=1.10E-10), demonstrating a significant improvement in quality of life with patisiran compared to placebo. Similar results were observed in the PP population.

[0166] Similar to mNIS+7, the effect of patisiran on Norfolk QOL-DN was seen as early as 9 months (LS mean difference of -15.0 points, 95% CI: -19.8, -10.2). The effect of patisiran on Norfolk QOL-DN was observed across patient subgroups defined by age, sex, race, geographic region, TTR genotype, neuropathy severity, disease stage, and prior use of TTR tetramer stabilizers.

[0167] Patisiran treatment also significantly improved NIS-W (-17.9 points LS mean difference, 95% CI: -22.3, -13.4, p=1.40E-13); R-ODS (+9.0 points LS mean difference, 95% CI: 7.0, 10.9, p=4.07E-16); and 10-meter walk test (+0.311 m / sec LS mean difference, 95% CI: 0.23, 0.26) compared to placebo at 18 months. 39, p=1.88E-12); BMI (LS mean difference of +115.7 kg / m² × g / L, 95% CI: 82.4, 149.0, p=8.83E-11); and COMPASS-31 (LS mean difference of -7.5 points, 95% CI: -11.9, -3.2, p=0.0008). Improvements were observed across patient subgroups defined by age, sex, race, geographic region, TTR genotype, neuropathy severity, disease stage, and prior use of TTR tetramer stabilizers.

[0168] As shown in the table below, statistical significance was achieved for all secondary endpoints at 18 months. Separation was observed for all secondary endpoints except COMPASS-31 at 9 months. Reference ranges were as follows: NIS-W: 0 (better) to 192 (worse); R-ODS: 0 (worse) to 48 (better); COMPASS 31: 0 (better) to 100 (worse).

[0169] [Table 16]

[0170] Decreased serum TTR Serum TTR concentrations were measured in study participants. Patisiran-treated patients experienced a mean reduction in serum TTR of 77.7% (minimum -38%, maximum 95%), compared with only a 5.8% reduction in placebo (minimum -57%, maximum 43%). The effect of patisiran on serum TTR was observed across patient subgroups defined by age, sex, genotype, and prior use of TTR tetramer stabilizers. Greater TTR reductions also correlated with improvements in both the mNIS+7 score, R value 0.52 (95% CI: -0.62, -0.41), and the Norfolk QoL-DN score, R value -0.40 (95% CI: -0.51, -0.27). The data are presented graphically in Figure 7.

[0171] Greater TTR reductions correlated with improved change in mNIS+7 (R value 0.52 [95% CI: -0.62, -0.41]). Greater TTR reductions correlated with improved change in Norfolk QoL-DN (R value -0.40 [95% CI: -0.51, -0.27]). The relationship between serum TTR reduction and mNIS+7 score at 18 months is shown in the graph in Figure 8.

[0172] PND score and FAP stage Patients were assessed for polyneuropathy disorder (PND) score and familial amyloidotic polyneuropathy (FAP) stage as described herein. PND score was determined as follows: PND I: Ability to walk is maintained, paresthesia; PND II: Ability to walk is reduced, but able to walk without a cane or crutch; PND IIIA: Walking with one cane or crutch; PND IIIB: Walking with two canes or crutches; PND IV: Wheelchair-bound or bedridden. FAP stage was as follows: FAP I: Ability to walk is intact; FAP II: Assistance required for ambulation; FAP III: Wheelchair-bound or bedridden.

[0173] There was a shift in both PND scores and FAP status at 18 months, as shown in Figure 9. Treatment with patisiran resulted in either stabilization or improvement in PND scores and FAP stage.

[0174] Skin biopsy: nerve fiber density and cutaneous amyloid content Random skin biopsies were performed on study participants. Nerve fiber density and skin amyloid content were determined. Approximately 50% of placebo patients who underwent baseline skin biopsies did not have an 18-month follow-up sample due to dropout. This, along with substantial variability, limited interpretation of the results. However, the results showed that patisiran treatment attenuated the decrease in intraepidermal nerve fiber density (IENFD) compared with placebo (nominal p-value significant), and there were no significant changes in sweat gland nerve fiber density (SGNFD) or skin amyloid content with patisiran treatment compared with placebo (data not shown).

[0175] Maintenance of patisiran efficacy Participants in the 18-month double-blind study were treated with patisiran for 12 months. The results are shown graphically in Figure 10 and demonstrate maintenance of patisiran's effect on mNIS+7 out to 30 months, as well as evidence of efficacy in patients previously receiving placebo.

[0176] Study participants in the 24-month study were treated with patisiran for 12 months. The results are shown graphically in Figure 11 and demonstrate maintenance of patisiran's effect on mNIS+7 out to 36 months.

[0177] Cardiac subgroup analysis The cardiac subpopulation, e.g., patients with cardiomyopathy, is described above. Generally, these were patients with a heart wall thickness of 13 mm or greater and no evidence of hypertension or valvular heart disease. The cardiac subpopulation consisted of 36 patients (46.8%) in the placebo subpopulation and 90 patients (60.8%) in the patisiran subpopulation. The total number of patients in the cardiac subpopulation was 126, or 56% of the patients in the study.

[0178] Exploratory cardiac-related endpoints, such as cardiac markers and / or echocardiographic parameters, were determined in the overall population, and the results are shown in the table below.

[0179] [Table 17]

[0180] Patisiran-treated patients showed stabilization of NT-proBNP compared with placebo patients (patisiran: increase of 12.5 pmol / L, placebo: increase of 227.2 pmol / L). The difference in NT-proBNP between patisiran-treated and placebo patients was -214.6 pmol / L (p=0.0024).

[0181] [Table 18]

[0182] Improvements were also seen in left ventricular (LV) wall thickness and longitudinal strain in patients receiving patisiran. LV wall thickness decreased by 0.1 cm compared to baseline in patisiran-treated patients, compared with only 0.007 cm in placebo patients (p=0.0173). LV longitudinal strain also stabilized in patisiran patients, increasing by only 0.08% compared with 1.46% in placebo patients (p=0.0154).

[0183] Example 5: Overview In some embodiments, the methods described herein are used to treat hereditary transthyretin-mediated amyloidosis with cardiomyopathy and polyneuropathy (hATTR) in a human patient in need thereof by administering patisiran to the patient at a dose of 0.3 mg siRNA per kg of body weight in a formulation as set forth in Table 1, wherein patisiran is administered intravenously once every 21 days or 3 weeks. The methods result in a reduction in the modified Neuropathy Impairment Score (mNIS+7) composite neurological impairment score from the subject's baseline score prior to administration of patisiran.

[0184] In some embodiments, the methods described herein are used to treat hereditary transthyretin-mediated amyloidosis with cardiomyopathy (hATTR) in a human patient in need thereof, the methods comprising administering patisiran to the patient at a dose of 0.3 mg siRNA per kg body weight in a formulation as set forth in Table 1, wherein patisiran is administered intravenously once every 21 days or 3 weeks.

[0185] In some embodiments, the methods described herein are used to reduce the modified Neuropathy Impairment Score (mNIS+7) composite neurological impairment score in a human patient being treated for Hereditary Transthyretin-Mediated Amyloidosis with Cardiomyopathy and Polyneuropathy (hATTR), the method comprising administering patisiran to the patient at a dose of 0.3 mg siRNA per kg of body weight in a formulation as set forth in Table 1, wherein patisiran is administered intravenously once every 21 days or 3 weeks, and the method results in a reduction in the modified Neuropathy Impairment Score (mNIS+7) composite neurological impairment score from baseline as determined at 18 months, where baseline is the patient's mNIS+7 score prior to administration of patisiran.

[0186] In some embodiments, the method results in an improvement compared to baseline in one or more endpoints selected from the group consisting of Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (QOL-DN); NIS-W; Rasch-built Integrated Disability Scale (R-ODS); 10-meter walk test; modified body mass index (mBMI); and COMPASS-31 score. In some embodiments, the method results in an improvement in all endpoints. In some embodiments, the method results in an improvement in Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (QOL-DN); and COMPASS-31 score and 10-meter walk test.

[0187] In some embodiments, the patient is administered a premedication such as dexamethasone, oral paracetamol / acetaminophen, diphenhydramine, hydroxyzine, fexofenadine, cetirizine, ranitidine, famotidine, or other IV histamine H1 or H2 receptor antagonist. In some embodiments, the premedication is administered about 1 hour before patisiran. In some embodiments, the patient is further administered an oral daily dose of the USDA Recommended Daily Allowance of vitamin A. In some embodiments, the patient is also administered a tetramer stabilizer such as tafamidis or diflunisal.

[0188] In some embodiments, patients treated with the methods of the present disclosure may be Caucasian; may reside in North America; may be 65 years of age or older; may be male; may have FAP stage I; may have FAP stage II; may have a baseline mNIS+7 score of 8 to 165; may have the Val30Met TTR mutation; may have one or more TTR mutations listed in Table X; may have echocardiographic evidence of cardiac amyloid pathology; and / or may have a long-term history of TTR tetramer stabilizer use. In some embodiments, administration of at least one drug is performed by the patient. In other embodiments, administration of at least one drug is performed by a medical professional.

[0189] While the present invention has been particularly illustrated and described with reference to preferred and various alternative embodiments, those skilled in the art will recognize that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0190] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A patisiran drug formulation for use in a method for treating cardiomyopathy in a human patient with hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis), comprising: The patisiran drug formulation contains, per ml: 2 mg of patisiran or 2.1 mg of patisiran sodium, wherein the patisiran is an siRNA consisting of a sense strand comprising the nucleotide sequence 5'-GuAAccAAGAGuAuuccAudTdT-3' and an antisense strand comprising the nucleotide sequence 5'-AUGGAAuACUCUUGGUuACdTdT-3', wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, c is 2'-O-methylcytidine, u is 2'-O-methyluridine, and dT is 2'-deoxythymidine; 13.0 mg of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA); 3.3 mg 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 6.2 mg cholesterol; 1.6 mg of α-(3′-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG2000-C-DMG); 0.2 mg potassium dihydrogen phosphate (anhydrous); 8.8 mg sodium chloride USP; 2.3 mg disodium hydrogen phosphate heptahydrate USP; and water for injection Including, The patisiran drug formulation is administered intravenously once every three weeks at a dose of 0.3 mg siRNA per kg of body weight, and the method results in stabilization or improvement in serum NT-proBNP levels and / or left ventricular (LV) strain and / or LV wall thickness compared to baseline as determined prior to administration of the patisiran drug formulation.

2. 2. The patisiran drug formulation of claim 1, wherein the hATTR amyloidosis is associated with cardiomyopathy and polyneuropathy, and the method results in a reduction in modified Neuropathy Impairment Score (mNIS+7) composite neurological impairment score from baseline as determined at 18 months, wherein baseline is the patient's mNIS+7 score prior to administration of the patisiran drug formulation.

3. 3. The patisiran drug formulation of claim 1 or 2, wherein the method results in stabilization or improvement of quality of life, exercise intensity, physical disability, walking speed, nutritional status, and / or autonomic symptoms compared to a baseline as determined prior to administration of the patisiran drug formulation.

4. 1. A patisiran drug formulation for use in a method for stabilizing or improving serum NT-proBNP levels and / or left ventricular (LV) strain and / or LV wall thickness in a human patient with hereditary transthyretin-mediated amyloidosis with cardiomyopathy (hATTR amyloidosis), comprising: The patisiran drug formulation contains, per ml: 2 mg of patisiran or 2.1 mg of patisiran sodium, wherein the patisiran is an siRNA consisting of a sense strand comprising the nucleotide sequence 5'-GuAAccAAGAGuAuuccAudTdT-3' and an antisense strand comprising the nucleotide sequence 5'-AUGGAAuACUCUUGGUuACdTdT-3', wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, c is 2'-O-methylcytidine, u is 2'-O-methyluridine, and dT is 2'-deoxythymidine; 13.0 mg of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA); 3.3 mg 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 6.2 mg cholesterol; 1.6 mg of α-(3′-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG2000-C-DMG); 0.2 mg potassium dihydrogen phosphate (anhydrous); 8.8 mg sodium chloride USP; 2.3 mg disodium hydrogen phosphate heptahydrate USP; and water for injection Including, The patisiran drug formulation is administered intravenously at a dose of 0.3 mg siRNA per kg of body weight once every three weeks, and the method results in stabilization or improvement of the serum NT-proBNP concentration and / or the left ventricular (LV) strain and / or the LV wall thickness, respectively, compared to a baseline as determined prior to administration of the patisiran drug formulation.

5. 3. The patisiran drug formulation of claim 2, wherein the change from baseline in the mNIS+7 score is -6.0 points.

6. 3. The patisiran drug formulation of claim 2, wherein the reduction from baseline in mNIS+7 score is also determined at 9 months.

7. The method comprises: a. Norfolk Quality of Life Questionnaire - Diabetic Neuropathy (QOL-DN); and b. NIS-W; and c. Rasch-Built Integrated Disability Scale (R-ODS); and d. 10-meter walk test (10-MWT); and e. Modified Body Mass Index (mBMI); and f. COMPASS-31 score 7. The patisiran drug formulation of any one of claims 1 to 6, which results in an improvement compared to baseline in one or more neuropathy-related clinical endpoints selected from the group consisting of:

8. 8. The patisiran drug formulation of claim 7, wherein the method results in an improvement in all of the neuropathy-related clinical endpoints.

9. 8. The patisiran drug formulation of claim 7, wherein the method results in improvements in Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (QOL-DN) and COMPASS-31 scores and 10-meter walk test.

10. 10. The patisiran drug formulation of any one of claims 1 to 9, wherein the method results in a decrease in the patient's serum percent TTR concentration compared to baseline as determined prior to administration of the patisiran drug formulation.

11. 11. The patisiran drug formulation of any one of claims 1 to 10, wherein the method results in stabilization or reduction of the patient's FAP stage compared to baseline as determined prior to administration of the patisiran drug formulation.

12. 12. The patisiran drug formulation of any one of claims 1 to 11, wherein the method results in a stabilization or reduction in PND scores compared to a baseline as determined before administration of the patisiran drug formulation.

13. 13. The patisiran drug formulation of any one of claims 1 to 12, wherein the method results in a decrease in intraepidermal nerve fiber density in a skin biopsy compared to a baseline as determined before administration of the patisiran drug formulation.

14. 14. The patisiran drug formulation of any one of claims 1 to 13, wherein the patient is administered the patisiran drug formulation for at least 12 months, 18 months, 24 months, 30 months, or 36 months.

15. 15. The patisiran drug formulation of any one of claims 1 to 14, wherein the patient is in need of treatment for hereditary transthyretin-mediated amyloidosis with cardiomyopathy (hATTR amyloidosis), and the method results in improvement or stabilization of cardiac markers and / or echocardiographic parameters compared to baseline as determined prior to administration of the patisiran drug formulation.

16. 16. The patisiran drug formulation of claim 15, wherein the cardiac marker is serum NT-proBNP concentration and the echocardiographic parameter is left ventricular (LV) strain or LV wall thickness.

17. 17. The patisiran drug formulation of any one of claims 1 to 16, wherein the method further comprises administering to the patient the following premedications: dexamethasone, oral paracetamol / acetaminophen, diphenhydramine, and ranitidine.

18. The method further comprises premedicating with: a. IV dexamethasone 10 mg, or equivalent; and b. Oral paracetamol / acetaminophen 500 mg, or equivalent; and c. IV histamine H1 receptor antagonist (H1 blocker): diphenhydramine 50 mg, or equivalent; and d. IV histamine H2 receptor antagonist (H2 blocker): ranitidine 50 mg 17. The patisiran drug formulation of any one of claims 1 to 16, further comprising administering to the patient:

19. 19. The patisiran drug formulation of claim 17 or 18, wherein the premedication is administered about 1 hour before each patisiran drug formulation administration.

20. 20. The patisiran drug formulation of any one of claims 1 to 19, wherein the method further comprises administering to the patient a daily oral dose of the USDA Recommended Daily Allowance of Vitamin A.

21. The patisiran drug formulation of any one of claims 1 to 20, wherein the method further comprises administering a tetramer stabilizer.

22. 22. The patisiran drug formulation of claim 21, wherein the tetramer stabilizer is tafamidis or diflunisal.

23. The patient: a. Is Caucasian; and / or b. Lives in North America; and / or c. 65 years of age or older; and / or d. is male; and / or e. FAP stage I; and / or f. FAP stage II; and / or g. Have a baseline mNIS+7 score of 8 to 165; and / or h. has the Val30MetTTR mutation; and / or i. have one or more TTR mutations listed in Table X; and / or j. Echocardiographic evidence of cardiac amyloid pathology; and / or k. have a history of long-term use of TTR tetramer stabilizers; The patisiran drug formulation according to any one of claims 1 to 22.

24. The patisiran drug formulation according to any one of claims 1 to 23, wherein administration is carried out over a period of 80 minutes.

25. The patisiran drug formulation of any one of claims 1 to 24, wherein the baseline is a mean value.

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