Treatment methods for transthyretin (TTR)-mediated amyloidosis

Abstract

The present invention provides a method for reducing or suppressing the increase of the Neuropathy Impairment Score (NIS) or Revised NIS (mNIS+7) in human subjects. [Solution] A method for reducing the neuropathic impairment score (NIS) or revised NIS (mNIS+7) of a human subject with TTR-related disorder, comprising the step of administering an effective amount of a transthyretin (TTR) inhibitor composition to a human subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority thereto of U.S. Provisional Patent Application No. 62 / 044,100, filed on 29 August 2014, and U.S. Provisional Patent Application No. 62 / 150,596, filed on 24 April 2015, both of which are invoked by reference in their entirety for any purpose. [Background technology]

[0002] Transthyretin (TTR) is a tetrameric protein primarily produced in the liver. When this protein tetramer becomes unstable due to a mutation in the TTR gene, monomer misfolding occurs, leading to aggregation and the formation of TTR amyloid fibrils (ATTR). Tissue deposition results in systemic ATTR amyloidosis (Non-Patent Literature 1; Non-Patent Literature 2; Non-Patent Literature 3). More than 100 TTR mutations have been reported, presenting with a variety of disease symptoms.

[0003] TTR amyloidosis manifests in various forms. 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 in the nervous system, the disease is called familial amyloid cardiomyopathy (FAC). A third major type of TTR amyloidosis is called pia mater / CNS (central nervous system) amyloidosis.

[0004] The most common mutations associated with familial amyloid polyneuropathy (FAP) and ATTR-associated cardiomyopathy are Val30Met (Non-Patent Document 4) and Val122Ile (Non-Patent Document 5), respectively.

[0005] Current treatment options for FAP focus on stabilizing or reducing the amount of circulating amyloid-forming proteins. Orthotopic liver transplantation has been reported to reduce mutant TTR levels (Non-Patent Literature 6) and improve survival in early-stage FAP patients, although wild-type TTR may continue to be deposited (Non-Patent Literature 7; Non-Patent Literature 8; Non-Patent Literature 9; Non-Patent Literature 10; Non-Patent Literature 11; Non-Patent Literature 12, forthcoming Transplantation).

[0006] Tafamidis and diflunisal can stabilize circulating TTR tetramers and slow disease progression (Non-Patent Literature 13; Non-Patent Literature 4; Non-Patent Literature 14; Non-Patent Literature 15). However, since symptoms continue to worsen during treatment in the majority of patients, the need for novel disease-modifying treatment options for FAP is highlighted.

[0007] For a description of dsRNAs that target TTRs, see, for example, (Patent Document 1) and (Patent Document 2). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] PCT / US2009 / 061381 specification (International Publication No. 2010 / 048228 brochure) [Patent Document 2] PCT / US2010 / 055311 specification (International Publication No. 2011 / 056883 brochure) [Non-patent literature]

[0009] [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

Summary of the Invention

Means for Solving the Problem

[0010] This specification describes a method for reducing or suppressing an increase in the neuropathy impairment score (NIS) or revised NIS (mNIS+7) of a human subject by administering an effective amount of a transthyretin (TTR) inhibitory composition, where the effective amount reduces the TTR protein concentration in the serum of the human subject to less than 50 μg / ml or reduces it by at least 80%. This specification also describes a method for adjusting the dosage of a TTR inhibitory composition in response to the treatment of an increase in NIS or familial amyloid polyneuropathy (FAP) by administering a TTR inhibitory composition to a subject having an increase in NIS or FAP and determining the TTR protein level of the subject having an increase in NIS or FAP. In some embodiments, when the TTR protein level is higher than 50 μg / ml, the dosage of the TTR inhibitory composition to be administered to the subject next is increased, and when the TTR protein level is less than 50 μg / ml, the dosage of the TTR inhibitory composition to be administered to the subject next is decreased. This specification also describes a formulated version of TTR inhibitory siRNA.

Brief Description of the Drawings

[0011] [Figure 1] It is a graph showing the relationship between the progression of ΔNIS or ΔmNIS+7 and the TTR concentration. [Figure 2] It is a graph showing the relationship between the progression of ΔNIS or ΔmNIS+7 and the TTR concentration.

Modes for Carrying Out the Invention

[0012] As described in further detail below, this specification discloses a method for reducing or suppressing the increase of the neuropathic impairment score (NIS) or revised NIS (mNIS+7) in human subjects by administering an effective amount of a transthyretin (TTR) inhibitory composition such that the effective amount reduces the serum TTR protein concentration to less than 50 μg / ml or by at least 80%. In one embodiment, the TTR inhibitory composition is patisirane. Patisirane is a TTR-specific small interfering ribonucleic acid (siRNA) formulated as hepatic-directed lipid nanoparticles (LNPs) for intravenous (IV) administration.

[0013] TTR inhibitor composition The methods described herein involve the administration of a TTR inhibitory composition. The TTR inhibitory composition may be any compound that reduces the concentration of TTR protein in the serum of a human subject. Examples include, but are not limited to, RNAi, such as siRNA. Examples of siRNA include siRNA that targets the TTR gene, such as patisirin (described in further detail below) and revusiran. Examples also include antisense RNA. For examples of antisense RNA that targets the TTR gene, see U.S. Patent No. 8,697,860.

[0014] TTR inhibitory compositions inhibit the 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 can be referenced to NM_000371. The sequence of mouse TTR mRNA can be referenced to NM_013697.2, and the sequence of rat TTR mRNA can be referenced to NM_012681.1.

[0015] The terms “silence,” “inhibit expression,” “downregulate expression,” and “suppress expression” refer, insofar as they refer to the TTR gene, to the extent that they refer to the TTR gene, to the extent that they refer to the TTR gene, to the extent that they refer to the TTR gene, and this suppression is manifested by a decrease in the amount of mRNA that can be isolated from the first group of cells or cells that have been treated to transcribe the TTR gene and inhibit its expression, compared to a second group of cells or cells (control cells) that are substantially identical to the first group of cells or cells but not treated. The degree of inhibition is usually,

[0016]

number

[0017] Alternatively, the degree of inhibition may be given by a decrease in a parameter functionally related to TTR gene expression, such as the amount of TTR gene-encoded protein secreted by cells, or a predetermined phenotype, such as a decrease in the number of cells exhibiting apoptosis. In principle, TTR gene silencing can be determined by any suitable assay in any cell constitutively or genetically engineered to express the target. However, if a reference is needed to determine whether a given dsRNA inhibits TTR gene expression to a predetermined degree and is therefore included in the present invention, the assays provided in the following examples will serve as such a reference.

[0018] RNAi In some embodiments, the methods described herein utilize a TTR inhibitory composition, such as RNAi, e.g., siRNA, or e.g., dsRNA, to inhibit the expression of the TTR gene. In one embodiment, the siRNA is a dsRNA that targets the TTR gene. The dsRNA comprises an antisense strand having a complementarity region complementary to at least a portion of the mRNA formed by the expression of the TTR gene, the complementarity region being less than 30 nucleotides long and generally 19 to 24 nucleotides long. The dsRNA of the present invention may further comprise one or more single-stranded nucleotide overhangs. TTR inhibitory siRNAs are described in International Patent Application No. PCT / US2009 / 061381 (International Publication No. 2010 / 048228) and International Patent Application No. PCT / US2010 / 055311 (International Publication No. 2011 / 056883) (both of which are incorporated herein by reference in their entirety).

[0019] In one embodiment, the TTR inhibitory composition is patisirane, which is described in more detail below. In another embodiment, the TTR inhibitory composition is levusirane, an siRNA specific to trivalent GalNAc carbohydrate cluster-conjugated TTR. A comprehensive description of levusirane can be found in International Patent Application No. PCT / US2012 / 065691 and U.S. Patent Application Publication No. 20140315835 (these are incorporated by reference in their entirety).

[0020] A dsRNA contains two RNA strands that are sufficiently complementary to hybridize to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a complementarity region that is substantially complementary to the target sequence, derived from the mRNA sequence formed during TTR gene expression, and the other strand (the sense strand) contains a region complementary to the antisense strand. Therefore, when combined under favorable conditions, these two strands hybridize to form a double-stranded structure. The term “antisense strand” refers to the strand of dsRNA containing a region substantially complementary to the target sequence. As used herein, the term “complementarity region” refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., the target sequence as defined herein. If the complementarity region is not perfectly complementary to the target sequence, the mismatch is most acceptable in the terminal regions, and if present, is generally in one or more terminal regions, e.g., within the range of 6, 5, 4, 3, or 2 nucleotides from the 5' and / or 3' ends. The term "sense strand," as used herein, refers to a strand of dsRNA containing a region substantially complementary to a region of the antisense strand. Generally, the double-stranded structure is 15–80, or 15–60, or 15–30, or 25–30, or 18–25, or 19–24, or 19–21, or 19, 20, or 21 base pairs long. In one embodiment, the double-stranded structure is 19 base pairs long. In another embodiment, the double-stranded structure is 21 base pairs long.

[0021] Each strand of dsRNA is generally 15–80, or 18–60, or 15–30, or 18–25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In another embodiment, each strand is 25–30 nucleotides long. Each strand of the double helix may have the same length or different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA may be the same or different.

[0022] dsRNA may contain one or more single-stranded overhangs consisting of one or more nucleotides. In one embodiment, at least one end of the dsRNA may contain 1 to 4, generally 1 or 2, single-stranded nucleotide overhangs. In another embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotide overhangs each at 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 each at its 3' and 5' ends beyond the antisense strand.

[0023] As used herein, unless otherwise specified, the term “complementary” refers to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under specific conditions to form a double-stranded structure, as those skilled in the art will understand when used to describe a first nucleotide sequence in comparison to a second nucleotide sequence. Such conditions may be stringent conditions, for example, which 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 be applied, such as physiologically relevant conditions that may be encountered in living organisms. Those skilled in the art will be able to determine the optimal set of conditions for examining the complementarity of the two sequences, depending on the final application of the hybridized nucleotides.

[0024] This includes base pairings of the first and second nucleotide sequences over the full length of an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred to herein as “substantially complementary” to the second sequence, these two sequences may be fully complementary, or they may form one or more, but generally four, three, or two or fewer, mismatched base pairs when hybridized, while retaining the ability to hybridize under conditions most relevant to their final application. However, if two oligonucleotides are designed to form one or more single-stranded overhangs when hybridized, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing a 21-nucleotide length of one oligonucleotide and a 23-nucleotide length of another oligonucleotide (where the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide) may still be referred to herein as “fully complementary.”

[0025] The “complementary” sequences as used herein also include, or may all be formed by, base pairs formed with non-Watson-Crick base pairs and / or non-naturally modified nucleotides, insofar as the above requirements for hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogstin base pairings.

[0026] The terms “complementary,” “fully complementary,” and “substantially complementary” may be used herein with respect to matching bases between the sense strand and antisense strand of a dsRNA, or between the antisense strand of a dsRNA and the target sequence, as will be understood from the context in which they are used.

[0027] As used herein, a polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding TTR) that includes the 5'UTR, open reading frame (ORF), or 3'UTR. For example, a polynucleotide is complementary to at least a portion of TTR mRNA if its sequence is substantially complementary to the undisrupted portion of the mRNA encoding TTR.

[0028] dsRNA can be synthesized by known standard methods in the art, as will be further discussed below, for example, by using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0029] modified dsRNA In some embodiments, the dsRNAs used in the methods described herein are chemically modified to enhance their stability. The nucleic acids characterizing 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. Let al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Specific examples of dsRNA compounds useful in the present invention include RNAs that have a modified backbone or that do not have a native internucleoside bond. As defined herein, dsRNAs with a modified backbone include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification and as is sometimes referred to in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides.

[0030] Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having the usual 3'-5' linkage, their 2'-5' linkage analogues, and those with inverted polarity where pairs of adjacent nucleoside units are linked to 3'-5'~5'-3' or 2'-5'~5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0031] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above include, but are not limited to, U.S. Patent 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; and U.S. Patent Nos. 5,4 U.S. Patent Nos. 5,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is incorporated herein by reference.

[0032] Modified dsRNA backbones that do not contain phosphorus atoms internally have backbones formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include those having morpholino bonds (partially formed 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 having mixed N, O, S, and CH2 constituent parts.

[0033] Representative U.S. patents that teach the preparation of the above oligonucleotides include, without limitation, U.S. Patent 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; U.S. Patent Nos. 5, U.S. Patent Nos. 489,677; U.S. Patent Nos. 5,541,307; U.S. Patent Nos. 5,561,225; U.S. Patent Nos. 5,596,086; U.S. Patent Nos. 5,602,240; U.S. Patent Nos. 5,608,046; U.S. Patent Nos. 5,610,289; U.S. Patent Nos. 5,618,704; U.S. Patent Nos. 5,623,070; U.S. Patent Nos. 5,663,312; U.S. Patent Nos. 5,633,360; U.S. Patent Nos. 5,677,437; and U.S. Patent Nos. 5,677,439, each of which is incorporated herein by reference.

[0034] In other suitable dsRNA mimetics, both the sugar and nucleoside bonds of the nucleotide unit, i.e., the backbone, are replaced with novel groups. The base unit is maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, a dsRNA mime, that has been shown to have outstanding 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-base is retained and is directly or indirectly bound to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, each of which is incorporated herein by reference. Further teachings on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0035] Another embodiment of the present invention is an oligonucleoside having a dsRNA having a phosphorothioate backbone, and a heteroatom backbone as described in U.S. Patent No. 5,489,677, referenced above, 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- [where the natural phosphodiester backbone is represented as -OPO-CH2-], and an amide backbone as described in U.S. Patent No. 5,602,240, referenced above. A dsRNA having a morpholino backbone structure as described in U.S. Patent No. 5,034,506, referenced above, is also preferred.

[0036] Modified dsRNA 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 1 to about 10) is particularly preferred. Other preferred dsRNAs include 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, RNA cleavage group, reporter group, intercalator, group that improves the pharmacokinetic properties of dsRNA, or group that improves the pharmacodynamic properties of dsRNA, and other substituents having similar properties. Preferred modifications include 2'-methoxyethoxy (also known as 2'-O-CH2CH2OCH3, 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 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples of this specification, 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 following examples of this specification.

[0037] Other preferred modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the dsRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide, or within the 2'-5' linked dsRNA and at the 5' position of the 5' terminal nucleotide. The DsRNA may also have sugar mimes such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include, without limitation, U.S. Patent 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; and 5,576,427 Examples include U.S. Patent 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, all of which share a common owner with the present application, and each of these is incorporated herein by reference in whole.

[0038] dsRNA may also include nucleoside base (often simply referred to as "bases" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleoside bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleoside bases 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 (pseudracil), 4 -Includes 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-daazaadenine (daazaguanin), and other synthetic and natural nucleic acid bases such as 3-deazaguanine and 3-deazaadenine. Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pp. 289-302, Crooke, STand Lebleu, B., ed., CRC Press, 1993. Certain of these nucleic acid bases 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, containing 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276–278), and is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0039] Representative U.S. patents teaching the above-mentioned modified nucleic acid bases and other modified nucleic acid bases include, but are not limited to, U.S. Patent No. 3,687,808, and U.S. Patents No. 4,845,205; U.S. Patents No. 5,130,30; U.S. Patent No. 5,134,066; U.S. Patent No. 5,175,273; U.S. Patent No. 5,367,066; U.S. Patent No. 5,432,272; U.S. Patent No. 5,457,187; U.S. Patent No. 5,459,255; and U.S. Patent No. 5,484,908. The following are examples of U.S. Patent 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 is U.S. Patent No. 5,750,692.

[0040] Pachishiran In one embodiment, the TTR inhibitory composition is patisiran. Patisiran is a small interfering ribonucleic acid (siRNA) specific for TTR 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 verify homology with WT TTR as well as any previously reported TTR mutations. After delivery to the liver by LNP-mediated means, patisiran targets TTR mRNA for degradation, thereby resulting in a potent and sustained decrease in mutant and WT TTR proteins via the RNAi mechanism.

[0041] The TTR siRNA (also known as ALN-18328) consists of a sense strand and an antisense strand containing the following sequences; lowercase letters indicate 2'-O-methyl type nucleotides. [Table 1]

[0042] The manufacturing process consists of synthesizing two single-stranded oligonucleotides of the double strand by conventional solid-phase oligonucleotide synthesis. After purification, the two oligonucleotides are annealed to form a double strand.

[0043] The patisiran drug formulation is a sterile formulation of TTR siRNA ALN-18328 and lipid excipients (DLin-MC3-DMA, DSPC, cholesterol, and PEG 2000 -C-DMG) in isotonic phosphate-buffered saline.

[0044] The formulation of patisiran is shown in Table 1 below. [Table 2]

[0045] In some embodiments, the patisiran drug formulation is provided in a container, such as a glass vial, in the following amounts per vial. [Table 3]

[0046] Patisiran solution for injection contains 2 mg / mL of TTR siRNA drug substance. The patisiran drug formulation is packaged in a 10 mL glass vial with a filling volume of 5.5 mL. This container sealing system consists of a United States Pharmacopeia / European Pharmacopeia (USP / EP) Type I borosilicate glass vial, a Teflon-coated butyl rubber stopper, and an aluminum flip-off cap.

[0047] Tetramer stabilizer In some embodiments, the methods described herein include co-administration of a tetramer stabilizer with another TTR inhibitor composition.

[0048] Tetramer stabilizers are compounds that bind to the TTR protein and stabilize the TTR tetramer. Mutations that destabilize the TTR tetramer cause TTR misfiles and aggregation.

[0049] 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., "Effects of tafamidis on reduced efficacy and safety in delayed-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).

[0050] Target and Diagnosis This specification discloses methods for reducing or suppressing the increase of the Neuropathy Disorder Score (NIS) or Revised NIS (mNIS+7) in human subjects, wherein the human subjects have TTR-related disorder. In some embodiments, TTR-related disorder is one of the diseases caused by mutations in the transthyretin (TTR) gene. In some embodiments, this disorder 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 disorder is called FAP. When the lesions are mainly in the heart and not in the nervous system, the disorder is called familial amyloid cardiomyopathy (FAC). A third major type of TTR amyloidosis is called pia mater / CNS (central nervous system) amyloidosis. ATTR develops in the autonomic nervous system.

[0051] In some embodiments, human subjects with TTR-related disorders have mutant TTR genes. More than 100 TTR mutations have been reported, presenting with a variety of 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)). More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.

[0052] In some embodiments, human subjects are selected to receive treatment for any form of TTR amyloidosis if they are adults (18 years of age or older) with a confirmed diagnosis of ATTR amyloidosis by biopsy and have mild to moderate neuropathy. In further embodiments, human subjects also have one or more of the following: Karnovsky Performance Status (KPS) ≥ 60%; Body Mass Index (BMI) 17-33 kg / m²2 Sufficient hepatic and renal function (aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 2.5 × upper limit of normal range (ULN), total bilirubin within the normal range, albumin > 3 g / dL, and international normalized ratio (INR) ≤ 1.2; serum creatinine ≤ 1.5 ULN); and negative serological tests for hepatitis B virus and hepatitis C virus.

[0053] In another embodiment, human subjects are excluded from treatment if they have undergone a liver transplant; undergone a planned surgery during treatment; are HIV-positive; have received an investigational drug other than tafamidis or diflunisal within the past 30 days; have a New York Heart Association heart failure classification >2; are pregnant or lactating; have been diagnosed with or suspected of having a systemic bacterial, viral, parasitic, or fungal infection; have unstable angina, uncontrolled, clinically significant cardiac arrhythmias; or have a history of severe reactions to liposomal formulations or have been diagnosed with hypersensitivity to oligonucleotides.

[0054] Neuropathy Disorder Score (NIS) The methods disclosed herein reduce or suppress the increase of the Neuropathy Impairment Score (NIS) in human subjects by administration of a transthyretin (TTR) inhibitor composition. NIS refers to a scoring method that measures muscle weakness, sensation, and reflexes, particularly in relation to peripheral neuropathy. The NIS score assesses muscle weakness using standard muscle groups (1 = 25% decrease, 2 = 50% decrease, 3 = 75% decrease, 3.25 = able to move against gravity, 3.5 = able to move without gravity, 3.75 = muscle spasm but immobile, and 4 = paralysis), standard muscle stretch reflex groups (0 = normal, 1 = low, 2 = absent), and touch pressure, vibration, joint position and movement, and pin pain (all scored for the index finger and big toe: 0 = normal, 1 = low, 2 = absent). The assessment is adjusted for age, sex, and physical fitness.

[0055] In one embodiment, the method for reducing the NIS score results in a reduction of at least 10% of the NIS score. In other embodiments, the method results in a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or at least 50% of the NIS score. In other embodiments, the method suppresses an increase in the NIS score; for example, the method results in a 0% increase in the NIS score.

[0056] Methods for determining the NIS in human subjects are well known to those skilled in the art and can be found below.

[0057] Dyck, PJ et al., "Longitudinal assessment of diabetic polyneuropathy using a composite score in the Rochester Diabetic Neuropathy Study cohort," Neurology 1997. 49(1):pgs.229-239).

[0058] Dyck PJ. "Detection, characterization, and staging of polyneuropathy: assessed in diabetics." Muscle Nerve. 1988 Jan;11(1):21-32.

[0059] Revised Neuropathy Impairment Score (mNIS+7) In some embodiments, the methods disclosed herein reduce or suppress the increase of the revised neuropathic impairment score (mNIS+7) in human subjects by administration of a transthyretin (TTR) inhibitor composition. As is well known to those skilled in the art, mNIS+7 refers to the assessment of neurological impairment (NIS) based on clinical tests combined with electrophysiological measurements of major and minor nerve fiber function (NCS and QST) and measurements of autonomic nervous system function (postural blood pressure).

[0060] The mNIS+7 score is a revised version of the NIS+7 score (equivalent to the NIS+7 tests). NIS+7 analyzes muscle weakness and muscle stretch reflexes. Five of the seven tests include nerve conduction attributes. These attributes are peroneal complex 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 variables such as age, sex, height, and weight. The remaining two of the seven tests include vibration detection threshold and heart rate reduction with deep breathing.

[0061] The mNIS+7 score is a revision of NIS+7 to take into account the use of Smart Somatotopic Quantitative Sensation Testing, novel autonomic assessments, and the use of compound muscle action potentials of the ulnar, fibular, and tibial nerves, as well as 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):pgs.121-128).

[0062] In one embodiment, the method for reducing the mNIS+7 score results in a reduction of at least 10% of the mNIS+7 score. In other embodiments, the method results in a reduction of at least 5, 10, 15, 20, 25, 30, 40%, or at least 50% of the mNIS+7 score. In other embodiments, the method suppresses an increase in the mNIS+7 score; for example, the method results in a 0% increase in the mNIS+7 score.

[0063] Serum TTR protein concentration The method described herein comprises the step of administering an effective amount of a transthyretin (TTR) inhibitor composition to a human subject, where 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 by any method known to those skilled in the art, for example, by an antibody-based assay, such as 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 substitute, for example, 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 following examples.

[0064] In some embodiments, the serum TTR protein concentration decreases to less than 50 μg / ml, or to less than 40 μg / ml, 25 μg / ml, or 10 μg / ml. In some embodiments, the serum TTR protein concentration decreases by 80%, or by 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or 95%.

[0065] AUC AUC refers to the area under the curve of the concentration of a drug, such as a TTR inhibitor, in the plasma bloodstream over time after a certain dose of the drug has been administered to a patient. AUC is affected by the absorption rate of the composition into the patient's plasma and its removal rate from the plasma. As is 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:

[0066] Predicted AUC = (D × F) / CL

[0067] (In the formula, D is the drug concentration, F is the measured bioavailability, and CL is the predicted clearance rate). Those skilled in the art will understand that the predicted AUC value includes an error in the range of ±3 to 4 times.

[0068] In some embodiments, data for determining AUC are obtained by taking blood samples from the patient at various time intervals after drug administration. In one embodiment, the mean AUC in the patient's plasma after administration of the TTR inhibitor composition is in the range of about 9000 to about 18000.

[0069] It is understood that plasma TTR concentrations can vary considerably 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, the peak plasma concentration (C max ) or time to reach maximum plasma concentration (T max ) or the area under the curve (AUC) from time point 0 to the last measurable concentration time point. last Values ​​such as the total area under the plasma concentration-time curve (AUC) may also vary depending on the subject. Due to this variability, the amount of compound, such as a TTR inhibitor composition, required to reach a "therapeutic effective dose" may vary depending on the subject.

[0070] Pharmaceutical composition The methods described herein include the administration of a TTR inhibitory composition, for example, an siRNA that targets the TTR gene, such as patisiran. In some embodiments, the TTR inhibitory composition is a pharmaceutical composition.

[0071] As used herein, “pharmaceutical composition” includes a TTR inhibitor composition and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Preferred inert diluents include sodium carbonate and calcium, sodium phosphate and calcium, and lactose, while corn starch and alginic acid are preferred disintegrants. Binders may include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If necessary, the tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0072] 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 local, transpulmonary, by inhalation or blowing of powder or aerosol, including with a sprayer; intratracheal, intranasal, epithelial and transdermal, oral or parenteral. Parenteral administration may be intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, for example, intraparenchymal, subarachnoid or intraventricular administration.

[0073] The composition can be delivered in a manner that targets specific tissues, such as the liver (e.g., hepatocytes of the liver). The pharmaceutical composition can be delivered directly to the brain by injection. The injection can be by stereotactic injection into a specific region of the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus), or the dsRNA can be delivered to multiple regions of the central nervous system (e.g., multiple regions of the brain and / or the spinal cord). The dsRNA can also be delivered to a diffusion region of the brain (e.g., diffusion delivery to the cerebral cortex).

[0074] In one embodiment, a dsRNA targeting TTR can be delivered using a cannula or other delivery device implanted at one end in a tissue, such as the brain, such as the substantia nigra, cortex, hippocampus, striatum, corpus callosum, or globus pallidus. The cannula may be connected to a reservoir containing the dsRNA composition. Flow or delivery may 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 a location away from the tissue, such as the abdomen, and delivery is achieved by a conduit leading from the pump or reservoir to the release site. Injection of the dsRNA composition into the brain may last for several hours or several days, such as 1, 2, 3, 5, or 7 days or longer. Delivery devices to the brain are described, for example, in U.S. Patent Nos. 6,093,180 and 5,814,014.

[0075] Dosage and timing of medication Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, prior treatment, the subject's overall health and / or age, and other pre-existing conditions, may influence the dosage and timing required for effective treatment of the subject. Furthermore, treatment of the subject with a therapeutically effective dose of the composition may include a single dose or a series of doses. The effective dosage and in vivo half-life of the TTR inhibitor compositions incorporated in this invention can be estimated using conventional methods or based on in vivo studies using appropriate animal models, as described in other parts of this specification.

[0076] Generally, a suitable dose of a TTR inhibitor pharmaceutical composition may range from 0.01 to 200.0 milligrams per kilogram of body weight per day, and generally from 1 to 50 mg per kilogram of body weight per day.

[0077] For example, the TTR inhibitory composition may be siRNA, and can be administered in doses 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. In another embodiment, the dosage is 0.15 mg / kg to 0.3 mg / kg. For example, the TTR inhibitory composition can be administered in doses of 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, or 0.3 mg / kg. In one embodiment, the TTR inhibitory composition is administered in a dose of 0.3 mg / kg.

[0078] The pharmaceutical composition (e.g., patisirane) may 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 dose can be compounded for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of the TTR inhibitor composition over several days. Sustained-release formulations are well known in the art and are particularly useful for drug delivery to specific sites, as can be used with the drug of the present invention.

[0079] In one embodiment, the TTR inhibitory composition is patisirane, with a dosage of 0.3 mg / kg, administered once every 21 days. In another embodiment, the effective dose is 0.3 mg / kg, administered once every 21 days by an infusion of 1 mL / min for 15 minutes, followed by 3 mL / min for 55 minutes, for a total of 70 minutes. In yet another embodiment, the effective dose is 0.3 mg / kg, administered in two doses every 21 to 28 days, either by an infusion of 3.3 mL / min for 60 minutes, or by an infusion of 1.1 mL / min for 15 minutes, followed by 3.3 mL / min for 55 minutes, for a total of 70 minutes.

[0080] The dosage of the TTR inhibitory composition can be adjusted in response to an increase in NIS or treatment of FAP by determining the subject's TTR protein level after administering the TTR inhibitory composition. If the TTR protein level is higher than 50 μg / ml, the dose of the TTR inhibitory composition administered to the subject should be increased; if the TTR protein level is less than 50 μg / ml, the dose of the TTR inhibitory composition administered to the subject should be decreased.

[0081] TTR inhibitor compositions can be administered in combination with other known agents effective in treating pathological processes mediated by the expression of target genes. In some embodiments, patisiran is administered with a tetramer stabilizer such as tafamidis or diflunisal. In any case, the administering physician may adjust the amount and timing of patisiran and / or tetramer stabilizer administration based on results observed using standard efficacy measurements known in the art or described herein. [Examples]

[0082] The following 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. While efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), some experimental error and deviation should naturally be tolerated.

[0083] The implementation of this invention will, unless otherwise specified, utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art. Such techniques are described in detail in the literature. For example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, 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 See Ed. (Plenum Press) Vols A and B (1992).

[0084] Example 1: Safety and efficacy of patisirane against TTR amyloidosis In a Phase I clinical trial, patisiran was found to reduce patients' TTR levels over a 28-day period. The results of this trial 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. The study design and summary of results are reproduced below.

[0085] This study was a multicenter, randomized, simple-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 18–45 years were eligible to participate if they were healthy (as determined by medical history, physical examination, and 12-lead electrocardiogram), had a BMI of 18.0–31.5, sufficient liver function and blood cell counts, and were not likely to be pregnant.

[0086] Participant groups (4 people per group) were randomly assigned in a 3:1 ratio to receive either patisiran or placebo (usually normal saline) at doses of 0.01–0.5 mg / kg. Patisiran was administered intravenously over 15 and 60 minutes, respectively. In this study, to reduce the risk of infusion-related reactions, patients received similar premedication the night before and on the day of infusion. These medications included dexamethasone, acetaminophen, diphenhydramine or cetirizine, and ranitidine.

[0087] The pharmacodynamic activity of patisiran was measured as reflected in serum TTR levels using enzyme-linked immunosorbent assay (ELISA) validated for all TTRs (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 prior to patisiran administration. Adverse events were monitored from the start of drug administration until day 28. Safety monitoring also included hematological evaluation, blood chemistry analysis, and thyroid function tests.

[0088] The plasma pharmacokinetics of TTR siRNA contained in patisiran were determined using a validated ELISA-based hybridization assay. siRNA detection and quantification were performed using the ATTO-Probe-HPLC assay (limit of quantification: 1.0 ng per ml) (Tandem Laboratories, Salt Lake City UT). Pharmacokinetic estimates were determined using WinNonlin (Pharsight, Princeton NJ).

[0089] Knockdown of TTR, vitamin A, and retinol-binding protein compared to baseline levels was measured (data not shown).

[0090] result At the two lowest patisiran doses, no significant change in TTR levels was observed (compared to placebo). However, substantial TTR knockdown was observed in all participants administered doses of 0.15–0.5 mg / kg (data not shown). The TTR knockdown was rapid, potent, and sustained at all three dose levels and was highly significant compared to placebo up to day 28 (P<0.001). Given the robust response observed at 0.15 and 0.3 mg / kg and the moderate gradual improvement at 0.5 mg / kg, only one participant received the 0.5 mg / kg dose.

[0091] The response kinetics, particularly at doses of at least 0.3 mg / kg, showed little variability among participants (data not shown), with a reduction of over 50% by day 3, reaching the lowest level by almost day 10, with suppression exceeding 50% continuing on day 28, and complete recovery by day 70. The maximum TTR knockdown in participants receiving 0.15 mg / kg, 0.3 mg / kg, and 0.5 mg / kg was 85.7%, 87.6%, and 93.8%, respectively. The mean lowest scores at doses of 0.15 mg / kg and 0.3 mg / kg were 82.3% (95% confidence interval (CI), 67.7–90.3) and 86.8% (95% CI, 83.8–89.3), respectively; these lowest scores showed little variability among participants when analyzed by absolute TTR level or percentage TTR knockdown, and were highly significant compared to placebo (P<0.001) (data not shown).

[0092] The duration of suppression appeared to depend on the degree of knockdown, with mean reductions at day 28 being 56.6% (95% CI, 11.6–78.7) and 67.1% (95% CI, 45.5–80.1) for participants receiving 0.15 mg / kg and 0.3 mg / kg, respectively, and a 76.8% reduction at day 28 for one patient receiving 0.5 mg / kg. The TTR knockdown observed in humans at a dose of 0.3 mg / kg was virtually identical to that observed in non-human primates at the same dose level (data not shown). These TTR reductions with patisiran correlated with changes in retinol-binding protein and vitamin A levels (data not shown).

[0093] The use of patisiran did not result in any significant changes in hematological, hepatic, or renal measurements or thyroid function, and there were no drug-related serious adverse events or discontinuation of the investigational drug due to adverse events (data not shown).

[0094] The plasma pharmacokinetic profile of patisiran showed that the peak plasma concentration of TTR siRNA and the area under the curve up to day 1 increased approximately proportionally to the dose in the tested dose range (data not shown).

[0095] Phenyriane's specificity To further demonstrate the specificity of patisiran's effect, the TTR 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) was also measured. 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 siRNA and not a nonspecific effect of the lipid nanoparticle formulation.

[0096] Further evidence supporting the specificity and mechanism of action of patisiran's pharmacodynamic effects was obtained by detecting predicted TTR mRNA cleavage products in circulating extracellular RNA using a 5'RACE (complementary DNA end rapid amplification) assay on blood samples obtained from participants administered a dose of 0.3 mg / kg. To collect blood samples, coagulated blood samples (from subjects before administration and 24 hours after administration) were centrifuged at 1200 × g for 20 minutes, and then serum was collected. The serum was centrifuged again at 1200 × g for 10 minutes to remove suspension cell material, and then frozen. The thawed serum was mixed with lithium chloride (final concentration 1 M) and incubated at 4°C for 1 hour. The samples were spun at 120,000 × g at 4°C for 2 hours to pelletize RNA, and total RNA was isolated from the pellet by Trizol extraction (Life Technologies, Grand Island, New York, USA) and isopropanol precipitation.

[0097] To detect TTR siRNA-mediated cleavage products, isolated RNA was used for ligation-mediated RACE PCR using the GeneRacer kit (Life Technologies). The 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 a Gene Racer GR5' forward primer and a TTR-specific reverse primer (5'-gcctttcacaggaatgttttattgtctctg-3') (SEQ ID NO: 4), which were complementary to the adapter. Nested PCR was performed using a GR5' nested primer and a TTR-specific reverse nested primer (5'-ctctgcctggacttctaacatagcatatgaggtg-3') (SEQ ID NO: 5). The 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.

[0098] TTR mRNA was detected in both pre-administration samples and samples obtained 24 hours after drug administration. Consistent with the RNAi mechanism, no predicted mRNA cleavage products were present in the pre-administration samples of all three participants, but they were present in the post-administration samples (data not shown).

[0099] LC / MS / MS assays for quantifying wild-type and mutant TTR in human serum were qualified and performed by Tandem Labs. Serum samples were digested with chymotrypsin, then treated by protein precipitation extraction, and analyzed by LC / MS / MS. Chymotrypsin 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. Endogenous peptide fragments (TTRW-1 and V30M-1) in human serum samples were calculated using standard calibration curve data obtained with stable isotope-labeled peptides (TTRW-1-D8 and V30M-1-D8). Linear calibration curves were constructed using 1 / x2 weighted least squares regression analysis with standard peak area ratios (i.e., TTRW-1-D8 relative to internal standard TTRW-L1-D16 and V30M-1-D8 relative to V30M-L1-D16). Using the qualified LC / MS / MS method, a limit of quantification (LLOQ) of 5 ng / ml was achieved on a standard curve in the range of 5–2500 ng / ml.

[0100] Example 2: Multiple dose studies on the safety and efficacy of patisiran therapy for familial amyloid polyneuropathy In this Phase II clinical trial, multiple doses of patisiran were administered to patients with TTR-mediated FAP to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple escalating intravenous doses of patisiran in these patients. The data were presented at the International Symposium on Familial Amyloidotic Polyneuropathy (ISFAP) in November 2013.

[0101] Eligible patients are adults (≥18 years of age) with a confirmed biopsy diagnosis of ATTR amyloidosis and mild to moderate neuropathy; a Karnovsky Performance Status (KPS) ≥60%; and a Body Mass Index (BMI) of 17-33 kg / m². 2Inclusion was defined as having sufficient hepatic and renal function (aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 2.5 × upper limit of normal range (ULN), total bilirubin within the normal range, albumin > 3 g / dL, and international normalized ratio (INR) ≤ 1.2; serum creatinine ≤ 1.5 ULN) and being serologically negative for hepatitis B and C viruses. Patients were excluded if they had undergone a liver transplant; had undergone surgery planned during this study; were HIV positive; had received an investigational drug other than tafamidis or diflunisal within the past 30 days; had a New York Heart Association heart failure classification > 2; were pregnant or lactating; had a known or suspected systemic bacterial, viral, parasitic, or fungal infection; had unstable angina, uncontrolled clinically significant cardiac arrhythmias; or had a history of severe reactions to liposomal formulations or hypersensitivity to oligonucleotides.

[0102] This was a multicenter, international, open-label, multi-dose escalation phase II study of patisiran in FAP patients. Three patient cohorts received two doses of patisiran, administered intravenously (IV). Cohorts 1-3 received two doses of patisiran at 0.01, 0.05, and 0.15 mg / kg every four weeks (Q4W), respectively; cohorts 4 and 5 both received two doses of patisiran at 0.3 mg / kg every four weeks (Q4W); and all patients in cohorts 6-9 received two doses of patisiran at 0.3 mg / kg every three weeks (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 intravenously at a rate of 3.3 mL / min over 60 minutes, or over 70 minutes using a microdosing regimen (1.1 mL / min over 15 minutes, followed by the remaining dose at 3.3 mL / min).

[0103] For all patients, total TTR protein levels in serum were evaluated using enzyme-linked immunosorbent assay (ELISA). In addition, wild-type and mutant TTR proteins in the serum of patients with the Val30Met mutation were individually and specifically measured 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, 23 (Q3W only); 28, 29 (Q4W only); 30 (Q4W only); 31 (Q3W only); 35, 38 (Q4W only); and at follow-up days 42, 49, 56, 112, and 208.

[0104] Plasma concentration-time profiles of TTR siRNA were constructed based on blood samples collected on day 0 and at the following time points: pre-administration (within 1 hour of planned administration initiation), end of infusion (EOI), 5, 10, and 30 minutes post-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 on days 35, 91, and 187 for the Q3W regimen. For cohorts 3–9, both free and encapsulated TTR siRNA were analyzed in blood samples from day 0, EOI, and 2 hours post-infusion. Serum TTR siRNA was analyzed using validated ATTO-Probe high-performance liquid chromatography (HPLC) assays (Tandem Laboratories, Salt Lake City, Utah, USA). PK analysis was performed using non-compartmental and / or compartmental assessments of TTR siRNA plasma concentration-time data, and PK parameter estimates were determined using the validated software program WinNonlin®. Post-administration analysis of urine samples was performed to assess excretion levels of TTR siRNA and renal clearance (CL). R ) was measured.

[0105] Serum vitamin A and retinol-binding protein (RBP) levels were measured by HPLC and turbidimetry, respectively, at the same time points specified for all TTRs (Biomins Specialized Medical Pathology, Lyon, France).

[0106] The mean and variance of TTR knockdown from baseline in the PP population were calculated (baseline was defined as the mean of all pre-administration values). PD data (natural logarithm transformed vs. baseline TTR) were analyzed using Tukey's post-hoc test for individual pairwise comparisons (between dose levels) with analysis of variance (ANOVA) and analysis of covariance (ANCOVA). The lowest TTR level was defined as the lowest level for each patient during the 28-day period after each dose administration (21-day period for the Q3W group) (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 levels and V30MTTR levels were examined by linear regression. The dose-proportionality of the patisirane component in PK parameters was determined using power model analysis. AEs were coded using the coding system of the Medical Dictionary for Regulatory Activities (MedDRA), version 15.0, and the 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 later. 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 Q4W, and 0.3 mg / kg Q3W groups, respectively.

[0107] Compared to the 0.01 mg / kg dose cohort, a significant decrease in TTR (p<0.001, post-hoc test after ANCOVA) was observed in the 0.3 mg / kg Q4W and Q3W cohorts after the first and second administrations of patisiran (data not shown). In patients with the Val30Met mutation, nearly the same degree of knockdown was observed for wild-type and mutant TTR (data not shown). Serum TTR knockdown levels were RBP(r 2 =0.89, p<10 -15 ) and vitamin A (r 2 =0.90, p<10 -15 This correlated strongly with a decrease in the circulation level of (data not shown).

[0108] Patients who took tafamidis or diflunisal had significantly increased baseline serum TTR levels compared to patients who did not take stabilizing therapy (p<0.001 ANOVA) (data not shown), but patisiran administration resulted in similar levels of TTR knockdown in both patient groups (data not shown).

[0109] Pharmacokinetics: The mean concentration of patisiran TTR siRNA decreased after EOI (data not shown), and there was no siRNA accumulation after the second dose on days 21 / 28. The stability of the circulating LNP preparation was demonstrated by the measurement of inclusion vs. uninclusion TTR siRNA concentrations after each dose. For both the first and second doses, the mean values ​​of peak plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC0-last) from 0 to the last measurable time point increased proportionally with the dose within the tested dose range. Cmax and AUC0-last after dose 1 and dose 2 were similar, and there was no accumulation. The median terminal half-life of patisiran on days 0 and 21 / 28 was 39-59 hours for doses >0.01 mg / kg, and there was relatively little change when comparing dose 1 and dose 2 for each dose cohort.

[0110] These Phase II data demonstrate that treatment of FAP patients with patisiran resulted in robust, dose-dependent, and statistically significant knockdown of serum TTR protein levels. Two consecutive doses of patisiran 0.3 mg / kg administered every 3-4 weeks resulted in a sustained mean TTR reduction of over 80%, with a maximum knockdown of 96% in the Q3W group. These knockdown rates are consistent with those observed in a single-dose escalation placebo-controlled Phase I trial of patisiran (Coelho et al. 2013a). Evidence from other systemic amyloid diseases indicates that clinical improvement or stabilization can be achieved by reducing disease-causing proteins by as little as 50% (Lachmann et al. 2003; Lachmann et al. 2007). The degree of TTR knockdown by patisiran was not affected by patients taking tafamidis or diflunisal, suggesting that these TTR stabilizers do not interfere with the pharmacological activity of patisiran. In patients with the Val30Met mutation, patisiran suppressed the production of both mutant and wild-type TTR; the latter remains amyloid-forming in patients with delayed-onset FAP after liver transplantation (Yazaki et al, 2003; Liepnieks et al, 2010).

[0111] Example 3: Reduction of neurological impairment measured by NIS and mNIS+7 by administration of patisiran. An open-label, expanded study was and will be conducted in FAP patients using the protocol described in Example 2. Administration of patisiran reduced both NIS and mNIS+7.

[0112] FAP patients who had previously received the drug in a Phase 2 trial were eligible and carried over to the Phase 2 OLE study. Clinical endpoints were assessed every 6 months, and the drug was administered at a dose of 0.30 mg / kg every 3 weeks for up to 2 years. The objectives of this study included the effect on neurological impairment (mNIS+7 and NIS), quality of life, mBMI, disability, motor function, grip strength, autonomic symptoms, nerve fiber density on skin biopsies, cardiac lesions (in the cardiac subgroup), and serum TTR levels.

[0113] The demographic characteristics of the patients are as follows: [Table 4]

[0114] The baseline characteristics included the following: [Table 5]

[0115] As shown in the table below, patisiran administration resulted in a decrease in serum TTR levels. Patisiran achieved a sustained serum TTR decrease of approximately 80%, with a further decrease of up to 88% between administrations. [Table 6]

[0116] As shown in the table below, patisiran administration resulted in a change of mNIS+7 when measured at 6 and 12 months. [Table 7]

[0117] As shown in the table below, patisiran administration resulted in changes in NIS at 6 and 12 months. [Table 8]

[0118] As shown in Figures 1 and 2, the relationship between the progression of ΔNIS or ΔmNIS+7 and TTR concentration was investigated by linear regression. TTR and mean pre-treatment trough levels [TTR] correlated with the change in mNIS+7 at 6 months.

[0119] NIS and mNIS+7 were measured at 0, 6, and 12 months. ΔNIS or ΔmNIS+7 at 0–6 months and 0–12 months were used as response variables. Predictor variables included two different TTR concentration measurements: area under the TTR protein concentration curve ("AUC"), and mean percentage knockdown compared to baseline at days 84 and 168 (for 0–6 month comparisons) and days 84, 168, 273, and 357 (for 0–12 month comparisons).

[0120] For all TTR measurements, "baseline" was defined as the mean of all pre-administration values. TTR AUC was calculated using the trapezoidal plotting method, starting from raw TTR concentration (μg / mL) and baseline values ​​(inserted on day 0) and extending to day 182 (for comparisons between 0 and 6 months) or day 357 (for comparisons between 0 and 12 months). Percentage knockdown relative to baseline was calculated at each planned time point. Linear regression was performed, and p-values ​​relevant to testing the null hypothesis that there is no association between the predictor and response variables were reported.

[0121] At 12 months, the mean mNIS+7 and NIS change were -2.5 and 0.4 points, respectively, which is comparable to the rapid increase in estimated mNIS+7 and NIS at 12 months (e.g., an increase of 10–18 points) from previous FAP studies in patient populations with similar baseline NIS. The favorable effect of patisiran on the progression of neuropathic impairment scores correlated with the degree of TTR reduction. This demonstrates that the reduction in serum TTR loading by patisiran leads to clinical benefit in FAP patients.

[0122] While the present invention is specifically illustrated and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various modifications of form and detail can be made thereto without departing from the spirit and scope of the invention.

[0123] All references, granted patents, and patent applications cited herein are incorporated by reference in their entirety for any purpose.

Claims

1. A method for reducing the neuropathic impairment score (NIS) or revised NIS (mNIS+7) in a human subject having TTR-related disorder, or for suppressing an increase in NIS or mNIS+7, the method comprising the step of administering an effective amount of a transthyretin (TTR) inhibitor composition to the human subject, wherein the effective amount reduces the serum TTR protein concentration to less than 50 μg / ml or reduces it by at least 80%.

2. A method for adjusting the dosage of a TTR inhibitor composition in accordance with the suppression of the decrease or increase of NIS or mNIS+7 in a human subject with TTR-related disorder, The steps include administering the TTR inhibitory composition to the subject having an increase in NIS or mNIS+7, The steps include determining the serum TTR protein concentration of the subject, If the serum TTR protein concentration is higher than 50 μg / ml, the next step is to increase the amount of the TTR inhibitory composition administered to the subject. A method that includes this.

3. The method according to claim 1 or 2, wherein the TTR inhibitory composition is siRNA, an antisense molecule, siRNA or an antisense molecule that targets the TTR gene, patisirane, or levusirane.

4. The method according to any one of claims 1 to 3, wherein the TTR-related disorder is familial amyloid polyneuropathy (FAP), FAP with a confirmed TTR mutation, familial amyloid cardiomyopathy (FAC), transthyretin-mediated amyloidosis (ATTR), or symptomatic polyneuropathy.

5. The method according to any one of claims 1 to 4, which results in a reduction of at least 10% of NIS or mNIS+7.

6. The method according to any one of claims 1 to 5, which results in suppressing the increase of NIS or mNIS+7.

7. The method according to any one of claims 1 to 6, wherein the serum TTR protein concentration is reduced to less than 40 μg / ml, 25 μg / ml, or 10 μg / ml.

8. The method according to any one of claims 1 to 7, wherein the serum TTR protein concentration is reduced by at least 85%, 90%, or 95%.

9. The method according to any one of claims 1 to 8, wherein the TTR inhibitory composition is patisirane, and the effective amount is 0.01 to 0.5 mg / kg, 0.15 to 0.3 mg / kg, or 0.3 mg / kg.

10. The method according to any one of claims 1 to 9, wherein the TTR inhibitory composition is patisirane, and the effective dose is 0.3 mg / kg administered once every 21 days by an infusion of 1 mL / min for 15 minutes, followed by 3 mL / min for 55 minutes, for a total of 70 minutes.

11. The method according to any one of claims 1, wherein the TTR inhibitory composition is patisirane, the effective dose is 0.3 mg / kg, and it is administered in two doses every 21 to 28 days by infusion of 3.3 mL / min over 60 minutes, or by infusion of 1.1 mL / min for 15 minutes followed by 3.3 mL / min for 55 minutes over 70 minutes.

12. The method according to any one of claims 1 to 11, wherein the TTR inhibitory composition is levushiran and the effective amount is 500 mg.

13. The method according to any one of claims 1 to 12, wherein the TTR inhibitory composition is administered intravenously.

14. The method according to any one of claims 1 to 13, wherein the TTR inhibitory composition is co-administered with a tetramer stabilizer, optionally tafamidis or diflunisal.

15. The method according to any one of claims 1 to 14, wherein the serum TTR protein concentration is determined by an immunochemistry-based assay, enzyme-linked immunosorbent assay (ELISA), an assay for determining vitamin A concentration, an assay for determining RBP concentration, or an assay for determining TTR mRNA concentration.

16. A method for reducing the neuropathic impairment score (NIS) or revised NIS (mNIS+7) of a human subject having TTR-related impairment, comprising the step of administering 0.3 mg / kg of patisiran as described in Table 1 to the human subject, wherein the patisiran is administered intravenously and the effective dose reduces the serum TTR protein concentration of the human subject to less than 50 μg / ml or by at least 80%, and the TTR concentration is determined by enzyme-linked immunosorbent assay (ELISA).

17. A method for reducing or suppressing the increase of a neuropathic impairment score (NIS) or revised NIS (mNIS+7) in a human subject having TTR-related disorder, comprising the steps of: intravenously administering 0.3 mg / kg of patisiran as described in Table 1 to the human subject once every 21 days; and reducing the serum TTR protein concentration to less than 50 μg / ml or by at least 80% as determined by ELISA.

18. A method for reducing or suppressing the increase of a neuropathic impairment score (NIS) or revised NIS (mNIS+7) in a human subject having a TTR-related disorder, the method comprising the step of administering an effective amount of a transthyretin (TTR) inhibitor composition to the human subject, wherein the effective amount results in a mean serum TTR AUC of 9,000 to 18,000 six months after treatment.

19. A composition containing patisirane as described in Table 1.

20. A composition comprising patisirane as described in Table 2.

21. A method for inhibiting transthyretin (TTR) expression in a human subject, comprising the step of administering an effective amount of the composition according to claim 19 or 20 to the human subject.

22. The method according to claim 21, wherein the composition is administered in a dose of 0.01 to 0.5 mg / kg.

23. The method according to claim 21, wherein the composition is administered in a dose of 0.15 to 0.3 mg / kg.

24. The method according to claim 21, wherein the composition is administered at a dose of 0.3 mg / kg.

25. The method according to claim 21, wherein the composition is administered once every 21 days at a dose of 0.3 mg / kg.

26. The method according to claim 21, wherein the composition is administered once every 21 days at a dose of 0.3 mg / kg by infusion of 1 mL / min for 15 minutes, followed by 3 mL / min for 55 minutes, for a total of 70 minutes.

27. The method according to claim 21, wherein the treatment results in a mean transthyretin level reduction in the range of 82.3 to 86.8% when determined by ELISA for serum transthyretin.

28. The method according to claim 21, wherein the human subject has a confirmed diagnosis by biopsy of transthyretin amyloidosis with mild to moderate neuropathy.

29. A method for treating transthyretin-mediated amyloidosis (ATTR) in a human subject, comprising the step of administering an effective amount of the composition according to claim 19 or 20 to the subject.

30. The method according to claim 29, wherein the composition is administered in a dose of 0.01 to 0.5 mg / kg.

31. The method according to claim 29, wherein the composition is administered in a dose of 0.15 to 0.3 mg / kg.

32. The method according to claim 29, wherein the composition is administered at a dose of 0.3 mg / kg.

33. The method according to claim 29, wherein the composition is administered once every 21 days at a dose of 0.3 mg / kg.

34. The method according to claim 29, wherein the composition is administered once every 21 days at a dose of 0.3 mg / kg by infusion of 1 mL / min for 15 minutes, followed by 3 mL / min for 55 minutes, for a total of 70 minutes.

35. The method according to claim 29, wherein the treatment results in a reduction of the neuropathic impairment score (NIS) of the subject.

36. The method according to claim 29, wherein the human subject has symptomatic polyneuropathy.

37. The method according to claim 29, wherein the human subject has been diagnosed with FAP having a confirmed TTR mutation.

38. A kit comprising the composition according to claim 19 or 20 and instructions for use.

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