REFERENCE TO SEQUENCE LISTING

[0001] This application incorporates by reference a Computer Readable Form of a Sequence Listing in ASCII text format submitted with this application, entitled 055920- 561001WO_SLST26.xml, was created on September 14, 2023, and is 38,493 bytes in size.

TECHNICAL FIELD

[0002] The present disclosure generally relates to a method of treating advanced hepatic fibrosis with a targeted lipid nanoparticle composition to inhibit production of heat shock protein 47 (HSP47).

BACKGROUND

[0003] Hepatic fibrosis can result from a vanety of etiologies including chronic HCV infection and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH). Patients with hepatic fibrosis have a higher risk of adverse clinical outcomes over time, such as cirrhosis, hepatic decompensation, and HCC. Additionally, mortality risk increases as fibrosis progresses such that patients with advanced fibrosis and cirrhosis have the highest all-cause and liver- related mortality risk. In recent years, direct-acting antiviral (DAA) regimens have become standard treatment for chronic HCV infection and they enable sustained virologic response (SVR) rates above 95% w ith 8-12-week regimens, even in patients with advanced fibrosis. How ever, despite HCV-SVR, adults with cirrhosis have an increased HCC risk and regular surveillance testing is advised.

[0004] Two key factors in fibrosis development, collagen dysregulation and imbalanced extracellular matrix (ECM) remodeling, are therapeutic targets that have been evaluated in preclinical and clinical studies. Collagen is a vital component of ECM and HSP47 is a molecular chaperone that binds triple-helical procollagen in the endoplasmic reticulum to prevent inappropriate collagen unfolding or aggregation. In preclinical models, HSP47 mRNA and protein expression are markedly increased in fibrotic tissue, suggesting a role for HSP47 in collagen accumulation and fibrosis progression.

[0005] Formulation (I) is a retinoid-conjugated lipid nanoparticle (LNP) containing HSP47 siRNA. The LNP contains a critical lipid targeting agent, DPD, which facilitates direction of the LNP to HSCs. There, retinoid-containing moieties on the LNP bind retinol binding protein (RBP) expressed on HSCs; in vitro studies that show strong binding of the DPD-containing LNP to human RBP support the mechanism of DPD-mediated LNP uptake by liver HSCs. Upon intracellular release, HSP47 siRNA recruits the RNA-induced silencing complex (RISC), which degrades HSP47 mRNA and prevents HSP47 protein translation. Thus, Formulation (I)- mediated HSP47 mRNA inhibition may reduce or reverse liver fibrosis by disrupting collagen formation and promoting HSC apoptosis, respectively.

[0006] Presented herein are results related to a method of treating advanced hepatic fibrosis with Formulation (I).

SUMMARY

[0007] Described herein is a method of treating advanced hepatic fibrosis with a targeted lipid nanoparticle composition to inhibit production of heat shock protein 47 (HSP47).

[0008] Accordingly, in a first aspect, the invention provides a method of treating advanced hepatic fibrosis in a subject with HCV, the method comprising administering Formulation (I) in an amount of 45 mg per dose per week or 90 mg per dose per week; wherein the advanced fibrosis is characterized by having a fibrosis stage of F3 or higher in the METAVIR fibrosis score, and wherein the subject has no detectable HCV RNA for at least 12 weeks.

[0009] In an embodiment, Formulation (I) comprises a small interfering RNA (siRNA) molecule and a lipid nanoparticle conjugated to a retinoid molecule, wherein the siRNA molecule silences heat shock protein 47 (HSP47) mRNA.

[0010] In an embodiment, Formulation (I) is administered for at least 12 weeks.

[0011] In an embodiment, Formulation (I) is administered intravenously.

[0012] In an embodiment, the method of treating advanced hepatic fibrosis in a subject with HCV comprises a reduction of at least one or more fibrosis stage in METAVIR fibrosis score determined by comparing a first biological sample comprising a liver biopsy from the subject with advanced hepatic fibrosis and a second biological sample comprising a liver biopsy after treatment from the same subject.

[0013] In an embodiment, the method of treating advanced hepatic fibrosis in a subject with HCV comprises a reduction of at least two or more stages in Ishak fibrosis score determined by comparing a first biological sample comprising a liver biopsy from the subject with advanced hepatic fibrosis and a second biological sample comprising a liver biopsy after treatment from the same subject.

[0014] In an embodiment, the method of treating advanced hepatic fibrosis in a subject with HCV comprises reducing HSP47 mRNA expression and HSP47 protein translation in hepatic stellate cells. [0015] In an embodiment, Formulation (I) is administered in an amount of 90 mg per dose per week for at least 12 weeks and comprises a reduction of at least one or more fibrosis stage in METAVIR fibrosis score by comparing a first biological sample comprising a liver biopsy from the subject with advanced hepatic fibrosis and a second biological sample comprising a liver biopsy after at least 12 weeks.

[0016] In an embodiment, the method of treating advanced hepatic fibrosis in a subject with HCV comprises reducing HSP47 mRNA expression in hepatic cells by at least 40%.

[0017] In an embodiment, the method of treating advanced hepatic fibrosis in a subject with HCV comprises reducing HSP47 protein translation in hepatic stellate cells by at least 10%. [0018] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0020] Figure 1 illustrates the NCT03420768 study design. Eligible patients were randomized 1: 1 :2 to receive placebo or Formulation (I) (45 mg or 90 mg) i.v. QW for 12 weeks. For screening, liver biopsies were performed within 8 weeks prior to or during the screening period. QW denotes once weekly.

[0021] Figures 2A-2B illustrate the METAVIR stage change from baseline to Week 12. Patients with >1 METAVIR stage improvement at Week 12 stratified by (2A) arm or (2B) baseline fibrosis stage, a = Odds-ratio and difference in response rates are shown for each arm compared with placebo; b = One F2 patient had an inadequate Week 12 biopsy specimen and was considered to have no fibrosis improvement. OR denotes odds-ratio; QW denotes once weekly.

[0022] Figure 3 illustrates the Ishak score change from baseline to Week 12. Patients with >2 Ishak score improvement at Week 12 stratified by study arm and baseline fibrosis stage, a = One F2 patient had an inadequate Week 12 biopsy specimen and was excluded from the analysis. QW denotes once weekly.

[0023] Figures 4A-4C illustrate the liver HSP47 mRNA and protein change from baseline to Week 12. (4A) Percentage of patients per arm with reduced HSP47 mRNA at Week 12. (4B,4C) Median percentage change in HSP47 mRNA (4B) and HSP47 protein (4C). a = Missing data n=l;. b = Missing data n=2. HSP47 denotes heat shock protein 47; PBO denotes placebo; QW denotes once weekly.

[0024] Figure 5 illustrates the geometric mean Formulation (I) plasma concentration. PK samples were collected at pre-, mid-, and post-infusion at Weeks 0, 1, 4, 6, and 11. Trough PK samples were collected pre-infusion at Weeks 2, 5, 7, and 8. Samples during a dosing interval were collected at 72 hours post-infusion following the doses at Weeks 0 and 6. Additional postinfusion PK samples were collected at 1 and 3 weeks following the Week 11 dose. PK denotes pharmacokinetics; QW denotes once weekly.

[0025] Figure 6 illustrates the liver pathology by METAVIR stage. The figure provides representative liver histology from patients staged with METAVIR F1-F4 fibrosis using trichrome staining, which stains type I collagen normally present in portal tracts and vessel walls and shows the presence and distribution of reactive fibrosis due to liver injury. Collagen appears “blue” and hepatocytes and other structures appear “red.”

[0026] Figures 7A-7B illustrate baseline median type III collagen serum biomarker concentrations by METAVIR stage. The figures represent median concentrations of (7A) PROCS and (7B) PIIINP across METAVIR stages at baseline. Bars represent minimum and maximum values and white lines inside the box plot median levels. PIIINP denotes N-terminal propeptide of type III procollagen; PRO-C3 denotes N-terminal propeptide of type III collagen. [0027] Figures 8A-8B illustrate baseline median serum fibroly sis biomarker concentrations by METAVIR stage. The figures represent median concentrations of (8A) C3M and (8B) TIMP-1 across METAVIR stages at baseline. Bars represent minimum and maximum values and white lines inside the box plot median levels. C3M denotes matrix metalloproteinase-9 degraded;

TIMP-1 denotes tissue inhibitor of metalloproteinases 1.

[0028] Figures 9A-9B illustrate categorical analysis of patients with METAVIR and qFIB improvement. The figures represent the percentage of patients who had (9 A) METAVIR score improvement or (9B) METAVIR score and qFIB improvement from baseline to Week 12. a = 1 patient (90 mg Formulation (I) QW; F2) had an inadequate Week 12 biopsy sample and was considered to have no fibrosis improvement. PBO denotes placebo; qFIB denotes quantitative assessment of liver fibrosis; QW denotes once weekly.

[0029] Figures 10A-10B illustrate categorical analysis of patients with Ishak and qFIB improvement. The figures represent the percentage of patients who had (10A) Ishak score improvement or (10B) Ishak score and qFIB improvement from baseline to Week 12. ^a Baseline Ishak scores shown are those assigned by the central pathologist. qFIB, quantitative assessment of liver fibrosis; QW, once weekly.

DETAILED DESCRIPTION

[0030] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. Also, as used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0031] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated compounds, which allows the presence of only the named compounds, along with any pharmaceutically carriers, and excludes other compounds.

[0032] Abbreviations as used herein, are defined as follows:

ADA anti-drug antibody

AE adverse event

AFP alpha-fetoprotein

ALT alanine aminotransferase

AST aspartate aminotransferase

BMD bone mineral density

C3M matrix metalloproteinase-derived collagen type Ill-specific neo-epitope peptide

CPA collagen proportionate area

DAA direct-acting antiviral

DILI drug-induced liver injury

DXA dual-energy X-ray absorptiometry

ECM extracellular matrix

HSC hepatic stellate cell HSP47 heat shock protein 47

INR international normalized ratio

IRR infusion-related reaction

LNP lipid nanoparticle

MELD model end stage liver disease

NAFLD nonalcoholic fatty liver disease

NASH nonalcoholic steatohepatitis

PD pharmacodynamics

PIIINP N-terminal propeptide of type III procollagen

PK pharmacokinetics

PRO-C3 N-terminal propeptide of type III collagen qFIB quantitative assessment of liver fibrosis

QW once weekly

RBP retinol -binding protein

RISC RNA-induced silencing complex

SAE serious adverse event siRNA small interfering ribonucleic acid

SVR sustained viral response

TEAE treatment-emergent adverse event

TIMP-1 1 issue inhibitor of matrix metalloproteinases 1

ULN upper limit of normal

[0033] As used herein “Formulation (I)” refers to a retinoid-conjugated lipid nanoparticle containing HSP47 siRNA. In an embodiment, the retinoid-conjugated lipid nanoparticle containing HSP47 siRNA relates to a double-stranded nucleic acid molecule comprising a sense strand and an anti-sense strand, a drug earner comprising a mixture of a lipid vesicle and a retinoid or retinoid conjugate.

[0034] In the present application, any number of retinoids or retinoid conjugates may be used if it demonstrates that it can be actively accumulated by hepatic stellate cells (the target cell). In an embodiment, the drug carrier may comprise a substance that improves incorporation into HSCs such as a retinol-binding protein.

[0035] As used herein, the drug carrier of the present application comprises a lipid vesicle. [0036] As used herein, the term “short interfering ribonucleic acid,” “silencing RNA” or “siRNA” refers to a class of double-stranded RNA that interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. As contemplated herein, the siRNA refers to the annealed oligonucleotide that is double-stranded.

[0037] As used herein, the term “sense region” refers to a nucleotide sequence of a siNA molecule complementary (partially or fully) to an antisense region of the siNA molecule. The sense strand of a siNA molecule can include a nucleic acid sequence having homology with a target nucleic acid sequence. The sense strand is complementary to the antisense strand of DNA, or template strand, which does not carry the translatable code in the 5' to 3' direction. As used herein, “sense strand” refers to nucleic acid molecule that includes a sense region and may also include additional nucleotides. Nucleotide positions of the sense strand are herein numbered 5' to 3' direction.

[0038] As used herein, the term “antisense region” refers to a nucleotide sequence of a siNA molecule complementary (partially or fully) to a target nucleic acid sequence. The antisense strand of a siNA molecule can optionally include a nucleic acid sequence complementary to a sense region of the siNA molecule. The antisense strand does not carry the translatable code in the 5' to 3' direction.

EXAMPLES

[0039] The following examples are offered for purposes of illustration, and are not intended to limit the scope of the claims provided herein. All literature citations in these examples and throughout this specification are incorporated herein by references for all legal purposes to be served thereby.

[0040] Composition

Table 1. Formulation for Injection

* citric acid, trisodium dehydrate, and alcohol are removed during the manufacturing process ** water for injection is removed during the lyophilization process

*** a dilute solution of sodium hydroxide may be used for buffer pH adjustment

[0041] In embodiments, Formulation (I) comprises an siRNA that may be, but is not limited to, siRNA 1, siRNA 2, siRNA 3, or siRNA 4. In embodiments, siRNA 1 is an antisense strand having SEQ. ID NO: 1 and a sense strand having SEQ. ID NO: 2. In embodiments, siRNA 2 is an antisense strand having SEQ. ID NO: 3 and a sense strand having SEQ. ID NO: 4. In embodiments, siRNA 3 is an antisense strand having SEQ. ID NO: 5 and a sense strand having SEQ. ID NO: 6. In embodiments, siRNA 4 is an antisense strand having SEQ. ID NO: 7 and a sense strand having SEQ. ID NO: 8.

[0042] In embodiments, a cationic lipid contemplated by the formulation is HEDC. In embodiments, HEDC has a formula according to:

[0043] In embodiments, a cationic lipid contemplated by the formulation is SI 04. In embodiments, SI 04 has a formula according to:

[0044] In embodiments, a zwitterionic phospholipid contemplated by the formulation is 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (“DOPE.”) In embodiments, DOPE has a formula according to:

[0045] In embodiments, a lipid contemplated by the formulation is 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy[polyethyleneglycol) -2000] (ammonium salt) (PEG-DMPE). In embodiments, PEG-DMPE has a formula according to:

[0046] In embodiments, a vitamin A targeting group contemplated herein a formula according

[0048] NCT03420768 was a Phase 2, randomized, double-blind, placebo-controlled, parallel- group study in adults with advanced hepatic fibrosis due to HCV who had achieved SVR (HCV- SVR; Fig. 1). Eligible patients had documented SVR (i.e., no detectable serum HCV RNA at least 12 weeks after completing antiviral therapy) for at least 1 year and advanced hepatic fibrosis (METAVIR F3 or F4) according to local pathology interpretation of liver tissue obtained within 8 weeks of screening. Exclusion criteria included Child-Pugh score >6, Model End Stage Liver Disease (MELD) score >12, or evidence of HCC based on serum concentration of alpha-fetoprotein (AFP) or imaging at screening. Additionally, patients were ineligible if they had a history of hepatic decompensation or transplantation, albumin <3.5 g/dL, international normalized ratio (INR) >upper limit of normal (ULN), or total bilirubin >ULN. The minimum allowable platelet count was initially 140,000/pL; however, to account for the frequency of thrombocytopenia in patients with cirrhosis, the protocol was amended toward the end of enrollment to lower the platelet count threshold to <100,000/pL. [0049] Randomization using interactive response technology followed a computer-generated block scheme that stratified patients by METAVIR fibrosis stage (F3 vs F4). Assignments were made 1: 1 :2 to receive placebo (0.3-0.6 mL INTRALIPID® in 100 mL 5% dextrose solution) or Formulation (I) (45 mg or 90 mg diluted in 0.9% saline to -100 mL) infused i.v. QW for 12 weeks (Weeks 0-11; Fig. 1). Pretreatment with antihistamines or hydrocortisone was allowed at the investigator’s discretion to reduce potential for infusion-related reactions (IRRs). Protocol amendments were made to evaluate and determine the optimal Formulation (I) infusion rate. Liver biopsies were performed at Week 12 and safety was monitored through Week 36. [0050] The primary endpoint was the proportion of patients with >1 METAVIR stage improvement in hepatic fibrosis at Week 12 as determined by histopathological interpretation of liver tissue obtained from biopsy. Key secondary endpoints were the proportion of patients with >2 Ishak stage improvement, Formulation (I) plasma concentrations, and safety at Week 36. Exploratory endpoints included changes from baseline to Week 12 in the following parameters: HSP47 mRNA and HSP47 protein in liver, fibrosis assessed by dual-photon microscopy (qFibrosis [qFIB]), and serum biomarkers of collagen formation (N-terminal propeptide of type III collagen [PRO-C3]), collagen degradation (matrix metalloproteinase-derived collagen type Ill-specific neo-epitope peptide [C3M]) and tissue inhibitor of matrix metalloproteinase [TIMP- 1]), and collagen turnover (N-terminal propeptide of type III procollagen [PIIINP]).

[0051] Baseline liver histology was analyzed to identify eligible study patients (i.e., those with METAVIR F3 and F4 fibrosis). Per the study protocol, the baseline biopsy specimens were subsequently reassessed to enable comparison with Week 12 biopsy specimens. Morphometric quantification of hepatic collagen in each biopsy specimen was performed on 4 pm-thick sections stained with picrosirius red; digital images of the sections were acquired using an Aperio ScanScope XT (Leica Biosystems, Vista, CA, USA) at 20x magnification. Collagen proportionate area (CPA) was defined as the percentage of the tissue section that was positive for red staining as determined by the Aperio Positive Pixel Count Algonthm. qFIB, a stain-free, fully automated, quantitative tissue imaging tool, was also used to assess fibrosis changes between baseline and Week 12 using 4 pm-thick tissue sections and second harmonic generation/two photon excitation fluorescence microscopy. Baseline blinded serum samples were analyzed for circulating concentrations of collagen and fibrosis biomarkers; data was stratified by METAVIR fibrosis stage. PRO-C3 and C3M were measured with competitive ELISAs developed and performed by Nordic Bioscience (Herlev, Denmark). PIIINP was measured using an ELISA developed by CisBio (Codolet, France) and performed by Nordic Bioscience (Herlev, Denmark), and TIMP-1 was measured using Luminex technology (Myriad RBM, Austin, TX, USA).

[0052] Magnetic resonance elastography (MRE) and FIBROSCAN® were used to assess liver stiffness at baseline as surrogate markers of liver fibrosis. All MRE images were analyzed by the central imaging facility. HSP47 protein was measured in 4 pm liver tissue sections by immunohistochemistry and HSP47 mRNA was measured in 5-10mm ² tissue sections using the HTG EDGESEQ™ system in liver tissue collected at baseline and Week 12. Blood PK samples were collected pre-, mid-, and post-infusion at Weeks 0, 1, 4, 6, 11, and additional post-dose samples were collected 1 and 3 weeks following the Week 11 dose. siRNA PK analysis was evaluated with a validated hybridization ELISA method that used complementary capture and detection oligonucleotides.

[0053] All treatment-emergent adverse events (TEAEs) were recorded through Week 36 and classified using MedDRA (v21.1). Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) at baseline and Week 36 by the central imaging facility. Anti-drug antibody (ADA) responses to Formulation (I) were measured in plasma using a 3-tier screening, confirmatory, and titer detection method where Formulation (I) (I) served as a capture and detection reagent for ADA. For these analyses, pre-infusion samples were collected from each patient on Weeks 1, 2, 4, and 12.

[0054] The study sample size was 61 patients randomized into 3 arms. Efficacy and safety analyses included all randomized patients who received at least 1 dose of study treatment. Biomarker and PK analyses included all patients who had received at least 1 dose of study treatment with at least 1 post-treatment biomarker measurement and available concentrationtime data, respectively. For the primary efficacy endpoint, a 95% exact CI and odds-ratios were used to estimate differences observed in the Formulation (I) vs placebo arms; those with a missing Week 12 histology score were considered to have no fibrosis improvement. The secondary efficacy endpoint was analyzed similarly to the primary endpoint except that patients without a Week 12 histology score were excluded. Safety analyses included all reported TEAEs through Week 36, which were listed and summarized by system organ class, preferred term, and arm. All exploratory endpoints were summarized using descriptive statistics by arm and study day, with change from baseline or percentage change from baseline reported as appropriate. Secondary and exploratory endpoints were not adjusted for multiplicity. Statistical Analysis Software (SAS®) version 9.4 was used for statistical analyses and tabulations.

[0055] Results [0056] All 61 patients who were enrolled and randomized 1 : 1 :2 to receive placebo (n= 15), 45 mg Formulation (I) (n=18), or 90 mg Formulation (I) (n=28) completed the treatment and follow-up periods. Most patients were white (92%) and Hispanic (53%) and the mean age was 60 years (Table 2). For all patients, the median time since SVR was 38 months; the 90 mg Formulation (I) arm had a median time of 46 months since SVR compared with 31 months for placebo and 32 months for the 45 mg Formulation (I) arm.

Table 2. Baseline patient demographics and characteristics ^a Two patients had a missing SVR date. BMI, body mass index; QW, once weekly; SVR, sustained virologic response; T2DM, type-2 diabetes mellitus.

[0057] All baseline fibrosis scores of enrolled patients were METAVIR F3 or F4; however, 32/61 (52%) of the baseline tissue samples as F1-F2 (Table 3). Most discrepancies between baseline fibrosis scores related to patients staged as F3 at screening.

Table 3. Baseline fibrosis stage by central read, liver biopsy tissue characteristics, and laboratory values

METAVIR fibrosis stage, n (%)

[0058] Almost all baseline biopsy specimens were >15 mm in length (placebo: 14/15, 93%; 45 mg Formulation (I): 16/18, 89%; 90 mg Formulation (I): 24/28, 86%) with >11 portal areas (58/61; 95%) (Fig. 6). Similarly, most Week 12 biopsy specimens were >15 mm in length (placebo: 15/15, 100%; 45 mg Formulation (I): 18/18, 100%; 90 mg Formulation (I): 21/28; 75%) with >11 portal areas (59/61; 97%). One patient (90 mg arm) had an inadequate Week 12 tissue sample. For the overall study population, baseline liver histology analysis generally revealed low levels of collagen and thin septa. Across METAVIR stages, median PRO-C3 and PIIINP concentrations were not appreciably different at baseline (Fig. 7); however, median C3M and TIMP-1 values increased as fibrosis stage increased (Fig. 8). Fibrosis parameters and liver- related laboratory values were similar across arms. The overall study population had preserved hepatic synthetic capacity, as evidenced by normal albumin and INR values.

[0059] Change in METAVIR stage at Week 12. Reduction in >1 METAVIR stage from baseline to Week 12 was observed for 2/15 (13%), 3/18 (17%), and 6/28 (21%) patients in the placebo, 45 mg Formulation (I), and 90 mg Formulation (I) arms, respectively (Fig. 2). The odds-ratios for response compared with placebo were 1.30 (95% CI: 0.13, 17.70; 45 mg arm) and 1.77 (95% CI: 0.26, 20.23; 90 mg arm). When stratified by baseline METAVIR score, 4/8 (50%) F4 patients treated with either dose of Formulation (I) had fibrosis improvement. Of the patients who achieved >1 METAVIR stage improvement at Week 12, 9/11 (82%) also showed an improvement in qFIB score, including 6/6 (100%) patients in the 90 mg Formulation (I) arm (Fig. 9).

[0060] Change in Ishak Score at Week 12. The secondary endpoint, a >2 Ishak score improvement from baseline to Week 12, was observed in 5 patients from the 90 mg Formulation (I) arm (Fig. 3). Most (3/5) had cirrhosis at baseline (i.e., an Ishak score of 5-6). Of patients who achieved >2 stage improvement in Ishak score, all 5 also had an improvement in fibrosis as determined by qFIB (Fig. 10).

[0061] From baseline to Week 12, liver HSP47 mRNA levels were reduced in 2/15 (13%), 8/18 (44%), 20/28 (71%) patients in the placebo, 45 mg Formulation (I), and 90 mg Formulation (I) arms, respectively (Fig. 4A). The median HSP47 mRNA percentage change (range) was 13.7% (-19.3%, 51.8%) for the placebo arm, -1.2% (—37.3%, 57.6%) for the 45 mg Formulation (I) arm, and -5.9% (-29.9%, 22.6%) for the 90 mg Formulation (I) arm (Fig. 4B). For HSP47 protein, the median percentage change from baseline to Week 12 (range) was 23.5% (-33.9%, 225.4%) for the placebo arm, -11.3% (-56.5%, 122.3%) for the 45 mg Formulation (I) arm, and -10.1% (-55.8%, 202.9%) for the 90 mg Formulation (I) arm (Fig. 4C). [0062] Pharmacokinetics. In patients who received either Formulation (I) dose, plasma concentrations increased during the infusion course and were higher at the end of the infusion compared with mid-infusion. Formulation (I) plasma concentrations decreased rapidly after the end of infusion. In general, dose-proportional plasma concentration increases were observed between the 45 mg and 90 mg Formulation (I) arms. No apparent Formulation (I) plasma accumulation occurred with weekly dosing based on similar peak and trough concentrations observed across weekly infusions (Fig. 5).

[0063] By Week 36, 37/61 (61%) patients experienced at least 1 AE, all of which were mild or moderate in intensity (Table 4). Twenty -five patients (41%) reported AEs that were considered possibly Formulation (I)-related; patients in either Formulation (I) arm had a higher frequency of Formulation (I)-related AEs compared with those who received placebo. The most common Formulation (I)-related AE was IRR, reported by 21/61 (34%) patients; all events were mild to moderate in intensity. The most common symptom associated with IRRs was back pain and/or spasms, reported by 17/21 patients. There were no reported events of anaphylaxis. Sixteen patients (45 mg Formulation (I) arm [n=3]; 90 mg Formulation (I) arm [n=13]) had at least 1 temporary infusion interruption due to an IRR; despite this, all patients completed all scheduled infusions. There were no reported Formulation (I)-related serious AEs, discontinuations due to AEs, or deaths. Additionally, there were no reported events of hepatic decompensation or laboratory changes indicative of potential drug-induced liver injury (DILI). At Week 36, no clinically meaningful changes were observed in BMD by DXA (data not shown).

Table 4. Safety Summary ^a Ankle fracture at Week 10; considered unrelated to study drug; ^b In the post-treatment follow up period, HCC was detected in 3 patients through surveillance. AE, adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; QW, once weekly; SAE, serious adverse event; ULN, upper limit of normal.

[0064] During the study, 3 patients (45 mg Formulation (I) arm [n=2]; 90 mg Formulation (I) arm [n=l]) had elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST) concentrations >3x ULN. For all 3 patients, the elevations were transient, not associated with symptoms or other liver-related laboratory abnormalities, and were considered unrelated to Formulation (I). During routine surveillance in the post-treatment follow-up period, HCC was detected in 3 patients; during the treatment period, 2 patients received 45 mg Formulation (I) and 1 received 90 mg Formulation (I).

[0065] Formulation (I) immunogenicity analyses are summarized in Table 5. At baseline, preexisting antibodies to Formulation (I) were identified in 1 patient each in the placebo and 90 mg Formulation (I) arms. During treatment, 2 patients (11%) in the 45 mg Formulation (I) arm developed ADA; the first patient tested positive on Weeks 2 and 4 and the second patient tested positive on Week 1. ADA was not detected at subsequent time points in either patient in the 45 mg arm. In the 90 mg Formulation (I) arm, ADA were detected in the same patient who was positive at baseline on Weeks 2 and 4 at the same titer.

Table 5. Summary of Formulation (I) immunogenicity analyses ^a Patients with an evaluable post-baseline ADA value, regardless of baseline value; ^b Patients with a post-treatment ADA positive sample while pre-treatment sample is ADA negative or missing; ^c Patients with post-treatment ADA positive sample titer that is >4X the pre-treatment sample titer when the pre-treatment sample is ADA positive; ^d Post-dose ADA positive results at >2 consecutive timepoints, where the first and last ADA positive results are >16 weeks apart; ^e Patients with a post-treatment ADA positive sample while pre-treatment sample is missing. ADA, anti-drug antibody; QW, once weekly.

[0066] In patients with hepatic fibrosis, the risk of hepatic decompensation, HCC, and mortality increases with advancing fibrosis stage. Though studies suggest that fibrosis reversal may improve clinical outcomes for patients with HCV, the HCC rate in patients with cirrhosis pre- and/or post-HCV-SVR can be as high as 2.3%- 12.5%. In this Phase 2 study of patients with HCV-SVR, 12-week treatment with Formulation (I) improved liver fibrosis in some patients, including those with cirrhosis.

[0067] Within this study population, patients had a median time of 3 years of HCV-SVR prior to the study. Eligible patients were those with advanced fibrosis, staged as METAVIR F3 or F4 at screening; however, when the baseline liver tissue specimens were restaged for enable comparison with Week 12 histology, patients were approximately staged 50% as Fl or F2. The discrepancies between the METAVIR staging were unexpected, particularly since the biopsy specimens were of adequate quality in terms of length and portal area inclusion. These results suggest that fibrosis staging post-HCV-SVR is complex and not entirely straightforward; further study of how to most accurately characterize post-SVR changes in fibrosis (e.g., less collagen, thin septa) in this patient population will be beneficial.

[006S] In an effort to learn more about the fibrosis characteristics of this patient population, exploratory assessments of collagen content and fibrosis biomarkers were performed. Lower CPA than had been previously reported was observed for patients with chronic HCV or NASH, but similar to that of patients with post-HCV-SVR fibrosis regression. Independent of METAVIR stage, median PRO-C3 concentrations for all arms were within range of those observed in healthy adults, who would be expected to have low levels of fibrogenesis. This suggests that these patients with HCV-SVR had low levels of fibrogenesis and that some fibrosis improvements occurred after SVR but prior to the baseline liver biopsy for this study. Patients with higher METAVIR stages had higher serum concentrations of C3M and TIMP-1 compared with patients with earlier stages of fibrosis. Although the biomarker ranges overlapped across METAVIR stages, these data suggest higher levels of fibrolysis in patients who have higher versus lower fibrosis scores, as assessed by the central pathologist in this study. By silencing HSP47 mRNA, Formulation (I) decreases fibrogenesis by reducing mature collagen formation; therefore, it is reasonable to hypothesize that Formulation (I) will have greater efficacy in patients with high levels of fibrogenesis compared with patients who have HCV-SVR, given the evidence that they have low levels of active fibrogenesis.

[0069] Treatment with Formulation (I) demonstrated target engagement by reducing liver HSP47 mRNA levels and decreasing liver HSP47 protein levels at Week 12 compared with placebo. However, maximal target engagement may not have been observed at the time of biopsy because biopsy collection was approximately 7 days after the last Formulation (I) dose. Notably, improvements in fibrosis were observed in some patients after Formulation (I) treatment. At Week 12, nearly 20% of patients who received Formulation (I), compared with 13% of patients who received placebo, had fibrosis improvement that was consistent with a decrease in METAVIR score by at least 1 stage. Additionally, half of patients in the 90 mg Formulation (I) group with cirrhosis at baseline had improved by at least 1 METAVIR stage. When Ishak score improvement was measured in this population, only patients in the 90 mg Formulation (I) 2 group (n=5) had achieved at least a 2-stage improvement and 3 of these patients had cirrhosis at baseline.

[0070] One drawback of staging fibrosis by manual interpretation is the potential for interreader variability. An automated tool that provides quantitative and reproducible histological analyses, qFIB, may act as a supplemental measure to evaluate liver biopsy specimens. Of the patients with METAVIR and Ishak score improvements, 82% or 100% of patients, respectively, were also shown to have improved fibrosis by qFIB.

[0071] Formulation (I) treatment for 12 weeks at 45 and 90 mg QW was generally safe and well-tolerated, and exhibited dose-proportional increases in exposure and no notable accumulation of Formulation (I) plasma concentrations with weekly dosing. Although the frequency of AEs was higher in the Formulation (I) treatment groups compared with placebo, all reported AEs were mild or moderate in intensity, and the majority were IRRs. During the study, no patients showed evidence of potential DILI or hepatic decompensation. HCC was detected in 3 patients during the follow-up period; these events were not deemed related to Formulation (I) treatment and were likely a reflection of the increased HCC risk in this patient population. Formulation (I) immunogenicity analyses revealed a low rate and titer of AD As. Only 2 patients in the 45 mg dose group exhibited AD As up to Week 4 and no patients in the 90 mg dose group were positive for AD As with respect to baseline. Together, the early, low-level ADA response, lack of ADA persistence in the 45 mg arm, and absence of baseline ADA in the 90 mg arm suggests that Formulation (I) has low immunogenicity. [0072] In summary, in this Phase 2 study of patients with HCV-SVR, 12-week Formulation (I) administered via weekly i.v. infusion improved METAVIR and Ishak scores, demonstrated target engagement, and was generally well-tolerated through Week 36.

[0073] The examples and embodiments described herein are for illustrative purposes only and in some embodiments, various modifications or changes are to be included within the purview of disclosure and scope of the appended claims.

INFORMAL SEQUENCE LISTING

Table 6. Sense and Anti-sense oligonucleotide sequences for HSP47 siRNA

[0074] Each Z and Z' is independently present or absent, but if present independently includes 1-5 consecutive nucleotides or non-nucleotide moieties or a combination thereof covalently attached at the 3'-terminus of the strand in which it is present.

[0075] Z" may be present or absent, but if present is a capping moiety covalently attached at the 5' terminus.