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| Targets |
Fazirsiran targets the alpha-1 antitrypsin (AAT) and Z-AAT messenger RNA (mRNA) transcripts. Upon administration, the N-acetylgalactosamine (GalNAc) moiety of fazirsiran binds with high affinity and specificity to asialoglycoprotein receptors (ASGPRs) that are abundantly expressed on the surface of hepatocytes. Following receptor-mediated endocytosis, the siRNA component is released into the cytoplasm where it incorporates into the RNA-induced silencing complex (RISC). The siRNA then binds to complementary sequences on AAT and Z-AAT mRNA through RNA interference (RNAi) mechanisms, leading to sequence-specific degradation of the target mRNA and inhibition of translation. This reduces synthesis of the misfolded Z-AAT protein, allowing the liver to clear previously accumulated Z-AAT protein aggregates. The ultimate biological targets are the SERPINA1 gene transcripts, and the reduction of Z-AAT is believed to decrease hepatic inflammation and fibrosis, thereby addressing the underlying cause of AATD-associated liver disease.
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| ln Vitro |
In vitro studies have demonstrated that fazirsiran effectively reduces AAT and Z-AAT mRNA and protein levels in hepatocyte cell lines. The siRNA component shows high specificity for its target sequences, with minimal off-target effects. Using human hepatoma cell lines such as Huh7 or primary human hepatocytes, treatment with fazirsiran results in dose-dependent reduction of AAT mRNA levels as measured by quantitative PCR (qPCR) and reduced AAT protein secretion measured by ELISA. The GalNAc conjugation enhances hepatocyte-specific uptake, with cell uptake studies showing significantly higher internalization of GalNAc-conjugated siRNA compared to unconjugated siRNA in ASGPR-expressing cells. In vitro potency assays demonstrate that fazirsiran achieves 70-90% target mRNA knockdown at nanomolar concentrations. The compound is not cytotoxic to hepatocytes at therapeutically relevant concentrations, with cell viability maintained above 90% as measured by MTT or ATP-based assays. Functional rescue of cellular phenotype includes reduced intracellular Z-AAT aggregates and decreased markers of endoplasmic reticulum (ER) stress.
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| ln Vivo |
In vivo studies in animal models and clinical trials have shown that fazirsiran effectively reduces serum and liver concentrations of Z-AAT. In preclinical studies using transgenic mouse models expressing human Z-AAT, subcutaneous administration of fazirsiran resulted in robust, dose-dependent reduction of Z-AAT mRNA and protein levels in the liver. In the Phase 2 SEQUOIA clinical trial (AROAAT2001), patients with homozygous ZZ AATD-associated liver disease received multiple subcutaneous doses of fazirsiran. One-year findings showed sustained, dose-dependent reductions in Z-AAT serum concentration and significant improvements in histological findings, including reduction in periodic acid-Schiff with diastase digestion (PAS+D) globule burden, which is a hallmark of AATD-associated liver disease. Follow-up studies demonstrated improved liver fibrosis scores and reduced markers of hepatocellular injury. The RNAi-mediated knockdown is durable, with effects persisting for weeks after a single dose. In the Phase 2 open-label extension, continued treatment maintained Z-AAT reduction and histological improvements. These findings support the potential of fazirsiran as a disease-modifying therapy for AATD-associated liver disease.
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| Enzyme Assay |
For in vitro enzyme/receptor binding studies, the ASGPR binding affinity assay is performed to confirm targeting specificity. Human ASGPR (hASGPR) protein is immobilized on a sensor chip. Varying concentrations of fazirsiran (or its GalNAc moiety) are flowed over the chip, and binding kinetics (association rate ka, dissociation rate kd) are measured by surface plasmon resonance (SPR). The equilibrium dissociation constant (KD) is calculated. Alternatively, competitive binding assays are performed using radiolabeled GalNAc or asialoorosomucoid (ASOR) as the tracer. For the RNAi mechanism study, the RNA-induced silencing complex (RISC) loading assay is performed. Cytosolic lysates from hepatocytes are incubated with a biotin-labeled double-stranded RNA that mimics the fazirsiran siRNA. Streptavidin pull-down is performed, and associated AGO2 protein (a core component of RISC) is detected by Western blotting. For nuclease stability assays, fazirsiran is incubated in human serum or liver homogenate at 37degC. Aliquots are taken at various time points (0, 1, 2, 4, 8, 24, 48 h), and intact siRNA is quantified by HPLC or LC-MS to determine the half-life of the compound in biological matrices.
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| Cell Assay |
For in vitro cell-based experiments, human hepatoma cell lines (e.g., Huh7, HepG2) or primary human hepatocytes are seeded in 6-well or 96-well plates at a density of 100,000-200,000 cells per well in appropriate culture medium supplemented with 10% FBS. Cells are cultured for 24-48 hours until reaching 60-80% confluence. Fazirsiran is added to the culture medium at concentrations ranging from 0.1 nM to 1000 nM. For uptake studies, cells are incubated with fluorescently labeled fazirsiran (e.g., Cy3-labeled) for 1-24 hours, and cellular fluorescence is visualized by confocal microscopy and quantified by flow cytometry. Competition assays are performed with excess free GalNAc or ASOR to confirm ASGPR-mediated uptake. For knockdown efficacy, cells are treated with fazirsiran for 48-72 hours. Total RNA is extracted, reverse transcribed, and AAT mRNA levels are measured by qPCR using TaqMan or SYBR Green assays. Culture supernatants are collected for measurement of secreted AAT protein by ELISA. For toxicity assessment, cell viability is measured using CellTiter-Glo or MTT assays after 72 hours of treatment. For ER stress markers, cell lysates are analyzed by Western blotting for BiP (GRP78), CHOP, and spliced XBP1. Apoptosis is assessed by caspase-3/7 activity or Annexin V-PI staining.
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| Animal Protocol |
For in vivo animal experiments, the Z-AAT transgenic mouse model (e.g., PiZ mouse expressing human Z-AAT) is used. Mice (8-12 weeks old, 20-30 g) are housed under standard conditions with ad libitum access to food and water. Fazirsiran is formulated in PBS or saline and administered by subcutaneous injection at doses ranging from 1 to 30 mg/kg. For dose-response studies, mice receive a single injection and are euthanized at various time points (days 1, 3, 7, 14, 21, 28, 56). For multiple-dose studies, weekly or monthly injections are given for 4-12 weeks. At the end of the study, animals are euthanized, and blood is collected by cardiac puncture for serum isolation. Liver tissue is harvested, weighed, and divided into portions for RNA extraction (flash-frozen in liquid nitrogen), protein analysis (homogenized in RIPA buffer), and histology (fixed in 10% formalin). AAT mRNA levels are measured by qPCR. Z-AAT protein levels in serum and liver homogenates are quantified by ELISA. Liver sections are stained with PAS+D to visualize globule burden, and with H&E for histological scoring of inflammation and fibrosis. Fibrosis is assessed by Sirius Red staining and quantified by image analysis. For pharmacokinetic studies, blood samples are collected at 0, 2, 4, 8, 24, 48, 72, 168, 336, 672 hours post-dose, and plasma siRNA concentrations are measured by hybridization ELISA or LC-MS.
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| ADME/Pharmacokinetics |
Pharmacokinetic studies of fazirsiran have been conducted in preclinical species and are ongoing in clinical trials. Following subcutaneous administration in rodents and non-human primates, fazirsiran is rapidly absorbed, with peak plasma concentrations (Cmax) achieved within 1-4 hours (Tmax). The GalNAc conjugation enhances liver-specific distribution, with the majority of the administered dose accumulating in hepatocytes via ASGPR-mediated uptake. The compound exhibits a long elimination half-life (t½) of several days to weeks in both animals and humans, typical for stabilized oligonucleotides. In animal studies, the half-life in liver tissue ranged from 7 to 21 days, supporting infrequent dosing intervals (e.g., monthly or quarterly). Plasma exposure increases in a dose-proportional manner. Plasma protein binding is high, typically >90%. Metabolism of fazirsiran occurs primarily via nucleases that sequentially shorten the siRNA from the ends; no cytochrome P450-mediated metabolism is involved. The primary route of elimination is through urine as degraded oligonucleotide fragments, with a smaller fraction excreted in feces. The compound is not a substrate, inhibitor, or inducer of CYP450 isozymes, suggesting low potential for drug-drug interactions. In patients with hepatic impairment, a Phase 1 study is underway to evaluate whether liver function affects the pharmacokinetics of fazirsiran.
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| Toxicity/Toxicokinetics |
The toxicological profile of fazirsiran has been evaluated in preclinical animal models and is being monitored in ongoing clinical trials. In repeated-dose toxicity studies in rodents and non-human primates, subcutaneous administration of fazirsiran at doses up to 30 mg/kg/week was generally well tolerated. The primary findings were dose-dependent, reversible increases in liver enzymes (ALT, AST) at higher doses, reflecting pharmacological target engagement rather than hepatotoxicity. No significant adverse effects were observed on kidney function, hematology, or histopathology of major organs. In the Phase 1 and Phase 2 clinical trials (including the SEQUOIA study), fazirsiran has shown an acceptable safety and tolerability profile. The most common adverse events reported were mild to moderate injection site reactions (erythema, pain, swelling), fatigue, and headache. No serious adverse events related to fazirsiran have been reported in the published clinical data. No evidence of off-target RNAi effects or immune-mediated reactions (e.g., complement activation) has been observed. The favorable safety profile is attributed to the hepatocyte-specific targeting via ASGPR, which concentrates the drug in the intended organ while minimizing systemic exposure. Long-term safety is being evaluated in ongoing Phase 3 open-label extension studies. As with all oligonucleotide therapeutics, renal function is monitored during treatment, but no nephrotoxicity has been reported to date. The compound is contraindicated in patients with known hypersensitivity to any component.
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| References | |
| Additional Infomation |
Fazirsiran is an investigational RNA interference (RNAi) therapeutic being developed for the treatment of alpha-1 antitrypsin deficiency (AATD)-associated liver disease. AATD is a rare, inherited genetic disorder caused by mutations in the SERPINA1 gene, most commonly the Z mutation, leading to the production of a misfolded Z-AAT protein that accumulates in hepatocytes, causing inflammation, fibrosis, cirrhosis, and an increased risk of hepatocellular carcinoma. Fazirsiran is designed to degrade both wild-type AAT and Z-AAT mRNA, thereby reducing the synthesis of the Z-AAT protein and allowing the liver to clear existing protein aggregates. The compound consists of a cholesterol-conjugated RNAi trigger and a melittin-derived peptide conjugated to N-acetylgalactosamine (NAG) as an excipient to promote endosomal escape. The GalNAc moiety targets the asialoglycoprotein receptor (ASGPR) on hepatocytes for liver-specific delivery. Fazirsiran is being developed by Arrowhead Pharmaceuticals in collaboration with Takeda Pharmaceuticals. The compound has received Fast Track designation from the FDA for the treatment of AATD-associated liver disease. Phase 2 clinical trials (SEQUOIA, NCT03945292) have demonstrated dose-dependent reductions in Z-AAT levels and improvements in liver histology. Phase 3 trials (including a long-term open-label extension) are ongoing to confirm safety and efficacy. Fazirsiran is not yet approved for marketing in any country. A Phase 1 study is also investigating the pharmacokinetics of fazirsiran in patients with normal liver function compared to those with mild, moderate, or severe hepatic impairment (NCT06165341). If approved, fazirsiran would be the first RNAi-based therapy specifically for AATD-associated liver disease.
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| Molecular Formula |
C493H610F11N163NA43O312P43S6
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| Molecular Weight |
16532.90
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| CAS # |
2175009-09-1
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| Appearance |
Solid powder
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| Synonyms |
ARO-AAT sodium
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.0605 mL | 0.3024 mL | 0.6049 mL | |
| 5 mM | 0.0121 mL | 0.0605 mL | 0.1210 mL | |
| 10 mM | 0.0060 mL | 0.0302 mL | 0.0605 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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