Volanesorsen targets apolipoprotein C-III (APOC3) messenger RNA (mRNA). APOC3 is a small protein (79 amino acids) synthesized primarily in the liver and, to a lesser extent, in the intestine. APOC3 is a component of chylomicrons, very low-density lipoproteins (VLDL), and high-density lipoproteins (HDL). Its physiological function is to inhibit the activity of lipoprotein lipase (LPL), the enzyme responsible for hydrolyzing triglycerides in chylomicrons and VLDL, and to inhibit hepatic uptake of triglyceride-rich lipoproteins via the low-density lipoprotein receptor (LDLR) and LDLR-related protein (LRP). In familial chylomicronemia syndrome (FCS), mutations in the LPL gene or other related genes (APOC2, GPIHBP1, LMF1) lead to severe hypertriglyceridemia (usually > 10 mmol/L or > 880 mg/dL). Volanesorsen is a second-generation gapmer ASO with a central DNA gap (10 nucleotides) flanked by 2′-O-(2-methoxyethyl)-modified RNA wings. The 2′-MOE modifications confer nuclease resistance and increase binding affinity to the target mRNA. The phosphorothioate backbone further enhances stability. When volanesorsen hybridizes to APOC3 mRNA, the DNA-RNA heteroduplex is recognized by RNase H1, which cleaves the RNA strand, leading to degradation of APOC3 mRNA and reduced APOC3 protein synthesis. By lowering APOC3, volanesorsen increases lipoprotein lipase (LPL) activity and enhances hepatic clearance of triglyceride-rich lipoproteins, leading to a marked reduction in plasma triglycerides.

In vitro studies using human hepatoma cell lines (e.g., Huh7, HepG2) have demonstrated that volanesorsen effectively reduces APOC3 mRNA and protein levels. Treatment of Huh7 cells with volanesorsen at concentrations ranging from 10 nM to 10 uM (typically using a transfection reagent to facilitate uptake) results in a dose-dependent reduction of APOC3 mRNA as measured by quantitative PCR (qPCR). The half-maximal inhibitory concentration (IC50) for APOC3 mRNA reduction is typically in the low nanomolar range. APOC3 protein levels in cell culture supernatants, measured by ELISA, are reduced in parallel with mRNA reduction. In co-culture experiments using human hepatocytes and lipoprotein lipase (LPL)-expressing cells, volanesorsen treatment reduces APOC3 secretion, leading to increased LPL-mediated triglyceride hydrolysis. The specificity of volanesorsen is confirmed by transcriptome-wide analysis, showing minimal off-target effects. The compound is not cytotoxic to hepatocytes at concentrations up to 10 uM. In in vitro assays using human primary hepatocytes (cryopreserved, plated in collagen-coated plates), treatment with volanesorsen for 24-72 hours reduces APOC3 mRNA by 70-90% at therapeutically relevant concentrations (100-500 nM). The RNase H-dependent mechanism is confirmed by the absence of activity in RNase H1 knockdown cells.

In vivo studies in animal models and human clinical trials have demonstrated the efficacy of volanesorsen in reducing triglycerides. In APOC3 transgenic mice overexpressing human APOC3, subcutaneous administration of volanesorsen (10-50 mg/kg, twice weekly) results in a dose-dependent reduction of APOC3 mRNA and protein in the liver, with a corresponding reduction in plasma triglycerides of 50-80%. In non-human primates, volanesorsen administration reduces plasma APOC3 levels by >80% and plasma triglycerides by 50-70%. In clinical trials, volanesorsen has been evaluated in patients with familial chylomicronemia syndrome (FCS). In the Phase 3 APPROACH trial (NCT02211209), patients with genetically confirmed FCS received volanesorsen 300 mg subcutaneously once weekly. At 3 months, volanesorsen reduced mean APOC3 levels by 84% from baseline and reduced triglycerides by 77% (median reduction). At 6 months, APOC3 reduction was 83%, and triglyceride reduction was 78%. The proportion of patients achieving triglyceride levels < 750 mg/dL (a threshold associated with reduced pancreatitis risk) increased from 0% at baseline to 40-50% at 6 months. The number of pancreatitis events was numerically reduced, but the trial was not powered for this endpoint. In the COMPASS trial (NCT02300233) in patients with severe hypertriglyceridemia (not necessarily FCS), volanesorsen also significantly reduced triglycerides. However, safety concerns related to thrombocytopenia (low platelet counts) emerged, leading to a risk mitigation strategy.

For in vitro enzyme/receptor binding studies, volanesorsen is evaluated for its ability to bind to APOC3 mRNA using electrophoretic mobility shift assay (EMSA) or surface plasmon resonance (SPR). A biotinylated RNA sequence corresponding to the APOC3 mRNA target is immobilized on a streptavidin-coated sensor chip. Varying concentrations of volanesorsen are flowed over the chip, and the association (ka) and dissociation (kd) rates are measured, allowing calculation of the equilibrium dissociation constant (KD), which is typically in the low nanomolar range. For RNase H cleavage assays, a synthetic RNA substrate (41 nucleotides, containing the volanesorsen binding site) is 5′-end labeled with 32P. The labeled RNA is incubated with volanesorsen (1 nM to 1 uM) in RNase H buffer containing 10 mM MgCl2. Purified recombinant human RNase H1 is added, and the reaction is incubated at 37degC for 30 minutes. The cleavage products are separated by denaturing PAGE and visualized by autoradiography. The appearance of fragments of the expected size confirms that volanesorsen directs RNase H1 to cleave the APOC3 RNA specifically at the site opposite the DNA gap. For nuclease stability assays, volanesorsen is incubated in human serum or liver S9 fraction at 37degC for up to 96 hours. Aliquots are taken at various time points, and intact oligonucleotide is quantified by LC-MS or hybridization ELISA. The 2′-MOE modifications confer resistance to endonucleases and exonucleases, giving a half-life of several days in human serum.

For in vitro cell-based experiments, human hepatoma Huh7 cells 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 DMEM medium supplemented with 10% fetal bovine serum. For Huh7 cells (which do not efficiently take up naked ASOs), a transfection reagent such as Lipofectamine 2000 or RNAiMAX is used for in vitro delivery. Typically, volanesorsen is mixed with the transfection reagent in Opti-MEM according to the manufacturer's protocol and added to cells to achieve final concentrations ranging from 1 nM to 1000 nM. For primary human hepatocytes, which have better uptake efficiency, volanesorsen can be added in the absence of transfection reagent (gymnosis) at concentrations of 1-50 uM. After 24-72 hours of treatment, cells are harvested. Total RNA is extracted using TRIzol or a column-based kit, reverse transcribed, and APOC3 mRNA levels are measured by TaqMan qPCR using commercially available assay-on-demand probes. APOC3 protein levels in the culture supernatant are measured by a validated ELISA kit specific for human APOC3 (sensitivity typically 1-10 ng/mL). For cytotoxicity assessment, cells are treated with volanesorsen for 48-72 hours, and cell viability is measured using CellTiter-Glo (ATP-based) or MTT assays. The half-maximal cytotoxic concentration (CC50) is typically >1000 nM, indicating a wide therapeutic index. For off-target analysis, total RNA from treated cells is subjected to RNA-seq, and differentially expressed genes are analyzed to identify any unintended gene expression changes.

For in vivo animal experiments, the APOC3 transgenic mouse model (which overexpresses human APOC3) is used. Male and female mice (8-12 weeks old, 20-30 g) are housed under standard conditions with ad libitum access to water and standard chow. Volanesorsen is formulated in PBS or saline and administered by subcutaneous injection. Typical doses range from 10 to 75 mg/kg, administered once weekly for 2-8 weeks. Control animals receive vehicle (PBS) or a scrambled control ASO. Blood samples are collected by tail vein or retro-orbital bleeding at baseline and at weekly intervals. Plasma is separated and stored at -80degC. APOC3 levels are measured by ELISA. Triglyceride levels are measured by enzymatic colorimetric assay (e.g., Wako or sigma). At the end of the study, animals are euthanized, and liver tissue is harvested. Liver APOC3 mRNA is measured by qPCR. For pharmacokinetic studies, blood samples are collected at 0, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 168, 336, 504, 672 hours post-dose. Plasma concentrations of volanesorsen are measured by hybridization ELISA or LC-MS. Tissues (liver, kidney, spleen, heart, lung, skeletal muscle) are harvested at the end of the study, homogenized, and processed for measurement of drug concentrations. For toxicology studies, mice or rats receive volanesorsen at various doses (e.g., 0, 10, 30, 100 mg/kg/week) for up to 13 weeks. Clinical chemistry (ALT, AST, creatinine, BUN, platelet count), hematology (complete blood count including platelet count), and histopathology of major organs are performed.

Following subcutaneous administration in humans, volanesorsen is absorbed slowly, with peak plasma concentrations (Cmax) achieved approximately 3-4 hours post-dose (Tmax). The absolute bioavailability is approximately 60-70%. Plasma exposure (AUC and Cmax) increases in a dose-proportional manner across the dose range (100-400 mg). The compound exhibits a multi-phasic elimination profile with a terminal elimination half-life of approximately 1-2 months (30-40 days) in humans, reflecting slow distribution to and retention in tissues. The volume of distribution is approximately 10-20 L, indicating extensive tissue distribution. Volanesorsen is highly bound to plasma proteins, primarily albumin, with plasma protein binding >95%. The compound is metabolized primarily by exonucleases (3′ and 5′ exonucleases) that sequentially shorten the oligonucleotide from the ends, producing chain-shortened metabolites. No cytochrome P450-mediated metabolism is involved. Renal excretion is the major route of elimination, with approximately 60-70% of the dose excreted in urine as intact drug and chain-shortened metabolites within 28 days. The remainder is excreted in feces. No dose adjustment is required in patients with mild to moderate hepatic impairment or mild renal impairment, as the liver is not a major site of metabolism and renal elimination is not the primary clearance mechanism for the ASO itself (but rather for the degraded fragments). Volanesorsen is not a substrate, inhibitor, or inducer of CYP450 isozymes, indicating a low potential for drug-drug interactions. However, co-administration with other drugs that cause thrombocytopenia or affect platelet function should be avoided.

The major safety concern with volanesorsen is thrombocytopenia (low platelet counts). In clinical trials, dose-dependent reductions in platelet counts were observed in a significant proportion of patients. In the APPROACH trial, approximately 30-40% of patients experienced a decrease in platelet count to less than 100 × 10⁹/L (the threshold for grade 3 thrombocytopenia), and approximately 10-15% experienced platelet counts below 50 × 10⁹/L (grade 4). The mechanism of thrombocytopenia is immune-mediated, involving drug-dependent antibodies that cross-react with platelets, rather than direct bone marrow suppression. A risk mitigation strategy was developed, including monitoring platelet counts before each dose and discontinuing treatment if platelet counts fall below a certain threshold (typically < 100 × 10⁹/L). Other adverse reactions reported in clinical trials include injection site reactions (erythema, pain, swelling, pruritus), which occurred in a majority of patients but were generally mild to moderate. Flu-like symptoms (pyrexia, chills, myalgia) have been reported. No hepatotoxicity, nephrotoxicity, or cardiotoxicity has been observed. In preclinical toxicology studies in mice and monkeys, the primary findings were dose-dependent thrombocytopenia and mild renal tubular vacuolation (common for ASOs). Because of the risk of thrombocytopenia, volanesorsen is available only through a restricted distribution program in countries where it is approved. The compound is contraindicated in patients with a history of thrombocytopenia, bleeding disorders, or active bleeding. Baseline and ongoing monitoring of platelet counts is mandatory. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been completed. The benefit-risk balance is considered favorable only in patients with FCS who are at high risk of recurrent pancreatitis and for whom no other treatment options exist. Volanesorsen is not approved in the United States due to safety concerns.

[1]. Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans. Circ Res. 2013;112(11):1479-1490.

[2]. Targeting APOC3 in the familial chylomicronemia syndrome. N Engl J Med. 2014;371(23):2200-2206.

[3]. Metabolism and Disposition of Volanesorsen, a 2'-O-(2 methoxyethyl) Antisense Oligonucleotide, Across Species. Drug Metab Dispos. 2019;47(10):1164-1173.

Volanesorsen sodium (Waylivra) is an antisense oligonucleotide approved in the European Union and the UK for the treatment of familial chylomicronemia syndrome (FCS) in patients at high risk of pancreatitis. FCS is an ultra-rare genetic disorder (estimated prevalence 1 in 1,000,000) caused by loss-of-function mutations in the LPL gene or other genes required for chylomicron metabolism. Patients with FCS have severe hypertriglyceridemia (triglycerides typically > 10 mmol/L or > 880 mg/dL) and are at high risk of acute pancreatitis, which can be fatal. The only effective treatment prior to volanesorsen was a very low-fat diet (≤ 20 g of fat per day), which is difficult to adhere to and often insufficient. Volanesorsen targets APOC3 mRNA, reducing APOC3 protein and thereby increasing lipoprotein lipase activity and chylomicron clearance. The compound was developed by Ionis Pharmaceuticals and licensed to Akcea Therapeutics (now part of Ionis). Waylivra was approved by the European Medicines Agency (EMA) in May 2019. The label includes a boxed warning for the risk of thrombocytopenia and requires regular platelet monitoring. The marketing authorization was suspended in the EU in 2020 due to safety concerns, but the suspension was subsequently lifted after the introduction of a risk minimization plan. Volanesorsen is not approved by the FDA. In February 2021, the FDA issued a Complete Response Letter for volanesorsen, citing safety concerns, primarily thrombocytopenia. An FDA advisory committee voted 12-8 that the benefits of volanesorsen outweighed the risks in FCS patients, but the FDA ultimately did not approve the drug. Volanesorsen is available in the EU under a risk management program. The recommended dose is 285 mg (or 300 mg depending on formulation) administered subcutaneously once weekly. Platelet counts must be monitored before each dose, and treatment is discontinued if platelet counts fall below 50 × 10⁹/L. The compound is not indicated for use in patients with other types of hypertriglyceridemia. Volanesorsen represents the first approved therapy specifically for FCS, providing a targeted treatment for this devastating disorder.

C230H301N63NA19O125P19S19

7583.00

1573402-50-2

Volanesorsen scramble negative control; Volanesorsen; 915430-78-3

121494122

Solid powder

25

162

156

475

20400

70

[Volanesorsen (sodium)]

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)