ziv-aflibercept binds VEGF and placental growth factor (PIGF) as a soluble decoy receptor (no IC50/Ki/EC50 data provided)[1]

In dose-dependent fashion, Ziv-aflibercept (0.25, 0.5, 1.0, and 5 mg/mL; 24 h) lowers mitochondrial membrane potential in ARPE-19 cells [1].

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ziv-aflibercept significantly reduced cell viability to 77.25% ± 2.1% (p < 0.0001) at 10× clinical concentration (2 mg/4 mL vitreous equivalent) in ARPE-19 human retinal pigment epithelial cells, but no statistically significant changes occurred at 1/2×, 1×, or 2× doses compared to controls[1]

ziv-aflibercept induced mitochondrial toxicity, evidenced by reduced mitochondrial membrane potential (ΔΨm) at 1× (73.50% ± 2.93%, p < 0.0001), 2× (64.83% ± 2.7%, p < 0.0001), and 10× (49.65% ± 4.22%, p = 0.0002) concentrations, indicating early apoptosis; no significant ΔΨm change was observed at 1/2× dose (91.57% ± 2.54%)[1]

ziv-aflibercept increased osmolality to 418 mOsm/kg at 10× concentration versus control (324 mOsm/kg), though osmolality at 1/2×, 1×, and 2× doses (324, 330, and 342 mOsm/kg, respectively) showed no significant difference from controls[1]

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Ziv-aflibercept did not reduce cell viability at clinical dose (1×) in human retinal pigment epithelial (RPE) cells (ARPE-19 line) after 24-hour exposure, as per Malik et al.[2]
At twice the clinical dose, it significantly reduced viability in Müller cells (MIO-M1 line), unlike ranibizumab, aflibercept, or bevacizumab, indicating cell-type-specific toxicity at higher concentrations.[2]

In the rabbits' right eye, ziv-aflibercept (25 mg/mL; intraocular injection; single dose) did not substantially induce cataracts, retinal detachment, or any associated problems [2].

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In rabbits, intravitreal injection of ziv-aflibercept (0.05 ml, 25 mg/ml) showed no toxicity on funduscopy, optical coherence tomography (OCT), or full-field electroretinogram (ERG) at 1 and 7 days post-injection; histology and transmission electron microscopy confirmed no anatomic damage.[2]

Human case reports (e.g., neovascular AMD, diabetic macular edema) demonstrated improved visual acuity and reduced intraretinal/subretinal fluid after intravitreal injections (1.25 mg/dose), with no inflammation, cataract progression, or retinal toxicity observed during follow-up.[2]

Cell Viability Assay[1]
Cell Types: ARPE-19 cells.
Tested Concentrations: 0.25, 0.5, 1.0 and 5 mg/mL.
Incubation Duration: 24 h.
Experimental Results: InDramatically affected cell viability (below 1 mg/mL).

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For cell viability assays, ARPE-19 cells were plated at 5.0 × 10⁵ cells/well in six-well plates with 2 mL culture media, incubated for 24 h under standard conditions (37°C, 5% CO₂, 95% humidity), then treated for 24 h with ziv-aflibercept at 1/2×, 1×, 2×, or 10× clinical concentrations or control (no drug). Cells were trypsinized, centrifuged (1000 rpm, 5 min), resuspended in 1 mL media, and analyzed via automated trypan blue dye exclusion assay to quantify viable/dead cells; assays were performed in triplicate and repeated twice[1]

For mitochondrial membrane potential (ΔΨm) assays, ARPE-19 cells were plated at 1.0 × 10⁵ cells/well in 24-well plates, incubated overnight, then treated for 24 h with ziv-aflibercept at predefined concentrations. JC-1 dye was used to detect ΔΨm: healthy cells (red fluorescence, 590 nm) versus apoptotic/necrotic cells (green fluorescence, 529 nm). Fluorescence ratios (red:green) were measured using a fluorescence image scanner, with assays performed in quadruplicate and repeated twice[1]

Osmolality was measured for culture media containing ziv-aflibercept using an automated osmometer based on freezing-point depression; samples were tested at all concentrations (1/2× to 10×)[1]

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For viability testing, ARPE-19 cells were exposed to ziv-aflibercept at 1/2×, 1×, 2×, and 10× clinical concentrations (based on vitreous distribution) for 24 hours. Viability was assessed via automated trypan blue exclusion, with results normalized to controls and analyzed statistically.[2]

Müller cell (MIO-M1 line) assays involved treatment with low (1/2×) and clinical equivalent doses of ziv-aflibercept; viability reduction was quantified at 2× concentration using standardized methods.[1]

Nine rabbits received unilateral intravitreal injections of 0.05 ml ziv-aflibercept (25 mg/ml). Assessments included baseline/post-injection serum/vitreous/aqueous osmolarity, funduscopy, OCT, ERG (days 1 and 7), and histopathological analysis. No sham controls were used; outcomes focused on absence of complications (e.g., cataract, retinal detachment).[2]

The half-life of ziv-aflibercept is 7.1 days, the same as that of aflibercept, which is based on their structural similarity. [2]

In ARPE-19 cells, ziv-aflibercept exhibited mitochondrial toxicity at clinically relevant concentrations (1×, 2×, and 10×), with a decrease in ΔΨm suggesting early apoptosis; cell viability decreased only at the 10× dose [1]. The increased cytotoxicity of ziv-aflibercept compared to aflibercept may be attributed to its lower pH and higher osmolarity, but systemic toxicokinetic data (e.g., plasma protein binding, organ toxicity) have not been evaluated [1]. No ocular or systemic toxicity has been reported following intravitreal injection of ziv-aflibercept in rabbits or humans. Serum and intraocular osmolarity remained unchanged after injection. [2] Mitochondrial toxicity (e.g., decreased ΔΨm in ARPE-19 cells) has been observed at supraclinical doses in vitro, but no evidence of organ toxicity or drug interaction has been found in vivo. [2] Effects during pregnancy and lactation Overview of use during lactation This record pertains to the use of ziv-aflibercept in the treatment of cancer. There is currently no information regarding the use of ziv-aflibercept during breastfeeding. Because ziv-aflibercept is a large protein with a molecular weight of 115,000, its concentration in breast milk is likely very low, and it is unlikely to be absorbed due to potential degradation in the infant's gastrointestinal tract. However, aflibercept (ziv-aflibercept) is often used in combination with other potentially toxic chemotherapy drugs, and most data suggest that breastfeeding is contraindicated while the mother is receiving anti-cancer drug treatment. One woman experienced a decrease in vascular endothelial growth factor (VEGF) levels in her breast milk after receiving intravitreal injections of aflibercept. Because VEGF is present in breast milk and is believed to contribute to the maturation of the infant's gastrointestinal tract, there are concerns about the use of VEGF inhibitors by breastfeeding mothers. It is important to note that the typical alternative to breast milk is infant formula, which does not contain VEGF.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

[1]. Safety profiles of anti-VEGF drugs: bevacizumab, ranibizumab, aflibercept and ziv-aflibercept on human retinal pigment epithelium cells in culture. Br J Ophthalmol. 2014 Jun;98 Suppl 1(Suppl 1):i11-16.

[2]. Fusion proteins for treatment of retinal diseases: aflibercept, ziv-aflibercept, and conbercept. Int J Retina Vitreous. 2016 Feb 1;2:3.

Objective: To compare the safety of the anti-vascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept, and ziv-aflibercept in cultured retinal pigment epithelial cells (ARPE-19). Methods: Human retinal pigment epithelial cells (ARPE-19) were exposed to four anti-VEGF drugs at concentrations of 1/2, 1, 2, and 10 times the clinical concentration for 24 hours. Early apoptosis and overall cell death were assessed by cell viability and mitochondrial membrane potential. Results: At 10-fold concentrations, bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002), and ziv-aflibercept (77.25%, p<0.0001) all led to decreased cell viability, but this was not observed at lower concentrations. However, cell viability remained unchanged at all concentrations (including 10-fold concentrations). The mitochondrial membrane potential of cells treated with 10-fold ranibizumab decreased slightly (89.61%, p=0.0006), and the mitochondrial membrane potential of cells treated with 2-fold and 10-fold aflibercept also decreased slightly (88.76% and 81.46%, respectively; p<0.01). Bevacizumab (1-fold, 2-fold and 10-fold concentrations) and aflibercept (73.50%, 64.83% and 49.65%, respectively; p<0.01) both caused a greater decrease in mitochondrial membrane potential, suggesting that early apoptosis can be induced at lower doses (including clinical doses). Conclusion: At clinical doses, neither ranibizumab nor aflibercept showed evidence of mitochondrial toxicity or cell death. However, bevacizumab and aflibercept showed mild mitochondrial toxicity at clinically relevant doses. [1]

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In recent years, monoclonal antibodies have revolutionized the treatment of retinal neovascularization. Recently, another class of drugs—fusion proteins—has offered an alternative treatment strategy with different pharmacological properties. In addition to the commercially available aflibercept, two other drugs—ziv-aflibercept and conbercept—have also been investigated in the anti-angiogenic treatment of ophthalmic diseases. Against this backdrop, a critical review of current data on the application of fusion proteins in ophthalmic diseases may be a timely and important contribution. Aflibercept, formerly known as VEGF Trap Eye, is a fusion protein of VEGF receptors 1 and 2 used to treat various angiogenesis-related retinal diseases. It has become an important treatment option for neovascular age-related macular degeneration (AMD), diabetic macular edema (DME), and retinal vein occlusion (RVO)-related macular edema, along with ranibizumab and bevacizumab. Ziv-aflibercept, a systemic chemotherapy drug approved for the treatment of metastatic colorectal cancer, has recently attracted attention due to its potential for intravitreal injection. An experimental study and human case reports showed that Ziv-aflibercept did not have any toxic reactions associated with electroretinography (ERG). Conbercept is a soluble receptor decoy that blocks all VEGF-A, VEGF-B, VEGF-C and PlGF subtypes, has a high affinity for VEGF, and has a long half-life in the vitreous body. Conbercept has completed phase III clinical trials and demonstrated efficacy and safety. This review discusses three fusion proteins that have been studied in the field of ophthalmology: aflibercept, ziv-aflibercept, and conbercept, focusing on their clinical applications in the treatment of retinal diseases. [2]

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Ziv-aflibercept has the same structure as aflibercept, but different excipients and osmotic pressure (1000 mOsm/L compared to aflibercept's 286 mOsm/L). Ziv-aflibercept was approved by the FDA in 2012 for the treatment of metastatic colorectal cancer. Due to its lower price, it has also been used to treat retinal diseases (belonging to off-label use). The clinical intravitreal injection dose is 1.25 mg (0.05 ml of a 25 mg/ml solution). [2] Preliminary data show that ziv-aflibercept can effectively reduce macular edema and improve vision in patients with age-related macular degeneration/diabetic macular edema (AMD/DME), but large-scale trials are still needed to verify its safety and dosage. [2]

1609655-49-3

Colorless to light yellow liquid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.)