Aflibercept inhibits vascular endothelial growth factor (VEGF) signaling by acting as a soluble decoy receptor for VEGF-A, VEGF-B, and placental growth factor (PlGF) [1]

Aflibercept (500 μg/mL; 24 h and 7 d) shows no toxicity on RPE cells, neither in MTT-assay nor in trypan blue exclusion assay[1]. Aflibercept (500 μg/mL; 24 h) shows a statistically significant effect on wound healing compared with control in the confluent RPE cell layer with three wounds[1]. Aflibercept (500 μg/mL; 7 d) displays a significantly diminished phagocytosis of opsonised latex beads compared to untreated control[1]. Aflibercept (1 and 10 μg/mL; 10 h) inhibits VEGF signaling by reducing VEGF-regulated processes, such as permeability and angiogenesis[2].

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Aflibercept (500 µg/mL) showed no cytotoxicity on primary porcine RPE cells after 24 h or 7 days. Cell viability remained at 97.8±3.0% (24 h) and 102.4±0.9% (7 days) in MTT assays, and 97.9±7.3% (24 h) and 97.2±17.4% (7 days) in trypan blue exclusion assays.[1]

At clinically relevant concentrations (500 µg/mL), Aflibercept significantly impaired RPE wound healing in scratch assays, reducing wound closure to 70.3±10.3% vs. control (87.3±8.4%, p<0.01). No effect was observed at 125 µg/mL (72.2±6.1% vs. control 74.2±5.5%).[1]

Aflibercept (500 µg/mL) reduced phagocytosis of opsonized latex beads by RPE cells (28.3±2.9 beads/cell vs. control 42.8±4.3 beads/cell, p<0.001), comparable to bevacizumab (30.2±5.2 beads/cell).[1]

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Aflibercept (1–10 µg/mL) reduced rVEGF-A-induced permeability in human umbilical vein endothelial cells (HUVECs) by 18.8–27.1% (p<0.01–0.0001), measured via FITC-dextran transwell assay. Tube formation was dose-dependently inhibited, confirming anti-angiogenic activity.[2]

In primary porcine RPE cells, Aflibercept (500 µg/mL) impaired wound healing (70.3±10.3% closure vs. control 87.3±8.4%, p<0.01) and reduced phagocytosis of opsonized beads (28.3±2.9 beads/cell vs. control 42.8±4.3, p<0.001).[2]

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Treatment of HUVECs with Aflibercept (50 nM, 24 h) significantly reduced intracellular NO levels (measured by DAF-FM fluorescence) and increased ROS accumulation (detected via DHE staining), linked to upregulated NOX1/NOX4 protein expression[3]

Western blot analysis revealed that Aflibercept (50 nM, 24 h) decreased phosphorylation of Akt (Ser473) and eNOS (Ser1177), suppressed CAT-1 transporter expression, and reduced intracellular L-arginine concentration. [3]

Aflibercept (10 mg/kg; 3 hours after middle cerebral artery occlusion (MCAO)) reduces stroke-induced VEGF-A and VEGFR2 expression, brain edema, and BBB disruption, and improves post-stroke survival in obese mice [ 2]. Aflibercept (18.2 mg/kg and 36.4 mg/kg; once intravenously) affects blood pressure, ROS and eNOS production in mice [3].

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In diet-induced obese stroke mice, Aflibercept (10 mg/kg IV, single dose at 3h post-MCAO) reduced mortality (17% vs. IgG control 40%), hemorrhagic transformation (27% vs. 43%), and brain swelling (18% vs. 28%, p<0.01). Blood-brain barrier disruption (IgG/dextran extravasation) was significantly attenuated (p<0.05).[2]

Neurological scores improved at 1/3 days post-stroke (p<0.05). No benefit was observed in nonobese mice with malignant infarction, confirming comorbidity-selective efficacy.[2]

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Single-dose intravenous Aflibercept (18.2 or 36.4 mg/kg) in C57BL/6 mice induced acute, dose-dependent hypertension (peak SBP elevation on day 2), impaired endothelium-dependent relaxation (EDR) in response to acetylcholine, and reduced aortic NO bioavailability[3]

Long-term administration (5 doses over 14 days) caused sustained hypertension, increased aortic ET-1 levels, elevated ROS (via NOX1/NOX4), and persistently suppressed p-Akt/p-eNOS signaling and CAT-1 expression[3]

Wound healing (scratch assay): Confluent primary porcine RPE cells were scratched with a toothpick to create wounds. After washing, cells were treated with Aflibercept (125 µg/mL or 500 µg/mL) in phenol red-free DMEM with 10% FBS. Wound closure was quantified microscopically at 24 h using AxioVision software.[1]

Phagocytosis assay: RPE cells treated with Aflibercept (500 µg/mL) for 7 days were incubated with FITC-labeled latex beads opsonized with porcine photoreceptor outer segments for 4 h. Phagocytosis was quantified by counting internalized beads per cell via fluorescence microscopy.[1]

Cellular uptake: RPE cells treated with Aflibercept (125 µg/mL or 500 µg/mL) for 1 h to 7 days were fixed, permeabilized, and stained with goat anti-human AlexaFluor555 antibody. Intracellular localization was analyzed by fluorescence microscopy, showing vesicular uptake (≥82.5% of cells positive) persisting for 7 days.[1]

Animal/Disease Models: Male C57BL/6 mice [3]
Doses: 18.2 mg/kg and 36.4 mg/kg
Route of Administration: intravenous (iv) (iv)injection; 18.2 mg/kg and 36.4 mg/kg Primary
Experimental Results: Mouse blood pressure increased rapidly and dose-dependently Elevated, endothelium-dependent relaxation (EDR) is Dramatically impaired, leading to NADPH oxidase 1 (NOX1) and NADPH oxidase 4 (NOX4)-mediated ROS production, reducing protein kinase B (Akt) and endothelial oxidation Activation of nitrogen synthase (eNOS) simultaneously reduces nitric oxide (NO) production and increases ET-1 levels in the mouse aorta.

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Obese C57BL/6 mice (HFD for 8–12 weeks) underwent 30-min transient MCAO. Aflibercept (10 mg/kg) or IgG control was administered intravenously 3h post-occlusion. Outcomes (infarct volume, swelling, BBB integrity) were assessed at 72h.[2]

For severe stroke in lean mice: 40-min MCAO followed by identical Aflibercept dosing (10 mg/kg IV, 3h post-occlusion).[2]

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Male C57BL/6 mice (8–10 weeks) received intravenous Aflibercept (18.2 or 36.4 mg/kg) as a single dose (short-term) or five doses at 2-day intervals (long-term). Doses were calculated based on human-to-mouse conversion (human equivalent: 4 mg/kg)[3]

For L-arg co-administration studies, mice were pretreated orally with L-arg (0.5 or 1.0 g/kg, twice daily) 3 days before Aflibercept injection and continued until endpoint[3]

Absorption, Distribution, and Excretion
In patients with neovascular (wet) age-related macular degeneration, the mean peak plasma concentration of free aflibercept was reached within 1–3 days after intravitreal injection of aflibercept (2 mg per eye); this concentration was estimated to be more than 100 times lower than the aflibercept concentration required to achieve systemic VEGF half-maximal binding. Two weeks after intravitreal injection, the concentration of free aflibercept in plasma was undetectable. No accumulation of aflibercept in plasma was observed after repeated intravitreal injections (i.e., every 4 weeks).
Absorption
Following intravitreal injection of 8 mg aflibercept in one eye, the mean (standard deviation) Cmax of free aflibercept in plasma was 0.30 (0.27) mg/L, and the median time to reach peak plasma concentration was 2.9 days. Following monthly intravitreal injections of aflibercept for the first three months, plasma accumulation of free aflibercept was extremely low (mean accumulation ratio 1.2); no further accumulation was subsequently observed. In patients with wet age-related macular degeneration (AMD), retinopathy of prematurity (RVO), and diabetic macular edema (DME), the mean Cmax of free aflibercept in plasma was 0.02 mcg/mL (range: 0 to 0.054 mcg/mL), 0.05 mcg/mL (range: 0 to 0.081 mcg/mL), and 0.03 mcg/mL (range: 0 to 0.076 mcg/mL), respectively, reached within 1 to 3 days after intravitreal injection of 2 mg per eye. Free aflibercept was undetectable in plasma in all patients two weeks after administration.
Volume of Distribution
The volume of distribution of free aflibercept after intravenous administration is approximately 7 liters.
Clearance
In cancer patients, the estimated clearance rates of free aflibercept and bound aflibercept 1 hour after intravenous infusion of 2 to 9 mg/kg aflibercept every 2 or 3 weeks are 0.88 L/day and 0.14 L/day, respectively. The clearance rate of free aflibercept in healthy subjects is similar, but the clearance rate of bound aflibercept is slightly faster (0.19 L/day). Patients with low albumin levels or high alkaline phosphatase levels also typically exhibit accelerated clearance of free aflibercept.

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Metabolism/Metabolites
Aflibercept is a therapeutic protein, and drug metabolism studies have not been conducted. The expected elimination pathways of aflibercept include: target-mediated clearance via binding to free endogenous VEGF, and metabolism via proteolysis.

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Biological Half-Life
For the intravitreal injection formulation, the half-life is estimated to be 7.13 days. For the intravenous formulation, after an intravenous injection of 4 mg/kg every two weeks, the elimination half-life of free ziv-aflibercept is approximately 6 days (range 4-7 days).

single intravenous injection (10 mg/kg) did not induce hypertension or leukopenia in mice. Reduced hemorrhagic transformation (27% vs. 43% in controls) indicated no increased risk of bleeding. [2]
In retinal pigment epithelial cells, aflibercept at 500 µg/mL showed no cytotoxicity (MTT/trypan blue assay showed >97% cell viability at 24 hours/7 days). [1]

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Aflibercept induced hypertension in 42.4% of patients and hypertensive crisis in 17.4% (clinical data). In mice, it caused vascular dysfunction by inhibiting the eNOS/NO signaling pathway, excessive ROS production, and elevated ET-1. [3]

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Toxicity Summary
Identification and Use: Intravitreal injection of aflibercept is intended for the treatment of neovascular (wet) age-related macular degeneration. Human Exposure and Toxicity: Aflibercept is more effective in improving vision in cases of poor initial visual acuity. Patients receiving aflibercept had a higher progression-free survival than those in the placebo group. The most common adverse reactions observed were anemia, diarrhea, and neutropenia. For patients with metastatic colorectal cancer whose disease has progressed after oxaliplatin-based therapy, the aflibercept plus FOLFIRI regimen is a safe and effective treatment option. It has not been proven to be superior to other anti-angiogenic therapies. For patients who have previously received bevacizumab and/or ranibizumab injections, aflibercept is a valuable alternative treatment. In patients who had previously received ranibizumab and/or bevacizumab injections every 4–6 weeks, visual stability and improved ocular anatomy were observed after initiation of aflibercept treatment. At 52 weeks, patients receiving 2 mg aflibercept every 8 weeks, 2 mg aflibercept every 4 weeks, or 0.5 mg ranibizumab every 4 weeks showed mean changes in visual acuity (defined as the number of letters of increase or decrease in visual acuity compared to baseline) of 7.9–8.9 letters, 7.6–10.9 letters, and 8.1–9.4 letters, respectively. Animal studies: In monkeys treated with intravitreal aflibercept, erosion and ulceration of the nasal turbinate respiratory epithelium were observed after intravitreal injections of 2 mg or 4 mg aflibercept per eye. The systemic exposure (AUC) following intravenous administration of the lowest evaluated dose (3 mg/kg) of aflibercept in monkeys was approximately 1500 times that following intravitreal injection of 2 mg aflibercept in humans. All changes were reversible within 20 weeks after treatment cessation. In rabbit models, all evaluated doses of aflibercept resulted in fetal malformations. In a rabbit model, the systemic exposure (AUC) after administration of the lowest evaluable dose (0.1 mg/kg) was approximately 10 times that of the systemic exposure after intravitreal injection of 2 mg in humans. Aflibercept is designed to exert a local ocular effect via intravitreal injection. Intravitreal injection allows the drug to penetrate all layers of the retina, thus minimizing systemic effects. No cytotoxic effects were observed in studies exposing cultured corneal endothelial cells to different concentrations of aflibercept. However, there is interest in the possibility of overdose due to slight misalignment of the pre-filled syringe plunger. These small measurement errors may result in the injected drug volume being twice the intended volume. Since the market launch of aflibercept, some cases of elevated intraocular pressure have been caused by overdose. Unfortunately, there is currently no known antidote for aflibercept overdose. Strict adherence to the instructions for use can minimize dosing errors and prevent toxicity from intravitreal injection of aflibercept.

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Effects during pregnancy and lactation
◉ Overview of medication use during lactation
This record pertains to the use of aflibercept via intravitreal injection. Aflibercept inhibits vascular endothelial growth factor (VEGF). Aflibercept is a large protein molecule with a molecular weight of 115,000. Due to its potential for partial destruction in the infant's gastrointestinal tract and poor oral absorption, it is unlikely to be absorbed and is not expected to have systemic effects on the infant. In one mother who received an intravitreal injection, trace amounts of aflibercept were detected in breast milk on only one day out of four days. Aflibercept is approved for intravitreal injection to treat retinopathy of prematurity in infants. The risk to the breastfed infant appears to be very low. Concerns have been raised regarding the use of VEGF inhibitors by breastfeeding mothers because human milk contains VEGF, and VEGF is believed to contribute to the maturation of the infant's gastrointestinal tract. However, 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
A woman with diabetic macular edema received an intravitreal injection of 2 mg aflibercept one week postpartum. She was not breastfeeding. Breast milk samples were collected before injection and on days 1 to 4 post-injection. VEGF levels decreased from 10.6 mcg/L at baseline to 4.9 mcg/L on day 1 and remained at that level for the next 3 days.

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Adverse Reactions
Adverse reactions associated with aflibercept use include eye irritation, vitreous detachment, transient blurred vision, eyelid swelling, and conjunctival hemorrhage. Serious adverse reactions associated with aflibercept administration and injection procedures include retinal detachment, traumatic cataract, thromboembolic events, and increased intraocular pressure. These complications occurred in less than 0.1% of cases with intravitreal injections of aflibercept.
A transient increase in intraocular pressure is expected within 60 minutes after administration and returns to baseline levels within minutes after injection. In patients with glaucoma and other ocular comorbidities, intraocular pressure may take longer to return to normal. Patients should be aware of common and serious adverse reactions and know when to inform their doctor.

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Human Toxicity Excerpt
/Human Exposure Studies/ The relative efficacy and safety of intravitreal injections of aflibercept, bevacizumab, and ranibizumab for the treatment of diabetic macular edema are not yet clear. Methods: We randomly assigned 660 adult patients (mean age 61 ± 10 years) with diabetic macular edema involving the macula from 89 clinical centers to receive intravitreal injections of aflibercept 2.0 mg (224 subjects), bevacizumab 1.25 mg (218 subjects), or ranibizumab 0.3 mg (218 subjects). The study drugs were administered as frequently as every 4 weeks according to the protocol-specified algorithm. The primary endpoint was the mean change in visual acuity at 1 year. Results: From baseline to 1 year, the mean letter score of visual acuity (range 0 to 100, with higher scores indicating better visual acuity; 85 points is approximately equal to 20/20) improved by 13.3 points in the aflibercept group, 9.7 points in the bevacizumab group, and 11.2 points in the ranibizumab group. Although the improvement in the aflibercept group was greater than that in the other two drugs (aflibercept vs. bevacizumab, P<0.001; aflibercept vs. ranibizumab, P=0.03), this difference was not clinically significant because it was primarily observed in the eye with the poorer baseline visual acuity (interaction P<0.001). When the initial visual acuity letter score was between 78 and 69 (equivalent to approximately 20/32 to 20/40) (representing 51% of the subjects), the aflibercept group showed an average improvement of 8.0 points, the bevacizumab group 7.5 points, and the ranibizumab group 8.3 points (p-values for pairwise comparisons between groups were >0.50). When the initial visual acuity letter score was below 69 points (approximately 20/50 or worse), the aflibercept group showed an average improvement of 18.9 points, the bevacizumab group 11.8 points, and the ranibizumab group 14.2 points (p-values for comparison between the aflibercept and bevacizumab groups were <0.001, between the aflibercept and ranibizumab groups were 0.003, and between the ranibizumab and bevacizumab groups were 0.21). There were no significant differences among the study groups in the incidence of serious adverse events (P=0.40), hospitalization rate (P=0.51), mortality rate (P=0.72), or incidence of major cardiovascular events (P=0.56). Conclusion: Intravitreal injection of aflibercept, bevacizumab, or ranibizumab can improve visual acuity in patients with central diabetic macular edema, but the relative efficacy depends on baseline visual acuity. When the initial visual acuity loss is mild, there is no significant difference in mean efficacy among the study groups. Aflibercept is more effective in improving visual acuity in cases of poor initial visual acuity. PMID: 25692915
/Human Exposure Studies/ This review summarizes the pharmacology, pharmacokinetics, efficacy, and safety of aflibercept in combination with the FOLFIRI regimen for the treatment of metastatic colorectal cancer (mCRC) that is resistant to or has progressed on oxaliplatin-containing regimens… Aflibercept is a selective vascular endothelial growth factor antagonist that was evaluated as monotherapy for mCRC in a phase II study and in combination with the FOLFIRI regimen in a phase III trial. The patient response to aflibercept monotherapy did not reach statistical significance. The results indicate that prior bevacizumab treatment did not affect the patient's response to aflibercept. A phase 3 clinical trial compared the safety and efficacy of ziv-aflibercept plus FOLFIRI versus placebo plus FOLFIRI in patients with metastatic colorectal cancer (mCRC) whose disease had progressed after oxaliplatin-based therapy. The median overall survival was 13.5 months in the ziv-aflibercept group and 12.06 months in the placebo group (hazard ratio [HR] = 0.817, 95% confidence interval [CI] = 0.713 to 0.937). Progression-free survival was also higher in the ziv-aflibercept group than in the placebo group (HR = 0.758; 95% CI = 0.661 to 0.869). The most common adverse reactions were anemia, diarrhea, and neutropenia. For mCRC patients whose disease has progressed after oxaliplatin-based therapy, the ziv-aflibercept combined with FOLFIRI regimen is a safe and effective treatment option. Its superiority over other anti-angiogenic therapies has not been demonstrated. PMID: 24259608
/Human Exposure Study/ Intravitreal injection of aflibercept, a fusion protein with high affinity for vascular endothelial growth factor, may provide an alternative therapy for exudative age-related macular degeneration. Preclinical studies and early and late-stage clinical trials have shown that aflibercept's high binding affinity may confer more durable activity and higher efficacy compared to ranibizumab or bevacizumab. This retrospective study included 266 eyes from 249 patients with exudative age-related macular degeneration who received aflibercept after treatment with bevacizumab and/or ranibizumab. Mean subfoveal retinal thickness and mean logMAR visual acuity, measured by spectral-domain optical coherence tomography (SD-OCT), were calculated at 1, 3, 6, and 12 months after the first aflibercept injection. Subgroup analyses were performed on eyes that had received at least 5 bevacizumab and/or ranibizumab injections within 6 months prior to aflibercept treatment, and on eyes that had received at least 10 injections within 12 months prior to aflibercept treatment. These eyes had received a mean of 14.7 (range 1–43) ranibizumab and/or bevacizumab treatments before initiating aflibercept treatment. Mean subfoveal retinal thickness decreased from 300 μm to 275 μm at 1 month (p<0.001) and remained stable at 6 months. Mean logMAR visual acuity improved from 0.60 (equivalent to 20/80 on the Snellen chart) to 0.54 (equivalent to 20/70, p = 0.01) at 1 month and stabilized at 0.55 (equivalent to 20/70 on the Snellen chart, p = 0.11, n = 251) at 6 months. In 82 eyes (mean total of 18.1 injections) that had received at least 5 injections within 6 months prior to aflibercept treatment, subfoveal retinal thickness improved from 296 μm to 279 μm at 1 month (p < 0.0001) and remained stable at 6 months (p < 0.0001). Visual acuity remained unchanged (0.48 [20/61] at 1 month, baseline 0.49 [20/62], p = 0.634; 0.51 [20/65] at 6 months, p = 0.601). In 50 eyes (mean total of 21.8 injections) that had received at least 10 injections within the 12 months prior to aflibercept treatment, the mean subfoveal retinal thickness decreased by 17 μm at 1 month (p = 0.0007) and remained unchanged at 6 months (p = 0.013). Similarly, visual acuity remained unchanged (0.46 at 1 month [20/56], baseline 0.44 [20/56], p = 0.547; 0.50 at 6 months [20/63], p = 0.2445). Aflibercept is a valuable treatment option for patients previously treated with bevacizumab and/or ranibizumab. In patients previously treated with ranibizumab and/or bevacizumab every 4–6 weeks, stable visual acuity and improved ocular anatomy were observed upon initiation of aflibercept treatment. PMID:24706352
/Human Exposure Study/ This study aimed to evaluate changes in intraocular pressure (IOP) in patients with neovascular age-related macular degeneration who had previously received intravitreal bevacizumab or ranibizumab after switching to aflibercept. This retrospective analysis included the medical records of the first 53 patients (53 eyes) who had received at least two 2 mg aflibercept injections (dissolved in 0.05 mL solution) up to March 6, 2013. These patients had previously received at least two 0.5 mg ranibizumab injections (dissolved in 0.05 mL solution), and some had previously received 1.25 mg bevacizumab injections (dissolved in 0.05 mL solution). The analysis was limited to each patient's first aflibercept treatment. All included cases had received ranibizumab as their last anti-vascular endothelial growth factor (VEGF) injection before switching to aflibercept. Each patient served as a self-control. Pre-treatment intraocular pressure (IOP) values for aflibercept (before bevacizumab or ranibizumab treatment) refer to the IOP before mydriasis at the first aflibercept injection visit. Statistical analysis was performed using Microsoft Excel. A total of 41 patients received ranibizumab treatment followed by aflibercept treatment; another 12 patients received both ranibizumab and bevacizumab treatments followed by aflibercept treatment. For each treatment sequence, IOP was calculated in three different ways during aflibercept treatment (post-treatment state): initial IOP measurement, final IOP measurement, and mean IOP during aflibercept treatment. Pooled data showed that the mean IOP before aflibercept treatment (pre-treatment state) was 14.87 mmHg, decreasing to 14.57 mmHg at the first measurement and 13.79 mmHg at the last measurement during treatment, with a mean IOP decreasing to 14.14 mmHg. This conclusion is based on a pooled analysis. The 95% confidence intervals for the differences in intraocular pressure (IOP) at the initial, final, and mean treatment levels (post-treatment minus pre-treatment) were -0.30 (-1.12 to 0.52), -1.08 (-1.83 to -0.32), and -0.73 (-1.30 to -0.17), respectively. During aflibercept treatment, the corresponding P-values for the initial, final, and mean IOP were 0.46, 0.006, and 0.01, respectively. In patients previously treated with ranibizumab and/or bevacizumab, switching to aflibercept resulted in a significant reduction in IOP. Aflibercept may have better IOP safety in patients who have previously received other anti-vascular endothelial growth factor therapies. PMID:25072648

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Non-human Toxicity Excerpt
/Experimental Animals: Acute Exposure/ In monkeys treated with intravitreal injections of aflibercept, erosion and ulceration of the nasal turbinate respiratory epithelium were observed at intravitreal injection doses of 2 or 4 mg per eye. At the no-observed-adverse-effect level (NOAEL) of 0.5 mg intraocularly in monkeys, the systemic exposure (AUC) was 56 times higher than the exposure observed after intravitreal injection of 2 mg in humans.
/Experimental Animals: Developmental or Reproductive Toxicity/ In a 6-month study in monkeys, the effects of weekly intravenous injections of aflibercept at doses ranging from 3 to 30 mg/kg on male and female fertility were evaluated. Amenorrhea or menstrual irregularities, accompanied by changes in female reproductive hormone levels and changes in sperm morphology and motility, were observed at all dose levels. Furthermore, female animals exhibited decreased ovarian and uterine weight, accompanied by impaired corpus luteum development and a reduced number of mature follicles. These changes were associated with uterine and vaginal atrophy. The no-observed-adverse-effect level (NOAEL) was not determined. Following intravenous administration of the lowest dose of aflibercept (3 mg/kg) evaluated in monkeys, the systemic exposure (AUC) was approximately 1500 times that observed after intravitreal injection of 2 mg in humans. All changes were reversible within 20 weeks of discontinuation of treatment.
/Experimental Animals: Developmental or Reproductive Toxicity/ Embryo-fetal toxicity was observed in pregnant rabbits during organogenesis by intravenous administration of ≥3 mg/kg every three days or subcutaneous administration of ≥0.1 mg/kg every six days. Adverse effects on the embryo-fetus included an increased incidence of post-implantation embryo loss and fetal malformations such as generalized edema, umbilical hernia, diaphragmatic hernia, gastroschisis, cleft palate, missing fingers (toes), intestinal atresia, spina bifida, meningocele, cardiac and major vascular defects, and skeletal malformations (fusion of vertebrae, sternum, and ribs; vertebral arch and rib hyperplasia; and incomplete ossification). The maternal no-adverse-effect level (NOAEL) in these studies was 3 mg/kg. In rabbits, all evaluated doses of aflibercept resulted in fetal malformations, with a fetal NOAEL of less than 0.1 mg/kg. The lowest dose evaluated in rabbits (0.1 mg/kg) resulted in a systemic exposure (AUC) approximately 10 times that observed after intravitreal injection of a 2 mg dose in humans.

[1]. Effects of aflibercept on primary RPE cells: toxicity, wound healing, uptake and phagocytosis. Br J Ophthalmol. 2014 Oct;98(10):1448-52.

[2]. Aflibercept, a VEGF (Vascular Endothelial Growth Factor)-Trap, Reduces Vascular Permeability and Stroke-Induced Brain Swelling in Obese Mice. Stroke. 2021 Aug;52(8):2637-2648.

[3]. The vascular endothelial growth factor trap aflibercept induces vascular dysfunction and hypertension via attenuation of eNOS/NO signaling in mice. Acta Pharmacol Sin. 2021 Sep;42(9):1437-1448.

Aflibercept can be internalized by RPE cells within 1 hour and remain in the cells for ≥7 days in the form of vesicles and reticular structures. This uptake is associated with reduced phagocytosis and is not associated with VEGF inhibition. [1] Clinical concentrations (500 µg/mL) of aflibercept impair RPE wound healing and phagocytosis, suggesting potential side effects of long-term treatment, including delayed retinal repair and photoreceptor degeneration. [1] Aflibercept is internalized by RPE cells (in vesicles/reticular structures) and remains for ≥7 days, which is associated with impaired phagocytosis. In obesity-related stroke, it downregulates ipsilateral VEGF-A/VEGFR2/NRP-1 expression (p<0.05). [2] FDA approved for the treatment of ocular neovascularization; it has the potential to treat obesity-related stroke edema due to its selective reduction of vascular permeability without altering the infarct foci. [2]

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Aflibercept is a fusion protein composed of the Ig domain of VEGFR1/R2 and the Fc region of human IgG1, used as a first-line monotherapy for cancer. It inhibits VEGF/PlGF-driven angiogenesis, but induces hypertension through disruption of the CAT-1/AKT/eNOS/NO pathway [3]
Mechanically, it reduces the bioavailability of NO, increases ET-1 and oxidative stress (NOX1/NOX4 mediated), and downregulates the cationic amino acid transporter CAT-1 [3]

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Drug indications
This ophthalmic drug is used to treat neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), and retinopathy of prematurity (ROP). The systemic injectable ziv-aflibercept, in combination with 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI regimen), is used to treat metastatic colorectal cancer that is resistant to oxaliplatin or has progressed after oxaliplatin treatment. Yesafili is indicated for adults to treat visual impairment caused by neovascular (wet) age-related macular degeneration (AMD) (see Section 5.1), macular edema secondary to retinal vein occlusion (branch RVO or central RVO) (see Section 5.1), visual impairment caused by diabetic macular edema (DME) (see Section 5.1), and visual impairment caused by myopic choroidal neovascularization (CNV) (see Section 5.1).
Eylea is indicated for adults to treat: neovascular (wet) age-related macular degeneration (AMD); visual impairment caused by macular edema secondary to retinal vein occlusion (branched or central retinal vein occlusion); visual impairment caused by diabetic macular edema (DME); and visual impairment caused by myopic choroidal neovascularization (myopic CNV).

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Therapeutic Uses
Review: Pharmacology, pharmacokinetics, efficacy, and safety of ziv-aflibercept in combination with FOLFIRI for the treatment of metastatic colorectal cancer (mCRC) that is resistant to or has progressed with oxaliplatin-based regimens… Ziv-aflibercept is a selective vascular endothelial growth factor antagonist that was evaluated as monotherapy for mCRC in a phase II study and in combination with FOLFIRI in a phase III trial. Patient response to ziv-aflibercept monotherapy did not reach statistical significance. Results indicate that response to ziv-aflibercept was not affected by prior bevacizumab treatment. A phase 3 clinical trial compared the safety and efficacy of ziv-aflibercept plus FOLFIRI versus placebo plus FOLFIRI in patients with metastatic colorectal cancer (mCRC) whose disease had progressed after oxaliplatin-based therapy. The median overall survival was 13.5 months in the ziv-aflibercept group and 12.06 months in the placebo group (hazard ratio [HR] = 0.817, 95% confidence interval [CI] = 0.713 to 0.937). Progression-free survival was also higher in the ziv-aflibercept group than in the placebo group (HR = 0.758; 95% CI = 0.661 to 0.869). The most common adverse events were anemia, diarrhea, and neutropenia. For mCRC patients whose disease has progressed after oxaliplatin-based therapy, ziv-aflibercept plus FOLFIRI is a safe and effective treatment option. Its superiority over other anti-angiogenic therapies has not been established. PMID: 24259608

Intravitreal injection of aflibercept, a fusion protein with high affinity for vascular endothelial growth factor, provides a novel treatment option for exudative age-related macular degeneration (AMD). Preclinical studies, as well as early and late-stage clinical trials, have shown that aflibercept's high binding affinity may confer more durable activity and higher efficacy compared to ranibizumab or bevacizumab. This retrospective study included 266 eyes from 249 patients with exudative AMD who received aflibercept after treatment with bevacizumab and/or ranibizumab. Mean subfoveal retinal thickness and mean minimum resolution logarithmic visual acuity (logMAR) were calculated at 1, 3, 6, and 12 months after the first aflibercept injection, as measured by spectral domain optical coherence tomography (SD-OCT). Subgroup analyses were performed on eyes that had received at least 5 bevacizumab and/or ranibizumab injections within 6 months prior to aflibercept treatment and eyes that had received at least 10 injections within 12 months prior to aflibercept treatment. These eyes had received a mean of 14.7 injections (range 1–43) of ranibizumab and/or bevacizumab before initiating aflibercept treatment. The mean subfoveal retinal thickness decreased from 300 μm to 275 μm at 1 month (p<0.001) and remained stable at 6 months. The mean logMAR visual acuity improved from 0.60 (equivalent to Snellen 20/80) to 0.54 (equivalent to Snellen 20/70, p=0.01) at 1 month and stabilized at 0.55 (equivalent to Snellen 20/70, p=0.11, n=251) at 6 months. In 82 eyes (mean total of 18.1 injections) that had received at least 5 injections within the 6 months prior to aflibercept treatment, subfoveal retinal thickness improved from 296 μm to 279 μm at 1 month (p<0.0001) and remained stable at 6 months (p<0.0001). Visual acuity remained unchanged (0.48 at 1 month [20/61], 0.49 from baseline [20/62], p = 0.634; 0.51 at 6 months [20/65], p = 0.601). In 50 eyes (mean total of 21.8 injections) that had received at least 10 injections within the 12 months prior to aflibercept treatment, the mean subfoveal retinal thickness decreased by 17 μm at 1 month (p = 0.0007) and remained stable at 6 months (p = 0.013). Furthermore, visual acuity remained unchanged (0.46 at 1 month of treatment [20/56], baseline 0.44 [20/56], p = 0.547; 0.50 at 6 months of treatment [20/63], p = 0.2445). Aflibercept is a valuable alternative treatment option for patients previously treated with bevacizumab and/or ranibizumab. In patients previously treated with ranibizumab and/or bevacizumab every 4–6 weeks, stable visual acuity and improved ocular anatomy were observed upon initiation of aflibercept treatment. PMID: 24706352

This study aimed to evaluate changes in intraocular pressure (IOP) after switching to aflibercept in patients with neovascular age-related macular degeneration (NVA) previously treated with intravitreal bevacizumab or ranibizumab. This is a retrospective case analysis that included patients who had received at least two 2 mg/0.05 mL aflibercept injections as of March 6, 2013, and who had previously received at least two 0.5 mg/0.05 mL ranibizumab injections, with some patients having previously received 1.25 mg/0.05 mL bevacizumab injections. The analysis was limited to each patient's first such treatment sequence. All included cases had their last anti-vascular endothelial growth factor injection before switching to aflibercept as ranibizumab. Each patient served as a self-control. Intraocular pressure (IOP) before aflibercept treatment (before bevacizumab or ranibizumab treatment) was the IOP value before mydriasis at the time of the first aflibercept injection. Statistical analysis was performed using Microsoft Excel. A total of 41 patients received ranibizumab treatment before switching to aflibercept, and another 12 patients received ranibizumab and bevacizumab treatment before switching to aflibercept. For each treatment sequence, intraocular pressure (IOP) in the treated eye during (and after) aflibercept treatment was calculated in three different ways: initial IOP, final IOP, and mean IOP during aflibercept treatment. Pooled data showed that the mean IOP before aflibercept treatment (pre-treatment state) was 14.87 mmHg, decreasing to 14.57 mmHg during the initial treatment, 13.79 mmHg during the final treatment, and 14.14 mmHg during the mean IOP. This inference is based on a pooled analysis. The 95% confidence intervals for the differences in initial, final, and mean IOP (post-treatment minus pre-treatment) were -0.30 (-1.12 to 0.52), -1.08 (-1.83 to -0.32), and -0.73 (-1.30 to -0.17), respectively. The corresponding p-values for initial, final, and mean IOP during aflibercept treatment were 0.46, 0.006, and 0.01, respectively. Studies have found that patients who previously received ranibizumab and/or bevacizumab treatment experienced a significant reduction in intraocular pressure after switching to aflibercept. Aflibercept may have better intraocular pressure safety for patients who have previously received other anti-vascular endothelial growth factor therapies. PMID:25072648

Intravitreal injection of aflibercept is used to treat neovascular (wet) age-related macular degeneration.

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Pharmacodynamics
The equilibrium dissociation constants (KD) of aflibercept with various human receptors are as follows: VEGF-A165 0.5 pM, VEGF-A121 0.36 pM, VEGF-B 1.92 pM, and PlGF-2 39 pM. In a randomized, placebo-controlled study, researchers evaluated the effect of intravenous injection of 6 mg/kg aflibercept every three weeks on the QTc interval in 87 patients with solid tumors. The results showed that, according to the Fridricia correction method, the mean QT interval did not change significantly from baseline (i.e., the change after placebo correction was greater than 20 ms). However, due to limitations in the study design, the possibility of a slight increase in the mean QTc interval (i.e., less than 10 ms) cannot be ruled out.

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Mechanism of Action
Aflibercept is a recombinant humanized fusion protein and an antagonist of vascular endothelial growth factor A (VEGF-A) and placental growth factor (PlGF). This drug is formed by fusing the extracellular domains of human vascular endothelial growth factor receptors (VEGFR) 1 and 2 with the Fc fragment of human immunoglobulin G1 (IgG1). Aflibercept is a soluble decoy receptor that binds to VEGF-A and PlGF and inhibits their biological activity. VEGF-A and PlGF are angiogenic factors that act as mitogenic factors, chemokines, and vascular permeability factors in endothelial cells. VEGF-A induces angiogenesis and increases vascular permeability, which appears to play a role in the pathogenesis and progression of neovascular (wet) age-related macular degeneration (AMD), a leading cause of blindness in the elderly in developed countries. Aflibercept's binding to VEGF-A and PIGF prevents these factors from binding to endogenous VEGF receptors (VEGFR-1 and VEGFR-2), thereby reducing angiogenesis and vascular permeability. Aflibercept exhibits a higher affinity for VEGF-A subtypes than for endogenous receptors. Even at low concentrations, aflibercept blocks the binding and activation of VEGF with VEGFR-1 and VEGFR-2. Aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor A (VEGF-A) and placental growth factor (PIGF) ligands. It can prevent these ligands from binding to endothelial cell receptors VEGFR-1 and VEGFR-2, thereby inhibiting angiogenesis and reducing vascular permeability. Ultimately, this will delay the progression of vision loss or metastatic colorectal cancer.

862111-32-8

Aflibercept;862111-32-8

Colorless to light yellow liquid

Aflibercept; Eylea; VEGF Trap; VEGF Trap-Eye; ...; 862111-32-8

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.)