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
Following intravitreal injection of aflibercept (2 mg in each eye) in patients with neovascular (wet) age-related macular degeneration, mean peak plasma concentration of free aflibercept was attained in 1-3 days; this concentration is estimated to be more than 100-fold lower than the concentration of aflibercept required to half-maximally bind systemic VEGF. Concentrations of free aflibercept in plasma were undetectable 2 weeks after intravitreal injection. No accumulation of aflibercept in plasma was observed following repeated intravitreal injections (i.e., once every 4 weeks).

Absorption
Following unilateral intravitreal administration of 8 mg aflibercept, the mean (SD) Cmax of free aflibercept in plasma was 0.30 (0.27) mg/L, and the median time to maximal concentration in plasma was 2.9 days. The accumulation of free aflibercept in plasma following three initial monthly intravitreal doses was minimal (mean accumulation ratio 1.2); subsequently, no further accumulation was observed. In patients with wet age-related macular degeneration (AMD), retinopathy of prematurity (RVO), and diabetic macular edema (DME), the mean Cmaxof free aflibercept in the 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 and was attained in 1 to 3 days following intravitreal administration of 2 mg per eye. The free aflibercept plasma concentrations were undetectable two weeks post-dosing in all patients.

Volume of Distribution
The volume of distribution of free aflibercept following intravenous (I.V.) administration of aflibercept is approximately 7 L.

Clearance
Following an hour of intravenous infusion of 2 to 9 mg/kg every 2 or 3 week in cancer patients, the clearances of free and bound aflibercept were estimated to be 0.88 L/day and 0.14 L/day respectively. Healthy subjects have a similar clearance of free aflibercept but slightly faster clearance of bound aflibercept (0.19 L/day). Patients with a low albumin or high alkaline phosphatase levels also typically exhibit faster clearance of free aflibercept.

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Metabolism / Metabolites
Aflibercept is a therapeutic protein and no drug metabolism studies have been conducted. Aflibercept is expected to undergo elimination through both target-mediated disposition via binding to free endogenous VEGF and metabolism via proteolysis.

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Biological Half-Life
For the intravitreal formulation, the half-life was estimated to be 7.13 days. For the intravenous formulation, following a dose of 4 mg per kg every two weeks administered intravenously, the elimination half-life of free ziv-aflibercept was approximately 6 days (range 4-7 days).

Single IV dose (10 mg/kg) caused no hypertension or leukopenia in mice. Hemorrhagic transformation was reduced (27% vs. 43% in controls), indicating no bleeding risk exacerbation.[2]

In RPE cells, 500 µg/mL Aflibercept showed no cytotoxicity (cell viability >97% at 24h/7d in MTT/trypan blue assays).[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 via eNOS/NO signaling suppression, ROS overproduction, and ET-1 elevation[3]

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Toxicity Summary
IDENTIFICATION AND USE: Aflibercept intravitreal injection is used for the treatment of neovascular (wet) age-related macular degeneration. HUMAN EXPOSURE AND TOXICITY: At worse levels of initial visual acuity, aflibercept was more effective at improving vision. Progression-free survival for patients receiving ziv-aflibercept was higher compared with placebo. The most common adverse effects observed were anemia, diarrhea, and neutropenia. Ziv-aflibercept is a safe and effective option in combination with FOLFIRI for the treatment of metastatic colorectal cancer in patients who progress on oxaliplatin-containing therapy. Superiority over other antiangiogenic treatment has not been established. Aflibercept is a valuable treatment alternative in patients previously treated with bevacizumab and/or ranibizumab injections. Stability of visual acuity and anatomic improvement on spectral-domain optical coherence tomography were observed after initiation of aflibercept treatment in those previously treated with ranibizumab and/or bevacizumab injections every 4-6 weeks. The mean change in visual acuity (defined as the number of letters of visual acuity gained or lost compared with baseline) at 52 weeks was a gain of 7.9-8.9, 7.6-10.9, or 8.1-9.4 letters in patients receiving aflibercept 2 mg every 8 weeks, aflibercept 2 mg every 4 weeks, or ranibizumab 0.5 mg every 4 weeks, respectively. ANIMAL STUDIES: Erosions and ulcerations of the respiratory epithelium in nasal turbinates in monkeys treated with aflibercept intravitreally were observed at intravitreal doses of 2 or 4 mg per eye. Intravenous administration of the lowest dose of aflibercept assessed in monkeys (3 mg per kg) resulted in systemic exposure (AUC) that was approximately 1500 times higher than the systemic exposure observed in humans after an intravitreal dose of 2 mg. All changes were reversible within 20 weeks after cessation of treatment. Aflibercept produced fetal malformations at all doses assessed in rabbits. Administration of the lowest dose assessed in rabbits (0.1 mg per kg) resulted in systemic exposure (AUC) that was approximately 10 times the systemic exposure observed in humans after an intravitreal dose of 2 mg.
Aflibercept is intended for intravitreal administration to exert local effects in the eye. IVI allows the medication to penetrate all retinal layers, minimizing systemic effects. No cytotoxic effects have been observed in studies that exposed cultured corneal endothelial cells to varying concentrations of aflibercept.
However, there has been an interest in the potential for drug overdose with intravitreal aflibercept therapy due to a slight misalignment of the plunger in prefilled syringes. These small mismeasurements can lead to delivering double the intended volume of medication. In part, aflibercept overdose accounts for a number of intraocular pressure elevations since its launch. Unfortunately, there is no known antidote for aflibercept overdose. Proper adherence to instructions for use can minimize dosing errors and prevent toxicity of intravitreal aflibercept.

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
This record refers to the use of intravitreal aflibercept. Aflibercept inhibits vascular endothelial growth factor (VEGF). Aflibercept is a large protein molecule with a molecular weight of 115,000, absorption is unlikely because it is probably partly destroyed in the infant's gastrointestinal tract and poorly absorbed orally, so systemic effects in infants are not expected. Aflibercept appeared in breastmilk in small amounts in only one of four days after intravitreal injection in one mother. Aflibercept is approved for intravitreal injection in preterm infants with retinopathy of prematurity. The risk to the nursing infant appears to be very low. Since VEGF is present in human milk and is thought to help in maturation of the infant’s gastrointestinal tract, concern has been raised about the maternal use of VEGF inhibitors during breastfeeding. However, the typical alternative to breastmilk is infant formula, which contains no VEGF.

◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.

◉ Effects on Lactation and Breastmilk
A woman with diabetic macular edema was given intravitreal aflibercept 2 mg one week postpartum. She was not breastfeeding her infant. Milk samples were obtained before the injection and on days 1 to 4 after the injection. VEGF levels were reduced from 10.6 mcg/L at baseline to 4.9 mcg/L on day 1, where it remained for the next 3 days.

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Adverse Effects
Adverse effects associated with aflibercept use include eye irritation, vitreous detachment, temporary blurred vision, eyelid swelling, and conjunctival hemorrhage. Serious adverse reactions related to the administration of aflibercept and the injection procedure include retinal detachment, traumatic cataracts, thromboembolic events, and increased intraocular pressure (IOP). These complications are observed in less than 0.1% of intravitreal injections with aflibercept.
A temporary rise in intraocular pressure within 60 minutes of administration can be expected and should return to baseline in minutes following injection. Intraocular pressure may take longer to normalize in patients with glaucoma and other ocular comorbidities. Patients should be educated about common and severe adverse effects and know when to notify their doctor.

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Human Toxicity Excerpts
/HUMAN EXPOSURE STUDIES/ The relative efficacy and safety of intravitreous aflibercept, bevacizumab, and ranibizumab in the treatment of diabetic macular edema are unknown. METHODS: At 89 clinical sites, we randomly assigned 660 adults (mean age, 61+/-10 years) with diabetic macular edema involving the macular center to receive intravitreous aflibercept at a dose of 2.0 mg (224 participants), bevacizumab at a dose of 1.25 mg (218 participants), or ranibizumab at a dose of 0.3 mg (218 participants). The study drugs were administered as often as every 4 weeks, according to a protocol-specified algorithm. The primary outcome was the mean change in visual acuity at 1 year. RESULTS: From baseline to 1 year, the mean visual-acuity letter score (range, 0 to 100, with higher scores indicating better visual acuity; a score of 85 is approximately 20/20) improved by 13.3 with aflibercept, by 9.7 with bevacizumab, and by 11.2 with ranibizumab. Although the improvement was greater with aflibercept than with the other two drugs (P<0.001 for aflibercept vs. bevacizumab and P=0.03 for aflibercept vs. ranibizumab), it was not clinically meaningful, because the difference was driven by the eyes with worse visual acuity at baseline (P<0.001 for interaction). When the initial visual-acuity letter score was 78 to 69 (equivalent to approximately 20/32 to 20/40) (51% of participants), the mean improvement was 8.0 with aflibercept, 7.5 with bevacizumab, and 8.3 with ranibizumab (P>0.50 for each pairwise comparison). When the initial letter score was less than 69 (approximately 20/50 or worse), the mean improvement was 18.9 with aflibercept, 11.8 with bevacizumab, and 14.2 with ranibizumab (P<0.001 for aflibercept vs. bevacizumab, P=0.003 for aflibercept vs. ranibizumab, and P=0.21 for ranibizumab vs. bevacizumab). There were no significant differences among the study groups in the rates of serious adverse events (P=0.40), hospitalization (P=0.51), death (P=0.72), or major cardiovascular events (P=0.56). CONCLUSIONS: Intravitreous aflibercept, bevacizumab, or ranibizumab improved vision in eyes with center-involved diabetic macular edema, but the relative effect depended on baseline visual acuity. When the initial visual-acuity loss was mild, there were no apparent differences, on average, among study groups. At worse levels of initial visual acuity, aflibercept was more effective at improving vision. PMID:25692915

/HUMAN EXPOSURE STUDIES/ Review pharmacology, pharmacokinetics, efficacy, and safety of ziv-aflibercept in combination with FOLFIRI for treatment of metastatic colorectal cancer (mCRC) resistant to or progressed following oxaliplatin-containing regimens.... Ziv-aflibercept, a selective vascular endothelial growth factor antagonist, was evaluated as monotherapy for treatment of mCRC in a phase 2 study and added to FOLFIRI in a phase 3 trial. Patient response to ziv-aflibercept as monotherapy did not reach statistical significance. Results suggest that response to ziv-aflibercept treatment is not influenced by prior bevacizumab therapy. A phase 3 trial compared the safety and efficacy of ziv-aflibercept plus FOLFIRI with placebo plus FOLFIRI in patients with mCRC who experienced disease progression on an oxaliplatin-containing regimen. Patients in the ziv-aflibercept arm had a median overall survival of 13.5 months, versus 12.06 months for those receiving placebo (hazard ratio [HR] = 0.817, 95% CI = 0.713 to 0.937). Progression-free survival for patients receiving ziv-aflibercept was higher compared with placebo (HR = 0.758; 95% CI = 0.661 to 0.869). The most common adverse effects observed were anemia, diarrhea, and neutropenia. Ziv-aflibercept is a safe and effective option in combination with FOLFIRI for the treatment of mCRC in patients who progress on oxaliplatin-containing therapy. Superiority over other antiangiogenic treatment has not been established. PMID:24259608

/HUMAN EXPOSURE STUDIES/ Intravitreal aflibercept, a fusion protein with high affinity for vascular endothelial growth factor, offers an alternative treatment for exudative age-related macular degeneration. Preclinical studies and early and late phase clinical trials suggest that aflibercept's high binding affinity may impart greater durability of activity and increased efficacy compared to ranibizumab or bevacizumab. A total of 266 eyes of 249 patients with exudative age-related macular degeneration who received aflibercept after treatment with bevacizumab and/or ranibizumab were included in a retrospective review. Mean central subfoveal thickness on spectral-domain optical coherence tomography and mean logarithm of the minimal angle of resolution (logMAR) visual acuity were calculated at 1, 3, 6, and 12 months after the first aflibercept injection. Subgroup analyses were performed in eyes receiving at least 5 bevacizumab and/or ranibizumab injections in the 6 months prior to aflibercept and in eyes receiving at least 10 injections in the 12 months prior to aflibercept. Eyes received an average of 14.7 (range 1-43) ranibizumab and/or bevacizumab treatments prior to initiation of aflibercept therapy. The mean central subfoveal thickness decreased from 300 to 275 um at 1 month (p<0.001) and was maintained at 6 months. Mean logMAR visual acuity improved from 0.60 (Snellen equivalent 20/80) to 0.54 (20/70, p = 0.01) at 1 month and was stable at 0.55 at 6 months (Snellen equivalent 20/70, p = 0.11, n = 251). In 82 eyes receiving at least 5 injections in the 6 months prior to aflibercept treatment (average of 18.1 injections total), the central subfoveal thickness improved from 296 to 279 um at 1 month (p<0.0001) and was maintained at 6 months (p<0.0001). Visual acuity did not change (0.48 [20/61] at 1 month compared to baseline, 0.49 [20/62], p = 0.634, and at 6 months 0.51 [20/65], p = 0.601). In 50 eyes receiving at least 10 injections in the 12 months prior to aflibercept treatment (average of 21.8 injections total), the mean central subfoveal thickness decreased by 17 um at 1 month (p = 0.0007) and was maintained at 6 months (p = 0.013). Again, visual acuity did not change (0.46 [20/56] at 1 month, baseline 0.44 [20/56], p = 0.547, and 0.50 [20/63] at 6 months, p = 0.2445). Aflibercept is a valuable treatment alternative in patients previously treated with bevacizumab and/or ranibizumab injections. Stability of visual acuity and anatomic improvement on spectral-domain optical coherence tomography were observed after initiation of aflibercept treatment in those preciously treated with ranibizumab and/or bevacizumab injections every 4-6 weeks. PMID:24706352

/HUMAN EXPOSURE STUDIES/ To assess for change in intraocular pressure (IOP) in neovascular age-related macular degeneration patients switched to aflibercept after receiving previous treatments of intravitreal bevacizumab or ranibizumab. This is a retrospective chart review of the first 53 patients (53 eyes) treated with at least 2 injections of 2 mg in 0.05 mL of aflibercept by March 6, 2013, after at least 2 previous injections of 0.5 mg in 0.05 mL of ranibizumab with or without previous injections of 1.25 mg in 0.05 mL of bevacizumab. The analysis was restricted to the first such sequence within each patient. The last previous anti-vascular endothelial growth factor injection before the switch to aflibercept was ranibizumab in all cases included in the study. Each person served as his or her own control. The pre-aflibercept IOP in the before state (treatment with bevacizumab or ranibizumab) was the preinjection IOP measure before dilation at the visit of the first aflibercept injection. Statistical analysis was performed using Microsoft Excel. There were 41 patients who were first treated with ranibizumab followed by aflibercept and 12 patients treated with ranibizumab and bevacizumab followed by aflibercept. For each of these sequences, IOP in the treated eye during treatment with aflibercept (the after state) was computed in 3 different ways: the first IOP, the last IOP, and the mean IOP for the period when treated with aflibercept. The pooled data showed a mean pre-aflibercept (the before state) IOP of 14.87 that decreased to a mean first IOP of 14.57, mean last IOP of 13.79, and a mean IOP of 14.14 during aflibercept treatment. The inference is based on the pooled analysis. The 95% confidence interval for the differences (after minus before) were -0.30 (-1.12 to 0.52), -1.08 (-1.83 to -0.32), and -0.73 (-1.30 to -0.17) for the first, last, and mean IOPs, respectively. The corresponding P values were 0.46 for the first, 0.006 for the last, 0.01 for the mean IOP during the aflibercept treatment period. Intraocular pressure was found to be significantly lower in patients switched to aflibercept after previous treatments with ranibizumab and/or bevacizumab. Aflibercept may have a more favorable IOP safety profile in patients previously on other anti-vascular endothelial growth factor treatments. PMID:25072648

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Non-Human Toxicity Excerpts
/LABORATORY ANIMALS: Acute Exposure/ Erosions and ulcerations of the respiratory epithelium in nasal turbinates in monkeys treated with aflibercept intravitreally were observed at intravitreal doses of 2 or 4 mg per eye. At the NOAEL of 0.5 mg per eye in monkeys, the systemic exposure (AUC) was 56 times higher than the exposure observed in humans after an intravitreal dose of 2 mg.

/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Effects on male and female fertility were assessed as part of a 6-month study in monkeys with intravenous administration of aflibercept at weekly doses ranging from 3 to 30 mg per kg. Absent or irregular menses associated with alterations in female reproductive hormone levels and changes in sperm morphology and motility were observed at all dose levels. In addition, females showed decreased ovarian and uterine weight accompanied by compromised luteal development and reduction of maturing follicles. These changes correlated with uterine and vaginal atrophy. A No Observed Adverse Effect Level (NOAEL) was not identified. Intravenous administration of the lowest dose of aflibercept assessed in monkeys (3 mg per kg) resulted in systemic exposure (AUC) that was approximately 1500 times higher than the systemic exposure observed in humans after an intravitreal dose of 2 mg. All changes were reversible within 20 weeks after cessation of treatment.

/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Aflibercept produced embryo-fetal toxicity when administered every three days during organogenesis to pregnant rabbits at intravenous doses >/= 3 mg per kg, or every six days at subcutaneous doses >/= 0.1 mg per kg. Adverse embryo-fetal effects included increased incidences of postimplantation loss and fetal malformations, including anasarca, umbilical hernia, diaphragmatic hernia, gastroschisis, cleft palate, ectrodactyly, intestinal atresia, spina bifida, encephalomeningocele, heart and major vessel defects, and skeletal malformations (fused vertebrae, sternebrae, and ribs; supernumerary vertebral arches and ribs; and incomplete ossification). The maternal No Observed Adverse Effect Level (NOAEL) in these studies was 3 mg per kg. Aflibercept produced fetal malformations at all doses assessed in rabbits and the fetal NOAEL was less than 0.1 mg per kg. Administration of the lowest dose assessed in rabbits (0.1 mg per kg) resulted in systemic exposure (AUC) that was approximately 10 times the systemic exposure observed in humans after an intravitreal dose of 2 mg.

[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 is internalized by RPE cells within 1 h and retained intracellularly for ≥7 days in vesicular and net-like structures. This uptake correlates with reduced phagocytic function, independent of VEGF inhibition.[1]

The impairment of RPE wound healing and phagocytosis by Aflibercept at clinical concentrations (500 µg/mL) suggests potential side effects in long-term therapy, including delayed retinal repair and photoreceptor degeneration.[1]
Aflibercept is internalized by RPE cells (vesicular/net-like patterns) and retained for ≥7 days, correlating with phagocytosis impairment. In obese stroke, it downregulates ipsilateral VEGF-A/VEGFR2/NRP-1 expression (p<0.05).[2]

FDA-approved for ocular neovascular diseases; repurposing potential for obesity-exacerbated stroke edema due to selective reduction of vascular permeability without infarct modification.[2]

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Aflibercept is a fusion protein comprising Ig domains of VEGFR1/R2 and Fc region of human IgG1, used as first-line monotherapy for cancers. It inhibits VEGF/PlGF-driven angiogenesis but triggers hypertension via CAT-1/AKT/eNOS/NO pathway disruption[3]

Mechanistically, it reduces NO bioavailability, increases ET-1 and oxidative stress (NOX1/NOX4-mediated), and downregulates cationic amino acid transporter CAT-1[3]

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Drug Indication
The opthalmic agent is used for the treatment of neovascular (Wet) age-related macular degeneration (AMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), and retinopathy of prematurity (ROP). The systemic injection, known as ziv-aflibercept, in combination with 5-fluorouracil, leucovorin, irinotecan-(FOLFIRI), is for the treatment of metastatic colorectal cancer that is resistant to or progressed following treatment with oxaliplatin.

Yesafili is indicated for adults for the treatment ofneovascular (wet) age-related macular degeneration (AMD) (see section 5. 1),visual impairment due to macular oedema secondary to retinal vein occlusion (branch RVO or central RVO) (see section 5. 1),visual impairment due to diabetic macular oedema (DME) (see section 5. 1),visual impairment due to myopic choroidal neovascularisation (myopic CNV) (see section 5. 1).
Eylea is indicated for adults for the treatment of: neovascular (wet) age-related macular degeneration (AMD); visual impairment due to macular oedema secondary to retinal vein occlusion (branch RVO or central RVO); visual impairment due to diabetic macular oedema (DME); visual impairment due to myopic choroidal neovascularisation (myopic CNV).

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Therapeutic Uses
Review pharmacology, pharmacokinetics, efficacy, and safety of ziv-aflibercept in combination with FOLFIRI for treatment of metastatic colorectal cancer (mCRC) resistant to or progressed following oxaliplatin-containing regimens.... Ziv-aflibercept, a selective vascular endothelial growth factor antagonist, was evaluated as monotherapy for treatment of mCRC in a phase 2 study and added to FOLFIRI in a phase 3 trial. Patient response to ziv-aflibercept as monotherapy did not reach statistical significance. Results suggest that response to ziv-aflibercept treatment is not influenced by prior bevacizumab therapy. A phase 3 trial compared the safety and efficacy of ziv-aflibercept plus FOLFIRI with placebo plus FOLFIRI in patients with mCRC who experienced disease progression on an oxaliplatin-containing regimen. Patients in the ziv-aflibercept arm had a median overall survival of 13.5 months, versus 12.06 months for those receiving placebo (hazard ratio [HR] = 0.817, 95% CI = 0.713 to 0.937). Progression-free survival for patients receiving ziv-aflibercept was higher compared with placebo (HR = 0.758; 95% CI = 0.661 to 0.869). The most common adverse effects observed were anemia, diarrhea, and neutropenia. Ziv-aflibercept is a safe and effective option in combination with FOLFIRI for the treatment of mCRC in patients who progress on oxaliplatin-containing therapy. Superiority over other antiangiogenic treatment has not been established. PMID:24259608

Intravitreal aflibercept, a fusion protein with high affinity for vascular endothelial growth factor, offers an alternative treatment for exudative age-related macular degeneration. Preclinical studies and early and late phase clinical trials suggest that aflibercept's high binding affinity may impart greater durability of activity and increased efficacy compared to ranibizumab or bevacizumab. A total of 266 eyes of 249 patients with exudative age-related macular degeneration who received aflibercept after treatment with bevacizumab and/or ranibizumab were included in a retrospective review. Mean central subfoveal thickness on spectral-domain optical coherence tomography and mean logarithm of the minimal angle of resolution (logMAR) visual acuity were calculated at 1, 3, 6, and 12 months after the first aflibercept injection. Subgroup analyses were performed in eyes receiving at least 5 bevacizumab and/or ranibizumab injections in the 6 months prior to aflibercept and in eyes receiving at least 10 injections in the 12 months prior to aflibercept. Eyes received an average of 14.7 (range 1-43) ranibizumab and/or bevacizumab treatments prior to initiation of aflibercept therapy. The mean central subfoveal thickness decreased from 300 to 275 um at 1 month (p<0.001) and was maintained at 6 months. Mean logMAR visual acuity improved from 0.60 (Snellen equivalent 20/80) to 0.54 (20/70, p = 0.01) at 1 month and was stable at 0.55 at 6 months (Snellen equivalent 20/70, p = 0.11, n = 251). In 82 eyes receiving at least 5 injections in the 6 months prior to aflibercept treatment (average of 18.1 injections total), the central subfoveal thickness improved from 296 to 279 um at 1 month (p<0.0001) and was maintained at 6 months (p<0.0001). Visual acuity did not change (0.48 [20/61] at 1 month compared to baseline, 0.49 [20/62], p = 0.634, and at 6 months 0.51 [20/65], p = 0.601). In 50 eyes receiving at least 10 injections in the 12 months prior to aflibercept treatment (average of 21.8 injections total), the mean central subfoveal thickness decreased by 17 um at 1 month (p = 0.0007) and was maintained at 6 months (p = 0.013). Again, visual acuity did not change (0.46 [20/56] at 1 month, baseline 0.44 [20/56], p = 0.547, and 0.50 [20/63] at 6 months, p = 0.2445). Aflibercept is a valuable treatment alternative in patients previously treated with bevacizumab and/or ranibizumab injections. Stability of visual acuity and anatomic improvement on spectral-domain optical coherence tomography were observed after initiation of aflibercept treatment in those preciously treated with ranibizumab and/or bevacizumab injections every 4-6 weeks. PMID:24706352

To assess for change in intraocular pressure (IOP) in neovascular age-related macular degeneration patients switched to aflibercept after receiving previous treatments of intravitreal bevacizumab or ranibizumab. This is a retrospective chart review of the first 53 patients (53 eyes) treated with at least 2 injections of 2 mg in 0.05 mL of aflibercept by March 6, 2013, after at least 2 previous injections of 0.5 mg in 0.05 mL of ranibizumab with or without previous injections of 1.25 mg in 0.05 mL of bevacizumab. The analysis was restricted to the first such sequence within each patient. The last previous anti-vascular endothelial growth factor injection before the switch to aflibercept was ranibizumab in all cases included in the study. Each person served as his or her own control. The pre-aflibercept IOP in the before state (treatment with bevacizumab or ranibizumab) was the preinjection IOP measure before dilation at the visit of the first aflibercept injection. Statistical analysis was performed using Microsoft Excel. There were 41 patients who were first treated with ranibizumab followed by aflibercept and 12 patients treated with ranibizumab and bevacizumab followed by aflibercept. For each of these sequences, IOP in the treated eye during treatment with aflibercept (the after state) was computed in 3 different ways: the first IOP, the last IOP, and the mean IOP for the period when treated with aflibercept. The pooled data showed a mean pre-aflibercept (the before state) IOP of 14.87 that decreased to a mean first IOP of 14.57, mean last IOP of 13.79, and a mean IOP of 14.14 during aflibercept treatment. The inference is based on the pooled analysis. The 95% confidence interval for the differences (after minus before) were -0.30 (-1.12 to 0.52), -1.08 (-1.83 to -0.32), and -0.73 (-1.30 to -0.17) for the first, last, and mean IOPs, respectively. The corresponding P values were 0.46 for the first, 0.006 for the last, 0.01 for the mean IOP during the aflibercept treatment period. Intraocular pressure was found to be significantly lower in patients switched to aflibercept after previous treatments with ranibizumab and/or bevacizumab. Aflibercept may have a more favorable IOP safety profile in patients previously on other anti-vascular endothelial growth factor treatments. PMID:25072648

Aflibercept intravitreal injection is used for the treatment of neovascular (wet) age-related macular degeneration.

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Pharmacodynamics
The equilibrium dissociation constants (K_D) for aflibercept for various human receptors are as follow: 0.5 pM for VEGF-A₁₆₅, 0.36 pM for VEGF-A₁₂₁, 1.92 pM for VEGF-B, and 39 pM for PlGF-2. The effect of 6 mg per kg intravenous aflibercept every three weeks on QTc interval was evaluated in 87 patients with solid tumors in a randomized, placebo-controlled study. No large changes in the mean QT interval from baseline (i.e., greater than 20 ms as corrected for placebo) based on the Fridericia correction method were detected in the study. However, a small increase in the mean QTc interval (i.e., less than 10 ms) cannot be excluded due to the limitations of the study design.

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Mechanism of Action
Aflibercept, a recombinant humanized fusion protein, is a vascular endothelial growth factor A (VEGF-A) and placental growth factor (PlGF) antagonist. The drug consists of portions of human VEGF receptor (VEGFR) 1 and 2 extracellular domains fused to the Fc portion of human immunoglobulin G1 (IgG1). Aflibercept acts as a soluble decoy receptor that binds to VEGF-A and PlGF and inhibits their biologic activity. VEGF-A and PlGF are angiogenic factors that can act as mitogenic, chemotactic, and vascular permeability factors for endothelial cells. VEGF-A induces neovascularization (angiogenesis) and increases vascular permeability, which appear to play a role in the pathogenesis and progression of the neovascular (wet) form of age-related macular degeneration, a leading cause of blindness in geriatric adults in developed countries. Binding of aflibercept to VEGF-A and PlGF prevents these factors from binding to endogenous VEGF receptors (i.e., VEGFR-1, VEGFR-2), reducing neovascularization and vascular permeability. The binding affinity of aflibercept for VEGF-A isoforms is higher than that of endogenous receptors; aflibercept blocks VEGF binding and activation of VEGFR-1 and VEGFR-2 even at low concentrations.

Ablibercept is a recombinant fusion protein that acts as a decoy receptor for the ligands, vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PIGF). It prevents these ligands to binding to endothelial receptors, VEGFR-1 and VEGFR-2, to suppress neovascularization and decrease vascular permeability. This ultimately will slow vision loss or the progression of 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.)