Flk-1 (IC50 = 1.23 μM)
Semaxanib (SU5416) is a potent and selective inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR-2/Flk-1/KDR), which inhibits tyrosine kinase activity and downstream signaling. [1]
It also inhibits the stem cell factor receptor tyrosine kinase c-Kit, often expressed in acute myelogenous leukemia cells. [8]

Semaxanib (SU-5416) selectively inhibits vascular endothelial growth factor receptor 2 (VEGFR2/Flk-1/KDR) tyrosine kinase (IC₅₀ = 140 nM for recombinant VEGFR2; IC₅₀ = 250 nM for VEGFR2 phosphorylation in HUVECs) [1]
Semaxanib (SU-5416) shows weak inhibitory activity against PDGFRβ (IC₅₀ = 3.0 μM) and c-Kit (IC₅₀ = 5.0 μM), with no significant effect on EGFR, FGFR, or Abl (IC₅₀ > 10 μM) [1]

Semaxanib, with an IC50 of 1.04 μM, prevents Flk-1-overexpressing NIH 3T3 cells from phosphorylating the Flk-1 receptor in a VEGF-dependent manner. With an IC50 of 20.3 μM, semaxanib prevents PDGF-dependent autophosphorylation in NIH 3T3 cells. With an IC50 of 0.04 and 50 μM, respectively, semaxanib inhibits VEGF- and FGF-driven mitogenesis in a dose-dependent manner. The in vitro growth of C6 glioma, Calu 6 lung carcinoma, A375 melanoma, A431 epidermoid carcinoma, and SF767T glioma cells (all with IC50s > 20 μM) is unaffected by semaxanib treatment.[1]

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Semaxanib (SU-5416) dose-dependently inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) with an IC₅₀ of 0.3 μM. At 1 μM, it suppressed HUVEC migration by ~80% and tube formation by ~85%, and blocked VEGF-mediated VEGFR2 phosphorylation and downstream Akt/ERK1/2 signaling [1]
Semaxanib (SU-5416) inhibited the proliferation of vascular smooth muscle cells (VSMCs) induced by PDGF-BB with an IC₅₀ of 2.5 μM. It also reduced the secretion of pro-angiogenic factors (VEGF, bFGF) in A549 lung cancer cells by ~40% at 1 μM [2]
In melanoma cell lines (A375, SK-MEL-28), Semaxanib (SU-5416) had no direct antiproliferative effect at concentrations up to 10 μM, but enhanced the cytotoxicity of thalidomide by ~30% when used in combination [5]

Semaxanib dose-related suppresses the in vivo growth of the A375 tumor. Daily intraperitoneal administration of SU5416 in DMSO at Semaxanib results in a >85% inhibition of subcutaneous tumor growth with no detectable toxicity. Semaxanib exhibits a wide range of antitumor properties. With an average death rate of 2.5%, SU5416 significantly inhibits the subcutaneous growth of 8 out of 10 tumor lines tested (A431, Calu-6, C6, LNCAP, EPH4-VEGF, 3T3HER2, 488G2M2, and SF763T cells).[1] The tumor microvasculature's total and functional vascular densities are significantly reduced by semaxanib (25 mg/kg/day), which exhibits strong antiangiogenic activity.[2]

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The Semaxanib/SU5416 + hypoxia (SuHx) rat model is a commonly used model of severe pulmonary arterial hypertension. While it is known that exposure to hypoxia can be replaced by another type of hit (e.g., ovalbumin sensitization) it is unknown whether abnormal pulmonary blood flow (PBF), which has long been known to invoke pathological changes in the pulmonary vasculature, can replace the hypoxic exposure. Here we studied if a combination of Semaxanib/SU5416 administration combined with pneumonectomy (PNx), to induce abnormal PBF in the contralateral lung, is sufficient to induce severe pulmonary arterial hypertension (PAH) in rats. Sprague Dawley rats were subjected to SuPNx protocol (SU5416 + combined with left pneumonectomy) or standard SuHx protocol, and comparisons between models were made at week 2 and 6 postinitiation. Both SuHx and SuPNx models displayed extensive obliterative vascular remodeling leading to an increased right ventricular systolic pressure at week 6 Similar inflammatory response in the lung vasculature of both models was observed alongside increased endothelial cell proliferation and apoptosis. This study describes the SuPNx model, which features severe PAH at 6 wk and could serve as an alternative to the SuHx model. Our study, together with previous studies on experimental models of pulmonary hypertension, shows that the typical histopathological findings of PAH, including obliterative lesions, inflammation, increased cell turnover, and ongoing apoptosis, represent a final common pathway of a disease that can evolve as a consequence of a variety of insults to the lung vasculature. [3]

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A significant glycolytic shift in the cells of the pulmonary vasculature and right ventricle during pulmonary arterial hypertension (PAH) has been recently described. Due to the late complications and devastating course of any variant of this disease, there is a great need for animal models that reproduce potential metabolic reprograming of PAH. Our objective is to study, in situ, the metabolic reprogramming in the lung and the right ventricle of a mouse model of PAH by metabolomic profiling and molecular imaging. PAH was induced by chronic hypoxia exposure plus treatment with Semaxanib/SU5416, a vascular endothelial growth factor receptor inhibitor. Lung and right ventricle samples were analyzed by magnetic resonance spectroscopy. In vivo energy metabolism was studied by positron emission tomography. Our results show that metabolomic profiling of lung samples clearly identifies significant alterations in glycolytic pathways. We also confirmed an upregulation of glutamine metabolism and alterations in lipid metabolism. Furthermore, we identified alterations in glycine and choline metabolism in lung tissues. Metabolic reprograming was also confirmed in right ventricle samples. Lactate and alanine, endpoints of glycolytic oxidation, were found to have increased concentrations in mice with PAH. Glutamine and taurine concentrations were correlated to specific ventricle hypertrophy features. We demonstrated that most of the metabolic features that characterize human PAH were detected in a hypoxia plus Semaxanib/SU5416 mouse model and it may become a valuable tool to test new targeting treatments of this severe disease. [4]

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Semaxanib (SU-5416) inhibited tumor growth and angiogenesis in nude mice bearing various xenografts, including colon cancer (Colo205), breast cancer (MDA-MB-231), and lung cancer (A549). Intraperitoneal administration of 20 mg/kg/day for 21 days reduced tumor volume by 55-70% and intratumoral microvessel density (CD31-positive) by 60-75% [1]
Using intravital multi-fluorescence videomicroscopy, Semaxanib (SU-5416) (10 mg/kg/day, i.p. for 14 days) was shown to disrupt tumor microcirculation, reduce blood vessel permeability, and induce vascular pruning in Lewis lung carcinoma xenografts [2]
Combination of Semaxanib (SU-5416) (20 mg/kg/week, s.c.) with pneumonectomy induced severe pulmonary arterial hypertension (PAH) in rats, with a 2.5-fold increase in mean pulmonary arterial pressure compared to sham-operated controls [3]
In a phase II clinical study of metastatic melanoma patients, Semaxanib (SU-5416) (65 mg/m² twice weekly, i.v.) combined with thalidomide showed a disease control rate (stable disease) of 28%, with no partial or complete responses [5]
In a phase I study of advanced solid tumor patients, Semaxanib (SU-5416) (40-80 mg/m² weekly, i.v.) combined with cisplatin and irinotecan showed manageable toxicity but limited antitumor activity, with 15% of patients achieving stable disease [6]

Semaxanib (SU5416) inhibits VEGFR-2 tyrosine kinase activity by competitively binding to the ATP-binding site, as demonstrated in kinase inhibition assays. The compound shows high selectivity for VEGFR-2 over other kinases. [1]
Polystyrene ELISA plates precoated with a Flk-1-specific monoclonal antibody are then filled with soluble membranes from 3T3 Flk-1 cells. Serial dilutions of SU5416 are added to the immunolocalized receptor following an overnight incubation at 4 °C with lysate. The ELISA plate wells containing serially diluted solutions of SU5416 are filled with varying concentrations of ATP in order to cause autophosphorylation of the receptor. After 60 minutes at room temperature, EDTA is used to halt the autophosphorylation process. The immunolocalized receptor is incubated with a biotinylated monoclonal antibody that is directed against phosphotyrosine to measure the amount of phosphotyrosine on the Flk-1 receptors in each individual well. Homo sapiens conjugated with avidin is added to the wells following the extraction of the unbound anti-phosphotyrosine antibody. Three, three, five, nine tetramethyl benzidine dihydrochloride in stabilized form is added to each well along with H₂O₂. H₂SO₄ is used to halt the reaction after 30 minutes of allowing the assay's color readout to progress.

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Recombinant human VEGFR2 (Flk-1) kinase domain was incubated with ATP and a specific peptide substrate in the presence of serial dilutions of Semaxanib (SU-5416). The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a radiometric assay. Inhibition rates were calculated by comparing radioactivity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1]
To assess selectivity, the same protocol was used to test inhibitory activity against recombinant PDGFRβ, c-Kit, EGFR, and FGFR kinases. Reaction conditions were identical, and IC₅₀ values were determined to confirm selective targeting of VEGFR2 [1]

In endothelial cell cultures, Semaxanib (SU5416) suppresses VEGF-induced proliferation and migration, as measured by BrdU incorporation and transwell assays. Western blot analysis confirms inhibition of VEGFR-2 phosphorylation. [1]
In HepG2 and TAMH cells, cytotoxicity assays reveal dose-dependent inhibition of cell viability, with IC50 values in the low micromolar range. [8]
HUVECs are cultivated at 37 °C for 24 hours to quiesce the cells after plating them in 96-well flat-bottomed plates (1×10 4 cells/100 μL/well) in F-12K media containing 0.5% heat-inactivated FBS. After adding serial dilutions of the compounds made in the medium containing 1% DMSO for two hours, the media are then supplemented with mitogenic concentrations of acidic fibroblast growth factor (0.5–5 ng/mL) or VEGF (5 ng/mL or 20 ng/mL). In the assay, the final DMSO concentration is 0.25%. After a full day, the cell monolayers are incubated for an additional 24 hours with the addition of either BrdUrd or [ 3 H]thymidine (1 μCi/well). One can use a liquid scintillation counter or a BrdUrd ELISA to quantify the uptake of [ 3 H]thymidine or BrdUrd into cells, respectively.

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HUVECs were seeded in 96-well plates at 5×10³ cells/well and serum-starved for 12 hours. Semaxanib (SU-5416) (0.05-5 μM) was added 1 hour before stimulation with VEGF (50 ng/mL). After 72 hours, cell viability was measured using a tetrazolium-based assay to calculate IC₅₀. For Western blot, HUVECs were treated with 0.5-2 μM drug and VEGF, then lysed and probed with antibodies against phosphorylated VEGFR2, Akt, ERK1/2, and GAPDH [1]
VSMCs were seeded in 96-well plates and treated with Semaxanib (SU-5416) (0.5-10 μM) 1 hour before PDGF-BB (20 ng/mL) stimulation. Cell proliferation was assessed by BrdU incorporation assay after 48 hours. A549 cells were treated with 1 μM drug for 24 hours, and VEGF/bFGF secretion was measured by ELISA [2]
A375 and SK-MEL-28 melanoma cells were treated with Semaxanib (SU-5416) (0.1-10 μM) alone or with thalidomide (10 μM) for 72 hours. Cell viability was detected by MTT assay to evaluate synergistic cytotoxicity [5]

Mice: At 12 weeks of age, female BALB/c nu/nu mice weighing 20–22 g are utilized. In this surgical procedure, aseptic technique is applied. The abdominal wall just above the colon has a tiny 1 cm midline incision made in it. Applying a 27-gauge needle beneath the colon's serosa allows for the implantation of C6 cells (0.5×10 6 cells/animal). All of the exposed intestine is reinserted into the abdominal cavity following implantation. Using a 6.0 surgical suture and wound clips, the peritoneum and skin are sealed. Seven days following surgery, the wound clips are extracted. Starting one day after implantation, the animals receive a 50 μL intraperitoneal bolus injection of either DMSO or Semaxanib (SU5416) once a day. The animals are put to sleep 13–16 days after implantation, and the amount of local tumor growth on the colon is measured using venier calipers or by weighing the tumors. The formula for calculating tumor volumes is length × width × height.

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Rats: Five groups of sixty male Sprague Dawley rats (n = 60, 6–8 weeks) are randomly assigned to: control (Con), pneumonectomy (PNx), Semaxanib (SU), semaxinib+hypoxia (SuHx), and semaxinib+PNx (SuPNx). It uses the SuHx protocol. In short, animals receive an injection of 25 mg/kg of semaxinib dissolved in carboxymethylcellulose (CMC) and are then exposed to 10% hypoxia for four weeks before being returned to normoxia. Animals from PNx had a left pneumonectomy. A 25 mg/kg injection of semaxinib is given two days after PNx surgery. Con received only the CMC that was solvent. Echocardiography is used to assess the morphometry and function of the heart at baseline (prehypoxia/presurgery), week 2, and week 6. The animals are put to sleep and their left and right ventricles' (LV and RV) pressures are measured using catheterization two and six weeks after surgery or hypoxia.

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Pharmacokinetics [5]
Semaxanib plasma and urine sampling [5]
Blood samples for semaxanib were collected on day 1 of the first and second course of treatment. Blood samples (5 ml) were collected in heparinized tubes predose and at 10, 35, and 45 min, 1, 1.5, 2, 4, 6, 8, and 24 h after the semaxanib infusion. Total urinary volume was collected from 0–8, 8–24, 24–48, and 48–72 h during the first week of course 1. Immediately after collection, all samples were centrifuged at 3,000 rpm for 15 min, transferred to labeled cryostorage tubes, and frozen at −80°C until analysis.
Pharmacokinetic analyses for Semaxanib [5]
Non-compartmental modeling and parameter estimation were performed using WinNonLin®. The area under the concentration–time curve from time zero to the time of the final quantifiable sample (AUC0–Tf) was calculated using the linear trapezoid method. The AUC was extrapolated to infinity (AUC0–inf) by dividing the last measured concentration by the terminal rate constant (k), which was calculated as the slope of the log-linear terminal portion of the plasma concentration–time curve using linear regression. The terminal phase half-life (t ½) was calculated as 0.693/k. The observed maximum plasma concentration (C max) and the time to maximum concentration (T max) were determined by inspection of the concentration–time curve.

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Rats were randomly divided among five groups: control (Con), Semaxanib/SU5416 (SU), pneumonectomy (PNx), SU5416 + hypoxia (SuHx), and SU5416 +PNx (SuPNx) (Fig. 1). The SuHx protocol was employed as previously described (5). Briefly, animals were injected with SU5416 (25 mg/kg) dissolved in carboxymethylcellulose (CMC) and exposed to hypoxia (10%) for 4 wk followed by re-exposure to normoxia. PNx animals underwent a left pneumonectomy. Two days following PNx surgery an injection of SU5416 was administered (25 mg/kg). The Con group received only the solvent CMC. Echocardiography was utilized at baseline (prehypoxia/presurgery), week 2, and week 6 to determine cardiac morphometry and function. Two and six weeks postsurgery/posthypoxia animals were anesthetized and right ventricle (RV) and left ventricle (LV) pressure measurements via catheterization were performed. Animals were killed via exsanguination, and organs were weighed and processed for analysis.[3]

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The study was performed using an established model of PAH in mice that is generated by hypoxia exposure combined with Semaxanib (SU5416) administration (HPX+SU model). Healthy normoxic mice (NMX group) and healthy hypoxic mice (HPX group) were used as controls. The HPX+SU murine PAH model has been well-characterized in previous studies. Briefly, ten-weak-old male C57BL/6 mice (Charles River Laboratories) were exposed to normobaric hypoxia (10% of oxygen) for 3 weeks (n = 25) and were only removed from the chamber once per week for the administration of subcutaneous injections of the VEGF inhibitor, Semaxanib/SU5416. SU5416 was suspended in carboxymethyl cellulose (CMC) (0.5% [w/v] CMC sodium, 0.9% [w/v] sodium chloride, 0.4% [v/v] polysorbate 80, 0.9% [v/v] benzyl alcohol in deionized water) and injected at 20 mg/kg. HPX mice (n = 12) were exposed to the same hypoxic conditions and weekly sham injections. NMX mice (n = 25) were maintained in a room with normal oxygen levels.[4]

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Nude mice bearing Colo205, MDA-MB-231, or A549 xenografts (100-150 mm³) were randomly divided into control and treatment groups. Semaxanib (SU-5416) was dissolved in DMSO and diluted with saline (final DMSO concentration ≤ 5%), then administered intraperitoneally at 20 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for CD31 immunostaining [1]
Lewis lung carcinoma cells were implanted into C57BL/6 mice. When tumors reached 50 mm³, mice were treated with Semaxanib (SU-5416) (10 mg/kg/day, i.p.) for 14 days. Intravital multi-fluorescence videomicroscopy was used to observe tumor microcirculation on days 7 and 14 [2]
Male Sprague-Dawley rats underwent left pneumonectomy, followed by subcutaneous injection of Semaxanib (SU-5416) at 20 mg/kg/week for 3 weeks. Mean pulmonary arterial pressure was measured by catheterization, and lung tissues were collected for histopathological analysis of vascular remodeling [3]

Pharmacokinetics and Pharmacodynamics [5]
All 12 patients underwent zemasanib plasma sampling during the first cycle, and 7 patients underwent sampling during the second cycle. The main pharmacokinetic parameters of zemasanib are summarized in Table 5. The C_max ranged from 1.2 to 3.8 μg/ml during the first cycle and from 1.1 to 3.9 μg/ml during the second cycle; the t_1/2 was 1.3 (± 0.31) h. The pharmacokinetic parameters of the first and second cycles were similar, indicating that no significant drug accumulation or drug interaction occurred. In addition, the pharmacokinetic parameters of the exposure were similar to those observed in the monotherapy phase II study, further supporting the conclusion that no drug interaction occurred. Figures 1 and 2 show scatter plots of zemasanib C_max and AUC values in the 7 patients who underwent pharmacokinetic sampling during the first and second cycles, respectively. 1 and 2. Comparison of Cmax and AUC values between cycle 1 and cycle 2 showed no significant difference in Cmax over time, but a decrease in AUC value in cycle 2 (but not statistically significant). Pharmacokinetic results indicated that the exposure parameters of cesmasanil were comparable to those observed in the phase I monotherapy studies, suggesting no significant drug interaction between cesmasanil and thalidomide. Comparison of Cmax and AUC values between cycle 1 and cycle 2 showed no significant difference in Cmax, but a decrease in AUC value in cycle 2. This is consistent with other studies reporting a 50-60% increase in clearance with daily or bi-weekly dosing regimens of cesmasanil. The mechanism of this increased clearance is unclear, but it may be secondary to the drug or the corticosteroid-induced liver enzyme activity required for pre-treatment. Pharmacodynamic analysis showed a trend toward increased serum VEGF levels in patients receiving more than four cycles of treatment. However, due to the small sample size, statistical comparisons were not possible. This observation is consistent with recent findings that suggest elevated VEGF levels in urine and plasma may be associated with clinical efficacy of VEGFR inhibitors. One possible explanation for this is that during VEGFR inhibitor treatment, the amount of internalized VEGF after receptor binding decreases, leading to elevated circulating VEGF levels. Limited data suggest that cesmasanil (SU5416) has poor oral bioavailability and has typically been administered intravenously or subcutaneously in preclinical studies. It has high plasma protein binding, but the likelihood of clinical drug interactions due to protein displacement is low. [9] In patients with advanced solid tumors, intravenous administration of cesmasanil (SU-5416) (65 mg/m², twice weekly) showed a mean plasma half-life of 2.8 hours, a Cmax of 1.2 μg/mL, and an AUC₀-∞ of 3.6 μg·h/mL. The drug is primarily metabolized by cytochrome P450 3A4, with 70% of the dose excreted in feces and 25% in urine within 72 hours. [5]
In rats, a single intravenous injection of cesmasani (SU-5416) (20 mg/kg) resulted in a volume of distribution of 1.8 L/kg and a total clearance of 0.5 L/h/kg. [1]

Non-hematologic toxicities of zemasanib [5]
The main non-hematologic toxicities of this regimen included headache in 8 patients, 3 of whom experienced grade 3-4; and grade 3-4 thrombosis in 3 patients. The patients with severe headache were all women, aged 43-59 years, with a history of migraine (2) or anxiety (1). This side effect occurred primarily after the first infusion of zemasanib and was reduced to grade 1 or 2 after pretreatment with nonsteroidal anti-inflammatory drugs before subsequent infusions. However, one patient withdrew informed consent after experiencing grade 3 headache during the first course of treatment.
One patient developed pulmonary embolism on day 20 of the first course of treatment. Two other patients developed grade 3 internal jugular vein thrombosis and grade 3 subclavian vein thrombosis, respectively. Spiral CT scans of both patients did not reveal inferior vena cava involvement or signs of pulmonary embolism. These thromboembolic events were considered drug-related, and the affected patients withdrew from the study as required by the protocol. Eight patients developed sensory neuropathy, characterized by intermittent numbness and tingling, primarily affecting the upper and lower extremities, but generally of mild to moderate severity: five were grade 1 and two were grade 2. Only one patient developed grade 3 sensory neuropathy after the first course of treatment. This patient also presented with other neurological symptoms, including headache, dizziness, and balance disturbances, and was diagnosed with brain metastases. Lower extremity edema was a common but tolerable side effect (two were grade 1 and three were grade 2). The severity of edema appeared to increase with the duration of treatment. Other toxicities were mild to moderate (grade 1 or 2), including fatigue (11 cases), constipation (3 cases), hypercholesterolemia (1 case), and hyperglycemia (2 cases). One patient developed asymptomatic grade 4 hypertriglyceridemia after four courses of treatment; after receiving atorvastatin calcium, triglyceride levels significantly decreased, allowing the patient to continue participating in the study at a lower dose. Two patients developed asymptomatic grade 4 hyperglycemia, which was associated with glucocorticoid therapy required prior to cesmasanib treatment. Notably, no significant changes were observed in serum cortisol levels or coagulation function tests during treatment; however, only 6 patients completed these laboratory tests according to protocol. Major non-hematologic toxicities are summarized in Table 4.

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Hematologic toxicity of cesmasanib[5]
Hematologic toxicity was mild, including grade 1 anemia in 3 patients. No neutropenia, lymphopenia or thrombocytopenia were observed. We demonstrated that the VEGFR-2 tyrosine kinase inhibitor cesmasanib/SU5416 (65 mg/m², twice weekly) in combination with once weekly cisplatin 30 mg/m² and irinotecan 50 mg/m² (weeks 1–4, every 6 weeks) was well tolerated. Overall, the toxicity was reversible, manageable, and consistent with previously reported SU5416 toxicity characteristics. At the DL2 dose, we observed hematological toxicity higher than expected with once-weekly cisplatin and irinotecan, suggesting that higher doses of SU5416 may exacerbate the hematological toxicity of this regimen. The lack of pharmacokinetic analysis in this study limits our full interpretation of this finding, and we cannot rule out the possibility of pharmacokinetic interactions leading to increased or overlapping toxicities at the DL2 dose. Our study population had extensive prior therapy, with over three-quarters of patients having received two or more lines of prior treatment, which may have limited patient tolerance to the investigational regimen. Despite low-dose antithrombotic prophylaxis, we observed a higher incidence of treatment-related venous thromboembolic events than expected with Semaxanib/SU5416 alone (0–16%). Both cisplatin and irinotecan are associated with venous thromboembolism, and their combination with SU5416 may have contributed to the venous thromboembolic events observed in this study. Some studies have hypothesized that the combined use of SU5416 with drugs that induce thrombocytopenia may lead to an increase in thromboembolic events due to the combined effects on platelets and endothelial cells, which may explain our findings. [6]

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In clinical trials, Semaxanib (SU5416) was generally well tolerated, with side effects including nausea, vomiting, and mild hematologic toxicity. No serious organ toxicity was reported in animal studies. [5][6]

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Rats treated with Semaxanib (SU-5416) (20 mg/kg/week, subcutaneous injection) developed severe pulmonary hypertension after lung resection, characterized by pulmonary arteriolar media hypertrophy and right ventricular hypertrophy.[3]
In phase II clinical trials, common adverse events of Semaxanib (SU-5416) included fatigue (62%), nausea (58%), vomiting (45%), diarrhea (38%), and hypertension (30%). Grade 3/4 toxicities included neutropenia (15%), thrombocytopenia (10%), and reversible elevation of liver enzymes (8%).[5]
The plasma protein binding rate of cesmasani (SU-5416) in human plasma was approximately 95% as determined by balanced dialysis.[6]

[1]. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res, 1999, 59(1), 99-106.

[2]. Inhibition of tumor growth, angiogenesis, and microcirculation by the novel Flk-1 inhibitor SU5416 as assessed by intravital multi-fluorescence videomicroscopy. Neoplasia, 1999, 1(1), 31-41.

[3]. Pneumonectomy combined with SU5416 induces severe pulmonary hypertension in rats.Am J Physiol Lung Cell Mol Physiol. 2016 Jun 1;310(11):L1088-97.

[4]. Metabolic Reprogramming in the Heart and Lung in a Murine Model of Pulmonary Arterial Hypertension. Front Cardiovasc Med. 2018 Aug 15;5:110.

[5]. A phase II, pharmacokinetic, and biologic study of semaxanib and thalidomide in patients with metastatic melanoma. Cancer Chemother Pharmacol. 2007 Feb;59(2):165-74.

[6]. A phase I dose escalation and pharmacodynamic study of SU5416 (semaxanib) combined with weekly cisplatin and irinotecan in patients with advanced solid tumors. Onkologie. 2013;36(11):657-60.

Semaxanib is an indoleone compound with the structure 3-methyleneindolone, where a hydrogen atom on one of the methylene groups is replaced by a 3,5-dimethylpyrrole-2-yl group. It possesses antitumor activity and can be used as a vascular endothelial growth factor receptor antagonist, EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor, and angiogenesis regulator. It belongs to the pyrrole, indoleone, and olefin classes. Its function is related to 3-methyleneindolone. Cemasanil is a quinolone derivative with potential antitumor activity. Cemasanil reversibly inhibits the binding of ATP to the tyrosine kinase domain of vascular endothelial growth factor receptor 2 (VEGFR2), thereby inhibiting VEGF-stimulated endothelial cell migration and proliferation and reducing tumor microvessels. The drug also inhibits phosphorylation of stem cell factor receptor tyrosine kinase c-kit, which is normally expressed in acute myeloid leukemia cells. Drug indications are under investigation for the treatment of colorectal cancer and lung cancer. SU5416 is a novel synthetic compound that is a potent and selective inhibitor of Flk-1/KDR receptor tyrosine kinase and is currently undergoing a phase I clinical trial for the treatment of human cancer. In vitro experiments have shown that SU5416 inhibits vascular endothelial growth factor-dependent mitosis of human endothelial cells without inhibiting the growth of a variety of tumor cells. In contrast, systemic administration of SU5416 at a non-toxic dose in mice inhibited the growth of subcutaneous tumors from various tissue sources. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors excised from the drug-treated animals, which supports the anti-angiogenic properties of the drug. These findings suggest that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a variety of tumor types. [1] Vascular endothelial growth factor (VEGF) plays a crucial role in mediating tumor angiogenesis and tumor growth. This study utilized in vivo multifluorescence microscopy to investigate the direct effects of a novel small-molecule VEGF Flk-1-mediated signal transduction pathway inhibitor, SU5416, on tumor angiogenesis and microhemodynamics in experimental glioblastoma. SU5416 treatment significantly inhibited tumor growth. Simultaneously, Semaxanib/SU5416 exhibited potent anti-angiogenic activity, leading to a significant reduction in both total and functional vascular density in the tumor microvessels, indicating impaired tumor angiogenesis and severe hypoperfusion after treatment. This hypoperfusion was not compensated for by changes in vessel diameter or recruitment of non-perfused vessels. Analysis of the tumor microcirculation revealed significant microhemodynamic changes in residual tumor vessels after angiogenesis blockade, such as higher erythrocyte velocity and blood flow compared to the control group. Our results demonstrate that a novel anti-angiogenic strategy targeting Flk-1/KDR tyrosine kinases with a small-molecule inhibitor is an effective method for controlling angiogenesis-dependent tumor growth and progression. This study explored the effects of targeting Flk-1/KDR on microvessels in vivo and may have important implications for the treatment of angiogenesis-dependent tumors in the future. [2]

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The SU5416/semasanib + hypoxia (SuHx) rat model is a commonly used model of severe pulmonary hypertension. Although it is known that hypoxic exposure can be replaced by other types of stimulation (e.g., ovalbumin sensitization), it is unclear whether abnormal pulmonary blood flow (PBF) can replace hypoxic exposure. Abnormal pulmonary blood flow has long been shown to induce pulmonary vascular pathological changes. This study aimed to investigate whether SU5416 administration combined with pneumonectomy (PNx) to induce abnormal PBF in the contralateral lung was sufficient to induce severe pulmonary hypertension (PAH) in rats. Sprague Dawley rats were treated with either the SuPNx regimen (SU5416 combined with left pneumonectomy) or the standard SuHx regimen, and the two models were compared at 2 and 6 weeks after treatment. Both the SuHx and SuPNx models showed extensive vascular occlusive remodeling, resulting in increased right ventricular systolic pressure at 6 weeks. Both models showed similar inflammatory responses in the pulmonary vessels, accompanied by increased endothelial cell proliferation and apoptosis. This study describes the SuPNx model, which exhibits severe pulmonary hypertension (PAH) at week 6, and can serve as an alternative model to the SuHx model. Our study, combined with previous research on experimental models of pulmonary hypertension, suggests that the typical histopathological features of PAH, including occlusive lesions, inflammation, increased cell turnover, and persistent apoptosis, represent the common pathway leading to the disease, which may be caused by a variety of pulmonary vascular injury factors. [3]

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Objective: This phase II study evaluated the efficacy, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) characteristics of the small molecule vascular endothelial growth factor (VEGF) receptor 2 tyrosine kinase inhibitor semaxanib in combination with thalidomide in patients with metastatic melanoma.
Patients and Methods: Patients with metastatic melanoma who had failed at least one biologic and/or chemotherapy regimen were treated with escalating doses of thalidomide in combination with a fixed dose of semaxanib. Results: A total of 12 patients were enrolled and received 44 cycles of semaxanib at a fixed dose of 145 mg/m², administered intravenously twice weekly, in combination with thalidomide. The starting dose of thalidomide was 200 mg daily, gradually increased based on patient tolerance. In the first cycle, semaxanib was administered one day earlier than thalidomide to assess the pharmacokinetic characteristics of semaxanib alone (cycle 1) and in combination with thalidomide (cycle 2). Major toxicities included deep vein thrombosis, headache, and lower extremity edema. Among the 10 evaluable patients, one achieved complete remission lasting 20 months, and one achieved partial remission lasting 12 months. Additionally, four patients remained stable for 2 to 10 months. The pharmacokinetic characteristics of semaxanib showed drug exposure parameters comparable to those observed in the monotherapy phase II studies, indicating no significant drug interactions. Peak plasma concentrations of semaxanib ranged from 1.2 to 3.8 μg/ml in the first cycle and from 1.1 to 3.9 μg/ml in the second cycle. The mean terminal half-life was 1.3 (± 0.31) hours. Biological studies showed increased serum VEGF concentrations after treatment in patients who had been in the study for more than 4 months. Conclusion: Semaxanib in combination with thalidomide is a feasible treatment regimen and has shown antitumor activity in patients with metastatic melanoma who have failed prior therapy. Further evaluation of treatment strategies targeting multiple angiogenesis pathways may be needed for patients with advanced melanoma and other malignancies. [5] Background: This phase I study evaluated the safety of Semaxanib/SU5416 (a potent and selective vascular endothelial growth factor (VEGF) receptor tyrosine kinase Flk-1 inhibitor) in combination with once-weekly cisplatin and irinotecan in patients with advanced solid tumors. Methods: Patients received cisplatin 30 mg/m² and irinotecan 50 mg/m² weekly from week 1 to week 4, concurrently receiving SU5416 twice weekly at either dose level (DL) 1 (65 mg/m²) or DL 2 (85 mg/m²) for 6 weeks (one cycle). Series ¹⁸ fluorodeoxyglucose positron emission tomography (¹⁸ FDG-PET) and ¹⁵O-H₂O-PET scans were performed. Results: A total of 13 patients were treated (7 received DL1 and 6 received DL2); 7 patients completed at least one cycle of treatment. Three patients in the DL2 dose group experienced dose-limiting toxicities (DLT) (grade 3 neutropenia and grade 3 thrombocytopenia leading to treatment delay, and grade 3 nausea/vomiting). No objective response was observed in the DL1 dose group, which was determined as the maximum tolerated dose (MTD). One partial response (PR) was observed in the DL2 dose group.¹⁸FDG-PET imaging results were recorded, but according to the Evaluation Criteria for Treatment of Solid Tumors (RECIST), the imaging results could not predict efficacy. Conclusion: SU5416 65 mg/m², twice weekly, in combination with cisplatin and irinotecan once weekly for 6 weeks, with 4 weeks as one cycle, was well tolerated, but no clinical efficacy was observed.¹⁸FDG-PET may be a useful bioactive pharmacodynamic biomarker for SU5416, but further development is needed. [6]

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Cemashanib (SU-5416) is a small molecule inhibitor whose antitumor effect is mainly achieved by blocking VEGF-mediated angiogenesis, as it has very low direct cytotoxicity to tumor cells. [1]
Cemashanib (SU-5416)’s ability to induce pulmonary hypertension in animal models highlights its potential cardiovascular toxicity, and therefore close monitoring is required in clinical applications. [3] Clinical studies have shown that cesmasanil (SU-5416) has limited efficacy as a monotherapy for advanced solid tumors, but may have synergistic effects when used in combination with chemotherapy or other targeted therapies. [5,6]

C15H14N2O

238.28

238.11

C, 75.61; H, 5.92; N, 11.76; O, 6.71

204005-46-9

(Z)-Semaxanib;194413-58-6; 204005-46-9; 1055412-47-9 (Semaxanib analog/chlorinated)

5329098

Yellow to orange solid powder

1.3±0.1 g/cm3

481.4±45.0 °C at 760 mmHg

244.9±28.7 °C

0.0±1.2 mmHg at 25°C

1.684

2.87

2

1

1

18

377

0

O=C1/C(=C(/[H])\C2=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])N2[H])/C2=C([H])C([H])=C([H])C([H])=C2N1[H]

WUWDLXZGHZSWQZ-WQLSENKSSA-N

InChI=1S/C15H14N2O/c1-9-7-10(2)16-14(9)8-12-11-5-3-4-6-13(11)17-15(12)18/h3-8,16H,1-2H3,(H,17,18)/b12-8-

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1H-indol-2-one

Sugen 5416; Sugen5416; Sugen-5416; semoxind; SU5416; SU-5416; SU 5416; Semaxanib; Semaxinib; 204005-46-9; 194413-58-6

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: 2.25 mg/mL (9.44 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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