VEGFR2/KDR (IC50 = 37 nM); VEGFR1/FLT1 (IC50 = 77 nM); VEGFR2/Flk1 (IC50 = 270 nM); PDGFRβ (IC50 = 580 nM); VEGFR3/FLT4 (IC50 = 660 nM)
Vatalanib (PTK-787; ZK-222584; CGP-79787) is described as a non-selective vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor [3].

Vatalanib also inhibits PDGFRβ, Flk, and c-Kit with IC50 values of 580 nM, 730 nM, and 270 nM, respectively. At an IC50 of 7.1 nM, vatalanib inhibits the thymidine incorporation that VEGF induces in HUVECs. It also suppresses VEGF-induced migration and survival of endothelial cells in the same dose range in a dose-dependent manner, without having an adverse effect on cells that do not express VEGF receptors[1]. According to a new study, vatalanib increases the levels of the protein Bax and decreases Bcl-xL and Bcl-2, which significantly inhibits the growth of hepatocellular carcinoma cells and enhances the IFN/5-FU-induced apoptosis[2].

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In human hepatocellular carcinoma (HCC) cell lines PLC/PRF/5 and HuH7, PTK/ZK (5 and 10 µM) combined with interferon-α (IFN, 0.5 µg/mL) and 5-fluorouracil (5-FU, 500 U/mL) significantly inhibited cell growth in a concentration-dependent manner compared to IFN/5-FU alone (P < 0.05). At 10 µM PTK/ZK, the addition reduced viable cell percentages by 22.2% in PLC/PRF/5 and 45.9% in HuH7 cells relative to IFN/5-FU alone. [2]
PTK/ZK (15 µM) combined with IFN/5-FU did not enhance the reduction of VEGF secretion compared to IFN/5-FU alone; both treatments significantly reduced supernatant VEGF to approximately 65–68% of control levels. [2]
PTK/ZK (5–10 µM) combined with IFN/5-FU increased the rate of apoptosis in PLC/PRF/5 cells in a concentration-dependent manner compared to IFN/5-FU alone (P < 0.05). Similar results were observed in HuH7 cells. [2]
Western blot analysis showed that PTK/ZK (10 µM) combined with IFN/5-FU decreased expression levels of anti-apoptotic proteins Bcl-xL and Bcl-2, and increased expression of pro-apoptotic protein Bax compared to control. The combination also modulated cell cycle-related proteins: in PLC/PRF/5 cells, p21 expression was significantly decreased; in HuH7 cells, p27 expression was increased. Cyclin D1 expression was decreased in PLC/PRF/5 cells. [2]
Cell cycle analysis revealed different responses between the two cell lines. In PLC/PRF/5 cells, PTK/ZK (10 µM) combined with IFN/5-FU increased the S-phase fraction and decreased the G0/G1 fraction over 72 hours. In HuH7 cells, the combination increased the G0/G1 fraction and decreased the S-phase fraction over 72 hours. [2]

Vatalanib results in dose-dependent suppression of the angiogenic response to VEGF and PDGF following a single oral dosage (25–100 mg/kg) in two models: one using growth factor implants, the other using tumor cell-driven angiogenesis. Vatalanib also inhibits the growth and metastases of multiple human carcinomas in nude mice within the same dose range, while having no discernible effect on bone marrow leukocytes or circulating blood cells[1].

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The study investigated the effect of Vatalanib (PTK-787; ZK-222584; CGP-79787) on tumor growth in a nude mouse model of human small cell lung cancer (SCLC) micrometastases following radiofrequency ablation (RFA).

Treatment with Vatalanib (PTK-787; ZK-222584; CGP-79787) significantly reduced tumor growth in the reference zones (RZ) of both the ablated lobe (right upper lobe, RUL) and the unablated lobe (right lower lobe, RLL). This was evidenced by a decrease in the pneumonic replacement area (PRA) value in the RFA + PTK/ZK group compared to the RFA group alone at days 1, 7, and 14 post-RFA. For example, in the RZ of the RUL at day 14, the PRA value was 20.7±3.01% in the RFA + PTK/ZK group versus 40.3±5.69% in the RFA group (p=0.014). In the RZ of the RLL at day 14, the PRA value was 12.1±3.12% in the RFA + PTK/ZK group versus 27.9±4.39% in the RFA group (p=0.009).

However, in the transition zone (TZ) surrounding the ablation zone, treatment with Vatalanib (PTK-787; ZK-222584; CGP-79787) did not significantly alter tumor growth. The PRA values in the TZ were similar between the RFA group and the RFA + PTK/ZK group at all time points (e.g., day 14: 52.6±9.89% vs. 54.5±10.1%, p=0.091) [3].

Every GST-fused kinase is cultured in buffer conditions that are optimized. ATP in a 30 μL total volume for 10 minutes at room temperature, either with or without the test drug vatalanib. To stop the reaction, add 10 μL of 250 mM EDTA[1].

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In 96-well plates coated with 1.5% gelatin, subconfluent HUVECs are seeded. A constant concentration of either VEGF (50 ng/mL), bFGF (0.5 ng/mL), or FCS (5%), with or without vatalanib, is added to the basal medium after 24 hours, replacing the growth medium. Additionally included as a control are wells devoid of growth factor. Cells are incubated for a further 24 hours after the addition of the BrdUrd labeling solution, and then they are fixed, blocked, and have peroxidase-labeled anti-BrdUrd antibody added. The 3,3',5,5'-tetramethylbenzidine substrate is then used to detect the bound antibody[1].

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Cell growth inhibition was assessed using the MTT assay. HCC cells were incubated with PTK/ZK (5 or 10 µM) and/or IFN (0.5 µg/mL) and 5-FU (500 U/mL) for 96 hours. Viability was measured at 570 nm and expressed as percentage of untreated controls. [2]
Apoptosis was measured by flow cytometry using annexin V-FITC and propidium iodide (PI) staining. After treatment, cells (1×10⁶) were incubated with binding buffer containing annexin V-FITC and PI for 15 minutes at room temperature, then analyzed on a flow cytometer. [2]
Cell cycle analysis was performed by flow cytometry. Cells were fixed in 70% cold ethanol for 4 hours, then stained with PI (50 µL of 1 mg/mL solution) and RNase for 30 minutes at 37°C. DNA content was analyzed using flow cytometry and cell cycle distribution determined using ModFIT software. [2]
VEGF concentration in cell culture supernatants was measured by human VEGF ELISA after 48 hours of treatment with PTK/ZK (15 µM) and/or IFN/5-FU. [2]
Western blot analysis was performed using standard protocols. Cells were lysed in RIPA buffer containing phosphatase inhibitor. Protein lysates were separated by SDS-PAGE, transferred to membranes, and probed with primary antibodies against Flt-1, KDR/Flk-1, Bcl-xL, Bcl-2, Bax, cyclin D1, p27, p21, and β-actin, followed by secondary antibodies. Band intensities were analyzed densitometrically and normalized to β-actin. [2]

On the dorsal flank of C57/C6 mice, a 0.5 mL porous Teflon chamber containing 0.8% w/v agar, 0.2% heparin (20 units/mL), growth factors (3 g/mL human VEGF, 2 g/mL human PDGF), or neither, is implanted s.c. Beginning one day prior to chamber implantation and continuing for five days thereafter, the mice are treated with either vehicle (water) or Vatalanib (12.5, 25 or 50 mg/kg dihydrochloride p.o. once daily). The treatment concludes with the mice's death and the removal of the chambers. Measurements of the tissue's hemoglobin content are used to determine the amount of blood present after the vascularized tissue surrounding the chamber is carefully removed and weighed[1].

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To evaluate the effect on tumor proliferation, Vatalanib (PTK-787; ZK-222584; CGP-79787) was administered to nude mice with established SCLC micrometastases.
The drug was dissolved in polyethylene glycol 400.
It was administered via intraperitoneal (i.p.) injections at a dose of 50 mg/kg, twice daily.
The treatment regimen began one day prior to the RFA procedure and continued until the mice were sacrificed at day 1, 7, or 14 following RFA [3].

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To evaluate the effect on tumor proliferation, Vatalanib (PTK-787; ZK-222584; CGP-79787) was administered to nude mice with established SCLC micrometastases.
The drug was dissolved in polyethylene glycol 400.
It was administered via intraperitoneal (i.p.) injections at a dose of 50 mg/kg, twice daily.
The treatment regimen began one day prior to the RFA procedure and continued until the mice were sacrificed at day 1, 7, or 14 following RFA [3].

Absorption, Distribution and Excretion
Absorption is rapid. Metabolism/Metabolites Metabolism is primarily through oxidation. Two pharmacologically inactive metabolites, CGP 84368/ZK 260120 and NVP AAW378/ZK 261557, have systemic exposures comparable to vataranilide and are the major contributors to total systemic exposure. Biological Half-Life Approximately 6 hours.

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No significant differences in body weight were observed among treatment groups in the xenograft study, suggesting no obvious adverse effects from the combination therapy (20 mg/kg PTK/ZK daily) during the 4-week treatment period. [2]

[1]. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res. 2000, 60(8.

[2]. Tyrosine kinase inhibitor PTK/ZK enhances the antitumor effects of interferon-α/5-fluorouracil therapy for hepatocellular carcinoma cells. Ann Surg Oncol. 2011, 18(2), 589-596.

[3]. Local recurrence of small cell lung cancer following radiofrequency ablation is induced by HIF-1α expression in the transition zone. Oncol Rep. 2016 Mar;35(3):1297-308.

Vatalanib belongs to the phthalazine class of compounds. Its structure consists of hydrogen atoms at positions 1 and 4 of the phthalazine molecule replaced by p-chlorophenylamino and pyridin-4-ylmethyl, respectively. It is a multi-target tyrosine kinase inhibitor that inhibits all VEGFR, PDGFR, and c-Kit subtypes. Vatalanib possesses multiple functions, including antitumor activity, EC 2.7.10.1 (receptor protein tyrosine kinase) inhibition, angiogenesis inhibition, and vascular endothelial growth factor receptor antagonism. It belongs to the phthalazine, pyridine, monochlorobenzene, and secondary amine classes of compounds. Vatalanib (PTK787/ZK-222584) is a novel oral anti-angiogenic molecule that inhibits all known vascular endothelial growth factor receptors. Vatalanib is being investigated for the treatment of solid tumors. Vatalanib is an orally bioavailable aniline phthalazine drug with potential antitumor activity. Vatalanib binds to and inhibits the activity of the protein kinase domains of vascular endothelial growth factor receptors 1 and 2; both of these receptor tyrosine kinases are involved in angiogenesis. The drug also binds to and inhibits related receptor tyrosine kinases, including platelet-derived growth factor (PDGF) receptors, c-Kit, and c-Fms.
See also: Vatalanib succinate (note moved to).
Indications

In combination with first-line and second-line chemotherapy for the treatment of metastatic colorectal cancer and non-small cell lung cancer (NSCLC).
Mechanism of Action

Valtalanib potently inhibits vascular endothelial growth factor (VEGF) receptor tyrosine kinases, enzymes that play a crucial role in angiogenesis, which contributes to tumor growth and metastasis.
Pharmacodynamics

Valtalanib is a novel oral angiogenesis inhibitor developed by Schering (in collaboration with Novartis). Vatalanib selectively inhibits the tyrosine kinase domains of vascular endothelial growth factor (VEGF) receptor, platelet-derived growth factor (PDGF) receptor, and c-KIT.

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Vatalanib (PTK-787; ZK-222584; CGP-79787) is a specific inhibitor that reduces the expression of vascular endothelial growth factor (VEGF), a key downstream effector of Hypoxia Inducible Factor-1α (HIF-1α) that plays a pivotal role in stimulating angiogenesis. The study used this compound to determine whether tissue angiogenesis is the driving force behind RFA-stimulated tumor growth in different lung zones [3].

C20H15CLN4

346.8129

346.098

C, 69.26; H, 4.36; Cl, 10.22; N, 16.15

212141-54-3

Vatalanib dihydrochloride;212141-51-0;Vatalanib succinate;212142-18-2

151194

White to off-white crystalline powder

1.3±0.1 g/cm3

587.8±50.0 °C at 760 mmHg

209-212ºC

309.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.711

3.8

1

4

4

25

407

0

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])C1C2=C([H])C([H])=C([H])C([H])=C2C(C([H])([H])C2C([H])=C([H])N=C([H])C=2[H])=NN=1

YCOYDOIWSSHVCK-UHFFFAOYSA-N

InChI=1S/C20H15ClN4/c21-15-5-7-16(8-6-15)23-20-18-4-2-1-3-17(18)19(24-25-20)13-14-9-11-22-12-10-14/h1-12H,13H2,(H,23,25)

N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine

PTK787/ZK 222584; CGP-7978; PTK787; PTK 787; PTK-787; ZK 222584; ZK222584; ZK-222584; CGP79787D; CGP 79787; CGP-797870; ZK-232934

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)

DMSO: 21.3~100 mg/mL (50.6~360.4 mM)
Ethanol: ~6 mg/mL (~14.3 mM)
Water: ~10 mg/mL (~23.8 mM)

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.)