VEGFR2 (IC50 = 0.2 nM); VEGFR3 (IC50 = 0.7 nM); c-Kit (IC50 = 14.8 nM); c-Kit (IC50 = 14.8 nM); c-Kit (IC50 = 14.8 nM)

Anlotinib shows high selectivity and inhibitory potency (IC 50 <1 nmol/L) for VEGFR2 in comparison to other tyrosine kinases, and it occupies the ATP-binding pocket of the VEGFR2 tyrosine kinase. With IC50 values of 0.2 and 0.7 nmol/L, respectively, anlotinib inhibits VEGFR2 and VEGFR3. With an IC50 value of 26.9 nmol/L, anlotinib has a lower inhibitory potency against VEGFR1. Anlotinib's IC50 values for inhibiting PDGFR-related kinases c-Kit and PDGFRβ are 14.8 and 115.0 nmol/L, in that order. Anlotinib has minimal impact, even at 2000 nmol/L, on the activity of other kinases such as c-Met, c-Src, EGFR, and HER2. In HUVEC, anlotinib has picomolar IC50 values that suppress VEGF-induced signaling and cell proliferation. Nevertheless, in vitro tumor cell proliferation must be directly inhibited by anlotinib at micromolar concentrations. Anlotinib strongly inhibits the migration and formation of tubes in HUVECs as well as the microvessel growth from rat aortic explants in vitro[1].

Anlotinib decreases vascular density in tumor tissue in vivo. Anlotinib, taken once daily orally, exhibits greater and more comprehensive antitumor efficacy in vivo when compared to the well-known tyrosine kinase inhibitor sunitinib. In certain models, it also leads to tumor regression in nude mice. In mice, it is well tolerated. Some TKIs require doses of 20–100 mg/kg to significantly inhibit tumor growth in mice, but anlotinib is effective at 1.5–1.6 mg/kg daily, which is significantly lower than those doses[1]. Anlotinib has demonstrated extensive activity in vivo against human tumor xenograft models of the non-small cell lung (Calu-3) and glioma (U87MG), liver (SMMC-7721), kidney (Caki-1), colon (SW-620), ovarian (SK-OV-3), and renal (Ana-1). When given orally to Sprague-Dawley rats and beagle dogs, anlotinib is quickly absorbed from the gastrointestinal tract. Oral bioavailability in rats ranges from 23–45%, while in dogs it ranges from 47–74%. Across both species, anlotinib has a wide distribution. When compared to plasma, anlotinib exposure levels in primary tissues like the lung, kidneys, liver, and heart are significantly higher in rats. The brain exposure level and the matching plasma level are similar. Anlotinib concentrates 2.4–2.6 times more in tumor tissue than in plasma in mice with tumors. Anlotinib has a relatively long t1/2 (96 ± 17 h) in humans that doesn't seem to depend on dosage[2]. Anlotinib's terminal half-life in dogs is 22.8±11.0 h, whereas in rats it is 5.1±1.6 h. The primary reason for this discrepancy seems to be the variation in total plasma clearance between the two species (dogs: 0.40±0.06 L/h/kg versus rats: 5.35±1.31 L/h/kg). Rather than α1-acid glycoprotein or γ-globulins, anlotinib is primarily bound to albumin and lipoproteins in human plasma[3].

The use of ELISA allowed for the determination of anlotinib's inhibitory activity against tyrosine kinases. ATP and tyrosine kinase reacted in reaction buffer (50 mmol/L HEPES pH 7.4, 50 mmol/L MgCl₂, 0.5 mmol/L MnCl₂, 0.2 mmol/L Na₃VO₄, 1 mmol/L DTT) and were incubated in 96-well plates coated with 20 μg/mL Poly(Glu,Tyr)4:1 for one hour at 37°C. HRP-conjugated anti-mouse IgG was added to the plate after the PY99 antibody had been incubated. Analyzer: A Synergy H4 Hybrid reader was used to measure absorbance at 490 nm following reaction with o-phenylenediamine solution and termination with addition of 2N H₂SO₄.

Cells were seeded in 96‐well plates and treated with serial dilutions of drugs. After a 72‐hour incubation, cell proliferation was evaluated by sulforhodamine B (SRB) assay.30 Potency of drugs in inhibiting cell proliferation was expressed as IC50 values, determined using GraphPad Prism version 5 curve‐fitting software[1]

---

Different test agent concentrations are applied to serum-starved HUVEC, Mo7e, U-87MG, and A431 cells for 1.5 hours. The cells are then stimulated for 10 minutes with VEGF (20 ng/mL), SCF-1 (2.5 ng/mL), PDGF-BB (10 ng/mL), or EGF (10 ng/mL). The designated antibodies are used to probe cell lysates.

human colon cancer SW620 xenograft model（Balb/cA-nude mice, 5-6 weeks old)
0.75, 1.5, 3 and 6 mg/kg
oral

---

Female nude mice (Balb/cA‐nude, 5‐6 weeks old), purchased from Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China), were housed in sterile cages under laminar airflow hoods in a specific pathogen‐free room with a 12‐hour light/12‐hour dark schedule, and fed autoclaved chow and water ad libitum. Human tumor xenografts were established by s.c. inoculating cells into the left axilla of nude mice. When tumor volumes reached 100‐200 mm3, mice were divided randomly into control and treatment groups. Control groups were given vehicle alone, and treatment groups received oral anlotinib or sunitinib daily. Tumor volume was calculated as (length × width2)/2. Tumor growth inhibition was calculated from the start of treatment by comparing changes in tumor volumes for control and treatment groups.[1]

---

Rat studies[3]
Rats were randomly assigned to four groups (five male and five female rats per group) to receive a single oral dose of anlotinib at 1.5, 3, or 6 mg/kg (via gavage) or a single intravenous dose at 1.5 mg/kg (from the tail vein). Serial blood samples (around 0.25 mL; before and 5, 15, and 30 min and 1, 2, 4, 6, 8, 11, and 24 h after dosing) were collected in heparinized tubes from the orbital sinuses of rats under isoflurane anesthesia and centrifuged at 1300×g for 10 min to yield plasma fractions. Rats under isoflurane anesthesia were killed by bleeding from the abdominal aorta at 1, 4, 8, and 24 h (three male and three female rats per time point) after a single oral dose of anlotinib at 3 mg/kg. [3]

---

Tumor-bearing mouse studies[3]
Female tumor-bearing mice were randomly assigned to three groups (20 mice per group) to receive a single oral dose of anlotinib at 0.75, 1.5, or 3 mg/kg (via gavage). Mice under isoflurane anesthesia were killed by bleeding from the orbital sinus at 2, 4, 8, and 24 h (five mice per time point) after dosing.

---

Dog study[3]
Dogs were randomly assigned to four groups (three male and three female dogs per group) to receive a single oral dose of anlotinib at 0.5, 1, or 2 mg/kg (via gavage) or a single intravenous dose at 0.5 mg/kg (from left forelimb vein).

[1]. Cancer Sci . 2018 Apr;109(4):1207-1219.

[2]. J Hematol Oncol . 2016 Oct 4;9(1):105.

[3]. Acta Pharmacol Sin . 2018 Jun;39(6):1048-1063.

Catequentinib Hydrochloride is the hydrochloride salt form of catequentinib, a receptor tyrosine kinase (RTK) inhibitor with potential antineoplastic and anti-angiogenic activities. Upon administration, catequentinib targets multiple RTKs, including vascular endothelial growth factor receptor type 2 (VEGFR2) and type 3 (VEGFR3). This agent may both inhibit angiogenesis and halt tumor cell growth.

C23H24CL2FN3O3

480.36

479.117

C, 57.51; H, 5.04; Cl, 14.76; F, 3.96; N, 8.75; O, 9.99

1360460-82-7

1058156-90-3;1360460-82-7 (HCl);

57380530

Solid powder

4

6

6

32

606

0

UUAKQNIPIXQZFN-UHFFFAOYSA-N

InChI=1S/C23H22FN3O3.2ClH/c1-13-9-15-16(27-13)3-4-19(22(15)24)30-18-5-8-26-17-11-21(20(28-2)10-14(17)18)29-12-23(25)6-7-23;;/h3-5,8-11,27H,6-7,12,25H2,1-2H3;2*1H

1-[[4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxyquinolin-7-yl]oxymethyl]cyclopropan-1-amine;dihydrochloride

AL3818 dihydrochloride; AL-3818 dihydrochloride; AL 3818 dihydrochloride; Anlotinib HCl; AL3818; Anlotinib dihydrochloride; Anlotinib HCl; 1360460-82-7; Anlotinib hydrochloride; AL3818 dihydrochloride; Catequentinib Hydrochloride; CATEQUENTINIB DIHYDROCHLORIDE; A3749M6582;AL 3818; AL-3818; Anlotinib; Catequentinib

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:≥ 60 mg/mL
Water:
Ethanol:

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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