α5β1 Integrin

Inhibits the adhesion of human umbilical vein endothelial cells (HUVECs) to fibronectin. The mechanism is that ATN - 161 can block the binding of integrin α5β1 to fibronectin, thereby inhibiting cell adhesion, which is detected by a cell adhesion assay [2]
When compared to control and single-agent therapy, the combination of ATN-161 plus 5-FU significantly reduces tumor cell proliferation (p<0.01). Furthermore, apoptotic (TUNEL-positive) tumor cells are significantly increased by combination therapy (p <0.03), while no increase in TUNEL-positive tumor cells is observed with single-agent therapy. After 48 hours of incubation, ATN-161 treatment significantly lowers the EC number (21% decrease) in comparison to the control group (p<0.03) [1]. ATN-161 did not stop proliferation in hCECs, but it did prevent VEGF-induced migration and capillary tube formation. Starting at 100 nM (P <0.001 vs. VEGF group), ATN-161 reduces the number of cells migrating in response to VEGF in a dose-dependent manner[2].

- When combined with 5 - FU, it can reduce colorectal liver metastases and improve the survival rate of mice. In a mouse model of colon cancer, ATN - 161 is administered subcutaneously at a dose of 100 μg per mouse per day, and 5 - FU is continuously infused intravenously. As a result, the number of liver metastases is significantly reduced, and the survival time of mice is prolonged [1]
- Inhibits angiogenesis in vivo. In a mouse model, after subcutaneous injection of ATN - 161, it can reduce the formation of new blood vessels in the tumor, which is evaluated by measuring the micro - vessel density of the tumor tissue [2]

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Preliminary studies using human colon cancer xenografts (HT29) lacking α5β1 demonstrate that ATN-161 treatment dramatically lowers tumor weight and vessel density[1]. Following laser photocoagulation, injection of ATN-161 inhibits choroidal neovascularization (CNV) leakage and neovascularization to a degree comparable to that of AF564[2].

For the cell adhesion assay of HUVECs, first, coat 96 - well plates with fibronectin. Then, add HUVECs suspended in serum - free medium to the wells, and add different concentrations of ATN - 161 at the same time. Incubate for a certain period at 37 °C, wash the unbound cells, and then fix and stain the adherent cells. Finally, measure the absorbance at a specific wavelength by a micro - plate reader to evaluate the adhesion ability of cells [2]

- In the experiment of reducing colorectal liver metastases in mice, dissolve ATN - 161 in saline, and administer it subcutaneously to mice at a dose of 100 μg per mouse per day. Continuously infuse 5 - FU intravenously at a dose of 25 mg/kg per day. Observe the number of liver metastases and the survival status of mice [1]
- In the angiogenesis inhibition experiment, dissolve ATN - 161 in saline, and subcutaneously inject it into mice. The specific dose is not mentioned. After a certain period, sacrifice the mice, take out the tumor tissue, and measure the micro - vessel density to evaluate the anti - angiogenesis effect [2]

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saline; 0.05-1 mg/kg/d; i.v.
BALB/c nu/nu mice

[1]. Inhibition of integrin alpha5beta1 function with a small peptide (ATN-161) plus continuous 5-FU infusion reduces colorectal liver metastases and improves survival in mice. Int J Cancer. 2003 Apr 20;104(4):496-503.

[2]. The antiangiogenic effects of integrin alpha5beta1 inhibitor (ATN-161) in vitro and in vivo. Invest Ophthalmol Vis Sci. 2011 Sep 14;52(10):7213-20.

ATN-161 is a pentapeptide derived from the fibronectin co-region. It exerts its anti-tumor and anti-angiogenic effects by blocking integrin-mediated signaling pathways, primarily targeting integrin α5β1. ATN-161 blocks the binding of integrin α5β1 to fibronectin, inhibiting cell adhesion and thus suppressing tumor angiogenesis and cancer cell metastasis. ATN-161 is a non-RGD-based integrin-binding peptide that targets both integrin α5β1 and αvβ3. It inhibits the migration and adhesion of specific integrins on activated endothelial cells, which play a key role in tumor angiogenesis. This strategy of simultaneously targeting tumor blood vessels and cancer cells themselves may be effective in both monotherapy and combination therapy. Since the expression of α5β1 integrin in cancer cells and its role in tumor angiogenesis are similar across various cancers, the therapeutic benefits of ATN-161 are expected to extend to multiple cancers.
The α5β1 inhibitor ATN-161 is a small peptide antagonist of α5β1 integrin with potential anti-tumor activity. ATN-161 selectively binds to and blocks the α5β1 integrin receptor, thereby preventing the binding of α5β1 integrins. This receptor blockade may lead to the inhibition of endothelial cell-interstitial interactions, endothelial cell-matrix interactions, angiogenesis, and tumor progression. Integrin α5β1 is expressed on endothelial cells and plays a key role in endothelial cell adhesion and migration.

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Drug Indications
Investigated for the treatment of brain cancer and unspecified cancers/tumors.

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Mechanism of Action
ATN-161 is a non-RGD-based integrin-binding peptide that targets α5β1 and αvβ3. It inhibits the migration and adhesion of specific integrins on activated endothelial cells, which play a key role in tumor angiogenesis. β-integrins, including β1, β3, and β5 isoforms, are present on endothelial cells and mediate endothelial cell-extracellular matrix interactions. Integrin α5β1 is expressed on activated endothelial cells and plays a key role in tumor angiogenesis, thus being particularly associated with cancer progression. Similarly, α5β1 integrin is also present on many tumor cells and plays a crucial role in cell adhesion and migration. Therefore, blocking this integrin can directly affect tumor progression and indirectly affect it by inhibiting angiogenesis.
Pharmacodynamics
ATN-161 is a non-RGD-based integrin-binding peptide that targets α5β1 and αvβ3 integrins. It inhibits the migration and adhesion of specific integrins on activated endothelial cells, which play a vital role in tumor angiogenesis. Since integrin overexpression and angiogenesis are common in various cancer types, this therapy is expected to be applicable to a wide range of cancers.

C23H35N9O8S

597.64

597.232

C, 46.22; H, 5.90; N, 21.09; O, 21.42; S, 5.36

262438-43-7

ATN-161 trifluoroacetate salt;904763-27-5; 904763-50-4 (mesylate); 262438-43-7; 904763-42-4 (HCl); 904763-58-2 (acetate); 904763-74-2 (sulfate)

9960285

N-acetyl-L-prolyl-L-histidyl-L-seryl-L-cysteinyl-L-asparaginamide; Ac-Pro-His-Ser-Cys-Asn-NH2

PHSCN; [acetyl]-PHSCN-[NH2]

Typically exists as solid at room temperature

1.4±0.1 g/cm3

1297.2±65.0 °C at 760 mmHg

738.3±34.3 °C

0.0±0.3 mmHg at 25°C

1.612

-3.52

9

10

15

41

1020

5

S([H])C([H])([H])[C@@]([H])(C(N([H])[C@]([H])(C(N([H])[H])=O)C([H])([H])C(N([H])[H])=O)=O)N([H])C([C@]([H])(C([H])([H])O[H])N([H])C([C@]([H])(C([H])([H])C1=C([H])N=C([H])N1[H])N([H])C([C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])([H])[H])=O)=O)=O)=O

MMHDBUJXLOFTLC-WOYTXXSLSA-N

InChI=1S/C23H35N9O8S/c1-11(34)32-4-2-3-17(32)23(40)29-14(5-12-7-26-10-27-12)20(37)30-15(8-33)21(38)31-16(9-41)22(39)28-13(19(25)36)6-18(24)35/h7,10,13-17,33,41H,2-6,8-9H2,1H3,(H2,24,35)(H2,25,36)(H,26,27)(H,28,39)(H,29,40)(H,30,37)(H,31,38)/t13-,14-,15-,16-,17-/m0/s1

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)

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.)