ETA ( IC50 = 0.055 nM )
Atrasentan (ABT-627, 0-50 μM) strongly inhibits the growth of LNCaP and C4-2b prostate cancer cells. When combined with Taxotere, ABT-627 causes a notably higher reduction in viable prostate cancer cells compared to when either drug is used alone. It also exhibits a higher degree of NF-κB DNA binding activity down-regulation[2]. Atrasentan significantly induces a number of CYPs and drug transporters (CYP3A4 is 12-fold induced at 50 μM, for example). It is a weak BCRP inhibitor (IC50 in MDCKII-BCRP cells = 59.8±11 μM) and a moderate P-gp inhibitor (IC50 in P388/dx cells = 15.1±1.6 μM)[3].

Atrasentan (ABT-627, 0-50 μM) strongly inhibits the growth of LNCaP and C4-2b prostate cancer cells. When combined with Taxotere, ABT-627 causes a notably higher reduction in viable prostate cancer cells compared to when either drug is used alone. It also exhibits a higher degree of NF-κB DNA binding activity down-regulation[2]. Atrasentan significantly induces a number of CYPs and drug transporters (CYP3A4 is 12-fold induced at 50 μM, for example). It is a weak BCRP inhibitor (IC50 in MDCKII-BCRP cells = 59.8±11 μM) and a moderate P-gp inhibitor (IC50 in P388/dx cells = 15.1±1.6 μM)[3].

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Atrasentan concentration-dependently inhibited the growth of androgen receptor (AR)-positive prostate cancer cell lines LNCaP and C4-2b, but showed minimal effect on the AR-negative PC-3 cell line.
In LNCaP cells, treatment with 10 µM, 25 µM, and 50 µM of Atrasentan for 72 hours reduced cell viability by 18%, 30%, and 60%, respectively.
In C4-2b cells, the same treatment resulted in 15%, 32%, and 56% reduction in cell viability.
Combination treatment with Atrasentan (25 µM) and docetaxel (Taxotere, 1 nM) for 72 hours resulted in significantly greater growth inhibition (~60-70% reduction in viability) and induction of apoptosis in LNCaP and C4-2b cells compared to either agent alone (~40% inhibition).
The pro-apoptotic effect of the combination was confirmed by histone-DNA ELISA and flow cytometry, showing an increased sub-G0-G1 fraction.
Mechanistically, Atrasentan treatment (25 µM, 72 hours) down-regulated constitutive nuclear factor-κB (NF-κB) DNA binding activity in C4-2b cells in a dose- and time-dependent manner.
Combination treatment led to greater down-regulation of NF-κB activity compared to single agents.
Western blot analysis showed that combination treatment with Atrasentan and docetaxel reduced the expression of anti-apoptotic proteins Bcl-2, Bcl-xL, and survivin, increased cleavage of PARP (a marker of apoptosis), and inhibited phosphorylation of Akt and its substrate GSK-3α/β in C4-2b cells.

Atrasentan (3 mg/kg, p.o.) suppresses the pressor response brought on by big endothelin-1 (1 nmol/kg) in pithed rats[1]. Aatrasentan (ABT-627, 10 mg/kg, i.p.) and Taxotere alone partially inhibited the growth of C4-2b tumors within the bone environment in the SCID-hu model[2].

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In a SCID-hu mouse model of experimental prostate cancer bone metastasis (using C4-2b cells implanted into human bone), Atrasentan (10 mg/kg, intraperitoneal injection daily) in combination with docetaxel (5 mg/kg, intravenous injection every 3rd day for a total of four doses) for 5 weeks resulted in a 90% reduction in tumor volume compared to the untreated control group.
The antitumor activity of the combination was significantly superior to that of either Atrasentan or docetaxel alone.
Serum prostate-specific antigen (PSA) levels were significantly decreased in the combination treatment group, correlating with reduced tumor burden.
Analysis of tumor tissues from the combination group showed down-regulation of NF-κB DNA binding activity and reduced protein expression of its downstream targets, survivin and Bcl-2, consistent with the in vitro findings.
No significant treatment-related toxicity was observed, as indicated by stable body weights of the mice throughout the study.

Atrasentan is used to treat and incubate the cells. After two PBS washes, they are lysed in an ice-cold lysis buffer [1 mM EGTA, 1 mM EDTA, 1.5 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium PPi, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/mL leupeptin, and 1 mM PMSF]. After centrifuging the extracts to get rid of any remaining cell debris, the bicinchoninic acid (BCA) protein assay reagent is used to measure the amount of protein in the supernatants. Proteins (150 μg) cross-linked to agarose hydrazide beads and incubated with mild rocking at 4°C for the entire night. The lysis buffer and kinase assay buffer (25 mM Tris (pH 7.5), 10 mM MgCl₂, 5 mM β-glycerol phosphate, 0.1 mM sodium orthovanadate, 2 mM DTT) are used to wash the immunoprecipitated products twice after Akt is selectively immunoprecipitated from the cell lysates. The products are then resuspended in 40 μL of kinase assay buffer that contains 200 μM ATP and 1 μg GSK-3α/β fusion protein. The addition of Lamelli SDS sample buffer stops the kinase assay reaction after it has been running for 30 minutes at 30°C. 10% SDS-PAGE is used to resolve the reaction products, and then an antiphosphorylated GSK-3α/β antibody is used for Western blotting. After separating 40 μg of protein from the lysate samples using 10% SDS-PAGE, anti-Akt antibody is used for Western blotting in order to determine the total amount of Akt.

In 96-well microtiter culture plates, the three prostate cancer cell lines (LNCaP, C4-2b, and PC-3) are seeded at a density of 3 × 10³ cells per well. Following an overnight incubation period, the medium is taken out and replaced with a new one that has been diluted from a 10-mM stock to contain varying concentrations of ABT-627 (0-50 μM). 20 μL of MTT solution (5 mg/mL in PBS) is added to each well after the drug has been incubated for 72 hours, and the wells are then incubated for an additional two hours. After the process is finished, the MTT formazan that was produced by metabolically viable cells is dissolved in 100 μL of isopropanol and the supernatant is aspirated. The absorbance is measured at 595 nm on a plate reader after the plates are mixed for 30 minutes on a gyratory shaker.

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Cell Culture and Growth Inhibition Assay (MTT): Human prostate cancer cell lines (LNCaP, C4-2b, PC-3) were seeded in 96-well plates. After overnight attachment, cells were treated with increasing concentrations of Atrasentan (0-50 µM) or a fixed concentration of Atrasentan (25 µM) alone or in combination with docetaxel (1 nM) for 72 hours. Cell viability was assessed by adding MTT solution. The formazan product was dissolved in isopropanol, and absorbance was measured at 595 nm.
Apoptosis Detection by ELISA: LNCaP and C4-2b cells were treated with Atrasentan (25 µM) and/or docetaxel (1 nM) for 72 hours. Cytoplasmic histone/DNA fragments were extracted and quantified using a cell death detection ELISA kit according to the manufacturer's instructions. Absorbance was measured at 405 nm.
Apoptosis Detection by Flow Cytometry: C4-2b cells were treated with Atrasentan (25 µM) and/or docetaxel (1 nM) for 48 hours. Cells were harvested, fixed in ethanol, stained with propidium iodide and RNase A, and analyzed by flow cytometry. The percentage of apoptotic cells was determined by calculating the fraction of cells in the sub-G0-G1 phase.
Protein Extraction and Western Blot Analysis: C4-2b cells were treated with Atrasentan (25 µM) and/or docetaxel (1 nM) for 72 hours. Cells were lysed, and protein extracts were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies against PARP, survivin, Bcl-2, Bcl-xL, Bax, phosphorylated Akt, total Akt, and β-actin.
Akt Kinase Activity Assay: Treated C4-2b cells were lysed. Akt was immunoprecipitated from the lysates using an immobilized Akt antibody. The immunoprecipitates were incubated with ATP and a GSK-3α/β fusion protein substrate. Phosphorylation of GSK-3α/β was detected by Western blot using a phospho-specific antibody.
Electrophoretic Mobility Shift Assay (EMSA): Nuclear extracts were prepared from treated C4-2b cells. The extracts were incubated with an IRDye-700-labeled NF-κB oligonucleotide probe. DNA-protein complexes were separated on a native polyacrylamide gel and visualized using an infrared imaging system.

Rats are orally given YM598 (0.3, 1, and 3 mg/kg), atrasentan (0.3, 1, and 3 mg/kg), or 0.5% methyl cellulose as a vehicle using a dosing cannula. 5 mL/kg is the dosage volume for both the test material and the vehicle. The rats are anesthetized with sodium pentobarbital about 20 minutes after the compounds are administered, and they are then pithed and ventilated 30 minutes after the dosage. Big endothelin-1 (1 nmol/kg) is injected intravenously and blood pressure is recorded about an hour after the compounds are taken orally. In these two experiments, linear regression analysis is used to determine the dose of test compound that causes 50% inhibition (ID50) of the big endothelin-1-induced increase in diastolic blood pressure.

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SCID-hu Mouse Model of Bone Metastasis: Male CB-17 SCID mice were implanted with a fragment of human fetal bone. After engraftment, C4-2b prostate cancer cells (1x10^6 cells in serum-free medium) were injected directly into the bone marrow space.
Treatment Regimen: Therapy began approximately 30 days after tumor cell inoculation, when bone implants showed signs of enlargement. Mice were randomized into four groups (n=7 per group): (1) Untreated control; (2) Atrasentan alone (10 mg/kg body weight, intraperitoneal injection, daily for 5 weeks); (3) Docetaxel alone (5 mg/kg body weight, intravenous injection, every 3rd day for a total of four doses); (4) Combination of Atrasentan and docetaxel, following the same schedules as individual treatments.
Tumor Measurement and Sample Collection: Tumor volume was measured twice weekly using calipers. Mice were euthanized one day after the last Atrasentan dose (5 weeks). Tumors were excised, weighed, and processed for histology (H&E staining) and molecular analysis (protein extraction). Serum was collected for PSA measurement.
Drug Formulation: The formulation for Atrasentan is not explicitly detailed beyond being dissolved appropriately for intraperitoneal injection. Docetaxel was obtained as a clinical formulation.

[1]. Superiority of YM598 over atrasentan as a selective endothelin ETA receptor antagonist. Eur J Pharmacol. 2004 Sep 13;498(1-3):171-7.

[2]. In vitro and in vivo molecular evidence for better therapeutic efficacy of ABT-627 combination in prostate cancer. Cancer Res. 2007 Apr 15;67(8):3818-26.

[3]. Interaction potential of the endothelin-A receptor antagonist atrasentan with drug transporters and drug-metabolising enzymes assessed in vitro. Cancer Chemother Pharmacol. 2011 Oct;68(4):1093-8.

Atrasentan belongs to the pyrrolidine class of compounds. Atrasentan is a substance under investigation for cancer treatment. It belongs to the class of endothelin-1 protein receptor antagonists. It is a novel selective endothelin A receptor antagonist (SERA). It is a pyrrolidine and benzodioxane derivative that acts as a receptor antagonist. It has therapeutic potential as an anti-tumor drug and for treating diabetic nephropathy.

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Drug Indications
It has been investigated for the treatment of prostate cancer and unspecified cancers/tumors.

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Atrasentan is a highly bioavailable, selective ETA receptor antagonist. By blocking the interaction between endothelin-1 (ET-1) and its ETA receptor, atrasentan inhibits the ET-1-mediated signaling pathway, thereby inhibiting the survival, proliferation, and osteoblastic bone metastasis of prostate cancer cells. This study provides preclinical evidence that atrasentan combined with the chemotherapy drug docetaxel demonstrates superior antitumor efficacy both in vitro and in vivo, particularly in an androgen receptor-positive prostate cancer model, compared to monotherapy. Mechanistically, this enhanced activity is associated with the synergistic downregulation of the Akt/NF-κB survival signaling pathway and its downstream anti-apoptotic targets (e.g., Bcl-2, survivin). These findings support potential clinical trials of atrasentan in combination with taxane chemotherapy for metastatic prostate cancer.

C29H38N2O6

510.62182

510.273

C, 68.21; H, 7.50; N, 5.49; O, 18.80

173937-91-2

Atrasentan hydrochloride; 195733-43-8; 178738-96-0 (sodium); 195704-72-4

159594

White to light yellow solid powder

1.188g/cm3

659.4ºC at 760mmHg

352.6ºC

2.76E-18mmHg at 25°C

4.631

1

7

12

37

734

3

O=C([C@H]1[C@H](C2=CC=C(OC)C=C2)N(CC(N(CCCC)CCCC)=O)C[C@@H]1C3=CC=C(OCO4)C4=C3)O

MOTJMGVDPWRKOC-QPVYNBJUSA-N

InChI=1S/C29H38N2O6/c1-4-6-14-30(15-7-5-2)26(32)18-31-17-23(21-10-13-24-25(16-21)37-19-36-24)27(29(33)34)28(31)20-8-11-22(35-3)12-9-20/h8-13,16,23,27-28H,4-7,14-15,17-19H2,1-3H3,(H,33,34)/t23-,27-,28+/m1/s1

(2R,3R,4S)-4-(1,3-benzodioxol-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid

ABT-627; (+)-A 127722; ABT627; ABT 627; ABT-627; NSC720763; A147627; Abbott 147627; trade name: Xinlay; A-147627

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.)