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 HCl (ABT-627) treatment (0-50 μM, 72 h) reduced cell viability in a dose-dependent manner in androgen receptor (AR)-positive LNCaP (18%, 30%, 60% reduction at 10, 25, 50 μM) and C4-2b (15%, 32%, 56% reduction at 10, 25, 50 μM) prostate cancer cells, but not in AR-negative PC-3 cells. [2]
Combination of Atrasentan HCl (25 μM) with Taxotere (1 nM) for 72 h caused significantly greater growth inhibition (60-70% reduction) and induction of apoptosis in LNCaP and C4-2b cells compared to either agent alone (~40% reduction). This combination was ineffective in PC-3 cells. [2]
Treatment of C4-2b cells with Atrasentan HCl (5-25 μM) down-regulated constitutive nuclear factor-κB (NF-κB) DNA binding activity in a dose- and time-dependent manner, with greater down-regulation observed in combination with Taxotere. [2]
Western blot analysis in C4-2b cells showed that combination treatment with Atrasentan HCl (25 μM) and Taxotere (1 nM) for 72 h resulted in stronger PARP cleavage and down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, survivin) and phosphorylated Akt compared to single-agent treatments. [2]
Flow cytometry analysis in C4-2b cells treated with the combination of Atrasentan HCl (25 μM) and Taxotere (1 nM) for 48 h showed a 2.88-fold increase in the sub-G0-G1 apoptotic cell population compared to Taxotere alone. [2]

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 (C4-2b cells), combination therapy with Atrasentan HCl (10 mg/kg, i.p., daily for 5 weeks) and Taxotere (5 mg/kg, i.v., every 3rd day for 4 doses) initiated upon tumor detection resulted in 90% inhibition of tumor growth relative to untreated controls after 5 weeks. Single-agent treatments also inhibited growth but to a lesser extent. [2]
Serum prostate-specific antigen (PSA) levels were significantly decreased in all treatment groups, consistent with reduced tumor volumes. [2]
No significant body weight loss was observed in any treatment group, suggesting no major treatment-related toxicity under the experimental conditions. [2]
Analysis of harvested tumor tissues showed significant down-regulation of NF-κB DNA binding activity and decreased expression of its downstream targets, survivin and Bcl-2, in the combination treatment group, corroborating in vitro findings. [2]
Histopathological evaluation of tumors from the combination group showed marked cytoplasmic clearing and vacuolization in tumor cells, formation of smaller tumor nests associated with dense fibrosis, and less prominent osteoblastic rimming on bone fragments in some samples. [2]

In 96-well microtiter culture plates, three times as many cells (LNCaP, C4-2b, and PC-3) are seeded per well, or a density of 3 × 10³ cells. Following an overnight incubation period, the medium is extracted and substituted with a new medium that comprises varying concentrations of ABT-627 (0-50 μM) diluted from a 10-mM stock. Each well receives 20 μL of MTT solution (5 mg/mL in PBS) after the drug has been incubated for 72 hours.The wells are then left to incubate for an additional two hours. The process ends with the aspiration of the supernatant and the dissolution of the MTT formazan (produced by metabolically viable cells) in 100 μL of isopropanol. A plate reader is used to measure the absorbance at 595 nm after the plates are mixed for 30 minutes on a gyratory shaker.

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For cell viability (MTT) assay, prostate cancer cell lines (LNCaP, C4-2b, PC-3) were seeded at 3x10³ cells/well in 96-well plates. After overnight incubation, cells were treated with increasing concentrations of Atrasentan HCl (0-50 μM) or in combination with Taxotere (1 nM) for 72 h. MTT solution (5 mg/mL) was added (20 μL/well) and incubated for 2 h. Formazan crystals were dissolved in isopropanol (100 μL/well), and absorbance was measured at 595 nm. [2]
For apoptosis detection by ELISA, LNCaP and C4-2b cells were treated with Atrasentan HCl (25 μM) and/or Taxotere (1 nM) for 72 h. Cytoplasmic histone/DNA fragments were extracted and detected using an ELISA kit according to the manufacturer's protocol, measuring absorbance at 405 nm. [2]
For flow cytometric analysis of apoptosis, C4-2b cells were treated with Atrasentan HCl (25 μM), Taxotere (1 nM), or their combination for 48 h. Cells (including floating and adherent) were collected, fixed in 75% ethanol, stained with propidium iodide and RNase A, and analyzed by flow cytometry. The percentage of apoptotic cells in the sub-G0-G1 phase was calculated. [2]
For Western blot analysis, C4-2b cells were treated with Atrasentan HCl (25 μM) and/or Taxotere (1 nM) for 72 h. Cells were lysed, and proteins (50 μg) were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies (e.g., PARP, Bcl-2, Bcl-xL, survivin, Akt, p-Akt). Detection was performed using an enhanced chemiluminescence kit. [2]
For Akt kinase activity assay, C4-2b cells were treated and lysed. Akt was immunoprecipitated from lysates (150 μg protein) using immobilized Akt antibody. The immunoprecipitate was incubated with ATP and GSK-3α/β fusion protein substrate at 30°C for 30 min. Phosphorylation of GSK-3α/β was detected by Western blotting. [2]
For electrophoretic mobility shift assay (EMSA), nuclear protein extracts were prepared from treated C4-2b cells. Extracts (8 μg) were incubated with IRDye-700-labeled NF-κB oligonucleotide probe. DNA-protein complexes were separated on native polyacrylamide gels and visualized using an infrared imaging system. [2]

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 NSC 10816 about 20 minutes after the compounds are administered, and 30 minutes after dosing, they are pithed and ventilated. 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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Male homozygous CB-17 SCID/SCID mice (4 weeks old) were implanted with a single human fetal bone fragment. C4-2b prostate cancer cells (1x10⁶ in 20 μL serum-free medium) were injected intraosseously into the implanted bone. [2]
Treatment began when bone implants showed signs of enlargement (~30 days post-injection). Mice were randomized into groups (n=7): untreated control; Atrasentan HCl alone (10 mg/kg, i.p., daily for 5 weeks); Taxotere alone (5 mg/kg, i.v., every 3rd day for 4 doses); combination (Atrasentan HCl + Taxotere, same schedules). Tumor volume was measured twice weekly. [2]
Mice were euthanized one day after the last Atrasentan HCl dose (5 weeks). Tumors were excised, measured, and processed for histology (H&E staining) and molecular analysis (nuclear protein extraction for EMSA, Western blot). Serum was collected for PSA measurement by ELISA. [2]
For tumor tissue nuclear protein extraction and EMSA, harvested tumors were minced and homogenized in ice-cold buffer. Nuclear pellets were suspended in high-salt extraction buffer, incubated on ice, and centrifuged. Supernatants containing nuclear proteins were collected for EMSA analysis as described in the Cell Assay section. [2]

No significant weight loss or treatment-related toxicities were observed in SCID-hu mice treated with atrasentan hydrochloride alone (10 mg/kg/day, intraperitoneal injection for 5 weeks), docetaxel alone (5 mg/kg, intravenous injection, every 3 days for 4 times), or a combination of both. [2]

[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 hydrochloride is the oral hydrochloride salt of pyrrolidine-3-carboxylic acid with potential antitumor activity. As a selective antagonist of the endothelin A (ETA) receptor, atrasentan selectively binds to the ETA receptor, thereby inhibiting endothelin-induced angiogenesis and tumor cell proliferation. It is a pyrrolidine and benzodioxane derivative that functions as an endothelin A receptor antagonist. It has therapeutic potential as an antitumor drug and for the treatment of diabetic nephropathy. Drug Indications: Treatment of nephropathy. Atrasentan hydrochloride (ABT-627) is a potent, orally bioavailable, and selective ETA receptor antagonist. Endothelin-1 (ET-1), produced by prostate cancer cells, binds to ETA receptors on bone marrow stromal cells, promoting osteogenic responses during bone metastasis. Blocking this interaction may inhibit bone metastasis and enhance the efficacy of docetaxel (Tasocor). [2] The mechanism is believed to be that ET-1 activates the PI3K-Akt pathway, which in turn activates NF-κB and upregulates anti-apoptotic genes (such as Bcl-2 and survivin). Atrasentan hydrochloride inhibits this pathway, downregulates NF-κB, and makes cancer cells more sensitive to apoptosis induced by taxotere. [2] The SCID-hu model used simulates the microenvironment of human bone metastasis, showing osteoblastic and osteolytic lesions similar to clinical disease. [2] A phase III clinical trial showed that atrasentan significantly delayed disease progression in patients with prostate cancer bone metastases compared to placebo. [2]

C29H39CLN2O6

547.08276

546.25

C, 63.67; H, 7.19; Cl, 6.48; N, 5.12; O, 17.55

195733-43-8

Atrasentan; 173937-91-2

159595

Off-white to gray solid powder

1.238g/cm3

659.4ºC at 760mmHg

352.6ºC

5.433

2

7

12

38

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.Cl

IJFUJIFSUKPWCZ-SQMFDTLJSA-N

InChI=1S/C29H38N2O6.ClH/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);1H/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;hydrochloride

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~28.57 mg/mL (~52.2 mM)
H2O: ~0.5 mg/mL (~0.9 mM)
0.1 M HCL: < 1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear 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 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 2: ≥ 2.5 mg/mL (4.57 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), clear 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 3: ≥ 2.5 mg/mL (4.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear 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 corn oil and mix evenly.

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Solubility in Formulation 4: 0.75 mg/mL (1.37 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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