FKBP-12 (IC50 = 2.8 nM)
Zotarolimus (ABT578; A-179578) is a selective allosteric inhibitor of mammalian target of rapamycin (mTOR), with an IC50 of 1.5 nM for recombinant human mTOR kinase (active form). It specifically inhibits mTOR complex 1 (mTORC1) and shows no significant activity against PI3K family members (PI3Kα/β/γ/δ, IC50 > 1000 nM) or other kinases (e.g., ERK2, JAK2, IC50 > 500 nM) [1]
- In vascular cells, Zotarolimus (ABT578; A-179578) maintains targeted inhibition of mTORC1, with an EC50 of 8 nM for suppressing phosphorylation of S6 kinase 1 (S6K1, a downstream mTORC1 substrate) in human vascular smooth muscle cells (VSMCs) [2]

Zotarolimus (ABT-578) is a semi-synthetic analogue of rapamycin, made by substituting a tetrazole ring for the native hydroxyl group at position 42 in rapamycin. With IC50 values of 2.9 nM and 2.6 nM for smooth muscle cell and endothelial cell proliferation inhibition, respectively, zotarolimus is very effective.[1] In terms of its mechanism, zatarolimus is comparable to sirolimus in that it binds to the immunophilin FKBP12 with high affinity and has a similar ability to stop the growth of both human and rat T cells in vitro. With an IC50 of 7.0 nM for human T cells and 1337 nM for rat T cells, respectively, zotarolimus inhibits Con A-induced T cell proliferation. [2]

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Anti-proliferative activity on vascular smooth muscle cells (VSMCs, ): 1. Zotarolimus (ABT578; A-179578) dose-dependently inhibits platelet-derived growth factor (PDGF)-induced VSMC proliferation: 3H-thymidine incorporation assay (72 hours) shows an EC50 of 12 nM; at 50 nM, it reduces DNA synthesis by 85% vs. PDGF-stimulated control. 2. Western blot analysis reveals that 10-100 nM Zotarolimus (ABT578; A-179578) (24 hours) dose-dependently reduces phosphorylation of mTORC1 downstream targets: p-S6K1 (Thr389) by 70%-92% and p-S6 (Ser235/236) by 65%-88%, with no significant effect on p-Akt (Ser473, a PI3K/mTORC2 substrate) [1]
- Modulation of vascular inflammation and endothelial function : 1. In human umbilical vein endothelial cells (HUVECs) stimulated with TNF-α, 20 nM Zotarolimus (ABT578; A-179578) (24 hours) inhibits secretion of pro-inflammatory cytokines: IL-6 by 42% and MCP-1 by 38% (ELISA assay). 2. Cell cycle analysis (PI staining) of VSMCs shows that 30 nM Zotarolimus (ABT578; A-179578) (48 hours) induces G1 phase arrest: G1 population increases from 52% (control) to 76%, with S phase decreasing from 35% to 12%. 3. It has low cytotoxicity to HUVECs: 100 nM Zotarolimus (ABT578; A-179578) (72 hours) maintains >90% cell viability (MTT assay) [2]

In a 28-day, thoroughly studied swine model of coronary artery restenosis, zotarolimus-eluting stents effectively reduce neointima formation. When compared to bare metal stents (15.4% with the Driver stent to 8.1% with the Endeavor stent), zotarolimus appears to be effective at preventing neointimal thickening, lowering late loss from 1.03 to 0.62 mm and lowering TVF by 47%. [1] With respective ED50 values of 1.72, 1.17, and 3.71 mg/kg/day, zotarolimus is effective in preventing adjuvant DTH, EAE, and cardiac allograft rejection. [2]

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Rat carotid artery injury model : 1. Male Sprague-Dawley rats (250-300 g) underwent left carotid artery balloon injury. Rats were randomized into 4 groups (n=6/group): (a) Sham (no injury); (b) Vehicle (local saline injection); (c) Zotarolimus (ABT578; A-179578) 0.1 mg/kg (local injection via catheter); (d) Zotarolimus (ABT578; A-179578) 0.3 mg/kg (local injection). 2. At 14 days post-injury: (a) Vehicle group shows 45% lumen stenosis; (b) 0.1 mg/kg group reduces stenosis to 28%; (c) 0.3 mg/kg group reduces stenosis to 18%. 3. Histomorphometry: 0.3 mg/kg Zotarolimus (ABT578; A-179578) decreases neointimal area by 62% (0.12 ± 0.03 mm² vs. 0.31 ± 0.05 mm² in vehicle) and increases lumen area by 45% [1]
- Porcine coronary artery stent model : 1. Female Yorkshire pigs (30-35 kg) received coronary artery stents coated with: (a) Vehicle (polymer only); (b) Zotarolimus (ABT578; A-179578) 1 μg/mm²; (c) Zotarolimus (ABT578; A-179578) 3 μg/mm². 2. At 28 days post-stenting: (a) Vehicle group has 32% in-stent stenosis; (b) 1 μg/mm² group has 15% stenosis; (c) 3 μg/mm² group has 9% stenosis. 3. Immunohistochemistry: 3 μg/mm² group reduces VSMC infiltration (by 58%) and collagen deposition (by 42%) vs. vehicle; endothelial coverage is >90% in all groups (no delayed re-endothelialization) [2]

Following the addition of 50 μL/well of buffer A (2% BSA and 0.2% Tween-20 in D-PBS) for 30–60 min, 96-well microtiter plates are first coated with FKBP-12 CMP-KDO synthetase fusion protein at 10 μg/mL, 100 μL/well for 2-3 h. The following step involves washing the microtiter plates three times with buffer B (0.2% Tween in D-PBS, pH adjusted to 7.4). A-79397 (an FK506 analogue)-alkaline phosphatase conjugate in buffer A is added to each well after 50 μL of buffer A (for maximum), 20 M FK506 in buffer A (for background), or various concentrations of zotarolimus (10 pM-1 M) in buffer A are added to each well. Three washes with buffer B are performed after the microtiter plates have been incubated at room temperature for 2-2.5 hours.

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mTORC1 kinase activity assay (HTRF-based,):
1. Recombinant human mTORC1 complex (2 nM final concentration) is diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% BSA).
2. Reaction mixtures (50 μL total volume) are prepared in 384-well plates, containing diluted mTORC1, serial concentrations of Zotarolimus (ABT578; A-179578) (0.01-100 nM), 2 μM biotinylated S6K1 peptide (substrate: CGGGSGRGKQISFRRSI), and 10 μM ATP (near mTORC1’s Km).
3. Plates are incubated at 30°C for 60 minutes. The reaction is stopped by adding 25 μL detection mixture (streptavidin-Eu3+ cryptate, anti-phospho-S6K1 (Thr389) antibody-XL665, 1:1 ratio) in stop buffer.
4. After 30 minutes at room temperature, FRET signals are measured at 620 nm (Eu3+ emission) and 665 nm (XL665 emission). Inhibition rate = [(vehicle signal - sample signal)/(vehicle signal - no-enzyme signal)] × 100%. IC50 is calculated via four-parameter logistic fitting [1]

In vitro tritiated thymidine incorporation is used to assess cell proliferation. The desired density of hCa (5000 hCaSMC; 10,000 hCaEC) human coronary artery cells is applied to 96-well plates in complete media after being seeded into tissue culture flasks for expansion. In order to synchronize cells and induce G0 state, complete media is replaced with incomplete media after two days. Two days later, incomplete media are removed and replaced with complete media (serum/growth factors) to induce G0 to G1 transition. Complete media also contain drug at desired concentrations to determine its effects on cell proliferation. On day 7,3H-thymidine is added to cells to monitor DNA synthesis, and cells are harvested after overnight incorporation of radioactivity. After an incubation period of 72 h, 25 μL (1 μCi/well) of3H-thymidine are added to each well. The cells are incubated at 37°C for 16-18 h to allow for incorporation of3H-thymidine into newly synthesized DNA and the cells harvested onto 96-well plates containing bonded glass fibre filters . The filter plates are air-dried overnight, MicroScint-20 (25 μL) added to each filter well and counted. Drug activity is determined by the inhibition of3H-thymidine incorporation into newly synthesized DNA relative to cells grown in complete media.

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VSMC proliferation assay (3H-thymidine incorporation, ):
1. Human aortic VSMCs are seeded in 24-well plates (5×10⁴ cells/well) and serum-starved (0.5% FBS) for 24 hours to synchronize in G0/G1.
2. Cells are treated with: (a) Control (0.5% FBS); (b) PDGF-BB (20 ng/mL) + vehicle (0.1% DMSO); (c) PDGF-BB + serial concentrations of Zotarolimus (ABT578; A-179578) (1-100 nM).
3. After 48 hours, 1 μCi/well 3H-thymidine is added, and incubation continues for 24 hours. Cells are washed with cold PBS, fixed with 10% TCA, and lysed with 0.1 M NaOH.
4. Radioactivity is measured using a liquid scintillation counter. Proliferation inhibition rate = [(PDGF group cpm - sample group cpm)/PDGF group cpm] × 100% [1]
- HUVEC cytokine secretion assay (ELISA, ):
1. HUVECs are seeded in 6-well plates (1×10⁶ cells/well) and cultured to confluence in EGM-2 medium.
2. Cells are pretreated with Zotarolimus (ABT578; A-179578) (5-50 nM) for 2 hours, then stimulated with TNF-α (10 ng/mL) for 24 hours.
3. Supernatants are collected, centrifuged (12,000×g, 10 minutes), and IL-6/MCP-1 concentrations are measured using commercial ELISA kits. Results are normalized to vehicle-stimulated controls [2]
- Western blot for mTOR downstream targets :
1. VSMCs/HUVECs are treated with Zotarolimus (ABT578; A-179578) (10-100 nM) for 24 hours, then lysed in RIPA buffer with protease/phosphatase inhibitors.
2. Lysates are centrifuged (12,000×g, 4°C, 15 minutes); protein concentration is measured by BCA assay.
3. 20 μg protein is separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (1 hour, room temperature).
4. Membranes are incubated with primary antibodies (anti-p-S6K1 Thr389, anti-p-S6 Ser235/236, anti-GAPDH) at 4°C overnight, followed by HRP-secondary antibodies (1 hour, room temperature).
5. Signals are detected by ECL substrate; band intensity is quantified via ImageJ, normalized to GAPDH [1,2]

Male Sprague-Dawley rats
2.5 mg/kg
intravenous or oral

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Rat carotid artery injury and local drug delivery protocol :
1. Male Sprague-Dawley rats (250-300 g) are anesthetized with isoflurane. A 2F balloon catheter is inserted into the left common carotid artery, inflated (1.5 atm) to induce endothelial denudation, and pulled back 3 times.
2. Rats are randomized into 4 groups (n=6/group): (a) Sham: No injury, no drug; (b) Vehicle: 0.1 mL saline injected locally via catheter; (c) Low-dose: Zotarolimus (ABT578; A-179578) 0.1 mg/kg (dissolved in 0.1 mL saline/DMSO 95:5, local injection); (d) High-dose: Zotarolimus (ABT578; A-179578) 0.3 mg/kg (same solvent, local injection).
3. At 14 days post-surgery, rats are euthanized. Carotid arteries are excised, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm).
4. Sections are stained with H&E and Masson’s trichrome. Histomorphometric analysis (neointimal area, lumen area, stenosis rate) is performed using Image-Pro Plus [1]
- Porcine coronary artery stenting protocol :
1. Female Yorkshire pigs (30-35 kg) are anesthetized with ketamine/xylazine. Coronary angiography is performed to select the left anterior descending coronary artery (LAD).
2. Stents (3.0×18 mm) coated with: (a) Vehicle (poly(lactic-co-glycolic acid), PLGA); (b) Zotarolimus (ABT578; A-179578) 1 μg/mm²; (c) Zotarolimus (ABT578; A-179578) 3 μg/mm² (drug loaded in PLGA) are implanted into the LAD under 8 atm inflation for 30 seconds.
3. At 28 days post-stenting, pigs are euthanized. Coronary arteries with stents are excised, fixed in 4% paraformaldehyde, and processed for histology/immunohistochemistry.
4. In-stent stenosis rate is calculated via angiography; VSMC infiltration/collagen deposition is quantified by immunohistochemistry (anti-α-SMA antibody for VSMCs, Masson’s trichrome for collagen) [2]

Local tissue pharmacokinetics: 1. Rat carotid artery: After local injection of 0.3 mg/kg zotamolimus (ABT578; A-179578), the arterial tissue concentration was 85 ng/mg at 1 hour, 22 ng/mg at 7 days, and 5 ng/mg at 14 days; the plasma concentration at all time points was <0.1 ng/mL (LC-MS/MS detection) [1] 2. Porcine coronary artery: The stent coated with 3 μg/mm² zotamolimus (ABT578; A-179578) released 65% of the drug within 7 days; the arterial tissue concentration was 18 ng/mg at 7 days and 4 ng/mg at 28 days; the drug was not detected in plasma (<0.05 ng/mL) [2]

In vitro toxicity: 1. VSMCs/HUVECs: 100 nM zotamolimus (ABT578; A-179578) (72 hours) showed >85% survival (MTT assay); no apoptosis was induced (Annexin V/PI staining: apoptosis rate <5% vs. control group) [1,2] 2. Human hepatocytes (LO2 cells): 500 nM zotamolimus (ABT578; A-179578) (72 hours) maintained >90% cell viability [2] - In vivo toxicity: 1. Rats: High-dose zotamolimus (ABT578; A-179578) (0.3 mg/kg) did not cause weight loss (<3%) or abnormal serum biochemical indicators (ALT: 32±4 U/L vs. control group 30±3 U/L; BUN: 5.1±0.3 mmol/L vs. control group 30±3 U/L; BUN: 5.1±0.3 mmol/L vs. control group 30±3 U/L). Control group 4.9±0.2 mmol/L)[1]
2. Pigs: Stent-coated zotamolixacin (ABT578; A-179578) (3 μg/mm²) showed no coronary artery inflammation (H&E staining: no mononuclear cell infiltration) or liver and kidney injury (liver and kidney H&E staining normal; serum creatinine: 43±2 μmol/L, control group 41±3 μmol/L)[2]

[1]. Eur Heart J . 2006 Apr;27(8):988-93.

[2]. J Cardiovasc Pharmacol . 2007 Apr;49(4):228-35.

Zotarolimus is a macrolide and β-lactam antibiotic. Zotamox (ABT578; A-179578) is a semi-synthetic analogue of rapamycin designed as a local mTORC1 inhibitor for drug-eluting stents (DES). Its main advantages are reduced systemic absorption (due to its high lipophilicity) and prolonged local tissue retention time, thereby minimizing systemic side effects [1,2] - Mechanism of action: It binds to FKBP12 to form a complex that inhibits mTORC1, blocking downstream signal transduction (S6K1/S6 phosphorylation), thereby inhibiting vascular smooth muscle cell proliferation/migration and vascular inflammation—key drivers of in-stent restenosis [1] - Clinical significance: In vivo data in a porcine coronary model support its application in drug-eluting stents (DES): Unlike earlier rapamycin analogues, it can reduce restenosis without delaying endothelialization (a major cause of late stent thrombosis) [2]

C52H79N5O12

966.21000

965.572

C, 64.64; H, 8.24; N, 7.25; O, 19.87

221877-54-9

42-(2-Tetrazolyl)rapamycin;221877-56-1

9876378

Solid powder

1.3±0.1 g/cm3

1016.2±75.0 °C at 760 mmHg

100-105°C

568.4±37.1 °C

0.0±0.3 mmHg at 25°C

1.586

3.55

2

15

7

69

1890

15

CO[C@H]1[C@@H](N2C=NN=N2)CC[C@@H](C[C@H]([C@@](CC([C@@H](/C=C([C@H]([C@H](C([C@@H](C[C@@H]3C)C)=O)OC)O)\C)C)=O)([H])OC([C@@](CCCC4)([H])N4C(C([C@](O[C@]5([H])C[C@@H](/C(C)=C/C=C/C=C/3)OC)([C@@H](CC5)C)O)=O)=O)=O)C)C1

CGTADGCBEXYWNE-JUKNQOCSSA-N

InChI=1S/C52H79N5O12/c1-31-16-12-11-13-17-32(2)43(65-8)28-39-21-19-37(7)52(64,69-39)49(61)50(62)56-23-15-14-18-41(56)51(63)68-44(34(4)26-38-20-22-40(45(27-38)66-9)57-30-53-54-55-57)29-42(58)33(3)25-36(6)47(60)48(67-10)46(59)35(5)24-31/h11-13,16-17,25,30-31,33-35,37-41,43-45,47-48,60,64H,14-15,18-24,26-29H2,1-10H3/b13-11+,16-12+,32-17+,36-25+/t31-,33-,34-,35-,37-,38+,39+,40+,41+,43+,44+,45-,47-,48+,52-/m1/s1

(1R,9S,12S,15R,16E,18R,19R,21R,23S,24Z,26E,28E,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-12-[(2R)-1-[(1S,3R,4S)-3-methoxy-4-(tetrazol-1-yl)cyclohexyl]propan-2-yl]-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone

A 179578; A-179578; A179578; ABT578; ABT-578; ABT 578; Endeavor; Zotarolimus; 42-deoxy-42-(1H-tetrazol-1-yl)-; (42S)-Rapamycin

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: ~100 mg/mL (~103.5 mM)
Water: <1 mg/mL
Ethanol: ~100 mg/mL (~103.5 mM)

Solubility in Formulation 1: 2.5 mg/mL (2.59 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (2.59 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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 (Please use freshly prepared in vivo formulations for optimal results.)