Estrogen receptor [DC50 = 2 nM]; CRBN
Vepdegestrant targets the estrogen receptor alpha (ERα), a key driver of cell growth in ER+ breast cancer . Its mechanism of action involves simultaneously binding to ER and the E3 ubiquitin ligase component cereblon (CRBN) . This interaction forms a ternary complex, leading to the ubiquitination of ER and its subsequent recognition and degradation by the proteasome. It is highly effective against both wild-type and clinically relevant, endocrine therapy-resistant ER mutants, such as ESR1 Y537S and D538G .

ARV-471, an estrogen receptor (ER) alpha PROTAC, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ˜ 2 nM. PROTAC-mediated ER degradation decreases the expression of classically-regulated ER-target genes (PR, GREB1, TFF) and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically-relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants[2].

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In vitro, vepdegestrant induces potent and rapid degradation of ER in multiple ER+ breast cancer cell lines. It achieves a half-maximal degradation concentration (DC50) of approximately 1 nM and a maximum degradation (Dmax) of over 95% . At a concentration of 100 nM, vepdegestrant reduces ER protein levels by more than 80% within 4 hours . It is equally potent against clinically relevant, ligand-independent ER mutants (e.g., Y537S, D538G) and effectively inhibits the proliferation of ER-dependent breast cancer cells .

In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant tumor volume regressions of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (˜130% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC-degrader[2].

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Vepdegestrant achieved substantial TGI (87%-123%) in MCF7 orthotopic xenograft models, better than those of the ET agent fulvestrant (31%-80% TGI). In the hormone independent (HI) mutant ER Y537S patient-derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regression and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant-induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib; the mTOR inhibitor everolimus; and the PI3K inhibitors alpelisib and inavolisib. Conclusions: Vepdegestrant achieved greater ER degradation in vivo compared with fulvestrant, which correlated with improved TGI, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.[3]

The primary non-cellular assay used to confirm direct binding of vepdegestrant to the estrogen receptor is the cell-free radioligand displacement assay. In this procedure, a recombinant estrogen receptor protein is incubated with a high-affinity radiolabeled ligand (such as estradiol). Vepdegestrant is then titrated into the reaction mixture at various concentrations. After an appropriate incubation period to reach equilibrium, the bound ligand is separated from the free ligand (typically using charcoal dextran or filtration methods). The amount of radiolabeled ligand remaining bound to the ER is measured using a scintillation counter. The ability of vepdegestrant to compete with and displace the radiolabeled ligand is directly correlated with a decrease in the radioactive signal, allowing researchers to calculate its half-maximal inhibitory concentration (IC50) and inhibitory constant (Ki) to quantify its binding affinity .

Western blot procedures[3]
All cell lines and uterine or xenograft tumor tissues were lysed/homogenized in RIPA lysis buffer and Halt protease inhibitors. ER protein levels in degradation assays were measured by standard western blot, in-cell western, or digital western analysis performed on either a WES or JESS instrument. See Supplementary Extended Methods for full methodologies.

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Cell growth inhibition assays[3]
Cell growth inhibition studies were conducted in 96-well plates at 2,000 cells/well with 3-fold serial dilution 8-point DRCs, unless otherwise stated. At day 5, cell viability was measured using Cell-Titer Glo and CTG data analyzed using GraphPad. Live-cell imaging proliferation and dose matrix drug combination assays are described in Supplementary Extended Methods.

Immature rat uterotrophic assay [3]
This model was conducted as previously described using immature female rats younger than postnatal day (PND) 30. Sprague–Dawley (SD) rats at PND 18 were dosed with 30 mg/kg vepdegestrant or 10 mg/kg AZD-9496 in vehicle of PEG400/2% Tween80, by oral gavage (per os, po) once daily for 3 days (qdx3), or a single subcutaneous (sc) dose of 100 mg/kg fulvestrant in a vehicle of 10% w/v ethanol, 10% w/v benzyl alcohol, and 15% w/v benzyl benzoate, made up to 100% w/v with castor oil (EBB/castor oil). Five animals were used per arm. Animals were euthanized and tissues harvested 24-hours post-last dose or on day 4 for fulvestrant/sc arms. Uterine weights were measured, flash frozen in liquid nitrogen, and stored at −80°C. ER levels were determined by western blot.

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MCF7 orthotopic xenograft model[3]
Eight- to 10-week-old female NOD/SCID mice were surgically implanted with a 0.36 mg 90-day release 17β-estradiol pellet subcutaneously. One to 2 days later, each mouse was injected with 5 × 106/200 µL MCF7 cells (ATCC) into one mammary fat pad. Cells were prepared in a 50/50 RPMI-1640 phenol red-free media/Corning Matrigel Membrane Matrix mix at 25 × 106 cells/mL. Dosing was initiated once tumors reached an average of 200 mm3. When oral combinations were dosed, vepdegestrant was dosed first and the second agent 30 to 60 minutes later. All oral agents (vepdegestrant, palbociclib, abemaciclib, ribociclib, inavolisib, alpelisib, and everolimus) were dosed at 5 mL/kg volume once daily for 28 days (qdx28) unless otherwise stated. Fulvestrant sc was dosed at 4 mL/kg twice per week (biw) for 2 weeks plus once per week (qw) for 2 weeks (biwx2, qwx2). Vehicles for the various compounds dosed in vivo are listed in Supplementary Table S5. Tumor volumes were measured twice per week in efficacy studies and calculated using (width2 × length)/2, in which all measurements are in millimeters (mm), and the tumor volume is in mm3. Body weights were recorded twice per week. In some drug combinatorial efficacy studies, some single-day dosing holidays (small black arrows in Fig. 6D and ​andE)E) were implemented on all arms if any body weight loss approached 10%. At study termination, mice were euthanized 18 hours post-last dose, and harvested tissue was snap-frozen on dry ice. TGI was calculated as follows, with tumor volume being expressed in mm3:

Administration and Dosage: Vepdegestrant is administered orally, once daily (QD), with food. The recommended phase 3 monotherapy dose is 200 mg once daily .
Absorption and Plasma Exposure: After a single oral dose of 200 mg, the geometric mean maximum plasma concentration (Cmax) is 630.9 ng/mL, and the 24-hour area under the plasma concentration-time curve (AUC0-24h) is 10,400 ng∙hr/mL. After multiple doses (once daily), the geometric mean Cmax increases to 1,056 ng/mL, and the 24-hour AUC increases to 18,310 ng∙hr/mL, indicating moderate accumulation with repeated dosing .
Metabolism: Vepdegestrant is metabolized via cytochrome P450 3A4 (CYP3A4). Based on in vitro studies, no human-specific metabolites were identified, and the metabolic profile in rats overlaps with that of humans .
Drug-Drug Interactions: Strong CYP3A4 inhibitors or inducers are contraindicated or require dose adjustments. Patients in clinical trials are excluded if receiving strong CYP3A4 inhibitors or inducers. Prior use is allowed if CYP3A4 inhibitors are stopped ≥7 days before enrollment and strong CYP3A4 inducers are stopped ≥14 days before enrollment .
Protein Binding and Clearance: In preclinical studies, hepatocyte clearance correlated more closely with in vivo rat PK data than liver microsomal clearance did. Physiologically based pharmacokinetic (PBPK) models developed in rats accurately simulate vepdegestrant's PK across fed and fasted states. Human models informed by in vitro ADME data closely mirrored post-oral dose plasma profiles .
Cross-Species and Ethnic Comparisons: No notable differences in pharmacokinetics were observed between Japanese patients and Western patients at the 200 mg QD dose

Dose-Limiting Toxicity (DLT): In a phase 1 study of Japanese patients with ER+/HER2- advanced breast cancer receiving 200 mg QD, no DLTs were observed during the first 28-day cycle .
Adverse Events (AEs): In the Japanese phase 1 study, 66.7% of patients experienced treatment-related adverse events (TRAEs). All TRAEs were grade 1 except one case of grade 2 anemia. No AEs led to dose reduction or discontinuation .
Phase 3 Safety Profile (VERITAC-2 trial): In a phase 3 trial comparing vepdegestrant (200 mg QD) with fulvestrant:
Adverse events occurred in 86.9% of vepdegestrant patients vs. 81.4% of fulvestrant patients .
The most common AEs with vepdegestrant were fatigue (26.6%), increased aspartate aminotransferase (14.4%), and increased alanine aminotransferase (14.4%) .
Grade 3-4 AEs occurred in 23.4% of vepdegestrant patients vs. 17.6% of fulvestrant patients. The most common grade 4 events with vepdegestrant were neutropenia (1.9%) and hypokalemia (1.9%) .
QT prolongation occurred in 9.9% of vepdegestrant patients without clinical sequelae .
Discontinuation due to AEs was low but higher for vepdegestrant (2.9%) compared to fulvestrant (0.7%) .
No deaths were attributed directly to either treatment .
Preclinical Safety Data: According to the Material Safety Data Sheet (MSDS), vepdegestrant is not classified as a hazardous substance or mixture under standard criteria .
CYP-Related Toxicity: Because vepdegestrant is primarily metabolized by CYP3A4, coadministration with strong CYP3A4 inhibitors or inducers is excluded in clinical trials to avoid toxicity due to altered exposure

[1]. Targeting estrogen receptor α for degradation with PROTACs: A promising approach to overcome endocrine resistance. Eur J Med Chem. 2020;206:112689.

[2]. Abstract P5-04-18: ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer.

[3]. Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor-Positive Breast Cancer. Cancer Res. 2023 Oct 2;83(19):3284-3304.

Vepdegestrant is an orally administered heterobifunctional molecule and a selective estrogen receptor (ER) α-targeting protein degrader, prepared using a proteolytic-targeting chimeric (PROTAC) technique, and possesses potential antitumor activity. Vepdegestrant consists of an ER α ligand and an E3 ubiquitin ligase recognition group. After oral administration, vepridogen targets and binds to the ER ligand-binding domain on ER α. The E3 ubiquitin ligase is recruited to ER via the E3 ubiquitin ligase recognition group, leading to ER α ubiquitination. This results in the proteasome ubiquitination and degradation of ER α. This reduces ER α protein levels, decreases the expression of ER α target genes, and blocks ER-mediated signal transduction. Ultimately, it inhibits the proliferation of ER α-overexpressing tumor cells. Furthermore, the degradation of ER α protein releases ARV-471, which can bind to other ER α target proteins. ER α is overexpressed in various cancers and plays a crucial role in cancer cell proliferation.

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The U.S. FDA has approved VEPPANU (vepdegestrant) for adults with ER+/HER2-, ESR1-mutated advanced or metastatic breast cancer following at least one line of endocrine therapy. This marks the first-ever approval of a PROTAC protein degrader therapy. In the pivotal Phase 3 VERITAC-2 trial, vepdegestrant demonstrated a statistically significant 43% reduction in the risk of disease progression or death compared to fulvestrant in patients with ESR1 mutations (median PFS: 5.0 vs. 2.1 months; HR 0.57). Majority of adverse events were low grade (Grade 1-2). The approval addresses a significant unmet need for patients with this aggressive form of breast cancer.

C45H49N5O4

723.9017

723.37845

C, 74.66; H, 6.82; N, 9.67; O, 8.84

2229711-68-4

2229711-08-2 (racemate)

134562533

White to off-white solid powder

6.4

2

7

7

54

1310

3

O([H])C1C([H])=C([H])C2=C(C=1[H])C([H])([H])C([H])([H])[C@]([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])[C@]2([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])N2C([H])([H])C([H])([H])N(C3C([H])=C([H])C4C(N([C@]5([H])C(N([H])C(C([H])([H])C5([H])[H])=O)=O)C([H])([H])C=4C=3[H])=O)C([H])([H])C2([H])[H])C([H])([H])C1([H])[H]

TZZDVPMABRWKIZ-XMOGEVODSA-N

InChI=1S/C45H49N5O4/c51-37-12-15-39-33(27-37)8-13-38(31-4-2-1-3-5-31)43(39)32-6-9-35(10-7-32)48-20-18-30(19-21-48)28-47-22-24-49(25-23-47)36-11-14-40-34(26-36)29-50(45(40)54)41-16-17-42(52)46-44(41)53/h1-7,9-12,14-15,26-27,30,38,41,43,51H,8,13,16-25,28-29H2,(H,46,52,53)/t38-,41+,43+/m1/s1

(3S)-3-[6-[4-[[1-[4-[(1R,2S)-6-hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl]phenyl]piperidin-4-yl]methyl]piperazin-1-yl]-3-oxo-1H-isoindol-2-yl]piperidine-2,6-dione

ARV-471; 2229711-68-4; Vepdegestrant; ARV-471 (protac); WC1U3R1YMI; ARV471; (S)-3-(5-(4-((1-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO: 110 mg/mL (151.95 mM)

Solubility in Formulation 1: ≥ 5.5 mg/mL (7.60 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 55.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2 mg/mL (2.76 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 20.0 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 3: 2 mg/mL (2.76 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)