Estrogen receptor

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].

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].

Western blot procedures[Clin Cancer Res. 2024 Aug 15;30(16):3549-3563.] 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[Clin Cancer Res. 2024 Aug 15;30(16):3549-3563.] 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 [Clin Cancer Res. 2024 Aug 15;30(16):3549-3563.]
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[Clin Cancer Res. 2024 Aug 15;30(16):3549-3563.]
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.

[1]. Targeting estrogen receptor \u03b1 for degradation with PROTACs: A\npromising approach to overcome endocrine resistance. Eur J Med Chem.\n2020;206:112689.

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

[3].https://ir.arvinas.com/static-files/7a4db470-3d7f-4d4b-98a4-481b8c573169

[4].https://doi.org/10.1158/1538-7445.SABCS21-PD13-08

[5].https://www.pfizer.com/news/press-release/press-release-detail/arvinas-and-pfizer-announce-protacr-protein-degrader-arv

Estrogen receptor alfa (ERα) is expressed in approximate 70% of breast cancer (BC) which is the most common malignancy in women worldwide. To date, the foremost intervention in the treatment of ER positive (ER+) BC is still the endocrine therapy. However, resistance to endocrine therapies remains a major hurdle in the long-term management of ER + BC. Although the mechanisms underlying endocrine resistance are complex, cumulative evidence revealed that ERα still plays a critical role in driving BC tumor cells to grow in resistance state. Fulvestrant, a selective estrogen receptor degrader (SERD), has moved to first line therapy for metastatic ER + BC, suggesting that removing ERα would be a useful strategy to overcome endocrine resistance. Proteolysis-Targeting Chimera (PROTAC) technology, an emerging paradigm for protein degradation, has the potential to eliminate both wild type and mutant ERα in breast cancer cells. Excitingly, ARV-471, an ERα-targeted PROTAC developed by Arvinas, has been in phase 1 clinical trials. In this review, we will summarize recent progress of ER-targeting PROTACs from publications and patents along with their therapeutic opportunities for the treatment of endocrine-resistant BC.[1]

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Vepdegestrant is an orally available hetero-bifunctional molecule and selective estrogen receptor (ER) alpha-targeted protein degrader, using the proteolysis targeting chimera (PROTAC) technology, with potential antineoplastic activity. Vepdegestrant is composed of an ER alpha ligand attached to an E3 ligase recognition moiety. Upon oral administration,vepdegestrant targets and binds to the ER ligand binding domain on ER alpha. E3 ligase is recruited to the ER by the E3 ligase recognition moiety and ER alpha is tagged by ubiquitin. This causes ubiquitination and degradation of ER alpha by the proteasome. This decreases ER alpha protein levels, decreases the expression of ER alpha-target genes and halts ER-mediated signaling. This results in an inhibition of proliferation in ER alpha-overexpressing tumor cells. In addition, the degradation of the ER alpha protein releases the ARV-471 and can bind to additional ER alpha target proteins. ER alpha is overexpressed in a variety of cancers and plays a key role in cancer cell proliferation.

C45H49N5O4

723.901671171188

723.378

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

2229711-08-2

2229711-68-4;

134579471

Typically exists as solid at room temperature

6.4

2

7

7

54

1310

2

C1CC2=C(C=CC(=C2)O)[C@H]([C@H]1C3=CC=CC=C3)C4=CC=C(C=C4)N5CCC(CC5)CN6CCN(CC6)C7=CC8=C(C=C7)C(=O)N(C8)C9CCC(=O)NC9=O

TZZDVPMABRWKIZ-MFTLXVFQSA-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

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

ARV471; (Rac)-Vepdegestrant; 2229711-08-2; 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; ARV471; vepdegrestrant; EX-A5021; starbld0007729; SCHEMBL20231167; ARV 471; ARV-471

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.)