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Prostate cancer (PCa) is one of the most common cancers in men. Castration-resistant prostate cancer (CRPC) continues to grow even when the testosterone levels are at or below the castrate level. CRPC can also be called hormone-refractory or hormone-resistant prostate cancer. The study found that ARD-2585 is a promising androgen receptor degrader for extensive investigations to treat advanced prostate cancer. In vivo drug efficacy evaluation and plasma stability studies showed that ARD-2585 can effectively inhibit the growth of Vcap (human prostate cancer cells） tumors, is very stable in liver microsomes and plasma, and has suitable PK parameters.
In the early stage of drug development, it’s essential to conduct plasma stability tests. By simulating the in vivo conditions, researchers can assess whether the drug or its metabolites may interact with other drugs or endogenous molecules in plasma, leading to unwanted effects. Plasma (blood) stability determination is a widely used, simple test in drug discovery screens.
Metastatic castration-resistant prostate cancer (mCRPC) encompasses a heterogeneous wide range of molecular tumor behavior and a high risk of progression and remains incurable and lethal. Androgen receptor antagonists, such as Enzalutamide, Apalutamide, and Darolutamide, are effective in treating mCRPC. However, patients treated with these androgen receptor antagonists ultimately develop drug resistance. In most tumors resistant to androgen receptor antagonists, the androgen receptor signaling continues to function and drives tumor growth and progression.
Some primary resistance mechanisms to androgen receptor antagonists include androgen receptor gene amplification, mutation, and expression of androgen receptor variants. Therefore, new therapeutic strategies are urgently needed to effectively target the androgen receptor signaling in tumors resistant to androgen receptor antagonists.
Proteolysis targeting chimeras (PROTACs)
Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules with three chemical elements: a ligand binding to a target protein of interest, a ligand binding to and recruiting E3 ubiquitin ligase complex, and a linker for conjugating these two ligands together. PROTAC is a chemical knockdown strategy that degrades the target protein through the ubiquitin-proteasome system. Unlike traditional inhibitors’ competitive- and occupancy-driven process, PROTACs are catalytic in their mode of action, which can promote target protein degradation at low exposures.
PROTACs have the potential to degrade the target pathogenic proteins and regulate the related signaling pathways, which traditional small molecule inhibitors or activators cannot achieve. PROTACs have gained significant attention from academia and the pharmaceutical and biotechnology industry as an efficient and novel technology.
PROTAC androgen receptor (AR) degraders
PROTAC-based androgen receptor degraders are bifunctional small molecules consisting of an androgen receptor ligand that binds to the androgen receptor protein and a ligand that binds to and recruits an E3 ligase complex, tethered together through a linker.
ARV-110 is the first PROTAC androgen receptor degrader advanced into human clinical trials and has demonstrated encouraging clinical activity and safety profile. ARV-110 is a first-in-class, orally active PROTAC protein degrader that selectively targets AR. ARV-110 degrades androgen receptor protein and inhibits cell growth at low nM concentrations in the LNCaP and VCaP cell lines.
In this study, researchers described their design, synthesis, and extensive evaluation of a series of PROTAC androgen receptor degraders. This led to the discovery of ARD-2585 as a potent, orally bioavailable, and efficacious PROTAC androgen receptor degrader. Through extensive optimization of the linker and modifications of the androgen receptor antagonist portion of the compounds, researchers have discovered a set of exceptionally potent and orally bioavailable androgen receptor degraders, exemplified by ARD-2585. ARD-2585 potently inhibits cell growth with IC50 values of 1.5 and 16.2 nM in the VCaP and LNCaP AR+ prostate cancer cell lines, respectively.
ARD-2585, a representative PROTAC androgen receptor degrader, is 30, 10, 300, and 1000 times more potent than ARV-110 in reducing the androgen receptor protein level in the VCaP, LNCaP, 22Rvl, and MDA-PCa-2b cell lines, respectively. ARD-2585 achieves excellent pharmacokinetics and 51% of oral bioavailability in mice. ARD-2585 is more efficacious than Enzalutamide in inhibiting VCaP tumor growth and does not cause any toxicity in mice. Therefore, ARD-2585 is a promising androgen receptor degrader for extensive investigations to treat advanced prostate cancer.
In vivo efficacy evaluation of ARD-2585
ARD-2585 effectively reduces androgen receptor protein in the VCaP tumor tissue. More importantly, ARD-2585 inhibits tumor growth at all three tested doses (10, 20, and 40 mg/kg). At the end of the treatment (day 37), ARD-2585 inhibits tumor growth by 54.9, 74.3, and 65.9% over the vehicle control group. Enzalutamide at 40 mg/kg inhibits tumor growth by 45.0%.
ARD-2585 at both 20 and 40 mg/kg is statistically more efficacious than Enzalutamide at 40 mg/kg in inhibiting tumor growth. In addition, both ARD-2585 and Enzalutamide were well tolerated in this efficacy experiment and did not cause animal weight loss or other signs of toxicity during the entire experiment.
Plasma stability studies of ARD-2585
Drugs must have sufficient stability in the body to exert a pharmacological effect over a reasonable period. A wide variety of compounds are unstable (degraded) when incubated in blood or plasma at rates inconsistent with the PK properties necessary for drug therapeutics. Plasma stability assay provides valuable insights into the drug’s pharmacokinetic properties, potential drug-drug interactions, and overall safety profile, helping researchers optimize drug dosing and improve therapeutic outcomes.
Typically, an appropriate analytical method, such as LC-UV or LC-MS, determines a compound’s plasma stability.
Researchers tested ARD-2585 for its plasma stability in five species (human, mouse, rat, dog, and monkey). ARD-2585 has excellent plasma stability in all 5 species with T1/2 > 120 min.
The in vitro stability of ARD-2585 was studied in human, mouse, rat, dog, and monkey plasmas. A test compound was dissolved in DMSO to a final concentration of 10 mM and then diluted to 10 μM in 0.1 M K/Mg buffer. 90 μL of prewarmed plasma at 37 °C was added to the wells of a 96-well plate before spiking them with 10 μL of 10 μM test compound to make the final concentration of the test compound to 1 μM.
The spiked plasma samples were incubated at 37 °C for 2 h. Reactions were terminated at 0, 5, 15, 30, 60, and 120 min by adding 400 μL of acetonitrile containing IS.
After quenching, the plates were shaken for 5 min at 600 rpm and stored at −20 °C if necessary before analysis by LC/MS.
Before LC/MS analysis, the samples were thawed at rt and centrifuged at 6000 rpm for 20 min. 100 μL of the supernatant from each well was transferred into a 96-well sample plate containing 100 μL water for LC/MS analysis. Procaine was used as a reference control compound for human, mouse, dog, and monkey plasma stability studies. Benfluorex was used as a reference control compound for rat plasma stability studies. The plasma stability assay was performed by Shanghai Medicilon.
ARD-2585 (Compound 43) is a very potent androgen receptor degrader. Researchers evaluated the pharmacokinetics in mice of six compounds. Specifically, ARD-2585 and Compound 45 have an excellent volume of distribution (Vss = 1.8–2.1 L/kg), a long half-life (t1/2 = 5.5–7.5 h), and a slow clearance (Cl = 0.2–0.3 L/h/kg) with intravenous administration.
With 5 mg/kg of oral administration, ARD-2585 achieves a Cmax of 1140 ng/mL and an AUC of 8254 h*ng/mL. With 3 mg/kg of oral administration, ARD-2585 reaches a Cmax of 484 ng/mL and an AUC of 8637 h*ng/mL. ARD-2585 has an oral bioavailability of 51%.
Researchers further carried out tissue distribution studies of ARD-2585 with a single oral administration at 20 mg/kg in mice bearing VCaP xenograft tumor. These tissue distribution data demonstrate that, consistent with its excellent PK profile, ARD-2585 is extensively distributed in tissues.
Summary
PROTACs opened a new chapter for novel drug development. PROTACs-induced targeted protein degradation has emerged as a novel therapeutic strategy in drug development and attracted the favor of academic institutions, large pharmaceutical enterprises, and biotechnology companies. PROTACs have been successfully employed in the degradation of different types of target proteins related to various diseases, including cancer, viral infection, immune disorders, neurodegenerative diseases, etc.
In this study, researchers report the discovery of exceptionally potent and orally bioavailable PROTAC androgen receptor degrader ARD-2585.
ARD-2585 is very stable in liver microsomes and plasma and has no hERG inhibition liability. It displays excellent PK parameters with intravenous and oral administration routes in mice and achieves extensive tissue distribution. Oral administration of ARD-2585 effectively reduces the androgen receptor protein in the VCaP xenograft tumor tissue in mice and inhibits VCaP tumor growth.
Therefore, ARD-2585 is a promising androgen receptor degrader in further extensive evaluations for the treatment of AR+ prostate cancer and other human diseases in which the androgen receptor plays a key role.
References:
[1] Weiguo Xiang, et al. Discovery of ARD-2585 as an Exceptionally Potent and Orally Active PROTAC Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer. J Med Chem. 2021 Sep 23;64(18):13487-13509. doi: 10.1021/acs.jmedchem.1c00900.
[2] Xiaojuan Jia, et al. Targeting androgen receptor degradation with PROTACs from bench to bedside. Biomed Pharmacother. 2023 Feb;158:114112. DOI: 10.1016/j.biopha.2022.114112
[3] Thi-Thao-Linh Nguyen, et al. Development of an LC-MS/MS Method for ARV-110, a PROTAC Molecule, and Applications to Pharmacokinetic Studies. Molecules. 2022 Mar 18;27(6):1977. doi: 10.3390/molecules27061977.
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#PROTAC
#pharmacokinetics
#androgen receptor
