Androgen Receptor[1].
Androgen receptor (AR). No IC50/Ki/EC50 values reported. [2][3]
Proxalutamide targets the androgen receptor (AR) as a potent antagonist. It binds to AR in target tissues, inhibiting androgen-induced receptor activation and promoting the formation of an inactive complex that cannot translocate to the nucleus. As a non-steroidal antiandrogen (NSAA), it competes with androgens such as testosterone and dihydrotestosterone for binding to the AR. The compound's oral bioavailability makes it suitable for chronic administration in prostate cancer and other AR-related diseases.

In biochemical assay, Proxalutamide (GT0918) more potently inhibits androgen binding with ARs ligand binding domain than Bicalutamide (11.4x) and MDV3100 (3.5x). In both hormone-sensitive (LNCaP) and CRPC (C4-2) cancer cells, Proxalutamide demonstrates stronger potency to block AR function of gene transcription than Bicalutamide (~5-10) and MDV3100 (2-5x) while maintaining full antagonism in CRPC cells. Proxalutamide impairs androgen stimulates AR translocation to cell nuclei hence blocks its binding DNA and shuts down the downstream oncogenic signaling. Moreover, Proxalutamide induces AR down regulation in prostate cancer cells. Proxalutamide not only inhibits proliferation of hormone-sensitive CaP cells, but also more potently inhibits proliferation of CRPC cells. In addition, Proxalutamide inhibits the growth of AR positive breast cancer cells. In contrast, Proxalutamide has minimum effects on the growth of AR-negative CaP cells (PC-3 and DU145), indicating it is a selective AR pathway inhibitor[1].

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In vitro, proxalutamide inhibits the proliferation of hormone-sensitive prostate cancer cells and more potently inhibits the proliferation of castration-resistant prostate cancer (CRPC) cells. The compound's anti-proliferative activity has been demonstrated in various prostate cancer cell lines. Its ability to inhibit AR signaling in both hormone-sensitive and castration-resistant settings makes it a promising candidate for prostate cancer therapy. Studies have also examined its effects on AR nuclear translocation and downstream gene expression.

The major pharmacokinetic parameters and statistical moment parameters are summarized. The tmax for the pHM-SD and conventional tablets are 0.9±0.4 h and 2.5±1.1 h, respectively, meaning that the pHM-SD tablets dissolve more quickly than the conventional tablets. Moreover, the difference between the tmax of the two treatments is statistically significant (p<0.05). The mean Cmaxand the AUC0-36 are 5.1±2.4 μg/mL and 38.3±8.2 μgh/mL for the pHM-SD tablets versus 3.1±1.5 μg/mL and 42.1±22.3 μgh/mL for conventional tablets, respectively. The relative bioavailability (frel) of the pHM-SD tablets is 125.6% of that for the conventional tablets on average, revealing that the bioavailability of the former is higher. The mean Proxalutamide (GT0918) half-life estimate from the pHM-SD tablets (7.9±2.2 h) was similar to that of the conventional tablets (8.4±0.5 h), remaining consistent with the following pharmacoki-netic theory: the extent and rate of absorption should not affect elimination[2].
In a double-blinded, randomized, placebo-controlled trial in mild to moderate COVID-19 patients (n=236), Proxalutamide (200 mg/day for 15 days) significantly increased viral clearance: on Day 7, 82% of subjects in the Proxalutamide group tested negative for SARS-CoV-2 by rtPCR (Ct>40) versus 31% in placebo group (p<0.001). In men, 81% vs 21%; in women, 85% vs 38%. [3]
The average time to clinical remission (including loss of taste/smell) was 4.2±5.4 days in the Proxalutamide group versus 21.8±13.0 days in placebo (p<0.001). By Day 7, 82.5% of Proxalutamide-treated subjects had fully recovered versus 24.2% in placebo. [3]
In vivo, proxalutamide has been investigated in preclinical models of prostate cancer and in clinical trials for prostate cancer and COVID-19. The compound's oral bioavailability and potent AR antagonism have been demonstrated in animal models. It has been studied in Phase 1/2/3 clinical trials for the treatment of prostate cancer, breast cancer, and the COVID-19 pandemic. In July 2021, it received approval in Paraguay for the treatment of COVID-19. The compound's ability to inhibit AR signaling in vivo supports its therapeutic potential.

In cell-free receptor binding assays, proxalutamide demonstrates high affinity binding to the androgen receptor. Radioligand displacement studies using purified AR or membrane preparations from cells expressing the receptor are used to determine the compound's binding affinity. The compound's ability to inhibit androgen-induced receptor activation is evaluated using biochemical assays that measure AR conformational changes and coactivator recruitment. These assays confirm the compound's mechanism as a competitive AR antagonist.

Cellular assays for proxalutamide involve evaluating its antagonist activity at the AR in prostate cancer cell lines. The compound's ability to inhibit androgen-induced AR nuclear translocation, DNA binding, and transcriptional activity is assessed using reporter gene assays. Anti-proliferative assays in hormone-sensitive and castration-resistant prostate cancer cells measure the compound's potency and efficacy. The compound's effects on AR target gene expression are also studied using qPCR and Western blot analysis.

Beagle Dogs[2] A single dose (25 mg Proxalutamide), randomized study with a two period crossover design is carried out to assess the pharmacoki-netics. Six healthy beagle dogs (9.3±0.7 kg) are randomly divided into two groups and fasted with free access to water overnight. Each group is orally administered with pHM-SDs tablet (Test) or a conventional tablet followed by 50 mL of water, respectively. The dogs obtain free access to water and food 6 h after drug administration. After a 1-week washout period, the groups are inverted and the administrations are repeated. A series of blood samples (1 mL) is collected in heparinized tubes using an indwelling cannula at pre-dose (-0.5 h) and post-dose (0.5, 1, 1.5, 2, 2.5 4, 6, 12, 24, 30, 36 and 48 h); these samples are gently mixed and centrifuged at 4000 rpm for 10 min within 1 h of collection[2].

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Proxalutamide (GT0918) formulation study in beagle dogs: Six healthy beagle dogs (9.3±0.7 kg) were used in a randomized two-period crossover design. Dogs were orally administered either pH-modified solid dispersion (pHM-SD) tablets (containing 25 mg GT0918) or conventional tablets, with a 1-week washout period. Blood samples (1 ml) were collected via indwelling cannula at pre-dose (-0.5 h) and at 0.5, 1, 1.5, 2, 2.5, 4, 6, 12, 24, 30, 36, and 48 h post-dose. Plasma was separated and stored at -20°C. Drug concentration was quantified by HPLC-MS/MS. [2]
Clinical trial for COVID-19: Double-blinded, randomized, placebo-controlled, prospective study. Subjects (n=236, 108 female, 128 male) with confirmed SARS-CoV-2 infection and mild symptoms (not requiring hospitalization) were randomized to receive either Proxalutamide (200 mg/day orally for 15 days or until full remission) or matching placebo. All subjects also received standard of care: nitazoxanide 500 mg twice daily after meals for 6 days, and azithromycin 500 mg/day one hour before meals for 5 days. Nasopharyngeal swabs were collected and SARS-CoV-2 status was determined by rtPCR (Cobas, Ct>40 negative) at baseline and on Day 7. Clinical remission was defined as absence of all COVID-19 symptoms (fever, shortness of breath, anosmia, ageusia, cough, runny nose, sore throat, headache, fatigue, muscle/joint pain, weakness, gastrointestinal symptoms, skin lesions). [3]
Animal models for proxalutamide include rodent xenograft models of prostate cancer. The compound is administered orally to evaluate its effects on tumor growth, AR signaling, and survival. Pharmacokinetic parameters are derived from plasma concentration-time curves after oral administration. Studies have examined the compound's tissue distribution, particularly its accumulation in AR-rich tissues such as the prostate. The compound's effects on serum testosterone and other hormonal parameters are also evaluated.

Proxalutamide (GT0918) has poor water solubility (<1 μg/ml in water at 25°C) and pH-dependent solubility (high under strongly acidic conditions, decreasing sharply as pH increases due to the basic pyridinyl group). Absolute bioavailability in mice was approximately 40%. [2]
In beagle dogs, after oral administration of conventional tablets (25 mg), pharmacokinetic parameters were: tmax 2.5±1.1 h, Cmax 3.1±1.5 μg/ml, t1/2 8.4±0.5 h, AUC0-48 42.1±22.3 μg·h/ml. For pH-modified solid dispersion tablets (GT0918/PVP/citric acid 1:2:2), parameters were: tmax 0.9±0.4 h (p<0.05 vs conventional), Cmax 5.1±2.4 μg/ml, t1/2 7.9±2.2 h, AUC0-48 38.3±8.2 μg·h/ml. Relative bioavailability of pHM-SD tablets was 125.6% of conventional tablets. [2]
Pharmacokinetic studies show that proxalutamide is orally bioavailable. The compound is soluble in DMSO at ≥60 mg/mL. Its oral bioavailability supports chronic dosing regimens for prostate cancer therapy. The compound's molecular weight and formula are not specified in the available literature. The compound is supplied at ≥98% purity. Standard pharmacokinetic studies in preclinical species have characterized its absorption, distribution, metabolism, and excretion profile.

In the COVID-19 clinical trial, treatment-emergent adverse events (TEAE) were reported more frequently in the Proxalutamide group (170 events in 171 subjects) than in placebo (99 events in 65 subjects). The most common TEAEs in the Proxalutamide arm were gastrointestinal: diarrhea (39), nausea (28), abdominal pain (19), abdominal discomfort (17), dyspepsia (20), heartburn (11), vomiting (4). Other events included fatigue (6), fever (1), disease progression (3), tachycardia (6), headache (5), anosmia (1), ageusia (0), arthralgia (0), back pain (3), pain in extremity (2), shortness of breath (5). No subjects in either arm discontinued the study due to TEAE. [3]
Toxicity data for proxalutamide are typical of androgen receptor antagonists. As a non-steroidal antiandrogen, the compound may cause side effects related to androgen deprivation, including hot flashes, fatigue, and sexual dysfunction. The compound has been evaluated in clinical trials for safety and tolerability. Standard safety precautions for handling pharmaceutical research compounds apply. The compound is for research use only and not for human use, except in approved clinical trial settings.

[1].Abstract 614: Proxalutamide (GT0918), a potent androgen receptor pathway inhibitor. Cancer Research. AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA.

[2].Microenvironmental pH-modified solid dispersions to enhance the dissolution and bioavailability of poorly water-soluble weakly basic GT0918, a developing anti-prostate cancer drug: preparation, characterization and evaluation in vivo. Int J Pharm. 2014 Nov 20;475(1-2):97-109.

[3].Proxalutamide Significantly Accelerates Viral Clearance and Reduces Time to Clinical Remission in Patients with Mild to Moderate COVID-19: Results from a Randomized, Double-Blinded, Placebo-Controlled Trial. Cureus. 2021 Feb; 13(2): e13492.

[4].Quantitative determination of proxalutamide in rat plasma and tissues using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2021 Feb 15;35(3):e9003.

[5].Metabolomic profiling to evaluate the efficacy of proxalutamide, a novel androgen receptor antagonist, in prostate cancer cells. Invest New Drugs. 2020 Oct;38(5):1292-1302.

Proxalutamide is being investigated in the clinical trial NCT03899467 (Safety and tolerability of Proxalutamide (GT0918) in patients with metastatic castration-resistant prostate cancer). Proxalutamide is an orally bioavailable androgen receptor (AR) antagonist with potential antitumor activity. After oral administration, proxalutamide binds to AR in target tissues, inhibiting androgen-induced receptor activation and promoting the formation of an inactive complex that cannot translocate to the nucleus. This prevents the binding and transcription of AR-responsive genes, which regulate the proliferation of prostate cancer cells. Furthermore, proxalutamide induces AR downregulation, thereby further blocking AR-mediated signaling. Ultimately, this leads to the inhibition of growth in prostate cancer cells expressing AR. Androgen receptors (AR) are overexpressed in prostate cancer and play a key role in prostate cancer cell proliferation. Mechanism of Action: Proxalutamide is an androgen receptor antagonist that inhibits androgen-induced receptor activation and leads to the formation of an inactive complex that cannot translocate to the nucleus. It can also induce androgen receptor downregulation, thereby further inhibiting androgen-induced receptor activation. The drug is being investigated for its potential anti-tumor activity and is being specifically studied for prostate cancer and COVID-19.

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Proxalutamide (GT0918) is a second-generation androgen receptor antagonist with a dual mechanism: direct AR antagonism and downregulation of AR expression, a mechanism not present in bicalutamide or enzalutamide. It is expected to be more effective and less toxic. It also lowers expression of ACE2, which may contribute to blocking SARS-CoV-2 entry into lung cells. [3] It is a developing anti-prostate cancer drug patented in the US and China. [2] The chemical structure is shown in Figure 1 of reference [2]: 4-[4,4-dimethyl-3-[6-[3-(2-oxazolyl)propyl]-3-pyridinyl]-5-oxo-2-thioxo-1-imidazolidinyl]-3-fluoro-2-(trifluoromethyl)-benzonitrile. [2]
Proxalutamide (GT0918) is an orally active potent androgen receptor antagonist. It can be used in the study for prostate cancer and COVID-19. It has been investigated in Phase 1/2/3 clinical trials for prostate cancer, breast cancer, and COVID-19. In July 2021, it received approval in Paraguay for the treatment of COVID-19. The compound is a non-steroidal antiandrogen (NSAA). The CAS number is 1398046-21-3. The compound is for research use only and not for human use except in approved clinical trials.

C24H19F4N5O2S

517.4986

517.119

C, 55.70; H, 3.70; F, 14.68; N, 13.53; O, 6.18; S, 6.20

1398046-21-3

60194102

Solid powder

4.3

0

10

6

36

894

0

KCBJGVDOSBKVKP-UHFFFAOYSA-N

InChI=1S/C24H19F4N5O2S/c1-23(2)21(34)32(17-9-6-14(12-29)19(20(17)25)24(26,27)28)22(36)33(23)16-8-7-15(31-13-16)4-3-5-18-30-10-11-35-18/h6-11,13H,3-5H2,1-2H3

4-(4,4-dimethyl-3-(6-(3-(oxazol-2-yl)propyl)pyridin-3-yl)-5-oxo-2-thioxoimidazolidin-1-yl)-3-fluoro-2-(trifluoromethyl)benzonitrile

Proxalutamide GT 0918 GT0918GT-0918

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.)