AR/androgen receptor

In this study, researchers describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC50, 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5α-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all[1].

TFM-4AS-1, but not Cl-4AS-1 exhibits SARM activities in OVX rats. [1]
In contrast, Cl-4AS-1 produced significant increases in uterine weight at all the doses that stimulated bone formation, indicating TFM-4AS-1 has tissue-selective effects in vivo.[1]
Treatments for 6 weeks with 3 mg/kg/day DHT sc or 10 mg/kg/day sc TFM-4AS-1 significantly increased LBM by 16.5 grams (118%) and 11.4 grams (81%) above that observed in vehicle-treated rats, respectively (Fig. 4E). The LBM increase caused by TFM-4AS-1 was significantly different than vehicle group but not from the DHT-treated group. DHT significantly decreased FM, whereas the reduction by TFM-4AS-1 was not significant (Fig. 4F). DHT again caused a ∼4-fold increase in uterus weight whereas TFM-4AS-1 did not. Thus TFM-4AS-1 showed anabolic activity without uterotrophic activity. [1]
Similar to the previous experiment (Fig. 4), DHT and TFM-4AS-1 significantly increased bone formation rate by 204 and 308%, respectively (Fig. 5A). In the same animals, DHT increased sebaceous gland mean area by 108% and uterus weight nearly 400% (Fig. 5, B and C). In contrast, 10 mpk TFM-4AS-1 increased gland mean area by 33% and did not increase uterus weight. These data indicate that TFM-4AS-1 has reduced effects on the pilosebaceous unit and the uterus at anabolic doses. [1]
Sexually mature males were castrated (orchiectomized, ORX) or sham-operated the day before treatment and dosed with TFM-4AS-1 or Cl-4AS-1 (10 mg/kg/day sc) for 7 days. In sham-operated rats, TFM-4AS-1 significantly reduced ventral prostate weights by 50%, demonstrating its antagonism of endogenous androgens (Fig. 5D). In castrated rats, TFM-4AS-1 caused a small (10%) but significant increase in ventral prostate weight. [1]
This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, researchers identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands[1].

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TFM-4AS-1 dosed sc for 24 days at 10 mg/kg/day increased the periosteal double-labeled surface, mineral apposition rate, and bone formation rate to levels similar to 3 mg/kg/day sc DHT, the lowest fully effective DHT dose as determined in pilot experiments.

Binding and Transcription Assays [1]
Binding and transactivation assays were performed with human breast carcinoma cell line MDA-MB-453, which expresses endogenous AR. AR binding assays were conducted with lysates from MDA-MB-453 cells or to the ligand binding domain of rhesus monkey (rhARLBD) fused in-frame to glutathione S-transferase (GST) and expressed in yeast. Radioligand competition binding assays with 0.5 nm [3H]methyltrienolone (R1881, a non-aromatizable AR agonist) were as described. Transactivation assays in 96-well plates used transient transfection of a modified mouse mammary tumor virus long terminal repeat promoter upstream of luciferase (MMTV-LUC)). This MMTV has two direct repeat copies of a consensus glucocorticoid receptor (GR) response element between positions −88 and −190; these sequences are also recognized by AR. Potencies of compounds were determined by calculating the inflection points of the sigmoidal dose response curves. The Emax values were calculated as percent of maximal activity at the highest dose tested relative to a full agonist (100 nm R1881 or DHT). Transrepression was assessed using the pGL2 luciferase reporter containing the human matrix metalloprotease-1 (MMP-1) promoter fragment (-179 to +63). The reporter was transfected with rhesus AR (rhAR) into 22RV1 prostate cancer cells, and activated by pretreatment with 100 nm 12-O-tetradecanoylphorbol-13-acetate. Repression was evaluated by measuring luciferase activity.

N/C Interaction [1]
The N/C interaction of rhAR was evaluated by a mammalian two-hybrid assay in CV1 cells. The Gal4-DNA binding domain was fused with the ligand binding domain (LBD) of rhAR (amino acids 637–895); a VP16 construct was fused with amino acids 1–513 of rhAR. Both plasmids were co-transfected with a luciferase reporter under the control of multiple Gal4-DBD binding sites. The N/C interaction was detected as a ligand-mediated increase of luciferase activity.

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Microarray and RNA Analyses [1]
Prostate microarray studies were performed as described. For cell culture studies, total RNA was extracted using TRIzol from duplicate 10-cm dishes of MDA-MB-453 cells treated 18 h. Microarray analysis was performed on 5 μg of total RNA. Data were normalized to achieve identical median fluorescence intensity of each array. For a transcript to be considered DHT-regulated, the probe must have corresponded to a gene annotated at Entrez Gene, and the hybridization signals must have tested different from vehicle control (p < 0.05, Rosetta error model and differed from vehicle controls by ≥1.5-fold in both duplicate 200 nm DHT samples. Gene expression data are the mean of the duplicates ± S.D. For quantitative RT-PCR, total RNA was collected and analyzed as described after 18 h of treatment. For studies involving cycloheximide, MDA-MB-453 cells in 10-cm dishes were pretreated for 30 min with 10 μg/ml cycloheximide, and the AR ligand was added at indicated concentrations for an additional 6 h.

Animal procedures were performed as described. Briefly, bone, body composition, and uterine studies were performed in ovariectomized (OVX) or sham-operated rats at age 6–9 months, 3 months post-surgery. Animals were also treated with the bone resorption inhibitor alendronate unless noted (5.6 μg/kg/week). Animals were randomized into groups of equal weights (n = 10–16) and compounds in 3% benzyl alcohol in sesame oil (vehicle) were given by subcutaneous (sc) injection for 24 days. Uteri were dissected at the cervix and weighed wet. Sebaceous gland area in dorsal skin sections was measured using BioQuant. Femurs were analyzed as described. The primary measurement, bone formation rate (BFR), was assessed by histological analysis of fluorochrome double-labeling at the periosteal surface of the distal femur. Calcein (10 mg/kg, SC) was administrated 12 and 3 days prior to the termination of the study. Fat and lean body mass composition changes were assessed by dual energy x-ray absorptiometry (DEXA). Statistical analysis was performed by Kruskal-Wallis non-parametric ANOVA followed by Student-Neuman-Keuls post-hoc testing for intergroup differences. Prostate and seminal vesicles were studied in 3–4 month-old 250–300 g rats after orchidectomy (ORX) or sham operation. Nine days following surgery, animals were injected (sc) daily with test compounds for 7 or 14 days. At the indicated times, animals were euthanized by CO2, and ventral prostates were weighed. Data were analyzed by Fisher's PLSD and ANOVA.[1]

[1]. Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands. J Biol Chem. 2009, 284,52.

Our experiments in castrated rats demonstrated that TFM-4AS-1 is a partial agonist that partially antagonizes both endogenous androgens and co-dosed DHT. However, poor solubility prevented us from testing whether it maintains tissue-selectivity at higher exposures. We identified a non-steroidal SARM, FTBU-1, which has no 5α-reductase activity and improved solubility. This compound closely mimics the in vitro transcriptional profile of TFM-4AS-1, albeit with higher agonistic activity (81% versus 55% MMTV transactivation). Like TFM-4AS-1, FTBU-1 had little effect on the uterus at anabolic doses and at exposures ≥8-fold above those required for anabolism FTBU-1 exhibited 50% less uterotrophic activity than DHT. While based on our experience with related compounds, we suspect that this uterotrophic effect is caused by the higher agonism.9 It remains possible that at high doses, tissue-selective SARMs could cause unwanted effects.[1]
The transcriptional profile of our SARMs is distinct from that of the SERMs. Raloxifene, an osteoprotective ER ligand that lacks the agonistic activities of estradiol in breast and uterus, and is an ER antagonist in transactivation assays but represses ER-controlled AP-1 binding sites. In contrast, TFM-4AS-1 is an agonist in transactivation assays and does not inhibit AP-1-mediated transcription of the MMP-1 reporter. Unlike the significant body of information describing the clinical properties of SERMs, little clinical information is available regarding the actions of SARMs. However, in a 12-week study in healthy postmenopausal subjects, an AR ligand, MK-0773, which was selected based on similarity to TFM-4AS-1, exhibited SARM-like properties by increasing LBM without affecting markers of skin virilization or endometrial proliferation. Thus the properties of SARMs described here might translate into patients and apply broadly to the discovery of new therapeutic androgens.[1]

C27H33F3N2O2

474.558337926865

474.249

188589-61-9

10277123

White to off-white solid powder

5.91

1

5

2

34

872

7

FC(C1C=CC=CC=1NC([C@H]1CC[C@H]2[C@@H]3CCC4[C@@](C=CC(N4C)=O)(C)[C@H]3CC[C@@]21C)=O)(F)F

YFBLEKKYWFJKBP-JZFZSVFHSA-N

InChI=1S/C27H33F3N2O2/c1-25-14-12-18-16(8-11-22-26(18,2)15-13-23(33)32(22)3)17(25)9-10-20(25)24(34)31-21-7-5-4-6-19(21)27(28,29)30/h4-7,13,15-18,20,22H,8-12,14H2,1-3H3,(H,31,34)/t16-,17-,18-,20+,22+,25-,26+/m0/s1

(1S,3aS,3bS,5aR,9aR,9bS,11aS)-6,9a,11a-trimethyl-7-oxo-N-[2-(trifluoromethyl)phenyl]-2,3,3a,3b,4,5,5a,9b,10,11-decahydro-1H-indeno[5,4-f]quinoline-1-carboxamide

TFM-4AS-1; 188589-61-9; (1S,3aS,3bS,5aR,9aR,9bS,11aS)-6,9a,11a-Trimethyl-7-oxo-N-[2-(trifluoromethyl)phenyl]-2,3,3a,3b,4,5,5a,9b,10,11-decahydro-1H-indeno[5,4-f]quinoline-1-carboxamide; SCHEMBL5241935; YFBLEKKYWFJKBP-JZFZSVFHSA-N;

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.)