In vitro assay of CYP17

The in vitro CYP17 inhibitory activity of Galeterone was evaluated using rapid acetic acid releasing assay, utilizing intact P450c17-expressing E. coli as the enzyme source. It involved the use of [21-3H]-17α-hydroxypregnenolone as the substrate, and CYP17 activity was measured by the amount of tritiated acetic acid formed during the cleavage of the C-21 side chain of the substrate. IC50 value was obtained directly from plots relating percentage inhibition versus inhibitor concentration over appropriate ranges.

Cell lines

LNCaP Cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

1 ~ 15 μM; 24 hrs

Applications

Compared with all Galeterone analogues, Galeterone was still the most effective compound in vitro, down-regulating >95% AR at the concentration of 15 μM.

Animal models

Male SCID mice bearing LAPC-4 tumors

Dosage form

0.15 mmol/kg; s.c.; b.i.d., for 31 days

Applications

Galeterone effectively inhibited the average volume of LAPC-4 tumors in SCID mice by more than 80%. After 31 days of Galeterone treatment, the final tumor weight was also significantly reduced by more than 50%.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

TOK-001 (Galeterone) is a novel small molecule that selectively target CYP17 and androgen receptor for prostate cancer treatment [1].

CYP17 and androgen receptor (AR) are two preferred target during the treatment of prostate cancer: The former is the prime enzyme that responsible for the production of cancer-inducing androgens [1], whereas the latter acts as transcription factor that increases the expression of androgen-responsive genes [2].

Galeterone is a small molecular therapeutics that designed to treat prostate cancer in three distinctive pathways: competitively inhibit the enzyme function of CYP17. Decreasing androgen-responsive genes expression by binding to AR, and downregulates AR population [3]. Using 293T cells transfected with CYP17, galeterone inhibited the lyase function of CYP17 with an IC50 value of 47 nM after 18 hrs of incubation [3]. In prostate cancer cell line assay, galeterone inhibited the proliferation of LNCaP and LAPC-4 with IC50 value of 6 and 3 μM, respectively [3].

In mice xenograft model that inoculated with LAPC4 cell lines, a daily subcutaneous injection of galeterone (0.15 mmol/kg, twice per day) reduced both the average volume and weight of tumour less by more than 80% and 50%, respectively in comparison with control group [3]. Galeterone is currently underwent phase III clinical trails for castration-resistant prostate cancer.

References:
[1]. Devore N M, & Scott E E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001. Nature, 2012, 482: 116-119.
[2]. Mallik I, Davila M, Tapia T, et al. Androgen regulates Cdc6 transcription through interactions between androgen receptor and E2F transcription factor in prostate cancer cells. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008,1783:1737-1744.
[3]. Bruno R D, Vasaitis T S, Gediya L. K, et al. Synthesis and biological evaluations of putative metabolically stable analogs of VN/124-1 (TOK-001): Head to head anti-tumor efficacy evaluation of VN/124-1 (TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model. Steroids, 2011,76: 1268-1279.