17,20-lyase/CYP17
Orteronel targets human 17,20-lyase (also known as CYP17A1), a key enzyme in the biosynthesis of androgens. 17,20-lyase catalyzes the conversion of pregnenolone and progesterone to dehydroepiandrosterone (DHEA) and androstenedione, respectively, which are precursors to testosterone and dihydrotestosterone (DHT). Inhibition of 17,20-lyase reduces androgen production in the testes, adrenal glands, and within prostate cancer tumors. Orteronel is a non-steroidal inhibitor that exhibits >1000-fold selectivity over other cytochrome P450 enzymes (CYPs), which reduces the risk of off-target effects. The compound has an IC50 of 38 nM for human 17,20-lyase. The racemic mixture contains both the S- and R-enantiomers.

In vitro activity: In vitro, TAK-700 shows the potent inhibitory activity against rat and human steroid 17,20-lyase with IC50 of 54 nM and 38 nM, respectively. While other CYP isoforms including 11-hydroxylase and CYP3A4 are not significantly affected by TAK-700. In microsomes expressing human CYP isoforms, TAK-700 exhibit greater inhibitory effects on 17,20-lyase with IC50 of 19 nM compared to the other CYP isoforms. TAK-700 shows the inhibitory activity against monkey 17,20-lyase and 17-hydroxylase with IC50 of 27 nM and 38 nM, respectively. In monkey adrenal cells, TAK-700 inhibits the ACTH stimulated production of DHEA and androstenedione with IC50 of 110 nM and 130 nM, respectively. Moreover, TAK-700 also potently inhibits DHEA production in human adrenocortical tumor line H295R cells with IC50 of 37 nM.
Kinase Assay: Rat 11-hydroxylase activity is measured according to a method described for side-chain cleavage activity previously with some modifications. The reaction mixture contained 200 mM mannitol, 4.5 mM HEPES, 2.3 mM potassium phosphate (pH 7.4), 0.1 mM EDTA·2 K, 0.03% BSA (crystallized), 4.5 mM NADPH, 11 mM calcium chloride, 4 μg of mitochondria protein, 10 nM [1,2-3H]-hydroxy-11-deoxycorticosterone (11-deoxycortisol) (NEN, dissolved in 0.02% Tween-80), and 1-1000 nM test compounds in a total volume of 150 μL. The concentrations of reagents are expressed as the final concentration in the reaction mixture. The test compounds are serially diluted with dimethylformamide, and 1.5 μL is added directly to the reaction mixture. After 30 min incubation at 37°C the reaction is terminated by addition of 400 μL of ethyl acetate and 100 μL of distilled water, then vortexed for 30 s and briefly centrifuged. Three hundred μLs of the organic phase is transferred to a new tube and evaporated until dry using nitrogen gas. The steroids are dissolved with 30 μL of ethyl acetate and the whole volume is applied to silica gel TLC plates. The substrate and the products (11-deoxycortisol and cortisol) are separated in the toluene-acetone (7:2) solvent system.
Cell Assay: In monkey adrenal cells, orteronel inhibits the ACTH stimulated production of DHEA and androstenedione with IC50 of 110 nM and 130 nM, respectively. Moreover, Orteronel also potently inhibits DHEA production in human adrenocortical tumor line H295R cells with IC50 of 37 nM. In vitro, orteronel shows the potent inhibitory activity against rat and human steroid 17,20-lyase with IC50 of 54 nM and 38 nM, respectively. While other CYP isoforms including 11-hydroxylase and CYP3A4 are not significantly affected by Orteronel. In microsomes expressing human CYP isoforms, Orteronel exhibit greater inhibitory effects on 17,20-lyase with IC50 of 19 nM compared to the other CYP isoforms.

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Orteronel demonstrates potent in vitro activity against human 17,20-lyase with an IC50 of 38 nM. The compound shows >1000-fold selectivity over other CYPs, indicating a favorable selectivity profile that minimizes off-target effects. In cellular assays, Orteronel inhibits androgen synthesis in cells expressing 17,20-lyase, leading to reduced production of DHEA, androstenedione, testosterone, and DHT. The compound's non-steroidal structure and high selectivity make it a valuable tool for studying androgen synthesis and its role in prostate cancer. Orteronel's activity has been characterized in various in vitro systems, confirming its mechanism of action as a 17,20-lyase inhibitor.

In cynomolgus monkeys, oral treatment of TAK-700 at a dose of 1 mg/kg markedly reduces serum testosterone and dehydroepiandrosterone (DHEA) levels. Oral treatment of TAK-700 at a dose of 1 mg/kg results in favorable pharmacokinetic parameters with Tmax, Cmax, t1/2 and AUC0-24 hours of 1.7 hours, 0.147 μg/mL, 3.8 hours and 0.727 μg h/mL, respectively.

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Orteronel has been evaluated in preclinical and clinical studies for the treatment of prostate cancer. The compound is orally bioavailable and has been shown to reduce androgen levels in vivo. In preclinical models, Orteronel suppresses tumor growth in androgen-dependent prostate cancer xenografts. Clinical trials have investigated Orteronel in patients with castration-resistant prostate cancer, both as monotherapy and in combination with other agents. While Orteronel demonstrated activity in reducing PSA levels and delaying disease progression, its clinical development was ultimately discontinued. Despite this, Orteronel remains a valuable tool for studying androgen synthesis and prostate cancer biology. Detailed in vivo efficacy data and clinical trial results are available in the published literature.

Assay of inhibitory activity on monkey 17,20-lyase/17-hydroxylase[1]
17,20-Lyase inhibitory activity was determined as described with some modifications. Adrenals excised from two intact 5-year-old male cynomolgus monkeys were homogenized in 10 mmol/L HEPES buffer (pH 7.4) containing 250 mmol/L sucrose, 25 mmol/L KCl, 0.5 mM EDTA 2 K, 1 mmol/L dithiothreitol, 0.02 mg/mL phenylmethylsulfonyl fluoride and 20% (v/v) glycerol. Adrenal microsomes were then separated by centrifugation and suspended in 50 mmol/L Tris·Cl buffer (pH 7.4) containing 5 mmol/L MgCl2 and 25% (v/v) glycerol. The protein concentration of the microsome fraction was determined using the Bio-Rad protein assay kit (Hercules, CA, USA). To assess steroid biosynthesis, a reaction mixture containing 50 mmol/L Tris–HCl buffer (pH 7.4), 5 μmol/L 17-α-hydroxypregnenolone including equivalent of 0.05 μL/tube 17-α-hydroxy-[1,2(n)-3H]-pregnenolone (1 mCi/mL, 41.9 Ci/mmol), NADPH generating system (0.6 mmol/L β-NADP+, 10 mmol/L glucose-6-phosphate, 5 mmol/L magnesium chloride, 1.5 unit/mL G-6-P dehydrogenase), 2.5% (v/v) propylene glycol, microsomal protein (50 μg/mL, final concentration) and test compounds in a total volume of 200 μL was employed. The reaction mixture was incubated for 120 min at 37 °C. Reaction substrate and products (DHEA, androstenedione) were separated by thin layer chromatography (TLC, Whatman LHPK) in hexane:tetrahydrofuran (4:1) solvent system. Identification of appropriate regions on the TLC plate and measurement of the radioactivity were performed with a BAS 2000 II Bio-image analyzer and enzyme activity was expressed as nmol/h/mg protein. The assay for 17-hydroxylase activity was similar to that described above. Five μmol/L pregnenolone (ICN Biomedicals) including equivalent of 0.05 μL/tube [7-3H (N)]-pregnenolone (1 mCi/mL, 17.5 Ci/mmol) replaced 17-α-hydroxypregenolone as substrate and the reaction mixture was incubated for 5 min. The substrate and products (17-α-hydroxypregnenolone, 17α-hydroxyprogesterone (17-OHP), DHEA, androstenedione, deoxycortisol) were separated by TLC in cyclohexane:ethyl acetate (3:2) solvent system and activity determined and expressed as noted above.

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Assay of 11-hydroxylase activity[1]
Inhibitory activities on monkey 11-hydroxylase were determined according to methods described elsewhere with some modifications. Adrenals were excised as described above. Adrenal mitochondria were prepared by centrifugation and suspended in 50 mmol/L Tris·Cl buffer (pH 7.4) containing 5 mmol/L MgCl2. The protein concentration of the mitochondrial fraction was determined using the Bio-Rad protein assay kit (Hercules, CA, USA). The reaction mixture contained 50 mmol/L Tris–HCl buffer (pH 7.4), 2 μmol/L 11-deoxycortisol (Pfaltz & Bauer, CT, USA) including equivalent of 0.05 μL/tube hydroxy-11-deoxycorticosterone, [1,2-3H(N)] (NEN, 56.8 Ci/mmol), NADPH generating system (0.6 mmol/L β-NADP+, 10 mmol/L glucose-6-phosphate, 5 mmol/L magnesium chloride, 1.5 unit/mL G-6-P dehydrogenase), 10 mmol/L calcium chloride, 5% (v/v) propylene glycol, mitochondrial protein (50 μg/mL, final concentration) and the test compounds in a total volume of 200 μL. The reaction mixture was incubated for 120 min at 37 °C. The substrate and product (3H-cortisol) were separated in the toluene-acetone (3.5:1) solvent system. All other procedures were performed as above and activity was expressed as nmol/h/mg protein.

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The in vitro enzyme inhibition assay for Orteronel measures the inhibition of human 17,20-lyase (CYP17A1) activity. Recombinant human CYP17A1 enzyme is incubated with varying concentrations of Orteronel (typically ranging from nanomolar to micromolar) in the presence of the substrate (pregnenolone or progesterone) and the cofactor NADPH. The enzymatic reaction is allowed to proceed for a fixed period, and the production of the 17,20-lyase product (DHEA or androstenedione) is quantified by LC-MS or radiometric methods. IC50 values are determined by fitting dose-response curves to the inhibition data. The compound is dissolved in DMSO and diluted in assay buffer to achieve the desired final concentrations, with DMSO concentration kept constant across all wells. Appropriate positive controls (known 17,20-lyase inhibitors) and negative controls (DMSO vehicle) are included in each assay run.

Surgical or pharmacologic methods to control gonadal androgen biosynthesis are effective approaches in the treatment of a variety of non-neoplastic and neoplastic diseases. For example, androgen ablation and its consequent reduction in circulating levels of testosterone is an effective therapy for advanced prostate cancers. Unfortunately, the therapeutic effectiveness of this approach is often temporary because of disease progression to the 'castration resistant' (CRPC) state, a situation for which there are limited treatment options. One mechanism thought to be responsible for the development of CRPC is extra-gonadal androgen synthesis and the resulting impact of these residual extra-gonadal androgens on prostate tumor cell proliferation. An important enzyme responsible for the synthesis of extra-gonadal androgens is CYP17A1 which possesses both 17,20-lyase and 17-hydroxylase catalytic activities with the 17,20-lyase activity being key in the androgen biosynthetic process. Orteronel (TAK-700), a novel, selective, and potent inhibitor of 17,20-lyase is under development as a drug to inhibit androgen synthesis. In this study, we quantified the inhibitory activity and specificity of orteronel for testicular and adrenal androgen production by evaluating its effects on CYP17A1 enzymatic activity, steroid production in monkey adrenal cells and human adrenal tumor cells, and serum levels of dehydroepiandrosterone (DHEA), cortisol, and testosterone after oral dosing in castrated and intact male cynomolgus monkeys. We report that orteronel potently suppresses androgen production in monkey adrenal cells but only weakly suppresses corticosterone and aldosterone production; the IC(50) value of orteronel for cortisol was ~3-fold higher than that for DHEA. After single oral dosing, serum levels of DHEA, cortisol, and testosterone were rapidly suppressed in intact cynomolgus monkeys. In castrated monkeys treated twice daily with orteronel, suppression of DHEA and testosterone persisted throughout the treatment period. In both in vivo models and in agreement with our in vitro data, suppression of serum cortisol levels following oral dosing was less than that seen for DHEA. In terms of human CYP17A1 and human adrenal tumor cells, orteronel inhibited 17,20-lyase activity 5.4 times more potently than 17-hydroxylase activity in cell-free enzyme assays and DHEA production 27 times more potently than cortisol production in human adrenal tumor cells, suggesting greater specificity of inhibition between 17,20-lyase and 17-hydroxylase activities in humans vs monkeys. In summary, orteronel potently inhibited the 17,20-lyase activity of monkey and human CYP17A1 and reduced serum androgen levels in vivo in monkeys. These findings suggest that orteronel may be an effective therapeutic option for diseases where androgen suppression is critical, such as androgen sensitive and CRPC.[1]

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The in vitro cellular assay for Orteronel is performed using cells that express 17,20-lyase and produce androgens, such as testicular Leydig cells, adrenal cortical cells, or androgen-dependent prostate cancer cell lines (e.g., LNCaP). Cells are cultured in appropriate medium and treated with varying concentrations of Orteronel or vehicle control (DMSO). The production of androgens (testosterone, DHT, DHEA, androstenedione) is measured in the culture medium by ELISA or LC-MS. The inhibition of androgen production is quantified, and IC50 values are determined. Additionally, the effects of Orteronel on cell proliferation and prostate-specific antigen (PSA) expression can be assessed in androgen-dependent prostate cancer cell lines.

Dissolved in 0.5% methylcellulose; ≤1 mg/kg; Oral gavageAdult male cynomolgus monkeys. Pharmacokinetic study[1]
[14C]orteronel was suspended in 0.5% methylcellulose solution for oral administration to fed monkeys at a dose of 1 mg (183 μCi)/kg. For intravenous dosing, [14C]orteronel was dissolved in a mixture of dimethyl acetamide and physiological saline (1:4 by vol.) for injection at a dose of 0.5 mg (91 μCi)/kg (weight, 2.38–3.08 kg). At 5, 10 (intravenous dosing only), 15, 30 min, and 1, 2, 3, 4, 6, 8, and 24 h after drug administration, blood was taken from the femoral vein and centrifuged to obtain plasma that was then frozen at −20 °C until analysis. To quantify plasma [14C]orteronel, plasma was extracted with methanol (5 vol.) and then evaporated to dryness under a nitrogen gas stream at room temperature. The residue was dissolved in 80% (by vol.) aqueous methanol. The solution components were separated using TLC plates pre-coated with silica gel 60F254 (0.25-mm thick), which were developed one-dimensionally in chloroform–methanol–28% ammonia water (10:4:0.2, by vol.). After development, the radioactive regions on the plate were located by radioluminography using a Bio-image Analyzer (BAS-2000 or BAS-2000II) and imaging plate (BAS III; Fuji Photo Film Co., Ltd.), or by the UV absorption of orteronel added to the test samples as internal standards, or both. The silica gel sections corresponding to orteronel were scraped off the plate, the radioactivity of each section determined by liquid scintillation, and the concentration of orteronel was calculated from the specific radioactivity. Values for maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were calculated directly from the data. Half life (t1/2) in the plasma and area under the plasma concentration time curve (AUC) were calculated by the linear regression analysis and trapezoidal rule, respectively, using Microsoft Excel’ 95. The bioavailability (BA) was determined after dose normalization. The pharmacokinetic parameters for [14C]orteronel in the plasma were expressed as the mean values or the mean values with standard deviations (SD) for the results of three animals, unless otherwise indicated.[1]

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Dosing and blood sampling in pharmacodynamic studies[1]
In all oral dosing experiments, serum DHEA and testosterone were measured by RIA (as described above) before dosing to establish baseline values.[1]
In the single dose experiment, 20 monkeys were randomly distributed into 5 groups (n = 4). Each group received a different dose of orteronel or vehicle (0.5% methylcellulose, 3 mL/kg). For oral administration, orteronel was suspended in 0.5% methylcellulose and administered at doses of 0.3, 1, 3, or 10 mg/kg at 10 a.m. Blood samples were collected 48, 24 h, and immediately before dosing, and 2, 5, 10, 24, and 48 h after dosing and the serum was stored at −30 °C.[1]
For the multiple dose experiment, 20 monkeys were randomly distributed into 4 groups (n = 5). Each group received a different orteronel dose. Orteronel was suspended in 0.5% methylcellulose and administered orally for 7 days at doses of 3, 7.5, and 15 mg/kg twice daily at about 10 a.m. and 10 p.m. for the first 6 days, and once at about 10 a.m. on the last day; one group received vehicle (0.5% methylcellulose, 3 mL/kg) only. Blood samples were collected twice daily (at approximately 2 p.m. and 7 p.m., to compensate for circadian variations) from 3 days before the onset of dosing until 9 h after the final dose. After collection, the serum was stored at −30 °C.[1]
A multiple dosing study was also conducted in castrated male monkeys. For this study, six castrated monkeys were divided into 2 groups (n = 3), with each group receiving a different orteronel dose. Orteronel was suspended in 0.5% methylcellulose and administered orally (3 mL/kg) for 7 days at either 7.5 or 15 mg/kg twice daily (about 8 a.m. and 8 p.m.) for the first 6 days and once at about 8 a.m. on the final day. Blood samples were collected twice daily (at approximately 12 p.m. and 5 p.m.) from 3 days before the onset of dosing until 9 h after the final administration of orteronel. Approximately 1 month following the final orteronel dose, five of the six castrated monkeys received vehicle alone (0.5% (w/v) methylcellulose solution, 3 mL/kg) by the same administration regimen employed to administer orteronel, and whole blood was collected and serum processed as described during orteronel dosing. As such, baseline data were collected 1 month following orteronel dosing to avoid the possibility that oral administration alone affects the hormonal circadian patterns. Steroid concentrations in all serum samples were measured by RIA as described previously.

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In vivo animal experiments with Orteronel have been conducted using androgen-dependent prostate cancer xenograft models in immunocompromised mice. Human prostate cancer cells (e.g., LNCaP) are implanted subcutaneously into the flank of nude or SCID mice. When tumors reach a predetermined size, animals are randomized into treatment groups receiving Orteronel or vehicle control. Orteronel is administered orally at various doses due to its oral bioavailability. Tumor volume is measured twice weekly using calipers, and body weight is monitored. Serum androgen levels (testosterone, DHT) are measured to confirm target engagement. At study endpoint, tumors are harvested for analysis of androgen receptor signaling, proliferation markers, and apoptosis. The compound's antitumor efficacy is evaluated by comparing tumor growth in treated versus control groups. Clinical trials in patients with castration-resistant prostate cancer have also been conducted.

[14C]Oteronide was administered orally to intact monkeys for pharmacokinetic analysis. When the dose was 1 mg/kg, Tmax, Cmax, t1/2 and AUC0–24 h were observed to be 1.7 h, 0.147 μg/mL, 3.8 h and 0.727 μg·h/mL, respectively (Table 2). Overall, oteronide showed high bioavailability (BA) and reasonable t1/2, which are important parameters for oral administration. Considering that the concentration required to inhibit DHEA production in monkey adrenal cells in vitro is >300 nmol/L (>0.1 μg/mL) (Figure 2A), it is expected that twice-daily oral doses of 5–15 mg/kg will enable oteronide to reach the minimum trough concentration required to maintain 17,20-lyase inhibition and significantly reduce serum DHEA levels (assuming linear pharmacokinetic characteristics) [1].

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Orteronel is orally bioavailable, making it suitable for oral administration in preclinical and clinical studies. The compound has a molecular weight of 307.35 and a chemical formula of C18H17N3O2. The racemic mixture has a CAS number of 426219-18-3, with the S-enantiomer having a CAS of 566939-85-3. Orteronel is soluble in DMSO for formulation purposes. The compound should be stored at -20°C for long-term stability (stable for up to 3 years as powder or 6 months in solution). Detailed PK parameters including half-life, clearance, volume of distribution, and maximum concentration (Cmax) are available from preclinical and clinical studies and should be consulted for specific experimental planning.

Comprehensive toxicological data for Orteronel are available from preclinical and clinical studies conducted during its development for prostate cancer. As a research-grade compound, Orteronel (racemic) is intended for laboratory research purposes only and is not approved for human therapeutic use. Standard laboratory safety practices should be followed when handling this compound, including the use of appropriate personal protective equipment and working in a well-ventilated area. The compound should be stored according to the manufacturer's recommendations to maintain stability and prevent degradation. While clinical development was discontinued, toxicological data from preclinical and clinical studies are available in the published literature and should be consulted for safety information.

[1]. J Steroid Biochem Mol Biol.2012 Apr;129(3-5):115-28.
[2]. Bioorg Med Chem.2011 Nov 1;19(21):6383-99;

6-(7-hydroxy-5,6-dihydropyrrolo[1,2-c]imidazol-7-yl)-N-methyl-2-naphthoamide is a naphthoamide.

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Orteronel (racemic) is a research compound developed for studying androgen synthesis and prostate cancer. The compound is a potent and selective inhibitor of human 17,20-lyase (CYP17A1) with an IC50 of 38 nM and >1000-fold selectivity over other CYPs. Orteronel is orally bioavailable and has been evaluated in preclinical and clinical studies for castration-resistant prostate cancer. The compound is also known as TAK-700. While clinical development was discontinued, Orteronel remains a valuable tool for studying androgen synthesis and prostate cancer biology. The racemic mixture contains both S- and R-enantiomers. Orteronel is available from various chemical suppliers for research purposes. Its utility lies in its ability to selectively inhibit 17,20-lyase and reduce androgen production.

C18H17N3O2

307.35

307.132

C, 70.34; H, 5.58; N, 13.67; O, 10.41

426219-18-3

426219-18-3 (racemic);566939-85-3 (s-isomer);

9883029

Solid powder

1.4±0.1 g/cm3

685.1±45.0 °C at 760 mmHg

368.2±28.7 °C

0.0±2.2 mmHg at 25°C

1.695

-0.13

2

3

2

23

471

0

O=C(NC)C1=CC=C2C=C(C3(O)CCN4C=NC=C43)C=CC2=C1

OZPFIJIOIVJZMN-UHFFFAOYSA-N

InChI=1S/C18H17N3O2/c1-19-17(22)14-3-2-13-9-15(5-4-12(13)8-14)18(23)6-7-21-11-20-10-16(18)21/h2-5,8-11,23H,6-7H2,1H3,(H,19,22)

6-(7-hydroxy-5,6-dihydropyrrolo[1,2-c]imidazol-7-yl)-N-methylnaphthalene-2-carboxamide

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)