17,20-lyase/CYP17

Orteronel has an IC50 of 110 nM for androstenedione and 110 nM for DHEA, which are both produced in monkey adrenal cells in response to ACTH stimulation. Also, with an IC50 of 37 nM, ortersteronel significantly reduces the synthesis of DHEA in human adrenocortical tumor line H295R cells[1]. With an IC50 of 54 nM for rat steroid 17,20-lyase and 38 nM for human steroid 17,20-lyase, respectively, orteronel has strong inhibitory activity against either enzyme in vitro. Orteronel does not appreciably impact CYP3A4 or 11-hydroxylase, among other CYP isoforms. Compared to the other CYP isoforms, Orteronel has a higher inhibitory impact on 17,20-lyase in microsomes expressing human CYP isoforms, with an IC50 of 19 nM1.

Favorable pharmacokinetic characteristics are obtained with ortionel (1 mg/kg, po), with Tmax, Cmax, t1/2, and AUC0-24 hours of 1.7 hours, 0.147 μg/mL, 3.8 hours, and 0.727 μg/mL, respectively[1]. Oral administration of 1 mg/kg of orally administered Orteronel significantly lowers the levels of DHEA and serum testosterone in cynomolgus monkeys[2].

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.

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]

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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Dissolved in 0.5% methylcellulose; ≤1 mg/kg; Oral gavage

Adult male cynomolgus monkeys.

[14C]orteronel was administered orally to intact monkeys for pharmacokinetic analysis. When a 1 mg/kg dose was administered, the 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). Collectively, orteronel showed high BA and reasonable t1/2 which are important parameters for oral dosing. Considering that concentrations >300 nmol/L (>0.1 μg/mL) were observed to be required to inhibit DHEA production in vitro in monkey adrenal cells (Fig. 2A), twice-daily oral dosing at 5–15 mg/kg was predicted to give minimum trough orteronel levels to maintain 17,20-lyase inhibition and produce a substantial reduction of serum DHEA levels (assuming linear pharmacokinetic characteristics).[1]

[1]. Orteronel (TAK-700), a novel non-steroidal 17,20-lyase inhibitor: effects on steroid synthesis in human and monkey adrenal cells and serum steroid levels in cynomolgus monkeys. J Steroid Biochem Mol Biol. 2012 Apr;129(3-5):115-28.

[2]. Discovery of orteronel (TAK-700), a naphthylmethylimidazole derivative, as a highly selective 17,20-lyase inhibitor with potential utility in the treatment of prostate cancer. From Bioorganic & Medicinal Chemistry (2011), 19(21), 6.

Orteronel is a member of the class of pyrroloimidazoles that is 6,7-dihydro-5H-pyrrolo[1,2-c]imidazole substituted by hydroxy and 6-(methylcarbamoyl)naphthalen-2-yl groups at position 7 (the 7S-stereoisomer). It is a non-steroidal 17,20-lyase inhibitor that suppresses androgen synthesis. It was previously in clinical development for the treatment of castration-resistant prostate cancer -- trial now discontinued. It has a role as an antineoplastic agent, a sterol biosynthesis inhibitor and an EC 1.14.99.9 (steroid 17alpha-monooxygenase) inhibitor. It is a naphthalenecarboxamide, a secondary carboxamide, a pyrroloimidazole and a tertiary alcohol.
Orteronel has been investigated for the treatment of Prostate Cancer.
Orteronel is an orally bioavailable non-steroidal androgen synthesis inhibitor of steroid 17alpha-monooxygenase (17,20 lyase) with potential antiandrogen activity. TAK-700 binds to and inhibits the steroid 17alpha-monooxygenase in both the testes and adrenal glands, thereby inhibiting androgen production. This may decrease androgen-dependent growth signaling and may inhibit cell proliferation of androgen-dependent tumor cells. The cytochrome P450 enzyme CYP17A1 (P450C17), localized to the endoplasmic reticulum (ER), exhibits both 17alpha-hydroxylase and 17,20-lyase activities, and plays a key role in the steroidogenic pathway that produces steroidal hormones, such as progestins, mineralocorticoids, glucocorticoids, androgens, and estrogens.

C18H17N3O2

307.35

307.132

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

566939-85-3

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

9796590

White to light brown 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

1

CNC(=O)C1=CC2=C(C=C1)C=C(C=C2)[C@]3(CCN4C3=CN=C4)O

OZPFIJIOIVJZMN-SFHVURJKSA-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)/t18-/m0/s1

(S)-6-(7-hydroxy-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-7-yl)-N-methyl-2-naphthamide

TAK700; TAK-700; TAK 700; (S)-Orteronel; (S)-6-(7-hydroxy-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-7-yl)-N-methyl-2-naphthamide; TAK700; UE5K2FNS92; Orteronel (s-isomer);

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)

Solubility in Formulation 1: ≥ 1.43 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.43 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1.43 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% methylcellulose: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)