17,20-lyase/CYP17

体外活性：在体外，TAK-700 对大鼠和人类固醇 17,20-裂合酶显示出有效的抑制活性，IC50 分别为 54 nM 和 38 nM。而其他 CYP 同工型（包括 11-羟化酶和 CYP3A4）不受 TAK-700 的显着影响。在表达人 CYP 同工型的微粒体中，与其他 CYP 同工型相比，TAK-700 对 17,20-裂解酶表现出更大的抑制作用，IC50 为 19 nM。 TAK-700 对猴 17,20-裂解酶和 17-羟化酶具有抑制活性，IC50 分别为 27 nM 和 38 nM。在猴肾上腺细胞中，TAK-700 抑制 ACTH 刺激的 DHEA 和雄烯二酮的产生，IC50 分别为 110 nM 和 130 nM。此外，TAK-700 还可有效抑制人肾上腺皮质肿瘤系 H295R 细胞中 DHEA 的产生，IC50 为 37 nM。激酶测定：根据先前描述的侧链裂解活性的方法并进行一些修改来测量大鼠11-羟化酶活性。反应混合物含有200mM甘露醇、4.5mM HEPES、2.3mM磷酸钾(pH 7.4)、0.1mM EDTA·2K、0.03％BSA(结晶)、4.5mM NADPH、11mM氯化钙、4μg线粒体蛋白、 10 nM [1,2-3H]-羟基-11-脱氧皮质酮（11-脱氧皮质醇）（NEN，溶解在 0.02% Tween-80 中）和 1-1000 nM 测试化合物，总体积为 150 μL。试剂的浓度表示为反应混合物中的最终浓度。将测试化合物用二甲基甲酰胺连续稀释，并将1.5μL直接添加至反应混合物中。 37°C 孵育 30 分钟后，加入 400 μL 乙酸乙酯和 100 μL 蒸馏水终止反应，然后涡旋 30 秒并短暂离心。将三百微升有机相转移至新管中并使用氮气蒸发直至干燥。将类固醇用 30 μL 乙酸乙酯溶解，并将整个体积涂在硅胶 TLC 板上。底物和产物（11-脱氧皮质醇和皮质醇）在甲苯-丙酮（7:2）溶剂体系中分离。细胞测定：在猴肾上腺细胞中，orteronel 抑制 ACTH 刺激的 DHEA 和雄烯二酮的产生，IC50 分别为 110 nM 和 130 nM。此外，Orteronel 还有效抑制人肾上腺皮质肿瘤系 H295R 细胞中 DHEA 的产生，IC50 为 37 nM。在体外，orteronel 对大鼠和人类固醇 17,20-裂合酶显示出有效的抑制活性，IC50 分别为 54 nM 和 38 nM。而其他 CYP 亚型，包括 11-羟化酶和 CYP3A4，则不受 Orteronel 的显着影响。在表达人 CYP 同工型的微粒体中，与其他 CYP 同工型相比，Orteronel 对 17,20-裂解酶表现出更大的抑制作用，IC50 为 19 nM。

在食蟹猴中，口服 1 mg/kg 剂量的 TAK-700 可显着降低血清睾酮和脱氢表雄酮 (DHEA) 水平。以 1 mg/kg 剂量口服 TAK-700 可产生良好的药代动力学参数，Tmax、Cmax、t1/2 和 AUC0-24 小时分别为 1.7 小时、0.147 μg/mL、3.8 小时和 0.727 μg·h/mL，分别。

猴17,20-裂合酶/17-羟化酶抑制活性测定[1]
17,20-裂合酶抑制活性如所述进行了一些修改。从两只完整的5岁雄性食蟹猴身上切下的肾上腺在10 mmol/L HEPES缓冲液（pH 7.4）中均质化，该缓冲液含有250 mmol/L蔗糖、25 mmol/L KCl、0.5 mM EDTA 2 K、1 mmol/L二硫苏糖醇、0.02 mg/mL苯甲基磺酰氟和20%（v/v）甘油。然后通过离心分离肾上腺微粒体，并将其悬浮在含有5 mmol/L MgCl2和25%（v/v）甘油的50 mmol/L Tris·Cl缓冲液（pH 7.4）中。使用Bio-Rad蛋白质测定试剂盒（Hercules，CA，USA）测定微粒体部分的蛋白质浓度。为了评估类固醇生物合成，反应混合物含有50 mmol/L Tris-HCl缓冲液（pH 7.4）、5μmol/L 17-α-羟基孕烯醇酮，包括相当于0.05μL/管17-α羟基-[1,2（n）-3H]-孕烯醇酮（1 mCi/mL，41.9 Ci/mmol）、NADPH生成系统（0.6 mmol/Lβ-NADP+，10 mmol/L葡萄糖-6-磷酸，5 mmol/L氯化镁，1.5单位/mL G-6-P脱氢酶）、2.5%（v/v）丙二醇、微粒体蛋白（50μG/mL，终浓度）和总体积为使用200μL。将反应混合物在37°C下孵育120分钟。在己烷：四氢呋喃（4:1）溶剂体系中，通过薄层色谱（TLC，Whatman LHPK）分离反应底物和产物（DHEA、雄烯二酮）。使用BAS 2000 II生物图像分析仪对TLC板上的适当区域进行鉴定并测量放射性，酶活性表示为nmol/h/mg蛋白质。 17-羟化酶活性的测定与上述类似。5μmol/L孕烯醇酮（ICN Biomedicals），包括相当于0.05μL/管[7-3H（N）]孕烯醇酮的当量（1 mCi/mL，17.5 Ci/mmol），取代17-α-羟基雷公藤酮作为底物，将反应混合物温育5分钟。在环己烷：乙酸乙酯（3:2）溶剂系统中，通过TLC分离底物和产物（17-α-Hydrophenolone，17α-羟基孕酮（17-OHP），DHEA，雄烯二酮，脱氧皮质醇），并如上所述测定和表示活性。

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11-羟化酶活性测定[1]
根据其他地方描述的方法，经过一些修改后，测定了对猴11-羟化酶的抑制活性。如上所述切除肾上腺。通过离心制备肾上腺线粒体，并将其悬浮在含有5 mmol/L MgCl2的50 mmol/L Tris·Cl缓冲液（pH 7.4）中。使用Bio-Rad蛋白质测定试剂盒（Hercules，CA，USA）测定线粒体部分的蛋白质浓度。反应混合物含有50 mmol/L Tris-HCl缓冲液（pH 7.4）、2μmol/L 11-脱氧皮质醇（美国康涅狄格州普法尔茨和鲍尔公司），包括相当于0.05μL/管羟基-11-脱氧皮质酮、[1,2-3H（N）]（NEN，56.8 Ci/mmol）、NADPH生成系统（0.6 mmol/Lβ-NADP+，10 mmol/L葡萄糖-6-磷酸，5 mmol/L氯化镁，1.5单位/mL G-6-P脱氢酶）、10 mmol/L氯化钙、5%（v/v）丙二醇、线粒体蛋白（50μG/mL，终浓度）和总体积为200μL。将反应混合物在37°C下孵育120分钟。底物和产物（3H-皮质醇）在甲苯-丙酮（3.5:1）溶剂体系中分离。所有其他程序均如上所述进行，活性以nmol/h/mg蛋白质表示。

控制性腺雄激素生物合成的手术或药物方法是治疗各种非肿瘤性和肿瘤性疾病的有效途径。例如，雄激素消融及其导致的循环睾酮水平的降低是晚期前列腺癌的有效治疗方法。不幸的是，这种方法的治疗效果往往是暂时的，因为疾病进展到“去势抵抗”(CRPC)状态，这种情况下的治疗选择有限。一种被认为是导致CRPC发生的机制是生殖腺外雄激素的合成以及这些残留的生殖腺外雄激素对前列腺肿瘤细胞增殖的影响。负责合成性腺外雄激素的一个重要酶是CYP17A1，它具有17,20-裂解酶和17-羟化酶的催化活性，其中17,20-裂解酶的活性是雄激素生物合成过程的关键。Orteronel (TAK-700)是一种新型的，选择性的，有效的17,20-裂解酶抑制剂，作为一种抑制雄激素合成的药物正在开发中。在本研究中，我们通过评估其对阉割和完整雄性食环猴口服给药后对CYP17A1酶活性、猴肾上腺细胞和人肾上腺肿瘤细胞中类固醇生成以及血清脱氢表雄酮(DHEA)、皮质醇和睾酮水平的影响，量化了orteronel对睾丸和肾上腺雄激素产生的抑制活性和特异性。我们报道，奥特龙能有效抑制猴子肾上腺细胞雄激素的产生，但仅微弱地抑制皮质酮和醛固酮的产生;奥特罗内酯对皮质醇的IC(50)值比对DHEA的IC(50)值高3倍。单次口服给药后，完整食蟹猴的血清脱氢表雄酮、皮质醇和睾酮水平迅速被抑制。在被阉割的猴子中，每天用奥特罗奈治疗两次，在整个治疗期间，DHEA和睾酮的抑制持续存在。在体内模型和与我们的体外数据一致，口服给药后血清皮质醇水平的抑制低于DHEA。在人CYP17A1和人肾上腺肿瘤细胞中，在无细胞酶测定中，orteronel抑制17,20-裂解酶活性的效力是17-羟化酶活性的5.4倍，在人肾上腺肿瘤细胞中，DHEA产生的效力是皮质醇产生的27倍，这表明在人与猴子中，17,20-裂解酶和17-羟化酶活性的抑制具有更大的特异性。综上所述，奥特罗内酯能有效抑制猴和人体内CYP17A1的17,20裂解酶活性，降低猴体内血清雄激素水平。这些发现表明，对于雄激素抑制至关重要的疾病，如雄激素敏感和CRPC，奥特罗奈可能是一种有效的治疗选择。[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]. 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-naphthalenecarboxamide is a naphthalenecarboxamide.

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

67.15

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)