17,20-lyase/CYP17

Orteronel 对雄烯二酮的 IC50 为 110 nM，对 DHEA 的 IC50 为 110 nM，这两种物质均由猴肾上腺细胞响应 ACTH 刺激而产生。此外，ortersteronel 的 IC50 为 37 nM，可显着减少人肾上腺皮质肿瘤系 H295R 细胞中 DHEA 的合成[1]。 Orteronel 对大鼠类固醇 17,20-裂解酶的 IC50 值分别为 54 nM，对人类固醇 17,20-裂解酶的 IC50 为 38 nM，在体外对这两种酶均具有很强的抑制活性。 Orteronel 不会明显影响 CYP3A4 或 11-羟化酶以及其他 CYP 亚型。与其他 CYP 异构体相比，Orteronel 对表达人 CYP 异构体的微粒体中的 17,20-裂解酶具有更高的抑制作用，IC50 为 19 nM1。

Ortionel（1 mg/kg，口服）获得了良好的药代动力学特征，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/mL。 1]。口服1 mg/kg Orteronel可显着降低食蟹猴体内DHEA和血清睾酮水平[2]。

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]. 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

67.15

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)

配方 1 中的溶解度: ≥ 1.43 mg/mL (4.65 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 14.3 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.43 mg/mL (4.65 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 14.3mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1.43 mg/mL (4.65 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 14.3 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 0.5% methylcellulose: 30 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。