依西美坦在乳腺癌治疗中的应用

乳腺癌是当前威胁女性健康的常见恶性肿瘤之一,大约有三分之二的乳腺癌是性激素依赖的,所以内分泌治疗在这类乳腺癌中起着非常重要的作用,目前第三代芳香化酶抑制剂在内分泌治疗中的作用倍受重视,依西美坦作为芳香化酶灭活剂在乳腺癌治疗中的作用也逐渐被人们认识。依西美坦是一种口服的甾体类芳香化酶灭活剂,它和内源性配体竞争芳香化酶的活性位点。然后代谢为反应性的中间介质,通过共价键结合,永久性地灭活芳香化酶。这种机制也称为自杀性抑制作用。非甾体类芳香化酶抑制剂与之不同,是可逆性地与芳香化酶的血红素部分结合。芳香化酶活性的…     

第3代芳香化酶抑制药治疗乳腺癌的研究进展

第3代芳香化酶抑制药治疗绝经后妇女乳腺癌具有较高的疗效和较好的耐受性，疗效明显优于标准的抗雌激素药物他莫昔芬和孕激素药物醋酸甲地孕酮，与非甾体类和甾体类药物之间无交叉耐药性，且严重不良反应较少见。目前已经应用于临床的第3代芳香化酶抑制药有阿那曲唑、来曲唑、依西美坦等。     

依西美坦有利于乳腺癌辅助治疗

一项涉及约 5 0 0 0名妇女的临床试验表明 ,辉瑞公司的芳香化酶抑制剂依西美坦 (exemestane ,Aro masin)可提高乳腺癌患者的无病存活率。由多个国家参与的对依西美坦的研究 (IES)证实了芳香化酶抑制剂在乳腺癌辅助治疗中的作用。据称用他莫昔芬治疗 2～ 3年后 ,临床医生应考虑换用依西美坦。尽管辉瑞公司声称正就该药用于乳腺癌早期治疗事宜与FDA进行商讨 ,但何时进行资料申请尚不清楚。参与IES研究的 4 472名绝经期妇女接受他莫昔芬治疗 2～ 3年 ,未出现病情恶化。受试者为雌激素受体阳性或受体状态未明的乳腺癌患者 ,每天随机接受依西…     

芳香化酶抑制剂在妇科内分泌治疗中的应用效果观察

目的探讨芳香化酶抑制剂在妇科内分泌治疗中的应用效果。方法选取本院2009年8月-2012年10月收治的74例妇科内分泌患者,针对所有患者的临床资料及实际情况进行分析．并对患者采用芳香化酶抑制剂进行治疗,主要包括依西美坦、阿那曲唑及来曲唑,评估患者的治疗效果。结果74例患者中,基本治愈39例．显效21例,好转9例,无效5例,治疗总有效率为93．2％;无效的5例患者主要是由于其心理因素影响．与医护人员的配合度不高所致。结论临床上应用芳香化酶抑制剂治疗妇科内分泌疾病的效果较为显著,对于促进患者病情的改善及生活质量提高具有重要意义,值得在临床推广应用。     

芳香化酶抑制剂治疗妇科内分泌疾病的效果分析

目的：探讨芳香化酶抑制剂在妇科内分泌疾病治疗中的应用价值。方法选取2010年8月~2012年8月在本院治疗的妇科内分泌患者90例,分别给予依西美坦（n=36）、阿那曲唑（n=28）和来曲唑（n=26）治疗,观察芳香化酶抑制剂的疗效、3组患者血清雌二醇（E2）抑制情况及不良反应发生情况。结果依西美坦组、阿那曲唑组和来曲唑组的治疗总有效率分别为94.44%、92.86%和92.31%,差异无统计学意义（P〉0.05）。治疗后3组患者血清E2水平明显下降,与治疗前比较差异有统计学意义（P〈0.05）;依西美坦组患者的血清E2抑制率及有效抑制率优于其他两组（P〈0.05）。3组均未出现严重不良反应;在体重增加上来曲唑组尤为显著（P〈0.05）。结论芳香化酶抑制剂可有效治疗妇科内分泌疾病,且药物耐受性好。     

英研究表明新药依西美坦可显著提高乳腺癌患者存活率

据英国《泰晤士报》2007年2月13日报道,英国科学家进行的一项研究显示,治疗乳腺癌的新药依西美坦(商品名为阿诺新)可显著提高患者的存活率。英国癌症研究会下属科学家对4700多名绝经后妇女进行的跟踪研究发现,乳腺癌患者在手术后使用目前比较有效的药物它莫西芬治疗,可使死亡率降低33%;如果在接受该药治疗两三年后改用依西美坦,死亡率可进一步降低17%。依西美坦是美国辉瑞公司生产的一种芳香化酶抑制剂。据悉,英国国家诊疗标准化研究所已推荐将依西美坦用作它莫西芬的替代药物。(消息来源:新华社上海信息网)英研究表明新药依西美坦可显著提…     

使用他莫昔芬后改服依西美坦作为辅助激素疗法治疗绝经期后乳腺癌：对机体组成及脂质的影响

近期的研究表明,相对于标准的5年他莫昔芬治疗,绝经后原发性乳腺癌患者在接受他莫昔芬2~3年治疗后,改用芳香化酶抑制剂依西美坦(exemes-tane)治疗,无瘤生存率得到改善。随机研究纵向评估了从他莫昔芬改服依西美坦后机体组成和脂质的变化。有研究证实体重和体脂(FM)过度增长与绝     

Aromasin在欧获准扩大适应症

药业巨头Pfizer公司称，他们的芳香化酶抑制剂Aromasin（exemestane，依西美坦）（Ⅰ）的新适应症——用于接受过手术并曾接受过2～3年常见抗癌药他莫昔芬（tamoxifen）（Ⅱ）治疗的绝经后早期乳癌患者的治疗，已经在欧洲得到批准，可以向病人开具处方。     

乳腺癌患者术后长期内分泌治疗的不良反应及治疗策略

内分泌治疗是乳腺癌患者术后治疗的重要手段，目前临床应用的主要药物有选择性雌激素受体调节剂他莫昔芬和芳香化酶抑制剂阿那曲唑、来曲唑及依西美坦。乳腺癌术后辅助内分泌治疗通常需要5年，部分患者可长达10年。他莫昔芬治疗引起的不良反应主要包括妇科和心血管系统不良反应。处理措施为定期检测子宫内膜及血压、血脂等指标，对症处理。芳香化酶抑制剂治疗引起的不良反应主要包括：（1）骨不良反应。处理措施为根据情况予以维生素D和钙剂治疗，必要时给予双膦酸盐类药物。（2）关节肌肉症状。病情较轻者可补充维生素D和钙剂，并进行适当体育锻炼；疼痛明显者可服用非甾体类抗炎药，也可以考虑停药3-4周。（3）心血管系统不良反应。处理措施为对症处理。     

芳香化酶抑制剂的研究进展

芳香化酶是雌激素生物合成过程中的一个关键酶,抑制芳香化酶的活性就可以抑制雌激素的生成,从而减少绝经后妇女乳腺癌等疾病的发病率。以芳香化酶为靶标的研究一直都是抑制剂研究的热点,目前已有多个芳香化酶抑制剂进入临床试验阶段或已上市,该文对近年来芳香化酶抑制剂的研究进行综述。     

Molecular basis for the interaction of four different classes of substrates and inhibitors with human aromatase

Aromatase cytochrome P450 (CYP19) converts androgen to estrogen. In this study, the interactions of four classes of compounds, 17beta-estradiol (the product of aromatase), 17-methyltestosterone (a synthetic androgen), dibenzylfluorescein (a synthetic substrate of aromatase), and coumestrol (a phytoestrogen), with aromatase were investigated through spectral analysis using purified human recombinant aromatase and site-directed mutagenesis studies using CHO cells expressing wild-type human aromatase or five aromatase mutants, E302D, D309A, T310S, S478T and H480Q. Spectral analysis showed that a type I binding spectrum was produced by the binding of 17-methyltestosterone to aromatase and a novel binding spectrum of aromatase was induced by dibenzylfluorescein. Mutagenesis experiments demonstrated that residues S478 and H480 in the beta-4 sheet play an important role in the binding of all four compounds. Computer-assisted docking of these compounds into the three-dimensional model of aromatase revealed that: (1) weak interaction between 17beta-estradiol and the beta-4 sheet of aromatase facilitates the release of 17beta-estradiol from the active site of aromatase; (2) 17-methyl group of 17-methyltestosterone affects its binding to aromatase; (3) dibenzylfluorescein binds to the active site of aromatase with its O-dealkylation site near the heme iron and residue T310; and (4) coumestrol binds to aromatase in a manner such that rings A and C of coumestrol mimic rings A and B of steroid. These structure-function studies help us to evaluate the structural model of aromatase, and to accelerate the structure-based design for new aromatase inhibitors.     

Effects of steroid D-ring modification on suicide inactivation and competitive inhibition of aromatase by analogues of androsta-1,4-diene-3,17-dione

Analogues of androsta-1,4-diene-3,17-dione (3a) in which the D ring is modified were prepared and tested as suicide inactivators and competitive inhibitors of human placental aromatase. As long as the five-membered ring is intact, modifications of the D ring such as reduction or removal of the carbonyl group or conversion to a gamma-butyrolactone cause a less than 6-fold decrease in affinity for and rate of inactivation of aromatase, compared to 3a. Thus, an oxygen atom at C-17 is not required for binding of these inhibitors to aromatase, suggesting that hydrogen bonding to the D-ring oxygen does not play a major role in binding. Opening the D ring converts the cyclopentane ring to an alkyl chain and causes a greater than 300-fold decrease in affinity; this can be partially reversed by shortening the chain length. These results are consistent with a model in which the free chain of the opened D ring adopts conformations that sterically interfere with binding of the inhibitor to the enzyme. These findings may have practical applications in drug design, by allowing the preparation of 17-deoxo analogues that have high affinity for aromatase but that are not subject to reduction of the 17-carbonyl group, which is a major mode of metabolism of 3a.     

Inhibitors of the P450 enzymes aromatase and lyase. Crystallographic and molecular modeling studies suggest structural features of pyridylacetic acid derivatives responsible for differences in enzyme inhibitory activity

Derivatives of 4-pyridylacetic acid are known to be inhibitors of the cytochrome P450 enzymes aromatase and lyase (17 alpha-hydroxylase/C17-20lyase), and are therefore of interest in the treatment of hormone dependent breast and prostate cancers. We report the determination of the crystal structure of one such derivative, the 4-tert-butyl cyclohexyl ester, and molecular modeling studies on two related inhibitors, the cyclohexyl ester and its alpha-methyl derivative. These latter two compounds show a marked difference in their relative activities against aromatase and lyase. Two models are proposed for the interaction of these molecules with the target enzymes on the basis of their ability to adopt conformations that partially mimic steroid substrates. From these models an explanation can be advanced for the fact that, compared with the unmethylated analogue, the (racemic) alpha-methylated compound is seven times poorer as an inhibitor of aromatase but seven times better as an inhibitor of lyase. The model proposed for binding to aromatase places the alpha-carbon of the ester group in the position occupied by C(2) of steroid substrates. In contrast, that proposed for binding to lyase places this atom in the position occupied by C(17) of steroid substrates. The introduction of steric bulk at C(2) is known to be unfavorable for aromatase inhibition, while its introduction at C(17) may lead to a better mimicry of the steroid D-ring and so improve lyase inhibition.     

Expression and purification of a recombinant form of human aromatase from Escherichia coli

Aromatase converts androgen to estrogen, a hormone that plays an important role in the development of breast cancer. Aromatase inhibitors have been shown to be a useful endocrine regimen for estrogen-dependent breast cancer. Structure-function studies of aromatase can generate critical structural information for designing highly potent and specific inhibitors. However, aromatase structure-function studies have been hampered by a lack of purified protein. In this report, we describe the construction and expression of a recombinant derivative of human aromatase in Escherichia coli using the pET vector system, and the purification of the enzyme by means of nickel-agarose affinity chromatography. We examined the expression of the full-length, Del-38, C-6xHis-tagged Del-38, and NC-6xHis-tagged Del-38 forms of aromatase. The recombinant aromatase without the first 38 amino acids from the amino-terminus (i.e. Del-38) was found to have a higher activity than the full-length enzyme. Moreover, the addition of two separate hexameric histidine tags at both the amino and the carboxyl-termini (i.e. NC-6xHis-tagged Del-38) increased the binding affinity of the recombinant enzyme to the nickel-agarose. The expressed aromatase (i.e. NC-6xHis-tagged Del-38 aromatase) was eluted from the nickel-agarose with 80 mM EDTA. The total aromatase activity of the 80 mM EDTA-eluted fractions was significantly higher than the detergent-solubilized protein extract, indicating a renaturation process during the nickel-agarose affinity chromatography. Purified aromatase exhibited a single band when analyzed by SDS-PAGE, and activity up to 5.8 nmol/mg/min was obtained using the tritiated water release assay. The K(m) value for androstenedione was determined to be 62+/-24 nM by enzyme kinetic analysis. The recombinant aromatase preparation was also characterized by reduced CO-difference spectral analysis, reaction product extraction assay, and inhibition studies using two aromatase inhibitors (letrozole and anastrozole). The results indicate that the recombinant aromatase from E. coli has catalytic properties identical to those of the enzyme expressed in human tissue and will be very useful for further structure-function studies of aromatase.     

Interactions of thiol-containing androgens with human placental aromatase

A series of thiol androgens were synthesized and investigated to characterize structural features important for the inhibition of aromatase. Analogues of androstenedione with thiol groups in either the 2 alpha-, 10 beta-, or 19-positions caused time-dependent inhibition of human placental aromatase. When their KI and kcat values were compared with those of 4-hydroxyandrost-4-ene-3,17-dione (4-OHa) and 10 beta-propargylestr-4-ene-3,17-dione (PED), the thiol androgen 10 beta-mercaptoestr-4-ene-3,17-dione (10 beta-SHnorA) proved to be the most potent suicide substrate. However, 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) was the best all-around inhibitor. All compounds except 19-SHA exhibited normal type I P-450 difference spectra with partially purified/solubilized, human placental aromatase. The Ks values for the series of compounds compared qualitatively to the KI values determined from the time and concentration-dependent inhibition experiments. 19-SHA induced split Soret peaks at 380 and 474 nm, which suggested binding of the 19-thiolate directly to the ferric iron of aromatase. This binding could be displaced by aminoglutethimide but not by androstenedione. The inhibitory activity of 19-SHA may be explained by two independent mechanisms: (1) suicide inactivation of aromatase in the ferrous state; and (2) a direct "hyper-type II" binding to the remaining portion of the cytochrome in the ferric state. A free thiol group was necessary for the suicide inhibitory activity of 19-SHA; time-dependent inactivation of aromatase by 19-(acetylthio)androst-4-ene-3,17-dione (19-SAcA) and 19-xanthogenylandrost-4-ene-3,17-dione (19-XanA) could be prevented if the microsomes were preincubated with a carboxyesterase inhibitor. Aromatase previously inactivated by either thiol androgens,4-OHA, or PED could not be reactivated after incubation with the disulfide reducing agent dithiothreitol, which suggests that a disulfide bond may not be involved in aromatase inactivation by these inhibitors.     

Update on the use of aromatase inhibitors in breast cancer

Estrogens are biosynthesised from androgens by the CYP450 enzyme complex called aromatase. Aromatase is expressed in the ovary, placenta, brain, bone, adipose tissue and breast tissue. In breast cancer, intratumoural aromatase is the source for local estrogen production in the tissue. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in estrogen-dependent breast cancer. The potent and selective third-generation aromatase inhibitors anastrozole, letrozole and exemestane were introduced to the market as endocrine therapy in postmenopausal patients failing anti-estrogen therapy alone, or multiple hormonal therapies. Anastrozole and letrozole are both non-steroidal aromatase inhibitors that compete with the substrate for binding to the enzyme active site. Exemestane is a mechanism-based steroidal inhibitor that mimics the substrate, is converted by the enzyme to a reactive intermediate, and results in inactivation of aromatase. These third-generation aromatase inhibitors are currently approved as first-line therapy for the treatment of postmenopausal women with metastatic estrogen-dependent breast cancer. The use of an aromatase inhibitor as initial therapy, or after treatment with tamoxifen, is now recommended as adjuvant hormonal therapy for postmenopausal women with hormone-dependent breast cancer. Several clinical studies of aromatase inhibitors focus on the use of these agents in the adjuvant setting, for the treatment of early breast cancer. Recently published results show improved responses with these agents compared with tamoxifen.     

Aromatase inhibition by 4 beta,5 beta-epoxides of 16 alpha-hydroxyandrostenedione and its 19-oxygenated analogs,potential precursors of estriol production in the feto-placental unit

To gain insight into the nature of the substrate binding site and the catalytic function of aromatase, we studied the inhibition of androstenedione aromatization by 4beta,5beta-epoxy-16alpha-hydroxyandrostenedione (4) and its 19-hydroxy and 19-oxo derivatives, 5 and 6, as well as the biochemical aromatization of these steroids in human placental microsomes. The 19-methyl and 19-oxo compounds, 4 and 6, were weak competitive inhibitors of aromatase, with apparent K(i) values of 246 microM and 270 microM, respectively, whereas the 19-hydroxy compound 5 inhibited aromatase in a non-competitive manner with the K(i) of 135 microM. The 19-methyl compound 4 inactivated aromatase in a time-dependent manner with k(inact) of 0.213 min(-1) in the presence of NADPH in air, but the other two did not cause it. The conversion of the three epoxides into estrogen, as well as 19-oxygenation of 19-methyl steroid 4 with human placental microsomes in the presence of NADPH in air, were not detected by gas chromatography-mass spectrometry. The present results are consistent with the two binding sites theory in the active site of aromatase.     

Structure-activity relationships of new A,D-ring modified steroids as aromatase inhibitors: design, synthesis, and biological activity evaluation

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New A,D-ring modified steroid analogues of formestane and testolactone were designed and synthesized and their biochemical activity was investigated in vitro in an attempt to find new aromatase inhibitors and to gain insight into their structure-activity relationships (SAR). All compounds tested were less active than formestane. However, the 3-deoxy steroidal olefin 3a and its epoxide derivative 4a proved to be strong competitive aromatase inhibitors (K(i) = 50 and 38 nM and IC50 = 225 and 145 nM, respectively). According to our findings, the C-3 carbonyl group is not essential for anti-aromatase activity, but 5alpha-stereochemistry and some planarity in the steroidal framework is required. Furthermore, modification of the steroidal cyclopentanone D-ring, by construction of a delta-lactone six-membered ring, decreases the inhibitory potency. From the results obtained, it may be concluded that the binding pocket of the active site of aromatase requires planarity in the region of the steroid A,B-rings and the D-ring structure is critical for the binding.     

Synthesis and biochemical studies of 19-oxygenated derivatives of 6alpha- and 6beta-methylandrostenediones as catalytic probes for the active site of aromatase

To gain insight into the binding aspects of 6alpha- and 6beta-methylandrostenediones (1 and 2), potent competitive inhibitors and effective substrates of aromatase, at the active site of the enzyme, we synthesized their 19-hydroxy and 19-oxo derivatives to determine their inhibition of aromatase activity as well as their aromatization rates in human placental microsomes. The 6beta- and 6alpha-methyl-19-ols 12 and 13 were produced from 19-(tert-butyldimethylsiloxy)androstenedione (6) in 6 steps in which the Grignard reaction of 5alpha,6alpha-epoxy steroid 8 with CH3MgBr was employed as a key reaction. Oxidation of the 19-ols 12 and 13 yielded the corresponding 19-als 14 and 15. The 6alpha-methyl steroids 13 and 15 were good competitive inhibitors of aromatase (Ki< or =100 nM), and their aromatization rates obtained by gas chromatography-mass spectrometric analysis were 110 and 205 pmol/min/mg protein, respectively. In contrast, the 6beta-methyl isomers 12 and 14 were non-competitive inhibitors, with Ki values of more than 500 nM, and they were aromatized at rates of 16 and 20 pmol/min/mg protein, respectively. These results reveal that there is a marked difference in binding to the active site between the 19-oxygenated 6alpha-methyl and 6beta-methyl inhibitors where the binding manner of the 6alpha-steroids, rather than the 6beta-isomers, is suitable as a substrate for aromatase.