Aromatase (IC50 = 15 nM)

With an IC50 of 15 nM, anastrozozole, a very simple achiral benzyltriazole derivative, inhibits human placental aromatase. It has 200 times the potency of aminoglutethimide (AG), twice the potency of 4-OHA, and one-third the potency of fadrozole in the same assay [1].

On day four, groups of immature (22-day-old) female rats received subcutaneous injections of androstenedione (AD) in peanut oil (30 mg/kg) for three days. On day four, the rats either received an oral injection of different doses of anastrozozole or no injection at all. After being dissected, the uterus was dried off and weighed. On days two or three of the cycle, an oral dose of 0.1 mg/kg of anastrozozole completely prevents ovulation. In immature rats, anastrozozole totally eliminated the uterotropic action of exogenous AD at the same daily dose (0.1 mg/kg). Oral doses of anastrozozole (0.1 mg/kg and above) administered twice daily in male pig-tailed monkeys decreased circulating estradiol concentrations by 50–60% [1].

In vitro aromatase inhibition assay[1]
Aromatase inhibition is measured using human placental microsomes and the method of Thompson and Siiteri with Testosterone (0.5 μM) as substrate. 11-hydroxylase inhibition is determined by measuring the conversion of [1,2,6,7-3H]-ll-deoxy- cortisol to cortisol using freshly prepared mitochondria from guinea pig, dog and cow adrenal glands. Reaction products areextracted into chloroform and separated by thin layer chromatography[1].

Establishment of resistant cell lines and culture conditions[2]
From the ER+ MCF-7-derived breast cancer cell line stably transfected with the human aromatase gene (MCF-7aro),22 a new anastrozole-resistant cell line denoted Res-Ana was established by exposing these MCF-7aro cells during 20 weeks to increasing concentrations (1, 3 and 5 µM) of anastrozole in Dulbecco's Modified Eagle Medium without phenol red, supplemented with 3% steroid-depleted, dextran-coated and charcoal-treated fetal calf serum (DCC medium) containing 25 nM 4-androstenedione (AD). The cells were purged in DCC medium for 4 days before each experiment described below. Media and treatment were changed every 2 days.[2]

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Cytotoxicity assay[2]
A total of 104 cells per well were plated in a 96-well plate and treated with AD combined with anastrozole, letrozole, 4-hydroxy-tamoxifen (OH-Tam), fulvestrant (ICI 182,780), MK-2206, rapamycin or a combination of treatments. Cell viability was assessed as previously described.

Aromatase inhibition. Groups of at least eight adult female rats (Alpk:AP~SD; Wistar derived), housed in controlled lighting (on 06.00-20.00 h) and temperature (24 + 2°C) and undergoing 4-day oestrous cycles, were treated p.o. with a single dose of anastrozole (0.01-0.1 mg/kg), fadrozole (0.01-0.1 mg/kg) or AG (5-20 mg/kg) on day 2 at 16.00 h or day 3 at 12.00 h. The presence or absence of eggs in the oviducts on day 1 of the next cycle was then determined. Ovulation was considered blocked when no eggs were found. Groups of eight immature (22-day-old) female rats were given AD (30 mg/kg) in arachis oil s.c. daily for 3 days with or without various doses of anastrozole p.o. On day 4 the uteri were dissected, blotted and weighed. Two groups of six mature male pigtailed monkeys (M. nernestrina) (body weights 11-21 kg) were used to compare effects of six dose levels (0.003, 0.01, 0.03, 0.1, 0.3 and 1.0 mg/kg) of anastrozole and fadrozole on plasma hormone concentrations. The drugs (in weights tailored to the individuals' body weights) were incorporated into peppermint candy and self administered twice daily (09.00 h and 16.00 h) by the monkeys. Each monkey was carefully observed for compliance; all doses were ingested. Blood samples were collected under ketamine sedation before the start of the study, after 7 days of treatment with the dosing vehicle (candy) and on the seventh day of treatment at each dose level. Blood collections were made at about the same time of day (15.00 h) on each occasion. Plasma was separated and stored at -20°C for hormone measurements (oestradiol, testosterone, cortisol and DOC). [1]

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Adrenal function. Effects of anastrozole, metyrapone, AG and fadrozole on adrenal weights were determined in male rats (150-180 g) treated for 7 days. Effects (adrenal weight and histology) of anastrozole in five male and five female rats treated for 14 days and in three male and three female Alderley Park beagle dogs treated for 21 days were also determined; plasma K ÷ was also monitored in the dogs. Effects on aldosterone secretion were determined in groups of six male rats. Blood samples were collected from the abdominal aorta under halothane anaesthesia 2 h after an oral dose of anastrozole (5-20 mg/kg) or fadrozole (0.1-5 mg/kg) and heparinized plasma separated and stored at -20°C for aldosterone assays. Induced mineralocorticoid activity, a marker of elevated adrenal DOC secretion, was determined in groups of five male rats. These were given a single oral or s.c. dose of vehicle or anastrozole (5-10 mg/kg) or fadrozole (1-5 mg/kg) and, 1 h later, 2.5 ml of physiological saline s.c. Pooled urine from each group was collected during the next 5 h and Na ÷ and K ÷ concentrations were measured by flame ionization photometry. From the latter values, log10 (10 [Na÷]/ [K÷]) was calculated for each group; this compensates for differences in urine volumes and, for the saline load administered, the value of this function for control rats is approximately unity. [1]

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Steroid hormone activities Oestrogenic/anti-oestrogenic activity was assessed in a standard 3-day uterotrophic assay in immature (22-day-old) female rats (eight per group), with oestradiol benzoate (0.5 pg/rat/day s.c.) as standard. Androgenic/anti-androgenic activity was assessed by measuring ventral prostate and seminal vesicle weights in immature (22-day-old) male rats (eight per group) after 7 days of treatment, with testosterone propionate (2.5 mg/kg/day s.c.) as ,~tandard. Progestogenic activity was assessed by the capacity to maintain pregnancy in rats (groups of five) ovariectomized on day 9 of pregnancy (day 1 = sperm-positive smear) and given a maintenance dose of oestradiol (0.1 #g/rat) s.c. daily. Treatment was given on days 9-15 inclusive, and the uterine contents inspected post-mortem on day 16. Glucocorticoid/antiglucocorticoid activity was assessed by measuring thymus weights in immature female rats (groups of six) after 4 days of treatment, with dexamethazone (5 pg/rat/day i.p.) as standard. [1]

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0.1 mg/kg; oral

Rats

Absorption, Distribution and Excretion
Anastrozole is rapidly absorbed and Tmax is typically reached within 2 hours of dosing under fasted conditions. Coadministration with food reduces the rate but not the overall extent of absorption - mean Cmax decreased by 16% and the median Tmax was extended to 5 hours when anastrozole was administered 30 minutes after ingestion of food, though this relatively minor alteration in absorption kinetics is not expected to result in clinically significant effects.
Hepatic metabolism accounts for approximately 85% of anastrozole elimination. Approximately 10% of the administered dosage is eliminated unchanged in the urine.
The volume of distribution of anastrozole into brain tissue in mice is 3.19 mL/g. Distribution into the CNS is limited due to the activity of P-gp efflux pumps at the blood brain barrier, of which anastrozole is a substrate.
Anastrozole's clearance is mainly via hepatic metabolism and can therefore be altered in patients with hepatic impairment - patients with stable hepatic cirrhosis exhibit an apparent oral clearance approximately 30% lower compared with patients with normal liver function. Conversely, renal impairment has a negligible effect on total drug clearance as the renal route is a relatively minor clearance pathway for anastrozole. In volunteers with severe renal impairment, renal clearance was reduced by 50% while total clearance was only reduced by approximately 10%.
Anastrozole is well absorbed into systemic circulation following oral administration. Plasma concentrations approach steady-state at about 7 days of once-daily dosing, and steady-state concentrations are approximately 3-4 times higher than concentrations achieved after a single dose of the drug. Food does not affect the extent of oral absorption of anastrozole.
Within the therapeutic plasma concentration range, anastrozole is 40% bound to plasma proteins.
Steady-state minimum plasma concentrations averaged 25.7 and 30.4 ng/mL, respectively, in white and Japanese postmenopausal women receiving anastrozole 1 mg daily for 16 days; serum estradiol and estrone sulfate concentrations were similar between the groups.
It is not known whether anastrozole is distributed into milk in humans.
For more Absorption, Distribution and Excretion (Complete) data for ANASTROZOLE (10 total), please visit the HSDB record page.

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Metabolism / Metabolites
Anastrozole is primarily metabolized in the liver via oxidation and glucuronidation to a number of inactive metabolites, including hydroxyanastrozole (both free and glucuronidated) and anastrozole glucuronide. Oxidation to hydroxyanastrozole is catalyzed predominantly by CYP3A4 (as well as CYP3A5 and CYP2C8, to a lesser extent) and the direct glucuronidation of anastrozole appears to be catalyzed mainly by UGT1A4. Anastrozole may also undergo N-dealkylation to form triazole and 3,5-Bis-(2-methylpropiononitrile)-benzoic acid. Labels for anastrozole state the main metabolite found in plasma following administration is triazole, but a recent pharmacokinetic study was unable to detect any products of N-dealkylation _in vitro_.
Anastrozole is extensively metabolized in the liver. Metabolism of anastrozole occurs via N-dealkylation, hydroxylation, and glucuronidation. Three metabolites of anastrozole have been identified in human plasma and urine: triazole, a glucuronide conjugate of anastrozole, and a glucuronide conjugate of hydroxyanastrozole. Triazole, the major circulating metabolite of anastrozole, lacks pharmacologic activity, and the aromatase inhibiting activity of anastrozole results principally from the parent drug. In addition, there are several minor metabolites of anastrozole, accounting for less than 5% of an administered dose, which have not been identified.
Hepatic. Metabolized mainly by N-dealkylation, hydroxylation, and glucuronidation to inactive metabolites. Primary metabolite is an inactive triazole.
Route of Elimination: Hepatic metabolism accounts for approximately 85% of anastrozole elimination. Renal elimination accounts for approximately 10% of total clearance.
Half Life: 50 hours

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Biological Half-Life
The elimination half-life of anastrozole is approximately 50 hours.
Following oral administration of anastrozole in postmenopausal women, a mean terminal elimination half-life of approximately 50 hours has been reported.

Toxicity Summary
Anastrozole selectively inhibits aromatase. The principal source of circulating estrogen (primarily estradiol) is conversion of adrenally-generated androstenedione to estrone by aromatase in peripheral tissues. Therefore, aromatase inhibition leads to a decrease in serum and tumor concentration of estrogen, leading to a decreased tumor mass or delayed progression of tumor growth in some women. Anastrozole has no detectable effect on synthesis of adrenal corticosteroids, aldosterone, and thyroid hormone. Organic nitriles decompose into cyanide ions both in vivo and in vitro. Consequently the primary mechanism of toxicity for organic nitriles is their production of toxic cyanide ions or hydrogen cyanide. Cyanide is an inhibitor of cytochrome c oxidase in the fourth complex of the electron transport chain (found in the membrane of the mitochondria of eukaryotic cells). It complexes with the ferric iron atom in this enzyme. The binding of cyanide to this cytochrome prevents transport of electrons from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted and the cell can no longer aerobically produce ATP for energy. Tissues that mainly depend on aerobic respiration, such as the central nervous system and the heart, are particularly affected. Cyanide is also known produce some of its toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinic dehydrogenase, and Cu/Zn superoxide dismutase. Cyanide binds to the ferric ion of methemoglobin to form inactive cyanmethemoglobin. (L97)

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Hepatotoxicity
Serum enzymes are reported to be elevated in 2% to 4% of women treated with anastrozole, but these elevations are usually mild, asymptomatic and self-limited, rarely requiring dose modification. There have been rare instances of clinically apparent liver injury associated with anastrozole therapy, typically arising within 1 to 4 months of starting treatment and having variable presentations but generally with a hepatocellular or mixed serum enzyme pattern (Case 1). Too few instances have been described in the literature to provide specific characteristics or clinical phenotype. Immunoallergic features (fever, rash, eosinophilia) were not mentioned in published cases, but low levels of autoantibodies were sometimes found. Recovery is usually rapid once anastrozole is stopped. There have been no cases of acute liver failure, chronic hepatitis or vanishing bile duct syndrome attributed to anastrozole use. Unlike tamoxifen, anastrozole has not been associated with development of fatty liver disease, although some degree of steatosis and steatohepatitis have been mentioned in descriptions of liver biopsies from acute cases. According to the product label, anastrozole has been linked to cases of hypersensitivity reactions and Stevens-Johnson syndrome as well as cases of hepatitis with jaundice.
Likelihood score: C (probable cause of clinically apparent liver injury).

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Protein Binding
Anastrozole is 40% protein bound in plasma and appears to be independent of plasma concentration.

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Toxicity Data
In rats, lethality is greater than 100 mg/kg.

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Interactions
Administration of a single 30 mg/kg or multiple 10 mg/kg doses of anastrozole to healthy subjects had no effect on the clearance of antipyrine or urinary recovery of antipyrine metabolites. Based on these in vitro and in vivo results, it is unlikely that co-administration of anastrozole 1 mg with other drugs will result in clinically significant inhibition of cytochrome P450 mediated metabolism.
In a study conducted in 16 male volunteers, anastrozole did not alter the pharmacokinetics as measured by Cmax and AUC, and anticoagulant activity as measured by prothrombin time, activated partial thromboplastine time, and thrombin time of both R- and S-warfarin.
Co-administration of anastrozole and tamoxifen in breast cancer patients reduced anastrozole plasma concentration by 27% compared to those achieved with anastrozole alone; however, the coadministration did not affect the pharmacokinetics of tamoxifen or N-desmethyltamoxifen.

[1]. Dukes M, et al. The preclinical pharmacology of "Arimidex" (anastrozole; ZD1033)--a potent, selective aromatase inhibitor. J Steroid Biochem Mol Biol. 1996 Jul;58(4):439-45.
[2]. Molecular characterization of anastrozole resistance in breast cancer: Pivotal role of the Akt/mTOR pathway in the emergence of de novo or acquired resistance and importance of combining the allosteric Akt inhibitor MK-2206 with an aromatase inhibitor. Int J Cancer. 2013 Oct 1;133(7):1589-602.

Therapeutic Uses
Antineoplastic
Anastrozole is indicated for the first-line treatment of postmenopausal woman with hormone receptor positive or hormone receptor unknown locally advanced or metastatic breast cancer. It is also indicated for treatment of advanced breast cancer in postmenopausal women with disease progression following tamoxifen therapy. /Included in US product label/
Anastrozole is an option for the neoadjuvant treatment of hormone receptorpositive, locally advanced breast cancer in postmenopausal women. Two phase 2, randomized, double-blind clinical trials found anastrozole to be at least as effective as tamoxifen in response rates and rates of improved surgery. A phase 2, unpublished abstract reported no differences between neoadjuvant anastrozole and chemotherapy (doxorubicin and paclitaxel) in response rates, number of patients qualifying for breast-conserving surgery, and 3-year disease-free survival. An international expert panel recommends neoadjuvant endocrine therapy in postmenopausal women who would benefit from preoperative chemotherapy but are ineligible to receive it. Anastrozole was well-tolerated. /Not included in US product label/
Anastrozole is not recommended for use in premenopausal women. Safety and efficacy have not been established. /Included in US product label/
For more Therapeutic Uses (Complete) data for ANASTROZOLE (8 total), please visit the HSDB record page.

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Drug Warnings
Among patients receiving adjuvant therapy, venous thromboembolic events occurred less frequently in patients receiving anastrozole than in those receiving tamoxifen (2 versus 4%); this included deep venous thrombosis (1 versus 2%). Ischemic cerebrovascular events also occurred less frequently in patients receiving anastrozole compared with those receiving tamoxifen (1 versus 2%). Ischemic cardiovascular disease was reported in 3% of such patients receiving anastrozole. Although angina pectoris was reported more frequently in patients receiving adjuvant therapy with anastrozole than in those receiving tamoxifen (about 2 versus 1%), the incidence of myocardial infarction was similar (0.8%).
Among patients receiving anastrozole as first-line therapy, thromboembolic disease was reported in 18 patients (4%), with 5 patients experiencing venous thrombosis (including pulmonary embolus, thrombophlebitis, and retinal vein thrombosis) and 13 patients experiencing coronary and/or cerebral thrombosis (including myocardial infarction, myocardial ischemia, angina pectoris, cerebrovascular accident, cerebral ischemia, and cerebral infarct). Despite its lack of estrogenic activity, there was no evidence of an increased incidence of myocardial infarction in patients receiving anastrozole compared with those receiving tamoxifen.
Among patients receiving anastrozole as second-line therapy, thromboembolic disease was reported in 3%, and thrombophlebitis occurred in 2-5%.
Among patients receiving adjuvant therapy, hot flushes (flashes) occurred less frequently in patients receiving anastrozole than in those receiving tamoxifen (35 versus 40%). Among patients receiving anastrozole as first-line or second-line therapy, hot flushes occurred in 26 or 13%, respectively.
For more Drug Warnings (Complete) data for ANASTROZOLE (35 total), please visit the HSDB record page.

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Pharmacodynamics
Anastrozole prevents the conversion of adrenal androgens (e.g. [testosterone]) to estrogen in peripheral and tumour tissues. As the growth of many breast cancers is stimulated and/or maintained by the presence of estrogen, anastrozole helps to treat these cancers by decreasing the levels of circulating estrogens. Anastrozole has a relatively long duration of action allowing for once daily dosing - serum estradiol is reduced by approximately 70% within 24 hours of beginning therapy with 1mg once daily, and levels remain suppressed for up to 6 days following cessation of therapy. The incidence of ischemic cardiovascular events was increased during anastrozole therapy and patients with pre-existing ischemic heart disease should consider the risks and benefits of anastrozole before beginning therapy. Anastrozole has also been reported to decrease spine and hip bone mineral density (BMD), so consideration should be given to monitoring of BMD in patients receiving long-term therapy.

C17H19N5

293.37

293.164

C, 69.60; H, 6.53; N, 23.87

120511-73-1

Anastrozole-d12;120512-32-5

2187

White to off-white solid powder

1.1±0.1 g/cm3

469.7±55.0 °C at 760 mmHg

81-82°C

237.9±31.5 °C

0.0±1.2 mmHg at 25°C

1.580

0.97

0

4

4

22

456

0

YBBLVLTVTVSKRW-UHFFFAOYSA-N

InChI=1S/C17H19N5/c1-16(2,9-18)14-5-13(8-22-12-20-11-21-22)6-15(7-14)17(3,4)10-19/h5-7,11-12H,8H2,1-4H3

2,2'-(5-((1H-1,2,4-triazol-1-yl)methyl)-1,3-phenylene)bis(2-methylpropanenitrile)

ZD-1033; ZD1033; ZD 1033; CCRIS 9352; HSDB 7462; ICI D1033; Anastrozole (ANAS); 120511-73-1; Arimidex; anastrazole; Anastrozol; ZD1033; 2,2'-(5-((1H-1,2,4-triazol-1-yl)methyl)-1,3-phenylene)bis(2-methylpropanenitrile); Asiolex; Trade name: Arimidex.

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: ≥ 2.5 mg/mL (8.52 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 25.0 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: ≥ 2.5 mg/mL (8.52 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 25.0 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: ≥ 2.5 mg/mL (8.52 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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