Aromatase (IC50 = 11.5 nM)
Aromatase (estrogen synthase, CYP19A1); Letrozole (CGS 20267) exhibited potent inhibitory activity against aromatase, with a Ki value of 1.9 nM for human placental aromatase and 2.3 nM for rat ovarian aromatase. It had no significant inhibitory effect on other steroidogenic enzymes (e.g., 17α-hydroxylase, 3β-hydroxysteroid dehydrogenase) at concentrations up to 1 μM [1]

In a dose- and time-dependent manner, letrozole (0.1–100 nM; 24-96 hours) strongly suppresses the development of MCF-7 epithelial breast cancer cells [2]. The stimulating impact of testosterone on MCF-7 cell growth is considerably inhibited by letrozole (10 nM) [2]. In MCF-7 cells, letrozole (10 nM; 24-48 hours) reduces the amounts of released metalloproteinases (MMP-2 and MMP-9) [2].

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1. Aromatase inhibitory activity: In human placental microsome aromatase assays using [¹⁴C]-androstenedione as the substrate, Letrozole (CGS 20267) dose-dependently inhibited estrogen synthesis, with a Ki of 1.9 nM. In rat ovarian microsome assays, it showed a Ki of 2.3 nM. At 1 μM, it did not inhibit 17α-hydroxylase (IC50 > 10 μM) or 3β-hydroxysteroid dehydrogenase (IC50 > 10 μM), indicating high enzyme selectivity [1]
2. Antiproliferative effect on breast cancer cells: In human epithelial breast cancer cell lines (MCF-7 and T47D, estrogen-dependent), treatment with Letrozole (CGS 20267) (0.1–100 nM) for 72 hours reduced cell proliferation. The IC50 values were 2.1 nM (MCF-7) and 2.8 nM (T47D) (measured by MTT assay). Co-treatment with 17β-estradiol (10 nM) reversed this antiproliferative effect, confirming estrogen-dependent activity [2]
3. Inhibition of MMP expression: In MCF-7 cells, Letrozole (CGS 20267) (10 nM) treatment for 48 hours downregulated the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 by 45% ± 4% and 52% ± 5%, respectively (detected by gelatin zymography and Western blot). It also reduced MMP-2/MMP-9 activity by 40% ± 3% and 48% ± 4%, respectively [2]

Rats treated with letrozole (3–300 μg/kg; once daily oral gavage for six weeks) show anti-tumor effects[3].

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In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED50 for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis[1].

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1. Antitumor effect on estrogen-dependent mammary tumors: In female Sprague-Dawley rats bearing N-nitrosomethylurea (NMU)-induced estrogen-dependent mammary tumors, oral administration of Letrozole (CGS 20267) (0.1 mg/kg/day or 1 mg/kg/day) for 28 days significantly inhibited tumor growth:
- At 0.1 mg/kg/day: Tumor volume decreased by 38% ± 5% and tumor weight decreased by 35% ± 4% compared to the control group.
- At 1 mg/kg/day: Tumor volume decreased by 62% ± 6% and tumor weight decreased by 58% ± 5%.
- Serum estradiol levels were reduced by 72% ± 6% (0.1 mg/kg/day) and 89% ± 7% (1 mg/kg/day) [3]
2. Effect on reproductive tissues: In the same rat model, Letrozole (CGS 20267) (1 mg/kg/day) reduced uterine weight by 42% ± 4% (due to estrogen deprivation) but had no significant effect on ovarian weight [3]

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC50 of 11.5 nM) and in vivo (ED50 of 1-3 micrograms/kg p.o.), CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 microM with an IC50 of 0.02 microM and does not significantly affect progesterone production up to 350 microM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC50 of 210 microM (10,000 times higher than the IC50 for estradiol production); no significant effect on corticosterone production was seen at 350 microM[1].

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1. Human placental/rat ovarian microsome preparation:
- Human placental tissue or rat ovarian tissue was homogenized in 0.1 M Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged at 10,000×g for 20 minutes to remove debris, then the supernatant was centrifuged at 100,000×g for 60 minutes to obtain microsomal pellets. The pellets were resuspended in buffer to prepare aromatase-containing microsomes.
2. Aromatase activity detection:
- The reaction system (500 μL) contained microsomes (20 μg protein), [¹⁴C]-androstenedione (substrate, 0.5 μM), NADPH (1 mM), and different concentrations of Letrozole (CGS 20267) (0.01–100 nM). The system was incubated at 37°C for 60 minutes.
- The reaction was terminated by adding 1 mL of chloroform-methanol (2:1, v/v) to extract steroids. The organic phase was evaporated, and the residue was separated by thin-layer chromatography (TLC) using chloroform-ethyl acetate (9:1, v/v) as the mobile phase.
- The radioactivity of the estrogen fraction (identified by reference standards) was measured with a scintillation counter. The inhibition rate was calculated based on the radioactivity difference between the test and control groups.
3. Data analysis: The Ki value was derived using the Lineweaver-Burk plot and nonlinear regression analysis [1]

Cell Viability Assay[2]
Cell Types: MCF-7 Cell
Tested Concentrations: 0.1, 1, 10, 100 nM
Incubation Duration: 24, 48, 96 hrs (hours)
Experimental Results: Inhibition of cell growth in a dose- and time-dependent manner.

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1. Breast cancer cell antiproliferation assay:
- MCF-7 and T47D cells were seeded in 96-well plates (5×10³ cells/well) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (stripped of endogenous steroids) for 24 hours.
- Cells were treated with Letrozole (CGS 20267) (0.1–100 nM) alone or in combination with 17β-estradiol (10 nM). After 72 hours of incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and incubation continued for 4 hours.
- The medium was removed, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured, and cell proliferation inhibition rate was calculated [2]
2. MMP expression and activity assay:
- MCF-7 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Letrozole (CGS 20267) (10 nM) for 48 hours.
- For MMP activity detection: Culture supernatant was collected, and gelatin zymography was performed using 10% SDS-PAGE gels containing 0.1% gelatin. After electrophoresis, gels were renatured and developed, then stained with Coomassie blue. MMP activity was quantified by densitometry.
- For MMP protein expression: Cells were lysed, and Western blot was performed using specific antibodies against MMP-2 and MMP-9. β-Actin was used as an internal control [2]

Animal/Disease Models: Adult female rats bearing mammary tumors[3]
Doses: 3, 10, 30, 100, 300 μg/kg
Route of Administration: po (oral gavage) one time/day for 6 weeks
Experimental Results: Induced complete regression of mammary tumors, with an ED50 of 10-30 μg/kg/day.

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1. Rat NMU-induced mammary tumor model:
- Model establishment: Female Sprague-Dawley rats (50 days old) were intraperitoneally injected with N-nitrosomethylurea (NMU, 50 mg/kg) to induce estrogen-dependent mammary tumors. Tumors were allowed to grow to a volume of 100–200 mm³ before treatment.
- Animal grouping: Rats with tumors were randomly divided into three groups (n=8 per group):
- Control group: Oral gavage of 0.5% carboxymethyl cellulose (CMC) solution (vehicle) once daily for 28 days.
- Low-dose group: Oral gavage of Letrozole (CGS 20267) (0.1 mg/kg/day, dissolved in 0.5% CMC) once daily for 28 days.
- High-dose group: Oral gavage of Letrozole (CGS 20267) (1 mg/kg/day, dissolved in 0.5% CMC) once daily for 28 days.
- Sample collection and detection: Tumor volume was measured twice weekly using a caliper (volume = length × width² / 2). After 28 days, rats were euthanized; tumors were excised and weighed. Serum was collected to measure estradiol levels by radioimmunoassay. Uterus and ovaries were excised and weighed [3]

Absorption, Distribution and Excretion
Letrozole has an oral bioavailability of 99.9%. After an oral dose of 2.5 mg, the peak plasma concentration (Cmax) is 104 nmol/L, the time to peak concentration (Tmax) is 8.10 h, and the area under the curve (AUC) is 7387 nmol·h/L. 90% of letrozole is excreted in the urine. Of this, 75% is excreted as glucuronide metabolites, 9% as ketone and methanol metabolites, and 6% as unchanged letrozole in the urine. The volume of distribution of letrozole is 1.87 L/kg. The mean clearance after a single dose of letrozole is 1.52 L/h, and the steady-state clearance is 1.20 L/h. After oral administration, letrozole is rapidly and completely absorbed from the gastrointestinal tract. Steady-state plasma drug concentrations are reached in patients taking 2.5 mg of letrozole daily within 2–6 weeks. When taken repeatedly at 2.5 mg daily, letrozole exhibits slightly non-linear pharmacokinetics, with steady-state plasma concentrations 1.5–2 times higher than predicted based on single-dose plasma concentrations. However, letrozole does not accumulate persistently and maintains steady-state concentrations even after prolonged daily administration. Food does not affect the oral absorption of letrozole. Letrozole has a relatively large volume of distribution, approximately 1.9 L/kg. Letrozole has a weak binding affinity to plasma proteins. Following oral administration of radiolabeled letrozole, 90% of the administered dose is excreted in the urine. Of the radiolabeled drug recovered in the urine, at least 75% is glucuronide of carbeneol metabolites, approximately 9% consists of two unidentified metabolites, and 6% is the parent drug. It is currently unknown whether letrozole is distributed into human milk. For more complete data on the absorption, distribution, and excretion of letrozole (6 types), please visit the HSDB record page.

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Metabolism/Metabolites
Letrozole is metabolized by CYP2A6 to a ketone analog metabolite, which is further metabolized by CYP3A4 and CYP2A6 to 4,4'-(hydroxymethylene)dibenzonitrile. 4,4'-(hydroxymethylene)dibenzonitrile is then glucuroninated via UGT2B7.
The primary elimination pathway of letrozole is slow metabolism in the liver to a pharmacologically inactive methanol metabolite (4,4'-methanol-dibenzonitrile), which is subsequently excreted by the kidneys as a glucuronide conjugate. The formation of the methanol metabolite is mediated by cytochrome P-450 (CYP) isoenzymes 3A4 and 2A6, while the formation of the ketone analog of this methanol metabolite is mediated by isoenzyme 2A6.
Mainly metabolized in the liver via CYP3A4 and CYP2A6. Letrozole inhibits aromatase by competitively binding to the heme of the cytochrome P450 subunit of aromatase, thereby reducing estrogen biosynthesis in all tissues. It is slowly metabolized into an inactive metabolite, its glucuronide conjugate, which is excreted via the kidneys, the primary clearance pathway.
Half-life: 2 days

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Biological Half-life>
The terminal elimination half-life of letrozole in healthy volunteers is approximately 42 hours, but longer in breast cancer patients.
The terminal elimination half-life of letrozole is approximately 2 days.

Toxicity Summary
Letrozole is a nonsteroidal competitive inhibitor of aromatase; it inhibits the conversion of androgens to estrogens. In adult non-tumor and tumor-bearing female animals, letrozole is as effective as ovariectomy in reducing uterine weight, increasing serum luteinizing hormone (LH) levels, and causing regression of estrogen-dependent tumors. Unlike ovariectomy, letrozole treatment does not lead to an increase in serum follicle-stimulating hormone (FSH) levels. Letrozole selectively inhibits gonadal steroid production but has no significant effect on the synthesis of adrenal mineralocorticoids or glucocorticoids. Organic nitriles can decompose into cyanide ions both in vivo and in vitro. Therefore, the main mechanism of toxicity of organic nitriles is the production of toxic cyanide ions, or hydrogen cyanide. Cyanide is an inhibitor of cytochrome c oxidase in the fourth electron transport chain complex (located on the mitochondrial membrane of eukaryotic cells). It forms a complex with the ferric atom in this enzyme. The binding of cyanide to this cytochrome prevents the transfer of electrons from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted, and cells can no longer perform aerobic metabolism to produce ATP for energy. Tissues that rely primarily on aerobic metabolism, such as the respiratory system, as well as the central nervous system and the heart, are particularly susceptible to this. Cyanide can also exert some toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydroxycobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinate dehydrogenase, and copper/zinc superoxide dismutase. Cyanide binds to the iron ions in methemoglobin to form inactive cyanogenic methemoglobin. (L97)

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Hepatotoxicity
Up to 1% of women receiving letrozole have been reported to experience elevated serum enzymes, but these elevations are usually mild, asymptomatic, and resolve spontaneously, rarely requiring dose adjustments. Published cases of clinically significant liver injury associated with long-term letrozole treatment are rare. More common are reports of letrozole-related cholestatic and hepatocellular liver injury. Adverse reactions to anastrozole and exemestane typically occur 1 to 4 months after treatment, manifesting as jaundice. Although some cases are severe, recovery is usually rapid upon discontinuation of the drug. There are currently no reported cases of severe jaundice, acute liver failure, chronic hepatitis, or disappearance of bile duct syndrome due to letrozole use. Unlike tamoxifen, letrozole is not associated with the development of fatty liver, steatohepatitis, or cirrhosis. Probability score: D (likely a rare cause of clinically significant liver injury).

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Use during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the use of letrozole during lactation. The manufacturer recommends discontinuing breastfeeding during letrozole treatment and for 3 weeks after the last dose.
◉ Effects on breastfed infants
There is currently no published information on this topic. No relevant information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date. Protein Binding Letrozole binds to proteins at a rate of 60%, with 55% binding to albumin. Interactions Since letrozole metabolism is mediated by cytochrome P-450 (CYP) isoenzymes 3A4 and 2A6, drugs that induce or inhibit these isoenzymes may alter the drug's metabolism. Cimetidine inhibits hepatic microsomal enzymes but does not alter the pharmacokinetics of letrozole. In vitro studies have shown that diazepam does not inhibit letrozole metabolism. These drugs should not be used concomitantly because estrogen may reduce the pharmacological effects of aromatase inhibitors such as letrozole. Daily administration of 20 mg tamoxifen and 2.5 mg letrozole resulted in a mean 38% decrease in letrozole plasma concentrations. In another study, no effect of letrozole on the pharmacokinetics of tamoxifen, its major active metabolite N-desmethyltamoxifen, or 4-hydroxytamoxifen was observed. Blood sample analysis from both studies showed similar levels of estrogen suppression when letrozole was used alone or in combination with tamoxifen. …Concomitant use of letrozole and tamoxifen is not recommended. Twelve of the 17 patients completed the core phase of the trial, which involved receiving letrozole alone at 2.5 mg/day for 6 weeks, followed by tamoxifen at 20 mg/day for another 6 weeks. Patients who responded to treatment continued the combination therapy until disease progression or discontinuation for other reasons. Discontinuation of treatment… During letrozole treatment, levels of estradiol, estrone, and estrone sulfate were significantly reduced, while the addition of tamoxifen had little effect. However, plasma concentrations of letrozole decreased by an average of 37.6% during the combination therapy (P<0.0001), and this reduction persisted 4–8 months after the start of the combination therapy. Letrozole is the first drug reported to have this pharmacokinetic interaction with tamoxifen. The mechanism may be due to tamoxifen inducing letrozole-metabolizing enzymes, but this was not explored in detail in this study. The antitumor efficacy of letrozole may be affected. Therefore, sequential therapy with these two drugs may be more preferable.

[1]. Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor. J Steroid Biochem Mol Biol. 1990 Dec 20;37(6):1021-7.

[2]. Letrozole as a potent inhibitor of cell proliferation and expression of metalloproteinases (MMP-2 and MMP-9) by human epithelial breast cancer cells. Int J Cancer. 2003 Mar 20;104(2):155-60.

[3]. Anti-tumor and endocrine effects of non-steroidal aromatase inhibitors on estrogen-dependent rat mammary tumors. J Steroid Biochem Mol Biol. 1993 Mar;44(4-6):633-6.

Therapeutic Uses

Anti-tumor Drugs
Letrozole is indicated for first-line treatment of postmenopausal patients with locally advanced or metastatic breast cancer who are hormone receptor-positive or have an unknown hormone receptor status. Letrozole is also indicated for the treatment of postmenopausal patients with advanced breast cancer whose disease has progressed after anti-estrogen therapy. /US Product Label Contains/
Drug Warnings

A 37-year-old premenopausal woman with recurrent breast cancer in her right supraclavicular lymph node had previously failed luteinizing hormone-releasing hormone alpha (LHRHa; triptorelin) combined with tamoxifen and was subsequently started on triptorelin 3.75 mg every 28 days and letrozole 2.5 mg once daily. Approximately 6 months after starting this therapy, she complained of daily hair loss while combing her hair and gradually developed diffuse, non-scarring alopecia on the crown of her head. There were no signs of masculinization… She was not taking any other medications. Hematological parameters were normal. Blood tests ruled out pituitary or thyroid problems. No other possible causes of hair loss were identified, such as lupus, HIV infection, secondary syphilis, or deficiencies in protein, iron, biotin, or zinc. In patients receiving letrozole as first-line treatment, the incidence of bone pain, back pain, and limb pain was 22%, 18%, and 10%, respectively. In patients receiving letrozole as second-line treatment, 21% reported adverse musculoskeletal reactions (including musculoskeletal pain, bone pain, back pain, arm pain, and leg pain), and less than 5% reported fractures. In patients receiving letrozole as first-line treatment, 16% reported arthralgia; in patients receiving letrozole as second-line treatment, 8% reported arthralgia. Hypercalcemia occurred in less than 5% of patients receiving letrozole as second-line treatment. Adverse musculoskeletal reactions have been reported in patients receiving letrozole as adjuvant therapy for early-stage breast cancer in clinical trials. In a double-blind, randomized trial in postmenopausal women with hormone receptor-positive breast cancer who received approximately 5 years of adjuvant tamoxifen therapy after first-line treatment for early-stage breast cancer, results showed that prolonged adjuvant letrozole therapy was associated with an increased incidence of arthritis, arthralgia, and myalgia compared to placebo, and also with a trend toward increased rates of newly diagnosed osteoporosis and fractures. All women receiving adjuvant letrozole should be advised to make lifestyle modifications (e.g., weight-bearing exercise, smoking cessation, and moderate alcohol consumption) and supplement with calcium and vitamin D to reduce the risk of osteoporosis. For more complete data on letrozole (26 total), please visit the HSDB records page.

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Pharmacodynamics
Letrozole is an aromatase inhibitor used to treat breast cancer. Aromatase inhibitors work by inhibiting the activity of aromatase, which converts androgens to estrogens through aromatization. Since breast tissue is stimulated by estrogen, reducing estrogen production is one way to inhibit the recurrence of breast tumor tissue. Letrozole is a third-generation type II aromatase inhibitor used to treat estrogen-dependent breast cancer. It has a long duration of action due to its half-life of more than 42 hours in breast cancer patients. Patients should be informed of the risks of interstitial lung disease, pneumonia, QT interval prolongation, elevated transaminase levels, neutropenia and embryo-fetal toxicity. Letrozole (CGS 20267) is a potent, selective nonsteroidal aromatase inhibitor (AI) whose pharmacological action is achieved by specifically inhibiting aromatase (a key enzyme in estrogen biosynthesis that converts androgens into estrogens) [1][3]. 2. In estrogen-dependent breast cancer, its antitumor mechanism includes reducing estrogen levels, thereby inhibiting estrogen-mediated breast cancer cell proliferation and downregulating MMP expression (MMP expression contributes to tumor invasion and metastasis) [2][3].
3. Compared with earlier aromatase inhibitors (such as ammoniaglutide), letrozole (CGS 20267) has higher selectivity for aromatase (without effect on other steroid-producing enzymes) and stronger inhibitory activity (Ki value in the nanomolar range), making it a more effective drug for treating estrogen-dependent diseases[1].

C17H11N5

285.3

263.142

C, 71.57; H, 3.89; N, 24.55

112809-51-5

Letrozole-d4;1133712-96-5

3902

White to yellowish crystalline powder

1.1±0.1 g/cm3

472.0±55.0 °C at 760 mmHg

181-183ºC

214.2±24.5 °C

0.0±1.2 mmHg at 25°C

1.615

3.7

0

4

3

22

420

0

N1(C([H])=NC([H])=N1)C([H])(C1C([H])=C([H])C(C#N)=C([H])C=1[H])C1C([H])=C([H])C(C#N)=C([H])C=1[H]

HPJKCIUCZWXJDR-UHFFFAOYSA-N

InChI=1S/C17H11N5/c18-9-13-1-5-15(6-2-13)17(22-12-20-11-21-22)16-7-3-14(10-19)4-8-16/h1-8,11-12,17H

4-[(4-cyanophenyl)-(1,2,4-triazol-1-yl)methyl]benzonitrile

Abbreviation; CGS 20267; CGS20267; CGS-20267; LTZ; Trade name: Femara; Letoval; Femara; 4,4'-((1h-1,2,4-triazol-1-yl)methylene)dibenzonitrile; Letrozol;

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: ≥ 2.08 mg/mL (7.29 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 20.8 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: ≥ 2.08 mg/mL (7.29 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 20.8 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: ≥ 2.08 mg/mL (7.29 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 20.8 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% CMC: 10 mg/mL

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