Letrozole In Plasma Research Articles

Development of a letrozole-based synchronization protocol for fixed-time artificial insemination in beef cattle

Experiments were done to determine ovulation synchrony following a 4-day letrozole treatment (Exp 1), compare the efficacy of a letrozole-based protocol with other commonly used synchronization protocols for FTAI (Exp 2), and test a new intravaginal letrozole-releasing device (LRD) with and without pre-synchronization (Exp 3). In Exp 1, heifers and lactating cows at random stages of the estrous cycle were given an LRD for 4 days, PGF at LRD removal, and GnRH at 48 or 60 h after PGF, or no GnRH. In Exp 2, heifers and lactating cows were assigned to three FTAI groups: i) LRD, ii) estradiol+progesterone, iii) 5-d Co-synch+PRID. In Exp 3, heifers were pre-synchronized with PGF or not (control) 8 days before insertion of either an X-LRD or T-LRD for 3 days; FTAI was done 48 h after device removal. In Exp 1, the variation in interval to ovulation in cows, but not heifers, in the GnRH 48-h group was less than half that in other groups (P < 0.05). In Exp 2, the P/AI was lower (P < 0.001) in the LRD group compared to the other groups. In Experiment 3, the X-LRD increased (P < 0.0001) plasma letrozole concentrations compared to the T-LRD; the pregnancy rate was not affected by pre-synchronization or the type of LRD. Although ovulation synchrony was achieved following LRD treatment, the LRD group had the lowest P/AI compared to other protocols, perhaps because of too short an interval between LRD removal and GnRH/FTAI. Drug delivery was enhanced with the new X-LRD.

Poloxamer188 composite electrospun poly L-lactic acid fibrous nonwoven: Sustained in vitro and in vivo release letrozole as a subcutaneous implant

Poloxamer188 composite poly L-lactic acid (PLLA) electrospun fibrous nonwovens (EFNW) were prepared by simple letrozole-poloxamer188-PLLA co-solution electrospinning and used as subcutaneous implants to sustained release letrozole. Contents of poloxamer188 and letrozole were varied to investigate the effect of the two compounds’ amounts on drug release behavior. Materials’ combination and drug incorporation in fibers were investigated by X-ray diffraction and differential thermal calorimetry. Letrozole contents in vitro and in vivo were all determined by high-performance liquid chromatography-ultraviolet spectrometry. Meanwhile, in order to analyze the mechanism of drug release behavior and show modulated drug release by electrospun fibers, letrozole-poloxamer188 solid dispersions (SDs) and letrozole-poloxamer188poly L-lactic acid-dichloromethane solution cast films (CFs) were also studied. Plasma letrozole concentration curves of subcutaneous implanting EFNW and daily oral administration of poloxamer188-letrozole SD on female rabbits were investigated and compared. Fibers of desirable morphology were obtained. During the whole release process of all EFNWs, water-insoluble letrozole could be released in the form of being dissolved. Letrozole release rates increased with increasing poloxamer188 content and decreased with increasing letrozole content. When letrozole and poloxamer188 content in fibers was 30% and 150% (EFNW-30-150%), respectively (with respect to PLLA in mass ratio), the release curve presented a desirable profile. After subcutaneous implant of EFNW-30-150%, the plasma concentration curve presented a later peak time and lower maximum value, and a comparable bioavailability with daily oral administration of poloxamer188-letrozole SD solution within 15 days. The research provided primary and encouraging information to use EFNW as a novel subcutaneous implant for sustained delivery of water-insoluble drugs.

Tailoring a new hyperbranched PEGylated dendrimer nano-polymer as a super-adsorbent for magnetic solid-phase extraction and determination of letrozole in biological and pharmaceutical samples

A novel hyperbranched magnetic dendrimer was successfully fabricated and utilized as an efficient adsorbent for solid-phase extraction of letrozole from different media (plasma, urine, and tablet matrices) in this study. Iron oxide nanoparticles as magnetic cores were modified with PEGylated block-dendrimer polymer branches. The Fe3O4@SiO2/DAG/MMA/ED/Gly-PEGylated was characterized by a Vibrating sample magnetometer (VSM), Thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and Scanning electron microscope (SEM) analyses. The average particle size was obtained as 47 ± 4.3 nm for the nano-adsorbent. The experimental parameters influencing the extraction efficiency of the letrozole, such as pH of the sample solution, and contact time were carefully examined and optimized. Moreover, the isotherm adsorption and kinetic studies demonstrated that the adsorption process was well fitted to Langmuir isotherm and Pseudo-first-order kinetic models, respectively. A maximum adsorption capacity of 20 mg g−1 was acquired based on the Langmuir model. Under optimal conditions, the suggested approach provided favorable linearity in the range of 0.1–15 μg L−1, low limits of detection (1.36 μg L−1), and quantification (4.54 μg L−1). The recoveries for spiking 1 μg L−1 of letrozole in plasma and urine samples and letrozole tablet were respectively, 75.6%, 98.4%, and 93.7%. Relative standard deviation values for plasma, urine, and tablet were respectively, obtained to be 1.1, 05, and 0.4. These results showed that the suggested technique was a potent analytical tool and a helpful alternative for common analysis of such medicines.

A new clinically-relevant rat model of letrozole-induced chronic nociceptive disorders

Among postmenopausal women with estrogen receptor-positive breast cancer, more than 80% receive hormone therapy including aromatase inhibitors (AIs). Half of them develop chronic arthralgia - characterized by symmetric articular pain, carpal tunnel syndrome, morning stiffness, myalgia and a decrease in grip strength - which is associated with treatment discontinuation. Only a few animal studies have linked AI treatment to nociception, and none to arthralgia. Thus, we developed a new chronic AI-induced nociceptive disorder model mimicking clinical symptoms induced by AIs, using subcutaneous letrozole pellets in ovariectomized (OVX) rats. Following plasma letrozole dosage at the end of the experiment (day 73), only rats with at least 90 ng/ml of letrozole were considered significantly exposed to letrozole (OVX + high LTZ group), whereas treated animals with less than 90 ng/ml were pooled in the OVX + low LTZ group. Chronic nociceptive disorder set in rapidly and was maintained for more than 70 days in the OVX + high LTZ group. Furthermore, OVX + high LTZ rats saw no alteration in locomotion, myalgia or experimental anxiety during this period. Bone parameters of the femora were significantly altered in all OVX rats compared to Sham+vehicle pellet. A mechanistic analysis focused on TRPA1, receptor suspected to mediate AI-evoked pain, and showed no modification in its expression in the DRG. This new long-lasting chronic rat model, efficiently reproduces the symptoms of AI-induced nociceptive disorder affecting patients' daily activities and quality-of-life. It should help to study the pathophysiology of this disorder and to promote the development of new therapeutic strategies.

Simultaneous quantification of palbociclib, ribociclib and letrozole in human plasma by a new LC-MS/MS method for clinical application.

A novel LC-MS/MS method was developed for the quantification of the new cyclin dependent kinase inhibitors (CDKIs) palbociclib and ribociclib and the aromatase inhibitor letrozole used in combinatory regimen. The proposed method is appropriate to be applied in clinical practice due to the simple and fast sample preparation based on protein precipitation, the low amount of patient sample necessary for the analysis (10 μL) and the total run time of 6.5 min. It was fully validated according to FDA and EMA guidelines on bioanalytical method validation. The linearity was assessed (R2 within 0.9992-0.9983) over the concentration ranges of 0.3-250 ng/mL for palbociclib, 10-10000 ng/mL for ribociclib and 0.5-500 ng/mL for letrozole that properly cover the therapeutic plasma concentrations. A specific strategy was implemented to reduce the carryover phenomenon, formerly known for these CDKIs. This method was applied to quantify the Cmin of palbociclib, ribociclib and letrozole in plasma samples from patients enrolled in a clinical study. The same set of study samples was analysed twice in separate runs to assess the reproducibility of the method by means of the incurred samples reanalysis. The results corroborated the reliability of the analyte concentrations obtained with the bioanalytical method, already proved by the validation process. The percentage differences were always within ±10% for all the analytes and the R2 of the correlation graph between the two quantifications was equal to 0.9994.

Letrozole concentration is associated with CYP2A6 variation but not with arthralgia in patients with breast cancer.

The aromatase inhibitor (AI) letrozole is a first-line drug in the adjuvant treatment of breast cancer in postmenopausal women. Adherence to AI therapy, including letrozole, remains problematic due to the development of debilitating AI-induced arthralgia. Letrozole is metabolized in the liver by CYP2A6. It remains unknown if plasma letrozole levels or CYP2A6 genetic variation is associated with the development of arthralgia. We enrolled 126 female breast cancer patients initiated on letrozole therapy and prospectively collected blood samples at baseline and two follow-up time points to determine letrozole plasma concentrations and CYP2A6 genotype. At each visit, participants completed two validated questionnaires to assess the severity of arthralgia symptoms. More than half (55%) of patients experienced a significant increase in their arthralgia symptoms after initiation of treatment. The clinical variables of body mass index (P = 0.0003) and age (P = 0.0430) were negatively and positively associated with plasma letrozole concentrations, respectively. CYP2A6 genotype was significantly associated with letrozole levels (P < 0.0001), and increased plasma letrozole levels were observed in patients with CYP2A6 reduced-function genotypes. Plasma levels of letrozole and CYP2A6 genotype were not significantly associated with a change in pain score from baseline. CYP2A6 genotype was a significant predictor of letrozole plasma levels, but was not associated with the development of arthralgia.

Abstract 4241: Preclinical studies of brain/brain tumor disposition and antitumor efficacy of the aromatase inhibitor letrozole

Abstract In this study, we investigated the potential role of aromatase (CYP19) as a target for the treatment of CNS malignancies, as well as brain disposition and anti-tumor efficacy of letrozole, an aromatase inhibitor, in Sprague Dawley rats. Cytotoxicity and aromatase activity of letrozole against human glioma cell lines were measured using MTT assay and Enzyme Immunoassay respectively. For brain and brain tumor PK of letrozole, rats with and without orthotopic implantation of C6 glioma received letrozole (4 - 12 mg/kg; i.v. andor oral). Dual probe intracerebral microdialysis was performed to determine the unbound extracellular fluid (ECF) letrozole concentrations. Serial ECF and blood samples were simultaneously collected over 8 hrs. µPET/CT imaging was performed using 18F FDG to evaluate changes in active tumor volumes pre- and post-treatment of letrozole. Brain tissues were collected at the end of the experiment for histological evaluations. All glioma cell lines included in this study expressed CYP19 and letrozole exerted marked cytotoxicity against these cells ( IC50s 0.1 - 3.5 μM). The relative brain distribution coefficients, measured as the ratio of the observed AUC in ECF and AUC of unbound letrozole in plasma (AUCecf/AUCp,ub) ranged from 0.31 - 0.98. Furthermore, the tumoral ECF levels of letrozole was 1.5 - 2 fold higher relative to tumor-free region of the brain, resulting in tumoral ECF Cmax values that were 10 and 35-fold higher than the observed IC50 value of 0.1 µM against C6 gliomas cells in culture. µPET/CT imaging showed a marked reduction of active tumor volume (75-90%) after 8-10 days of letrozole treatment (N=7). Oral administration of letrozole showed similar brain disposition and efficacy of letrozole. Immunohistochemical analysis of aromatase demonstrated selectivelymarkedly higher aromatase expression in tumoral regions of the brain as well asand a clear considerable reduction in aromatase expression in letrozole the treated rats relative to the treatment group as compared to the control group. Thus, employing multifaceted and cutting edge in vitro and in vivo methods, we conclude: a) aromatase is abundantly expressed in glioma cell lines examined, b) letrozole exerts marked cytotoxicity in these cells presumably due to aromatase inhibition, c) letrozole has selectively higher accumulation in tumoral region of the brain and d) In vivo µPET/CT studies show marked efficacy of letrozole on C6 glioma in a preclinical rat model. Note: This abstract was not presented at the meeting. Citation Format: Nimita Dave, Pankaj B. Desai, Gary A. Gudelsky, Kathleen LaSance, Lionel M.L. Chow, Xiaoyang Qi. Preclinical studies of brain/brain tumor disposition and antitumor efficacy of the aromatase inhibitor letrozole. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4241. doi:10.1158/1538-7445.AM2014-4241

Plasma Letrozole Concentrations in Postmenopausal Women With Breast Cancer Are Associated With CYP2A6 Genetic Variants, Body Mass Index, and Age

The associations between plasma letrozole concentrations and CYP2A6 and CYP3A5 genetic variants were tested in the Exemestane and Letrozole Pharmacogenomics (ELPH) trial. ELPH is a multicenter, open-label prospective clinical trial in women randomly assigned (n≈250 in each arm) to receive 2 years of treatment with either oral letrozole (2.5 mg/day) or oral exemestane (25 mg/day). CYP2A6 and CYP3A showed effects on letrozole metabolism in vitro. DNA samples were genotyped for variants in the CYP2A6 and CYP3A5 genes. Plasma letrozole concentrations showed high interpatient variability (>10-fold) and were associated significantly with CYP2A6 genotypes (P<0.0001), body mass index (BMI) (P<0.0001), and age (P=0.0035). However, CYP3A5 genotypes showed no association with plasma letrozole concentrations. These data suggest that CYP2A6 is the principal clearance mechanism for letrozole in vivo. CYP2A6 metabolic status, along with BMI and age, may serve as a biomarker of the efficacy of letrozole treatment or a predictor of adverse effects.

Novel extraction method based on the dispersion of the extraction solvent for extraction of letrozole from biological fluids

The analysis of drugs in various biological fluids is an important criterion for the determination of the physiological performance of a drug. Dispersive liquid–liquid microextraction (DLLME) technique was successfully used as a simple, rapid and sensitive sample preparation method for the determination of letrozole in biological fluids and water samples. Chloroform at microlitre volume level and acetone were used as extraction and disperser solvents, respectively. The influence of several variables (e.g. type and volume of disperser and extraction solvents, ionic strength, etc.) on the performance of the sample preparation step was carefully evaluated. Under the optimum conditions, a preconcentration factor of 120 was obtained from only 10.0 mL of a water sample. The calibration graph was linear in the range of 1–500 μg L−1 with a detection limit of 0.3 μg L−1. The relative standard deviation (RSD) for five replicate measurements of letrozole was 3.2%. The calibration graphs were linear in the range of 2.5–500 μg L−1 and 5.0–500 μg L−1 with detection limits of 0.7 μg L−1 and 1.5 μg L−1 in urine and plasma samples, respectively. The relative recoveries of letrozole in plasma, urine and tap water samples at spiking levels of 50 μg L−1, 30 μg L−1 and 5 μg L−1 were 87.4%, 92.0% and 96.0%, respectively. DLLME combined with HPLC-UV is a fast, simple and efficient method for the determination of letrozole in biological fluids and water samples.

Cytochrome P450 (CYP) 2A6 Genetic Variation Predicts Letrozole Plasma Concentrations in Postmenopausal Women with Breast Cancer.

Abstract Background: The potent aromatase inhibitor (AI) letrozole is effective in the treatment of postmenopausal women with steroid-hormone-receptor–positive breast cancer (metastatic, neoadjuvant and adjuvant). The beneficial and adverse effects of letrozole vary widely among patients, and differences in pharmacokinetics may contribute to this variability. Letrozole is primarily cleared by hepatic metabolism. Since CYP2A6 has been implicated in letrozole metabolism in vitro, we tested the influence CYP2A6 genetic variants on plasma concentrations of letrozole in breast cancer patients. Methods: Postmenopausal women with early stage hormone receptor-positive breast cancer were recruited into an ongoing multicenter randomized clinical trial to study the pharmacogenomics of two AIs, exemestane (25 mg/day) and letrozole (2.5 mg/day) given orally. Total accrual to the trial is anticipated to be 250 in each arm. Three months plasma letrozole concentrations were measured by HPLC from 146 patients who were randomized to the letrozole arm and multiple CYP2A6 variants were genotyped from genomic DNA. Results: Plasma concentrations of letrozole showed wide variability among patients (16.6 ng/ml to 141.2 ng/ml; 8.5-fold difference). Genotypes were grouped based in to normal (*1/*1), intermediate (*1/*9 and *1/*12, n=23) and slow (*9/*12, *1/*2, *2/*9, *1/*35, *1/*4E; n=12) metabolizers based on genotyped predicted phenotype. A statistically significant difference in letrozole concentrations was observed among the patients with the different genotype groups (p&amp;lt;0.0001; one-way ANOVA), with strong gene-dose effect relationship (r2=0.28; p&amp;lt;0.0001). Letrozole plasma concentrations were significantly higher in patients with the slow or intermediate genotypes than those with normal genotype (p &amp;lt; 0.0001; Dunn's post-test for multiple comparison correction).Conclusions: Polymorphisms in the CYP2A6 gene account for ∼28% of variability in letrozole concentrations in breast cancer patients. Prior genotyping for CYP2A6 variants may help to identify patients who benefit from letrozole and who experience adverse effects from the drug. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5160.

Synthesis and PET studies of [11C-cyano]letrozole (Femara), an aromatase inhibitor drug

Aromatase, a member of the cytochrome P450 family, converts androgens such as androstenedione and testosterone into estrone and estradiol, respectively. Letrozole (1-[bis-(4-cyanophenyl)methyl]-1H-1,2,4-triazole; Femara) is a high-affinity aromatase inhibitor (K(i)=11.5 nM) that has Food and Drug Administration approval for breast cancer treatment. Here we report the synthesis of carbon-11-labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon. Letrozole and its precursor (4-[(4-bromophenyl)-1H-1,2,4-triazol-1-ylmethyl]benzonitrile) were prepared in a two-step synthesis from 4-cyanobenzyl bromide and 4-bromobenzyl bromide, respectively. The [(11)C]cyano group was introduced via tetrakis(triphenylphosphine)palladium(0)-catalyzed coupling of [(11)C]cyanide with the bromo precursor. Positron emission tomography (PET) studies in the baboon brain were carried out to assess regional distribution and kinetics, reproducibility of repeated measures and saturability. Log D, the free fraction of letrozole in plasma and the [(11)C-cyano]letrozole fraction in arterial plasma were also measured. [(11)C-cyano]Letrozole was synthesized in 60 min with a radiochemical yield of 79-80%, with a radiochemical purity greater than 98% and a specific activity of 4.16+/-2.21 Ci/mumol at the end of bombardment (n=4). PET studies in the baboon revealed initial rapid and high uptake and initial rapid clearance, followed by slow clearance of carbon-11 from the brain, with no difference between brain regions. Brain kinetics was not affected by coinjection of unlabeled letrozole (0.1 mg/kg). The free fraction of letrozole in plasma was 48.9%, and log D was 1.84. [(11)C-cyano]Letrozole is readily synthesized via a palladium-catalyzed coupling reaction with [(11)C]cyanide. Although it is unsuitable as a PET radiotracer for brain aromatase, as revealed by the absence of regional specificity and saturability in brain regions such as amygdala, which are known to contain aromatase, it may be useful in measuring letrozole distribution and pharmacokinetics in the brain and peripheral organs.

HPLC Determination of Letrozole in Plasma Using Fluorescence Detection: Application to Pharmacokinetic Studies

A simple, rapid and sensitive HPLC method has been developed and validated for the analysis of letrozole in human plasma. The separation was achieved on a monolithic silica column using acetonitrile–phosphate buffer. A fluorescence detector was used for the quantitation with excitation and emission wavelengths at 230 and 295 nm. The assay enables the measurement of letrozole for therapeutic drug monitoring with a minimum quantification limit (LOQ) of 0.5 ng mL−1. The method involves a simple, one-step extraction procedure with complete recovery. Calibration was linear over the concentration range 0.5–80 ng mL−1. The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.

Double-blind, randomised, multicentre endocrine trial comparing two letrozole doses, in postmenopausal breast cancer patients

Letrozole is an orally competitive aromatase inhibitor. This double-blind, randomised, multicentre trial was carried out to evaluate the endocrine effects of two doses of letrozole, 0.5mg versus 2.5mg orally daily, in postmenopausal advanced breast cancer patients progressing after tamoxifen. The pharmacokinetics of letrozole was also assessed. 46 patients entered the trial, 22 on letrozole 0.5mg and 24 on 2.5mg. A significant suppression of oestrone and oestradiol levels was achieved by both letrozole doses. Neither letrozole dose induced any changes in cortisol and aldosterone production at rest or after Synacthen stimulation. Androstenedione, testosterone, 17α-OH progesterone, triiodothyronine (T3) thyroxine, (T4) and thyroid-stimulating hormone (TSH) plasma levels did not show any significant changes. Sex hormone binding globulin (SHBG), follicle-stimulating hormone (FSH) and luteinising hormone (LH) levels increased significantly over time. Plasma letrozole concentrations increased until reaching steady-state values after 1 month at the dose of 0.5mg and after 2 months at 2.5mg. In conclusion, both letrozole doses suppressed oestrogen levels without affecting adrenal activity.