Quantification of the aromatase inhibitor letrozole and its carbinol metabolite in mouse plasma by UHPLC-MS/MS.

A liquid chromatography - electrospray ionization-mass spectrometry (LC-ESI-MS) method was developed for the quantification of letrozole, a third-generation aromatase inhibitor, and its main carbinol metabolite (CM) in support of murine pharmacokinetic studies. Using polarity switching, simultaneous ESI-MS measurement of letrozole and CM was achieved in positive and negative mode, respectively. The assay procedure involved a one-step protein precipitation and extraction of all analytes from mouse plasma requiring only 5 µL of sample. Separation was optimized on an Accucore aQ column with gradient elution at a flow rate of 0.4 mL/min in 5 min. Two calibration curves per day over four consecutive measurement days showed satisfactory linear responses (r2 > 0.99) over concentration ranges of 5-1000 ng/mL and 20-2000 ng/mL for letrozole and CM, respectively. No matrix effect was found, and the mean extraction recoveries were 103-108 % for letrozole and 99.8-107 % for CM. Precision and accuracy within a single run and over four consecutive measurement days were verified to be within acceptable limits. Application of the developed method to preclinical pharmacokinetic studies in mice receiving oral letrozole at a dose 1 or 10 mg/kg revealed that the systemic exposure to letrozole was dose-, formulation-, and strain-dependent. These findings may inform the future design of preclinical studies aimed at refining the pharmacological profile of this clinically important drug.

A Phase 0/I Pharmacokinetic and Pharmacodynamics and Safety and Tolerability Study of Letrozole in Combination with Standard Therapy in Recurrent High-Grade Gliomas.

PURPOSE: High-grade gliomas (HGG) carry a poor prognosis, with glioblastoma accounting for almost 50% of primary brain malignancies in the elderly. Unfortunately, despite the use of multiple treatment modalities, the prognosis remains poor in this population. Our preclinical studies suggest that the presence of aromatase expression, encoded by CYP19A1, is significantly upregulated in HGGs. Remarkably, we find that letrozole (LTZ), an FDA-approved aromatase inhibitor, has marked activity against HGGs. PATIENTS AND METHODS: We conducted a phase 0/I single-center clinical trial (NCT03122197) to assess the tumoral availability, pharmacokinetics (PK), safety, and tolerability of LTZ in recurrent patients with HGG. Planned dose cohorts included 2.5, 5, 10, 12.5, 15, 17.5, and 20 mg of LTZ administered daily pre- and postsurgery or biopsy. Tumor samples were assayed for LTZ content and relevant biomarkers. The recommended phase 2 dose (R2PD) was determined as the dose that resulted in predicted steady-state tumoral extracellular fluid (ECF; Css,ecf) >2 µmol/L and did not result in ≥33% dose-limiting adverse events (AE) assessed using CTCAE v5.0. RESULTS: Twenty-one patients were enrolled. Common LTZ-related AEs included fatigue, nausea, musculoskeletal, anxiety, and dysphoric mood. No DLTs were observed. The 15 mg dose achieved a Css,ecf of 3.6 ± 0.59 µmol/L. LTZ caused dose-dependent inhibition of estradiol synthesis and modulated DNA damage pathways in tumor tissues as evident using RNA-sequencing analysis. CONCLUSIONS: On the basis of safety, brain tumoral PK, and mechanistic data, 15 mg daily is identified as the RP2D for future trials.

Super Magnetic Niosomal Nanocarrier as a New Approach for Treatment of Breast Cancer: A Case Study on SK-BR-3 and MDA-MB-231 Cell Lines.

In the present study, a magnetic niosomal nanocarrier for co-delivery of curcumin and letrozole into breast cancer cells has been designed. The magnetic NiCoFe2O4 core was coated by a thin layer of silica, followed by a niosomal structure, allowing us to load letrozole and curcumin into the silica layer and niosomal layer, respectively, and investigate their synergic effects on breast cancer cells. Furthermore, the nanocarriers demonstrated a pH-dependent release due to the niosomal structure at their outer layer, which is a promising behavior for cancer treatment. Additionally, cellular assays revealed that the nanocarriers had low cellular uptake in the case of non-tumorigenic cells (i.e., MCF-10A) and related high viability but high cellular uptake in cancer cell lines (i.e., MDA-MB-231 and SK-BR-3) and related low viability, which is evidenced in their high cytotoxicity against different breast cancer cell lines. The cytotoxicity of the letrozole/curcumin co-loaded nanocarrier is higher than that of the aqueous solutions of both drugs, indicating their enhanced cellular uptake in their encapsulated states. In particular, NiCoFe2O4@L-Silica-L@C-Niosome showed the highest cytotoxicity effects on MDA-MB-231 and SK-BR-3 breast cancer cells. The observed cytotoxicity was due to regulation of the expression levels of the studied genes in breast cancer cells, where downregulation was observed for the Bcl-2, MMP 2, MMP 9, cyclin D, and cyclin E genes while upregulation of the expression of the Bax, caspase-3, and caspase-9 genes was observed. The flow cytometry results also revealed that NiCoFe2O4@L-Silica-L@C-Niosome enhanced the apoptosis rate in both MDA-MB-231 and SK-BR-3 cells compared to the control samples. The findings of our research show the potential of designing magnetic niosomal formulations for simultaneous targeted delivery of both hydrophobic and hydrophilic drugs into cancer cells in order to enhance their synergic chemotherapeutic effects. These results could open new avenues into the future of nanomedicine and the development of theranostic agents.

Gender-based differences in brain and plasma pharmacokinetics of letrozole in sprague-dawley rats: Application of physiologically-based pharmacokinetic modeling to gain quantitative insights.

Based on the discovery that the estrogen synthase aromatase (CYP19A1) is abundantly expressed in high- grade gliomas, the aromatase inhibitor, letrozole is being investigated in pre-clinical models as a novel agent against this malignancy. Here, we investigated the systemic and brain pharmacokinetics of letrozole following single and steady state dosing in both male and female Sprague-Dawley rats. Furthermore, we employed physiologically-based pharmacokinetic (PBPK) modeling to gain quantitative insights into the blood-brain barrier penetration of this drug. Letrozole (4 mg/kg) was administered intraperitoneally daily for 5 days (for males) and 11 days (for females) and intracerebral microdialysis was performed for brain extracellular fluid (ECF) collection simultaneously with venous blood sampling. Drug levels were measured using HPLC and non-compartmental analysis was conducted employing WinNonlin®. Simcyp animal simulator was used for conducting bottom-up PBPK approach incorporating the specified multi-compartment brain model. Overall, marked gender-specific differences in the systemic and brain pharmacokinetics of letrozole were observed. Letrozole clearance was much slower in female rats resulting in markedly higher plasma and brain drug concentrations. At steady state, the plasma AUC 0-24 was 103.0 and 24.8 µg*h/ml and brain ECF AUC 0-12 was 24.0 and 4.8 µg*h/ml in female and male rats, respectively. The PBPK model simulated brain concentration profiles were in close agreement with the observed profiles. While gender-specific differences in letrozole PK are not observed in the clinical setting, these findings will guide the dose optimization during pre-clinical investigations of this compound. The PBPK model will serve as an important clinical translational tool.

Pharmacokinetics, safety, activity, and biomarker analysis of palbociclib plus letrozole as first-line treatment for ER+/HER2- advanced breast cancer in Chinese women.

PURPOSE: This phase 1, open-label, single-arm clinical trial evaluated pharmacokinetics, safety, and biomarker activity of palbociclib-letrozole as first-line treatment for estrogen receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer (ABC) in postmenopausal Chinese women to support palbociclib approval in China. METHODS: Patients received palbociclib 125 mg once daily (3/1 schedule) plus letrozole 2.5 mg once daily. Blood samples were collected predose and ≤ 120 h after single and multiple doses of palbociclib. The incidence and severity of adverse events were reported. Skin biopsy tissues and blood samples were collected for biomarker assessments. RESULTS: By 31 July 2018, 26 patients were enrolled. After single and multiple dosing, palbociclib maximum plasma concentration was 82.14 and 139.7 ng/mL, apparent clearance was 52.40 and 49.97 L/h, AUCτ was 1217 and 2501 ng∙h/mL, and t½ was 23.46 and 27.26 h, respectively. Levels of Ki67, retinoblastoma protein, and thymidine kinase decreased after palbociclib treatment. A similar safety profile as previously reported was observed. CONCLUSIONS: Pharmacokinetic and pharmacodynamic effects of palbociclib were well characterized in Chinese patients with ABC. Despite higher exposure, pharmacokinetic parameters were similar to those of a previously studied non-Asian population. No palbociclib dose adjustment based on Chinese ethnicity is needed. Palbociclib-letrozole had a manageable safety profile. CLINICAL TRIAL REGISTRATION: NCT02499146.

Abemaciclib in combination with endocrine therapy for East Asian patients with HR+, HER2- advanced breast cancer: MONARCH 2 & 3 trials.

This post hoc analysis of MONARCH 2 and MONARCH 3 assesses the efficacy, safety, and pharmacokinetics (PK) of abemaciclib in combination with endocrine therapy (ET) in East Asian patients with hormone receptor positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer. MONARCH 2 and MONARCH 3 are global, randomized, double-blind, phase 3 studies of abemaciclib/placebo + fulvestrant and abemaciclib/placebo + nonsteroidal aromatase inhibitor (NSAI, anastrozole or letrozole), respectively. The East Asian population comprised 212 (31.7%) of the 669 intent-to-treat (ITT) population in the MONARCH 2 trial and 144 (29.2%) of the 493 ITT patients in the MONARCH 3 trial. In the East Asian population, median progression-free survival (PFS) was significantly prolonged in the abemaciclib arm compared with placebo in both MONARCH 2 (hazard ratio [HR], 0.520; 95% confidence interval [CI], 0.362 to 0.747; P < .001; median: 21.2 vs 11.6 months) and MONARCH 3 (HR, 0.326; 95% CI, 0.200 to 0.531, P < .001; median: not reached vs 12.82 months). Diarrhea (MONARCH 2: 90%; MONARCH 3: 88%) and neutropenia (MONARCH 2: 68%; MONARCH 3: 58%) were the most frequent adverse events observed in the East Asian populations. Abemaciclib exposures and PK were similar in East Asians and the non-East Asian populations of both trials. Abemaciclib in combination with ET in the East Asian populations of MONARCH 2 and MONARCH 3 provided consistent results with the ITT populations, demonstrating improvements in efficacy with generally tolerable safety profiles for patients with HR+, HER2- advanced breast cancer.

Mechanisms of Herb-Drug Interactions Involving Cinnamon and CYP2A6: Focus on Time-Dependent Inhibition by Cinnamaldehyde and 2-Methoxycinnamaldehyde.

Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.

Simultaneous quantification of abemaciclib and letrozole in rat plasma: method development, validation and pharmacokinetic application.

Treatment through a combination of drugs involving cyclin D-dependent kinase inhibitors like abemaciclib and aromatase inhibitor like letrozole proved to be a potential therapeutic regimen and first-line treatment in estrogen receptor-positive breast cancer. In this study, we developed a simple and simultaneous RP-HPLC bioanalytical method for quantifying abemaciclib and letrozole in rat plasma. Abemaciclib and letrozole were separated on Zorbax Eclipse C18 column employing a gradient elution method comprising 10 mM ammonium acetate (pH 5) and acetonitrile as mobile phase. The method was found to have acceptable selectivity, accuracy (97.20-118.17%), precision (1.10-9.39%) and stability in the validation experiment performed as per the US Food and Drug Administration guidelines. The method sensitivity was low at a concentration level of 100 ng/ml. The applicability of the method has been verified through a single-dose oral pharmacokinetic study in rat. The developed method will be useful to quantitate the analytes in rat plasma samples of different preclinical studies including their pharmacokinetic drug-drug interactions in the future. To date, no method has been reported for the quantification of abemaciclib and letrozole simultaneously in any type of biological matrices. Therefore, this study makes a definite significant contribution in the field of bioanalytical research.

Capecitabine-Based Chemoendocrine Combination as First-Line Treatment for Metastatic Hormone-Positive Metastatic Breast Cancer: Phase 2 Study.

BACKGROUND: Preclinical studies have suggested a synergistic effect of tamoxifen and capecitabine in estrogen receptor-positive cell lines. We evaluated the safety and efficacy of first-line chemoendocrine treatment in patients with metastatic breast cancer. Biochemical assessment was performed of serum levels of thymidine phosphorylase enzyme (TP), serum tamoxifen, hydroxytamoxifen, and 5-fluorouracil in relationship to efficacy. PATIENTS AND METHODS: This prospective phase 2 interventional study studied patients with estrogen receptor-positive, HER2- metastatic breast cancer who received either tamoxifen/capecitabine or letrozole/capecitabine as first-line treatment. The dose of capecitabine provided at 2000 mg per day continuously as a fixed dose. RESULTS: Forty women with a median age of 49.3 years were enrolled. For the whole study group, median progression-free survival (PFS) was 10 months and median overall survival (OS) was 23.3 months. The overall response rate was 60% and the clinical benefit rate 82.5%. Progesterone receptor positivity was associated with significantly longer PFS (12 vs. 7 months, P = .021). The most frequent adverse events were palmar-plantar erythrodysesthesia (62.5%), fatigue (62.5%), diarrhea (30%), abdominal pain (12.5%), and constipation (10%). Changes in serum level of TP were not correlated to response to treatment, PFS, or OS. Higher serum levels of tamoxifen and hydroxytamoxifen were correlated with higher response rates and longer PFS but not OS. CONCLUSION: Chemoendocrine treatment is well tolerated, with no evidence of contradictory effects between the combination components. However, the efficacy data need more validation.

Palbociclib Plus Letrozole as First-Line Therapy in Postmenopausal Asian Women With Metastatic Breast Cancer: Results From the Phase III, Randomized PALOMA-2 Study.

PURPOSE: In PALOMA-2, palbociclib plus letrozole significantly improved progression-free survival (PFS) as initial treatment of estrogen receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer. We assessed the benefit of palbociclib plus letrozole in Asians. PATIENTS AND METHODS: Of 666 enrolled postmenopausal women with estrogen receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer (no prior treatment of advanced disease), 95 were Asian. Patients were randomly assigned 2:1 to receive palbociclib plus letrozole or placebo plus letrozole. The primary end point was investigator-assessed PFS. Secondary end points were overall survival, objective response, patient-reported outcomes, pharmacokinetics, and safety. RESULTS: Median PFS was significantly longer in Asian patients who received palbociclib plus letrozole versus placebo plus letrozole (25.7 months [95% CI, 19.2 months to not estimable] v 13.9 months [95% CI, 7.4 to 22.0 months]; hazard ratio, 0.49; 95% CI, 0.27 to 0.87; P = .007). The most common toxicities with palbociclib were hematologic and more frequent among Asians versus non-Asians: neutropenia (any grade, 95.4% v 76.8%; grade 3/4, 89.2% v 62.5%), leukopenia (43.1% v 38.3%; 32.3% v 23.5%), and thrombocytopenia (27.7% v 13.5%; 4.6% v 1.1%). No Asians had febrile neutropenia. Discontinuation rates as a result of adverse events were similar among Asian and non-Asian patients who received palbociclib plus letrozole (10.8% and 9.5%). In Asians, quality of life (QOL) was maintained with no significant differences observed between treatments from baseline in breast cancer-specific QOL and general health status scores. Change from baseline in EuroQol five dimensions index scores was significantly higher with palbociclib plus letrozole (0.013 v -0.069; P = .0132). Geometric mean palbociclib trough concentration values were higher in Asians versus non-Asians (93.8 v 61.7 ng/mL). CONCLUSION: Consistent with the overall study population, the addition of palbociclib to letrozole significantly improved PFS in Asians. Hematologic toxicities were more frequent in Asians versus non-Asians but manageable with early dose modifications while maintaining QOL.

Nintedanib plus letrozole in early breast cancer: a phase 0/I pharmacodynamic, pharmacokinetic, and safety clinical trial of combined FGFR1 and aromatase inhibition.

BACKGROUND: The combined use of a FGFR1 blocker and aromatase inhibitors is appealing for treating breast cancer patients with FGFR1 amplification. However, no pharmacodynamic studies have addressed the effects of this combined target modulation. We conducted a phase 0/I clinical trial in an adjuvant setting, with the goal of obtaining pharmacodynamic proof of the effects of combined aromatase and FGFR1 inhibition and to establish the RP2D for nintedanib combined with letrozole. PATIENTS AND METHODS: Women with early-stage luminal breast cancer were eligible for enrollment in the study. Dose level 1 was nintedanib (150 mg/bid) plus letrozole (2.5 mg/day) administered for a single 28-day cycle (DLT assessment period), followed by a classic 3 + 3 schedule. FGF23 and 17-B-estradiol levels were determined on days 0 and 15; pharmacokinetic parameters were assessed on days 1 and 28. Patients were allowed to continue treatment for 6 cycles. The primary study endpoint was a demonstration of FGFR1 modulation (defined as a 25% increase in the plasma FGF23 level). RESULTS: A total of 19 patients were enrolled in the study (10 in the expansion cohort following dose escalation). At the RP2D (nintedanib 200 mg/bid plus letrozole 2.5 mg/day), we observed a 55% mean increase in the plasma FGF23 level, and 81.2% of the patients had no detectable level of 17-B-estradiol in their plasma (87.5% of the patients treated with letrozole alone). Nintedanib and letrozole displayed a pharmacokinetic interaction that led to three- and twofold increases in their respective plasma concentrations. Most G3 toxic events (5 out of 6: 2 diarrhea and 3 hypertransaminasemia) occurred subsequent to the DLT assessment period. CONCLUSION: Combined treatment with nintedanib (200 mg/bid) plus letrozole (2.5 mg/day) effectively suppressed FGFR1 and aromatase activity, and these respective doses can be used as starting doses in any subsequent trials. However, drug-drug interactions may produce tolerability issues when these drugs are co-administered for an extended time period (e.g., 6 months). Patients enrolled in future trials with these drugs should be carefully monitored for their FGF23 levels and signs of toxicity, and those findings should guide individualized treatment decisions. TRIAL REGISTRATION: This trial was registered at www.clinicaltrials.gov under reg. # NCT02619162, on December 2, 2015.

Plasma and brain pharmacokinetics of letrozole and drug interaction studies with temozolomide in NOD-scid gamma mice and sprague dawley rats.

PURPOSE: The aromatase inhibitor, letrozole, is being investigated in experimental animal models as a novel treatment for high-grade gliomas (HGGs). To facilitate optimal dosing for such studies, we evaluated the plasma and brain pharmacokinetics (PK) of letrozole in NOD-scid gamma (NSG) mice, which are frequently employed for assessing efficacy against patient-derived tumor cells. Furthermore, we evaluated the potential PK interactions between letrozole and temozolomide (TMZ) in Sprague-Dawley rats. METHODS: NSG mice were administered letrozole (8 mg/kg; i.p) as a single or multiple dose (b.i.d, 10 days). Brain tissue and blood samples were collected over 24 h. Letrozole and TMZ interaction study employed jugular vein-cannulated rats (three groups; TMZ alone, letrozole alone and TMZ + letrozole). Intracerebral microdialysis was performed for brain extracellular fluid (ECF) collection simultaneously with venous blood sampling. Drug levels were measured employing HPLC and PK analysis was conducted using Phoenix WinNonlin®. RESULTS: In NSG mice, peak plasma and brain tissue letrozole concentrations (Cmax) were 3-4 and 0.8-0.9 µg/ml, respectively. The elimination half-life was 2.6 h with minimal accumulation following multiple dosing. In the drug interaction study, no PK changes were evident when TMZ and letrozole were given in combination. For instance, peak plasma and brain ECF TMZ levels when given alone were 14.7 ± 1.1 and 4.6 ± 0.6 µg/ml, respectively, and 12.6 ± 2.4 and 3.4 ± 0.8 µg/ml, respectively, when given with letrozole. CONCLUSIONS: These results will guide the optimization of dosing regimen for further development of letrozole for HGG treatment.

Pharmacokinetic Effects and Safety of Olaparib Administered with Endocrine Therapy: A Phase I Study in Patients with Advanced Solid Tumours.

INTRODUCTION: The PARP inhibitor olaparib is efficacious as monotherapy and has potential application in combination with endocrine therapy for the treatment of breast cancer. This phase I study assessed the safety and pharmacokinetic (PK) profiles of olaparib combined with tamoxifen, anastrozole or letrozole in patients with advanced solid tumours. METHODS: During part A, PK profiles were assessed in three consecutive treatment periods: (1) olaparib (tablet) 300 mg bid, days 1-5 followed by a 4-day washout; (2) cohort 1, tamoxifen 60 mg loading dose qd days 10-13, 20 mg qd days 14-26; cohort 2, anastrozole 1 mg qd days 10-19; cohort 3, letrozole 2.5 mg qd days 10-38; (3) as for period 2, with concomitant olaparib 300 mg bid for 5 days. Patients could then enter part B and receive olaparib monotherapy (300 mg bid continuously). Safety was assessed in parts A and B until 12 months after the last patient entered part B. RESULTS: Seventy-nine patients (20.3% with breast cancer) received treatment in part A; 72 completed part A and 69 entered part B. Anastrozole and letrozole had no effect on the PK profile of olaparib and vice versa. Co-administration with tamoxifen produced a modest decrease in exposure to olaparib [geometric least-squares mean (GLSmean) Cmax,ss and AUC0-τ decreased by 20% (90% CI 0.71-0.90) and 27% (0.63-0.84), respectively]. Exposure to tamoxifen was slightly increased when combined with olaparib [GLSmean Cmax,ss and AUC0-τ increased by 13% (1.06-1.22) and 16% (1.11-1.21), respectively]; however, the 90% CI fell within the 0.7-1.43 boundary and there were no changes in exposure to tamoxifen metabolites. The safety profile for olaparib alone and in combination with the antihormonal therapies was acceptable. CONCLUSIONS: The combination of olaparib and either anastrozole, letrozole or tamoxifen was generally well tolerated, with no clinically relevant PK interactions identified. FUNDING: AstraZeneca. CLINICAL TRIAL REGISTRATION: NCT02093351.