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Journal of Chinese Pharmaceutical Sciences
http://www.jcps.ac.cn
Bioequivalence and safety study of letrozole tablet in healthy Chinese postmenopausal women volunteers Yi Liu 1 , Yifan Zhang 2 , Qian Wang 1 , Wei Yang 1 , Xiaoyan Chen 2 , Shan Jing 1 , Libo Zhao 1 , Chunyan Zhang 1 , Lihui Wei 3 , Xiaoping Li 3 , Wanyu Feng 1 , Dafang Zhong 2* , Yi Fang 1* 1. Department of Pharmacy, Peking University People’s Hospital, Beijing 100044, China 2. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China 3. Department of Gynecology and Obstetrics, Peking University People’s Hospital, Beijing 100044, China
Abstract: Letrozole is an orally active aromatase inhibitor for the treatment of postmenopausal women with breast cancer. A singledose, randomized, openlabel, twoway crossover study was designed to compare the bioequivalence and safety of two formulations of letrozole (2.5 mg/tablet), including a newly developed generic formulation (test) and a branded formulation (reference) in a group of healthy Chinese postmenopausal women volunteers under fasting conditions. Blood samples were obtained before study drug administration and at 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 6.00, 8.00, 12.00, 24.00, 48.00, 72.00, 96.00, 144.00, 192.00 and 240.00 h after drug administration. Letrozole levels in plasma were analyzed using a liquid chromatographytandem mass spectrometry (LCMS/MS) method. The safety profile was evaluated by adverse events (AEs) record, and assessed by physical examination, vital signs, spontaneous reporting, and clinical laboratory results. A total of 30 healthy Chinese postmenopausal women were enrolled in this study; however, only 29 subjects were included in bioequivalence assessments due to serious adverse events (SAEs) in 1 subject. The 90% CIs for the lntransformed ratios of Cmax, AUC0–t, and AUC0–∞ were 99.55%–115.17%, 97.35%–103.50%, and 97.29%–103.96%, respectively. All values met the predetermined criteria for assuming bioequivalence. One subject (3.3%) experienced SAE who received the reference formulation and 10 subjects (33.3%) reported a total of 13 mild AEs (4 reported from 4 subjects who received the test formulation, and 9 reported from 6 subjects who received the reference formulation). In this singledose (2.5 mg) study, we found that the test and reference formulations of letrozole tablet met the regulatory definition for assuming bioequivalence in healthy Chinese postmenopausal women. Both formulations were generally well tolerated in the population studied. Chinese Clinical Trials registration number: ChiCTRTRC11001457. Keywords: Letrozole; Bioequivalence; Safety; LCMS/MS; Chinese postmenopausal women CLC number: R945.1
Document code: A
1. Introduction Letrozole, systematically named 4,4'[1H1,2,4triazol 1ylmethylene] bisbenzonitrile (CAS 112809515), is a third generation, competitive and highly specific nonsteroidal inhibitor of the aromatase enzyme system, which could block the conversion of androgens to estrogens in all tissues [1] . Suppression of estrogen synthesis by inhi bition of the aromatase enzyme system has been regarded as a crucial point in the treatment of estrogendependent breast cancers. Letrozole has been indicated for the hormonal treatment of advanced breast cancer in women with natural or artificially induced postmenopausal status, who have disease progression following antiestrogen therapy. Letrozole has been demonstrated to be associated with greater estrogen suppression effect compared with other aromatase inhibitors, such as anastrozole, exemestane, formestane, and aminoglutethimide [2,3] . Letrozole is rapidly and completely absorbed from Received date: 20121217. * Corresponding author. Tel.: 862150800738; 861066583834; Email:
[email protected];
[email protected] doi:10.5246/jcps.2013.02.027
Article ID: 1003–1057(2013)2–190–07
the gastrointestinal tract (mean absolute bioavailability is 99.9%) [4] . Food slightly decreases the rate of absorption (median Tmax: 1 h fasted versus 2 h fed; and mean Cmax: (129±20.3) nmol/L fasted versus (98.7±18.6) nmol/L fed), but the extent of absorption (AUC) is not changed. Plasma protein binding of letrozole is approximately 60%. The concentration of letrozole in erythrocytes is about 80% of that in plasma. The apparent volume of distribution at steady state is about (1.87±0.47) L/kg. Metabolic clearance to a pharmacologically inactive carbinol metabolite is the major elimination pathway of letrozole (CLm = 2.1 L/h), which is relatively slow compared to hepatic blood flow (about 90 L/h). Although the pharmacokinetics (PK) or bioavailability of letrozole has been previously examined in different populations [4–8] , including Chinese healthy men [9–11] , no data are available in healthy Chinese postmenopausal women subjects. The present study was designed to investigate the bioequivalence and safety properties of two formulations of letrozole 2.5 mg tablet, including a newly developed generic formulation (test) and a branded formulation (reference) in healthy Chinese postmenopausal women population. We established a validated LCMS/MS method
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for the determination and quantification of letrozole in human plasma. Our study was necessary before the marketing of this newly developed generic formulation in China.
2. Materials and methods 2.1. Clinical protocols The present study was conducted in accordance with the ethical standards for studies in humans of the Declara tion of Helsinki and its amendments [12] , the International Conference on Harmonisation Guideline for Good Clinical Practice [13] , and the Guideline for Good Clinical Principles recommended by the SFDA of China [14] . The study protocol, informedconsent form, and consent methods were approved by the Ethics Committee of Peking University People’s Hospital prior to subject screening and enrollment. The bioequivalence study in clinical trials was conducted at the laboratory of Peking University People’s Hospital, which was accredited by the National Center for Clinical Laboratories of China. 2.2. Formulations and subjects selection This study evaluated a test formulation (Trozet ® , Fresenius Kabi Oncology Ltd., Beijing, China; Lot No. 871AH00203; Expiration date, Mar. 2013) and a reference formulation (Femara ® , Novartis Pharma Stein AG, Switzerland; Lot No. SO803; Expiration date, Feb. 29 th , 2016) of lerozole at a dose of 2.5 mg/tablet. Healthy Chinese postmenopausal women volunteers participated in this study were enrolled in Phase I Clinical Trial Unit of Peking University People’s Hospital from Dec. 2011 to Apr. 2012. Healthy Chinese postmenopausal women aged older than 40 years old, with BMI between 18 and 25 kg/m 2 , weight at least 50 kg, not having experienced menstruation for at least one year, and having plasma folliclestimulating hormone (FSH) and estradiol levels in the postmenopausal range were eligible for this study. All subjects were assessed by medical history, physical examination, vital signs (body temperature, sitting blood pressure, pulse, and respiration rate), laboratory analysis (hematology, blood biochemistry, urinalysis, and hepatic function), 12lead ECG and chest radiography. Subjects were excluded from the study with any of the reasons, including unable to provide consent; had abnormal findings on ECG or measurement of vital signs; had any clinically significant abnormality based on medical and laboratory analysis; had positive results for hepatitis B, hepatitis C, syphilis or HIV on laboratory testing at screening; smoked; had a history of drug addiction or significant use of alcohol (defined as a history of alcoholism or moderate alcohol use); had consumed alcohol within 48 h before administration of the medicine; had a history of any allergic reactions; or had used drugs known to affect hepatic drug metabolism within 14 d before admini stration of study medication. Subjects were required to
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verify the absence of significant use of drugs or alcohol by both drug scanning and breath alcohol test before checkin. Subjects were also excluded if they had a history of any disease or condition that might compromise body systems (renal, hepatic, endocrine, pulmonary, central nervous, cardiovascular, immunological, dermatological, gastrointestinal or any other body system). Additional exclusion criteria included a history of asthma, known hypersensitivity or idiosyncratic reaction to letrozole or any related drugs. Subjects were instructed to abstain from using any medi cations for ≥14 d prior to and during the study. The subjects had been informed about the details, including the risks and benefits of this study, and provided written informed consent before participating in the study. All subjects were free to withdraw from the study at any time. 2.3. Study procedure This was a randomizedsequence, singledose, openlabel, twoperiod crossover bioequivalence study. Based on published data, the intrasubject coefficients of variation should be approximately 14% for AUC and 16% for Cmax individually [7] . Based on these data, as well as an expected Test/Reference ratio of AUC and Cmax within 0.90 and 1.10, the study was expected to have a power of at least 80% to show bioequivalence with 28 subjects. Considering of possible dropout and withdrawn during the study, we decided to include 30 subjects to ensure the test power. The order of administration of two formulations for each subject during the 2 study periods was determined according to a randomization schedule on the basis of body weight. Equal allocation of subjects to each sequence was ensured. The study personnel involved in the sample analysis were blinded from the randomization code during the entire study. Treatment phases were separated by a minimum 21day washout period, which was 5 to 7 times of the t1/2 of letrozole (~2 d). The subjects were confined to the hospital 11 h before drug administration and for 96 h after administration. Therefore, every subject had to stay for 5 consecutive nights in Phase I Clinical Trial Unit of Peking University People’s Hospital in each treatment period, and had to visit the trial facility at the 144 h (d 7), 192 h (d 9) and 240 h (d 11) after drug administration in each period. After an overnight fast for 10 h, the subjects received the drug in either formulation in sitting posture, taken with (240±2) mL of water. This activity was followed by a mouth check to assess administration compliance. Subjects were instructed to remain in a sitting or ambulatory posi tion for the first 3 h after administration. Thereafter subjects were allowed to engage in normal activities only, but were required to avoid any severe physical exertion. Water intake was permitted 2 h after treatment, and food intake was allowed 4 h after treatment. Alcoholic beverages, intense physical activity, and smoking were not allowed during the study. The standardized lunch and dinner (8 kcal/kg body weight; 55% carbohydrate, 15% protein, and 30% fat) were provided 4 h and 10 h after administration
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during their staying in wards, respectively. During study period out off the wards (about 41 d out of ward between the screening period, washout period, Period I and Period II), subjects need to keep delicate balance diet. Any diet that may cause allergy and highfat diet should be prohibited. For the measurement of letrozole concentrations in the plasma, blood samples (3 mL) were collected from a suitable forearm vein into vacuum heparinized tubes. Samples were obtained before study drug administration (baseline) and at 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 6.00, 8.00, 12.00, 24.00, 48.00, 72.00, 96.00, 144.00, 192.00 and 240.00 h after drug administration. Blood samples after 96.00 h were collected on an ambulatory basis. Before each blood sample was collected, heparin in the heparinlocked catheter was discarded with 0.5 mL of blood, and 3 mL of blood was collected into a vacuum tube. Plasma was immediately separated by centrifugation at 2000 g for 10 min below 10 °C, and was kept in icewater bath before and during separation. The separated plasma was then transferred to prelabeled polypropylene tubes in 2 aliquots [around 0.4 mL in first lot (0.8 mL for the predose sample) and the remaining blood in second lot] and stored at –80 °C until LCMS/MS analysis. After administration of the other formulation, blood samples were drawn and analyzed in the exact same manner. 2.4. Drug analysis In this study, a simple and validated method was used for the determination of letrozole in plasma by an LCMS/MS method. Letrozole (purity, 99.9%, Batch No. 1204100A1) and d4Letrozole (internal standard, IS, purity, 99.8%, Batch No. 1040005A1) were purchased from TLC Phar machem., Inc. (Ontario, Canada). HPLCgrade acetonitrile and methanol were supplied by Sigma (St. Louis, MO, USA). Ammonium acetate (HPLCgrade) was purchased from Fluka Company (Germany). The LC system (Agilent 1100, Waldbronn, Germany) was equipped with a quaternary pump (G1311A), a vacuum degasser (G1322A), a thermo stated column oven (G1316A), and an autosampler (G1367A). Chromatographic separation was achieved with a 100 mm×4.6 mm, 5.0 μm Capcell PAKC18 column (Shiseido Co., Ltd., Tokyo, Japan) protected with a 3.0 mm×4.0 mm, 5.0 μm C18 catridge guard (Phenomenex Inc., Torrance, California, USA). The mobile phase was composed of a mixture of methanol and 10.0 mM ammonium acetate (65:35, v/v) at a flow rate of 0.6 mL/min. MS detection was performed using a Thermo Electron Finnigan TSQ Quantum Ultra triple quadrupole instrument (San Jose, CA, USA) operated in negative ionization mode. The ionspray voltage was set at 3500 V and the source temperature was maintained at 320 °C. Nitrogen was used as the sheath gas (35 Arb) and auxiliary gas (10 Arb) to assist with nebulization and dissolution. For collisioninduced dissociation (CID), argon was used as the collision gas at a pressure of 1.2 mTorr. The optimized collision energies
(CEs) for transitions of letrozole and IS were both set at 25 eV. Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 284→(215+242) for letrozole and m/z 288→(219+246) for IS, respectively, with a scan time of 0.2 s per transition. Data acquisition and processing were powered by the Agilent ChemStation and Finnigan Xcalibur software 2.0.7 packages. Sample preparation involves a simple onestep protein precipitation without the requirement of an evaporation step. Briefly, frozen plasma samples from the subjects were thawed to room temperature. After vortexing, a 12.5 μL aliquot of the IS solution, 25 μL methanol–water (1:1, v/v) and 200 μL acetonitrile were added to 100 μL of plasma sample. The mixture was vigorously vortexed for 1 min and centrifuged at 11 000 g for 5 min. Finally, 20 μL supernatant was injected into the LC–MS/MS system for analysis. 2.5. Tolerability assessments Two physicians were available within the clinical facility when the subjects were housed (from checkin to checkout) and during ambulatory blood sampling in each period. Subjects might contact with investigators at any time during the study period. Tolerability of the drug in two formulations was determined by monitoring vital signs (body temperature, sitting blood pressure, pulse, and respiration rate) at baseline (before dosing), at approxi mately 2, 6, 12 h after administration, and at completion of the study. Physical examinations were performed at baseline and at completion of the study. Laboratory tests including hematology, blood biochemistry and urinalysis and 12lead ECGs were also performed at baseline, approximately 24 h after administration and completion of the study. Health status of subjects was asked at the time of clinical examination and during ambulatory samplings in each period. AEs were assessed at the time of each blood draw using direct observation, spontaneous reporting, and nonspecific questioning. Any undesirable sign, symptom, or medical conditions occurring after the study were recorded regardless of the suspected relationship to the study drug. AEs were graded as mild, moderate, or severe, and their relationship to the study drug was determined by the physicians as not related, probably not related, uncertain, possibly related, probably related, or definitely related. Important medical events that may result in death, life threatening, requiring hospitalization, leading to disability, or requiring medical intervention to prevent permanent impairment or damage may be considered as SAEs drug experience based on appropriate medical judgment. All AEs and SAEs were recorded in the source data and casereport form, and their relationship to the study drug was determined by the study physicians who were blinded to the randomization schedule. 2.6. Pharmacokinetic and statistical analysis PK analysis was conducted with a noncompartmental
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method using Phoenix WinNonlin version 6.1 (Pharsight Corporation, Mountain View, California). Cmax and Tmax were obtained directly from the concentrationtime curves of letrozole. PK properties were analyzed by noncompart mental PK data analysis based on an equation described by Shumakerusing PKCALC, a widelyused computer program for PK analysis [15] . AUC0−t was calculated according to the linear trapezoidal rule. AUC0–∞ was calculated as AUC0−t + C t/λz , where C t was the final concentration and λz was the slope of the linear regression of the lntransformed concentrationtime curve. The t1/2 of letrozole in plasma was calculated as 0.693/λz [16] . Descriptive statistics, including mean and SD, were used to summarize the PK data for two formulations. ANOVA was performed on the lntransformed PK parameters (AUC0−t, AUC0–∞, and Cmax). The model of ANOVA had fixed factors for sequence, treatment, and period and subject within sequence. The Wilcoxon signed rank test was used for the nonparametric analysis to determine differences in Tmax. As proposed by FDA, if the parametric 90% CI fell within a predetermined range of 80% to 125%, the two formulations were considered to meet the regulatory criteria for bioequivalence.
of quantitation (LLOQ) of this assay was 0.4 ng/mL and the intra and interday precisions were 5.0% and 12.9%, with relative error to be 7.4%. The intra and interday precision values for QC samples were 3.4%–5.5% and 1.6%–8.9%, respectively, with relative errors from –0.9% to 4.4%. The mean extraction recoveries were 70.7%–76.3%, and 79.6% for letrozole and IS, respectively. The analyte and IS were stable in an auto sampler at room temperature for ≥6 h, after preextraction at room temperature for 24 h, after three freezethaw cycles and after 116 d of storage at –20 °C and –80 °C, with relative standard deviations (RSDs) of 1.1%–1.8%, 2.4%–6.1%, 2.2%–4.1%, 2.8%–5.0% and 2.4%–2.5%, respectively. The accuracy and precision for dilution samples at 150 ng/mL were –3.5% and 2.0%, respectively. Dilution samples were diluted 5fold with blank plasma before sample analysis. Taken together, these results indicate that the established LCMS/MS method was simple, sensitive, accurate, and rapid, which could be used in bioequivalence study according to international guidelines [18,19] . The QC samples and the calibration curves were analyzed with processed test samples at intervals in each run. The results of the QC samples provided the basis for accepting or rejecting the run.
3. Results
3.2. Pharmacokinetic properties
3.1. Drug analysis
A total of 30 healthy Chinese postmenopausal women volunteers were enrolled in this study. One subject withdrew from the study because of SAE. Therefore, 29 subjects were included in the PK and bioequivalence analyses. All 30 subjects were included in the tolerability assessment. Demographic characteristics of the subjects were summarized in Table 1. The mean letrozole plasma concentrationtime curves after oral administration of single 2.5 mg tablet of formu lations in 29 healthy Chinese postmenopausal women volunteers are shown in Figure 1. The primary PK parameters of letrozole are listed in Table 2.
Table 1. Baseline demographic characteristics (Mean, SD) Safety study (n = 29)
Age (years)
54.8 (4.7)
54.9 (4.7)
Weight (kg)
56.6 (4.6)
56.4 (4.6)
Height (cm)
158.9 (4.9)
158.7 (4.8)
BMI (kg/m 2 )
22.4 (1.3)
22.4 (1.4)
Table 2. Comparison of PK parameter of letrozole Parameter
Test (Mean, SD)
Reference (Mean, SD)
Cmax (ng/mL)
47.2 (11.0)
44.8 (13.6)
Tmax (h)
1.49 (0.72)
1.66 (0.97)
AUC0–t (ng∙h /mL)
1840 (602)
1820 (596)
AUC0–∞ (ng∙h /mL)
2060 (820)
2040 (815)
t1/2z (h)
63.3 (25.2)
62.0 (25.8)
CL/F (L/h)
1.43 (0.62)
1.43 (0.58)
60 Plasma letrozole concentration (ng/mL)
Bioequivalence study (n = 30)
50 40 30 20
60 Plasma letrozole concentration (ng/mL)
The analytical method during validation process has been demonstrated to be suitable for the determination of letrozole in human plasma for bioequivalence study. This process in our study conformed strictly to the FDA guidance for the validation of bioanalytical methods [17] . Under the above conditions, the calibration curves were linear over the range of letrozole concentrations from 0.4 to 50 ng/mL in human plasma with a coefficient of correlation (r 2 ) at 0.9990. No peaks interfering with quantitation were observed throughout the validation process. The lower limit
Test Reference
50 40 30 20 10 0 0
1 2 3 t (h)
4 5
6
10 0 0 40 80 120 160 200 240 t (h)
Figure 1. Meanplasma concentrationtime curves of letrozole following a single 2.5 mg oral dose of a test or reference formulation (n = 29). The LLOQ was 0.4 ng/mL.
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3.3. Bioequivalence evaluation ANOVA was performed on the lntransformed data of Cmax, AUC0–t and AUC0–∞. On ANOVA, no sequence or formulation effects were observed for any PK properties. However, a significant period effect was observed for Cmax (P
