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Bioavailability and bioequivalence of two oral formulations of alendronate sodium 70 mg: An open-label, randomized, two-period crossover comparison in healthy Korean adult male volunteers
Clinical Therapeutics/Volume 31, Number 5, 2009
Bioavailability and Bioequivalence of Two Oral Formulations of Alendronate Sodium 70 mg: An Open-Label, Randomized, Two-Period Crossover Comparison in Healthy Korean Adult Male Volunteers Si-Youn Rhim, MD, PhD1; Jin-Hee Park, PhD2,3; Yoo-Sin Park, PhD2,3; Min-Ho Lee, MD, PhD4; Dong-Sun Kim, MD, PhD4; Leslie M. Shaw, PhD5; Seok-Chul Yang, MD, PhD6; and Ju-Seop Kang, MD, PhD2,3 1Division
of Pediatric Surgery, Department of Surgery, College of Medicine, Hanyang University, Seoul, Republic of Korea; 2Department of Pharmacology and Clinical Pharmacology Laboratory, College of Medicine, Hanyang University, Seoul, Republic of Korea; 3Institute of Biomedical Science, Hanyang University, Seoul, Republic of Korea; 4Department of Internal Medicine, College of Medicine, Hanyang University, Seoul, Republic of Korea; 5Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; and 6Department of Internal Medicine, College of Medicine, Seoul National University, Seoul, Republic of Korea ABSTRACT Background: Alendronate sodium is a bisphosphonate drug used to treat and prevent osteoporosis and several other bone diseases. A new formulation has been developed and is currently awaiting regulatory approval, pending findings on bioequivalence. Objectives: The aims of the present study were to compare the bioavailability and pharmacokinetic (PK) properties, and to determine the bioequivalence, of a test and reference formulation of alendronate sodium 70 mg in a healthy Korean adult male population. Methods: This open-label, randomized, 2-sequence, 2-period crossover study was carried out at Hanyang University Medical Center (Seoul, Republic of Korea). Healthy Korean adult male volunteers were randomly assigned to receive a single 70-mg dose of the test or reference formulation of alendronate sodium, administered with 240 mL of water, followed by a 7-day washout period and administration of the alternate formulation. The study drugs were administered after a 12-hour overnight fast. Serial blood samples were collected and adverse events were monitored by a clinical investigator via observation, personal interview, and vital signs (blood pressure, heart rate, and body temperature) over a 7-hour period (at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, and 7 hours) after drug administration. Plasma alendronate sodium concentrations were determined using a validated highMay 2009
performance liquid chromatographic–postcolumn fluorescence derivatization method, with visible detection in the range of 2 to 100 ng/mL and lower limit of quantification set at 2 ng/mL. PK properties, including AUC0–t, AUC0–∞, Cmax, Tmax, t1/2, and the elimination constant (ke), were determined using noncompartmental analysis. The formulations were considered bioequivalent if the 90% CI ratios for Cmax and AUC were within the predetermined interval of 80% to 125%, the regulatory definition set by the US Food and Drug Administration (FDA). Results: Twenty-three healthy male volunteers (mean [SD] age, 23.5 [2.0] years [range, 19–28 years]; height, 175.9 [5.4] cm [range, 162.0–185.0 cm]; and weight, 71.2 [9.5] kg [range, 61–96 kg]) were included in the study. No period or sequence effects were detected. The 90% CIs for the corresponding ratios of AUC0–t, AUC0–∞, and Cmax were 84.97 to 114.47, 86.09 to 115.59, and 82.37 to 110.71, respectively. Additionally, the mean (range) of Tmax was 1.09 hours (0.5–2.0 hours), and the mean (SD) of t1/2 and ke were 2.04 (0.97) hours and 0.34 (0.71) hour, respectively. The values for the test and reference formulations Accepted for publication March 11, 2009. doi:10.1016/j.clinthera.2009.05.001 0149-2918/$ - see front matter © 2009 Excerpta Medica Inc. All rights reserved.
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Clinical Therapeutics were within the FDA bioequivalence definition interval of 80% to 125%. No adverse events were reported in this study. Conclusions: Single doses of these formulations of alendronate sodium 70 mg met the criteria for bioequivalence. No statistically significant differences in AUC0–t, AUC0–∞, and Cmax were found in this healthy Korean adult male population. (Clin Ther. 2009;31: 1037–1045) © 2009 Excerpta Medica Inc. Key words: alendronate sodium, bioequivalence test, pharmacokinetics, HPLC–FLD method.
by the ingestion of food, especially products containing calcium. The mean plasma t1/2 is rapid (0.5–2 hours) due to rapid uptake of 20% to 60% of the absorbed fraction into the bone. The remainder is excreted into the urine. The t1/2 in bone is long (~300 days in rats and ≥1000 days in dogs), and the release of bisphosphonates occurs only after resorption of bone into which the compounds have been taken up.8 The aims of the present study were to compare the bioavailability and PK properties, and to determine the bioequivalence of a test and reference formulation, of alendronate sodium 70 mg in a healthy Korean adult male population.
INTRODUCTION The regulatory bioequivalence of 2 formulations of the same drug, particularly conventional drug products, can be determined based on the absence of significant differences in primary pharmacokinetic (PK) properties of bioavailability, such as the rates of absorption and elimination (Cmax and Tmax), and the extent of absorption or total amount of drug absorbed in the body (AUC).1 Alendronate sodium (sodium [4-amino-1-hydroxybutylidene] bisphosphonate) is indicated for the treatment of osteoporosis, hypercalcemia, and Paget’s disease of bone.2 Alendronate sodium is selectively accumulated in the bone, and its oral absorption is <1% of the administered dose in the fasted state.3 When a patient is undergoing treatment with alendronate sodium, normal bone tissue develops, and the drug is deposited in the bone matrix in a pharmacologically inactive form.2 For optimal action, sufficient calcium and vitamin D are needed in the body to promote normal bone development.2 The analytical methods used in the determination of alendronate sodium have been reported in previous studies.3,4 However, because alendronate sodium is not metabolized and is renally excreted, the number of methods for use with serum or plasma is limited.5 Consequently, many PK studies have relied on urine, rather than plasma, in the determination of alendronate sodium concentrations.6,7 These analytical methods, however, require complex derivatization processes and labor-intensive preparation as part of the sample cleanup steps. Very long run times are also required, mainly due to the presence of derivative byproducts along with the derived analysis. Bisphosphonates, including alendronate sodium, are not metabolized and intestinal absorption is poor (<10%).2,8 Furthermore, the intestinal absorption rate is reduced 1038
SUBJECTS AND METHODS Subjects Healthy Korean adult male subjects were eligible to be enrolled in the study. Inclusion criteria consisted of unremarkable results on medical history, physical examination, and clinical laboratory tests (vital signs, serum chemistry and hematology, and urinalysis). Subjects with HIV or hepatitis B were excluded from the study. Concurrent medications, including over-thecounter drugs, and the consumption of alcohol were not allowed from 2 weeks before administration of the first dose until the end of the study period. Subjects were informed about the aims and the risks of the study, and written informed consent was obtained from all volunteers before screening.
Study Design The study protocol was approved by the institutional review board at Hanyang University Medical Center, Seoul, Republic of Korea. The study was carried out at Hanyang University Medical Center and was performed in accordance with the revised Declaration of Helsinki9 and the Good Clinical Practice guideline.10 The study was conducted in an open-label, randomized, 2-sequence, 2-period crossover design with a 7-day washout period. Using a table of random numbers, subjects were assigned to receive a single 70-mg dose of either the test (expiration date: 02/20/2010) or reference* (expiration date: 05/15/2011) alendronate sodium formulation, followed by the washout period, and then the alternate formulation. Both drug *Trademark: Fosamax ® (MSD Korea [Merck] Ltd., Seoul, Republic of Korea).
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S.-Y. Rhim et al. formulations were supplied free of charge by their respective pharmaceutical company.
Drug Administration Protocol and Blood Sample Collection Subjects were admitted to the hospital at 5 pm the evening before administration of the first dose of study drug and received a standardized regular-calorie meal (920 ± 20 kcal: 65% carbohydrate; 20% protein; and 15% fat). The drug administration protocol in the study was similar to the dosing instructions for alendronate sodium, which require that the patient be in the fasting state and the drug be administered with water. Therefore, after a 12-hour overnight fast, subjects received a single 70-mg dose of either formulation (test or reference) with 240 mL of tap water. Food and beverage intake (other than water, which was allowed 2 hours after study drug administration) was not permitted until 4 hours after study drug administration. Subjects received a standardized lunch and dinner according to a regular time schedule. Throughout the study, subjects were prohibited from consuming alcohol or beverages containing xanthine derivatives, engaging in intense physical activity, and smoking. The subjects remained under continuous medical supervision at the study site to monitor for adverse events. Venous blood samples (9 mL) were collected in EDTA-coated tubes (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, New Jersey) at baseline (before study drug administration) and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, and 7 hours after study drug administration. The vein was maintained through a heparin-lock catheter (Korea Vaccine Co., Ltd., Seoul, Republic of Korea). Plasma was immediately separated at 3000 rpm (1000g) for 10 minutes in a refrigerated centrifuge and was transferred to properly labeled 3-mL microfuge tubes (Eppendorf, Hamburg, Germany) and stored at −70°C until analysis.
HPLC With Postcolumn Fluorescence Derivatization and Method Development Plasma concentrations of alendronate sodium were determined using a modified high-performance liquid chromatographic–postcolumn fluorescence derivatization (HPLC–FLD) method11 that was validated using established international guidelines.1 In the previous method, a 4.6 × 150-mm internal diameter (ID), 5.0-μm particle size column, 1.5 mL/min flow rate, and temperature of 35°C were used.11 However, the May 2009
modifications in the present study included a thinner separation column (2.0 × 150-mm ID), slower flow rate (0.35 mL/min), and lower temperature (25°C). All solvents were HPLC grade; other chemicals and reagents were analytical grade. Alendronate sodium (molecular weight [mw], 325.124 kDa; purity, 98.96%; Eczacibasi Pharmaceuticals Manufacturing Co., Istanbul, Turkey) and internal standard (IS) pamidronate (mw, 235.06992 kDa; purity, 99.00%; Calbiochem Co., an affiliate of Merck KGaA, Darmstadt, Germany) were provided by Schnell Korea Pharma Co., Ltd., Seoul, Republic of Korea. The HPLC–FLD conditions were optimized for the column, mobile phase, and sample preparation. The HPLC–FLD (Varian Inc., Palo Alto, California) system consisted of a pump (ProStar 230, Varian Inc.), an autosampler (ProStar 410, Varian Inc.), a fluorescence detector (RF-10AXL, Shimadzu Corporation, Kyoto, Japan), and peak simple data system software version 6.3 (Star LC Workstation, Varian Inc.). The separation of compounds was performed in an analytical column (Capcell Pak C18, Shiseido Company, Ltd., Tokyo, Japan; 2.0 × 150-mm ID, 5.0-μm particle size) and eluted with a mobile phase consisting of mixed organic solution (acetonitrile:methanol = 1:1 vol/vol, solvent A) and buffer (25-mM citric acid: 25-mM sodium pyrophosphate = 1:1 vol/vol, without pH adjustment, solvent B). Run times for solvents A and B were 32:68 vol/vol for 0 to 9 minutes, 60:40 vol/ vol for 9 to 16 minutes, and 32:68 vol/vol for 16 to 24 minutes. The analyses were conducted at 25°C, and a flow rate of 0.35 mL/min, with excitation and emission wavelengths of 260 nm and 310 nm, respectively, and the injection volume was 50 μL. The column was equilibrated for 1 minute before each subsequent sample was injected. No significant chromatographic interferences at the retention times of alendronate sodium or IS were observed (Figure 1). The peak area was measured for calculation of the peak area ratio of alendronate sodium to IS, and the concentrations were calculated. Each analytical process required ~25 minutes. The calibration curves were validated over a concentration range of 5 to 100 ng/mL for alendronate sodium in human plasma, visible detection in the range of 2 to 100 ng/mL, and lower limit of quantification set at 2 ng/mL using 1.0 mL of plasma.
Sample Preparation and Processing The procedures described here were applied to subject samples as well as extraction of samples for cali1039
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B 3
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Volts (units)
Volts (units)
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Figure 1. Chromatograms (A) of blank plasma, (B) with internal standard (IS) (12.5 μg/mL), (C) with alendronate sodium (50 ng/mL) and IS (12.5 μg/mL), and (D) of human plasma taken 1 hour after single oral dose of alendronate sodium 70 mg spiked with IS (12.5 μg/mL).
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S.-Y. Rhim et al. bration curve and quality control (QC) processes. By adding the distilled water, the concentration of the stock solution of alendronate sodium was 1 mg/mL. Separate solutions were prepared for the calibration standards and QC samples. To obtain working solutions of 1000, 500, 200, 100, 50, and 20 ng/mL, 300 μL of these solutions was diluted immediately before use with purified water. Additionally, to obtain 100, 50, 20, 10, 5, and 2 ng/mL for each analyte, the solutions were mixed with 2.7 mL of blank serum. The sample was prepared by mixing 3.0 mL of serum and 100 μg/mL of the IS solution in a tapered tube and adding 6% trichloroacetic acid solutions, followed by vortex mixing for 1 minute. Then, a mixture of 0.1 M KH2PO4 (0.2 mL), 1 M CaCl2 (0.2 mL), and 1 M NaOH (0.4 mL) was successively added. The sample was stirred (5Roll Mixer, Hyunil Lab-Mate Co., Seoul, Republic of Korea) for 10 seconds and centrifuged (VS-6000, Vision Co., Seoul, Republic of Korea) at 3000 rpm (1000g) for 5 minutes, and the precipitate was carefully transferred to a tapered tube without an upper solution layer. After removal of the liquid phase, the deposition was dissolved completely in 0.5 mL of 0.2 M acetic acid and 3 mL of purified water was added. The precipitation was repeated 3 times. The resulting precipitate was dissolved in 1 mL of 0.2 M acetate buffer (pH 6.0) and 2 mL of acetic acid, diluted with 3 mL of purified water. The sample was loaded on a Bond Elute DEA cartridge (Varian Inc.) and washed twice with 0.5 mL of water. The cartridge was then eluted with 1 mL of 0.2 M sodium citrate, and 270 μL of the eluent was taken for derivatization. The derivatization procedure involved adding 0.2 mL of 1 M sodium carbonate buffer (pH 11.9) to 270 μL of the eluent, and adding 0.2 mL of 9-fluorenyl methyl chlorformate solution (1 mg in 4 mL of acetonitrile). After 3 minutes, 0.2 mL of 1 M citric acid was added to stop the reaction and 50 μL of the sample was injected into the HPLC–FLD system. The calibration standards and blanks were freshly prepared in duplicate for each assay and were extracted together with plasma and QC samples. To assess intra- and interassay precision, accuracy, and recovery for method validation, QC samples were prepared by spiking control human plasma with 2 (lower limit of quantification), 5, 10, 20, 50, and 100 ng/mL of alendronate sodium. Intraday precision and accuracy were determined by repeated analyses of the group of standards on 1 batch (n = 5). Interday precision and accuracy were deterMay 2009
mined by repeated analyses on 5 consecutive days (n = 5 series per day). One QC sample set (consisting of 4 alendronate sodium concentrations [2, 5, 50, and 100 ng/mL]) was analyzed in a preparation batch of 50 samples using the same equipment, reagents, and staff in a 24-hour period.
Tolerability Adverse events (nausea, vomiting, diarrhea, abdominal bloating, gastric pain, musculoskeletal and joint pain, headache, and fatigue) were monitored by a clinical investigator (D.-S.K.) via observation, personal interview, and vital signs (blood pressure, heart rate, and temperature) over a 7-hour period after study drug administration. All adverse events were recorded on the clinical record form per subject up to 1 week after the study.
Pharmacokinetic and Statistical Analysis Noncompartmental analysis for deriving PK properties was performed using PK Solutions version 2.0 (Summit Research Services, Montrose, Colorado). The actual times of blood sample collection were used for PK analyses of alendronate sodium. Cmax and Tmax were obtained directly from the plasma concentration– time curve after a single 70-mg dose of alendronate sodium (test or reference) was administered (Figure 2). AUC0–t was calculated using the linear trapezoidal method. AUC0–∞ was calculated as AUC0–t and the ratio of the Ct/ke, where Ct was the last measured concentration and ke was the slope of the linear regression of the log-transformed plasma concentration time in the terminal phase. The plasma elimination t1/2 was calculated as 0.693/ke. AUC0–t, AUC0–∞, and Cmax were the primary variables used to assess bioequivalence between the test and reference formulations. Bioequivalence between the 2 formulations was determined by calculating the 90% CIs for AUC0–t, AUC0–∞, and Cmax values for the test and reference formulations, using log-transformed data. A power analysis, with a power of 80% to detect a difference, indicated that a sample of 23 adult volunteers was of adequate size to detect statistically significant differences between test and reference formulations. A 2-way analysis of variance (ANOVA) was conducted to test for period, sequence, and group effects. Significance was set at P < 0.05. To access bioequivalence assessment between 2 formulations, in accor1041
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Test Reference
Plasma Concentrations of Alendronate (ng/mL)
100 90 80 70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
7
Time (h) Figure 2. Mean (SD) plasma concentration–time plots of alendronate sodium 70 mg after a single oral dose of test and reference formulations in healthy Korean adult male volunteers (N = 23). No significant differences were observed.
dance with current US Food and Drug Administration guidelines,1,12,13 the formulations were considered bioequivalent if the 90% CIs for AUC0–t, AUC0–∞, and Cmax were within the predetermined range of 80% to 125%.
RESULTS Thirty-eight subjects were recruited for participation, and a total of 24 subjects met all study criteria and were enrolled; 1 subject, however, withdrew before study drug administration. Therefore, a total of 23 healthy male volunteers (mean [SD] age, 23.5 [2.0] years [range, 19–28 years]; height, 175.9 [5.4] cm [range, 162.0–185.0 cm]; and weight, 71.2 [9.5] kg [range, 61–96 kg]) completed both treatment periods of the study with no protocol violations.
Alendronate Sodium Quantification in Plasma The analytical method for alendronate sodium quantification in plasma samples found good specificity, sensitivity, linearity, precision, and accuracy over the entire range of clinically significant and therapeuti1042
cally achievable plasma concentrations, thereby enabling its use in bioequivalence trials. The linearity was observed within the range of 2 to 100 ng/mL (alendronate sodium; y = 0.0096 ∙ x − 0.0006, r2 = 0.9999; x = plasma concentration, y = peak area ratio). The intraday precision ranged from 0.23% to 1.90%, whereas intraday accuracy ranged from 98.1% to 101.3%. Interday precision ranged from 0.74% to 1.78%, whereas interday accuracy ranged from 98.0% to 102.1%. The extraction recovery of alendronate sodium was determined in its QC samples in 5 replicates. The mean absolute recovery was 90.26%, whereas the relative recovery ranged from 96.4% to 103.4%.
Pharmacokinetic Properties The mean (SD) plasma concentration–time profiles of the test and reference formulations of alendronate sodium are shown in Figure 2. Both formulations were readily absorbed from the gastrointestinal tract, and alendronate sodium was measurable at the first sampling time (0.25 hour) in the 23 volunteers. At the first sampling time, alendronate sodium apVolume 31 Number 5
S.-Y. Rhim et al. peared in plasma in both groups; mean Cmax values were 37.17 and 38.83 ng/mL, and Tmax values were 1.09 and 1.24 hours for the test and reference formulations, respectively. The plasma concentrations gradually decreased, with a mean terminal t1/2 of 1.85 and 2.04 hours for the test and reference formulations, respectively, and then decreased rapidly but were detected up to 7 hours later. The mean alendronate sodium plasma concentration was 4.36 ng/mL. As shown in Table I, after adjusting for weight, the mean AUC0–t values were 1.38 and 1.40 ng ∙ h/mL/kg for the test and reference formulations, respectively, and the mean AUC0–∞ values were 1.54 and 1.55 ng ∙ h/mL/kg for the test and reference formulations, respectively. The ANOVA for the ratio of AUC0–t, AUC0–∞, and Cmax values for the test and reference formulations, using log-transformed data, are shown in Table II. ANOVA assessment found no significant product, group, or period effect in the present study. The 90% CIs for the ratio of AUC0–t, AUC0–∞, and Cmax values (84.97–114.47, 86.09–115.59, and 82.37–110.71, respectively) for the test and reference formulations were within the established regulatory interval of 80% to 125%.
Tolerability Both tablet formulations of alendronate sodium were well tolerated by all volunteers. No adverse events were reported during the study or up to 1 week after the study.
DISCUSSION Because alendronate sodium systemically disappears rapidly (0.5–2 hours after oral administration),8 resulting in low plasma bioavailability (0.7%) in the fasted state, its PK properties have been previously reported based on urinary excretion data.14 Moreover, the oral bioavailability of this drug is <2% in all species including humans.15 Thus, few studies have been conducted on the plasma concentrations of alendronate sodium after oral administration.16 Therefore, using a validated HPLC–FLD method with lower limit of quantification of 2 ng/mL, the present study was designed to examine the PK properties in plasma after a 70-mg oral dose of alendronate sodium. In a previous study assessing PK properties after oral administration of alendronate 70 mg, Cmax, Tmax, t1/2, and AUC0–t were 40.94 ng/mL, 1.0 hour, 1.67 hour, and 118.55 ng ∙ h/mL, respectively, in 4 healthy Koreans.11 Although the alendronate formulations in the present
Table I. Pharmacokinetic (PK) properties (weight corrected) of alendronate sodium formulations (N = 23).* Data are mean (SD) unless otherwise specified. PK Parameter
Test
Reference†
AUC0–t OHtIN-LH
AUC0–∞ OHtIN-LH
Extrapolation (AUCt–∞ tLH"6$0–∞ tLH ‡
0.16 (0.10)
0.16 (0.08)
Cmax OHN-tLH
Tmax, mean SBOHF
I
o
o
t1/2 I
ke I
ke = elimination rate constant. * No significant between-treatment differences were found. † Trademark: Fosamax ® (MSD Korea [Merck] Ltd., Seoul, Republic of Korea). ‡ Weight-corrected ratio of AUC t–∞ to AUC0–∞.
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Clinical Therapeutics
Table II. Analysis of variance of 2 oral formulations of alendronate sodium 70 mg after single-dose administration. 2.
Test: Reference* Ratio
90% CI
AUC0–t
o
AUC0–∞
o
Cmax
o
PK Parameter
PK = pharmacokinetic. * Trademark: Fosamax ® (MSD Korea [Merck] Ltd., Seoul, Republic of Korea). 6.
study are different from that in the previous study, after adjusting for mean (SD) weight (71.2 [9.5] kg), similarities were found in the present data in Cmax (38.83 ng/mL), Tmax (1.24 hour), t1/2 (1.85 hour), and AUC0–t (99.68 ng ∙ h/mL).
Limitations This study had some limitations because it included only males, healthy volunteers, and single-dose administration. A larger scale study including women is needed to assess the PK properties of alendronate sodium.
CONCLUSIONS In this small study in healthy male Koreans, no statistically significant differences in AUC0–t, AUC0–∞, and Cmax were found between the test and reference formulations of alendronate sodium. The single 70-mg dose of these formulations met the regulatory criteria for bioequivalence in this fasting, adult volunteer population.
ACKNOWLEDGMENT This work was supported by the cluster research fund of Hanyang University (HY-2008-C).
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64%FQUPG)FBMUIBOE)VNBO4FSWJDFT 'PPEBOE%SVH Administration (FDA), Center for Drug Evaluation BOE3FTFBSDI $%&3 (VJEBODFGPSJOEVTUSZ#JPBWBJMB CJMJUZ BOE CJPFRVJWBMFODF TUVEJFT GPS PSBMMZ BENJOJTUFSFE
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S.-Y. Rhim et al. UXP BMFOESPOBUF PODFXFFLMZ GPSmulations. Arzneimittelforschung. 2006; 56:84–89. 1PSSBT "( )PMMBOE 4% (FSU[ #+ Pharmacokinetics of alendronate. Clin Pharmacokinet o -JO+) 3VTTFM( (FSU[#1IBSNBDPLJnetics of alendronate: An overview. Int J Clin Pract. 1999;101(Suppl):18–26.
Address correspondence to: Ju-Seop Kang, MD, PhD, Department of Pharmacology & Institute of Biomedical Science, College of Medicine, Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea. E-mail: [email protected] May 2009
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Bioavailability and Bioequivalence of Two Oral Formulations of Alendronate Sodium 70 mg: An Open-Label, Randomized, Two-Period Crossover Comparison in Healthy Korean Adult Male Volunteers Si-Youn Rhim, MD, PhD1; Jin-Hee Park, PhD2,3; Yoo-Sin Park, PhD2,3; Min-Ho Lee, MD, PhD4; Dong-Sun Kim, MD, PhD4; Leslie M. Shaw, PhD5; Seok-Chul Yang, MD, PhD6; and Ju-Seop Kang, MD, PhD2,3 1Division
of Pediatric Surgery, Department of Surgery, College of Medicine, Hanyang University, Seoul, Republic of Korea; 2Department of Pharmacology and Clinical Pharmacology Laboratory, College of Medicine, Hanyang University, Seoul, Republic of Korea; 3Institute of Biomedical Science, Hanyang University, Seoul, Republic of Korea; 4Department of Internal Medicine, College of Medicine, Hanyang University, Seoul, Republic of Korea; 5Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; and 6Department of Internal Medicine, College of Medicine, Seoul National University, Seoul, Republic of Korea ABSTRACT Background: Alendronate sodium is a bisphosphonate drug used to treat and prevent osteoporosis and several other bone diseases. A new formulation has been developed and is currently awaiting regulatory approval, pending findings on bioequivalence. Objectives: The aims of the present study were to compare the bioavailability and pharmacokinetic (PK) properties, and to determine the bioequivalence, of a test and reference formulation of alendronate sodium 70 mg in a healthy Korean adult male population. Methods: This open-label, randomized, 2-sequence, 2-period crossover study was carried out at Hanyang University Medical Center (Seoul, Republic of Korea). Healthy Korean adult male volunteers were randomly assigned to receive a single 70-mg dose of the test or reference formulation of alendronate sodium, administered with 240 mL of water, followed by a 7-day washout period and administration of the alternate formulation. The study drugs were administered after a 12-hour overnight fast. Serial blood samples were collected and adverse events were monitored by a clinical investigator via observation, personal interview, and vital signs (blood pressure, heart rate, and body temperature) over a 7-hour period (at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, and 7 hours) after drug administration. Plasma alendronate sodium concentrations were determined using a validated highMay 2009
performance liquid chromatographic–postcolumn fluorescence derivatization method, with visible detection in the range of 2 to 100 ng/mL and lower limit of quantification set at 2 ng/mL. PK properties, including AUC0–t, AUC0–∞, Cmax, Tmax, t1/2, and the elimination constant (ke), were determined using noncompartmental analysis. The formulations were considered bioequivalent if the 90% CI ratios for Cmax and AUC were within the predetermined interval of 80% to 125%, the regulatory definition set by the US Food and Drug Administration (FDA). Results: Twenty-three healthy male volunteers (mean [SD] age, 23.5 [2.0] years [range, 19–28 years]; height, 175.9 [5.4] cm [range, 162.0–185.0 cm]; and weight, 71.2 [9.5] kg [range, 61–96 kg]) were included in the study. No period or sequence effects were detected. The 90% CIs for the corresponding ratios of AUC0–t, AUC0–∞, and Cmax were 84.97 to 114.47, 86.09 to 115.59, and 82.37 to 110.71, respectively. Additionally, the mean (range) of Tmax was 1.09 hours (0.5–2.0 hours), and the mean (SD) of t1/2 and ke were 2.04 (0.97) hours and 0.34 (0.71) hour, respectively. The values for the test and reference formulations Accepted for publication March 11, 2009. doi:10.1016/j.clinthera.2009.05.001 0149-2918/$ - see front matter © 2009 Excerpta Medica Inc. All rights reserved.
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Clinical Therapeutics were within the FDA bioequivalence definition interval of 80% to 125%. No adverse events were reported in this study. Conclusions: Single doses of these formulations of alendronate sodium 70 mg met the criteria for bioequivalence. No statistically significant differences in AUC0–t, AUC0–∞, and Cmax were found in this healthy Korean adult male population. (Clin Ther. 2009;31: 1037–1045) © 2009 Excerpta Medica Inc. Key words: alendronate sodium, bioequivalence test, pharmacokinetics, HPLC–FLD method.
by the ingestion of food, especially products containing calcium. The mean plasma t1/2 is rapid (0.5–2 hours) due to rapid uptake of 20% to 60% of the absorbed fraction into the bone. The remainder is excreted into the urine. The t1/2 in bone is long (~300 days in rats and ≥1000 days in dogs), and the release of bisphosphonates occurs only after resorption of bone into which the compounds have been taken up.8 The aims of the present study were to compare the bioavailability and PK properties, and to determine the bioequivalence of a test and reference formulation, of alendronate sodium 70 mg in a healthy Korean adult male population.
INTRODUCTION The regulatory bioequivalence of 2 formulations of the same drug, particularly conventional drug products, can be determined based on the absence of significant differences in primary pharmacokinetic (PK) properties of bioavailability, such as the rates of absorption and elimination (Cmax and Tmax), and the extent of absorption or total amount of drug absorbed in the body (AUC).1 Alendronate sodium (sodium [4-amino-1-hydroxybutylidene] bisphosphonate) is indicated for the treatment of osteoporosis, hypercalcemia, and Paget’s disease of bone.2 Alendronate sodium is selectively accumulated in the bone, and its oral absorption is <1% of the administered dose in the fasted state.3 When a patient is undergoing treatment with alendronate sodium, normal bone tissue develops, and the drug is deposited in the bone matrix in a pharmacologically inactive form.2 For optimal action, sufficient calcium and vitamin D are needed in the body to promote normal bone development.2 The analytical methods used in the determination of alendronate sodium have been reported in previous studies.3,4 However, because alendronate sodium is not metabolized and is renally excreted, the number of methods for use with serum or plasma is limited.5 Consequently, many PK studies have relied on urine, rather than plasma, in the determination of alendronate sodium concentrations.6,7 These analytical methods, however, require complex derivatization processes and labor-intensive preparation as part of the sample cleanup steps. Very long run times are also required, mainly due to the presence of derivative byproducts along with the derived analysis. Bisphosphonates, including alendronate sodium, are not metabolized and intestinal absorption is poor (<10%).2,8 Furthermore, the intestinal absorption rate is reduced 1038
SUBJECTS AND METHODS Subjects Healthy Korean adult male subjects were eligible to be enrolled in the study. Inclusion criteria consisted of unremarkable results on medical history, physical examination, and clinical laboratory tests (vital signs, serum chemistry and hematology, and urinalysis). Subjects with HIV or hepatitis B were excluded from the study. Concurrent medications, including over-thecounter drugs, and the consumption of alcohol were not allowed from 2 weeks before administration of the first dose until the end of the study period. Subjects were informed about the aims and the risks of the study, and written informed consent was obtained from all volunteers before screening.
Study Design The study protocol was approved by the institutional review board at Hanyang University Medical Center, Seoul, Republic of Korea. The study was carried out at Hanyang University Medical Center and was performed in accordance with the revised Declaration of Helsinki9 and the Good Clinical Practice guideline.10 The study was conducted in an open-label, randomized, 2-sequence, 2-period crossover design with a 7-day washout period. Using a table of random numbers, subjects were assigned to receive a single 70-mg dose of either the test (expiration date: 02/20/2010) or reference* (expiration date: 05/15/2011) alendronate sodium formulation, followed by the washout period, and then the alternate formulation. Both drug *Trademark: Fosamax ® (MSD Korea [Merck] Ltd., Seoul, Republic of Korea).
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S.-Y. Rhim et al. formulations were supplied free of charge by their respective pharmaceutical company.
Drug Administration Protocol and Blood Sample Collection Subjects were admitted to the hospital at 5 pm the evening before administration of the first dose of study drug and received a standardized regular-calorie meal (920 ± 20 kcal: 65% carbohydrate; 20% protein; and 15% fat). The drug administration protocol in the study was similar to the dosing instructions for alendronate sodium, which require that the patient be in the fasting state and the drug be administered with water. Therefore, after a 12-hour overnight fast, subjects received a single 70-mg dose of either formulation (test or reference) with 240 mL of tap water. Food and beverage intake (other than water, which was allowed 2 hours after study drug administration) was not permitted until 4 hours after study drug administration. Subjects received a standardized lunch and dinner according to a regular time schedule. Throughout the study, subjects were prohibited from consuming alcohol or beverages containing xanthine derivatives, engaging in intense physical activity, and smoking. The subjects remained under continuous medical supervision at the study site to monitor for adverse events. Venous blood samples (9 mL) were collected in EDTA-coated tubes (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, New Jersey) at baseline (before study drug administration) and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, and 7 hours after study drug administration. The vein was maintained through a heparin-lock catheter (Korea Vaccine Co., Ltd., Seoul, Republic of Korea). Plasma was immediately separated at 3000 rpm (1000g) for 10 minutes in a refrigerated centrifuge and was transferred to properly labeled 3-mL microfuge tubes (Eppendorf, Hamburg, Germany) and stored at −70°C until analysis.
HPLC With Postcolumn Fluorescence Derivatization and Method Development Plasma concentrations of alendronate sodium were determined using a modified high-performance liquid chromatographic–postcolumn fluorescence derivatization (HPLC–FLD) method11 that was validated using established international guidelines.1 In the previous method, a 4.6 × 150-mm internal diameter (ID), 5.0-μm particle size column, 1.5 mL/min flow rate, and temperature of 35°C were used.11 However, the May 2009
modifications in the present study included a thinner separation column (2.0 × 150-mm ID), slower flow rate (0.35 mL/min), and lower temperature (25°C). All solvents were HPLC grade; other chemicals and reagents were analytical grade. Alendronate sodium (molecular weight [mw], 325.124 kDa; purity, 98.96%; Eczacibasi Pharmaceuticals Manufacturing Co., Istanbul, Turkey) and internal standard (IS) pamidronate (mw, 235.06992 kDa; purity, 99.00%; Calbiochem Co., an affiliate of Merck KGaA, Darmstadt, Germany) were provided by Schnell Korea Pharma Co., Ltd., Seoul, Republic of Korea. The HPLC–FLD conditions were optimized for the column, mobile phase, and sample preparation. The HPLC–FLD (Varian Inc., Palo Alto, California) system consisted of a pump (ProStar 230, Varian Inc.), an autosampler (ProStar 410, Varian Inc.), a fluorescence detector (RF-10AXL, Shimadzu Corporation, Kyoto, Japan), and peak simple data system software version 6.3 (Star LC Workstation, Varian Inc.). The separation of compounds was performed in an analytical column (Capcell Pak C18, Shiseido Company, Ltd., Tokyo, Japan; 2.0 × 150-mm ID, 5.0-μm particle size) and eluted with a mobile phase consisting of mixed organic solution (acetonitrile:methanol = 1:1 vol/vol, solvent A) and buffer (25-mM citric acid: 25-mM sodium pyrophosphate = 1:1 vol/vol, without pH adjustment, solvent B). Run times for solvents A and B were 32:68 vol/vol for 0 to 9 minutes, 60:40 vol/ vol for 9 to 16 minutes, and 32:68 vol/vol for 16 to 24 minutes. The analyses were conducted at 25°C, and a flow rate of 0.35 mL/min, with excitation and emission wavelengths of 260 nm and 310 nm, respectively, and the injection volume was 50 μL. The column was equilibrated for 1 minute before each subsequent sample was injected. No significant chromatographic interferences at the retention times of alendronate sodium or IS were observed (Figure 1). The peak area was measured for calculation of the peak area ratio of alendronate sodium to IS, and the concentrations were calculated. Each analytical process required ~25 minutes. The calibration curves were validated over a concentration range of 5 to 100 ng/mL for alendronate sodium in human plasma, visible detection in the range of 2 to 100 ng/mL, and lower limit of quantification set at 2 ng/mL using 1.0 mL of plasma.
Sample Preparation and Processing The procedures described here were applied to subject samples as well as extraction of samples for cali1039
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B 3
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Volts (units)
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Figure 1. Chromatograms (A) of blank plasma, (B) with internal standard (IS) (12.5 μg/mL), (C) with alendronate sodium (50 ng/mL) and IS (12.5 μg/mL), and (D) of human plasma taken 1 hour after single oral dose of alendronate sodium 70 mg spiked with IS (12.5 μg/mL).
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S.-Y. Rhim et al. bration curve and quality control (QC) processes. By adding the distilled water, the concentration of the stock solution of alendronate sodium was 1 mg/mL. Separate solutions were prepared for the calibration standards and QC samples. To obtain working solutions of 1000, 500, 200, 100, 50, and 20 ng/mL, 300 μL of these solutions was diluted immediately before use with purified water. Additionally, to obtain 100, 50, 20, 10, 5, and 2 ng/mL for each analyte, the solutions were mixed with 2.7 mL of blank serum. The sample was prepared by mixing 3.0 mL of serum and 100 μg/mL of the IS solution in a tapered tube and adding 6% trichloroacetic acid solutions, followed by vortex mixing for 1 minute. Then, a mixture of 0.1 M KH2PO4 (0.2 mL), 1 M CaCl2 (0.2 mL), and 1 M NaOH (0.4 mL) was successively added. The sample was stirred (5Roll Mixer, Hyunil Lab-Mate Co., Seoul, Republic of Korea) for 10 seconds and centrifuged (VS-6000, Vision Co., Seoul, Republic of Korea) at 3000 rpm (1000g) for 5 minutes, and the precipitate was carefully transferred to a tapered tube without an upper solution layer. After removal of the liquid phase, the deposition was dissolved completely in 0.5 mL of 0.2 M acetic acid and 3 mL of purified water was added. The precipitation was repeated 3 times. The resulting precipitate was dissolved in 1 mL of 0.2 M acetate buffer (pH 6.0) and 2 mL of acetic acid, diluted with 3 mL of purified water. The sample was loaded on a Bond Elute DEA cartridge (Varian Inc.) and washed twice with 0.5 mL of water. The cartridge was then eluted with 1 mL of 0.2 M sodium citrate, and 270 μL of the eluent was taken for derivatization. The derivatization procedure involved adding 0.2 mL of 1 M sodium carbonate buffer (pH 11.9) to 270 μL of the eluent, and adding 0.2 mL of 9-fluorenyl methyl chlorformate solution (1 mg in 4 mL of acetonitrile). After 3 minutes, 0.2 mL of 1 M citric acid was added to stop the reaction and 50 μL of the sample was injected into the HPLC–FLD system. The calibration standards and blanks were freshly prepared in duplicate for each assay and were extracted together with plasma and QC samples. To assess intra- and interassay precision, accuracy, and recovery for method validation, QC samples were prepared by spiking control human plasma with 2 (lower limit of quantification), 5, 10, 20, 50, and 100 ng/mL of alendronate sodium. Intraday precision and accuracy were determined by repeated analyses of the group of standards on 1 batch (n = 5). Interday precision and accuracy were deterMay 2009
mined by repeated analyses on 5 consecutive days (n = 5 series per day). One QC sample set (consisting of 4 alendronate sodium concentrations [2, 5, 50, and 100 ng/mL]) was analyzed in a preparation batch of 50 samples using the same equipment, reagents, and staff in a 24-hour period.
Tolerability Adverse events (nausea, vomiting, diarrhea, abdominal bloating, gastric pain, musculoskeletal and joint pain, headache, and fatigue) were monitored by a clinical investigator (D.-S.K.) via observation, personal interview, and vital signs (blood pressure, heart rate, and temperature) over a 7-hour period after study drug administration. All adverse events were recorded on the clinical record form per subject up to 1 week after the study.
Pharmacokinetic and Statistical Analysis Noncompartmental analysis for deriving PK properties was performed using PK Solutions version 2.0 (Summit Research Services, Montrose, Colorado). The actual times of blood sample collection were used for PK analyses of alendronate sodium. Cmax and Tmax were obtained directly from the plasma concentration– time curve after a single 70-mg dose of alendronate sodium (test or reference) was administered (Figure 2). AUC0–t was calculated using the linear trapezoidal method. AUC0–∞ was calculated as AUC0–t and the ratio of the Ct/ke, where Ct was the last measured concentration and ke was the slope of the linear regression of the log-transformed plasma concentration time in the terminal phase. The plasma elimination t1/2 was calculated as 0.693/ke. AUC0–t, AUC0–∞, and Cmax were the primary variables used to assess bioequivalence between the test and reference formulations. Bioequivalence between the 2 formulations was determined by calculating the 90% CIs for AUC0–t, AUC0–∞, and Cmax values for the test and reference formulations, using log-transformed data. A power analysis, with a power of 80% to detect a difference, indicated that a sample of 23 adult volunteers was of adequate size to detect statistically significant differences between test and reference formulations. A 2-way analysis of variance (ANOVA) was conducted to test for period, sequence, and group effects. Significance was set at P < 0.05. To access bioequivalence assessment between 2 formulations, in accor1041
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Test Reference
Plasma Concentrations of Alendronate (ng/mL)
100 90 80 70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
7
Time (h) Figure 2. Mean (SD) plasma concentration–time plots of alendronate sodium 70 mg after a single oral dose of test and reference formulations in healthy Korean adult male volunteers (N = 23). No significant differences were observed.
dance with current US Food and Drug Administration guidelines,1,12,13 the formulations were considered bioequivalent if the 90% CIs for AUC0–t, AUC0–∞, and Cmax were within the predetermined range of 80% to 125%.
RESULTS Thirty-eight subjects were recruited for participation, and a total of 24 subjects met all study criteria and were enrolled; 1 subject, however, withdrew before study drug administration. Therefore, a total of 23 healthy male volunteers (mean [SD] age, 23.5 [2.0] years [range, 19–28 years]; height, 175.9 [5.4] cm [range, 162.0–185.0 cm]; and weight, 71.2 [9.5] kg [range, 61–96 kg]) completed both treatment periods of the study with no protocol violations.
Alendronate Sodium Quantification in Plasma The analytical method for alendronate sodium quantification in plasma samples found good specificity, sensitivity, linearity, precision, and accuracy over the entire range of clinically significant and therapeuti1042
cally achievable plasma concentrations, thereby enabling its use in bioequivalence trials. The linearity was observed within the range of 2 to 100 ng/mL (alendronate sodium; y = 0.0096 ∙ x − 0.0006, r2 = 0.9999; x = plasma concentration, y = peak area ratio). The intraday precision ranged from 0.23% to 1.90%, whereas intraday accuracy ranged from 98.1% to 101.3%. Interday precision ranged from 0.74% to 1.78%, whereas interday accuracy ranged from 98.0% to 102.1%. The extraction recovery of alendronate sodium was determined in its QC samples in 5 replicates. The mean absolute recovery was 90.26%, whereas the relative recovery ranged from 96.4% to 103.4%.
Pharmacokinetic Properties The mean (SD) plasma concentration–time profiles of the test and reference formulations of alendronate sodium are shown in Figure 2. Both formulations were readily absorbed from the gastrointestinal tract, and alendronate sodium was measurable at the first sampling time (0.25 hour) in the 23 volunteers. At the first sampling time, alendronate sodium apVolume 31 Number 5
S.-Y. Rhim et al. peared in plasma in both groups; mean Cmax values were 37.17 and 38.83 ng/mL, and Tmax values were 1.09 and 1.24 hours for the test and reference formulations, respectively. The plasma concentrations gradually decreased, with a mean terminal t1/2 of 1.85 and 2.04 hours for the test and reference formulations, respectively, and then decreased rapidly but were detected up to 7 hours later. The mean alendronate sodium plasma concentration was 4.36 ng/mL. As shown in Table I, after adjusting for weight, the mean AUC0–t values were 1.38 and 1.40 ng ∙ h/mL/kg for the test and reference formulations, respectively, and the mean AUC0–∞ values were 1.54 and 1.55 ng ∙ h/mL/kg for the test and reference formulations, respectively. The ANOVA for the ratio of AUC0–t, AUC0–∞, and Cmax values for the test and reference formulations, using log-transformed data, are shown in Table II. ANOVA assessment found no significant product, group, or period effect in the present study. The 90% CIs for the ratio of AUC0–t, AUC0–∞, and Cmax values (84.97–114.47, 86.09–115.59, and 82.37–110.71, respectively) for the test and reference formulations were within the established regulatory interval of 80% to 125%.
Tolerability Both tablet formulations of alendronate sodium were well tolerated by all volunteers. No adverse events were reported during the study or up to 1 week after the study.
DISCUSSION Because alendronate sodium systemically disappears rapidly (0.5–2 hours after oral administration),8 resulting in low plasma bioavailability (0.7%) in the fasted state, its PK properties have been previously reported based on urinary excretion data.14 Moreover, the oral bioavailability of this drug is <2% in all species including humans.15 Thus, few studies have been conducted on the plasma concentrations of alendronate sodium after oral administration.16 Therefore, using a validated HPLC–FLD method with lower limit of quantification of 2 ng/mL, the present study was designed to examine the PK properties in plasma after a 70-mg oral dose of alendronate sodium. In a previous study assessing PK properties after oral administration of alendronate 70 mg, Cmax, Tmax, t1/2, and AUC0–t were 40.94 ng/mL, 1.0 hour, 1.67 hour, and 118.55 ng ∙ h/mL, respectively, in 4 healthy Koreans.11 Although the alendronate formulations in the present
Table I. Pharmacokinetic (PK) properties (weight corrected) of alendronate sodium formulations (N = 23).* Data are mean (SD) unless otherwise specified. PK Parameter
Test
Reference†
AUC0–t OHtIN-LH
AUC0–∞ OHtIN-LH
Extrapolation (AUCt–∞ tLH"6$0–∞ tLH ‡
0.16 (0.10)
0.16 (0.08)
Cmax OHN-tLH
Tmax, mean SBOHF
I
o
o
t1/2 I
ke I
ke = elimination rate constant. * No significant between-treatment differences were found. † Trademark: Fosamax ® (MSD Korea [Merck] Ltd., Seoul, Republic of Korea). ‡ Weight-corrected ratio of AUC t–∞ to AUC0–∞.
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Table II. Analysis of variance of 2 oral formulations of alendronate sodium 70 mg after single-dose administration. 2.
Test: Reference* Ratio
90% CI
AUC0–t
o
AUC0–∞
o
Cmax
o
PK Parameter
PK = pharmacokinetic. * Trademark: Fosamax ® (MSD Korea [Merck] Ltd., Seoul, Republic of Korea). 6.
study are different from that in the previous study, after adjusting for mean (SD) weight (71.2 [9.5] kg), similarities were found in the present data in Cmax (38.83 ng/mL), Tmax (1.24 hour), t1/2 (1.85 hour), and AUC0–t (99.68 ng ∙ h/mL).
Limitations This study had some limitations because it included only males, healthy volunteers, and single-dose administration. A larger scale study including women is needed to assess the PK properties of alendronate sodium.
CONCLUSIONS In this small study in healthy male Koreans, no statistically significant differences in AUC0–t, AUC0–∞, and Cmax were found between the test and reference formulations of alendronate sodium. The single 70-mg dose of these formulations met the regulatory criteria for bioequivalence in this fasting, adult volunteer population.
ACKNOWLEDGMENT This work was supported by the cluster research fund of Hanyang University (HY-2008-C).
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64%FQUPG)FBMUIBOE)VNBO4FSWJDFT 'PPEBOE%SVH Administration (FDA), Center for Drug Evaluation BOE3FTFBSDI $%&3 (VJEBODFGPSJOEVTUSZ#JPBWBJMB CJMJUZ BOE CJPFRVJWBMFODF TUVEJFT GPS PSBMMZ BENJOJTUFSFE
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S.-Y. Rhim et al. UXP BMFOESPOBUF PODFXFFLMZ GPSmulations. Arzneimittelforschung. 2006; 56:84–89. 1PSSBT "( )PMMBOE 4% (FSU[ #+ Pharmacokinetics of alendronate. Clin Pharmacokinet o -JO+) 3VTTFM( (FSU[#1IBSNBDPLJnetics of alendronate: An overview. Int J Clin Pract. 1999;101(Suppl):18–26.
Address correspondence to: Ju-Seop Kang, MD, PhD, Department of Pharmacology & Institute of Biomedical Science, College of Medicine, Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea. E-mail: [email protected] May 2009
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