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Pharmacokinetic properties and bioequivalence of two formulations of arbidol: An open-label, single-dose, randomized-sequence, two-period crossover study in healthy chinese male volunteers

Background: Arbidol is an antiviral drug indicated for the prevention and treatment of all types of influenza infection and some other kinds of acute respiratory infections, specifically against influenza groups A and B, and severe acute respiratory syndrome. It is used to help prevent influenza infection as long as necessary with little risk for influenza mutation rendering it less effective. Objective: The aim of this study was to compare the pharmacokinetic properties and tolerability, and to determine bioequivalence, of a newly developed generic dispersible tablet formulation (test) and a branded capsule formulation (reference) of arbidol 200 mg in healthy Chinese fasted male volunteers. Methods: This open-label, single-dose, randomized-sequence, 2-period crossover study was conducted in healthy native Chinese male volunteers. Eligible subjects were randomly assigned in a 1:1 ratio to receive a single 200-mg dose of the test or reference formulation, followed by a 1-week washout period and administration of the alternate formulation. The study drugs were administered after a 12-hour overnight fast. After the study drug administration, serial blood samples were collected for 72 hours after administration. Plasma drug concentrations were determined using high-performance liquid chromatography coupled with tandem mass spectrometry. Several pharmacokinetic pararameters, including Cmax, Tmax, t½, AUC0-t, and AUC0-∞, were determined from the plasma concentrations of the 2 formulations of arbidol using noncom-partmental analysis. The formulations were to be considered bioequivalent if the log-transformed ratios of Cmax and AUC were within the predetermined bioequivalence range of 80% to 125% established by the State Food and Drug Administration (SFDA) of the People's Republic of China. Tolerability was assessed by monitoring vital signs (blood pressure, heart rate, temperature, and electrocardiography), laboratory analysis (hematology, blood biochemistry, hepatic function, and urinalysis), and subject interview on adverse events. Results: Twenty subjects were enrolled and completed the study (mean [SD] age, 21.1 [1.1] years; weight, 64.7 [5.1] kg; and height, 172.3 [3.1] cm). Neither period nor sequence effect was observed. The main pharmacokinetic properties with the test and reference formulations were as follows: Cmax, 417.4 (107.6) and 414.8 (95.1) ng/mL, respectively (P = NS); median (range) Tmax, 0.63 (0.25–1.0) and 0.75 (0.5–1.5) hours (P = 0.035); AUC0-t, 2033.6 (564.9) and 1992.0 (483.3) ng/mL/h (P = NS); AUC0-∞, 2285.4 (597.7) and 2215.2 (604.0) ng/mL/h (P = NS); and t1/2, 6.9 (4.2) and 6.1 (5.2) hours (P = NS). The 90% CIs for the log-transformed ratios of Cmax, AUC0-t, and AUC0-∞ were 91.7% to 109.7%, 91.0% to 112.8%, and 92.0% to 116.3%, respectively (all, P < 0.05), which were within the predetermined range for bioequivalence. No adverse events were found on analysis of vital signs or laboratory tests or reported by subjects in this study. Conclusion: In this study in healthy Chinese male volunteers, the dispersible tablet formulation and the 200-mg capsule formulation of arbidol met the SFDA's regulatory definition of bioequivalence based on the rate and extent of absorption.

Healthy Chinese male nonsmoking volunteers were selected for this study. All subjects signed an informed-consent form after they were given the detailed information about the aims, restrictions, and adverse events that could be experienced as a result of receiving the study drug. Before study entry, subjects underwent an interview regarding their occupation, smoking and drinking habits, and medical history; routine physical examination, including vital sign monitoring (blood pressure, heart rate, temperature, and electrocardiography); and laboratory analysis (ie, hematology, blood biochemistry, hepatic function, and urinalysis) to ensure that they were healthy enough to take part in the study.
Subjects who had a history or evidence of a renal, gastrointestinal, hepatic, or hematologic abnormality; any acute or chronic disease; or an allergy to any chemicals were excluded. Subjects who had used drugs of any kind within the 2 weeks prior to the start of or during the study were excluded, as were those with alcohol abuse (>28 U of alcohol per week [1 U = 285 mL of beer]).

Study Drug Administration
Enrolled subjects were randomized in a 1:1 ratio, using a table of random numbers, to receive a single 200-mg dose of arbidol as a dispersible tablet (test) formulation* (lot no. 20050901; expiration date, September 2007) followed by a capsule (reference) formulation † (lot no. 050701; expiration date, June 2007), or vice versa. Subjects were hospitalized at 8 pm and given a standardized evening meal (200-~400 g of cooked rice or other staple food, ~400 g of vegetables, ~50 g of meat, and 250 mL of water). At 7 am after an overnight fast (as required by the State Food and Drug Administration [SFDA] of the People's Republic of China), subjects were administered a single 200-mg dose of arbidol (test or reference formulation) with 250 mL of water and then fasted for 4 hours. A standardized lunch (the same as the standardized evening meal) and a standardized evening meal were served at 4 and 9 hours, respectively, after study drug administration. No other food was permitted during the period; water was available 2 hours after study drug administration.

INTRODUCTION
Arbidol is an antiviral drug first marketed in Russia in 1993 and in China in 2006. It is indicated for the prevention and treatment of influenza A and B, respiratory syncytial virus, severe acute respiratory syndrome (including exacerbation of bronchitis and pneumonia), secondary immunodeficiency, overall therapy for chronic bronchitis, pneumonia, and recurrent herpesvirus infections, and for the prevention of postoperative infections and stabilization of immune status. [1][2][3][4][5] Arbidol might prevent contact and entry of viruses into cells by inhibiting the fusion of viral lipid membranes with cell membranes. It has also been reported that arbidol has interferon-inducing activity, activated phagocytic activity of macrophages, and stimulated humoral and cell-mediated immunity, and that it heightens the body's ability to fight infection. [6][7][8][9][10] T max with a single 50-mg dose of arbidol has been reported to be 1.2 hours; with 100 mg, 1.5 hours. Arbidol is metabolized in the liver. The t 1/2 of the drug in the body is 17 to 21 hours. Approximately 40% is excreted unchanged, mostly (38.9%) through the bile, with an insignificant amount (0.12%) excreted through the kidneys. Within the first 24 hours after administration, 90% of the drug has left the body. 11 The aim of the present study was to compare the pharmacokinetic properties and determine bioequivalence of two 200-mg formulations of arbidol: a newly developed generic formulation (dispersible tablet; test) and a branded capsule formulation (reference) in healthy Chinese fasted male volunteers.

SUBJECTS AND METHODS Study Design and Subject Selection
This open-label, single-dose, randomized-sequence, 2-period crossover study was conducted at the First Affiliated Hospital of China Medical University, Shenyang, People's Republic of China. The study was conducted according to the principles of the Declaration of Helsinki and its amendments 12 for biomedical research involving human subjects and the principles of the Good Clinical Practice guideline. 13 The clinical protocol and the informed-consent form were approved by the local ethics committees at China Medical University.

Clinical Therapeutics
achieved on a C 18 column (100 × 2.0-mm internal diameter [ID], 3-μm particle size, Shiseido Co., Ltd., Tokyo, Japan), which was preceded by the use of a guard column (C 18 , 10 × 4.6-mm ID, Phenomenex, Inc., Torrance, California) at a column temperature of 20°C. The mobile phase was prepared by mixing methanol:5% formic acid at a ratio of 72:28 (vol/vol), an injection volume of 2 μL, and a flow rate of 0.3 mL/min. Separation was conducted under isocratic conditions, and the total running time was within 3 minutes.
The electrospray ionization source was performed in the selected ion monitoring mode. The [M + H + ], m/z 479 for arbidol, and the [M + H + ], m/z 286 for the IS, using diazepam as the IS, were selected as detecting ions, respectively. The MS operating conditions were optimized as follows: drying gas (nitrogen gas), 2.0 L/ min; curved desolvation line temperature, 250°C; atomization gas (nitrogen gas), 1.5 L/min; and detector voltage, 1.4 kV. Analytical data were processed using HPLC-MS/MS Solution 3.0 (Shimadzu Biotech, Nakagyoku, Japan), and the peak area was used for quantification.

Sample Preparation
Frozen human plasma samples were thawed at ambient temperature. Plasma samples (500 μL) were spiked with 50 μL of IS (diazepam 10 μg/mL), 50 μL of methanol, 400 μL of 0.04-mol/L Na 2 CO 3 , and 3 mL of diethyl ether and vortex-mixed for 3 minutes. After centrifugation (2000g, 4°C, 5 minutes), the organic phase was transferred to another dry clean tube and evaporated to dryness in a thermostatically controlled water-bath at 40°C in a slight stream of nitrogen. The residue was dissolved in 100 μL of methanol, with 2 μL used for HPLC-MS/MS analysis. Sample extraction was protected from light.

Method Validation and Quantification
To investigate the specificity and selectivity of the HPLC-MS/MS method, blank human plasma samples, spiked plasma samples, and subject samples were prepared and analyzed. Chromatography findings are shown in Figure 1. The arbidol and diazepam retention times were 1.7 and 2.8 minutes, respectively, suggesting that no endogenous peaks at the retention time of arbidol and IS interfered with the determination.
The calibration curves of arbidol in human plasma were obtained using 6 calibration standards, each of which was prepared in triplicate and extracted together with blank samples and quality control (QC) After a 1-week washout period, subjects returned to the clinical unit, where the alternative formulation was administered and blood samples were drawn as before so as to complete the crossover design.

Blood Sample Collection
In this study, blood samples (5 mL) were collected from a suitable forearm vein using an indwelling catheter into heparin-containing tubes before (0 hour; baseline) and 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2, 4, 7, 12, 24, 48, and 72 hours after study drug administration. Just before each blood sample was collected, heparin in the heparin-locked catheter was discarded with 0.5 mL of blood. All of the blood samples were immediately centrifuged at 1000g for 10 minutes at room temperature (20°C) for separating plasma, and plasma samples were stored at -20°C until quantitative analysis for the determination of plasma arbidol concentrations. The sample collection and handling processes were protected from sunlight.

Determination of Plasma Arbidol Concentrations
A method of the determination of arbidol concentration in plasma using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was used and has been described elsewhere. 14,15

Chemicals and Materials
Arbidol hydrochloride and diazepam (as the internal standard [IS]) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, People's Republic of China (purity >99.5%). The deionized water was purified using the Milli-Q water system (Millipore Research and Development, Bedford, Massachusetts). Methanol (Merck KGaA, Darmstadt, Germany) was of highperformance LC grade. Formic acid (Tedia Co., Inc., Fairfield, Ohio), sodium carbonate (Na 2 CO 3 , Shandong Aona Chemical Co. Ltd., Liuyuan, People's Republic of China), and diethyl ether (Sigma-Aldrich Corporation, St. Louis, Missouri), were all of analytical grade and commercially available. Blank plasma was obtained from a blood center in Shenyang, People's Republic of China.

Clinical Therapeutics
bioequivalence study according to the international guidelines. 16,17 Tolerability The investigators included 2 clinicians, a clinical pharmacist, and ≥2 nurses who monitored the subjects for adverse events. These events were also determined using subject interview (subjective chief complaints by subjects, objective observation by clinicians) regarding the potential presence of adverse events, such as allergic reaction, nausea, diarrhea, and dizziness 18,19 at any time during the study, and by comparing the results of vital signs monitoring (blood pressure, heart rate, and body temperature) and laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) at baseline, 12 hours after study drug administration, 7 days after completion of the study (follow-up visit), and for 6 additional months thereafter (self-report by subjects).

Pharmacokinetic and Statistical Analyses
Pharmacokinetic analysis was performed by using DAS version 2.0, authorized by the Chinese Pharmacology Society, Beijing, People's Republic of China. Data obtained from individual volunteers were subjected to noncompartmental pharmacokinetic analysis. 16,17,20 AUC 0-t , AUC 0-∞ , C max , T max , t 1/2 , and elimination rate constant (k e ) were determined in each volunteer. C max and T max were obtained directly from the observed data. k e was obtained as the slope of the linear regression of the log-transformed concentration versus time samples. The peak area was used for quantification. QC samples were used to assess the intraday and interday precision, accuracy, and recovery, prepared by spiking control human plasma with 10-, 25-, 100-, 200-, 500-, and 1000-ng/mL arbidol. The linearity was observed within the range of 10 to 1000 ng/mL, with a correlation coefficient <0.9965 (n = 6). The lower limit of quantification of arbidol was 10 ng/mL.
The intraday and interday variations and recoveries for arbidol determination in human plasma are shown in Table I.
The short-term stability of extracted arbidol samples for 24 hours at room temperature and the freezethaw cycles stability of frozen samples (frozen at -20°C, thawed at room temperature [20°C], freezethaw for 3 cycles, 2 hours per cycle) were determined to ensure that the samples were stable during the handling and analysis processes. Long-term storage stability of plasma samples at -20°C was determined after 45 days to ensure that the stability of frozen samples at storage temperature (-20°C) could not affect reliability of the results on HPLC-MS/MS analysis. The results of the stability of arbidol in the human plasma (freeze-thaw for 3 cycles and short-term stability) and long-term storage (-20°C, 45 days) are shown in Table II. Arbidol was stable for 24 hours at room temperature (20°C) in extracted plasma samples, for 45 days at -20°C, and within 3 freeze-thaw cycles in plasma samples.
The analytical method for arbidol quantification in plasma samples was validated and applied to the were enrolled in the study. All subjects completed both treatment periods, with no protocol violations. No abnormalities were found in clinical or biochemical parameters on comparison of baseline versus end-ofstudy assessments.
The mean plasma concentration-time profiles of the 2 formulations after administration of a single oral dose of 200-mg arbidol are shown in Figure 2. The 90% CIs of the ratios (test/reference) for the logtransformed C max , AUC 0-t , and AUC 0-∞ values, as well data in the terminal portion of the curve. t 1/2 was calculated as 0.693/k e . AUC 0-t was calculated according to the trapezoidal rule. AUC 0-∞ was calculated using the following formula 16 : where C t was the last measurable concentration.
To test the bioequivalence of the test and reference formulations, analysis of variance (ANOVA) for the crossover design was conducted on log-transformed C max , AUC 0-t , and AUC 0-∞ . The ratios of the logtransformed C max , AUC 0-t , and AUC 0-∞ were calculated using the F score. The probability of exceeding the limits of acceptance for bioequivalence established by the SFDA (80%-125%) was obtained using two 1-sided t tests, as described by Schuirmann 21 and the US Food and Drug Administration. 22 The formulations were to be considered bioequivalent if the logtransformed ratios (test/reference) of C max , AUC 0-t , and AUC 0-∞ were within the predetermined bioequivalence range of 80% to 125% and if P was <0.05 for the 90% CIs. 21

Clinical Therapeutics
Chinese populations. The different k e results might have been related to the differences between the races of the studied populations. In the present study, the T max of the test formulation was significantly shorter compared with that of the reference formulation (P < 0.05). This finding might have been the result of differences in the preparation technologies between the tablet (test) and capsule (reference) formulations. The results would be instructive for administration in patients in the different states of illness in clinical practice. The present study had a few limitations that should be considered. First was the small sample size; singledose, open-label design; and assessment of clinical relevance of the adverse events by the investigators. The pharmacokinetic data in this study were obtained only from healthy Chinese male volunteers; the pharmacokinetic properties of arbidol might be different in women and in infected patients in clinical practice. Because this study was conducted in fasted subjects, the results cannot be applied to predict the pharmacokinetic properties of arbidol concurrently administered with food. The methods used for the assessment of the tolerability were not perfect, and the sample size was not large enough to observe all of the possible adverse events. The sample size, 20 volunteers, is typical in bioequivalence studies according to the SFDA guideline. 16 However, a larger sample size may be needed to assess tolerability and to represent pharmacokinetic properties in the Chinese population in future studies.
as the probability of exceeding the limits of acceptance for bioavailability and the power of the test are shown in Table IV. The 90% CIs for the ratios of C max , AUC 0-t , and AUC 0-∞ were 91.7% to 109.7%, 91.0% to 112.8%, and 92.0% to 116.3%, respectively, which were within the range of 80% to 125%. All P values were <0.05.

Tolerability
No adverse events were observed by clinicians, found on the analyses of vital sign monitoring or laboratory analysis, or reported by subjects during or after the study.

DISCUSSION
The findings from the present study with regard to T max and t 1/2 (~0.63 and ~6 hours, respectively) were consistent with those from a review by Liu et al, 15 who reported that the T max of arbidol was ~40 minutes, and t 1/2 was ~7.5 hours after study drug administration. Glushkov 11 had reported that T max was 1.2 hours after the administration of a 50-mg dose, T max was 1.5 hours after the administration of a 100-mg dose, and t 1/2 was 17 to 21 hours. The product information of the arbidol capsule formulation 19 mentions that T max was 1.63 hours and t 1/2 was ~11 to 17 hours.
The comparison of our findings with those from previously published studies suggests that the rate and extent of absorption were different between the dispersible tablet and capsule formulations, and the elimination rate was different between Russian and