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An open-labeled, randomized, cross-over bioequivalence study of Seratrodast 80mg under fasting condition

Dr.Bhupesh Dewan, Raghuram Chimata

RUNNING TITLE: Bioequivalence Study of Seratrodast Tablet Formulations ABSTRACT: Seratrodast is a novel thromboxane A2 (TXA2) receptor antagonist used as an add on therapy in the management of asthma. This bioequivalence study was carried out to determine the pharmacokinetic profile of seratrodast in plasma in an open labeled, two treatment, two period, two sequence, single dose, comparative, randomized, two way crossover design under non fed condition with 14 days washout period in 26 healthy male subjects. Blood samples were collected at intervals up to 72 hours as per the approved protocol. Concentrations of Seratrodast in plasma were analyzed by a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method. A non-compartmental model was used for pharmacokinetic analysis. The mean ( S.D.) values of the pharmacokinetic parameters (test vs. reference) were Cmax (5.38 0.77 vs. 5.25 0.74 g/ml), AUC0-t (80.41 20.11 vs. 76.67 21.02 g.h/ml), AUC0-inf (99.78 23.27 vs.97.32 26.61 g.h/ml), and t1/2 (22.66 6.69 vs. 22.87 8.92 h). The mean times ( S.D.) to attain maximal plasma concentrations (tmax) of seratrodast were 3.48 0.61 and 3.56 0.70 h for test and reference formulations respectively.The 90% of confidence intervals (C.I.s) for the test/reference ratio of mean Cmax, AUC0-t, and AUC0-inf were within the acceptable range of 80.00 to 125.00. Both the formulations were well tolerated. In conclusion, the two formulations were bioequivalent and could be used interchangeably. Key words: Seratrodast; TXA2; Pharmacokinetics; Bioequivalence; LC/MS/MS

INTRODUCTION: Seratrodast, ()-7-(3,5,6-trimethyl 1,4-benzoquinon-2-y1)-7-phenylheptanoic acid (Figure 1) rimethylis an orally active quinone derivative and a potent TXA2 receptor antagonist (1). Seratrodast has been established to be useful as an add on therapy for asthma (2). Seratrodast has been shown to inhibit bronchoconstriction induced by TXA2, prostaglandin D2 (PGD2), 9,11choconstriction prostaglandin F2 (9,11-PGF2) prostaglandin F2 (PGF2), leukotriene D4 (LTD4) and platelet), activating factor (PAF) (3-5), and has been observed to affect both immediate and late , asthmatic responses (6-7). Seratrodast has been reported to reduce airway hyper hyperresponsiveness to various stimuli like allergens, PAF, LTC4, D4 and B4, bradykinin, stimuli, endothelin, endotoxin and ozone (1, 6-9).

Figure 1: Chemical structure of Seratrodast [4] In clinical studies in humans, seratrodast has been shown to markedly improve clinical parameters of bronchial asthma. In patients with asthma, the oral administration of seratrodast reduces bronchial hyper-responsiveness ( responsiveness (10). Treatment with seratrodast has been shown to significantly improve symptom scores, peak expiratory flow (PEF), diurnal variation in PEF and bronchial responsiveness. These improvements have been associated with significant reduction in the number of activated eosi eosinophils in the bronchial mucosa, suggesting that he seratrodast acts by modulating infiltration of activated eosinophils into the airways and release of pro-inflammatory chemokines like RANTES (regulated and normal T cell inflammatory expressed and secreted) and macrophage inflammatory protein (MIP)-1 (11). Seratrodast inflammato significantly reduces daytime asthma symptoms and daytime supplemental use of 2adrenoceptor agonists. Seratrodast has been observed to alter physicochemical properties of sputum. It decreases the albumin concentratio , eosinophil cationic protein (ECP) levels and concentration, dynamic viscosity of sputum. Seratrodast simultaneously decreases sputum production and . improves nasal mucociliary clearance (12). Seratrodast is presently approved in Japan as a tablet ( This product is not available in (2). Europe, USA or India. The objective of the present study was to compare the pharmacokinetic profiles of Seratrodast formulation manufactured by Zuventus Healthcare Ltd., India using indigenously developed active pharmaceutical ingredient (API) as well as the formulation, API) with commercially available Bronica, of Takeda Pharmaceutical Co., Ltd., Japan. MATERIALS AND METHODS: Drugs: The active pharmaceutical ingredient along with the test formulation (batch number FD/31A/12; manufacturing dat April 2012) was indigenously manufactured by Zuventus ; date Healthcare Ltd., India. The reference product Bronica (batch number 0113, expiry date , March 2014) was manufactured by Takeda Pharmaceuticals Co., Ltd., Japan. Each film ) coated tablet contained Seratrodast equivalent to 80 mg. The study was conducted at Drug trodast 0 Monitoring Research Institute, Mumbai, India and it was sponsored by Zuventus Healthcare , Ltd., India.

Study Subjects: Guidelines drawn up by the Institutional Review Board (IRB), which met the requirements of the U.S. code of Federal Regulations, the Canadian MRC guidelines and Declaration of Helsinki, Tokyo 2004 as well as the ethical norms laid down by the Indian Council of Medical Research (ICMR), New Delhi, India, 2006 were followed during the study (13, 14). Twenty six healthy male subjects, including 2 subjects as standby to replace dropouts, were included in the study. The protocol was approved by an independent ethics committee. All participants gave a written informed consent prior to participation, which had an approval of the independent ethics committee, after they had been informed of the nature and details of the study in a language (both written and verbal) which they understood. Subject inclusion criteria included age between 18-45 years, non-smoker and Asian adult male of Indian origin with no evidence of underlying disease, medical disorders/impairments (hepatic, renal, cardiac, gastrointestinal tract and psychiatric), no vital sign abnormalities, no clinically significant abnormal values during pre-study screening, acceptable ECG, no consumption of drugs for two weeks prior to the study, and no participation in any bioavailability or bioequivalence study at least 3 months prior to the present study. The exclusion criteria included history of hypersensitivity to the study product or related products, abnormalities in vital sign (systolic blood pressure < 90 or > 140 mm Hg or diastolic blood pressure < 50 or > 90 mm Hg or heart rate < 50 bpm or > 100 bpm), significant medical illness or conditions known to interfere with absorption, distribution, metabolism and excretion of the study drugs, clinically significant cardiovascular, gastrointestinal, hepatic, renal, pulmonary, hematological, neurological or endocrinal disease, significant clinical illness during 3 weeks prior to day one of the study or hospitalization during 3 months prior to the commencement of the study, history of smoking, alcohol abuse, drug dependency, use of any systemic medications including over the counter (OTC) drugs within 7 days prior to day one of the study, HIV or hepatitis positive subjects, and subjects who had donated blood (350 ml) within last 3 months prior to the study. Study design: The study was performed as an open labeled, randomized, two-way, two-period, twotreatment, single dose cross-over bioequivalence study, under non fed condition, and the treatments were separated by a wash-out period of 14 days. Each subject was assigned a unique identification number. All the subjects arrived at the study center at least 13 hours prior to the start of the study. They were housed in an air-conditioned facility and were given a standard dinner, which was finished at least 10 hours before dosing in each period of the study. After an overnight fasting period of 10 hours, the subjects were administered the medications as per the randomization schedule, for the test or the reference products, with 240 ml of plain drinking water. The intake of the study formulations was closely monitored by a physician and the oral cavity was checked properly to ensure completion of the administration process. Subjects were instructed to remain seated upright for the first 2.0 hours after dosing. No meal was allowed until four hours after dosing. Drinking water was restricted from 1 hour before dosing till 1 hour after dosing and ad libitum thereafter. Lunch, snacks and dinner were served as per the scheduled time. All the subjects were abstained from any xanthine-containing food or beverages or alcoholic products for 72 hours prior to formulation administration and throughout the sampling schedule during each period. Subjects were informed not to take any drug at least 14 days prior to the study. No concomitant medication was permitted during the study period.

Blood Sampling: Blood samples (4 ml) were collected through an indwelling cannula placed in a forearm vein using coded, sterile vacutainers containing ethylenediamine tetraacetic acid (EDTA) as an anticoagulant. Blood samples were obtained immediately prior to dosing (predose sampling, 0.00 h) and at 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00, 10.00, 11.00, 12.00, 14.00, 16.00, 24.00, 48.00 and at 72.00 hours after dosing. Blood samples were centrifuged at 3500 rpm (at 0-4 C) for five minutes. The plasma was separated and stored frozen at -20C 5C until assayed. During the study periods, all the subjects were under medical supervision. Vital signs were examined at scheduled time as per the protocol. Analytical procedure: A validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was used for determination of seratrodast concentration in human plasma. Equipment used was SILHTc HPLC model of Shimadzu and API 3000 MS/MS model of MDS-Sciex. Column type used was BDS Hypersil (150 mm 4.6 mm, i.d.) 5 and the mobile phase used was Methanol:Ammonium formate buffer (2mM) at pH 7.5 (90:10). The data acquisition system used was Analyst 1.4.2 of Applied Bio System. Procedures of validation and acceptance criteria were based on FDA Bio-analytical Method validation guidelines (15). The bioanalytical method was validated for sensitivity, specificity, linearity, accuracy and precision (repeatability and reproducibility), percent recovery and stability of samples (freezethaw stability, bench-top stability, autosampler stability, short-term and long-term stability of stock solution and internal standard). The linearity range of the analytical method was 0.353 g/ml to 35.317 g/ml for Seratrodast. Plasma samples were withdrawn from the deep freezer as per planned activity and allowed to thaw at room temperature. The thawed samples were vortexed to ensure complete mixing of contents. Aliquot of 200l of plasma sample was drawn into pre-labeled tubes, to which internal standard stock dilution was added. The samples were vortexed to ensure complete mixing. Methyl tert-butyl ether was added and the samples were vortexed again for 10 min. The samples were centrifuged at 4C for 5 min at 5000 rpm, after which the organic layer was separated, evaporated at 50C for 15 min under nitrogen gas and reconstituted with 2 ml of mobile phase. The reconstituted solution was then transferred into the auto sampler for analysis. Pharmacokinetic analysis: All pharmacokinetic parameters were determined by non-compartmental method model using SAS Software (version 9.2). Values below quantification limit (<0.353 g/ml) were set to zero for calculation purposes. The maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) were taken directly from observed concentration vs. time data. The elimination rate constant (Kel) was estimated by a non-linear least square regression analysis of the individual concentrations observed as a function of time during the elimination phase. The apparent elimination half life (t) was obtained by dividing 0.693 by Kel. The area under the curve (AUC) of seratrodast in plasma from time zero to last quantifiable time point (t), AUC0-t , was calculated using the linear trapezoidal rule. The AUC from time zero to infinity, AUC0-inf, was calculated from the sum of AUC0-t and Clast/Kel, where Clast is the last measurable concentration of seratrodast in plasma. The test and the reference formulations were considered to be bioequivalent if the calculated 90% confidence intervals (C.I.s) for the log transformed ratios (test/reference) of the Cmax, AUC0-t, and AUC0-inf were within the bioequivalence criteria range of 80.00-125.00 as established by the Central Drug Standard Control Organization (CDSCO), India; US Food and Drug Administration (US FDA) and European Medicines Evaluation Agency (EMEA).

Statistical Analysis: Statistical analysis for establishing bioequivalence was performed using the statistical package of SAS 9.2. PROC GLM was used for the estimation of least square mean differences (test-reference) of the test and reference products on the log-transformed pharmacokinetic parameters Cmax, AUC0-t and AUC0-inf and the corresponding standard errors of the differences were computed. The plasma concentrations at each sampling time points and pharmacokinetic parameters were tabulated for each subject and product combination, together with descriptive statistics including mean, standard deviation, coefficient of variation, median and range for each product at each scheduled sampling time point. Analysis of variance (ANOVA) was performed (=0.05) on the log-transformed pharmacokinetic parameters Cmax, AUC0-t and AUC0-inf. The analysis of variance model included sequence, subjects nested within sequence, period and treatment as factors. Each analysis of variance included calculation of least-square means, adjusted differences between formulation means and the standard error associated with these differences. The significance of the sequence effect was tested using the subjects nested within the sequence as the error term. Ratio analysis was performed on geometric mean of test and reference for log transformed data. Consistent with the two one-sided tests for bioequivalence, 90% confidence intervals were constructed for geometric mean of test and reference of the log-transformed Cmax, AUC0t and AUC0-inf. RESULTS: Twenty six healthy male subjects, including 2 subjects as standby to replace dropouts, were included in the study. No dropouts were observed in the study. Thus twenty six adult males with a mean age (S.D.) of 29.77 6.60 years (range 19-41 years), a mean weight of 63.95 6.99 kg (range 49.10-77.30 kg) and a mean height of 166.92 6.09 cm (range 156.40-180.50 cm) completed the study. The two formulations were well tolerated by the subjects. No adverse event was observed during both the periods of the study in any of the subjects. Both clinical and laboratory parameters of all subjects showed no clinically significant changes. Figure 2 shows the plots of mean serum concentrations of seratrodast vs. time. Both the formulations were rapidly absorbed and detected from 0.50 hour in plasma.
Mean conc. of Seratrodast (g/ml) 6.000 4.000 2.000 0.000 0 1 2 3 4 6 8 Time (hrs) 10 12 16 48

Mean Linear Plot: Seratrodast conc. vs. time

Seratrodast 80mg (Test)

Figure 2: Mean plasma concentration vs. time curves of the test and reference tablets, each containing 80mg Seratrodast Mean pharmacokinetic parameters of seratrodast for the test and the reference formulation, in 26 healthy Indian subjects are presented in Table 1.

Table 1: Mean pharmacokinetic parameters of the test and the reference formulations, each containing 80 mg Seratrodast
Parameters Cmax (g/ml) AUC0-t (g.h/ml) AUC0-inf (g.h/ml) tmax (h) Kel (h-1) t (h) Test (mean S.D.) 5.38 0.77 80.41 20.11 99.78 23.27 3.48 0.61 0.034 0.012 22.66 6.69 Reference (mean S.D.) 5.25 0.74 76.67 21.02 97.32 26.61 3.56 0.70 0.038 0.022 22.87 8.92

The results of ANOVA for log transformed data of Cmax, AUC0-t and AUC0-inf showed statistically significant variation for subject nested sequence (P<0.05) and no statistically significant variation for effect of sequence, period and period (P>0.05). The intra-subject variation, calculated using mean square error obtained from the logarithmically transformed Cmax, AUC0-t and AUC0-inf values, were 12.53, 17.03 and 20.57% respectively. Additionally the 90% C.I.s for the ratios of mean Cmax, AUC0-t, and AUC0-inf were within the range of 80.00 to 125.00 (using log transformed data), meeting the regulatory criterion for bioequivalence as mentioned above. Table 2 represents the ratio (test/reference), 90% confidence interval and the intra-subject variations of the Cmax, AUC0-t, and AUC0-inf. Table 2: Ratio (Test/Reference), 90% confidence interval and intra-subject variation following the administration of 80 mg seratrodast tablets Ratio (Test/Reference %) 102.43 105.32 103.82 90% Confidence Interval (log transformed data) 96.54 108.69 97.20 114.12 94.26 114.35 Intrasubject Variability (log transformed data %) 12.53 17.03 20.57

Parameters Cmax AUC0-t AUC0-inf

For overall extent of absorption, both the formulations were equivalent, with test/reference formulation ratios of both AUC0-t and AUC0-inf very close to 100%. Based on the plasma levels of the 26 completed subjects, the mean relative bioavailability of seratrodast was 105.32% as compared with the reference. The pharmacokinetic data for each subject are illustrated in Table 3. Table 3: Individual pharmacokinetic parameters of seratrodast 80 mg tablets
Subject No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Cmax (g/ml) T R 5.709 6.043 5.730 5.081 6.306 5.253 5.121 4.946 5.388 5.643 5.798 5.763 5.470 5.220 5.314 5.466 5.274 4.643 5.077 4.920 5.877 5.668 5.353 5.054 5.084 4.523 4.460 4.292 tmax (h) T R 3.0 4.0 3.5 3.5 4.0 3.0 2.5 4.0 3.0 2.5 4.0 3.0 4.0 3.0 3.5 4.0 3.0 3.5 2.5 3.5 3.5 4.0 5.0 3.0 3.5 3.0 4.0 4.0 AUC0-inf (g.h/ml) T R 121.6248 144.8662 84.4466 61.7960 151.5447 122.5141 83.7534 67.7096 109.5716 111.3017 65.9065 104.5380 76.3624 107.5553 77.8105 82.8004 90.9857 93.5469 90.2350 77.3445 112.2295 112.6999 76.3350 119.5947 146.3265 113.1865 82.1775 47.0114 t(h) T R 23.3673 30.2578 15.9977 8.4688 32.7571 34.1810 18.0528 12.8280 29.2158 31.5351 13.8897 27.4688 10.0488 30.2544 12.5340 17.9079 17.1270 29.4204 17.5157 17.6973 28.2872 27.7939 12.7854 30.9643 26.0180 38.4411 28.5395 6.9593

15 16 17 18 19 20 21 22 23 24 25 26

5.191 5.568 5.430 7.429 6.006 3.981 5.060 3.589 5.384 5.910 6.234 4.220

6.157 6.542 5.707 4.916 4.560 5.622 4.049 4.551 7.087 4.585 4.337 5.874

2.5 4.0 3.0 3.0 4.0 3.5 3.0 4.0 4.0 3.0 3.5 4.0

3.5 2.0 4.0 5.0 4.0 3.5 3.5 4.0 4.0 3.5 2.5 5.0

80.2367 127.6900 120.3094 116.1491 83.3342 98.9126 111.2683 73.5653 110.3343 75.7537 102.6056 124.7994

128.6953 78.3293 133.0323 96.8265 73.2306 99.8076 71.0902 65.1101 150.7655 82.2749 84.8798 99.8999

17.5027 28.0157 28.9895 23.7414 19.2290 31.3880 32.0985 20.5669 25.3581 22.7968 23.4315 29.9150

35.9972 7.7492 26.0848 13.2006 20.6561 30.5946 18.7631 20.1427 23.4271 17.1822 18.0104 18.6681

(T=Test Formulation, R=Reference Formulation) DISCUSSION: This is the first bioequivalence study of seratrodast in India. This study assessed the bioequivalence of 80 mg seratrodast tablet formulation with Bronica 80 mg tablet of Takeda Pharmaceuticals Co., Ltd., Japan. The active pharmaceutical ingredient along with the test formulation (batch number FD/31A/12; manufacturing date April 2012) was indigenously manufactured by Zuventus Healthcare Ltd., India. Seratrodast has a great potential for the treatment of bronchial asthma. The information on the pharmacokinetic parameters of the formulations is warranted. The measured AUC and Cmax values following oral administration of both formulations (test and reference) maintained 90% confidence interval within 80.00-125.00 for the log transformed values, suggesting that the two formulations were bioequivalent. The estimated tmax, Cmax, and t of the test and the reference formulations in this study are consistent with previous investigations (16). Seratrodast was found to be well tolerated in the present study. No adverse effects were reported or observed in any of the subjects. In summary this study has demonstrated the bioequivalence of the 80 mg seratrodast tablet manufactured by Zuventus Healthcare Ltd., India and the reference product, Bronica manufactured by Takeda Pharmaceuticals Co., Ltd., Japan. Hence it can be concluded that the two formulations can be used interchangeably in clinical settings.
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