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 Table of Contents  
Year : 2014  |  Volume : 2  |  Issue : 2  |  Page : 160-164

A randomized two-way crossover comparative pharmacokinetic study of two different tablet formulations containing ilaprazole in healthy human Indian volunteers

1 Bioequivalence Study Centre, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India
2 Department of Pharmacology, Midnapore Medical College and Hospital, Paschim Medinipur, West Bengal, India

Date of Web Publication11-Nov-2014

Correspondence Address:
Tapan Kumar Pal
Bioequivalence Study Centre, Department of Pharmaceutical Technology, Jadavpur University, Kolkata - 700 032
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2321-4848.144324

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Background: Proton Pump Inhibitors (PPI) are observed to be great healer in gastroesophageal reflux disorder (GERD) and duodenal ulcer. Quantification of the drugs in human plasma by validated bioanalytical method are very important to determine pharmacokinetic parameters for undergoing comparative study with standard available formulations to make the newer one commercially available. Objective: The objective of this study was to determine the relative bioavailability of Ilaprazole, a novel PPI comparing the test formulation to the reference one according to standard regulatory guidelines. Materials and Methods: The bioequivalence of two tablet formulations, one as reference and other as test containing 10 mg of ilaprazole [CAS No. 172152-36-2] was studied in 12 healthy Indian volunteers. This was a single dose, twoperiod and randomized crossover study separated with a washout period of one week. Plasma samples for pharmacokinetic analysis were collected before dosing and at pre-specified time points after dosing. The concentration of ilaprazole in plasma was determined by a validated HPLC-UV method using theophylline as internal standard. The formulations were compared using the parameters Area under the plasma concentration-time curve (AUC 0-t ), Area under the plasma concentration-time curve from zero to infinity (AUC 0-͵), Peak plasma concentration (C max ), and time to reach peak plasma concentration (t max ). Results: Mean AUC 0-t of test and reference product were calculated to be 2627.793 ± 154.989 ng h ml−1 and 2555.905 ± 225.916 ng h ml−1 , with a C max of 347.459 ± 48.175 ng h ml−1 . While mean AUC 0-͵ of test and reference product were calculated to be 2733.334 ± 242.438 ng h ml−1 and 2728.716 ± 284.408 ng h ml−1 . Conclusion: The results of this investigation indicated no statistically significant differences between the logarithmic transformed AUC 0-͵ and C max values of the two preparations. The 90% confidence interval for the ratio of the logarithmic transformed AUC 0-t , AUC 0-͵ and C max were 2 within the bioequivalence limit of 80-125% and the relative bioavailability of test formulation was 102.81% to that of reference formulation. The results of this study in healthy human volunteers of 27.92 ± 5.12 yrs (average age), 171.28 ± 6.85 cm (average height) and 66.43 ± 5.21 kg (average weight) support the use of the 10 mg dose tablet newly formulated.

Keywords: Bioequivalence, HPLC-UV, ilaprazole, pharmacokinetics

How to cite this article:
Dan S, Choudhury H, Sarkar P, Gorain B, Barik A, Ghosh B, Pal TK. A randomized two-way crossover comparative pharmacokinetic study of two different tablet formulations containing ilaprazole in healthy human Indian volunteers . Arch Med Health Sci 2014;2:160-4

How to cite this URL:
Dan S, Choudhury H, Sarkar P, Gorain B, Barik A, Ghosh B, Pal TK. A randomized two-way crossover comparative pharmacokinetic study of two different tablet formulations containing ilaprazole in healthy human Indian volunteers . Arch Med Health Sci [serial online] 2014 [cited 2023 Mar 29];2:160-4. Available from: https://www.amhsjournal.org/text.asp?2014/2/2/160/144324

  Introduction Top

Proton pump inhibitors (PPIs) have been used therapeutically for many years and shown great efficacy in accelerating ulcer healing. The newer molecule ilaprazole {2-[(4-methoxy-3-methyl)-2-pyridinyl]methylsulfinyl-5-(1H pyrrol- 1-yl)-1H-benzimidazole[CAS No. 172152-36-2, a novel PPI, is found to significantly prevent the development of reflux oesophagitis and gastric secretion in a dose-dependent manner as an outcome of one clinical study on patients with gastroesophageal reflux disorder (GERD) and the newer PPI has also shown efficacy in treatment of duodenal ulcer in a detailed comparative study on patients with DU. [1],[2] Currently, researches are focused on more potent PPIs. Some preclinical studies have shown that ilaprazole might be such a new substitute. [3] It is usually rapidly absorbed after oral administration. Ilaprazole might be dominantly metabolized in the liver by CYP3A and partly by CYP2C19. In all, in vitro and in vivo evidence supporting CYP3A and CYP2C19 both the types display roles on the systemic elimination of ilaprazole, which has been demonstrated by the study of Y. Li et al.2008. [4] Previous work done on bioequivalence study of ilaprazole by validated LCMS/MS method have shown the values of t 1/2 , AUC 0-͵, C max are 4.5 h, 500-900 ng h mL−1 , 90-102 ng mL−1 , respectively, on ilaprazole 5 mg oral administration to healthy human volunteers. The drug is primarily excreted in the urine as drug metabolites and unchanged drug to some extent. [5]

A rapid, sensitive, cost effective high performance liquid chromatography (HPLC) method with ultraviolet (UV) detection technique has been developed for the pharmacokinetic study of ilaprazole in human plasma. The method was reproducible and validated as per the guidelines.

Bioavailability and bioequivalence issues have been an increasing concern to drug regulatory authorities for assessment of the safety and efficacy of drug products. As the number of synonymous drug products increase, special attention in bioavailability issues becomes a major concern. [6] Bioequivalence of two formulations of the same drug comprises equivalence with respect to the rate and extent of absorption while the area under plasma concentration-time curve (AUC) generally serves as the characteristic of the extent of absorption. [7] No single parameter reliably measures the rate of absorption. For instance, the maximal drug concentration (C max ) has been widely used, but it depends more on the fraction absorbed than the rate of absorbed; the time of maximal drug concentration (t max ) depends on both absorption and elimination rates. [8]

The objective of the present study was to evaluate the relative bioavailability of tablet ilaprazole (containing ilaprazole enteric-coated tablet 10 mg) in comparison with Noltec tablet 10 mg (containing ilaprazole enteric-coated tablet 10 mg) in 12 healthy male adult human Indian volunteers under fasting condition.

  Materials and Methods Top

Materials and reagents

Acetonitrile (ACN), ethyl acetate were purchased from Merck (MERCK India Ltd., Mumbai) and Spectrochem (Spectrochem Pvt. Ltd., Mumbai, India) supplied potassium dihydrogen phosphate buffer. All solvents used were of HPLC grade. Other chemicals and reagents of analytical grade were used throughout the study. HPLC grade water with a resistivity of 18 MΩ was taken from a Milli-Q gradient system of Millipore (Elix, Milli-Q A10 Academic, Bedford, MA, USA). The pooled blank human plasma with EDTA-K 3 anticoagulant was collected from Clinical Pharmacological Unit (CPU) of Bioequivalence Study Centre, Jadavpur University, Kolkata (India).

Products studied

Test product

Tablet Ilaprazole (containing ilaprazole enteric-coated tablet 10 mg) manufactured by M/S Precise Chemipharma Pvt. Ltd., Malwa Unit No.108, ES Patanwala Industrial Estate, LBS Marg, Ghatkopar (W), Mumbai - 400 086, India [Batch No.: IZ-004, Mfg. Date: 03/2011, Exp. Date: 02/2013].

Reference product

Noltec Tablet 10 mg (containing ilaprazole enteric-coated tablets 10 mg) manufactured by II-Yang Pharma, Korea [Batch No.: H001, Mfg. Date: Mar/2009, Exp. Date: 24/02/2013].

Study design

Healthy, normal twelve Indian human volunteers were enrolled for this study. The volunteers were clinically examined by the doctor and after confirming that they did not have any history of smoking, alcohol consuming, and participation on any other study in previous 2-3 months, they were included in the study. Generally, the informed consent process of the volunteers was followed as per the protocol after obtaining approval by the Drugs Controller General of India (DCGI), New Delhi and Institutional Ethical Committee (IEC) of Jadavpur University, Kolkata (India). Average age, height, and weight of the volunteers were 27.92 ± 5.12 years, 171.28 ± 6.85 cm, and 66.43 ± 5.21 kg, respectively. Informed consent was obtained from all the subjects prior to the start of this randomized, single dose, fasting, two-period, two-sequence crossover study with one-week washout period. [9],[10] The study was carried out in compliance with Good Clinical Practice (GCP) and Declaration of Helsinki.

Drug administration and sample collection

All 12 volunteers, as stated before under the heading "Study design," were assembled in the CPU. After overnight fasting of 10 h, they received their individual drug as per randomization. They received either the test drug or the reference drug with 240 mL of water. The volunteers must be ensured that they did not consume caffeinated or alcoholic beverages at least 72 h prior to drug administration or during the study days. Usually, the sampling time should extend to at least three terminal elimination half-lives of the active ingredient. The time periods between sampling should not exceed one terminal half-life. [11],[15]

A total of 14 blood samples were collected at 0 h (before drug administration) and 0.5, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 6.00, 10.00, 18.00, 24.00, and 36.00 h (after drug administration) in the test tubes containing EDTA at each time point. Breakfast, lunch, and dinner were provided after 4 h, 8 h, and 14 h, respectively, after drug ingestion. On study days, volunteers were permitted normal activities, excluding strenuous exercise.

Collected blood samples were centrifuged immediately; plasma was separated and stored in frozen condition at −20 0 C with appropriate labeling of volunteer code no., study date, and collection time till analysis.

Sample preparation

In a stoppered test tube, 1.0 mL of plasma was taken. To this 0.1 mL of Internal Standard (IS; theophylline 2000 ng mL−1 ) was added and mixed for 30 s. Following this, 6.5 mL of ethyl acetate was added and mixed for 10 min and centrifuged for 5 min at 4000 rpm. Approximately 5 ml of the organic layer was separated and evaporated in presence of nitrogen atmosphere at low temperature (40°C-45°C). The residue was reconstituted with 200 μL mobile phase and an aliquot of 20 μL was injected in HPLC System. Peak areas of ilaprazole and IS were recorded for statistical analysis and interpretation of data.

Chromatographic conditions

Processed and reconstituted plasma samples were analyzed for ilaprazole by HPLC. The HPLC system was of Shimadzu, Japan with a BDS Hypersil C 18 (250 × 4.6 mm, 5 μ particle size) column. The wave length for detection was 274 nm and Spinchrom integrating software was used. Mobile phase consisting of ACN with 10 mM potassium dihydrogen phosphate (pH 7) in 20:80 (v/v) ratio with a 1.0 mL/min flow rate were set. The method was validated as per the standard guidelines. [13]


Serial dilutions (100-5000 ng mL−1 ) of the ilaprazole stock solution were prepared using mobile phase. Each of these dilutions (0.1 mL) was added to 0.9 mL screened plasma to furnish concentrations in the range 10-500 ng mL−1 . These samples were processed as stated previously and analyzed by HPLC. The ratio of areas of the ilaprazole and theophylline (IS) peak were plotted against the concentration of ilaprazole [Figure 1].
Figure 1: Calibration plot for ilaprazole in acetonitrile – 10mM potassium dihydrogen phosphate (pH 7) 20:80 (v/v) after spiking of plasma with the pure drug. Regression equation, y = 0.0373x + 0.4997, correlation coefficient, r, 0.9995

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Method Validation

The method was validated for accuracy, precision, sensitivity, recovery, linearity, and stability in accordance with current updated version of FDA guidelines, using theophylline as IS. [12] Linearity, precision, and accuracy were tested by analysis of five batches of spiked plasma quality-control (QC) samples. Each batch of spiked plasma samples included one complete set of calibration standards (comprising six different concentrations, blank, and blank with internal standard) and QC samples at low (30 ng mL−1; LQC), middle (200 ng mL−1; MQC), and high (400 ng mL−1; HQC) concentrations.

Within-run and between-run precision values (%CV) ranged from 5.062% to 9.570%. Within-run and between-run accuracy values (% nominal) were 92.40% for LQC, 94.36% for MQC, and 93.22% for HQC samples. Recovery after extraction was calculated to be 95.69-98.32% by comparing the peak areas of the plasma LQC, MQC, and HQC samples to the absolute peak area of the unextracted standards containing the same concentrations of ilaprazole. The stability of LQC, MQC, and HQC samples were determined after three freeze thaw cycles comparing against freshly thawed samples of the same concentration. The stability of ilaprazole ranges between 98.56-98.97% after three cycles.

Application to comparative pharmacokinetics study

The validated HPLC-UV method is successfully applied to the bioequivalence study of ilaprazole evaluating the relative bioavailability of tablet ilaprazole (containing ilaprazole enteric-coated tablet 10 mg) in comparison with Noltec Tablet 10 mg (containing ilaprazole enteric-coated tablet 10 mg) in 12 healthy male adult human Indian volunteers under fasting condition.

Pharmacokinetic analysis

Plasma levels of ilaprazole 10 mg (both test and reference) for every volunteer at each time point were plotted to obtain time-plasma concentration curves for the study preparations. The mean pharmacokinetic parameters of bioavailability calculated for this single-dose study were maximum plasma concentration (C max ), time to maximum plasma concentration (t max ), the area under plasma concentration vs. time curve (AUC 0-t ), the area under plasma concentration vs. time curve 0 to infinity (AUC 0-͵), elimination half life (t 1/2 ), and elimination rate constant (K el ) from the analytical data.

The last seven quantifiable levels were used to determine K el . AUC 0-t was calculated from the measured levels, from time zero to the time of last quantifiable level, by the linear trapezoidal rule. AUC 0-͵ was calculated according to the following formula: AUC 0-͵ = AUC 0-t + C last /K el , where C last is the last quantifiable plasma level. t 1/2 was calculated using the conventional formula 0.693/K el . Tolerability of ilaprazole was assessed by monitoring and subjects interview regarding the potential presence of adverse events.

Statistical analysis

For each subject, descriptive statistics were used to summarize the estimated pharmacokinetic parameters. For the purpose of bioequivalence analysis, AUC 0-t , AUC 0-͵, and C max values were considered primary variables. Their log-transformed data were analyzed by an analysis of variance (ANOVA), including treatment, period, and subject. The bioequivalence analysis was made according to guidance of Committee for Proprietary Medicinal Products (CPMP): "test" product was considered bioequivalent to "reference" product if 90% confidence interval (CI) for the ratio between each parameter fell within the predetermined equivalence range of 80-125%. [14]

  Results Top

The maximum plasma concentrations of 328.640 + 57.710 ng mL−1 (C max ) at the time 4.667 + 0.985 h (t max ) were found for the reference product whereas the C max and T max for the test product containing 10 mg of Ilaprazole were 347.459 + 48.175 ng mL−1 and 4.833 + 1.030 h, respectively.

Administration of the Reference preparation, Noltec Tablet 10 mg, produced the AUC 0-t 2555.905 ± 225.916 ng hr mL−1 , whereas administration of the test preparation produced AUC 0-t 2627.793 ± 154.989 ng hr mL−1 [Table 1]. Mean plasma concentration versus time curves after administration of "reference" and "test" products to healthy subjects are shown in [Figure 2]. The plasma t 1/2 for reference and test preparations was 5.386 ± 0.767 h and 5.501 ± 0.689 h, respectively.
Table 1: Mean pharmacokinetic parameters of Ilaprazole enteric coated tablet 10 mg for test and reference product (± SD, n = 12)

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Figure 2: Mean (n = 12) plasma concentration-time profiles after administration of "test" and "reference" formulations in healthy Indian volunteers

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On the basis of comparison of the AUC 0-t for ilaprazole after single-dose administration, the relative bioavailability of the test preparation ilaprazole was 102.81% that of the reference preparation, Noltec Tablet 10 mg The 90% confidence interval for C max , values is represented in the [Table 1]. The observed t max values for "test" product were within the acceptable limits (±20% of the mean values of "reference" product).

  Discussion Top

The aim of bioequivalence study is to assure interchangeability between an innovator and a generic formula in terms of efficacy and safety. When a pharmacological effect is difficult to measure, the plasma levels of a drug may be used as an indicator of clinical activity. Therefore, ilaprazole plasma levels obtained in this study suggest an equal clinical efficacy of the two brands tested and provide pharmacokinetic data from Indian population. The developed bioanalytical method for the determination of ilaprazole in human plasma was sensitive, fast, and accurate.

It has been observed from the present study that the subjects formed a homogeneous population in terms of age, weight, and body mass index. Ilaprazole was well-tolerated, and there were no dropouts as well as no discomforts after administration to the volunteers.

From the literature review, it has been learnt that ilaprazole represents itself in two enantiomers in the body, i.e., d-ilaprazole and l-ilaprazole but both enantiomers possess the PPI activity. Therefore, imbalance between these two forms does not result in failure of drug action. This is proved from the successful application of this non-enantiomer selective HPLC-UV method on the pharmacokinetic study. Almost equal elimination rate, t 1/2 , and t max of the reference and test products indicate bioequivalence, and it shows no effect of the variation in pharmaceutical formulation on the pharmacokinetic parameters of the drug.

  Conclusion Top

The 90% CI of C max , AUC 0-t and AUC 0-͵ were in the acceptable range of 80-125% ANOVA (subject, period, treatment) was applied to the C max , ln C max , AUC 0-t , and ln AUC 0-t values. There was no statistically significant difference for the treatment values. Both formulations were equal in terms of rate and extent of absorption. On the basis of pharmacokinetic parameters studied, it can be concluded that the "test" product is bioequivalent with the "reference" product.

  Acknowledgement Top

The authors are thankful to Department of Science and Technology (DST), New Delhi (India) for providing us the necessary instrumental facilities and M/S Precise Chemipharma Pvt. Ltd., Mumbai, India for sponsoring this bioequivalence study. Author Shubhasis Dan is also thankful to UGC-BSR for providing followship.

  References Top

1.Kumar D, Singh GP. Comparative study of Omeprazole, Rabeprazole and Ilaprazole for their efficacy in patient suffering from reflux esophagitis with symptoms. IOSR J Dent Med Sci2014;13:7-12.  Back to cited text no. 1
2.Bohidar NP, Krishna K, Panda BK, Patel C. Ilaprazole: Is this a superior proton pump inhibitor for duodenal ulcer? Trop Gastroenterol 2013;34:95-8.  Back to cited text no. 2
3.Wang L, Zhou L, Lin S, Hu H, Xia J. A new PPI, Ilaprazole compared with omeprazole in the treatment of duodenal ulcer: A randomized double-blind multicenter trial. J Clin Gastroenterol 2011;45:322-9.  Back to cited text no. 3
4.Li Y, Zhang W, Guo D, Zhou G, Zhou H, Xiao Z. Pharmacokinetics of the new proton pump inhibitor ilaprazole in Chinese healthy subjects in relation to CYP3A5 and CYP2C19 genotypes. Clin Chim Acta 2008;391:60-7.  Back to cited text no. 4
5.Tan Z, Ouyang D, Chen Y, Zhou G, Cao S, Wang Y, Peng X, et al. Development and validation of a LC-MS/MS method for the determination of ilaprazole enantiomers and its application to a bioequivalence study in healthy Chinese volunteers. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:2072-6.  Back to cited text no. 5
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8.Schulz HU, Steinijans VW. Striving for standards in bioequivalence assessment: A review. Int J Clin Pharmacol Ther Toxicol 1992;30 Suppl 1:S1-6.  Back to cited text no. 8
9.Farolfi M, Power JD, Rescigno A. On the determination of bioequivalence. Pharmacol Res 1999;39:1-4.  Back to cited text no. 9
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11.Mandal U, Jayakumar M, Ganesan M, Senthil DR, Pal TK, Gowda VK. Bioequivalence Study on quetiapine fumarate tablets by HPLC. Indian Drugs 2005; 42(6); 353-356.   Back to cited text no. 11
12.U. S. Food and Drug Administration. Guidance for Industry: Bioavailability and Bioequivalence Studies for orally Administered Drug Products- General Considerations. Rockville, MD: Center for Drug Evaluation and Research. 2000. www.fda.gov/downloads/Drugs/.../Guidances/ucm070124.pdf  Back to cited text no. 12
13.Gowda KV, Rajan DS, Mandal U, Selvan PS, Sam Solomon WD, Bose A. Evaluation of bioequivalence of two formulations containing 100 miligrams of aceclofenac. Drug Dev Ind Pharm 2006;32:1219-25.   Back to cited text no. 13
14.EMEA. Note for guidance on the investigation of bioavailability and bioequivalence CPMP/EWP/QWP/1401/98; 2002.  Back to cited text no. 14
15.Pal TK, Nandi U, Gorain B, Choudhury H, Roy B. Development and validation of a high-performance liquid chromatographic method with ultraviolet detection for quantitation of lornoxicam in human plasma: Application to bioequivalence study. J Pharm Res 2011;4(9).  Back to cited text no. 15


  [Figure 1], [Figure 2]

  [Table 1]

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