Bioequivalence Study of Two Formulations of Ibuprofen Arginine Granules in Healthy Chinese Adult Subjects

Abstract

Objective

To evaluate the bioequivalence and safety of the test and reference formulations of ibuprofen arginine granules (400 mg) in healthy Chinese adult subjects under fasting and fed conditions.

Methods

This was a randomized, open-label, single-center, single-dose, two-treatment, two-sequence, two-period crossover bioequivalence study conducted in separate fasting and fed cohorts. A total of 84 healthy Chinese adults were enrolled, including 34 subjects in the fasting cohort and 50 subjects in the fed cohort. Subjects were randomized to receive either the test formulation followed by the reference formulation or the reverse sequence, with a washout period of at least 2 days between treatments. Plasma ibuprofen concentrations were measured using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Pharmacokinetic parameters were calculated using non-compartmental analysis, and bioequivalence was assessed based on geometric mean ratios (GMRs) and corresponding 90% confidence intervals (CIs) for Cmax, AUC0-t, and AUC0-∞.

Results

Under fasting conditions, the GMRs (90% CIs) of the test versus reference formulation for AUC0-t, AUC0-∞, and Cmax were 1.0008 (0.9698–1.0327), 1.0023 (0.9710–1.0346), and 0.9825 (0.9383–1.0288), respectively. Under fed conditions, the corresponding values were 0.9916 (0.9755–1.0081), 0.9776 (0.9432–1.0133), and 0.9982 (0.9431–1.0567). All 90% CIs were within the predefined bioequivalence acceptance range of 80.00%-125.00%. No serious adverse events, deaths, or study discontinuations due to adverse events occurred. All treatment-emergent adverse events were mild and resolved without sequelae.

Conclusion

The test and reference formulations of ibuprofen arginine granules were bioequivalent under both fasting and fed conditions in healthy Chinese adult subjects. Both formulations were generally safe and well tolerated, with comparable safety profiles.

Introduction

Ibuprofen is a widely used arylpropionic acid non-steroidal anti-inflammatory drug to be applied in clinical practice, with strong anti-inflammatory, anti-rheumatic, antipyretic and analgesic effects.1 For a long time in the past, ibuprofen has been the cornerstone of pain management, especially for peripheral pain conditions such as rheumatoid arthritis, osteoarthritis and musculoskeletal disorders. It reduces the generation of key mediators of inflammation and pain at the inflammatory site - prostaglandins (PG) by inhibiting cyclooxygenase (COX-1 and COX-2), thereby alleviating tissue congestion, swelling and sensitivity to peripheral nerve pain. However, long-term use of ibuprofen carries risks such as gastrointestinal complications, renal function impairment and cardiovascular problems.

Ibuprofen arginine granules have been approved in China for the relief of pain and fever. The addition of L-arginine enhances the solubility and absorption rate of ibuprofen.2 A new formulation of ibuprofen arginine granules has been developed to support generic drug registration in China. According to the National Medical Products Administration (NMPA) Technical Guidelines3 for Human Bioequivalence Studies of Chemical Generic Drugs, demonstration of bioequivalence is required to establish therapeutic equivalence between a generic product and its reference product. Similar crossover bioequivalence studies4 have been widely applied to support generic drug development and regulatory approval in Chinese populations. Therefore, comparative pharmacokinetic evaluation under both fasting and fed conditions is necessary before regulatory approval can be granted.

The present study was designed to evaluate the bioequivalence of a test formulation of ibuprofen arginine granules and the reference formulation following a single oral dose of 400 mg in healthy Chinese adult subjects under fasting and fed conditions. Safety and tolerability of the two formulations were also assessed.

Materials

Investigational Products

The test formulation was ibuprofen arginine granules manufactured and supplied by Haleon (Suzhou) Pharmaceutical Co., Ltd. (former Wyeth Pharmaceutical Co., Ltd) at a quantity of 400mg ibuprofen per sachet. The reference formulation was ibuprofen arginine granules (under the product name of Spedifen^®) manufactured by Hainan Zambon Pharmaceutical and supplied by Wyeth Pharmaceuticals at a quantity of 400mg ibuprofen per sachet.

Instruments

The experimental instruments include the LC-20AD High Performance Liquid Chromatography Device supplied by Shimadzu Corporation of Japan and the API 4500 mass spectrometer manufactured by AB Sciex of the USA

Selection of Subjects

This study was registered with the Clinical Trials registry (http://www.clinicaltrials.gov; NCT05737069). It was approved by the Clinical Trial Ethics Committee of Chengdu City No. 5 People’s Hospital [Approval No.: EC approval 2022–021 (medicine)-01]. A valid informed consent was obtained from all subjects before any research procedure. This study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice (GCP) principles.

Eligibility Criteria

Healthy Chinese subjects (male and female) aged range of 18–50 years with a body mass index (BMI) range of 19 to 26 kg/m², body weight of male was ≥50 kg, and that female was ≥45 kg. Subjects with a negative polymerase chain reaction (PCR) or antigen test result for novel coronavirus pneumonia (COVID-19) were able to provide a valid informed consent and willing and able to follow all study procedures were included in the study.1. Subjects with any significant abnormality in medical history (allergies, prolonged QT interval, tests for drug abuse or positive on a urine screening) were excluded. Other main exclusions were as follow: subjects smoked or regularly used tobacco or nicotine-containing products within 6 months before screening or refused to discontinue the use of tobacco or nicotine-containing products during the study.

Sample Size Calculation

Sample size was estimated by PASS software (Version 11.0.7 or higher; NCSS, Kaysville, UT, USA). Sample size of fasting cohort, GMR was 0.95, α was 0.05, power was 90%, intrasubject CV = 23%, and the equivalent confidence limit was set to 80.00 to 125.00%, the estimated sample size for the fasting cohort was 32 subjects. The sample size of the fed cohort, GMR was 0.95, α was 0.05, power was 90%, intrasubject CV = 28% and the equivalent confidence limit was set to 80.00 to 125.00%, the estimated sample size for the fed cohort was 46 subjects.

Study Design and Blood Sample Collection

This was a randomized, open-label, single-center, single-dose, two-treatment, two-sequence, two-period crossover bioequivalence study conducted in separate fasting and fed cohorts. A total of 34 healthy subjects were enrolled in the fasting cohort and 50 healthy subjects were enrolled in the fed cohort. Subjects were randomized in a 1:1 ratio to receive either the test formulation followed by the reference formulation (TR sequence) or the reverse sequence (RT sequence). A washout interval of at least 2 days was applied between treatment periods. The terminal elimination half-life of ibuprofen arginine is approximately 1.5–2 h; therefore, a washout period of at least 2 days (>20 half-lives) was considered sufficient to eliminate potential carry-over effects.

In the fasting cohort, blood collection time points included within 1 hour prior drug administration (0h) and 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, 120 min, 2.5, 3, 4, 6, 8, and 12 hours after administration. In the fed cohort, blood collection time points included within 1 hour before administration (0h) and 10, 30, 45, 60, 75, 90, 120 minutes (min), 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10 and 12 hours after administration. K2-EDTA anticoagulated blood collection tubes were used for every blood collection time, and approximately 3.0 mL of venous blood was collected at each timepoint.

In the fed cohort, subjects received a standardized high-fat, high-calorie breakfast containing approximately 800–1000 kcal, including about 500–600 kcal from fat (>50% of total calories), approximately 150 kcal from protein, and approximately 250 kcal from carbohydrates. Subjects completed the meal within 30 min and received study medication 30 min after meal completion.

Within 60 minutes after blood sample collection, plasma separation was performed through centrifugation at 700xg for 10 minutes at 2–8°C. The plasma sample was then divided into two aliquots of 1 mL. The two plasma samples were placed in 2 mL cryovials and stored directly in a −80°C (−60°C to −90°C) refrigerator until subsequent measurement procedures. Plasma ibuprofen concentrations were quantified using a validated LC-MS/MS method. The assay had a calibration range of 0.1–80.0 μg/mL with a lower limit of quantification (LLOQ) of 0.1 μg/mL.

Statistical Processing

Pharmacokinetic parameters, including Cmax, AUC0-t, AUC0-∞, Tmax, and t1/2, were calculated using a noncompartmental approach with Phoenix WinNonlin Version 8.0 (Certara, USA). Descriptive statistical analyses were performed using SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA). Bioequivalence analyses were conducted separately for the fasting and fed cohorts. The primary PK parameters (Cmax, AUC0-t, and AUC0-∞) were natural log-transformed and analyzed using an analysis of variance (ANOVA) model including treatment, period, and sequence as fixed effects and subject nested within sequence as a random effect. Tmax was analyzed using a nonparametric method, and the Hodges-Lehmann estimate of the treatment difference together with the corresponding 90% confidence interval was calculated. Geometric mean ratios (GMRs) and corresponding 90% confidence intervals (CIs) were calculated. Bioequivalence was concluded if the 90% CIs were entirely within the predefined acceptance range of 80.00%-125.00%.

Results

Subjects Disposition

84 healthy Chinese adult subjects were enrolled in this study (34 and 50 subjects were enrolled in the fasting and fed cohorts, respectively). The average age (±SD) of the subjects was 29.6 (±7.44) and 28.2 (±6.54) in the fasted and fed cohorts, respectively. All enrolled subjects completed the study and were included in the pharmacokinetic and bioequivalence analyses. Demographic characteristics were generally comparable between cohorts. (Table 1).

Table 1.

Demographic Characteristics of Study Participants

Characteristic	Fasting Cohort (N=34)	Fed Cohort (N=50)
Age (years), mean ± SD	29.6 ± 7.44	28.2 ± 6.54
Male, n (%)	25 (73.5)	32 (64.0)
Female, n (%)	9 (26.5)	18 (36.0)
Height (cm), mean ± SD	167.2 ± 7.39	167.0 ± 7.58
Weight (kg), mean ± SD	63.0 ± 7.91	60.8 ± 7.83
BMI (kg/m2), mean ± SD	22.5 ± 1.74	21.7 ± 1.74

Pharmacokinetic Parameters

With no observed protocol deviations, violations and premature study discontinuations, all randomized subjects (ie, 17 and 25 subjects for corresponding treatments sequences and 34 and 50 subjects in total for fasted and fed cohorts, respectively) were included in various analysis populations including safety set (SS), Pharmacokinetic Concentration Analysis Set (PKCS), Pharmacokinetic Parameter Analysis Set (PKPS) and Bioequivalence Analysis Set (BES).

Please refer to Table 2 and Table 3 for the randomized population, SS, PKCS, PKPS and BES data of the 34 randomized subjects in the fasting cohort and 50 randomized subjects in the postprandial cohort.

Table 2.

Analysis Sets-All Subjects in the Fasting Cohort

	Test Formulation-Reference	Reference Formulation-Test	Total
	n (%)	n (%)	n (%)
Randomized subjects	17 (100.0)	17 (100.0)	34 (100.0)
Safety set (SS)	17 (100.0)	17 (100.0)	34 (100.0)
Pharmacokinetic Concentration Analysis Set (PKCS)	17 (100.0)	17 (100.0)	34 (100.0)
Pharmacokinetic Parameter Analysis Set (PKPS)	17 (100.0)	17 (100.0)	34 (100.0)
Bioequivalence Analysis Set (BES)	17 (100.0)	17 (100.0)	34 (100.0)

Note: n=Number of subjects in a specific category, calculated as a percentage (%) based on the number of subjects enrolled.

Table 3.

Analysis Sets-All Subjects in the Postprandial Cohort

	Test Formulation-Reference	Reference Formulation-Test	Total
	n (%)	n (%)	n (%)
Randomized subjects	25 (100.0)	25 (100.0)	50 (100.0)
Safety set (SS)	25 (100.0)	25 (100.0)	50 (100.0)
Pharmacokinetic Concentration Analysis Set (PKCS)	25 (100.0)	25 (100.0)	50 (100.0)
Pharmacokinetic Parameter Analysis Set (PKPS)	25 (100.0)	25 (100.0)	50 (100.0)
Bioequivalence Analysis Set (BES)	25 (100.0)	25 (100.0)	50 (100.0)

Note: n=Number of subjects in a specific category, calculated as a percentage (%) based on the number of subjects enrolled.

Bioequivalence Evaluation

The pharmacokinetic parameters of ibuprofen following administration of the test and reference formulations are summarized in Table 4 and Table 5. Under both fasting and fed conditions, the test and reference formulations exhibited similar pharmacokinetic profiles, with comparable values of Cmax, AUC0-t, AUC0-∞, Tmax, λz, and t1/2.

Table 4.

Pharmacokinetic Parameters Under Fasting Conditions

Parameter	Test Formulation (N=34) Mean ± SD	Reference Formulation (N=34) Mean ± SD
Cmax (μg/mL)	55.11 ± 8.75	56.02 ± 8.50
Tmax (h)	0.33 (0.25, 0.33)	0.33 (0.33, 0.50)
AUC0-t (h·μg/mL)	124.63 ± 25.02	124.80 ± 26.30
AUC0-∞ (h·μg/mL)	127.45 ± 26.89	127.39 ± 27.82
λz (h−1)	0.318 ± 0.040	0.323 ± 0.038
t1/2 (h)	2.21 ± 0.29	2.17 ± 0.26
%AUCex (%)	2.04 ± 1.20	1.90 ± 0.85

Table 5.

Pharmacokinetic Parameters Under Fed Conditions

Parameter	Test Formulation (N=50) Mean ± SD	Reference Formulation (N=50) Mean ± SD
AUC0-t (h·μg/mL)	116.15 ± 22.39	117.13 ± 23.00
AUC0-∞ (h·μg/mL)	127.29 ± 25.29	132.53 ± 35.30
Cmax (μg/mL)	34.95 ± 8.90	35.06 ± 8.90
Tmax (h)	1.50 (0.75, 3.00)	1.50 (0.75, 2.50)
λz (h−1)	0.278 ± 0.081	0.270 ± 0.074
t1/2 (h)	2.78 ± 0.92	2.87 ± 0.86
%AUCex (%)	8.9 ± 10.3	9.8 ± 11.5

Upon analyzing the area under the plasma concentration-time curve from time, zero to last observed concentration at time t (AUC_0-t), area under the plasma concentration-time curve from time zero to time infinity (AUC_0-∞) and observed maximum plasma concentration (C_max) results of ibuprofen in fasted and fed states, it was found that the GMRs for test and reference products and their 90% CIs for the primary PK parameters AUC_0-t, AUC_{0- ∞}, and C_max in the fasted cohort were 1.0008 (0.9698, 1.0327), 1.0023 (0.9710, 1.0346), and 0.9825 (0.9383, 1.0288), respectively. Intra-subject CVs were 7.7% for AUC_0-t, 7.7% for AUC_{0- ∞}, and 11.3% for C_max. In the fed cohort, the GMRs for test and reference products and their 90% CIs for the primary PK parameters AUC_0-t, AUC_{0- ∞}, and C_max were 0.9916 (0.9755, 1.0081), 0.9776 (0.9432, 1.0133), and 0.9982 (0.9431, 1.0567), respectively. Intra-subject CVs for AUC_0-t, AUC_{0- ∞}, and C_max were 4.9%, 9.8%, and 17.1%, respectively. The 90% CIs of the GMRs were all within the regulatory acceptance interval of 0.8000 to 1.2500, demonstrating bioequivalence of the test and reference formulations under fasting and fed conditions.

A total of 50 and 34 subjects participated in the study administering 400mg of the test and reference formulations of oral ibuprofen arginine granules in the fed and fasting state, respectively. After calculation and analysis of GMRs and their 90 CIs for all primary PK parameters (AUC_0-t, AUC_{0- ∞} and C_max) of the test formulation and the reference formulation. It demonstrated that 90% CIs of GMRs for test and reference products all within the equivalence range requirements (80.00% −125.00%) for bioequivalence in the guidelines for human bioavailability and bioequivalence tests of pharmaceutical formulations.5,6

The study indicated that the test formulation and the reference formulation of ibuprofen arginine granules 400mg were bioequivalent in healthy Chinese adult subjects. The average plasma concentration-time curves on linear and semi-logarithmic scale are shown in Figure 1 and 2 respectively (fasted cohort: Figures 1a and 2a; fed cohort: Figures 1b and 2b).

Figure 1.

(A) Mean plasma concentration (±SD)-versus-time plot - linear scale - pharmacokinetic concentration analysis set - fasted cohort. (B) Mean plasma concentration (±SD)-versus-time plot - linear scale - pharmacokinetic concentration analysis set - fed cohort. Green indicates Test Product (T) and Blue indicated Reference Product (R).

Figure 2.

(A) Mean plasma concentration-versus-time plot - semi-log scale - pharmacokinetic concentration analysis set - fasted cohort. (B) Mean plasma concentration-versus-time plot - semi-log scale - pharmacokinetic concentration analysis set - fed cohort. Green indicates Test Product (T) and Blue indicated Reference Product (R).

Safety Evaluation

All the randomized subjects (ie, 34 and 50 subjects in the fasted and fed cohorts) were administered 400 mg dosages of the experimental drug (ibuprofen arginine granules) in the form of the test formulation or reference formulation. The incidence of AEs is summarized in Table 6 and Table 7.

Table 6.

Summary of Fasting Cohort AEs

	Test (n=34)			Reference (n=34)			Total (n=34)
	N	n	%	N	n	%	N	n	%
AEs	8	6	17.6	6	5	14.7	14	10	29.4
TEAE	8	6	17.6	6	5	14.7	14	10	29.4
SAEs	0	0		0	0		0	0
Test drug associated TEAE	1	1	2.0	1	1	2.9	2	2	5.9
Test drug associated SAE	0	0		0	0		0	0
TEAE caused study termination	0	0		0	0		0	0

Table 7.

Summary of Fed Cohort AEs

	Test (n=50)			Reference (n=50)			Total (n=50)
	N	n	%	N	n	%	N	n	%
AEs	13	11	22.0	11	10	20.0	24	19	38.0
TEAE	13	11	22.0	11	10	20.0	24	19	38.0
SAEs	0	0		0	0		0	0
Test drug associated TEAE	0	0		4	3	6.0	2	2	6.0
Test drug associated SAE	0	0		0	0		0	0
TEAE caused study termination	0	0		0	0		0	0

Upon conducting rigorous tests for the fasting cohort, we made the following observations pertaining to the 34 subjects: Whereby they were grouped according to formulations, the test formulation had 8 adverse events in 6 subjects and a 17.6% incidence rate of adverse reactions, while the reference formulation had 6 adverse events in 5 subjects and a 14.7% incidence rate of adverse reactions. Meanwhile, upon conducting tests for the postprandial cohort, we made the following observations pertaining to the 50 subjects: Whereby they were grouped according to formulations, the test formulation had 13 adverse events in 11 subjects and a 22% incidence rate of adverse reactions, while the reference formulation had 11 adverse events in 10 subjects and a 20% incidence rate of adverse reactions.

It is worth noting that no serious adverse events occurred in this study, and most adverse events were mild and had a good prognosis. In addition, the test formulation and the reference formulation showed similarities in adverse drug reactions, and there was no significant difference in their incidence rates.

In conclusion, the test formulation of ibuprofen arginine granules was bioequivalent to the reference formulation under both fasting and fed conditions in healthy Chinese adults. Both formulations demonstrated comparable pharmacokinetic characteristics and were generally safe and well tolerated.

Discussion

Ibuprofen arginine granules is a commonly used nonsteroidal anti-inflammatory drug (NSAID) indicated for the relief of fever and mild-to-moderate pain associated with conditions such as headache, toothache, dysmenorrhea, musculoskeletal pain, and minor injuries.2,7 As an orally administered formulation widely used in clinical practice, demonstration of bioequivalence is essential to ensure therapeutic equivalence between generic and reference products.

In the present study, the bioequivalence of the test and reference formulations of ibuprofen arginine granules was evaluated in healthy Chinese adults under both fasting and fed conditions. The results demonstrated that the 90% confidence intervals (CIs) for the geometric mean ratios of Cmax, AUC0-t, and AUC0-∞ were all within the predefined bioequivalence acceptance range of 80.00%-125.00%, confirming bioequivalence between the two formulations under both conditions.

Ibuprofen arginine is a salt formulation developed to improve the solubility and absorption of ibuprofen. Compared with conventional ibuprofen formulations, the addition of arginine may facilitate a faster dissolution rate and more rapid drug absorption, contributing to a quicker onset of analgesic action.2,5 This rapid absorption profile is consistent with published evidence suggesting that ibuprofen arginine may provide onset of analgesic activity within approximately 15–30 minutes after administration, a characteristic that supports its use in acute pain management.6,8,9 In the present study, the pharmacokinetic profiles of the test and reference formulations were highly comparable, supporting their pharmaceutical equivalence and interchangeability.

Food intake is known to affect the absorption characteristics of ibuprofen. Consistent with previous reports, absorption was slower under fed conditions, as reflected by a longer Tmax compared with fasting administration.1,2 However, the extent of drug exposure, represented by AUC0-t and AUC0-∞, remained comparable between the two formulations, indicating that food primarily influenced the rate rather than the extent of absorption. Importantly, these effects did not affect the bioequivalence conclusions.

Both formulations were generally safe and well tolerated. No serious adverse events, deaths, or study discontinuations due to adverse events occurred during the study. All treatment-emergent adverse events were mild in severity and resolved without sequelae and showed no clinically meaningful differences between formulations. These findings are consistent with the established safety profile of ibuprofen reported in previous studies.7

Several limitations should be acknowledged. First, the study was conducted in healthy Chinese adults, and therefore the findings may not fully represent patients requiring analgesic or antipyretic treatment. Second, only single-dose administration was evaluated. Nevertheless, the study design complied with current regulatory requirements and was adequate to demonstrate comparable pharmacokinetic performance between the two formulations.

Overall, the test formulation demonstrated pharmacokinetic equivalence to the reference formulation under both fasting and fed conditions, supporting its use as a bioequivalent alternative to the reference formulation for generic drug registration in China.

Funding Statement

2025 Chengdu Municipal Health Commission - Chengdu University of Traditional Chinese Medicine Joint Innovation Fund Project: “FA/HA Dual Receptor-Mediated Drake Red Dimer-Luteolin Hybrid Nanomedicine for Remodeling Macrophage M1/M2 Polarization in the Treatment of Acute Lung Injury”. Project Number: WXLH202501284.

Data Sharing Statement

The individual deidentified participant data underlying the results reported in this article, together with the study protocol and statistical analysis plan, may be made available upon reasonable request to the corresponding author and subject to sponsor review and applicable data protection regulations.

Ethics Approval and Consent to Participate

This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of Chengdu Fifth People’s Hospital (Approval No. EC approval 2022-021 (medicine)-01). The trial was registered at ClinicalTrials.gov (NCT05737069). Written informed consent was obtained from all participants before any study-specific procedures.

Disclosure

Pete Zeng, Marianna Armogida, Umesh Gupta, Amy Liu (at the time the study was conducted), Billy Franks, Sophia Jiang, and Tracy Lu are employees of Haleon and may hold stocks or stock options in the company. They participated in study design, oversight of study conduct, data review, interpretation of findings, and manuscript preparation. The authors report no other conflicts of interest related to this work.