Keywords: Bioequivalence, COX-2 inhibitor, etoricoxib, generics
Pharmacokinetics and bioequivalence of generic etoricoxib in healthy volunteers
Introduction and Study Objectives
Etoricoxib (ETO), which has the chemical formula 5-chloro-6’- methyl-3-[4-(methylsulfonyl) phenyl]-2, 3’-bipyridine, see Figure 1, is a highly selective non-steroidal cyclooxygenase (COX)-2 inhibitor with a molecular weight of 358.84 g/mol. Cyclooxy- genase catalyses the production of prostaglandins and exists in two isoforms, namely COX-1 and COX-2. Inhibition of COX-2 reduces the production of prostaglandins, leading to anti- infl ammatory and analgesic effects.COX-2 also plays a role in ovulation, implantation and closure of the ductus arteriosus in newborns, and regulates certain functions of the renal and cen-
Author for correspondence: Dr Chuei Wuei Leong, Formulation and R&D Technologies, Duopharma Innovation Sdn. Bhd., No.2 Jalan Saudagar U1/16 Zon Perindustrian Hicom Glenmarie Seksyen, U1 Shah Alam, 40150 Shah Alam, Selangor, Malaysia
Submitted: 19 May 2021; Revised: 26 August 2021; Accepted: 26 August 2021; Published online first: 8 September 2021
Shalini Harikrishnan
1, BSc; Ka-Liong Tan
2, DPhil; Kar Ming Yee
3, BPharm; Alia Shagari Ahmad Shukri
1, MSc; N Ramana Reddy
4, MBBS; Chewy Wuei Leong
3, PhD
Introduction/Study Objectives: A bioequivalence study was performed to compare the pharmacological profi le of innovator etoricoxib (ETO) with a newly developed generic ETO, both in a 120 mg tablet formulation. A dissolution study was conducted to optimize the formulation process before evaluating physical changes in the active pharmaceutical ingredient and the formulated product.
Method: This was a randomized, open-label, balanced, two-treatment, two-period, two-sequence, single-dose, two-way cross- over, truncated bioequivalence study involving a washout period of ten days. A total of 26 healthy male volunteers were recruited.
The pharmacokinetic profi le of the test formulation was compared with the reference formulation.
Results/Discussion: The pharmacokinetic parameters of ETO were calculated based on the plasma drug concentration-time profi le using non-compartmental analysis to determine its safety profi le and tolerability. The Test/Reference (T/R) ratio of ETO was 104.36% (90% confi dence interval (CI): 98.30%–110.80%) for area under curve (AUC)0-72 while the T/R ratio of maximum con- centration (Cmax) was 101.39% (92.15%–111.56%). The 90% CI of the Cmax and AUC0-72 of ETO were within acceptable bioequiva- lence limits of 80%–125%. All values were within the predetermined limits of the Association of Southeast Asian Nation (ASEAN) bioequivalence guidelines.
Conclusion: The test formulation was therefore found to be bioequivalent with respect to the reference drug, according to ASEAN bioequivalence guidelines.
tral nervous systems such as the induction of fever, perception of pain and cognition [1].
ETO has been shown to produce dose-dependent inhibition of COX-2 at doses up to 150 mg/day. However, it does not inhibit the synthesis of gastric prostaglandin or exert any effects on platelet function [2]. Thus, it can be ingested orally to relieve the acute pain associated with rheumatoid arthritis, psoriatic arthri- tis, osteoarthritis, gout, back pain, headache, or infl ammation secondary to dental surgery [3].
In terms of pharmacokinetics, ETO shows good oral absorption with an absolute bioavailability of approximately 100%. Peak plasma concentration, maximum concentration (Cmax), of 3.6 µg/
mL was reached one hour after the administration of 120 mg ETO in fasting adults [4] with an area under curve (AUC) of 37.8 µg·hr/mL. Furthermore, the pharmacokinetics of ETO are linearly related to the clinical dose range [4]. Onset of action of ETO can occur from 24 minutes after administration. Approxi- mately 92% of administered ETO is bound to human plasma protein with a concentration range of 0.05 µg/mL–5 µg/mL. At steady state, the volume of distribution (Vdss) is approximately 120 L in humans.
ETO undergoes extensive metabolism; fewer than 1% is excreted as the parent compound in urine. ETO metabolism is catalysed by cytochrome P450 (CYP) enzymes before forming 6’-hydroxy- methyl derivatives. Further oxidation of the 6’-hydroxymethyl derivative leads to the formation of the principal metabolites from ETO metabolism, i.e. the 6’-carboxylic acid derivatives of Figure 1: Chemical structure of etoricoxib
S
O
N N
CI O
ETO. These principal metabolites act as weak COX-2 inhibitors with minimal or no measurable activity. None of the principal metabolites are COX-1 inhibitors.
A generic drug should have the same dosage, strength, route of administration, safety profi le, quality, and performance charac- teristics as the innovator product [5]. Generic versions of ETO are often preferable in view of their equivalence to the innova- tor and their availability at a lower cost. A bioequivalence (BE) study in compliance with the Association of Southeast Asian Nation (ASEAN) Guidelines is required to establish therapeutic equivalence between the generic and innovator formulations before any new generic products can be registered under the National Pharmaceutical Regulatory Agency in Malaysia. There- fore, this study aimed to determine the BE of a generic ETO ( 120 mg, tablet formulation) in comparison to the innovator product, Arcoxia® (120 mg, tablet formulation).
Methods
Subjects & study design
This was a randomized, open-label, balanced, two-treatment, two-period, two-sequence, single-dose, two-way crossover, truncated trial with a washout period of 10 days. The study recruited 26 healthy male volunteers aged between 18–45 years with a body weight of ≥ 45 kg and body mass index (BMI) rang- ing from 18.5 to 30.0 kgm-2. The mean age, height, weight, and BMI of the participants were 32.69 ± 6.30 years, 169.5 ± 6.74 cm, 71.07 ± 10.05 kg, and 24.7 ± 2.6 kg/m2, respectively. Research- ers monitored the contraindications, hypersensitivity, poten- tial risk to ETO. Vital signs (blood pressure, pulse rate) were measured at pre-dose, 1.00, 3.00, 9.00, 25.00, 33.00 and 50.00, hours after dosing.. Table 1 shows the demographic characteris- tics of the subjects, who were selected based on predetermined eligibility criteria. Their medical history was obtained and physi- cal examination was performed to record blood pressure, radial pulse rate, body temperature and respiratory rate. Electrocardio- gram and other clinical laboratory evaluations were carried out, including evaluations of HIV 1 & 2 antibody status, hepatitis B surface antigen, hepatitis C virus antibodies, tests for venereal disease, and tests for common drugs of abuse (amphetamine, barbiturates, benzodiazepines, morphine, tetrahydrocannabi- nols and cocaine) 21 days prior to the study commencing.
This study (No.152-18) followed the International Confer- ence on Harmonisation Good Clinical Practice and the ASEAN guidelines on the conduct of BE studies [6]H. All subjects were
informed about the objectives, procedures, and potential risks of participation in the study and all subjects signed an informed consent form before enrolling in the study. The trial protocol received approval from the Maarg Independent Ethics Commit- tee, an independent ethics committee regulated by the Indian Drug and Cosmetic Act and the 2008 Declaration of Helsinki.
Analysis was performed by RA Chem Pharma Limited, Hyder- abad, India (Clinical Research and Biosciences Division).
Test products
The test product, etoricoxib Tablet 120 mg (Batch No.1908296PB) was manufactured by Duopharma Manufacturing Bangi Sdn Bhd, Malaysia. The reference product, Arcoxia® ETO tablet 120 mg (Lot No. 83882064) was manufactured by Frosst Iberica, SA, Spain.
Dissolution test
A dissolution study was conducted to compare the test and reference products prior to the BE study. It was performed using the Electrolab dissolution test system, in accordance with United States Pharmacopeia (USP) general methods. A high- performance liquid chromatography system (Agilent, 1260 Infi n- ity, India) equipped with model software was used to quantify the samples. The Metrohm model AG/913 (India) was used to determine the pH of all solutions with 0.45 µm nylon mem- branes procured from Millipore, India.
Treatment phase and blood sampling
Subjects underwent at least ten hours of fasting before sam- pling. A total of 23 sampling points were planned. A pre-dose sample was collected as the baseline. Subjects received either a single dose of test product or reference product along with 240 mL of water. Blood samples were collected at 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.50, 4.00, 6.00, 8.00, 10.00, 12.00, 24.00, 36.00, 48.00 and 72.00 hours post-administration. The samples were centrifuged at 4,000 rpm for 10 minutes at 4°C. Plasma samples were transferred to the bioanalytical department in dry ice and stored at -80°C until analysis.
Analysis of drug concentration
Liquid chromatography tandem mass spectrometry (LC-MS/MS) was conducted to analyse the samples. The analyte ETO and its internal standard etoricoxib D4 were separated with the analyti- cal column Phenomenex, C18, 50 x 4.6 mm, 5 µm. This method produced a linear response in the plasma ETO concentration in the K2EDTA blood tube over a concentration range of 10.28–
5479.52 ng/mL. The method has been validated for its selec- tivity, intra-batch and inter-batch precision, accuracy, recovery, stability, linearity, and sensitivity. Liquid-liquid extraction was completed using Phenomenex, C18, 50 x 4.6 mm, 5 µm column to extract ETO from the plasma.
Statistical analysis
Statistical analysis was performed using SAS® software version 9.4 (SAS Institute Inc. Cary NC, USA) using the non-compart- mental method in Phoenix® WinNonlin (Version 8.0) to iden- tify the pharmacokinetic values of Cmax and AUC of the drug.
ANOVA analysis was performed on the Ln-transformed phar- macokinetic parameters using the General Linear Model (PROC Table 1: Demographic characteristics summary of the subjects
Gender (n) Mean (Range)
Male 24
Female 0
Ethnicity South Indian
Age (Yr) 32.69 (20.00–42.00)
Weight (Kg) 71.07 (53.10–98.90)
BMI (Kg/m2) 24.70 (19.80–29.40)
reference products). The highest intra-subject coeffi cient of vari- ation was found to be 15.3% for Cmax.
A similar study was previously conducted in Saudi Arabia [4]. In this study, the AUC0-72 for the test product was 23067.3 ± 8978.36 as compared to 23478.2 ± 9719.32 for the reference product. The Cmax was 1923.900 ± 466.83 for the test product and 1986.143 ± 614.41 for the reference product. As the BE was within a range GLM). The BE of the test formulation was
established within a 90% CI such that the rela- tive means of Cmax and AUC0-72 were expected to fall within 80%–125% of that of the refer- ence formulation.
Results and Discussion
Comparative dissolution was conducted at pH 1.2, 4.5, and 6.8. At pH 1.2, more than 85% of the active ingredient dissolved within 15 minutes. At pH 4.5 and pH 6.8, the similar- ity factors were 43.8 and 50.8, respectively.
The accepted similarity factor range is above 50 [6]. At pH 4.5, the dissolution of the test formulation behaves differently due to its variation in excipient compositions compared to the reference. Tables 2 and 3 compare the dissolution profi le of the test formulation against the reference formulation.
To determine the test and reference prod- uct’s comparative bioavailability, a BE study must be performed. We therefore recruited 26 healthy male subjects of South Asian heritage who fulfi lled the inclusion criteria. All subjects received both the test and reference products and were included in the pharmacokinetic and safety analysis. No serious adverse events (SAEs) were reported. However, two adverse events (AEs) were reported: fever and head- ache not associated with nausea and vomit- ing during fi rst phase (of the crossover) after the administration of reference product, and fever not associated with any other symptoms during the fi rst phase after the administration of test product. Both AEs were categorised as mild. No clinical biochemistry values or vital signs met the predefi ned criteria of Potential Clinical Importance.
Table 4 shows the pharmacokinetic param- eters for the test and reference products. In this study, the pharmacokinetic parameters of the ETO tablet were assessed based on the plasma concentrations of ETO. The T/R ratios of ETO were 104.36% (90% CI inter- val: 98.30%-110.80%) for AUC0-72 and 101.39%
(92.15%–111.56%) for Cmax. The Tmax for the test and reference products were 1.063 and 1.156 hours, respectively. Furthermore, intra- subject variability was low for both Cmax and AUC0-72 as the CV (%) did not exceed 30%
for any parameter. The AUC0-72 and Cmax of the test formulation passed the acceptance criteria for BE as the 90% CI of the AUC0-
72 and Cmax of the test and reference formulations fell within the range of 80% - 125% according to guidelines. The AUC0-72 and Cmax of ETO were also within an 80 - 125% range.
Figure 5 shows the mean plasma concentration versus time after the oral administration of 120 mg ETO tablet (both the test and
Table 3: Results of dissolution data in pH 1.2 (HCl buffer), pH 4.5 (phosphate buffer) and pH6.8 (phosphate buffer) for reference formulation
Dissolution by HPLC
ETO reference formulation Time point (Minutes)
5 10 15 20 30 45 60
Etoricoxib dissolved (%) with respect to label claim
pH1.2 Mean 100 99 99 99 99 100
Maximum 103 101 103 101 101 102
Minimum 97 98 96 97 97 98
SD 2 1 2 1 1 1
pH4.5 Mean 20 24 27 29 33 38 42
Maximum 21 25 29 31 37 45 50
Minimum 19 23 26 27 30 34 36
SD 1 1 1 1 2 4 5
pH6.8 Mean 18 22 24 29 33 36 37
Maximum 19 23 26 31 35 41 41
Minimum 17 20 23 26 30 32 33
SD 1 1 1 1 1 2 2
Table 2: « Results of dissolution data in pH 1.2 (HCl buffer), pH 4.5 (phosphate buffer) and pH6.8 (phosphate buffer) for test formulation
Dissolution by HPLC
ETO test formulation Time point (Minutes)
5 10 15 20 30 45 60
Etoricoxib dissolved (%) with respect to label claim
pH1.2 Mean 95 95 96 96 95 96
Maximum 96 96 96 96 97 97
Minimum 94 94 95 95 94 95
SD 1 1 1 1 1 1
pH4.5 Mean 36 39 40 42 44 47 48
Maximum 38 41 42 44 46 49 51
Minimum 35 37 39 40 43 45 47
SD 1 1 1 1 1 1 1
pH6.8 Mean 32 33 35 36 38 40 42
Maximum 37 35 37 38 40 43 46
Minimum 28 29 30 31 32 34 36
SD 2 2 2 2 2 2 2
of 80%–125%, the test product was deemed to be bioequivalent to the innovator [4]. In comparison, our study reported that the 90% CI observed for Cmax was 92.15%–111.56%, higher than the 89.76%–106.81% reported in the Saudi Arabian study. Similarly, the AUC0-72 of 98.30%–110.80% in the current study is also slightly higher than the 95.72%–102.48% reported in the previous study [4]. The difference could be due to the ETO formulations and study populations.
A further study administered a 60 mg formulation of ETO among healthy volunteers in Mexico [7]. The geomet- ric mean ratios of Cmax and truncated AUC0-72 were 99.55%–119.33% and 95.97%–103.06% respectively. Thus, the test product was also deemed bio- equivalent to the reference product in this study. Furthermore, a single dose study of ETO 60 mg was conducted in 24 healthy subjects in Bangladesh [8].
In this study, the AUC0-120 reported was 85.37 - 107.74% with a Cmax of 85.54%–111.98%, indicating that the test and reference formulation of ETO met the regulatory criteria for BE.
Finally, a similar study comparing the reference product (Arcoxia) against a test product produced by PT Dexa Medica in 26 healthy subjects was conducted in Indonesia. The results also showed bioequivalence, with a AUC0-72 of 98.70%–108.32% and a Cmax of 100.18%–119.18%.
The current study had a suffi cient number of subjects to ensure adequate statistical power to prove the equiva- lency of the test product to the refer- ence product. However, the study has some limitations. Due to recruitment capacity, we could only include male volunteers. This is because the use of ETO is not recommended in women who are trying to conceive due to evi- Table 4. Following oral administration of a single tablet of test or reference drug
Parameter Test formulation Mean ± SD
Reference formulation Mean ± SD
Geometric Mean Ratio CV Geometric Ratio
Cmax (ng/mL) 2687.698 ± 697.634 2625.192 ±569.968 1.012 1.263
AUC0–72 (ng·h/mL) 50407.792 ± 12234.127 48028.655 ± 11570.431 1.051 0.965
tmax (h) 1.063 ± 0.924 1.156 ± 0.691 0.803 1.432
Data are presented as arithmetic mean± standard deviation (SD). CV, intrasubject coeffi cient of variation; Cmax, maximum observed plasma concentration; AUC0–72, area under the concentration–time curve from time zero to time of last quantifi able plasma concentration; t1/2, elimination half-life;
Figure 2: Comparative dissolution profi le in pH 1.2
Dissolution profiles in pH 1.2 HCl buffer, using basket,100 RPM, 900 mL
0 10 20 30 40 50 60 70 80 90 100 110
0 10 20 30 40 50 60
% Drug dissolved
Time (mins)
ETO Test Formulation ETO Reference Formulation
Figure 3: Comparative dissolution profi le in pH 4.5
Dissolution profiles of Etoricoxib in pH 4.5 phosphate buffer, using basket, 100 RPM, 900 mL
0 10 20 30 40 50 60 70 80 90 100 110
0 10 20 30 40 50 60
% Drug dissolved
Time (mins)
ETO Test Formulation ETO Reference Formulation
dence of an increased risk of miscarriage, and no women who are unable to conceive, e.g. post hysterectomy, volunteered to participate in the study [10]
Conclusion
This study intended to assess the biological equivalence of a newly developed etoricoxib (120 mg, tablet formulation) in
comparison to the innovator prep- aration, Arcoxia® (120 mg, tablet formulation). Based on the study results, we conclude that the new etoricoxib 120 mg tablet meets bio- equivalence guidelines.
Funding sources
This work was fi nancially supported by a Duopharma R & D fund (Pur- chase Order No. 4100225052). Phar- macological evaluation support was provided by KL Tan, DPhil during an industrial internship, funded by Duopharma Biotech Berhad.
Disclosure
This study received ethical approval from the independent ethics commit- tee with permits from India Central Drugs Standard Control Organisation.
Informed consent was obtained from all subjects.
Prior presentations: None.
Competing interest: This work was fi nancially supported by the Duopharma R & D fund. KL Tan received a subsidy from Duopharma Biotech Berhad, Malaysia during an industrial internship at Universiti Sains Islam Malaysia (USIM) for a bioequivalence study of ETO.
Provenance and peer review: Not com- missioned; externally peer reviewed.
Authors
Nishalini Harikrishnan1, BSc Ka-Liong Tan2, DPhil Kar Ming Yee3, BPharm Alia Shaari Ahmad Shukri1, MSc N Ramana Reddy4, MBBS Chuei Wuei Leong*,3, PhD
1Outsource R&D, Duopharma Inno- vation Sdn. Bhd., No. 2 Jalan Sau- dagar U1/16 Zon Perindustrian Hicom Glenmarie Seksyen, U1 Shah Alam, 40150 Shah Alam, Selangor, Malaysia
2Pharmacology Unit, Faculty of Medi- cine & Health Sciences, Universiti Sains Islam Malaysia, Level 13, Menara B, Persiaran MPAJ, Jalan Pandan Utama, Pandan Indah, 55100 Kuala Lumpur, Malaysia
3Formulation and R&D Technologies, Duopharma Innovation Sdn.
Bhd., No. 2 Jalan Saudagar U1/16 Zon Perindustrian Hicom Glen- marie Seksyen, U1 Shah Alam, 40150 Shah Alam, Selangor, Malaysia Figure 4: Comparative dissolution profi le in pH 6.8
Dissolution profiles of Etoricoxib in pH 4.5 phosphate buffer, using basket, 100 RPM, 900 mL
0 10 20 30 40 50 60 70 80 90 100 110
0 10 20 30 40 50 60
% Drug dissolved
Time (mins)
ETO Test Formulation ETO Reference Formulation
Figure 5: Mean plasma concentrations versus time profi les of ETO in human subjects (n = 24) after single-dose oral administration of 120 mg of Etoricoxib tablet of the test drug and the reference drug
Mean Plasma Concentartion vs Time Profile [Linear Scale, Etoricoxib, N = 24]
0 500 1,000 1,500 2,000 2,500
0 10 20 30 40 50 60 70 80
Concentration (ng/mL)
Time (hr)
ETO Test Formulation ETO Reference Formulation
4RA Chem Pharma Limited, Clinical Research and Biosciences Division, Plot-No.26 & 27, Technocrat Industrial Estate (TIE), Balanagar, Hyderabad 500037, India
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