358 Original Article
Bioequivalence of Two Pregabalin 300 mg capsules (Neurexal and Lyrica®) in Healthy Human Volunteers
Affiliations
Key words ▶ pharmacokinetics ● ▶ bioequivalence ● ▶ pregabalin ●
A. Al-Ghazawi1, N. Idkaidek1, 2, E. Daccache3, J.-C. Sarraf3, S. Kyriacos3 1
Triumpharma LLC; Clinical Evaluation Centre, Amman, Jordan College of Pharmacy, University of Petra, Amman, Jordan 3 Research & Development Department, Pharmaline, Jdeidet-El-Metn, Lebanon 2
Abstract
▼
The pharmacokinetics of 2 brands of pregabalin 300 mg capsules were compared in 23 healthy human volunteers after a single oral dose in a randomized cross-over study. The study protocol was prepared with relevance to the requirements set in the US FDA and the EMA guidances for conduction of bioequivalence studies. Reference (Lyrica®, Pfizer, France) and test (Neurexal, Pharmaline, Lebanon) products were administered to fasted volunteers. Blood samples were collected up to 48 h and assayed for pregabalin
Introduction
▼
received 23.05.2013 accepted 18.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1361127 Published online: December 4, 2013 Drug Res 2014; 64: 358–362 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. Kyriacos Pharmaline R & D Manager P.O. Box 90201 Jdeidet-El-Metn Lebanon Tel.: + 961/9/440 901 Fax: + 961/9/448 418 [email protected]
Pregabalin is an alpha-2-delta (α-2-δ) ligand that has analgesic, anxiolytic and anticonvulsant activity. It is a structural derivative of the inhibitory neurotransmitter gammaaminobutyric acid (GABA). However it does not bind directly to GABAA, GABAB, or benzodiazepine receptors. Pregabalin binds with high affinity to the alpha2delta site (an auxiliary subunit of voltage-gated calcium channels) in central nervous system tissues. Although the mechanism of action of pregabalin has not been fully elucidated, results with genetically modified mice and with compounds structurally related to pregabalin (such as gabapentin) suggest that binding to the alpha2-delta subunit may be involved in pregabalin's anti-nociceptive and antiseizure effects in animals, [1, 2]. Pregabalin is indicated in the management of central and peripheral neuropathic pain including diabetic peripheral neuropathy, postherpetic neuralgia, fibromyalgia and neuropathic pain associated with spinal cord injury. It is also indicated to treat generalized anxiety disorder and as adjunctive therapy for adult patients with partial onset seizures [3].
Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
using a validated LC-MS/MS method. The pharmacokinetic parameters AUC0–t, AUC0–∞, Cmax, Tmax, T1/2 and elimination rate constant were determined from plasma concentration-time profile by non-compartmental analysis method using WinNonlin V5.2. The analysis of variance did not show any significant difference between the 2 formulations and 90 % confidence intervals fell within the acceptable range for bioequivalence: 80–125 %. It was concluded that the 2 brands exhibited comparable pharmacokinetic profiles and that Pharmaline’s Neurexal is bioequivalent to Lyrica® of Pfizer, France.
Pregabalin is described chemically as (S)-3(aminomethyl)-5-methylhexanoic acid. The molecular formula is C8H17NO2 and the molecular weight is 159.23. The chemical structure of ▶ Fig. 1. pregabalin is shown in ● According to the Biopharmaceutics classification system [4], pregabalin is a Class 1 drug as it exhibits a high solubility and high permeability [1, 5]. As such it is eligible to biowaiver (i. e., bioequivalence study based on in vitro dissolution test rather than in vivo study). The in vitro method (dissolution test) and the in vivo method (the concentration of the active ingredient or metabolite in an accessible biological fluid is measured as a function of time in humans) are, according to the US FDA and EMA, the most recommended methods to assure that formulations perform in an equivalent manner and thus demonstrate that they are therapeutic equivalent [6, 7]. However, health regulatory authorities in some countries do not accept biowaivers and still require an in vivo bioequivalence study for all types of molecules. This bioequivalence study was therefore performed in vivo on the highest strength available. The medicine is available as hard-shell capsules containing 25, 50, 75, 100, 150, 200, 225, and 300 mg of pregabalin.
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Authors
Original Article 359
Fig. 1 Chemical structure of pregabalin.
Materials and Methods
▼
Study products Investigational product: Neurexal – Pregabalin 300 mg Capsules Batch no.: 72010 Expiry Date: Feb/2014 Manufacturer: Pharmaline, Lebanon Reference product: Lyrica® 300 mg Capsules Batch no.: 0402060 F Expiry Date: May/2013 Manufacturer: Pfizer, France
Objectives
▼
This study was conducted to assess the bioequivalence of two pharmaceutical products: Neurexal 300 mg Capsules (Test product/Pharmaline s.a.l, Lebanon) and Lyrica® 300 mg Capsules (Reference product/Pfizer, France). The study protocol was prepared with relevance to the requirements set in the USFDA guidance for conduction of bioequivalence studies [6] and the EMA CPMP (CPMP/EWP/QWP/1401/98 Rev. 1/Corr [7].
Twenty six (26) healthy, Caucasian adult males participated in this comparative study at Triumpharma, Amman, Jordan. The sample size calculation was based on 20 % intra-subject variability of AUC and 80 % power to detect 20 % difference between the two formulations [9]. The mean age was 28 ± 6.3 years with a range of 18–37 years old, mean body weight was 77 ± 14.1 kg with a range of 59–102 kg, mean body height was 179 ± 6.7 cm with a range of 165–194 cm and body mass index was 23.64 ± 3.586 kg/m2 with a range of 18.51–29.93 kg/m2. The volunteers had medical history and physical examination within the range of clinical acceptability, and laboratory results (hematology, blood biochemistry, and urine analysis) within normal ranges. All subjects were instructed to abstain from taking any drug including vitamins and herbal supplements for 14 days prior to first dosing and during the study period. They were informed about the aim and risks of the study by the clinical investigator and then signed a written informed consent statement before entering the study. The study was carried out in accordance with the principles enunciated in the Declaration of Helsinki resolved in Helsinki in 1964 and amended in Seoul, 2008 [10], the ICH harmonized tripartite guideline regarding Good Clinical Practice [11] and the local requirements of the Jordan Food and Drug Administration in relation to human rights and confidentiality [12]. Before the start of the study, the protocol was approved by the Institutional Review Board (IRB) of Triumpharma, Amman, Jordan.
Drug administration and sample collection The study was designed as single dose, 2-treatment, 2-sequence, 2-period, and complete crossover design performed under fasting conditions. Subjects were admitted to Triumpharma clinical site approximately 12 h prior to study drug administration, until 24 h after dosing. The subjects returned to the site to give the 48.00 h sample as per schedules time. Following an overnight fasting of at least 10 h, the study subjects were given single dose of either formulation (reference or test) of pregabalin 300 mg with 240 ml of water. No food was allowed until 4 h after dose administration. Lunch and dinner were given to all volunteers according to a time schedule; breakfast was served after the 24 h sample was collected. Water intake was allowed 1h after the dosing; there was no restriction on water intake after that. The volunteers were continuously monitored throughout the confinement period of study. They were not permitted to lay down or sleep for the first 4h after the dose. Blood samples were collected in each study period before (0 h) and at 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, 5.00, 6.00, 8.00, 10.00, 12.00, 24.00 and 48.00 h after dosing. For each sample, 8 ml of blood for pregabalin assay were drawn into lithium heparinated tubes through indwelling cannula. Blood samples were centrifuged at 3 400 rpm for 5 min. The resulting plasma was immediately stored at − 20 °C Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
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Study design Pregabalin steady-state pharmacokinetics are similar in healthy volunteers, patients with epilepsy receiving anti-epileptic drugs and patients with chronic pain. Pregabalin is rapidly absorbed when administered in the fasted state, with peak plasma concentrations occurring within 1 h following both single and multiple dose administration. Pregabalin oral bioavailability is estimated to be over 90 % and is independent of dose. Following repeated administration, steady state is achieved within 24–48 h. The rate of pregabalin absorption is decreased when given with food resulting in a decrease in Cmax by approximately 25–30 % and a delay in Tmax to approximately 2.5 h. However, administration of pregabalin with food has no clinically significant effect on the extent of pregabalin absorption. In preclinical studies, pregabalin has been shown to cross the blood brain barrier in mice, rats, and monkeys. Pregabalin has been shown to cross the placenta in rats and is present in the milk of lactating rats. In humans, the apparent volume of distribution of pregabalin following oral administration is approximately 0.56 l/kg. Pregabalin is not bound to plasma proteins. Pregabalin undergoes negligible metabolism in humans. Following a dose of radiolabelled pregabalin, approximately 98 % of the radioactivity recovered in the urine was unchanged pregabalin. The N-methylated derivative of pregabalin, the major metabolite of pregabalin found in urine, accounted for 0.9 % of the dose. In preclinical studies, there was no indication of racemisation of pregabalin S-enantiomer to the R-enantiomer. Pregabalin is eliminated from the systemic circulation primarily by renal excretion as unchanged drug. Pregabalin mean elimination half-life is 6.3 h. Pregabalin plasma clearance and renal clearance are directly proportional to creatinine clearance. Pregabalin pharmacokinetics are linear over the recommended daily dose range. Inter-subject pharmacokinetic variability for pregabalin is low (about 28 %). Multiple dose pharmacokinetics are predictable from single-dose data. Therefore, there is no need for routine monitoring of plasma concentrations of pregabalin, [3, 8].
360 Original Article
Table 1 Linearity, accuracy, precision and stability data for the analytical method validation for the determination of pregabalin in plasma. Regression equa-
Concentration range (μg/
Plasma sample Intra-batch (n = 6) Inter-batch (n = 12) Short-term stability (n = 6) Freeze and thaw stability (n = 6)
Number of points
Correlation coefficient
Precision ( %)
8 0.30 μg/ml Accuracy ( %) Precision ( %) 92.67 2.99 95.67 5.33 104.74 6.69
0.9995 6.00 μg/ml Accuracy ( %) Precision ( %) 96.15 0.83 98.73 3.32 – –
0.03 12.5 μg/ml Accuracy ( %) Precision ( %) 90.86 1.28 93.95 3.95 100.27 2.49
ml) 0.1–15.00 0.10 μg/ml Accuracy ( %) Precision ( %) 96.00 7.71 97.00 10.00 – – –
–
93.43
9.41
until assayed. After a washout period of 7 days the study was repeated in the same manner to complete the crossover design.
Analysis for parent drug Pregabalin and the internal standard (Pregabalin-13C3) were extracted from human plasma samples by direct precipitate extraction using methanol. 50 microliters of internal standard Pregabalin 13C3 working solution (20.0 μg /mL) were added to 200 μl of plasma sample and vortexed for 10 s. Then 100 μL of extraction buffer (ammonium acetate 0.1 M) was added and vortexed 10 s. After adding 1 200 μL of precipitation solvent (methanol) and vortexing for 3.0 min, the samples were centrifuged for 5.0 min at high rpm speed. 200 microliters were transferred from the supernatant into the auto sampler vial inserts. Five microliters were injected to the column. Analysis was performed using a method fully developed and validated at Triumpharma Bio-analytical laboratory. The study plasma samples were analyzed for pregabalin using a validated LC-MS/MS system (LCsystem 04 & LC- system 05). All solvents used were of HPLC grade and were purchased from Tedia (USA). Other chemicals and reagents were of analytical grade. Pregabalin was obtained from Pharmaline whereas the internal standard Pregabalin-13C3 was from TRC, Ontario, Canada. The LC-MS-MS consisted of liquid chromatographic system (Agilent 1 260 infinity, USA), coupled with a triple quadrupole spectrometer (API 3000) from Applied Biosystems, MDS Sciex, Canada, equipped with ESI source for the ionization (positive ionization mode). Integration was done using the Analyst 1.5.2 software (Applied Biosystems MDS Sciex, Canada). Chromatographic separation was performed using Inertsil C8 (5 μm) (150*4.6 mm) column from GL sciences, Japan. The mobile phase consisted of a methanol and water mixture (75:25) with 0.2 % formic acid and eluted at a rate of 0.80 (mL/min) with splitter (2/3) out. The auto-sampler temperature was 15 °C whereas the column temperature was 30 °C. Detection was done by multi reaction monitoring (MRM) mode, using the positive mode. The ion transition (m/z) for Pregabalin was: 160.4/97.2. The ion transition for the internal standard (Pregabalin-13C3) was: 163.2/100.2. The peak area was measured, and the peak area ratio of drug to internal standard and the concentration were calculated by Analyst software. The method was validated as per international guidelines [13, 14]. Method development and validation were conducted in accordance with International Guidelines [13, 14]. Under the described conditions, the lower limit of quantitation from 200 μl plasma was 0.1 μl/ml for pregabalin. 6 calibration curves each consisting of a blank, zero and 8 non-zero standards prepared in human plasma were chromatographed. Linearity was evaluated by calAl-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
–
–
101.36
2.36
culating the linear regression (product moment correlation coefficient, r), and by evaluating the back calculated concentrations of the calibration standards. Results of the calibration curve lin▶ Table 1. earity are summarized in ● The relationship between concentration and peak area ratio was found to be linear within the range of 0.1–15.00 μg/ml. Accuracy and precision were verified using quality control samples at low, medium, high concentration as well as at the LLOQ. Results are ▶ Table 1. reported in ● The intra-day accuracy of the method for pregabalin ranged from 90.86 to 103 %, while the intra-day precision ranged from 0.83 to 15.70 %. The inter-day accuracy for pregabalin ranged from 93.95 to 98.73 % while the inter-day precision ranged from 3.32 to 10 %. Absolute recovery – percentage ratio of the concentrations in an extracted plasma sample with the reference sample of the same concentration which was dissolved in the same solution as extracted sample – was between 43.06 and 56.37 % for pregabalin and 51.18 % for the internal standard. Accuracy ranged between 87.20 and 95.18 %. Short-term temperature stability study demonstrates that pregabalin in plasma is stable for ▶ Table 1. 6 h on the bench at room temperature, as shown in ● Stability studies showed that pregabalin in the plasma samples was stable when stored frozen for 45 days at − 20 °C. Freeze and thaw stability test was determined after four cycles: after storage at − 20 °C for 24 h, samples were completely thawed unassisted at room temperature; samples were then refrozen for at least 12–24 h under the same conditions; the freeze–thaw cycle was repeated three more times. The stability results indicate that Pregabalin is stable under fourth freezing and thawing ▶ Table 1. cycles at − 20 °C, as shown in ●
Pharmacokinetic analysis Pharmacokinetic analysis was performed using the WinNonlin® Computer Program Version 5.2 (Pharsight, USA). The elimination rate constant (λz) was obtained as the slope of the linear regression of the log-transformed concentration values vs. time data in the terminal phase. Elimination half-life (T1/2) was calculated as 0.693/λz. Area under the plasma concentration vs. time curve (AUC0–t), from time (0) to the last measurable concentration (t), was calculated by the linear trapezoidal method. The area under the plasma concentration versus time curve from time (0) to infinity (AUC0–∞) was calculated as the sum of the AUC0–t plus the ratio of the last measurable plasma concentration to the elimination rate constant.
Statistical analysis The pharmacokinetic parameters AUC0–t, AUC0–∞ and Cmax were considered as primary variables. Two-way analysis of variance
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tion
Original Article 361
Table 2 Pharmacokinetic parameters of pregabalin for 2 brands (mean ± standard deviation, n = 23). Pharmacokinetic
Neurexal 300 mg
Lyrica® 300 mg
Parameter
capsules (Test)
capsules (Reference)
Cmax (ng/ml) AUC 0–t (ng*hr/ml) AUC 0–∞ (ng*hr/ml) Tmax (hr) T1/2 (hr) Kelimination (hr-1) (AUC 0–t/AUC 0–∞) %
6.348 ± 1.2551 42.852 ± 6.1030 44.996 ± 6.8205 1.39 ± 0.543 5.34 ± 1.007 0.1340 ± 0.02404 95.40 ± 2.489
6.393 ± 1.0922 42.971 ± 6.5899 45.352 ± 7.0601 1.43 ± 0.802 5.39 ± 0.812 0.1313 ± 0.01820 94.81 ± 3.003
Assessment Parameter Point Estimate ( %) Lower limit ( %) Upper limit ( %) Power
Cmax 99.01 91.12 107.57 0.9958
AUC0–t
AUC0–∞
100.06 97.60 102.57 1.000
99.41 97.43 101.43 1.000
Fig. 2 Mean plasma concentration ( ± SD) of pregabalin after oral administration of single dose of 2 brands to 23 healthy human volunteers.
for crossover design was used to assess the effect of formulations, periods, sequences and subjects on these parameters. Difference between two related parameters was considered to be statistically significant for p-value equal to or less than 0.05. Parametric 90 % confidence intervals based on the ANOVA of the mean test/reference (T/R) ratios of AUCs and Cmax were computed, [15–17].
Results and Discussion
▼
Out of the 26 volunteers, 23 subjects completed the study. One subject did not tolerate the drug as he experienced adverse events after dosing period I. Before admission in period II, another subject was withdrawn from the study for medical reasons (he had a fever) and a third one was withdrawn from the study for medical reasons (he took some medications). Samples for all the subjects who completed the clinical part of the study were analyzed and included in the pharmacokinetic analysis and bioequivalence assessment for Pregabalin. Pregabalin was well tolerated; unexpected incidents that could have influenced the outcome of the study did not occur. All volunteers were discharged in good health. Both formulations were readily absorbed from the gastrointestinal tract and pregabalin was measurable at the first sampling time (0.5 h) in all volunteers. The mean-concentration-time pro▶ Fig. 2. file of pregabalin for the 2 formulations is shown in ● Means of peak concentration of 6.348 and 6.393 ug/ml for pregabalin were attained at means of 1.39 and 1.43 h after drug administration and then declined rapidly. No pregabalin was detectable at 48 h. ▶ Table 2 reports the pharmacokinetic parameters of pregabalin ● for the 2 brands which highlights the closeness of the results. Mean and standard deviation of the three pharmacokinetic parameters of the 2 formulations did not differ significantly. More variability in Cmax is expected as measurement relies on one single point and not on several points as with the area under the curve. The relative bioavailability of Neurexal on the basis of
Lyrica® is 100.21 ± 7.188 % for AUC0–t, 99.49 ± 5.790 % for AUC0–∞, and 101.40 ± 24.412 % for Cmax. For bioequivalence assessment, the test and reference products are considered bioequivalent if the 90 % confidence interval of the geometric mean of the logtransformed data of the test/reference ratio percentage for Pregabalin fall within 80.00–125.00 % for each of the primary end ▶ Table 3, confidence intervals for the pripoints. As reported in ● mary pharmacokinetic parameters Cmax, AUC0–t and AUC0–∞ were 91.1–107.5 %, 97.6–102.5 % and 97.4–101.4 % respectively. They were all within the accepted 80–125 % range. Analysis of variance (ANOVA) for these parameters, after logtransformation of the data, reveals no statistically significant difference between the 2 formulations, with p-value greater than 0.05. The ANOVA analysis of the drug demonstrates that the sequence, product and period effect for all bioequivalence metrics did not influence the outcome of the study. Our results confirm the application of the biowaiver, as per US FDA and EMA guidelines [7, 18] for pregabalin since it falls into class 1 drug according to the biopharmaceutics classification system. The in vivo method confirms the bioequivalence of the two formulations. In vitro dissolution data in different media (results not published), demonstrate the similarity of the reference and test formulations. A review of the literature reveals that there are no incidents of Type I errors in the use of in vitro testing to assess the bioequivalence of Class 1 drugs in the USA and EU [19]. Advantages of in vitro testing includes among other avoidance of unnecessary human experiments, reduction in timelines in the drug development process and in the cost of developing drug products. Policies of waiving in vivo bioequivalence studies should be part of the legal framework of all regulatory agencies.
Conclusion
▼
The Bioequivalence of Neurexal 300 mg Capsules (Test Product/ Pharmaline s.a.l, Lebanon) and Lyrica® 300 mg Pregabalin Capsules (Reference Product/Pfizer, France) following the administration of a single dose of 300 mg Pregabalin to healthy adults under fast conditions was demonstrated. Both products Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
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Table 3 Statistical Analysis: 90 % confidence intervals of log transformed data of pregabalin, n = 23.
can be considered therapeutic equivalent, and thus they can be substituted for each other without any adjustment in dose or other additional therapeutic monitoring.
Conflict of Interest
▼
Authors of the manuscript do not have conflict of interest to declare.
References 1 European Medicines Agency. H-C-546. Lyrica (pregabalin) European Public Assessment Report, Scientific Discussion. EMEA 2004. 2 Gajraj N. Pregabalin: Its Pharmacology and Use in Pain Management. Anesthesia and Analgesia 2007; 105: 1805–1815 3 Lyrica (pregabalin). Summary of Product Characteristics.New York, NY: Pfizer, Inc., August 2012Available at www.medicines.org.uk/emc/ medicine/14651/ 4 Amidon GL, Lennernäs H, Shah VP et al. A Theoretical Basis for a Biopharmaceutic Drug Classification: the Correlation of In Vitro Drug Product Dissolution and In Vivo Bioavailability. Pharm Res 1995; 12: 413–420 5 Cook J, Addicks W, Wu Y. Application of the Biopharmaceutical Classification System in Clinical Drug Development – An Industrial View. AAPS J 2008; 10: 306–310 6 Centre for Drug Evaluation and Research. Guidance for Industry: Bioavailability and bioequivalence studies for orally administered drug products-general consideration. Food and Drug Administration, Rockville, MD: 2003 7 European Medicines Agency. Guideline on the Investigation of Bioequivalence EMA CPMP, (CPMP/EWP/QWP/1401/98 Rev. 1/Corr*). EMA, London: January 2010
Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
8 Bockbrader HN, Radulovic LL, Posvar EL et al. Clinical Pharmacokinetics of Pregabalin in Healthy Volunteers. J Clin Pharmacol 2010; 50: 941–950 9 Randinitis EJ, Posvar EL, Alvey CW et al. Pharmacokinetics of Pregabalin in Subjects with Various Degrees of Renal Function. J Clin Pharmacol 2003; 43: 277–283 10 Declaration of Helsinki” as amended in Seoul 2008; Available at www. ich.org 11 The European Agency for the Evaluation of Medicinal Products (EMEA). Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95).EMEA, London: July 2002 12 JFDA Clinical Studies Law no. 2 for the year 2011; Available at: www. jfda.jo 13 Centre for Drug Evaluation and Research. Guidance for Industry: Bioanalytical Method Validation Guidelines. Food and Drug Administration, Rockville, MD: 2001 14 European Medicines Agency. Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009). EMEA, London: 2009 15 Schuirmann DJ. A Comparison of the Two-One Sided Tests Procedure and the Power for Assessing the Equivalence of Average Bioavailability. J Pharmacokinet Biopharm 1987; 500: 657–680 16 Chow SC, Liu JP. Design and Analysis of Bioavailability and Bioequivalence Studies. 3rd editionNew York: Chapman & Hall, CRC Biostatistics Series, CRC Press, 2008 17 Steinijans V, Diletti E. Statistical Analysis of Bioavailability Studies: Parametric and Nonparametric Confidence Intervals. Eur J Clin Pharmacol 1983; 24: 127–136 18 Centre for Drug Evaluation and Research. Guidance for Industry: Waiver of In Vivo Bioavailability and Bioequivalence Studies for ImmediateRelease Solid Oral Dosage Forms based on a Biopharmaceutics Classification System. Food and Drug Administration, Rockville, MD: 2000 19 Polli J. In Vitro Studies are Sometimes Better than Conventional Human Pharmacokinetic In Vivo Studies in Assessing Bioequivalence of Immediate-Release Solid Oral Dosage Forms. AAPS J 2008; 10: 289–299
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362 Original Article
Bioequivalence of Two Pregabalin 300 mg capsules (Neurexal and Lyrica®) in Healthy Human Volunteers
Affiliations
Key words ▶ pharmacokinetics ● ▶ bioequivalence ● ▶ pregabalin ●
A. Al-Ghazawi1, N. Idkaidek1, 2, E. Daccache3, J.-C. Sarraf3, S. Kyriacos3 1
Triumpharma LLC; Clinical Evaluation Centre, Amman, Jordan College of Pharmacy, University of Petra, Amman, Jordan 3 Research & Development Department, Pharmaline, Jdeidet-El-Metn, Lebanon 2
Abstract
▼
The pharmacokinetics of 2 brands of pregabalin 300 mg capsules were compared in 23 healthy human volunteers after a single oral dose in a randomized cross-over study. The study protocol was prepared with relevance to the requirements set in the US FDA and the EMA guidances for conduction of bioequivalence studies. Reference (Lyrica®, Pfizer, France) and test (Neurexal, Pharmaline, Lebanon) products were administered to fasted volunteers. Blood samples were collected up to 48 h and assayed for pregabalin
Introduction
▼
received 23.05.2013 accepted 18.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1361127 Published online: December 4, 2013 Drug Res 2014; 64: 358–362 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. Kyriacos Pharmaline R & D Manager P.O. Box 90201 Jdeidet-El-Metn Lebanon Tel.: + 961/9/440 901 Fax: + 961/9/448 418 [email protected]
Pregabalin is an alpha-2-delta (α-2-δ) ligand that has analgesic, anxiolytic and anticonvulsant activity. It is a structural derivative of the inhibitory neurotransmitter gammaaminobutyric acid (GABA). However it does not bind directly to GABAA, GABAB, or benzodiazepine receptors. Pregabalin binds with high affinity to the alpha2delta site (an auxiliary subunit of voltage-gated calcium channels) in central nervous system tissues. Although the mechanism of action of pregabalin has not been fully elucidated, results with genetically modified mice and with compounds structurally related to pregabalin (such as gabapentin) suggest that binding to the alpha2-delta subunit may be involved in pregabalin's anti-nociceptive and antiseizure effects in animals, [1, 2]. Pregabalin is indicated in the management of central and peripheral neuropathic pain including diabetic peripheral neuropathy, postherpetic neuralgia, fibromyalgia and neuropathic pain associated with spinal cord injury. It is also indicated to treat generalized anxiety disorder and as adjunctive therapy for adult patients with partial onset seizures [3].
Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
using a validated LC-MS/MS method. The pharmacokinetic parameters AUC0–t, AUC0–∞, Cmax, Tmax, T1/2 and elimination rate constant were determined from plasma concentration-time profile by non-compartmental analysis method using WinNonlin V5.2. The analysis of variance did not show any significant difference between the 2 formulations and 90 % confidence intervals fell within the acceptable range for bioequivalence: 80–125 %. It was concluded that the 2 brands exhibited comparable pharmacokinetic profiles and that Pharmaline’s Neurexal is bioequivalent to Lyrica® of Pfizer, France.
Pregabalin is described chemically as (S)-3(aminomethyl)-5-methylhexanoic acid. The molecular formula is C8H17NO2 and the molecular weight is 159.23. The chemical structure of ▶ Fig. 1. pregabalin is shown in ● According to the Biopharmaceutics classification system [4], pregabalin is a Class 1 drug as it exhibits a high solubility and high permeability [1, 5]. As such it is eligible to biowaiver (i. e., bioequivalence study based on in vitro dissolution test rather than in vivo study). The in vitro method (dissolution test) and the in vivo method (the concentration of the active ingredient or metabolite in an accessible biological fluid is measured as a function of time in humans) are, according to the US FDA and EMA, the most recommended methods to assure that formulations perform in an equivalent manner and thus demonstrate that they are therapeutic equivalent [6, 7]. However, health regulatory authorities in some countries do not accept biowaivers and still require an in vivo bioequivalence study for all types of molecules. This bioequivalence study was therefore performed in vivo on the highest strength available. The medicine is available as hard-shell capsules containing 25, 50, 75, 100, 150, 200, 225, and 300 mg of pregabalin.
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Authors
Original Article 359
Fig. 1 Chemical structure of pregabalin.
Materials and Methods
▼
Study products Investigational product: Neurexal – Pregabalin 300 mg Capsules Batch no.: 72010 Expiry Date: Feb/2014 Manufacturer: Pharmaline, Lebanon Reference product: Lyrica® 300 mg Capsules Batch no.: 0402060 F Expiry Date: May/2013 Manufacturer: Pfizer, France
Objectives
▼
This study was conducted to assess the bioequivalence of two pharmaceutical products: Neurexal 300 mg Capsules (Test product/Pharmaline s.a.l, Lebanon) and Lyrica® 300 mg Capsules (Reference product/Pfizer, France). The study protocol was prepared with relevance to the requirements set in the USFDA guidance for conduction of bioequivalence studies [6] and the EMA CPMP (CPMP/EWP/QWP/1401/98 Rev. 1/Corr [7].
Twenty six (26) healthy, Caucasian adult males participated in this comparative study at Triumpharma, Amman, Jordan. The sample size calculation was based on 20 % intra-subject variability of AUC and 80 % power to detect 20 % difference between the two formulations [9]. The mean age was 28 ± 6.3 years with a range of 18–37 years old, mean body weight was 77 ± 14.1 kg with a range of 59–102 kg, mean body height was 179 ± 6.7 cm with a range of 165–194 cm and body mass index was 23.64 ± 3.586 kg/m2 with a range of 18.51–29.93 kg/m2. The volunteers had medical history and physical examination within the range of clinical acceptability, and laboratory results (hematology, blood biochemistry, and urine analysis) within normal ranges. All subjects were instructed to abstain from taking any drug including vitamins and herbal supplements for 14 days prior to first dosing and during the study period. They were informed about the aim and risks of the study by the clinical investigator and then signed a written informed consent statement before entering the study. The study was carried out in accordance with the principles enunciated in the Declaration of Helsinki resolved in Helsinki in 1964 and amended in Seoul, 2008 [10], the ICH harmonized tripartite guideline regarding Good Clinical Practice [11] and the local requirements of the Jordan Food and Drug Administration in relation to human rights and confidentiality [12]. Before the start of the study, the protocol was approved by the Institutional Review Board (IRB) of Triumpharma, Amman, Jordan.
Drug administration and sample collection The study was designed as single dose, 2-treatment, 2-sequence, 2-period, and complete crossover design performed under fasting conditions. Subjects were admitted to Triumpharma clinical site approximately 12 h prior to study drug administration, until 24 h after dosing. The subjects returned to the site to give the 48.00 h sample as per schedules time. Following an overnight fasting of at least 10 h, the study subjects were given single dose of either formulation (reference or test) of pregabalin 300 mg with 240 ml of water. No food was allowed until 4 h after dose administration. Lunch and dinner were given to all volunteers according to a time schedule; breakfast was served after the 24 h sample was collected. Water intake was allowed 1h after the dosing; there was no restriction on water intake after that. The volunteers were continuously monitored throughout the confinement period of study. They were not permitted to lay down or sleep for the first 4h after the dose. Blood samples were collected in each study period before (0 h) and at 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, 5.00, 6.00, 8.00, 10.00, 12.00, 24.00 and 48.00 h after dosing. For each sample, 8 ml of blood for pregabalin assay were drawn into lithium heparinated tubes through indwelling cannula. Blood samples were centrifuged at 3 400 rpm for 5 min. The resulting plasma was immediately stored at − 20 °C Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
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Study design Pregabalin steady-state pharmacokinetics are similar in healthy volunteers, patients with epilepsy receiving anti-epileptic drugs and patients with chronic pain. Pregabalin is rapidly absorbed when administered in the fasted state, with peak plasma concentrations occurring within 1 h following both single and multiple dose administration. Pregabalin oral bioavailability is estimated to be over 90 % and is independent of dose. Following repeated administration, steady state is achieved within 24–48 h. The rate of pregabalin absorption is decreased when given with food resulting in a decrease in Cmax by approximately 25–30 % and a delay in Tmax to approximately 2.5 h. However, administration of pregabalin with food has no clinically significant effect on the extent of pregabalin absorption. In preclinical studies, pregabalin has been shown to cross the blood brain barrier in mice, rats, and monkeys. Pregabalin has been shown to cross the placenta in rats and is present in the milk of lactating rats. In humans, the apparent volume of distribution of pregabalin following oral administration is approximately 0.56 l/kg. Pregabalin is not bound to plasma proteins. Pregabalin undergoes negligible metabolism in humans. Following a dose of radiolabelled pregabalin, approximately 98 % of the radioactivity recovered in the urine was unchanged pregabalin. The N-methylated derivative of pregabalin, the major metabolite of pregabalin found in urine, accounted for 0.9 % of the dose. In preclinical studies, there was no indication of racemisation of pregabalin S-enantiomer to the R-enantiomer. Pregabalin is eliminated from the systemic circulation primarily by renal excretion as unchanged drug. Pregabalin mean elimination half-life is 6.3 h. Pregabalin plasma clearance and renal clearance are directly proportional to creatinine clearance. Pregabalin pharmacokinetics are linear over the recommended daily dose range. Inter-subject pharmacokinetic variability for pregabalin is low (about 28 %). Multiple dose pharmacokinetics are predictable from single-dose data. Therefore, there is no need for routine monitoring of plasma concentrations of pregabalin, [3, 8].
360 Original Article
Table 1 Linearity, accuracy, precision and stability data for the analytical method validation for the determination of pregabalin in plasma. Regression equa-
Concentration range (μg/
Plasma sample Intra-batch (n = 6) Inter-batch (n = 12) Short-term stability (n = 6) Freeze and thaw stability (n = 6)
Number of points
Correlation coefficient
Precision ( %)
8 0.30 μg/ml Accuracy ( %) Precision ( %) 92.67 2.99 95.67 5.33 104.74 6.69
0.9995 6.00 μg/ml Accuracy ( %) Precision ( %) 96.15 0.83 98.73 3.32 – –
0.03 12.5 μg/ml Accuracy ( %) Precision ( %) 90.86 1.28 93.95 3.95 100.27 2.49
ml) 0.1–15.00 0.10 μg/ml Accuracy ( %) Precision ( %) 96.00 7.71 97.00 10.00 – – –
–
93.43
9.41
until assayed. After a washout period of 7 days the study was repeated in the same manner to complete the crossover design.
Analysis for parent drug Pregabalin and the internal standard (Pregabalin-13C3) were extracted from human plasma samples by direct precipitate extraction using methanol. 50 microliters of internal standard Pregabalin 13C3 working solution (20.0 μg /mL) were added to 200 μl of plasma sample and vortexed for 10 s. Then 100 μL of extraction buffer (ammonium acetate 0.1 M) was added and vortexed 10 s. After adding 1 200 μL of precipitation solvent (methanol) and vortexing for 3.0 min, the samples were centrifuged for 5.0 min at high rpm speed. 200 microliters were transferred from the supernatant into the auto sampler vial inserts. Five microliters were injected to the column. Analysis was performed using a method fully developed and validated at Triumpharma Bio-analytical laboratory. The study plasma samples were analyzed for pregabalin using a validated LC-MS/MS system (LCsystem 04 & LC- system 05). All solvents used were of HPLC grade and were purchased from Tedia (USA). Other chemicals and reagents were of analytical grade. Pregabalin was obtained from Pharmaline whereas the internal standard Pregabalin-13C3 was from TRC, Ontario, Canada. The LC-MS-MS consisted of liquid chromatographic system (Agilent 1 260 infinity, USA), coupled with a triple quadrupole spectrometer (API 3000) from Applied Biosystems, MDS Sciex, Canada, equipped with ESI source for the ionization (positive ionization mode). Integration was done using the Analyst 1.5.2 software (Applied Biosystems MDS Sciex, Canada). Chromatographic separation was performed using Inertsil C8 (5 μm) (150*4.6 mm) column from GL sciences, Japan. The mobile phase consisted of a methanol and water mixture (75:25) with 0.2 % formic acid and eluted at a rate of 0.80 (mL/min) with splitter (2/3) out. The auto-sampler temperature was 15 °C whereas the column temperature was 30 °C. Detection was done by multi reaction monitoring (MRM) mode, using the positive mode. The ion transition (m/z) for Pregabalin was: 160.4/97.2. The ion transition for the internal standard (Pregabalin-13C3) was: 163.2/100.2. The peak area was measured, and the peak area ratio of drug to internal standard and the concentration were calculated by Analyst software. The method was validated as per international guidelines [13, 14]. Method development and validation were conducted in accordance with International Guidelines [13, 14]. Under the described conditions, the lower limit of quantitation from 200 μl plasma was 0.1 μl/ml for pregabalin. 6 calibration curves each consisting of a blank, zero and 8 non-zero standards prepared in human plasma were chromatographed. Linearity was evaluated by calAl-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
–
–
101.36
2.36
culating the linear regression (product moment correlation coefficient, r), and by evaluating the back calculated concentrations of the calibration standards. Results of the calibration curve lin▶ Table 1. earity are summarized in ● The relationship between concentration and peak area ratio was found to be linear within the range of 0.1–15.00 μg/ml. Accuracy and precision were verified using quality control samples at low, medium, high concentration as well as at the LLOQ. Results are ▶ Table 1. reported in ● The intra-day accuracy of the method for pregabalin ranged from 90.86 to 103 %, while the intra-day precision ranged from 0.83 to 15.70 %. The inter-day accuracy for pregabalin ranged from 93.95 to 98.73 % while the inter-day precision ranged from 3.32 to 10 %. Absolute recovery – percentage ratio of the concentrations in an extracted plasma sample with the reference sample of the same concentration which was dissolved in the same solution as extracted sample – was between 43.06 and 56.37 % for pregabalin and 51.18 % for the internal standard. Accuracy ranged between 87.20 and 95.18 %. Short-term temperature stability study demonstrates that pregabalin in plasma is stable for ▶ Table 1. 6 h on the bench at room temperature, as shown in ● Stability studies showed that pregabalin in the plasma samples was stable when stored frozen for 45 days at − 20 °C. Freeze and thaw stability test was determined after four cycles: after storage at − 20 °C for 24 h, samples were completely thawed unassisted at room temperature; samples were then refrozen for at least 12–24 h under the same conditions; the freeze–thaw cycle was repeated three more times. The stability results indicate that Pregabalin is stable under fourth freezing and thawing ▶ Table 1. cycles at − 20 °C, as shown in ●
Pharmacokinetic analysis Pharmacokinetic analysis was performed using the WinNonlin® Computer Program Version 5.2 (Pharsight, USA). The elimination rate constant (λz) was obtained as the slope of the linear regression of the log-transformed concentration values vs. time data in the terminal phase. Elimination half-life (T1/2) was calculated as 0.693/λz. Area under the plasma concentration vs. time curve (AUC0–t), from time (0) to the last measurable concentration (t), was calculated by the linear trapezoidal method. The area under the plasma concentration versus time curve from time (0) to infinity (AUC0–∞) was calculated as the sum of the AUC0–t plus the ratio of the last measurable plasma concentration to the elimination rate constant.
Statistical analysis The pharmacokinetic parameters AUC0–t, AUC0–∞ and Cmax were considered as primary variables. Two-way analysis of variance
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tion
Original Article 361
Table 2 Pharmacokinetic parameters of pregabalin for 2 brands (mean ± standard deviation, n = 23). Pharmacokinetic
Neurexal 300 mg
Lyrica® 300 mg
Parameter
capsules (Test)
capsules (Reference)
Cmax (ng/ml) AUC 0–t (ng*hr/ml) AUC 0–∞ (ng*hr/ml) Tmax (hr) T1/2 (hr) Kelimination (hr-1) (AUC 0–t/AUC 0–∞) %
6.348 ± 1.2551 42.852 ± 6.1030 44.996 ± 6.8205 1.39 ± 0.543 5.34 ± 1.007 0.1340 ± 0.02404 95.40 ± 2.489
6.393 ± 1.0922 42.971 ± 6.5899 45.352 ± 7.0601 1.43 ± 0.802 5.39 ± 0.812 0.1313 ± 0.01820 94.81 ± 3.003
Assessment Parameter Point Estimate ( %) Lower limit ( %) Upper limit ( %) Power
Cmax 99.01 91.12 107.57 0.9958
AUC0–t
AUC0–∞
100.06 97.60 102.57 1.000
99.41 97.43 101.43 1.000
Fig. 2 Mean plasma concentration ( ± SD) of pregabalin after oral administration of single dose of 2 brands to 23 healthy human volunteers.
for crossover design was used to assess the effect of formulations, periods, sequences and subjects on these parameters. Difference between two related parameters was considered to be statistically significant for p-value equal to or less than 0.05. Parametric 90 % confidence intervals based on the ANOVA of the mean test/reference (T/R) ratios of AUCs and Cmax were computed, [15–17].
Results and Discussion
▼
Out of the 26 volunteers, 23 subjects completed the study. One subject did not tolerate the drug as he experienced adverse events after dosing period I. Before admission in period II, another subject was withdrawn from the study for medical reasons (he had a fever) and a third one was withdrawn from the study for medical reasons (he took some medications). Samples for all the subjects who completed the clinical part of the study were analyzed and included in the pharmacokinetic analysis and bioequivalence assessment for Pregabalin. Pregabalin was well tolerated; unexpected incidents that could have influenced the outcome of the study did not occur. All volunteers were discharged in good health. Both formulations were readily absorbed from the gastrointestinal tract and pregabalin was measurable at the first sampling time (0.5 h) in all volunteers. The mean-concentration-time pro▶ Fig. 2. file of pregabalin for the 2 formulations is shown in ● Means of peak concentration of 6.348 and 6.393 ug/ml for pregabalin were attained at means of 1.39 and 1.43 h after drug administration and then declined rapidly. No pregabalin was detectable at 48 h. ▶ Table 2 reports the pharmacokinetic parameters of pregabalin ● for the 2 brands which highlights the closeness of the results. Mean and standard deviation of the three pharmacokinetic parameters of the 2 formulations did not differ significantly. More variability in Cmax is expected as measurement relies on one single point and not on several points as with the area under the curve. The relative bioavailability of Neurexal on the basis of
Lyrica® is 100.21 ± 7.188 % for AUC0–t, 99.49 ± 5.790 % for AUC0–∞, and 101.40 ± 24.412 % for Cmax. For bioequivalence assessment, the test and reference products are considered bioequivalent if the 90 % confidence interval of the geometric mean of the logtransformed data of the test/reference ratio percentage for Pregabalin fall within 80.00–125.00 % for each of the primary end ▶ Table 3, confidence intervals for the pripoints. As reported in ● mary pharmacokinetic parameters Cmax, AUC0–t and AUC0–∞ were 91.1–107.5 %, 97.6–102.5 % and 97.4–101.4 % respectively. They were all within the accepted 80–125 % range. Analysis of variance (ANOVA) for these parameters, after logtransformation of the data, reveals no statistically significant difference between the 2 formulations, with p-value greater than 0.05. The ANOVA analysis of the drug demonstrates that the sequence, product and period effect for all bioequivalence metrics did not influence the outcome of the study. Our results confirm the application of the biowaiver, as per US FDA and EMA guidelines [7, 18] for pregabalin since it falls into class 1 drug according to the biopharmaceutics classification system. The in vivo method confirms the bioequivalence of the two formulations. In vitro dissolution data in different media (results not published), demonstrate the similarity of the reference and test formulations. A review of the literature reveals that there are no incidents of Type I errors in the use of in vitro testing to assess the bioequivalence of Class 1 drugs in the USA and EU [19]. Advantages of in vitro testing includes among other avoidance of unnecessary human experiments, reduction in timelines in the drug development process and in the cost of developing drug products. Policies of waiving in vivo bioequivalence studies should be part of the legal framework of all regulatory agencies.
Conclusion
▼
The Bioequivalence of Neurexal 300 mg Capsules (Test Product/ Pharmaline s.a.l, Lebanon) and Lyrica® 300 mg Pregabalin Capsules (Reference Product/Pfizer, France) following the administration of a single dose of 300 mg Pregabalin to healthy adults under fast conditions was demonstrated. Both products Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
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Table 3 Statistical Analysis: 90 % confidence intervals of log transformed data of pregabalin, n = 23.
can be considered therapeutic equivalent, and thus they can be substituted for each other without any adjustment in dose or other additional therapeutic monitoring.
Conflict of Interest
▼
Authors of the manuscript do not have conflict of interest to declare.
References 1 European Medicines Agency. H-C-546. Lyrica (pregabalin) European Public Assessment Report, Scientific Discussion. EMEA 2004. 2 Gajraj N. Pregabalin: Its Pharmacology and Use in Pain Management. Anesthesia and Analgesia 2007; 105: 1805–1815 3 Lyrica (pregabalin). Summary of Product Characteristics.New York, NY: Pfizer, Inc., August 2012Available at www.medicines.org.uk/emc/ medicine/14651/ 4 Amidon GL, Lennernäs H, Shah VP et al. A Theoretical Basis for a Biopharmaceutic Drug Classification: the Correlation of In Vitro Drug Product Dissolution and In Vivo Bioavailability. Pharm Res 1995; 12: 413–420 5 Cook J, Addicks W, Wu Y. Application of the Biopharmaceutical Classification System in Clinical Drug Development – An Industrial View. AAPS J 2008; 10: 306–310 6 Centre for Drug Evaluation and Research. Guidance for Industry: Bioavailability and bioequivalence studies for orally administered drug products-general consideration. Food and Drug Administration, Rockville, MD: 2003 7 European Medicines Agency. Guideline on the Investigation of Bioequivalence EMA CPMP, (CPMP/EWP/QWP/1401/98 Rev. 1/Corr*). EMA, London: January 2010
Al-Ghazawi A et al. Bioequivalence of Two Pregabalin … Drug Res 2014; 64: 358–362
8 Bockbrader HN, Radulovic LL, Posvar EL et al. Clinical Pharmacokinetics of Pregabalin in Healthy Volunteers. J Clin Pharmacol 2010; 50: 941–950 9 Randinitis EJ, Posvar EL, Alvey CW et al. Pharmacokinetics of Pregabalin in Subjects with Various Degrees of Renal Function. J Clin Pharmacol 2003; 43: 277–283 10 Declaration of Helsinki” as amended in Seoul 2008; Available at www. ich.org 11 The European Agency for the Evaluation of Medicinal Products (EMEA). Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95).EMEA, London: July 2002 12 JFDA Clinical Studies Law no. 2 for the year 2011; Available at: www. jfda.jo 13 Centre for Drug Evaluation and Research. Guidance for Industry: Bioanalytical Method Validation Guidelines. Food and Drug Administration, Rockville, MD: 2001 14 European Medicines Agency. Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009). EMEA, London: 2009 15 Schuirmann DJ. A Comparison of the Two-One Sided Tests Procedure and the Power for Assessing the Equivalence of Average Bioavailability. J Pharmacokinet Biopharm 1987; 500: 657–680 16 Chow SC, Liu JP. Design and Analysis of Bioavailability and Bioequivalence Studies. 3rd editionNew York: Chapman & Hall, CRC Biostatistics Series, CRC Press, 2008 17 Steinijans V, Diletti E. Statistical Analysis of Bioavailability Studies: Parametric and Nonparametric Confidence Intervals. Eur J Clin Pharmacol 1983; 24: 127–136 18 Centre for Drug Evaluation and Research. Guidance for Industry: Waiver of In Vivo Bioavailability and Bioequivalence Studies for ImmediateRelease Solid Oral Dosage Forms based on a Biopharmaceutics Classification System. Food and Drug Administration, Rockville, MD: 2000 19 Polli J. In Vitro Studies are Sometimes Better than Conventional Human Pharmacokinetic In Vivo Studies in Assessing Bioequivalence of Immediate-Release Solid Oral Dosage Forms. AAPS J 2008; 10: 289–299
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362 Original Article
