The goal of our research was to compare the pharmacokinetics and evaluate the bioequivalence of two brands of cephradine 500 mg capsules in 24 normal Korean volunteers. The plasma samples were acquired at 13 time points for 8 h after administration. The concentrations of cephradine in human plasma were measured by a high-performance liquid chromatography (HPLC). Isocratic mobile phase which consisted of acetonitrile, methanol, and 20 mM potassium phosphate (15/5/80, v/v/v, pH 3.48) w as used to separate the analytical column cosmosil cholester (250 × 4.6 mm, 3 μm). Analytes were detected in ultraviolet (260 nm). The novel analytical method was described as simple sample preparation, a short retention time (less than 6 min) and making it suitable for use in clinical trials. Pharmacokinetic parameters, such as AUC0-t (20.54 vs 18.42 μg·h/mL), AUC0-infinity (21.22 vs 19.14 μg·h/mL), Cmax (12.69 vs 12.81 μg/mL), Tmax (1.22 vs 0.92 h), half-life (1.02 vs 1.13 h), extrapolation (3.22 % vs 3.75%), and Ke (0.73 vs 0.69 h–1) were determined for the reference and test drugs in plasma. Pharmacokinetic parameters with a 90% confidence interval were 87 % - 95% for AUC0-t and 91 % - 115% for Cmax. They were satisfied within the bioequivalence range 80 % - 125% of the KFDA guidelines. Therefore, our HPLC method was well applied in a bioequivalence and pharmacokinetic study of two formulations in normal subjects.
Cephradine ((6R, 7R)-7-{[(2R)-2-amino-2-cyclohexa-1, 4-dien-1-ylacetyl] amino}-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid) is a first generation cephalosporin antibiotic. It has broad spectrum antibacterial activity against gram-positive and gram-negative microorganisms and acts through inhibiting bacterial cell wall synthesis. Cephradine is used to treat upper respiratory infections, ear infections, skin infections, and urinary tract infections. It is rapidly absorbed from the gastrointestinal tract and has low plasma protein binding (6% - 20%) [
Several analytical methods have been reported to measure cephradine in biological fluids. These methods include spectrophotometry [
Since the Drug Price Competition and Patent Term Restoration Act were implemented in 1984, the US Food and Drug Administration (FDA) have established bioavailability and bioequivalence requirements for generic substitution [
Furthermore, we performed a comparison of basic PK parameters such as AUC, Cmax, Tmax and t1/2 of cephradine for Korean subjects and the other races, since previous PK studies [
Cephradine and ofloxacin (Internal Standard (IS)) were purchased from Sigma-Aldrich Co. (St. Louis, Mo, USA). HPLC grade acetonitrile and methanol were purchased from J.T. Baker (Philipsburg, NJ, USA). Water was purified using a Milli-Q purification system (Millipore Co., MA, USA). All other chemicals and solvents were HPLC-analytical grade. The reference drug was Yuhan Cephradine capsules 500 mg (Yuhan Pharm Co. Ltd. Seoul, Korea), and the test drug was Korus cephradine capsules 500 mg (Hankook Korus Pharm Co. Ltd. Seoul, Korea).
The HPLC system consisted of an isocratic solvent delivery pump (Model 510 pump, Waters Scientific Co., USA), an autosampler (Model 717 Plus, Waters Scientific Co.) an analytical column cosmosil cholester (250 × 4.6 mm, 3 µm) (Nacalai, Kyoto, Japan) and a variable wavelength ultraviolet detector (Model 486 Tunable Absorbance Detector, Waters Scientific Co.) set at 260 nm. Data was acquired and processed with Empower 3.0 software. The mobile phase consisted of acetonitrile, methanol, and 20 mM potassium phosphate (15/5/80, v/v/v, pH 3.48). The flow rate of the mobile phase was 1.3 mL/min and a sample volume of 50 μL was injected into the chromatography system.
Primary stock solutions of cephradine and IS were prepared in 50% aqueous methanol (1:1 methanol/water, v/v) to final concentrations of 1 mg/mL and 200 μg/mL, respectively, and both were kept at −70˚C. A set of seven non-zero calibration standards, ranging from 0.3 ~ 50 μg/mL, was prepared for blank human plasma with an appropriate amount of cephradine. The quality control (QC) samples were prepared in blank plasma at cephradine concentrations of 0.3 (Lower Limit of Quantification (LLOQ)), 1 (low-middle), 10 (high-middle) and 50 μg/mL (high). Blank plasma was tested before spiking, to ensure that there was no endogenous interference before measuring the retention times of cephradine and IS. Frozen human plasma was thawed at room temperature and centrifuged at 3000 rpm for 10 min at 4˚C to precipitate the sediment. A 500-μL aliquot of plasma was transferred to a screw cap glass tube with 50 μL of IS working solution (IS, 200 μg/mL) and 100 μL of acetonitrile/perchloric acid (1:1, v/v). The mixture was vortexed for 15 sec. After centrifugation at 12,000 rpm for 5 min, the supernatant was transferred to a 2 ml tube and centrifuged again. Following centrifugation, the supernatant was transferred to an autosampler vial and an aliquot (50 μL) was injected into the HPLC system.
The assay was validated according to the FDA guidance on validation of bioanalytical methods [
To evaluate the applicability of this method, a randomized, two-period, and crossover design was used to study the pharmacokinetics and bioequivalence of two types of cephradine in 24 normal Korean subjects.
The study was performed according to the World Medical Association Declaration of Helsinki (1997) for biomedical research involving human subjects in 2007 [
( AUMC ∞ = ∫ 0 t C t ⋅ t ⋅ d t + C p ⋅ t λ z 2 ). The mean residence time (MRT) of cephradine
in the body was calculated as AUMC/AUC, with λz being the Ke [
The molecular structures of cephradine and ofloxacin (IS) were indicated in
The following chromatograms were shown in
A calibration curve was constructed from the peak area ratios of cephradine against IS by using a double-blank sample (plasma sample without cephradine
or IS) and seven calibration standard concentrations (0.3 ~ 50 μg/mL). The standard calibration curve was indicated good linearity within the range of 0.3 - 50 μg/mL by least-squares regression analysis (y = 0.0521, x − 0.0022, r2 = 0.9999, 1/x2 weighting). Intra- and inter-day precision and accuracy were determined by analyzing QC samples against the calibration curve, on the same day (n = 5) and on different days (n = 5). As indicated in
The proposed method was applied to the determination of cephradine in plasma samples for the purpose of establishing the pharmacokinetic and bioequivalence study of 500 mg cephradine formulations in 24 healthy Korean volunteers [
The PK parameters for the reference and test drug obtained were described as follows. The first sampling time of 24 subjects were measurable at 0.25 h after oral administration of cephradine. The profiles of the plasma cephradine concentration vs time were shown in
The PK parameters of cephradine were shown in
Applied Method | LOD | Linearity | Run times | Sample type |
---|---|---|---|---|
Cho HY et al. 2002 [ | 0.2 μg/mL | 0.2 - 30.0 μg/mL | 10 min | Human plasma |
Johnson VM et al. 2000 [ | 0.2 μg/mL | 0.2 - 30.0 μg/mL | 18 min | Human plasma |
Shoaib MH et al. 2008 [ | 0.4 μg/mL | 1 - 12 μg/mL | 28 min | Human plasma |
Nominal Concentration | Precision (%CV) | Accuracy (%) | ||
---|---|---|---|---|
(μg/mL) | Intra-day | Inter-day | Intra-day | Inter-day |
0.3 (LLOQ) | 4.15 | 6.22 | 99.71 | 97.48 |
1 (low) | 3.71 | 4.83 | 105.53 | 99.07 |
10 (middle) | 4.75 | 5.43 | 105.65 | 99.86 |
50 (high) | 3.27 | 4.56 | 103.01 | 97.86 |
LLOQ = lower limit of quantification, CV = coefficient of variation.
In order of the reference and test formulation of cephradine, Cmax were 12.69 and 12.81 μg/mL and Tmax were 1.22 and 0.92 h, respectively. The mean AUC0-t values were 20.54 and 18.42 μg∙h/mL and the mean AUC0-∞ values were 21.22 and 19.14 μg∙h/mL. In addition, the other PK parameters of two formulations of 500 mg cephradine after oral administration were as follows. The mean AUMCt
Subject | Gender (M/F) | Age (years) | Height (cm) | Weight (kg) | Sequence |
---|---|---|---|---|---|
1 | M | 24 | 173.1 | 65.6 | T-R |
2 | M | 26 | 175 | 50.6 | T-R |
3 | M | 26 | 174.5 | 66.3 | T-R |
4 | M | 33 | 173.3 | 55.2 | T-R |
5 | F | 30 | 157.1 | 47 | R-T |
6 | M | 23 | 175 | 62 | R-T |
7 | M | 26 | 171.7 | 60.4 | R-T |
8 | M | 19 | 178.8 | 84 | R-T |
9 | M | 25 | 176 | 66 | R-T |
10 | M | 27 | 181.7 | 69 | T-R |
11 | F | 35 | 159.3 | 55.8 | R-T |
12 | M | 25 | 176 | 92 | R-T |
13 | M | 24 | 176 | 74 | R-T |
14 | M | 23 | 173 | 75 | R-T |
15 | M | 25 | 168.6 | 61 | T-R |
16 | M | 23 | 170.2 | 61.5 | T-R |
17 | M | 24 | 174.5 | 68.5 | T-R |
18 | M | 24 | 179.7 | 79.3 | T-R |
19 | M | 27 | 176.6 | 78.6 | R-T |
20 | M | 29 | 169.7 | 62.1 | T-R |
21 | M | 24 | 177.5 | 87.5 | T-R |
22 | M | 25 | 177.9 | 75.8 | R-T |
23 | M | 35 | 167.9 | 68.7 | R-T |
24 | M | 28 | 167.5 | 72.7 | T-R |
values were 39.34 and 28.56 μg∙h2/mL, and AUMC∞ values were 39.93 and 33.47 μg∙h2/mL for the reference and test drug. The mean MRT value obtained for the reference and test drug were 1.86 and 1.73 h, respectively. These parameters almost overlapped between the test and reference drugs (
The 90 % CI of the test/reference percentage ratio were 90.6% (86.64% - 97.74%) for AUC0-t and 102.3% (91.06% - 114.84%) for Cmax, both of which were within the bioequivalence limits of 80% - 125% for the percentage ratio of product averages. These results support that the two branded formulations of 500 mg cephradine capsules were bioequivalent. Since we evaluated the minimum number of healthy adult subjects, this study has no consideration for all ages and gender.
Parameters | Reference drug | Test drug |
---|---|---|
AUCt (μg∙h/mL) | 20.54 ± 4.30 | 18.42 ± 2.96 |
AUC∞ (μg∙h/mL) | 21.22 ± 4.38 | 19.14 ± 3.06 |
Extrapolated AUC (%) | 3.22 ± 1.05 | 3.75 ± 1.73 |
AUMCt (μg∙h2/mL) | 35.34 ± 13.56 | 28.56 ± 7.88 |
AUMC∞ (μg∙h2/mL) | 39.93 ± 15.37 | 33.47 ± 9.19 |
Vd (L) | 36.68 ± 11.25 | 45.18 ± 20.68 |
MRT (h) | 1.86 ± 0.39 | 1.73 ± 0.29 |
Cmax (μg/mL) | 12.69 ± 3.27 | 12.81 ± 2.80 |
T1/2α (h) | 0.29 ± 0.15 | 0.40 ± 0.27 |
Tmax (h) | 1.22 ± 0.57 | 0.92 ± 0.32 |
kα (h−1) | 3.23 ± 2.64 | 2.46 ± 1.58 |
T1/2β (h) | 1.02 ± 0.31 | 1.13 ± 0.57 |
λz (ke, h−1) | 0.73 ± 0.17 | 0.69 ± 0.19 |
CL (L/h) | 25.36 ± 5.26 | 27.83 ± 4.57 |
AUCt = area under the plasma concentration-time curve from time 0 to time t, AUC∞ = area under the plasma concentration-time curve from time 0 to infinite time, AUMCt = area under the first moment of the plasma concentration-time curve from time 0 to time t, AUMC∞ = area under the first moment of the plasma concentration-time curve from time 0 to infinite time, Vd = the apparent volume of distribution, MRT = the mean residence time, Cmax= the maximal plasma concentration, T1/2α = the half-life of absorption, Tmax = the time to maximal plasma concentration, kα = the distribution rate constant, T1/2β = the half-life of elimination, λz(ke) = the elimination rate constant, CL= clearance.
We developed a fast HPLC method to measure cephradine in human plasma. It was based on removing protein by precipitation, and used a high resolution column and a fast isocratic flow rate. This method had similar precision and accuracy to previous methods, whereas, it had indicated more short analysis times (cephradine [4.13 min], IS [5.55 min]) than previous analysis methods. The use of a method with a short retention time was important, particularly for large-scale clinical trials, because it allowed its high throughput. The method was successful in a strictly-controlled bioequivalence and pharmacokinetic study of two brands of cephradine 500 mg capsules in 24 normal subjects.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP; Ministry of Science, ICT & Future Planning) (No. 2017R1A6A3A11033084).
The authors declare no conflicts of interest regarding the publication of this paper.
Kim, H.-J., Kim, S.-H., Kim, S., Ahn, J.-S. and Kang, J.-S. (2018) Clinical Pharmacokinetic and Bioequivalence Studies of Two Brands of Cephradine in Healthy Korean Using HPLC Method. Pharmacology & Pharmacy, 9, 279-292. https://doi.org/10.4236/pp.2018.97022