The authors have declared that no competing interests exist.
Conceived and designed the experiments: JL XX TT. Performed the experiments: JL HC YL XC Yan Zhao Yunfan Zhao. Analyzed the data: JL XX. Contributed reagents/materials/analysis tools: JL. Wrote the paper: JL XX.
Amoxicillin is a commonly used antibiotic which has a short half-life in human. The frequent administration of amoxicillin is often required to keep the plasma drug level in an effective range. The short dosing interval of amoxicillin could also cause some side effects and drug resistance, and impair its therapeutic efficacy and patients’ compliance. Therefore, a three-pulse release tablet of amoxicillin is desired to generate sustained release
The pulsatile release tablet consists of three pulsatile components: one immediate-release granule and two delayed release pellets, all containing amoxicillin. The preparation of a pulsatile release tablet of amoxicillin mainly includes wet granulation craft, extrusion/spheronization craft, pellet coating craft, mixing craft, tablet compression craft and film coating craft. Box–Behnken design, Scanning Electron Microscope and
Single factor experiments identify four important factors of the formulation, namely, coating weight of Eudragit L30 D-55 (X1), coating weight of AQOAT AS-HF (X2), the extrusion screen aperture (X3) and compression forces (X4). The interrelations of the four factors were uncovered by a Box–Behnken design to help to determine the optimal formulation. The immediate-release granule, two delayed release pellets, together with other excipients, namely, Avicel PH 102, colloidal silicon dioxide, polyplasdone and magnesium stearate were mixed, and compressed into tablets, which was subsequently coated with Opadry® film to produce pulsatile tablet of amoxicillin.
This study has demonstrated the interrelation of factors affecting the pulsatile formulation of amoxicillin using a Box–Behnken design. The three-pulse release tablets of amoxicillin were proven to generate pulsatile release
Amoxicillin is one of the most common semi synthetic antibiotics with broad antibacterial spectrum. The half-life of amoxicillin is about 60 minutes in human [
Different from a single pulse formulation, a multiple pulse formulation delivers more than one pulse concomitantly. Each pulse corresponds to a specific lag time [
A pulse release tablet of amoxicillin was first developed by the MinddleBrook Company in the United States. The drug was called MOXATG® and approved by FDA in 2008. The extended release tablet consists of three pulsatile components: one immediate-release pulse and two types of delayed release pellet. The pulsatile release is controlled by the pH-sensitive coating. The pH-sensitive coating is dissolved gradually with the increase of pH along the gastrointestinal tract. A gradual increase in pH is observed through the human small intestine: pH 5 ~ 6 at the duodenum, pH 6 ~ 6.6 at the jejunum, and pH 6.5 ~ 8 at the ileum [
The physiologically based pharmacokinetic model (PBPK) is a kind of
The goal of this investigation was to prepare an oral pulsatile release tablet of amoxicillin as a once daily oral dosage form. Moreover, the investigation was to study the internal cause among the formulated factors of the pulsatile release tablet by the single factor experimental design and the Box–Behnken design. The pharmacokinetic behavior of this formulation was evaluated by both the animal model and PBPK technique.
Amoxicillin tryhydrate was supplied by the United Laboratories Co., Ltd (ZhuHai, China). The working standard of amoxicillin trihydrate (85.8% purity) and ampicillin trihydrate (Internal standard, IS) (
Chemical structure of (a) amoxicillin trihydrate, (b) ampicillin trihydrate is internal standard.
The amoxicillin tablet was designed to consist of three kinds of pulses, including the pulsatile immediate-release (the pulse Ⅰ) and two pulsatile delayed-releases (the pulse Ⅱ and Ⅲ) (
The pulse Ⅰ was prepared by the wet granulation craft. The pulse Ⅰ was made of amoxicillin, excipient Avicel MCC, PVPP and the binder HPMC E5 solution. Amoxicillin, Avicel MCC, and PVPP which were firstly mixed for 3 min at 300 rpm by the wet-mixing granulator, and were subsequently mixed with HPMC E5 solution as the binder through a 40-mesh sieve. Finally, the wet granule was dried in an oven at 50°C for 3 h.
The extrusion/spheronization process is a well-accepted method of producing pellets [
The obtained pellet core underwent further craft to produce pulse II and III pellets. The pulse Ⅱ was prepared by coating Eudragit L30 D-55. The coating suspension contained TEC (equaling to 100% of the dry polymer of Eudragit L30 D-55, w/w) as plasticizer, talc as anti-plastering aid agents. The procedure of coating was as follows. The dispersions were diluted with distilled water to achieve 20% content of dry polymer. And other materials were added slowly to the coating fluid with 500 rpm of stirring for at least 1 h. The pellets coated with HPMC were then coated with different coating weight of Eudragit L30 D-55 polymer (respectively 10%, 20%, 30% based on weight HPMC pellets, w/w) by a fluid-bed coater [
The pulse Ⅲ was prepared by AQOAT AS-HF coating. The coating suspension contains AQOAT AS-HF, sodium lauryl sulfate (equaling to 0.5% of the dry polymer of AQOAT AS-HF, w/w) being the polymer to increase solubility of AQOAT AS-HF, TEC (equaling to 100% of the dry polymer of AQOAT AS-HF, w/w) as plasticizer and talc as anti-plastering aid agents. The procedure of coating was as follows. Sodium lauryl sulfate and TEC were dissolved in the water. AQOAT AS-HF was slowly added to the solution to achieve a dry polymer content of 7%. Talc was slowly added to the solution and stirred at 500 rpm for more than 2 h. The pellets coated with HPMC were then coated with different coating weight of AQOAT AS-HF polymer (respectively 5%, 15%, 25%, based on weight HPMC pellets, w/w) by a fluid-bed coater [
Box–Behnken design was used to optimize the tablet formulation. According to the results of the single-factor experiments, coating weight of Eudragit L30 D-55 (X1), coating weight of AQOAT AS-HF (X2), the extrusion screen aperture (X3) and compression forces (X4) were chosen as the main factors. The independent variables studied (X1, X2, X3 and X4) and the responses in the experimental design were shown in
Factors | Levels | ||
-1 | 0 | 1 | |
X1: coating weight of Eudragit L30 D-55 (%) | 10 | 20 | 30 |
X2: coating weight of AQOAT AS-HF (%) | 5 | 15 | 25 |
X3: extrusion screen aperture (mm) | 0.3 | 0.4 | 0.6 |
X4: compression forces (KN) | 8 | 13 | 18 |
Response | Constraints | ||
R1: cumulative release at 2.25h (%) | 49.5 ~ 57.5 | ||
R2: cumulative release at 4.5h (%) | 79.0 ~ 85.0 |
NO. | X1 | X2 | X3 | X4 | R1 | R2 |
---|---|---|---|---|---|---|
1 | 30 | 15 | 0.3 | 13 | 40.6 | 74.6 |
2 | 20 | 5 | 0.6 | 13 | 43.8 | 64.8 |
3 | 30 | 15 | 0.4 | 8 | 43.6 | 79.8 |
4 | 20 | 15 | 0.4 | 13 | 54.1 | 83.9 |
5 | 20 | 25 | 0.3 | 13 | 53.4 | 70.5 |
6 | 20 | 15 | 0.4 | 13 | 54.1 | 83.9 |
7 | 10 | 15 | 0.6 | 13 | 42.8 | 73.6 |
8 | 10 | 15 | 0.4 | 18 | 60.9 | 87.8 |
9 | 20 | 15 | 0.4 | 13 | 53.9 | 83.6 |
10 | 30 | 25 | 0.4 | 13 | 41.8 | 65.9 |
11 | 20 | 25 | 0.6 | 13 | 43.6 | 58.9 |
12 | 20 | 15 | 0.4 | 13 | 54.3 | 84.2 |
13 | 30 | 15 | 0.6 | 13 | 39.2 | 60.9 |
14 | 10 | 5 | 0.4 | 13 | 61.2 | 95.7 |
15 | 10 | 15 | 0.4 | 8 | 63.9 | 90.7 |
16 | 20 | 15 | 0.3 | 18 | 60.2 | 89.8 |
17 | 20 | 5 | 0.4 | 8 | 55.9 | 90.7 |
18 | 20 | 5 | 0.3 | 13 | 52.8 | 91.2 |
19 | 30 | 5 | 0.4 | 13 | 40.5 | 67.9 |
20 | 30 | 15 | 0.4 | 18 | 42.9 | 74.3 |
21 | 20 | 15 | 0.3 | 8 | 56.8 | 83.2 |
22 | 20 | 15 | 0.4 | 13 | 54.3 | 83.7 |
23 | 10 | 25 | 0.4 | 13 | 65 | 76.3 |
24 | 20 | 15 | 0.6 | 8 | 39.5 | 69.3 |
25 | 10 | 15 | 0.3 | 13 | 63.5 | 83.9 |
26 | 20 | 25 | 0.4 | 8 | 53.2 | 72.3 |
27 | 20 | 5 | 0.4 | 18 | 60.2 | 90.7 |
28 | 20 | 15 | 0.6 | 18 | 45.3 | 72.9 |
29 | 20 | 25 | 0.4 | 18 | 64.1 | 74.3 |
The pulseⅠ, Ⅱ, Ⅲ and other excipients were compressed by different compression forces (8KN, 13KN, 18KN) using a single-punch tableting machine (Riva Mini Press MⅡ, Riva, Germany) with a punch of 22 mm diameter by Box–Behnken design in
The tablets were prepared by film-coating of Opadry® (Colorcon). The coating suspension was prepared by adding Opadry® powder to the purified water with 500 rpm stirring for at least 45 min. The amoxicillin containing tablets were coated using the Opadry® suspension by a coating pan (BY300, Shanghai precision instruments Co., Ltd, Shanghai, China). The conditions were given as follows: the temperature was 35 ~ 40°C, the spray rate was 3 ~ 6 mL/min, the atomization pressure was 1.5 bar and weightening ratio of film coating was 3%. The film-coated tablets were saved in sealed pockets and closed containers.
The dissolution test was performed using USP apparatus II (Paddle) with 900 mL solution at 37 ± 0.5°C in 3 stages: 50 mM potassium phosphate monobasic buffer at pH 4.0 (0–2 h), pH 6.0 (2–4 h) and pH 6.8 (4 h and beyond), with a rotating speed of 75 rpm, at designated sampling time points: 0.25, 0.5, 1, 2, 2.25, 2.5, 3, 4, 4.25, 4.5, 5 and 6h [
The surface morphology of coated pellets before and after the compression was investigated by field emission scanning electron microscope (FESEM). The samples were sputter coated with a thin layer of silver under Argon atmosphere, and scanned by the FESEM (Zeiss Sigma, Germany).
Six male adult beagle dogs had an age range of 6 ~ 10 months and a weight range of 9.0 ~ 11.0 kg. The principles of Laboratory Animal Care were approved by the department of laboratory animal research at China State Institute of Pharmaceutical Industry (License Number: SYXK (Shanghai) 2014–0018). The use of animals was approved by the Animal Management and Ethics Committee of China State Institute of Pharmaceutical Industry. Prior to the test, all the dogs were fed chow and sterilized tap water in a standard laboratory.
The multi-pulsatile release tablets and common tablets (250 mg, Kunming Yuanrui Pharmaceutical Co., Ltd., China) were used in the pharmacokinetic and the bioequivalence studies. A single multi-pulsatile release tablet (775 mg) or three common tablets (750 mg) was administered orally after a meal by the design of a randomized crossover study. The foreleg venous blood samples were collected into vacuum tubes immediately before and after the drug administration at the following time points: 0.5, 1, 1.5, 2, 2.5, 3, 4, 4.5, 5, 6, 8, 10 and 12 h. The blood samples were processed by centrifugation at 3600 rpm and at 4°C for 10 min. The obtained plasma samples were stored at −80°C until analysis.
The plasma was thawed at room temperature. To all the standards (25 μL) or QC samples (25 μL), 50 μL of ampicillin trihydrate (IS) and 225 μL of blank plasma were added, and vortexed for 30 s. Then, methanol (0.7 mL) was added to the plasma samples, being subsequently vortexed for 30 s. The mixture was centrifuged at 12000 rpm at 4°C for 3 min. Then the supernatant was transferred into autosamper vials and subject to the Liquid chromatography–mass spectrometry / mass spectrometry (LC-MS/MS) analysis.
The HPLC system consisted of a model CBM-20A system controller, a model LC-20AD pump, a model SIL-20AHT auto-injector, a model CTO-20A column oven, and a model C-R7A plus integrator (Shimadzu, Kyoto, Japan). A Triple Quadrupole Mass Spectrometer (LCMS-8030, applied Shimadzu, Kyoto, Japan) was used for the LC-MS-MS analyses and detection. The MS was operated in positive ion detection mode [
The temperature of the vaporizer was set at 400°C. The drying gas (N2) flow rate was 15 L/min. The nebulizer gas (N2) flow rate was 3 L/min. The mass spectrometer was operated at unit mass resolution with a dwell time of 100 ms per transition. Collision energy for amoxicillin was set at -11 V and -20 V for IS. Quantification was performed using multiple reactions monitoring (MRM) of the transition ions m/z 366.00 → m/z 349.10 and m/z 350.00 → m/z 106.10 for amoxicillin and IS respectively.
A concentration-time curve was plotted. The AUC0-12 h and AUC0-∞ was calculated by the trapezoidal rule [
The Simcyp® software (Simcyp Simulator, Version 13.2, Certara USA, Inc., USA) was used to establish the PBPK model of amoxicillin for human and beagle dog based on the physical and chemical properties of amoxicillin and the pharmacokinetics parameters reported in literature as summarized in
Name | Input value | Source |
---|---|---|
Mol Weight (g·mol-1) | 365.400 | Literature [ |
Compound Type | Ampholyte | Literature [ |
Solubility (mg·mL-1) |
3.4 | Literature [ |
pKa1 |
2.800 | Literature [ |
pKa2 |
7.200 | Literature [ |
log Po:w |
-0.580 | Literature [ |
Peff,man (10−4 cm·s-1) |
1.47 | Literature [ |
B/P |
0.67 | Predicted by Simcyp |
fu value |
0.63 | Literature [ |
CLpo(L·h-1·Kg-1) |
0.045 | Literature [ |
a, Measured values
b, Optimized values
c, Predicted values
log Po:w, the oil-water partition coefficients; Peff,man, Human jejunum permeability; B/P, Blood/plasma ratio; fu value, Fraction unbound in plasma; CLpo, oral clearance.
The Box–Behnken design statistical analysis Box–Behnken design was performed using Software Design-Expert (Design-Expert 8.0.6, Stat-Ease, Inc., USA) and Origin Pro Software (Origin Pro 8.5.1, Origin Lab Co., Ltd., USA). The pharmacokinetic studies statistical analysis data and results were performed by a two one-sided t-test, and a P value < 0.05 was accepted as significant by DAS (DAS Version 2.0, Shanghai, China).
The excipient Avicel MCC PH 101 or PH 301 was chosen due to their capacity to enhance the buffering capacity of pulse Ⅰ, and that is crucial to the tablet compression procedure. The concentration of the binder HPMC E 5 solution was fixed at 5%, 10% and 15% considering the particle size distribution of granules. The composition of different formulation of the pulseⅠwe examined was shown in
formulation | F1 | F2 | F3 | F4 | F5 | F6 |
---|---|---|---|---|---|---|
Amoxicillin(%) | 69 | 69 | 69 | 69 | 69 | 69 |
Avicel PH 301(%) | 25 | 25 | 25 | / | / | / |
Avicel PH 101(%) | / | / | / | 25 | 25 | 25 |
HPMC E5(%) | 4 (5% |
4 (10% |
4 (15% |
4 (5% |
4 (10% |
4 (15% |
PVPP(%) | 2 | 2 | 2 | 2 | 2 | 2 |
500μm >PSD>105μm(%) | 64.2 | 80.1 | 67.1 | 59.2 | 70.7 | 66.5 |
angle of repose (°) | 36.3 | 27.8 | 34.6 | 41.4 | 35.2 | 35.6 |
F1 ~ F6, is formula 1 ~ formula 6 respectively; PSD, particle size distribution.
* the solution concentration of HPMC E5.
It can be seen from the
It is crucial to maintain the humidity to some extent during the process of the extrusion. This is also important to get the uniformity of the pellets [
Box–Behnken designs [
R1 = +92.76396–1.31586*x1-0.43943*x2+49.93199*x3-3.19882*x4-6.25000E-003*x1*x2+3.49828*x1*x3+0.01150*x1*x4-0.21897*x2*x3+0.033000*x2*x4+0.98276*x3*x4-0.027583*x1^2+9.41667E-003*x2^2–188.88889*x3^2+0.092667*x4^2 (R2 = 0.9361); R2 = +95.69340+0.10771*x1-1.30345*x2+150.19789*x3-2.44979*x4+0.043500*x1*x2-0.20690*x1*x3-0.013000*x1*x4+2.34828*x2*x3+1.00000E-002*x2*x4-6.89655E-003*x3*x4-0.030250*x1^2–0.045500*x2^2–258.61111*x3^2+0.10100*x4^2 (R 2 = 0.9361);
The interaction among the independent variables (X1, X2, X3 and X4) in the equations and the interaction between the independent variables and the dependent variables (R1, R2). The coefficients before (X1 ~ X4) indicated the independent variables were dependent on each other. The coefficients with more than one factor term and those with higher order terms represented interaction terms and quadratic relationships [
X1: coating weight of Eudragit L30 D-55; X2: coating weight of AQOAT AS-HF; X3: the extrusion screen aperture; X4: compression forces; R1 and R2: the cumulative release at 2.25h and 4.5h.
When the extrusion screen aperture (X3) and the compression forces (X4) were fixed values, R1 or R2 were increasing as the coating weight of Eudragit L30 D-55 (X1) and the coating weight of AQOAT AS-HF (X2) were decreasing in the
A, effect of X2, X3 variables on the response R1 and R2; B, effect of X2, X4 variables on the response R1 and R2; C, effect of X1, X4 variables on the response R1 and R2; D, effect of X1, X3 variables on the response R1 and R2; E, effect of X3, X4 variables on the response R1 and R2; F, effect of X1, X2 variables on the response R1 and R2.
Evaluating indexes | Observed | Predicted |
---|---|---|
R1 | 51.7 | 54.4 |
R2 | 83.9 | 83.9 |
Different compression forces influenced coating the film integrity during the compression progress. The ideal coating should be stable and strong enough and keep its physical continuity to have a large extension before being destroyed [
After preliminary investigation, a speed of 75 rpm was chosen to investigate the dissolution for the comparison of uncompression and different pression force (
FESEM photos are shown in
Scanning electron micrographs of pellets: (A), (B) uncoated pellets; (C), (D) L30D-55 coated pellets; (E), (F) AS-HF coated pellets; (G), (H) after pressed with 50% pellets under compression force of 13 KN.
Linearity of the calibration curve was yielded in the concentration range from 105.60 to 83633.90 ng·mL-1 (r2 ≥ 0.996). The intra-day and inter-day precision (n = 6) was all less than 5.0%. The mean recoveries of amoxicillin at QC concentrations (316.80, 34847.46 and 66907.11ng·mL-1) were 91.7 ± 2.7%, 93.4 ± 3.9% and 92.6 ± 3.2% (n = 3), respectively. In storage for 3 days at −80°C and three freeze–thaw cycles, amoxicillin remained stable in plasma. And, the results manifested that the bioanalytical method was qualified to quantify the plasma concentration for the pharmacokinetic studies.
Extrapolation method for AUC0-∞ was estimated by a linear regression model from the last three data points, which has been log-transformed, in the elimination phase. Cmax was received from the actual surveyed concentration without interpolation. Plasma concentration time profiles of amoxicillin in the beagle dogs given pulsatile release tablets and reference tablets are shown in
A: amoxicillin pulse tablets; R: amoxicillin tablets
Pharmacokinetic parameters | Unit | Amoxicillin pulse tablets | Amoxicillin tablets(common tablets) |
---|---|---|---|
AUC0-12 h /Dose | ×10−6 mL-1·h | 141.17 ± 25.41 | 133.38 ± 25.14 |
AUC0-∞ /Dose | ×10−6 mL-1·h | 141.17 ± 25.41 | 133.38 ± 25.14 |
Tmax | h | 2.08 ±0.49 | 1.25 ± 0.27 |
Cmax /Dose | ×10−6 mL-1 | 46.76 ± 6.75 | 75.56 ± 8.34 |
t1/2 | h | 1.67 ±0.27 | 1.30 ± 0.38 |
MRT0→12 h | h | 3.01 ±0.19 | 2.05 ± 0.28 |
AUC0-12 h, area under the concentration time curve from zero to 12 h; AUC0-∞, area under the concentration time curve from zero hour to infinity; Tmax, time point of maximum plasma concentration; Cmax, maximum plasma concentration; t1/2, elimination half-life; MRT0→12 h, the average retention time.
The Simcyp® software is a platform and database for the ‘bottom-up’ mechanistic modelling and simulation dealing with the processes of oral absorption, tissue distribution, metabolism and excretion of drugs and drug candidates in healthy and disease populations [
Predicted | Observed [ |
Fold error | |
---|---|---|---|
Cmax (μg·mL-1) | 9.10 | 10.57 | 1.16 |
AUC0-∞ (μg·mL-1·h) | 34.30 | 33.28 | 1.03 |
Tmax | 1.68 | 1.75 | 1.04 |
beagle dog | human | |||||
---|---|---|---|---|---|---|
Predicted | Observed | Fold error | Predicted | Observed | Fold error | |
Cmax (μg·mL-1) | 29.64 | 35.07 | 1.18 | 7.14 | 6.62 | 1.08 |
AUC0-∞ (μg·mL-1·h) | 105.09 | 105.87 | 1.01 | 34.09 | 29.79 | 1.14 |
Tmax | 1.16 | 2.08 | 1.79 | 2.64 | 3.14 | 1.19 |
Experimental results manifested that an extrusion/spheronization and different pH sensitive coating materials preparation is successfully applied to a pulsatile release tablet to get pulsatile release in