Gastric-State-Controlled Bioequivalence Studies
Establishing bioequivalence is difficult for drugs with high inter-subject variability or strong dependence on the physiological state of the gut.
Bioequivalence is the absence of a significant difference in the rate and extent of drug absorption of a test product when compared to a reference product.1 Bioequivalence is assessed using statistical analysis of pharmacokinetic measures, such as area under the curve (AUC). Researchers calculate 90% confidence intervals for the ratio of the averages (population geometric means) of the data points for the test and reference products. It is well known that establishing bioequivalence is difficult for drugs with high inter-subject variability or strong dependence on the physiological state of the gut, especially pH and motility. This applies to many of the class I drugs, which are highly soluble and permeable, according to the newest biopharmaceuticals classification system.2 Clavulanic acid and amoxicillin are classic examples.
Table 1. Pharmacokinetic Means (SEM) and T-test of AUC0-t and Cmax
Amoxicillin is a beta-lactam antibiotic with a wide antibacterial spectrum. Clavulanic acid has comparatively low antibacterial activity but irreversibly inhibits a broad spectrum of beta-lactamase enzymes. The elimination half-lives of amoxicillin and clavulanic acid are 1 to 1.5 h, with peak plasma levels after 1 to 2.5 h following oral administration.3 Our data suggest that the inter-subject variability in the pharmacokinetic parameters of clavulanic acid is 25 to 50%. Therefore, a minimum set of 24 subjects is needed for a bioequivalance study with 80% statistical power to detect a 20% mean difference with 95% confidence. (The power of a statistical test is the probability of correctly rejecting the null hypothesis.)
We believe we are the first to investigate the role of gastric state on these important and costly studies conducted in humans. We compared two independent bioequivalence studies of an amoxicillin-clavulanic acid combination to examine the effect of controlling gastric state on bioequivalence.
Figure 1. Mean (±SD) for Amoxicillin Serum Concentrations (µg/mL) After 250 mg Oral Dose
MATERIALS AND METHODS
We tested amoxicillin-clavulanic acid combination tablets (from the same lots bought on the Jordanian market) against GlaxoSmithKline's Augmentin, the reference drug. All reagents were obtained from Sigma Chemical Company, USA. Both test and reference products met
in vitro
USP compendial standards.
We investigated two outcomes: the bio-equivalence of the test and reference tablets and the impact of gastric state on bioequivalence. A total of 24 healthy, male subjects gave written informed consent to participate in the study, which was approved by the Institutional Review Board of the study site, Istiklal Hospital in Amman, Jordan. Subjects were judged healthy based on medical history, physical examination, complete blood count, and serum chemistry. In addition, all subjects were medication-free, including over-the-counter drugs, for seven days prior to the study day. However, as one subject was dropped due to vomiting, 23 subjects completed the trial and formed the basis for our analysis.
Table 2. Analysis of the Test-to-Reference Ratios of AUC0-t and Cmax
EXPERIMENTAL PROCEDURE
First, we tested the uncontrolled gastric state. All subjects fasted overnight for ten hours. Then, each subject took an oral 250 + 125 mg dose of amoxicillin-calvulanic acid, followed by 240 mL tepid water. Blood samples were collected using an indwelling catheter before the initial dose and at 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8 and 10 h after dosing. Samples were centrifuged at 3,000 rpm for 5 min and then stored at -20°C until analysis. Stability studies show that amoxicillin is stable in plasma for two weeks when stored at -20°C, and clavulanic acid is stable in plasma for three weeks when stored at -70°C. (For more details, see "Analytical Procedures".)
Controlled Gastric State. We found an interesting procedure in the literature that can bring subjects to a consistent gastric state. The gastric emptying rate of a 200-mL, zero-calorie, phenol-red solution was found to be substantially greater than that of 50-mL solution, suggesting that gastric emptying of larger volumes is governed by stomach volume rather than stomach contractile activity or motility.4,5 In addition, the overall mean emptying time is faster using the 200-mL solution. Additionally, the stomach distension produced by a large volume of aqueous solution with no calories reduces variability in the gastric emptying rate significantly, compared to a smaller volume.4,5
In our second trial, the gastric state for all subjects was adjusted to the same baseline. The 23 subjects fasted for ten hours and then drank 200 mL of tepid water exactly one hour before dosing — which does not violate FDA rules concerning bioequivalence study protocols. This volume of water changes gastric motility from a more variable, fasted-state to a less-variable state similar to a fed state.4-6
PHARMACOKINETIC AND STATISTICAL DATA ANALYSIS
The following pharmacokinetic parameters were calculated by non-compartmental analysis: the areas under the curve from administration to last quantifiable measurement (AUC
0-t
) and to infinity (AUC
0-∞
), maximum plasma concentration (C
max
), time to maximum plasma concentration (T
max
), elimination rate (K
el
) and half life. 90% confidence intervals and analysis of variance were calculated according to CDER guidance methods.
1
All data analyses were performed with Kinetica2000 software.
7
Figure 2. Mean (+SD) Clavulanic Acid Serum Concentrations (µg/mL) After 125 mg Oral Dose
RESULTS AND DISCUSSION
Plots of mean serum drug concentrations are shown in Figures 1 and 2. Table 1 shows the pharmacokinetic means of the crucial parameters in bioequivalence decision: AUC
0-t
and C
max
.
We suspected that controlling gastric state would reduce intra-subject variability by changing gastric motility from a more variable, fasted state to a less-variable, fed-like motility, thus eliminating gastric emptying and motility factors. In addition, pre-dosing with water can help stabilize acid-labile products and allow a small amount of water to remain in the stomach so that when tablets start to dissolve, there is sufficient volume to overcome any small differences in inherent dissolution rates.4-6
However, in our two independent studies, inter-subject variability was essentially the same after controlling the gastric state. We use a paired t-test and look for p-values less than 0.05. When comparing gastric states, we found significantly different Cmax for reference amoxicillin and AUC0-t for reference clavulanic acid, but none of the other comparisons were statistically significant. A possible explanation of these results is that we had too small a sample, so small differences in gastric state did not affect the outcome. Table 2 shows that with p-values far above 0.05, we successfully demonstrated the bio-equivalence of the two drugs for both controlled and uncontrolled gastric states.
Bioequivalance is a function of all the factors and variables involved in the study, and the more variables we control, the better our study design. Therefore, even though the outcome of both the controlled and uncontrolled studies was the same, we suggest controlling gastric state using the methods described here. Using this procedure increases the probability that any treatment effect reflects a significant difference between the two formulations.
In conclusion, we devised a way to control subjects' gastric state to the same baseline without violating study protocol or FDA guidelines. The technique is simple but could save drug companies a lot of money and risk. We suggest it be adopted for bioequivalance studies, especially studies of highly variable drugs and class-I drugs that show gastric state absorption dependency.
ACKNOWLEDGEMENTS
We thank the staff, nurses, and technicians in International Pharmaceutical Research Center and Istiklal Hospital for their help and cooperation. Triumpharma, LLC and International Pharmaceutical Research Center supported this work.
REFERENCES
1.FDA, CDER, Population and individual bioequivalence working group of the biopharmaceutics coordinating committee in the office of pharmaceutical science. Guidance for industry: statistical approaches to establishing bioequivalence. 2001. Available at:
www.fda.gov/cder/guidance/3616fnl.htm
.
2 Amidon GL, Lennernäs H, Shah VP, and Crison JR. 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.
3. Gold Standard Multimedia Inc. Clinical Pharmacology USA, 1995. Available at URL: www.cp.gsm.com.
4. Oberle RL, Chen T-S, Lloyd C, Barnett J, Owyang C, Meyer J, et al. The influence of the interdigestive migrating myoelectric complex on the gastric emptying of liquids. Gastroenterology 1990; 99(5):1275-1282.
5. Amidon GL. The role of the BCS in regulatory requirements of bioequivalance. Presentation on new EU and FDA notes for guidance on BA/BE. 2003 April; Lisbon Portugal.
6. Rhie JK, Hayashi Y, Welage LS, Frens J, Wald RJ, Barnett JL, et al. Drug marker absorption in relation to pellet size, gastric motility and viscous meals in humans. Pharm. Res. 1998; 15(2): 233-238.
7. InnaPhase Corp. Kinetica2000 manual, Version 3.0. 2000; Philadelphia, PA.
ANALYTICAL PROCEDURES
A rapid and sensitive high-performance liquid chromatographic (HPLC) method for the determination of amoxicillin in human plasma was validated using Cefadroxil (an antibiotic) as the internal standard. Sample preparation consists of adding the internal standard and precipitating plasma protein using 5% perchloric acid. The resulting mixture is centrifuged, and the supernatant is then chromatographed on a Lichrospher RP-18 column at room temperature. The mobile phase was pumped at 1.5 mL/min and consists of acetonitrile and phosphate buffer in the ratio of 1 to 9.
Detection of amoxicillin and the internal standard is achieved by monitoring the absorbance of ultraviolet light at 230 nm. The peak-area ratio of drug to internal standard and the concentration is calculated by Millennium32 software (Version 3.00, Waters, USA). The relationship between concentration and peak-area ratio was found to be linear within the range from 0.50 to 18.00µg/mL for amoxicillin. The limit of quantitation from 0.1 mL of plasma for amoxicillin was 0.50 µg/mL.
The intra-day (day vs. night) accuracy of the method for amoxicillin ranged from 92.00 to 101.33%, while the intra-day precision ranged from 3.19 to 8.52%. The inter-day (one day vs. the next) accuracy ranged from 96.00 to 102.79%, while the inter-day precision ranged from 3.53 to 9.60%. The absolute recovery of amoxicillin was 101.25% and the absolute recovery of the internal standard (cefadroxil) was 99.39%, while the relative recovery of amoxicillin ranged from 104.13 to 105.9 %.
A rapid and sensitive HPLC method for the determination of clavulanic acid in human plasma was validated using metronidazole (an antibiotic) as the internal standard. Sample preparation consists of adding the internal standard and then precipitating plasma protein using methanol. The resulting mixture is centrifuged, and the supernatant is diluted with mobile phase and then chromatographed on a Nucleosil 100 C18 AB 4 x 125 column (4 mm i.d. and 5 µm particle size) at room temperature. The mobile phase is pumped at 1.5 mL/min and consists of acetonitrile, methanol, and sodium acetate buffer in ratios of 1 to 1 to 8 with a final pH of 2.85.
Detection of clavulanic acid and the internal standard is achieved by monitoring the absorbance of ultraviolet light at 311 nm. The peak-area ratio of drug to internal standard and the concentration are calculated by Class VP-5 software (Version 5.03, Shimadzu, Japan). The relationship between concentration and peak-area ratio was found to be linear within the range from 0.10 to 5.00 µg/mL for clavulanic acid. The limit of quantitation from 0.5 mL of plasma for clavulanic acid was 0.10 µg/mL.
The intra-day accuracy of the method for clavulanic acid ranged from 89.33 to 98.90%, while the intra-day precision ranged from 3.64 to 13.40 %. The inter-day accuracy ranged from 87.00 to 96.08%, while the inter-day precision ranged from 5.99 to 13.54 %. The absolute analytical recovery of clavulanic acid was 105.82%, and the absolute analytical recovery of the internal standard (metronidazole) was 92.66%, while the relative analytical recovery of clavulanic acid ranged from 88.00 to 94.40%.
Corresponding author Nasir M. Idkaidek, Ph.D., is professor at the College of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan, and also head of clinical studies at Triumpharma, LLC, Amman, Jordan, +962.2.7201000, fax +962.2.7095019, dekaidek@just.edu.jo, nasir@triumpharma.com. Ahmad Al-Ghazawi, Ph.D., is the general manager of Triumpharma, LLC. Naji Najib, Ph.D., is professor at the College of Pharmacy, Jordan University of Science and Technology, and general manager of International Pharmaceutical Research Center, Amman, Jordan.
