A Comparative Single-Dose Bioequivalence Study of Two Enteric Coated Aspirin Brands Among Healthy Volunteers

 

Abeer A. Zeitoun, PharmD

Jean G. Dib, PharmD

Mohammad Mroueh, PhD

 

School of Pharmacy, Lebanese American University, Byblos, Lebanon

 

KEY WORDS: Acetylsalicylic acid (ASA), bioequivalence, bioavailability, HPLC

ABSTRACT

Objective: Aspicot® is an enteric-coated aspirin that is being extensively used in the Middle East, including Lebanon where this drug is manufactured, without any clinical in vivo implication showing or confirming its bioequivalence. For this reason, this investigation was carried out to evaluate the in vitro dissolution as well as the bioavailability and pharmacokinetic properties of 2 tablet oral dosage forms of enteric-coated aspirin, Aspirin protect® and Aspicot®, in a single dose of 200 mg among healthy volunteers.

Method: Twelve healthy volunteers (7 males, 5 females) were enrolled in the study; each received a single dose of each drug in an open randomized 2-way crossover study with a washout period of 7 days. Venous blood samples were obtained before each administration and at 10 different times (0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 12 h after dosing). The plasma concentrations of acetylsalicylic acid were then determined using sensitive High Performance Liquid Chromatography (HPLC) assay. For the purpose of bioequivalence analysis, area under the plasma concentration-time curve (AUC_0-∞) and maximum concentration (C_max) were considered primary variables and time to C_max (T_max) as a secondary variable.

Results: The 2 products were closely related in terms of their in vitro compendial requirements. Moreover, the difference in peak serum concentration, corresponding peak time, and area under serum concentration-time curve for the 2 products were not statistically significant (P >0.05).

Conclusion: The results were within acceptable bioequivalence ranges. These findings suggest that the 2 products are bioequivalent in terms of bioavailability and pharmacokinetic effects on healthy volunteers.

INTRODUCTION

After almost a century of clinical use, aspirin remains one of the world’s most extensively used drug. Aspirin, or acetylsalicylic acid (ASA), was used as an analgesic during the time of Hippocrates, and their antipyretic effects have been recognized for more than 200 years. Acetylsalicylic acid was introduced in the late 1890s and has been used to treat a variety of inflammatory conditions; however, the antiplatelet activity of this agent was not recognized until almost 70 years later.^1,2

Aspirin exerts its effect primarily by interfering with the biosynthesis of cyclic prostanoids, which include thromboxane A2 (TXA2), prostacyclin, and other prostaglandins. These prostanoids are generated by the enzymatically-catalyzed oxidation of arachidonic acid, which is itself derived from membrane phospholipids. Arachidonic acid is metabolized by the enzyme prostaglandin (PG) H-synthase, which through its cyclooxygenase (COX) and peroxidase activities, results in the production of PGG2 and PGH2 respectively. PGH2 is then metabolized by specific synthases into prostaglandins: PGD2, PGE2, PGF2a, PGI2 (prostacyclin), and TXA2, all of which mediate specific cellular functions.1,3,4,5

Despite continuing efforts to develop new analgesic and antipyretics with improved clinical tolerability, aspirin remains the most widely used drug of this class. The oral administration of conventional aspirin tablets is the most commonly used form; however, it entails drawbacks such as gastric irritation.

To minimize this problem, pharmaceutical formulations have been developed that are especially designed to avoid the contact of large drug particles with discrete areas of the gastric wall. Such formulations include solution (by using either rapidly water-soluble salt such as lysine ASA or amino acid l-ornithine) and buffered ASA using effervescent carbonated-buffered formulations.6 Other formulations were formulated in an attempt to decrease side effects, mainly gastrointestinal toxicity, such as enteric-coated tablets, sustained-release, and topical formulations (such as suppositories).^1,5,7 Different studies proved that these different formulations would have different bioequivalence and bioavailability.5–7

The most common side effect of regular aspirin intake is its action on the stomach resulting in dyspepsia, gastric and duodenal erosions, occult bleeding, and hemorrhage. Dyspepsia is a relatively frequent disorder in patients but does not correlate with gastric bleeding. To reduce dyspepsia, aspirin was given with meals or antacids with the hope that by buffering the acid in the stomach, gastric irritation and blood loss will be reduced. However, using an antacid with aspirin has a significant effect on plasma levels of salicylate. The increase in the urinary pH produced an increase in the renal clearance of salicylate and thus produces reduced plasma level of the salicylate level. Besides, dyspepsia, if not avoided, would decrease the gastric emptying, which would alter the level of aspirin absorbed. Enteric-coated aspirin was reported to decrease such a problem.1,5,7,8

As for gastric toxicity, attempts have been made to decrease it by pharmacologic manipulation. Enteric-coated formulations have been demonstrated to produce relatively selective inhibition of platelet TXA2 production with minimal direct gastric irritation and thus might have less gastric toxicity. Additionally, gastrointestinal blood loss has been shown to be less with enteric-coated aspirin than uncoated ones. Nevertheless, because the mechanism of action of enteric-coated aspirin still leads to the systemic inhibition of COX, coated aspirin would still be associated with gastric bleeding compared with placebo.1,5,7,8

In aspirin plain forms, the absorption of acetylsalicylic acid takes place rapidly and completely after oral administration. As a result of the acid-resistant layer of enteric-coated aspirin tablets, the active substance is not released in the stomach but rather in the alkaline medium of the intestine, mainly from the upper small intestine; therefore, absorption of the ASA is delayed by 3 to 6 hours and the time to achieve the mean peak of aspirin concentration is 4 to -6 hours. Thus, the corresponding times to achieve peak salicylic acid concentrations are 8 to 14 hours.9 As for the steady-state levels, it is reached after 5 to 7 days of therapy.7

After oral administration of aspirin, it was found to follow first-order kinetics. Many factors affect the rate of absorption such as the rate of gastric emptying, volume of blood, concurrent administration of other drugs, posture, exercise, and pH of stomach content.5,7,9,10

Once absorbed, aspirin is rapidly hydrolyzed to salicylic acid with a half-life of 15 to 20 minutes. Thus, from this stage onward the pharmacokinetics of aspirin are predominantly dependent on the salicylate moiety. Salicylic acid plasma protein binding is concentration-dependent (66–98%). Volume of distribution ranges from 9.6 to 12 L in adults.5 After the administration of high doses, ASA is detectable in the cerebral, spinal, and synovial fluid. It crosses the placenta and passes into breast milk.

ASA is converted into its main metabolite salicylic acid during and after absorption. The acetyl group of ASA begins to be split off hydrolytically even during passage through the gastrointestinal mucosa, but mainly this process takes place in the liver. The elimination kinetics of ASA is dose-dependent after Michales-Menten kinetics, because the metabolism is limited by the capacity of liver enzymes. The elimination half-life varies between 2 to 3 hours after low doses to approximately 12 hours after usual analgesic doses.

Enteric-coated aspirin (Aspicot®) is being extensively used among different patients as an over-the-counter drug in the Middle East, including Lebanon where this drug is being manufactured by Pharmaline” (a Lebanese pharmaceutical industry), without any clinical in vivo studies showing or confirming its bioequivalence. Moreover, the erratic absorption of enteric-coated aspirin and the unpredictable bioavailability has been reported in several studies accounting for large unpredictable variation in the blood levels after oral administration of the drug.1,7,11,12 These reasons we’ve mentioned strongly support the need for a bioequivalence study between 2 different enteric-coated aspirin formulations (Pharmaline” enteric-coated aspirin [Aspicot®] versus Bayer” enteric-coated aspirin [Aspirin protect®]).

SUBJECTS AND METHODS

Subjects

Fifteen healthy volunteers (8 males, 7 females) participated in this study. Twelve (7 males, 5 females) completed the study. Three dropped out as a result of noncompliance. All the subjects were considered healthy after a complete physical examination, urinalysis, hematology, and blood chemistry analysis that were done 1 week before the trial. All subjects signed an informed consent before study initiation. The study was approved by the Ethics and Research Committee for Clinical Investigation at Notre Dam Des Secours Hospital (NDDSH), Byblos, Lebanon, and was carried out according to the ethical principles.

Study Drugs

The tested drug was a Lebanese 100-mg tablet of enteric-coated aspirin (Lot number 3040 2, Pharmaline® expiration date February 2004). The reference formulation was a German 100 mg enteric-coated aspirin (Lot number GRFCA 1, Bayer©, expiration date January 2005).

In vitro Dissolution Rate

The dissolution tests were carried out on both formulations using the basket method according to US Pharmacopeia (USP) XXIV guidelines, operated at 100 rpm in a dissolution bath at 37˚ C ± 0.5˚C for the acid and the buffer stage. Hydrochloric acid (0.1 N) was used as the dissolution medium to provide a pH similar to that of the gastric juice. As for the buffer, 0.1 hydrochloric acid and 0.2 M tribasic sodium phosphate (3:1) adjusted with 2 N hydrochloric acid or 2 N sodium hydroxide to a pH of 6.8 ± 0.05 similar to that of the gastrointestinal. Samples were withdrawn and filtered after 2 hours with the acidic medium and after 90 minutes in the buffer medium. Dissolved acetylsalicylic acid was determined from ultraviolet absorbencies at the wavelength of 280 nm for the acidic medium and 165 nm for the buffer medium.

Study Design

The study was carried out in an open randomized 2-way crossover design with blind determination of drug plasma concentrations. The washout period between formulations was 1 week. All volunteers received a single 200-mg dose, after fasting for at least 10 hours, during each of the 2 trial periods. The human bioavailability study was performed at NDDSH, Byblos, Lebanon. Analysis of the acetylsalicylic acid plasma concentrations level by HPLC was conducted at the Lebanese American University, School of Pharmacy, Byblos, Lebanon.

Within the 14 days before the first administration, every subject verified his compliance with the inclusion and exclusion criteria. Table 1 shows the mean demographic data of the 12 volunteers who completed the study. One week before enrollment a complete physical examination and biochemical screening was performed. Subjects with a history of bleeding disorders, cardiac, renal, hepatic, and gastrointestinal diseases, those undergoing surgery or pregnant, and those with drug allergy were excluded from the study. Volunteers were asked to abstain from using ethanol, caffeine, chocolate, and tea for at least 24 hours before each dose. They also were asked to abstain from taking any prescription drugs for 2 months before the study or any other drug, including vitamins, for at least 1 week before and until the end of the study. Subjects fasted from 10 hours before and until 3 hours after the administration of the drug. Enteric-coated aspirin was administered with 200 mL of water. During the 2 trial periods of the study, all subjects abstained from smoking and exercise. The dietary regimen was similar for all subjects in both trial periods.

Collection and Storage of Samples

Venous blood samples were collected immediately before and 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 12 hours after drug administration. The collected blood samples were immediately chilled; the plasma was separated by centrifugation in a refrigerated centrifuge, and set at 4˚C with a rate of 3500 rpm for 10 minutes. The separated plasma was then immediately shock-frozen using liquid nitrogen and then stored at –70˚C until assay. The stability of these samples at –70˚C is 3 months.13

Sample Preparation

Frozen plasma sample were thawed in an ice water bath. An amount of 200 mL plasma was added to 200 mL of the internal standard solution (5 mg/mL 2-methyl benzoic acid [MBA] in a 50:50 mixture of 0.2 M hydrochloric acid and 0.2 M orthophosphoric acid) and then vortex-mixed for 1 to 2 seconds. The resulting pH of the mixture was approximately 2.7. Acidification of the internal standard was needed to prevent the hydrolysis of ASA. An amount of 400 mL acetonitrile was then added to precipitate protein and then was mixed. After 15 minutes at 4˚C the mixture was centrifuged for 1 minute to separate the precipitated proteins. The supernatant was then transferred into 2 mL Eppendorf tubes containing 100 mg sodium chloride. The suspension was then vortex-mixed briefly and incubated at 4˚C for 10 minutes and then centrifuged again for 1 minute. All dilutions in plasma were prepared keeping the tubes in an ice water bath.20

Acetylsalicylic acid and salicylic acid serum concentrations were determined by the internal standard method using peak concentration (acetylsalicylic acid, MBA) and (salicylic acid, MBA).

Analysis of Enteric-Coated Aspirin Plasma Samples

Plasma concentrations of acetylsalicylic acid were measured in a blinded fashion by HPLC.20 The HPLC consisted of a double-piston 2-pump system (Waters, model 510, USA), an injector (Rheodyne, USA) with 50 mL loop size, a variable ultraviolet detector wavelength monitor set at 273 nm (Waters, model 486, USA), an integrator (Waters, model 746, USA), and a prepacked stainless steel column mBondapack C18, 4-mm silica (3.9 ¥ 300 mm) column. The system was operated at room temperature. The mobile phase consisted of water/phosphate buffer (H₃PO₄/KH₂PO₄, pH 2.5)/acetonitrile in a ratio of (35/40/25) by volume. The phosphate buffer was used in the mobile phase to keep the ASA in the nonionized state, which will allow the ASA to remain in the organic phase on extraction. The mobile phase was degassed under vacuum using a 0.45 mm ¥ 47 membrane filter paper (Nylon 66, Supelco Inc, Bellifonate, USA) and a degassing system (Wheaton, USA). The flow rate was 2 mL/min.

Pharmacokinetic Parameters and Statistical Analysis

The pharmacokinetic characteristics of enteric-coated aspirin were determined from the plasma concentration-time data. Peak plasma concentration (C_max) and time to maximum plasma concentration (T_max) were determined directly from raw data. The area under the curve (AUC_0-t, from 0 to last measured concentration) was calculated by the trapezoidal method.

Data were assessed using Jandel Sigmastat Statistical Software version 2.0 1995, (Jandel Corp., San Rafael, CA) using the Mann-Whitney Rank Serum Test (a nonparametric comparison) and 95% confidence interval analysis with a minimum level for significant difference set at P <0.05. All data were reported as mean ± standard deviation.

RESULTS

The in vitro biopharmaceutical characteristics of the 2 formulations were similar as shown by their dissolution performance in acidic and buffer medium (Table 2). Dealing with enteric-coated tablets, the expected dissolution is to occur in the buffer medium simulating the upper intestinal medium. Therefore, little or minimal dissolution is expected to occur in the acid stage that simulate the gastric medium. Results were manifested by the 2 brands with no significant difference.

The clinical study was completed within 4 weeks. Enteric-coated aspirin was well tolerated by subjects, and no adverse events occurred during the study. Mean pharmacokinetic parameters for the 12 subjects for the enteric-coated aspirin tested formulation and the reference formulation are shown in Table 3. The time course of mean acetylsalicylic acid concentrations after 200 mg for both formulations is presented in Figure 1. The maximum concentration was reached for both preparations within 5 hours.

For the C_max values, there was no significant difference in the bioavailability of the test formulation compared with the reference formulation (23.66 ± 16.26 mg/mL vs 21.73 ± 11.33 mg/mL); the standard 95% confidence interval (CI) was 9.19 to 6.41. The mean (± SD) values for AUC_0-t were 66.2 ± 42.9 µg/mL for the tested formulation versus 64.8 ± 38.0 for the reference formulation. The standard 95% CI was 24.3 to 21.5. No significant period effect was detected. The mean (± SD) for T_max were 4.9 ± 2.4 hours for the tested formulation and 4.6 ± 1.5 hours for the reference formulation. The standard 95 % CI was 1.33 to 0.85 with statistically no significant difference.

DISCUSSION

Proving 2 drug products (of the same active ingredient) to be therapeutically equivalent entails a similarity in rate and extent to which a drug in a dosage form becomes available for biologic absorption.14 Area-under-the-curve is accepted as a good indicator of extent of absorption, whereas C_max and T_max are considered estimators of the rate of absorption. Two internationally recognized organizations (U.S. Food and Drug Administration and European Agency for the Evaluation of Medicinal Products) have proposed that bioequivalence can only be assumed when the characteristic parameters of bioavailability show no more than a defined difference.15,16 These differences depend on the nature of the drug, the patient population, and the clinical end point.

The rapid hydrolysis of ASA was a major handicap in all steps. This was minimized by working under low temperature at all times: using liquid nitrogen to shock-freeze the samples and stop hydrolysis, thawing in chilled ice water, and centrifuging in a refrigerated centrifuge set at 4˚C. Such methods helped us bypass the need to add an enzyme inhibitors (such as potassium fluoride or physostigmine) to plasma to enzymatically inhibit hydrolysis of ASA. According to earlier investigations such preservatives are not very efficient, because enzymatic hydrolysis in plasma overlaps with chemical hydrolysis.17,18 Therefore, immediate acidifying and/or cooling techniques used in this study, including storage at –70˚C after sampling, are the best steps to prevent degradation of ASA in plasma.

Limit of detection of this technique proved to be 5 mg/mL acetylsalicylic acid in plasma when 200 mL of plasma was analyzed. This highlights its sensitivity. Other techniques in the literature had higher detection limits, thus were less sensitive. Another issue is the need for such a small amount of plasma. Limitations of this study include the small number of volunteers, thus implementing a great variation in our standard deviation.

Lack of statistical significant differences in AUC values, C_max and, T_max between the 2 products indicate that the 2 formulations are closely similar in terms of their pharmacokinetic properties and bioavailability. This suggests that the in vivo dissolution and the absorption rate are closely identical for the 2 products. Furthermore, this in vivo finding is consistent with the in vitro release pattern.

CONCLUSION

Based on the in vivo pharmacokinetic results obtained, this study suggests that the 2 products of enteric-coated aspirin included in this investigation are bioequivalent. Thus, Aspicotâ and Aspirin Protectâ might be considered interchangeable based on the pharmacokinetic effect.

REFERENCES

1. Awtry AE, Loscalzo J: Aspirin. Circulation 101:1206–1218, 2000.

2. Abdel-Rhaman MS, Reddi AS, Curro FA, et al: Bioavailability of aspirin and salicylamide flowing oral co-administration in human volunteers. Canadian Journal of Physiology and Pharmacology 69:1436–1442, 1991.

3. Hawaky CJ: COX-2 inhibitors. Lancet 353:307–314, 1999.

4. Fitzgerald GA: Mechanisms of platelet activation: thromboxane A2 as an amplifying signal for other agonists. Am J Cardiol 68:11B–15B, 1991.

5. Clissold SP: Aspirin and related derivatives of salicylic acid. Drugs 32(suppl 4):8–26, 1986.

6. Patrono C, Collar B, Dalen J, et al: Platelet-active drugs: the relationship among dose, effectiveness, and side effects. Chest 114:470S–488S, 1998.

7. Lopez-Farre A, Caramelo C, Esteban A, et al: Effect of aspirin on platelet–neutrophil interactions: role of nitric oxide and endothelin-1. Circulation 91:2080–2088, 1995.

8. Hussein S, Andrew NP, Mulcahy D, et al: Aspirin improves endothelial dysfunction in atherosclerosis. Circulation 97:716–720, 1998.

9. Ridker PM, Cushman M, Stamper MJ, et al: Inflammation, aspirin, and the risks of cardiovascular disease in apparently healthy men. N Engl J Med 336:973–979, 1997.

10. Jimenz AH, Stubbs ME, Tofler GH, et al: Rapidity and duration of platelet suppression by enteric coated aspirin in young healthy volunteers men. Am J Cardiol 69:258–262, 1992.

11. Weksler BB, Pett SB, Alonso D, et al: Differential inhibition by aspirin of vascular and platelet prostaglandin synthesis in atherosclerotic patients. N Engl J Med 308:800–805, 1983.

12. Tohgi H, Konno S, Tamura K, et al: Effects of low to high doses of aspirin on platelet aggregability and metabolites of thromboxane A2 and prostacyclin. Stroke. 23:1400–1403, 1992.

13. Kees F, Jehnich D, Grobecker H: Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography. Journal of Chromatography B 677:172–177, 1996.

14. Chow CS, Liu JP: Design and analysis of bioavailability and bioequivalence studies. New York: Marcel Dekker; 1992:1–2.

15. US Food and Drug Administration (FDA): Guidance: statistical procedure for bioequivalence studies using standard 2-treatment cross-over design. Statement under 21 CRF 10.9. Rockville, MD: US Department of Health and Human Services; 1992.

16. Committee for Proprietary Medicinal Products (CPMP) Working Party on the Efficacy of Medicinal Products: Note for guidance: investigation of bioavailability and bioequivalence. Brussels; 1991.

17. Kwong TC: Salicylate measurement: Clinical Usefulness and Methodology, Crit Rev Clin Lab Sci 25:137–159, 1987.

18. Kwong I, Adams N, Young N: Rapid determination of salicylate in serum on acentrifugal analyzer. Clin Biochem 17:170–172, 1984.

 

 

Table 1. Demographic Characteristics of the 12
Study Participants

 

                                                   Age       Body weight    Height

                                                   (y)              (kg)            (cm)

 

Mean ± standard deviation   21.6 ± 2.9    71 ± 13.3    173 ± 7.9

 

Minimum                                     18.0             53.0           162.0

 

Maximum                                    30.0             89.0           185.0

 

 

Table 2. In Vitro Dissolution Analysis

 

Drug release        Aspicot®     Aspirin Protect®     Specification

(dissolution)        (100 mg)            (100 mg)           (USP XXIV)

Acid stage            1.36%                1.79%           Maximum 10% 

 

Buffer stage        99.55%              99.11%           Minimum 80%

 

Table 3. Pharmacokinetic Parameters of the Test Formulation* and Reference Formulation† of Enteric-Coated Aspirin in the 12 Study Participants

                                  AUC_0-t                 C_max                    T_max

                                (µg/mL)            (µg/mL)                (h)

Test formulation

   Mean ± SD         66.2 ± 42.9       23.6 ± 16.3         4.9 ± 2.4

 

   Minimum                   21.6                   8.1                     1

 

   Maximum                176.5                 59.7                   10

 

   95% CI                     24.3                  9.19                  1.33

 

Reference formulation

   Mean ± SD         64.8 ± 38.0       21.7 ± 11.3         4.9 ± 1.5

 

   Minimum                   24.6                   9.8                     2

 

   Maximum                160.2                 44.3                    8

 

   95% CI                     21.5                  6.41                  0.85

 

*Trademark: Aspicot® 100 mg (Pharmaline©, Lebanon).

†Trademark: Aspirin Protect® 100 mg (Bayer©, Germany).

Auc_0-t, area under the plasma concentration-time curve from zero to time t; C_max, maximum concentration; T_max, time to Cmax; t1/2, half-life.

 

 

Figure 1. Average ASA concentration versus time of brand.