A pharmacokinetic comparison of three pharmaceutical formulations of nimesulide in healthy volunteers

Background/Aim. Switching the patient from one pharmaceutical formulation of the same drug to another, may lead to therapeutic inadequancy in some cases. To minimize the risk, careful pharmacokinetic studies are desired in the pre-registration period and afterwards. Methods. A randomized, crossover design with one-week wash-out period between each dose was applied. Serum samples, obtained before dosing and at various appropriate time points up to 15 hours, were analyzed for nimesulide content by a high-performance liquid chromatographic method with ultraviolet (UV) detection. The pharmacokinetics and relative bioavailability of three different pharmaceutical formulations containing nimesulide, manufactured by the same pharmaceutical factory, were studied prospectively in 12 healthy subjects of both sexes. A single 100-mg oral dose of nimesulide was given to the volunteers in the form of conventional tablets, mouth dissolving tablets or as a suspension. Analysis of variance, power analysis, 90% confidence intervals, and two one-sided tests were used for the statistical analysis of pharmacokinetic parameters. Results. The tolerability of all preparations was excellent. The respective confidence intervals of the ratios of geometric means of Cmax and AUC of nimesulide were out of acceptable limits either for conventional tablets in comparison with suspension or for mouth dissolving tablets when compared with conventional tablets. A comparison of mouth dissolving tablets with suspension showed a statistically significant difference between Cmax values (suprabioavailability of mouth dissolving tablets), while the point estimate of the ratio of geometric means of AUC was 0.945 with the corresponding 90% confidence interval of 0.902−0.991. At the 5% level of significance, there were no differences between the formulations under the study in times elapsed to peak serum concentrations, as revealed by the non-parametric Wilcoxon signed ranks test. Conclusion. Only a 90% confidence interval for the relative differences of log-transformed AUC values of nimesulide absorbed from mouth dissolving tablets vs. suspension was included in the 80% to 125% interval proposed by the Food and Drug Administration (FDA). On that basis, mouth dissolving tablets (Nimulid-MDTM) were considered bioequivalent to NimulidTM suspension according to the extent of drug absorption. Concerning the comparable amounts of nimesulide available in the systemic circulation after application of these formulations the one might not expect therapeutic failure after switching the patient from one to another.


Introduction
Nimesulide is a synthetic sulfonanilide derivative.It belongs to a class of non-steroidal anti-inflammatory drugs (NSAID) which selectively inhibit cyclooxygenase-2 but, also, interfere with the production/action of mediators other than prostaglandins such as enzymes, toxic oxygen derivatives, cytokines, platelet-activating factor and histamine (1, 2).A combination of a variety of mechanisms makes nimesulide a NSAID with marked anti-inflammatory, analgesic and antipyretic activities (2,3).Several recent experimental studies have also demonstrated a significant neuroprotective effect of nimesulide on chronic cerebral hypoperfusion (4), global cerebral ischemia (5,6), diffuse traumatic brain injury (7,8) or cerebral infarction induced by permanent middle cerebral artery occlusion (9).The pharmacokinetic profile of orally administered nimesulide is characterized by rapid and complete absorption, the rate and the extent of which do not differ significantly whether the drug was administered in a tablet or in a suspension form.The time to reach maximal concentration was reported to vary from 1.9h in children to more than 2.5h in healthy volunteers (3).
Nimesulide is 99% bound to human plasma proteins, mainly albumins and it is principally distributed in the extracellular fluid compartment.The drug undergoes extensive metabolism in the liver.Metabolic biotransformation can occur at both the phenoxy ring moiety and the aromatic nitro group.The principal metabolite is 4-hydroxynimesulide, which contributes to the anti-inflammatory activity of the compound.Another degradation product, 2-(4'-hidroxyphenoxy)-4-N-acetylamino-methansulfonanilide, was recently identified in human urine and it is produced in much lower concentrations.
Only 1% to 3% of the dose is excreted unchanged in the urine, while the metabolites are eliminated mainly via the urine (70%) or the feces (20%) in both free and conjugated form.The terminal elimination half-life of nimesulide is shorter in children than in adults (approx.2.4h vs. up to 5h) while the elimination half-times of metabolites are nearly 2-times higher than that of the parent compound (3).
The purpose of this study was to evaluate the tolerability, pharmacokinetic property and comparative bioavailability of the three different pharmaceutical formulations of nimesulide, all manufactured by the drug company Panacea Biotec Ltd. from India, that are available at the Serbian market.Particular attention has been made to the pharmacokinetic profile of flavoured dispersible tablets (MD tablets) with fast mouth dissolving characteristics thereby providing immediate relief of the drug (10).Although this had not been a classic bioequivalence study, it was of therapeutic interest to compare their bioavailability with that of the liquid formulation (suspension) which is usually thought to be a 100% bioavailable.

Methods
A single-dose, open-label, randomized, six-sequence, three-period crossover study design was used to evaluate the bioavailability of nimesulide, prepared either as a 100mg conventional tablets (Nimulid™ tablets), 100-mg mouth dissolving tablets (Nimulid-MD™ tablets) or a 50 mg/5 ml suspension (Nimulid™ suspension).Twelve subjects of both sexes in good physical condition, as determined by the complete medical and laboratory examinations before the study, were enrolled and provided written informed consent prior to any study related procedure.The study was approved by the Drug Commission and the Ethics Committee of the Military Medical Academy, on September 20, 2001.
The enrolled subjects were randomly assigned to one of the six sequence groups such that upon the completion of the study each subject received all three regimens.Dosing in each of the three consecutive periods was separated by 7-day washout period.A single dose of 100 mg nimesulide (one conventional tablet, one MD tablet or a 10-ml volume of suspension) was given with 200 ml of non-carbonated mineral water following an overnight fast of at least 10 hours.

Assay method
The High Performance Liquid Chromatography (HPLC) set was equipped with a pump (Model 2150, LKB, Bromma, Sweden), an automatic sample system (Model AS-100, Bio-Rad Laboratories, Inc., Hercules, CA, USA), a variable wavelength UV detector (Model 1801, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and an integrator (Model 2221, LKB, Bromma, Sweden).For the acquisition and integration of analytical data, a Bio-Rad Value Chrom ™ software, operated by Pentium microprocessor, was used.Separations were performed on a reverse phase column (BioSil C18 HL, 4.6 x 250 mm; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at the ambient temperature.
The HPLC assay for determination of unchanged nimesulide was fully developed and completely validated in our laboratory.Its development, however, had been based on the data published by Gupta (11) but due to differences in the equipment and materials being on disposal, it had to be generally modified.
The HPLC analysis was performed by using a mobile phase consisted of 0.01M ammonium-dihydrogen-orthophosphate and acetonitrile (1:1), previously filtrated and degassed by a membrane degasser.The flow rate was 1.0 ml/min, an injector loop volume was 20 μl, and an UV detector wavelength set at 300 nm throughout the assay.Under these conditions the retention time of nimesulide was about 6.47 min.All the chemicals were of HPLC, and p.a. purity, and had been purchased commercially.
After being refrigerated, the samples were allowed to melt spontaneously at room temperature.Aliquots of 0.5 ml of serum were dispensed into glass tubes and were deproteinized using 0.5 ml of acetonitrile.The mixture was strongly vortexed for 60 seconds to ensure adequate mixing and afterwards centrifuged at 8000 g for 2 minutes.The obtained supernatant amounting 20-μl was injected into the HPLC system and further analyzed using the validated HPLC-UV method.Standard solutions (range of concentrations 0.05−10.0µg/ml) and spiked serum samples (range of concentrations 0.1−5.0µg/ml) were also determined under the same assay conditions.

Pharmacokinetic analysis
The pharmacokinetic parameters of nimesulide were estimated using non-compartmental techniques.The peak plasma concentration (C max ) and the time elapsed to peak concentration (t max ) were obtained directly from the data.The elimination rate constant (k e ) was obtained from the slope of the terminal log-linear phase of the semilog plot of concentration versus time.Half-life (t 1/2 ) was calculated as ln2/k e (ln2 = 0.693).The area under the nimesulide serum concentration-time curve (AUC 0-15 ) was computed using the linear trapezoidal rule while the area under the serum concentration-time curve from time 0 to the infinite time (AUC 0-∝ ) was calculated as the sum of AUC 0-15 and C t /k e , where t was the time of the last measurable concentration (C t ) and k e was the elimination rate constant.
Parameters C max , t max and AUC 0-∝ were accepted as the main variables, while the values of AUC 0-15 , residual areas [AUC 0-15 /AUC 0-∝ * 100], k e and t 1/2 served as the secondary pharmacokinetic objectives.

Statistical analysis
The following main pharmacokinetic parameters, which completely describe the rate and the extent of absorption of nimesulide, derived from the individual serum concentration-time profiles were subjected to statistical analysis: C max , AUC 0-∝ and t max .The comparison of secondary kinetic variables was only descriptive.
Following logarithmic transformation of AUC 0-∝ and C max the values were subjected to analysis of variance (ANOVA) including terms for subjects, treatment (sequence) and period.For evaluation of bioequivalence the point estimates and 90% confidence intervals for the difference between test and reference formulations were constructed using the residual mean square error, obtained from the multifactorial ANOVA.The point estimates and the 90% confidence intervals were then back transformed to give estimates of the ratio of the geometric means and the corresponding 90% confidence intervals for the ratios of the two formulations in the comparison.A nonparametric test (Wilcoxon Signed Rank's Test) was performed for t max .
Bioequivalence between the formulations was accepted if the back transformed 90% confidence intervals for the geometric mean ratios od AUC 0-∝ and C max had follen within 0.80-1.25 range (12−14) and if the differences in t max between the two formulations had been not statistically different (p > 0.05).

HPLC method
Under the described assay conditions, linearity was observed in serum standard curves of nimesulide over the range of 0.05 -10.0 µg/ml with a correlation coefficient greater than 0.999.The lower limit of quantification (LOQ) of unchanged nimesulide was 0.1 µg/ml while the limit of detection was 0.05 µg/ml of serum.Concentrations below LOQ were reported as 0.0 µg/ml.The mean recovery of extraction of nimesulide from serum samples ranged from 93% to 109%.The same working standard solutions had been stable during a 5-day working period when kept in the refrigerator.

Study in healthy volunteers
A total of 12 subjects, whose demographic characteristics are summarized in Table 1, were selected to participate in the study.There were no differences in their age, weight or height that might compromise the validity of the planned pharmacokinetic trial.
The mean serum concentration-time profile of nimesulide absorbed from the 3 different pharmaceutical preparations is shown in Figure 1.Marked differences were observed between the amounts of drug that had reached the systemic circulation during the period of 15 hours.However, the tolerability of all three preparations containing nimesulide was reported as excellent.
The relevant pharmacokinetic parameters of nimesulide for each preparation are listed in Table 2.As it can be seen, the residual area (relation between AUC 0-15 and AUC 0-∝ ) of nimesulide (7.3% suspension, 5.5% conventional tablets, 5.0% MD tablets) accounted for less than 20% of the area from time 0 to the time of the last measurable concentration.Therefore, the stated criterion (AUC 0-15 /AUC 0-∝ * 100 > 80%) was fulfilled and the residual area had no sizeable impact on the calculation of AUC 0-∝ and, thus, on bioavailability.The rate of nimesulide absorption from the tested pharmaceutical formulations, as well as their half-lives of the terminal phase, agreed very well after the application of the three nimesulide-containing preparations.Marked differences in the maximal concentrations were noted between MD tablets and the other two formulations following the same dosages of the drug.
The statistical evaluation of the main pharmacokinetic variables, which describe the rate and the amount of absorption of nimesulide, is presented in Table 3.The respective point estimates of the ratios of geometric means of logtransformed C max and AUC 0-∝ of nimesulide were 0.655 and 0.498 (conventional tablets vs. suspension), 1.244 and 0.945 (MD tablets vs. suspension), and 1.827 and 2.103 (MD tablets vs. conventional tablets).The corresponding 90% confidence intervals were 0.260−0.607,0.335−0.603,1.004−1.542,0.902-0.991,1.358−2.457and 1.575−2.810,respectively.For the median t max values, at a 5% level of significance, there were no significant differences between the formulations in the study, as revealed by the nonparametric Wilcoxon signed ranks test.

Discussion
The analysis of the assay data indicated that the chosen HPLC-UV method was simple, precise and enough accurate for performing a valid bioequivalence study.According to the literature, its sensitivity was comparable to the results of the HPLC methodologies that had been applied so far to determine nimesulide concentration by other investigators (15−20).As expected, however, the HPLC method we used was less sensitive than liquid chromatography coupled to tandem mass spectrometry (21).
Twelve subjects were arbitrarily chosen to participate into the study.That number corresponded to the smallest sample that is accepted as sufficient to assess bioequivalence (22,23) and might represent a reliable number of participants for the bioequivalence decision.The post-study calculations based on the log-ANOVA error data [AUC 0-∝ CV-intra = 6.68%; suspension vs. MD tablets] revealed a sample size of 7 subjects to be quite enough to show the difference of 20% between the AUC 0-∝ values of the test and the reference articles.The type I and type II errors would not exceed 5% and 20%, respectively.
The overall pharmacokinetic profile of nimesulide in the present study, independently from the pharmaceutical formulation being used, was close and in agreement with the data previously published for oral nimesulide preparations in the relevant publications (3, 15−18, 24−29).However, on the basis of maximum serum concentrations and AUC 0-∝ values of nimesulide it was concluded that they had been significantly different after the administration of suspension and conventional tablets.Oppositely, the total amount of nimesulide (AUC 0-∝ ) absorbed from the suspen-sion and MD tablets did not differ significantly with the power (derived from ANOVA) greater than 0.999.
Only 90% confidence intervals for AUC 0-∝ geometric mean ratios of MD tablets, when compared to suspension, were included in the 80% to 125% interval proposed by the U.S. Food and Drug Administration (12,13) and the difference between t max values was statistically insignificant.On that basis, tablets Nimulid-MD™ were considered bioequivalent to suspension Nimulid™ according to the extent of absorption of nimesulide.The differences in C max values, indicating suprabioavailability of MD tablets according to the rate of absorption, were judged not to be therapeutically important since, nimesulide is not a medicine which pharmacodynamic action is closely related to the level of maximal concentrations in the blood.

Conclusion
From the pharmacokinetic point of view, concerning the total amount of drug that is available at the site of action, suspension and MD tablets could be marked as interchangeable by a physician and might not be expected to produce therapeutic failure after switching the patient from one to another.An increased maximal concentration in the serum after MD tablets would definitely not increase the toxicity level of nimesulide due to its wide therapeutic window in patients.

Fig. 1 −
Fig. 1 − Mean (±SE) concentration vs. time profiles of nimesulide in serum after oral administration of a 100-mg dose of suspension Nimulid TM , conventional tablets Nimulid TM or Nimulid-MD TM tablets to 12 healthy volunteers of booth sexes.

Table 1 Demographic data of subjects (n = 12)
† SE -standard error of the mean, ‡ CV -coefficient of variation

Table 2 *Mean (± SE) † values of pharmacokinetic parameters of nimesulide after the ingestion of a single 100-mg dose of conventional tablets Nimulid™, mouth dissolving tablets Nimulid-MD™ and suspension Nimulid™
F o r m u l a t i o n † SE -standard error of the mean