Sunday, June 21, 2009

Impact of pharmaceutical dosage forms on the pharmacokinetics of roxithromycin in healthy human volunteers

Objectives: The impact of two different pharmaceutical preparations on the pharmacokinetics of roxithromycin was investigated in healthy human volunteers.

Methods: The degradation kinetics and products of roxithromycin were investigated in simulated gastric fluid and simulated intestinal fluid. Two oral dosage forms of roxithromycin were employed: enteric-coated pellets and dispersible tablets.

Results: The degradation half-time of roxithromycin in simulated gastric fluid was 0.23 h, and three main degradation products were characterized. In contrast, roxithromycin was stable in simulated intestinal fluid and remained unchanged after a 1.00 h incubation. The roxithromycin enteric-coated pellets exhibited higher bioavailability and a more potent serum antibacterial activity than the dispersible tablets.

Conclusions: The type of oral dosage forms of roxithromycin altered its pharmacokinetics. Whether or not this affects the in vivo antibacterial efficacy requires further study.

Keywords: stability , macrolides , pharmaceutical preparations


Roxithromycin ((E)-erythromycin-9-[O-[(2-methoxyethoxy)methyl]oxime]) is an orally administered macrolide and an ether-oxime derivative of erythromycin. It has an in vitro antibacterial spectrum analogous to the parent compound, including many pathogens causing soft tissue, skin, respiratory and urogenital tract infections.1,2

Although the presence of the oxime ether side chain at the 9-position confers better acid stability to the erythronolide ring, by preventing formation of inactive 6,9,9,12-spiroketal derivatives, the antibacterial activity of roxithromycin is reduced at pH values below 7.4.3 For example, incubation in the presence of carbon dioxide resulted in an increase in the minimum inhibitory concentration for 50% of anaerobe isolates tested (MIC50) and 90% of anaerobe isolates tested (MIC90) from 0.25 and 16.0 mg/L without added CO2 to 2.0 and 64.0 mg/L in the presence of 10% CO2, respectively.4,5 This suggests that roxithromycin might be unstable in an acidic environment, perhaps because of conversion into inactive forms, although other possibilities would include reduced uptake of antibiotic by bacteria at low pH values due to decreased lipophilicity.

In combination with proton pump inhibitors, roxithromycin concentrations in both gastric juice and gastric tissue were significantly increased compared with roxithromycin administered alone, contributing to the synergic beneficial action in the eradication therapy of Helicobacter pylori.6,7 Proton pump inhibitors suppress the production of stomach acid and the resultant higher pH might lead to increased roxithromycin stability.

Therefore, we assume that the proper dosage form of roxithromycin should avoid releasing the drug rapidly in the stomach, but promptly liberate the drug upon reaching the small intestine to allow for good oral absorption. The gastric emptying time is approximately 0.5–3 h in humans.

To confirm the above-mentioned considerations, the degradation kinetics and products of roxithromycin in the simulated gastric fluid were examined. The stability in the simulated intestinal fluid was also investigated for comparison with that in the simulated gastric fluid and as the main release site of roxithromycin enteric-coated pellets. Moreover, the absorption and residence time is longer in the intestine than in the stomach for all drugs. Finally, the impact of dosage forms on the bioavailability of roxithromycin was studied in healthy human volunteers.

 Materials and methods


Roxithromycin was kindly provided by the Huatai Drug Research Institute (Shenyang, China). The two degradation products, including decladinose roxithromycin and (Z)-roxithromycin, were synthesized using a previously published procedure and these were provided by the Department of Medicinal Chemistry (Shenyang Pharmaceutical University, Shenyang, China).8 Their identity was confirmed by examining their nuclear magnetic resonance spectra (NMR) and mass spectra (MS), which were identical to the reported data.8 Roxithromycin dispersible tablets (Hayao Pharm. Group, Haerbin, China) containing 75 mg of roxithromycin, and roxithromycin enteric-coated pellets (Wancheng Pharm. Co., Dongguan, China) containing 25 mg of roxithromycin, were used.

Characterization of roxithromycin degradation in simulated gastric fluid and simulated intestinal fluid using high performance liquid chromatography (HPLC)-tandem MS

The simulated gastric fluid (pH 1.2) consisted of HCl and the simulated intestinal fluid (pH 6.8) consisted of 0.05 M phosphate buffered solution, prepared according to the Chinese Pharmacopoeia 2000.9 The content of roxithromycin was measured by HPLC-tandem MS (Waters, MA, USA) using a slight modification of the method of Zhong et al.10 Positive-ion mode was selected. The spray voltage was 4.20 kV. The capillary voltage was set as 30 V and its temperature was 180°C. The HPLC eluate was nebulized using nitrogen at a flow rate of 0.75 L/min and an auxiliary gas flow of 0.15 L/min. The separation was carried out using a Kromasil ODS column (5 µm, 150 mm x 4.0 mm ID; Hi-Tech Scientific Instrument Corp., Tianjin, China) at ambient temperature. The mobile phase consisted of acetonitrile/methanol/10 mM ammonium acetate (53:10:37) at a flow rate of 0.3 mL/min. At designated times, the degradation of roxithromycin (4 mg/L) in the simulated gastric fluid or the simulated intestinal fluid (200 µL) was terminated by addition of 300 µL of 0.1 M Na2CO3. Firstly, 100 µL of clarithromycin (4 mg/L, internal standard) was added to the mixture followed by extraction with 3 mL of chloroform. After centrifugation at 3000 g for 5 min, the organic layer was collected and evaporated to dryness under nitrogen at 30°C.8,11 The resultant residues were reconstituted in 0.5 mL of mobile phase and a 20 µL aliquot was injected into the HPLC apparatus. The standard curve was linear between 0.05 and 10 mg/L. The limit of sensitivity of the assay was 0.02 mg/L in both the simulated gastric fluid and the simulated intestinal fluid. The mean intra- and inter-assay coefficients of variation were less than 5%. Each set of experiments was conducted in triplicate.

Pharmacokinetics of two roxithromycin dosage forms

Approval for this study was obtained from the ethics committee of Liaoning Provincial Hospital. Eighteen Chinese adult male volunteers participated, with ages ranging from 19 to 28 years. Subjects were not allowed to drink alcohol or smoke during the study. All subjects completed the study, and no adverse events were recorded, indicating that roxithromycin was well tolerated.12 Subjects were randomly assigned to one of two crossover experiments at a single oral dose of 150 mg with a 7 day washout period. Venous blood (5 mL) was collected from an indwelling catheter in the forearm, at the designated time points: 0, 0.33, 0.67, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 11.0, 15.0, 24.0, 36.0 h. Blood samples were placed in plain tubes, immediately cooled on ice, centrifuged at 12 000 g for 3 min at –4°C, and then stored in plastic tubes at –20°C until assayed. Roxithromycin concentrations in serum were determined by a validated agar plate diffusion microbiological assay with Micrococcus luteus (B) 28001 (provided by the Department of Microbiology, Shenyang Pharmaceutical University, Shenyang, China) as a test strain. The standard curve was produced in Hanks' balanced salt solution containing 40% blank pooled serum and was linear between 0.4 and 6.4 mg/L. The limit of sensitivity of the assay was 0.4 mg/L. The mean intra- and inter-assay coefficients of variation were less than 6%.

Data analysis

The maximum roxithromycin serum concentration (Cmax) and the time to reach the peak concentration (Tmax) were both obtained as directly measured values. The apparent terminal elimination rate constant (kel) was calculated from the slope of the logarithm of the serum concentration versus time over the last four sampling time points. The area under the serum concentration–time curve (AUC0–36) was calculated by the trapezoidal method. The relative bioavailability (F%) was calculated by the following equation, F% =(AUCEP·doseDT)/(AUCDT·doseEP), where EP stands for enteric-coated pellets and DT for dispersible tablets.

 Results and discussion

The degradation of roxithromycin in the simulated gastric fluid followed pseudo-first-order kinetics. On the other hand, roxithromycin was quite stable in the simulated intestinal fluid, and remained unchanged after a 1.0 h incubation. The percentage roxithromycin remaining in the simulated gastric fluid was 32%, 17%, 11% and 4%, at 0.25, 0.5, 0.75 and 1.00 h after incubation, respectively. The first-order rate constant was 3.00 h–1 and the degradation half-time was 0.23 h in the simulated gastric fluid, suggesting that roxithromycin was unstable and decomposed quickly. Provided that the degradation products possess zero or low antibacterial activity compared with parent roxithromycin, this may mean that a dosage form that releases roxithromycin rapidly in the stomach may not produce the desired therapeutic effect.

The HPLC chromatograms and MS spectra of roxithromycin and its degradation products in the simulated gastric fluid after a 5 min incubation are shown in Figure 1. Their structures were identified by comparison of the HPLC retention time and electrospray ionization (ESI) MS with that of synthetic standards.8 Roxithromycin had a retention time of 10.6 min, and produced a quasi-molecular ion [M + H]+ at m/z 837. Degradation product 1 (D1) had a retention time of 7.1 min and a quasi-molecular ion [M + H]+ at m/z 837. Two compounds displayed HPLC-MS2 spectra of fragment ions at m/z 679, 558 and 522, resulting from loss of cladinose (–158 u), the oxime ether side chain (–121 u) and desosamine (–157 u), respectively.8,10,11 The above data strongly suggest that D1 was an isomer of roxithromycin. Following comparison of the chromatographic and MS characteristics of a (Z)-roxithromycin standard, D1 was identified as (Z)-roxithromycin. Degradation product 2 (D2) had a retention time of 5.7 min and a quasi-molecular ion [M + H]+ at m/z 679. Furthermore, the D2 HPLC-MS2 spectra of fragment ions were at m/z 558, 540 (–18 u, loss of water) and 522, and the chromatographic and MS characteristics of D2 were the same as a decladinose roxithromycin standard,8 indicating that D2 is decladinose roxithromycin. Degradation product 3 (D3) had a retention time of 4.5 min and a quasi-molecular ion [M + H]+ at m/z 679. The D3 HPLC-MS2 spectra of fragment ions were the same as D2, suggesting that D3 is probably (Z)-decladinose roxithromycin.8,10,11

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Figure 1.. Representative HPLC chromatograms and ESI MS2 spectra of roxithromycin and its degradation products in simulated gastric fluid after incubation with roxithromycin for 5 min at 37°C.

After a 1.00 h incubation in the simulated gastric fluid, almost no roxithromycin and (Z)-roxithromycin (D1) were measurable in the simulated gastric fluid, and the remaining degradation product was entirely decladinose roxithromycin (D2).

It has been reported that, compared with roxithromycin, the antibacterial activity of (Z)-roxithromycin was only 50% against Micrococcus luteus (B) 28001 and 33% against Bacillus subtilis (B) 63501 and Bacillus pumliu (B) 632002, and that the antibacterial activity of decladinose roxithromycin was almost zero.8,13 Therefore, we concluded that dosage forms of roxithromycin would exert a strong effect on its pharmacokinetics, because the gastric transit time is usually not less than 0.5 h in humans.

Currently, there are commercial roxithromycin dispersible tablets capable of releasing drug rapidly following oral administration. In order to confirm our hypothesis, the comparative pharmacokinetics of roxithromycin dispersible tablets and enteric-coated pellets was studied in healthy male human volunteers. In addition, a microbiological assay was selected to determine the antibacterial concentrations and to investigate overall antibacterial activity in serum relative to roxithromycin and the impact of different dosage forms on this. The antibacterial activity consisted of roxithromycin and its active metabolites in humans. It has been reported that the metabolites in humans include decladinose roxithromycin, erythromycin-oxime, N-, O-, and N,O-di-demethylated roxithromycin, as well as the (Z)-isomers of roxithromycin and the above corresponding metabolites, and that the (Z)-isomers exhibit low antibacterial activity.8,13 Above all, isomerization from the (E)- to the (Z)-isomer only occurred in gastric conditions but not in other organs, and the (Z)-isomer metabolites were derived from the (Z)-isomers formed during the presystemic gastric acid degradation.11 Hence, microbiological assay is a suitable method to measure the effect of roxithromycin rapid release in the stomach on the bioavailability, and also on the overall serum antibacterial activity relative to roxithromycin.

The pharmacokinetic parameters after administration of a single 150 mg dose of either roxithromycin dispersible tablets or enteric-coated pellets were calculated, and the serum concentration–times curves are shown in Figure 2. Tmax was 2.83 ± 0.99 h and 1.43 ± 0.84 h for roxithromycin enteric-coated pellets and dispersible tablets, respectively, indicating that dispersible tablets released roxithromycin more rapidly than enteric-coated pellets. Also, the Cmax and AUC0~36 of roxithromycin enteric-coated pellets were 5.07 ± 0.95 mg/L and 60.85 ± 11.23 mg·h/L, respectively, which was greater than those of the dispersible tablets (3.95 ± 1.52 mg/L, 42.70 ± 16.28 mg·h/L). Furthermore, the relative bioavailability of roxithromycin enteric-coated pellets to dispersible tablets was estimated as 143%. The 90% confidence intervals of two one-sided tests for the percentage ratios with a significance level ({alpha}) of 0.05, were 130.6–155.5 for AUC0~36, and 107.7–152.9 for Cmax, respectively. Hence, the two roxithromycin formulations were not bioequivalent from a statistical standpoint, as these values were outside the interval proposed by the Chinese Pharmacopoeia (80–125 for the AUC and 70–143 for the Cmax, respectively).14 These results support the assumption that dosage forms releasing roxithromycin rapidly in the stomach exhibit relatively poor oral absorption compared with the enteric-coated preparations. Accordingly, the type of oral dosage form of roxithromycin has a significant effect on its pharmacokinetics, which should be taken into consideration when designing roxithromycin oral delivery systems. The potential therapeutic implications of this remain to be studied.

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Figure 2.. Roxithromycin serum concentration–time profiles for either enteric-coated pellets (open circles) or dispersible tablets (filled circles) following oral administration to healthy humans volunteers at a single dose of 150 mg. Each value is the mean ± SD (n=18).


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