INTRODUCTION

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Although theophylline (TP) has severe adverse effects, it is widely used as an antiasthmatic agent because of its therapeutic benefit. In humans, TP is metabolized mainly by cytochrome P-450 in liver, and its major metabolites, 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), and 1-methyluric acid (1-MU), are excreted in the urine (9, 27) (Fig. 1). It is known that CYP1A2 is the major enzyme responsible for TP metabolite formation (2, 22, 23, 28). Consequently, TP clearance is largely controlled by CYP1A2, and changes in the activity and content of this enzyme have a significant effect on the elimination of TP (10). CYP1A2 is known to be induced by smoking and by many drugs, and there is considerable interindividual variation in the level of this enzyme (6, 26, 29). Therefore, in the clinical usage of TP, which has a narrow therapeutic index, it is of the utmost importance that constant concentrations of TP in blood should be maintained by continuous monitoring and selection of the appropriate dose for each individual. Moreover, since asthma is often complicated by respiratory infections, the combined use of new quinolone antimicrobials and TP may often be necessary. Therefore, attention must be paid to the effect of new quinolone antimicrobials on TP metabolism during their concomitant use.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Metabolic pathways of TP in humans.

In 1984, Wijnands et al. first reported the severe clinical adverse effects that occur with the concomitant use of TP and a new quinolone, enoxacin (ENX), and demonstrated that blood TP concentrations were markedly increased by such concomitant use (30). CYP1A2 is a major cytochrome P-450 enzyme, along with CYP3A4 and CYP2C, in human liver microsomes (25), and this enzyme metabolizes a number of drugs (26, 32). Moreover, drugs which specifically inhibit CYP1A2 also induce drug interactions, even if they are not substrates for the enzyme. At present, ENX, ciprofloxacin (CPFX), and tosulfloxacin (TFLX) are examples of quinolones known to interact with TP metabolism via inhibition of CYP1A2 (3, 4, 12).

DU-6859a, ()-7-[(7S)-7-amino-5-azaspiro[2,4] heptan- 5-yl]-8-chloro-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4- dihydro-4-oxo-3-quinolinecarboxylic acid sesquihydrate (Fig. 2), is a new quinolone antimicrobial which is effective against aerobic and anaerobic gram-positive and -negative bacteria, Chlamydia spp., and Mycoplasma spp. It also exhibits antibacterial activity markedly superior to that of conventional new quinolones against quinolone-resistant methicillin-resistant Staphylococcus aureus, Pneumococcus spp., and Pseudomonas spp. (24). A pharmacokinetic study has shown that DU-6859a is well absorbed and has high oral bioavailability, good tissue distribution, and favorable elimination half-life properties that should make this compound an effective new drug (16, 19).

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Chemical structure of DU-6859a.

Prior to our investigation of in vivo drug interactions of DU-6859a with TP, the effect of DU-6859a on TP metabolism in an in vitro metabolic system with human liver microsomes was investigated by high-performance liquid chromatography (HPLC) quantitation of the 1-MX and 1,3-DMU metabolites, and the possibility of drug interaction was further investigated in vivo by comparison with other known drug analogs. In the clinical study, the effect of DU-6859a on the metabolism of TP at steady state was investigated with repeated administration of DU-6859a at clinical dosages, and the relationship between the in vivo findings and the in vitro results was evaluated.

	MATERIALS AND METHODS

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Chemicals. DU-6859a, ENX, CPFX, TFLX, norfloxacin (NFLX), and levofloxacin (LVFX) were synthesized in this laboratory. Sustained-release oral TP tablets (Theodur; 200 mg of TP per tablet) were obtained from Nikken Chemicals Co., Ltd. (Tokyo, Japan). TP, 1,3-DMU, 1-MU, 3-MX, and 1-MX standards were purchased from Sigma Chemical Co. (St. Louis, Mo.). NADP, glucose-6-phosphate (G-6-P), and G-6-P dehydrogenase were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan), and tetrabutylammonium hydrogen sulfate was obtained from Wako Pure Chemical Industries Co., Ltd. (Tokyo, Japan). Other reagents were of analytical grade.

Subjects. After being informed of the nature and risks of the study and giving written informed consent for participation, 12 adult male volunteers were enrolled in the study. The study protocol was approved by the Ethics Committee of Kyushu Clinical Pharmacology Institute, and the study was performed in accordance with the Good Clinical Practice Guidelines. The subjects' ages ranged from 20 to 26 years, and they weighed from 59 to 70 kg. All subjects were judged to be healthy, based on a physical examination and standard biochemical, hematological, and urinalysis screening tests prior to the study. They were nonsmokers and had no history of drug hypersensitivity or drug-induced gastrointestinal disorders. No xanthine-containing food or alcoholic beverages were consumed, and excessive exercise was prohibited throughout the study.

Study design. The study type was a single crossover, with each subject serving as his own control. Healthy adults in groups of six were administered TP orally 30 min after a meal at a dosage of 200 mg twice daily (b.i.d.) at 12-h intervals for 9 days (single dose on the final day). DU-6859a was administered orally on day 5 after the administration of TP at a dosage of 50 or 100 mg b.i.d. for 5 days (single dose on the final day). For serum TP measurement (control), blood samples were collected on the day prior to DU-6859a administration (day 4 of TP administration) and on days 3 (day 7 of TP administration) and 5 (day 9 of TP administration) of DU-6859a administration at 0, 1, 2, 3, 4, 8, 10, and 12 h after administration. The blood samples were left at room temperature for 1 h and then centrifuged to separate the serum. Urine samples (12-h collection) were collected on days 4 (control), 7, and 9 of TP administration. The serum and urine samples were then stored at 20°C until analysis.

In vitro study. Human liver microsomes were purchased from Human Biologics, Inc. (Phoenix, Az.). Information including medication history, cause of death, and presence of viral infections was provided by the vendor and deemed suitable for these studies. Cytochrome P-450 content and enzyme activities were determined by methods reported previously (20, 21). Protein concentrations were measured by the method of Lowry et al., with bovine serum albumin as a standard (13). Each quinolone (final concentration of 0.05 to 2.0 mM), an NADPH-generating system (3.3 mM G-6-P, 1.3 mM NADP, 0.4 U of G-6-P dehydrogenase), 0.2 mM TP, and 3 mg of microsomes were added to 0.1 M phosphate buffer (pH 7.4) and diluted to 1 ml. After a 5-min preincubation, the reaction was initiated by addition of NADP. After 30 min, the reaction was stopped by addition of 0.8 ml of 2% ZnSO₄. An internal standard (-hydroxyethyl TP) was added to the reaction mixture, which was then centrifuged at 3,000 rpm for 10 min. The separated supernatant was stored at 20°C until analysis.

Method of analysis. Serum TP concentrations were determined by fluorescence polarization immunoassay (18). Urinary TP and its metabolites were determined by HPLC. TP and each of its metabolites were measured with a Nucleosil 7 C₁₈ (4.6 by 250 mm) column. Separation of TP and its metabolites was achieved with a linear gradient of solutions A (10 mM sodium acetate [pH 4.8], 0.5% tetrahydrofuran [THF]) and B (85% 10 mM sodium acetate [pH 4.8], 0.5% THF, 15% acetonitrile) at a flow rate of 1.5 ml min¹ for over 16 min. Detection was performed by UV A₂₈₀. TP metabolites present after the in vitro reaction were determined by HPLC as reported previously (11). Serum DU-6859a was determined by HPLC with photolysis-fluorescence detection (1).

Statistical analysis. Comparison between the respective values of pharmacokinetic parameters (maximum concentration of drug in serum [C_max] and area under the concentration-time curve [AUC]) and urinary TP metabolites for TP alone and a group receiving concomitant DU-6859a was made by repeated-measures analysis of variance. The differences were evaluated by use of a test based on pairwise differencesthe nonparametric Wilcoxon paired test. Statistical significance was assumed for p values of <0.05.

	RESULTS

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In vitro inhibition of TP metabolism in human liver microsomes. As shown in Table 1, the effects of new quinolones, including DU-6859a, on the formation of 1-MX and 1,3-DMU from TP were investigated with human liver microsomes in the presence of an NADPH-generating system. The inhibition potential for 1-MX formation was highest in ENX (50% inhibitory concentration [IC₅₀], 145 µM), followed by CPFX, NFLX, and TFLX, in descending order. DU-6859a showed some inhibition (IC₅₀, 5.08 mM), but to a lesser extent than the quinolones. LVFX did not significantly inhibit the metabolism of TP.

Serum TP concentration profile after concomitant administration of DU-6859a. Following administration of TP (200 mg b.i.d. for 9 days), alone or together with DU-6859a (50 or 100 mg b.i.d. for 5 days), to six healthy subjects, serum TP concentrations were measured to determine the effect of DU-6859a on TP metabolism (Fig. 3). Serum TP concentrations were not affected by the administration of DU-6859a at 50 mg b.i.d. for 5 days, and TP concentrations resulted in a slight but statistically insignificant increase in serum TP concentrations following 5 days of administration of DU-6859a at 100 mg b.i.d. Pharmacokinetic parameters were calculated by determining the serum TP concentrations on days 3 and 5 of DU-6859a administration (Tables 2 and 3). The AUCs after the 50- and 100-mg b.i.d. dosages were 98.48 and 99.77 µg ml¹ · h, respectively, and were not significantly different from those prior to DU-6859a administration. Both the C_max and AUC parameters were increased approximately only 1.1-fold over control levels.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Concentrations of TP in serum following repeated oral administration of TP at dosages of 200 mg b.i.d. for 9 days and coadministration of DU-6859a (50 or 100 mg) b.i.d. for 5 days. Data are means ± standard deviations for six subjects.

Serum DU-6859a concentrations after concomitant administration of DU-6859a and TP. The DU-6859a concentration in serum was measured on days 3 and 5 of administration. C_maxs after 5 days of the 50- and 100-mg doses were 0.49 and 0.86 µg ml¹, respectively (Table 4) and were therefore proportional to the given doses. No significant differences were detected in the serum drug levels between days 3 and 5 of administration at either the 50- or 100-mg dose.

Urinary excretion of TP and its metabolites after concomitant administration of DU-6859a. Urinary excretion of TP and its metabolites was measured on day 4 of TP administration (the day prior to initiation of DU-6859a treatment) and on days 3 and 5 (final day) of the DU-6859a treatment. No changes in excretion levels of TP, 3-MX, 1-MU, or 1,3-DMU were found after the administration of 50 mg of DU-6859a; however, the administration of 100 mg of DU-6859a resulted in decreases in all urinary TP metabolites, and this decrease was more evident on day 5 than on day 3 (Fig. 4).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Urinary excretion of TP and its metabolites after repeated oral administration of TP at dosages of 200 mg b.i.d. for 9 days and coadministration of DU-6859a (50 or 100 mg) b.i.d. for 5 days. Data are means ± standard deviations for six subjects. *, P < 0.05.

	DISCUSSION

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It has been well established in human liver microsomes that TP is metabolized by cytochrome P-450, particularly the CYP1A2 enzyme, to 1,3-DMU, 3-MX, and 1-MU (5, 14). Since it has been demonstrated that renal clearance and protein binding of TP remain unchanged with the concomitant administration of other drugs, the TP-drug interaction can be estimated by monitoring the effect of these drugs on TP metabolism by CYP1A2 (15, 31). In order to elucidate the inhibitory effects of the new quinolone, DU-6859a, on TP metabolism in vitro, we chose a substrate concentration of 0.2 mM TP, corresponding to approximately four to eight times the levels used clinically (5 to 10 µg ml¹ [25 to 50 µM]). Although no data have yet been reported regarding liver/blood TP ratios following administration, the high hepatic clearance of TP suggests that hepatic drug levels may be much higher than blood drug levels. The high TP concentrations used in this study, therefore, were expected to correspond to the hepatic TP concentrations.

The effects of DU-6859a on TP metabolism in human liver microsomes indicate that this transformation, especially the formation of 1-MX, was slightly inhibited at the IC₅₀ of 5 mM (higher DU-6859a concentrations than those believed to be present in human liver). In addition, other drugs previously known to either interact or not with TP were also examined, and it was demonstrated that DU-6859a is a relatively weak inhibitor of TP metabolism in vitro. On the basis of this finding, DU-6859a was administered concomitantly with TP at clinical doses to healthy adult subjects, and the inhibition of TP metabolism in these subjects was then examined by measuring the levels of TP in serum and its urinary metabolites. As a result, it was necessary to perform pharmacokinetic analyses using more detailed concentration points in order to extrapolate the in vitro results to the in vivo system. Serum TP levels remained unchanged with the DU-6859a dosage of 50 mg b.i.d. for 5 days, but at the 100-mg b.i.d. dosage, serum TP levels increased 1.1-fold over control levels. This small increase appears to reflect the weak in vitro inhibitory effect of DU-6859a (Table 1). Furthermore, following administration of DU-6859a at 100 mg b.i.d. for 5 days, there was a significant decrease in levels of 1,3-DMU, 1-MU, and 3-MX in urine which correlated well with the increase in levels of TP in blood. Concentrations of DU-6859a in plasma were also measured, and both the AUC and C_max increased in a dose-dependent manner, with a final C_max of 0.86 µg ml¹ following completion of the 5-day regimen at 100 mg b.i.d. These findings indicate the linear pharmacokinetics of DU-6859a in the dose levels. This C_max is lower than the concentration used in the in vitro study, indicating that in this instance, the in vitro results may not necessarily predict in vivo results.

In the microsomal study, DU-6859a and the other quinolone antimicrobials were examined for any effects on the formation of 1-MX from TP. LVFX resulted in minimal inhibition of this reaction, while ENX, CPFX, and TFLX each demonstrated clear inhibition of 1-MX formation. The strongest inhibition occurred with ENX, CPFX, and TFLX, in that order. These three drugs exhibited strong interactions with TP in clinical trials, and those interactions follow the same rank order (17), consistent with these in vitro observations. Therefore, the inhibition of 1-MX formation by DU-6859a observed may be significant and suggests that the slight increases in TP concentrations observed clinically may be due in part to the intrinsic inhibitory effect of DU-6859a. However, minimal drug-drug interactions were observed both in vivo and in vitro, demonstrating that human liver microsomes can be useful in the prediction of clinically relevant drug interactions.

We found that when DU-6859a was concomitantly administered with TP, the formation of TP metabolites decreased, as evidenced by decreased urinary excretion. The increases in serum TP levels observed after DU-6859a dosing may be due to the decreased formation of 1,3-DMU, a major metabolite of TP. The effect of various quinolones, including DU-6859a, on 1,3-DMU formation in human liver microsomes was investigated, and the results demonstrate that the inhibition by quinolones of 1,3-DMU formation was less than that observed for 1-MX formation. The most potent inhibition of 1,3-DMU formation was with ENX, and all the other drugs tested resulted in relatively weak inhibition. Recent studies have demonstrated that CYP2E1 and -3A4 are also involved in the formation of the 1,3-DMU metabolite, and the affinities of TP vary for these enzymes (8, 22). This multiplicity in metabolic pathways may explain the absence of inhibition in the in vitro system at the specified concentrations of these drugs. Urinary metabolites of TP, 1,3-DMU, 1-MU, and 3-MX, were identified in the clinical trial, but 1-MX was found only in the human liver microsome study. These differences suggest that cytochrome P-450 is not involved in the metabolic transformation of 1-MX to 1-MU, and as previously described, this reaction is likely due to the action of xanthine oxidase (7).

In conclusion, we have paired an in vitro microsomal study with an in vivo examination of the potential interactions between DU-6859a and TP. DU-6859a was shown in the clinical study to have limited but significant effects on the metabolism of TP, and this effect was confirmed with the human liver microsomal system. Although the drug interactions observed clinically were weak, the interactions could still be detected with the in vitro system, thereby establishing that in some instances, in vitro studies with human liver microsomes are useful in the assessment of drug interactions. The weak interactions found in vitro are probably made even less significant in vivo by other variables. As a result, it appears that potential adverse effects attributed to inhibition of TP metabolism by DU-6859a are unlikely when this quinolone is coadministered at clinical doses.