Macrolide–theophylline interactions: no role for the inhibition of cytochrome P4501A2

Many clinical resources for healthcare providers continue to list erythromycin and clarithromycin as inhibitors of cytochrome P4501A2 (CYP1A2) [1]. This classification is based on the pharmacokinetic interactions of these drugs with theophylline, the magnitudes of which are dependent on macrolide dose and the duration of treatment (either no pharmacokinetic changes, or decreases in theophylline clearance by 10–40%; reviewed by Periti et al. and Westphal) [2, 3]. Since theophylline is metabolized primarily by CYP1A2 at clinically relevant concentrations (f_mCYP1A2 estimated as approximately 0.8) [4], it is assumed that perturbations in metabolic clearance must result from altered CYP1A2 activity. However, available data suggest that macrolides are poor inhibitors of CYP1A2. In vitro, the 7-hydroxylation of (R)-warfarin by recombinant CYP1A2 was not inhibited by erythromycin or roxithromycin [5], and high concentrations of various macrolides reduced the activity of CYP1A2 in human liver microsomal preparations by ≤10% in competitive inhibition studies [6]. Furthermore, the in vivo indices of CYP1A2 activity, oral caffeine clearance and 6 h paraxanthine to caffeine plasma concentration ratio, were unchanged following clarithromycin dosing in healthy volunteers (500 mg b.i.d. × 7 days) [7].

Drug–drug interactions involving macrolides are typically attributed to mechanism-based inactivation (MBI) of CYP3A enzymes [8]. This type of inhibition is readily detected in vitro by observing time-dependent inhibition (TDI) (CYP activity is reduced by a ‘pre-incubation step’; reviewed by Polasek and Miners [9]). However, the possibility that weak TDI of CYP1A2 by macrolides may contribute to their interactions with theophylline has not been thoroughly explored. To address this, a conventional in vitro approach ([10]) was used in our laboratory to evaluate potential TDI of recombinant (Escherichia coli-expressed) and human liver microsomal CYP1A2 by troleandomycin, erythromycin, clarithromycin, roxithromycin and azithromycin (0.1–1000 µM). Phenacetin O-deethylation was used to measure CYP1A2 activity. The relative contribution of CYP1A2 in human liver microsomes to this reaction was estimated to exceed 90% (see Polasek et al. [10] for details of the experimental method). As evident from Figure 1, all five macrolides were poor inhibitors of phenacetin O-deethylation using either enzyme source (≤25% inhibition at the highest concentration tested), and TDI was not observed. Consistent with previous reports, fluvoxamine (negative control) did not demonstrate TDI, whereas furafylline (5 µM) and clorgyline (5 µM) (positive controls) both reduced phenacetin O-deethylation rate with time, so that approximately 50% of control activity remained following a 10-min pre-incubation. The lack of TDI seen here with all five macrolides supports recent data for erythromycin, which failed to demonstrate TDI of CYP1A2 using a similar experimental approach [11].

Figure 1.

Lack of time-dependent inhibition of phenacetin O-deethylation (CYP1A2) by macrolides. Macrolides (1000 µM) were pre-incubated with either (a) human liver microsomes or (b) Escherichia coli-expressed recombinant CYP1A2 and nicotinamide adenine dinucleotide phosphate-regenerating system for between 0 and 60 min prior to assay for residual CYP1A2 activity according to Polasek et al. Each point in (a) represents the mean of six determinations (from H6, H7, H10, H12, H13 and H40 microsomes), whereas points in (b) represent the means of duplicate determinations. Troleandomycin (–▪–); Erythromycin (–◊–); Clarithromycin (); Roxithromycin (–▵–); Azithromycin (–•–)

If competitive inhibition and MBI of CYP1A2 are excluded, what explains the macrolide–theophylline interactions? It is possible that CYP3A enzymes play a greater role in the metabolism of theophylline in vivo than predicted from in vitro studies. The poor correlation between the 6-h paraxanthine to caffeine plasma ratio and the oral clearance of theophylline [12] suggests that, although the disposition of caffeine and theophylline are mediated primarily by CYP1A2, there are additional CYP (mostly CYP2E1 and CYP3A) involved in the in vivo disposition of theophylline [7, 13]. Indeed, CYP3A may be the principal CYP implicated when CYP1A2 activity is low, a hypothesis substantiated by Tjia et al., who demonstrated partial restoration of theophylline metabolism when human liver microsomal CYP1A2 was fully inhibited by α-naphthoflavone [14]. Furthermore, the 8-hydroxylation pathway can be activated by nifedipine and α-naphthoflavone in microsomes from liver donors treated with dexamethasone, suggestive of allosteric effects on CYP3A [15]. Intriguingly, the rank order of macrolide interaction potential with theophylline corresponds to that of midazolam, a drug exclusively eliminated by CYP3A, i.e. troleandomycin > erythromycin ≈ clarithromycin > roxithromycin > azithromycin for both affected drugs [2, 8]. Similarly, verapamil and diltiazem, also mechanism-based inactivators of CYP3A (but not CYP1A2 [11]) that interact with midazolam, cause between 10 and 25% reduction in theophylline clearance [16]. Despite these observations, the actual extent of CYP3A involvement in the in vivo metabolism of theophylline remains unclear. In fact, other studies with the prototype CYP3A inhibitors ketoconazole [17] and grapefruit juice [18], and the CYP3A substrate nifedipine [19], have demonstrated essentially no effect on theophylline clearance.

Another explanation is that macrolides compete with theophylline uptake into hepatocytes via organic anion transporter 2, as shown recently for erythromycin and clarithromycin [20]. It is also known that macrolides inhibit P-glycoprotein [21] and various organic anion transporting polypeptides [22]; however, the degree to which these transporters influence the disposition of theophylline is still unknown (T. Yamamoto, personal communication). Inhibition of CYP2E1 by macrolides is another possibility, although no data are available to support this line of reasoning.

It is therefore proposed that the pharmacokinetic interactions between older macrolides and theophylline probably arise via a combination of potent CYP3A inactivation and the inhibition of theophylline uptake into hepatocytes. Substantial evidence is now available to exclude CYP1A2 inhibition as the molecular mechanism responsible for the macrolide–theophylline interactions, and clinical resources for healthcare providers should be updated accordingly.