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Aims To evaluate the antimalarial agent quinine as a potential in vivo probe for hepatic cytochrome P450 (CYP) 3A4 activity.
Methods Ten healthy adult volunteers received, by randomized crossover design, either a single oral dose of quinine sulphate (600 mg) alone, or quinine sulphate (600 mg) plus the CYP3A4 inhibitor troleandomycin (TAO; 500 mg every 8 h). Plasma and urine samples were collected before quinine administration, and up to 48 h thereafter, then analysed by h.p.l.c. for both quinine and its CYP3A4-generated metabolite, 3-hydroxyquinine. During both phases, the erythromycin breath test (ERMBT) was administered at specific times to assess hepatic CYP3A4 activity.
Results Compared with control, TAO treatment significantly decreased the mean time-averaged ERMBT result by 77% (95% CI, 68, 85%), the mean apparent oral clearance of quinine (CL/F ) by 45% (95% CI, 39, 52%), and the mean apparent formation clearance of 3-hydroxyquinine (CL3-OH) by 81% (95% CI, 76, 87%). There was no correlation between the TAO-mediated percent decrease in the time-averaged ERMBT result and the percent decrease in CL/F or in CL3-OH. When TAO and control treatments were analysed separately, there were no significant correlations between the time-averaged ERMBT result and CL/F, CL3-OH, or single plasma quinine concentration at 12, 24, and 48 h.
Conclusions Quinine may be a useful probe to detect inhibition of liver CYP3A4 activity within an individual. Further studies are needed to determine whether it can provide a quantitative measure of CYP3A4 activity suitable for intersubject comparison.
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In adult humans, cytochrome P450 (CYP) 3A4 is the most abundant CYP isoform expressed in small intestinal epithelial cells (enterocytes) and liver. CYP3A4 metabolizes a vast number of currently used drugs [1, 2]. There are large inter-patient differences in CYP3A4 content and catalytic activity in both liver and intestine [3–5]. This variability appears to account in part for inter-patient differences in dosing requirements of some CYP3A4 drug substrates. The prototypical CYP3A4 substrates cyclosporin and midazolam are such examples [6–8]. CYP3A4 also plays a potentially important role in the biotransformation of harmful dietary contaminants, including mycotoxins (e.g. aflatoxin B1), pyrizolidine alkaloids, and arylhydrocarbons (e.g. benzo[a]pyrene) [9, 10]. A logical but largely untested hypothesis is that genetic mutations leading to altered CYP3A4 gene expression may account for inter-individual differences in susceptibility to toxicity from these xenobiotics .
To evaluate this hypothesis, a means of measuring CYP3A4 activity in large numbers of individuals is required. It may also be desirable in some circumstances to measure selectively hepatic and not intestinal CYP3A4 activity, as intestinal CYP3A4 content can be greatly influenced by dietary manipulations. For example, consumption of a single glass of grapefruit juice causes a significant reduction in intestinal CYP3A4 activity but has little or no influence on hepatic CYP3A4 activity . Dietary differences may explain in part why the relative activities of CYP3A4 in liver and intestine do not correlate within an individual . Thus if the goal of a study is to identify individuals with genetically altered CYP3A4 activity, it might be desirable to measure selectively hepatic CYP3A4 activity only. To date, indirect measurement of hepatic CYP3A4 activity involves intravenous injection of the probe (e.g. midazolam or the erythromycin breath test). It would obviously be desirable to develop a safe oral probe that could selectively measure liver CYP3A4 activity.
Quinine is one of the most widely prescribed drugs for the treatment of resistant Plasmodium falciparum malaria . It is also used for the treatment of leg cramps in the elderly. In healthy subjects, the absolute oral bioavailability of quinine is very high (∼90%) , indicating that the drug is nearly completely absorbed into the systemic circulation following oral administration and undergoes little first-pass extraction. In addition, grapefruit juice had no effect on the oral pharmacokinetics of quinine , confirming that first-pass metabolism of quinine by the intestine is negligible. The fraction of a dose of quinine excreted unchanged in the urine, however, is low (12%), and only small amounts may be excreted into the bile , indicating that the drug undergoes extensive metabolism. This is most likely to occur in the liver and after it reaches the systemic circulation. Moreover, using human liver microsomes, it was shown that the metabolism of quinine to a major metabolite, 3-hydroxyquinine, is catalysed primarily by CYP3A4 [17, 18]. Based on these observations, we hypothesized that if hepatic CYP3A4 is the rate-limiting step in the elimination of quinine, this drug might serve as a safe and inexpensive oral probe for hepatic CYP3A4 activity.
This hypothesis was tested in healthy volunteers using two approaches. First, the effect of the mechanism-based, selective CYP3A4 inhibitor troleandomycin (TAO) [19, 20] on the disposition of quinine and 3-hydroxyquinine was examined. If hepatic CYP3A4 is rate limiting in the elimination of quinine, the apparent oral clearance of quinine and the apparent formation clearance of 3-hydroxyquinine should decrease significantly. Second, correlations were examined between hepatic CYP3A4 activity (as measured by the erythromycin breath test) and the following pharmacokinetic measures of quinine: apparent oral clearance, terminal elimination half-life, and single plasma concentrations at various times.
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A preliminary study was conducted in three cancer patients to determine the optimal timing of TAO administration to maintain maximal inhibition of hepatic CYP3A4 activity. The maximum decrease in the ERMBT result occurred 2 h after TAO administration (Figure 1, Subject 1), and at least 70% inhibition was observed at 8 h (Subjects 1 and 2). CYP3A4 activity appeared to have recovered to baseline by 24–44 h (Subjects 1 and 3). Based on these results, the current study was designed such that TAO was given as a pre-treatment 2 h before the administration of quinine and every 8 h thereafter until the end of the study phase.
Figure 1. Time course of the effect of a single oral dose (500 mg) of troleandomycin (TAO) on hepatic CYP3A4 activity, as measured by the ERMBT, in three female cancer patients. □ subject 1; ● subject 2; ▴ subject 3.
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Shown in Figure 2 (upper panel) are typical ERMBT results, obtained at three (control) or five (TAO) different occasions over the 48 h study period, for a representative subject (Subject F). Also shown (lower panel) is the ERMBT result profile for the one atypical subject (Subject G). This subject experienced side-effects (nausea and headaches) from TAO after two consecutive doses and vomited after taking the third dose. Accordingly, TAO was discontinued, but the subject soon felt better, and the pharmacokinetic portion of the study was completed without further TAO treatment. During the control phase, the mean ERMBT result was unchanged over the time course (Table 1). In contrast, during the TAO phase, the mean ERMBT result significantly decreased by 95% (95% CI, 93, 98%) 2 h after the first dose of TAO and then increased at the end of the 8 h TAO dosing interval (i.e. 6 h after quinine was given) (Table 1). The mean ERMBT result then decreased at 24 h (i.e. 2 h after the TAO dose), but rose again at 48 h (also 2 h after the TAO dose) (Table 1). Exclusion of Subject G from the TAO phase did not change the mean ERMBT values at the first three time points (−2, 0, and 6 h relative to quinine administration; see Table 1) but did decrease the mean ERMBT values at the last two time points (to 0.25 and 0.82 at 24 and 48 h, respectively). TAO treatment significantly decreased the mean time-averaged ERMBT by 77% (95% CI, 68, 85%) (Figure 3). This value was essentially unchanged if Subject G was excluded (80%; 95% CI, 76, 85%). All tests of statistical significance remained unchanged with Subject G excluded from the analysis.
Figure 2. Time course of the effect of multiple doses of troleandomycin (TAO, 500 mg) on hepatic CYP3A4 activity (ERMBT) in a typical subject (Subject F) and in the one atypical subject (Subject G) in whom TAO was discontinued due to side-effects. ○ represents the control phase (quinine alone), ● represents the TAO phase (quinine + TAO), and arrows represent times when TAO was given.
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Table 1. Time course of the ERMBT result obtained from 10 healthy subjects given quinine alone (control phase) or quinine + TAO (TAO phase).
| || || || Time relative to quinine administration || || |
| Subject || −2 h || 0 h || 6 h || 24 h || 48 h |
| Quinine alone || || || ERMBT (% of 14 C exhaled h −1 ) || || |
|A|| ||2.40|| ||1.92||1.95|
|B|| ||1.77|| ||1.55||1.45|
|C|| ||1.66|| ||2.59||2.23|
|D|| ||2.40|| ||1.92||1.95|
|E|| ||2.93|| ||3.99||3.31|
|F|| ||2.83|| ||2.28||2.61|
|G|| ||2.93|| ||2.55||2.59|
|H|| ||3.29|| ||3.19||3.21|
|I|| ||2.24|| ||2.77||1.77|
|J|| ||2.01|| ||1.81||1.85|
|Mean ± s.d.|| ||2.45 ± 0.54|| ||2.46 ± 0.73||2.29 ± 0.62|
|95% CI|| ||2.11, 2.78|| ||2.00, 2.91||1.91, 2.68|
| Quinine + TAO || || ||ERMBT (% of 14C exhaled h−1)|| || |
|Mean ± s.d.||2.48 ± 0.65||0.12 ± 0.13*||0.75 ± 0.35*||0.43 ± 0.60*||0.98 ± 0.57*|
|95% CI||2.08, 2.89||0.04, 0.20||0.53, 0.97||0.06, 0.80||0.62, 1.33|
Figure 3. Effect of TAO treatment on the time-averaged ERMBT result (upper panel), apparent oral clearance (CL/F) of quinine (middle panel), and apparent formation clearance of 3-hydroxyquinine (CL3-OH) (lower panel). Bars and error bars denote the average and standard deviation, respectively. Asterisks (*) indicate that the mean value from the TAO phase statistically differed from that in the control phase (paired Student's t-test, P < 0.0001).
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Representative plasma concentration vs time profiles for quinine and 3-hydroxyquinine after a single oral dose of quinine sulphate (600 mg) for the two treatment phases (control and TAO) are shown in Figure 4. The corresponding profiles for subject G from 24 to 48 h (i.e. when the ERMBT indicated no inhibition of CYP3A4) were not obviously different from those in the other nine subjects (not shown). However, there were only three data points to consider. Therefore, Subject G was included in the statistical analysis of the pharmacokinetic data.
Figure 4. Plasma concentration vs time profiles of quinine and 3-hydroxyquinine following administration of a single oral dose of quinine sulphate (600 mg) to a representative subject (Subject B). Open symbols represent the control phase (quinine alone); solid symbols represent the TAO phase (quinine + TAO).
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With TAO treatment, quinine concentrations were higher, especially during the later time points, as compared to the control phase (Figure 4, upper panel). The formation of 3-hydroxyquinine was less during the TAO phase than in the control phase throughout the time course (Figure 4, lower panel). Pharmacokinetic measurements for quinine and 3-hydroxyquinine are summarized in Table 2 and 3, respectively. Values obtained from the control phase agreed with those previously reported for healthy volunteers [16, 23–25]. Compared with control, TAO significantly increased the mean quinine AUC(0,∞) and Cmax by 90% (95% CI, 66, 113%) and 26% (95% CI, 16, 36%), respectively, and decreased the mean CL/F by 45% (95% CI, 39, 52%) (Table 2, Figure 3). In addition, TAO significantly prolonged the mean terminal elimination half-life (t½) of quinine by 63% (95% CI, 49, 77%) and increased the mean percentage of the quinine dose excreted unchanged in the urine (0–48 h) by 101% (95% CI, 63, 138%). There were no significant differences in mean values for tmax and ClR of quinine between the control and TAO phases (Table 2).
Table 2. Pharmacokinetics of quinine following a single oral dose (600 mg) of quinine sulphate to 10 healthy subjects during two treatment phases: control (quinine alone) and TAO (quinine + TAO). Data are presented as mean ± s.d. (95% CI).
| Measure || Control phase || TAO phase |
|AUC(0,∞) (mg l−1 h)||63 ± 10 (57–69)||118 ± 27* (102–135)|
| C max (mg l−1)|| 3.2 ± 0.5 (2.9–3.4)|| 3.8 ± 0.6** (3.5–4.2))|
| t max (h)|| 3.3 ± 1.8 (2.2–4.4)|| 3.6 ± 2.4 (2.1–5.1)|
|CL/F (l h−1)|| 9.8 ± 1.8 (8.7–10.9)|| 5.3 ± 1.0* (4.6–5.9)|
| t ½ (h)||12.3 ± 1.7 (11.2–13.3)||20.0 ± 4.5* (17.2–22.8)|
| f e,Q (0,48 h)||10.1 ± 2.4 (8.6–11.6)||19.6 ± 5.5* (16.2–23.1)|
|CLR (l h−1)||0.97 ± 0.21 (0.84–1.10)||1.03 ± 0.35 (0.82–1.25)|
Table 3. Pharmacokinetics of 3-hydroxyquinine following a single oral dose (600 mg) of quinine sulphate to 10 healthy subjects during two treatment phases: control (quinine alone) and TAO (quinine + TAO). Data are presented as mean ± s.d. (95% CI).
| Measure || Control phase || TAO phase |
|AUC(0,∞) (mg l−1 h)|| 7.7 ± 3.1 (5.8–9.5)|| 3.4 ± 2.2* (2.0–4.7)|
| C max (mg l−1)||0.32 ± 0.11 (0.25–0.38)||0.08 ± 0.03* (0.06–0.09)|
| t max (h)|| 3.2 ± 1.8 (2.0–4.3)|| 9.4 ± 8.2 (4.3–14.5)|
| t ½ (h)||14.3 ± 2.6 (12.7–15.9)||25.7 ± 11.7** (18.5–33.0)|
| f e,3-OH || 7.1 ± 1.6 (6.1–8.2)|| 2.5 ± 1.2* (1.7–3.3)|
|CL3-OH (l h−1)||0.69 ± 0.17 (0.59–0.80)||0.13 ± 0.07* (0.09–0.17)|
During both phases of the study, 3-hydroxyquinine displayed formation rate-limited kinetics (Figure 4), as its average terminal elimination half-life (14.3 and 25.7 h for control and TAO phases, respectively) was not significantly different from that for quinine (12.3 and 20.0 h for control and TAO phases, respectively) (Tables 2 and 3). Compared with control values, coadministration of TAO with quinine resulted in significant decreases in the average AUC(0,∞) (58%; 95% CI, 51, 65%), Cmax (75%; 95% CI, 70, 81%), the percentage of the dose excreted in the urine as 3-hydroxyquinine (65%; 95% CI, 55, 74%), and apparent formation clearance (81%; 95% CI, 76, 87%) of 3-hydroxyquinine (Table 3). The mean terminal elimination t½ of 3-hydroxyquinine was significantly prolonged during the TAO phase. Although not significant, the mean tmax increased nearly three-fold (Table 3).
When data from the control and TAO phases were analysed separately, there were no significant correlations between the time-averaged ERMBT result and the following: CL/F; single plasma quinine or 3-hydroxyquinine concentration at 12, 24, and 48 h; CL3-OH; and the metabolite-to-parent AUC ratio. There were also no correlations between the ERMBT result at 24 and 48 h and the corresponding plasma concentrations. When data from the two treatment groups were pooled, statistically significant correlations existed between the time-averaged ERMBT result and each of the aforementioned indices. However, these correlations resulted solely from the two distinct clusters created by control and TAO-treated subjects. Within subjects, there were no correlations between the percent TAO-mediated decreases in the ERMBT result and quinine CL/F, or between the percent decreases in the ERMBT result and CL3-OH.
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Previous studies have established that TAO is a potent and selective inhibitor of human hepatic CYP3A4 in vivo, as shown by considerable reductions (>75%) in the ERMBT result  and systemic clearances of the CYP3A4 substrates midazolam and alfentanil . The current study, utilizing the ERMBT, demonstrated that TAO administered every 8 h resulted in continuous potent inhibition of CYP3A4 activity throughout the entire 48 h period. The inhibitory effect appeared to abate slightly at the end of the first TAO dosing interval (i.e. 6 h after quinine was administered) (Table 1), suggesting that dosing intervals greater than 8 h would result in less inhibition. The inhibitory effect also appeared to abate between 24 and 48 h (Table 1). In rats, TAO treatment has been shown to increase CYP3A mRNA production and presumably CYP3A enzyme synthesis . If this is also the case in humans, this mechanism could account for the gradual rise in CYP3A4 activity and may limit the usefulness of TAO for chronic inhibition.
During the TAO phase, the mean apparent oral clearance (CL/F) of quinine significantly decreased by 45% compared with control. Since quinine is essentially completely absorbed into the systemic circulation , and TAO had only a modest effect on mean Cmax and tmax, the reduction in CL/F by TAO treatment was primarily due to inhibition of the systemic clearance of quinine. Therefore, because the mean renal clearance was also unchanged, the effect of TAO was likely to be on the hepatic clearance of quinine. The mean apparent formation clearance of 3-hydroxyquinine (CL3-OH) was also significantly reduced by TAO treatment (81%), consistent with previous in vitro studies demonstrating that the formation of 3-hydroxyquinine is mainly catalysed by CYP3A4 [17, 18]. Thus, the present study demonstrates that hepatic CYP3A4 accounts, on average, for at least 45% of quinine CL/F and up to 81% of CL3-OH. Correspondingly, this infers that up to 55% of CL/F may be non-CYP3A4 mediated. At least seven metabolites of quinine have been detected in human urine, with three of these being identified as 3-hydroxyquinine, 2′-oxoquininone, and quinine glucuronide . The enzymes responsible for the latter two pathways are not known. The conclusion that hepatic CYP3A4 is a major pathway for the elimination of quinine is also consistent with the in vivo observation of a six-fold increase in quinine CL/F following pretreatment with rifampicin , a prototypical CYP3A4 inducer. However, it is possible, that intestinal CYP3A4-mediated metabolism could become important in the disposition of quinine after treatment with rifampicin (or other CYP3A4 inducers).
Mirghani et al. reported that coadministration of quinine with the CYP3A4 inhibitor ketoconazole led to a significant decrease (31%) in both the mean quinine CL/F and 3-hydroxyquinine AUC(0,∞). The greater percent reductions observed in the current study suggest that TAO is more effective than ketoconazole as an inhibitor of CYP3A4 in vivo. It is also likely that TAO is a more selective inhibitor than ketoconazole, as the latter has been shown to inhibit the secretory (efflux) transporter P-glycoprotein (P-gp) [29–31]. Located on the apical (lumenal) surface of human enterocytes , P-gp acts to extrude its substrates from the interior of the enterocyte back into the intestinal lumen, often leading to a decrease in the fraction of the dose absorbed. There is considerable overlap between CYP3A4 and P-gp substrates , potentially rendering it difficult to distinguish the effects of ketoconazole on the two proteins. In contrast to ketoconazole, we have found that TAO does not inhibit P-gp in the human intestinal cell line Caco-2 . Collectively, these observations suggest that TAO may be superior to ketoconazole as an experimental means of selectively inhibiting human CYP3A4 activity in vivo.
It is not known whether quinine is hydroxylated by polymorphically expressed CYP3A5, which shares 84% amino acid sequence homology with CYP3A4 . If so, variability in the expression of CYP3A5 may have influenced the changes in quinine disposition. Interestingly, half of the subjects were African-American, who have been reported to possess at least one CYP3A5 allele at a greater frequency compared with Caucasians . TAO inhibits CYP3A5 to a much lesser extent compared to CYP3A4 (K.E. Thummel & D.J. McConn, University of Washington, personal communication). Therefore, if quinine were metabolized by CYP3A5, the TAO-mediated decrease in quinine CL/F in CYP3A5 expressors would be predicted to be less compared with nonexpressors. Since the current group of subjects was neither genotyped nor phenotyped for CYP3A5, the influence of CYP3A5 expression on quinine disposition cannot be ascertained at present.
There is large inter- and intra-individual variation in human CYP3A4 activity, between 10-fold and 40-fold, depending on the method used and the population studied [4, 5, 7]. This large variability in CYP3A4 activity accounts for much of the inter- and intra-patient variations in the disposition of many CYP3A4 substrates . The search for a suitable probe (biomarker) to assess hepatic CYP3A4 activity has therefore been intense. Many CYP3A4 probes have been proposed, including the ERMBT , the ratio of endogenous cortisol to 6β-hydroxycortisol , and midazolam systemic clearance [7, 8]. The utility, suitability, and limitation(s) of each have been discussed elsewhere in detail [6, 38].
It was anticipated that results from the current study would substantiate the use of quinine as the first oral probe capable of quantifying hepatic CYP3A4 activity. In support of this, following TAO treatment, both the mean CL/F of quinine and CL3-OH decreased in parallel with the mean time-averaged ERMBT result (Figure 3), suggesting that quinine may be suitable to assess inhibition of hepatic CYP3A4. However, within an individual, there were no correlations between the percent decrease in either quinine CL/F or CL3-OH and the percent decrease in the ERMBT result. In addition, time-averaged ERMBT results from the control or TAO phase did not correlate with either quinine CL/F or CL3-OH. The absence of such correlations may not necessarily mean that no correlation existed, as the sample size (10) could only detect a strong correlation (r > 0.8) with 85% probability. It should also be noted that the different CYP3A4 probes generally fail to correlate with one another, and multiple factors may account for these lack of correlations [39, 40]. Future studies will examine whether quinine CL/F, or other surrogates, can predict the CL/F of other CYP3A4 substrates, such as midazolam.
In summary, using TAO as a chemical ‘knockout’ for CYP3A4, we have confirmed that a major pathway for the metabolism of quinine in vivo is mediated by hepatic CYP3A4. Compared with ketoconazole, TAO appears to be the experimental treatment of choice to ablate CYP3A4 activity in vivo. Because CYP3A4 plays a prominent role in quinine elimination, quinine may serve as a useful probe to assess within-subject inhibition of liver CYP3A4 activity in certain circumstances. For example, this approach can be easily performed in patients already receiving quinine as treatment or prophylaxis for malaria. Further studies are needed to determine whether quinine can provide a quantitative measure of CYP3A4 activity suitable for intersubject comparison.