1 Introduction
Hydroxychloroquine (HCQ) is still an important medicine for patients with rheumatic diseases and has a long, albeit declining, tradition in the treatment of malaria. In addition, there was much interest in HCQ when searching for a remedy in the early coronavirus disease 2019 (COVID-19) pandemic [
1]. Polypharmacy, reflecting comorbidity, is common in patients with rheumatic diseases [
2‐
4]. In a survey among rheumatologists, interfering comorbidities and pharmacological management were identified as clinically relevant situations in the management of difficult-to-treat rheumatoid arthritis not covered by current guideline recommendations [
5].
In rheumatic arthritis and systemic lupus erythematosus, cardiovascular diseases or mood disorders are common comorbidities and present a challenge to medical management [
4‐
6]. Research into drug–drug interactions (DDIs) is important for the co-treatment of rheumatic diseases and their comorbidities. Common cardiovascular drugs and anti-thrombotic medicines are cytochrome P450 (CYP) 3A4 substrates: examples include atorvastatin, simvastatin, dronedarone, eplerenone and ticagrelor. The β
1-selective beta blockers metoprolol and nebivolol are CYP2D6 substrates. Also, many antidepressants belong to the group of CYP2D6 substrates, such as venlafaxine, amitriptyline, doxepin, nortriptyline and selective serotonin re-uptake inhibitors, such as fluvoxamine, fluoxetine and paroxetine [
7].
Although it is assumed that HCQ may have clinically relevant interactions with other drugs metabolized by CYP isozymes, there is surprisingly little evidence on HCQ’s interaction potential from clinical trials. In 2020, several reviews of the potential DDIs of HCQ were published, which largely extrapolated their recommendations from chloroquine trial data (e.g. [
8]). From the COVID-19 pandemic, there is an episodic report on elevated clarithromycin blood concentrations under treatment with HCQ in two patients, but in vitro assessments by the same authors did not support the hypothesis that the parent drug HCQ inhibits CYP3A4 activity [
9]. However, the inhibitory potential of HCQ metabolites was not analysed in this study. Later, in vitro evidence showed that HCQ metabolites inhibit CYP3A4 [
10]: only in 2023, a detailed in vitro study showed that HCQ is metabolized by CYP2D6, CYP3A4 and CYP2C8, and also suggested that HCQ and its metabolites might inhibit CYP2D6 [
10].
In a clinical trial, initiated during the COVID-19 pandemic, we investigated HCQ as a victim drug of a suspected proton-pump inhibitor-mediated increase in gastric pH in healthy volunteers but found no significant effect of pantoprazole on HCQ exposure [
11]. To our knowledge, there is only one other DDI trial with HCQ in humans. That trial evaluated the interaction of HCQ with metoprolol and dextromethorphan in healthy volunteers and indicated that HCQ weakly inhibits CYP2D6 [
12].
In this paper, we report the effects of a single 400 mg dose of HCQ on specific CYP3A and CYP2D6 substrates in healthy volunteers. As part of a clinical DDI trial that explored the effect of pantoprazole on HCQ absorption as a primary endpoint [
13], healthy volunteers were administered microdoses of the CYP3A substrate midazolam and the CYP2D6 substrate yohimbine at baseline and after a single dose of HCQ [
14,
15]. The secondary endpoint of the trial was to evaluate HCQ as a potential inhibitor of CYP3A and CYP2D6 activity in humans.
4 Discussion
We detected no significant increase of yohimbine exposure by a single dose of HCQ, indicating that HCQ is not a clinically relevant CYP2D6 inhibitor. In vitro, HCQ and its metabolites, mainly desethylchloroquine, have been reported to competitively and reversibly inhibit CYP2D6 activity, albeit only mildly [
10]. In the only clinical trial on the CYP2D6-inhibiting properties of HCQ so far, CYP2D6-inhibiting properties of HCQ were evaluated after an 8-day course of 400 mg HCQ twice daily in seven healthy volunteers [
12]: after administration of HCQ, the AUC of the CYP2D6 substrate metoprolol increased by 65% and
Cmax by 72%. Interestingly, the urinary metabolic ratio of dextromethorphan did not show any significant change. The effect on metoprolol was consistent for the six homozygous extensive (normal) CYP2D6 metabolizers, while the heterozygous extensive (intermediate) metabolizer did not show any relevant change in exposure. In our trial including more participants with a greater variation of genotypes, we did not observe an effect in the CYP2D6 genotype subgroups. Because of the large variation of yohimbine exposure due to the genotype, also a large effect would have been visible. We administered 400 mg HCQ as a single dose on the day of CYP phenotyping. Assuming a low affinity of HCQ to CYP2D6, this single dose of HCQ may have resulted in too low blood concentrations to exert perpetrator properties on yohimbine.
The exposure of the CYP3A substrate midazolam increased by 25% in all participants after the intake of HCQ. However, this observation was driven by the subgroup receiving pantoprazole, in which the AUC of midazolam was increased by as much as 46%. The partial AUC2–4 h for 1-OH-midazolam, the active metabolite of midazolam, increased by 17% under HCQ in all. This tended to be the case in the pantoprazole group, while there was clearly no effect in the control group. Concurrently, the ratio between the partial AUC of midazolam and 1-OH-midazolam also increased significantly after the combined intake of HCQ and pantoprazole (indicating reduced production of 1-OH midazolam by CYP3A), while it remained unchanged in the control group receiving only HCQ. The increased ratio strongly suggests that the metabolism of midazolam was inhibited by pantoprazole (leading to increased absorption due to reduced first-pass metabolism or to slower elimination) and that the exposure change was not primarily caused by gastric pH changes although a contribution cannot be completely excluded.
From clinical drug interaction trials with various CYP3A4 substrates, pantoprazole is not known as a perpetrator [
22,
23]. However, in vitro pantoprazole inhibits CYP3A4-mediated midazolam metabolism at relatively high concentrations [
24], which we confirmed with another CYP3A4 inhibition assay also in vitro.
Midazolam is a well-absorbed compound with moderate to high pre-systemic extraction ratio; administered in therapeutic or microdoses, it has an oral bioavailability of approximately 25% [
25]. Intestinal first-pass metabolism contributes approximately 50% to the overall first-pass metabolism of oral midazolam [
26], indicating that modulation of intestinal CYP3A can substantially increase midazolam bioavailability. In our trial, 40 mg pantoprazole was administered with approximately 200 ml of fluid, yielding local concentrations in the gut that likely exceeded 500 µM. In vitro, pantoprazole reduced CYP3A activity by 75% at concentrations of 100 µM, suggesting that in the first hours after oral administration, local pantoprazole concentrations may be high enough to substantially inhibit CYP3A in the gut. Because pantoprazole peak concentrations are reached approximately 2–3 h after oral administration [
27] and may be considerably delayed by food [
28], it may well be that at the time of midazolam administration, i.e. 4 h after pantoprazole administration, local intestinal concentrations were still in the inhibitory range. Peak serum concentrations were much lower (2.5 µg/ml equalling 6.5 µM [
29]) and associated with less CYP3A inhibition, explaining that pantoprazole is not known as a CYP3A inhibitor from daily practice.
Limitations: Because HCQ steady state is only reached after 4 months [
30], our trial was designed with a single-dose administration of HCQ. Although we did not find a short-term inhibitory effect of HCQ on CYP3A, we cannot exclude the possibility that time-dependent CYP3A inhibition develops with longer-term HCQ treatment. The active metabolites desethylchloroquine, didesethylchloroquine,and desethylhydroxychloroquine (DHCQ) are time-dependent inhibitors of CYP3A4 in vitro [
10]. This could explain the elevated clarithromycin exposure that was reported for two patients with COVID-19 treated with HCQ [
9]. In this context, the CYP2D6 genotype could theoretically indirectly influence time-dependent CYP3A4 inhibition in longer-term HCQ treatment via the formation of DHCQ: A Korean trial found that the DHCQ:HCQ ratio is lower in carriers of CYP2D6*10 polymorphisms who have reduced CYP2D6 activity [
31].
Yohimbine AUC0–6 h was highly variable due to the different CYP2D6 genotypes. This makes it difficult to find a statistically significant change between baseline and intervention. To mitigate this problem, we analysed the genotype subgroups, but those were small, which presents another statistical challenge. We cannot exclude that there might have been an effect in a larger group.
Furthermore, the microdosing method for CYP phenotyping using probe drugs has been validated in fasting condition. In the present trial, HCQ tablets had to be swallowed with a meal, so CYP microdosing could not take place in a fasting state but only 3 h after the intake of HCQ and the standardized meal. Administering an oral standard dose of midazolam 1 h after a meal delays the peak plasma concentration by 0.9 h as compared with the fasting state, and decreases midazolam exposure [
32]. Midazolam AUC
2–4 h in this trial was at the lower end of the range known from previous trials in our department, and we are aware of a mild food effect from another trial [
33]. However, the identical food–drug interval was also followed at baseline. So, even though the absorption might have been slightly different from the fasting state, comparability between the trial parts was ensured.
In conclusion, HCQ did not inhibit metabolism by CYP2D6 and CYP3A in the short term. Whether or not longer treatment, which leads to higher concentrations of parent compound and potentially interacting metabolites, modulates these isozymes differently still needs to be shown. Concurrently, we found evidence that pantoprazole can act as an inhibitor of CYP3A4 and moderately changes midazolam exposure. This was unexpected, because no clinically relevant perpetrator properties with other CYP3A substrates have been described for pantoprazole so far. However, CYP3A inhibitor characteristics have repeatedly been described in vitro. Whether this is due to the particular timing and is only observed with CYP3A substrates with quantitatively relevant intestinal metabolism remains to be investigated.