Cyp3a4 Perpetrator

[Dardo Hameed]

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Typically, concentration-response curves are based upon nominal inducer concentrations for in-vitro-to-in-vivo extrapolation of CYP3A4 induction. The limitation of this practice is that it assumes the hepatocyte culture model is a static system. We assessed whether correcting for: 1) changes in perpetrator concentration in the induction medium during the incubation period, 2) perpetrator binding to proteins in the induction medium, and 3) nonspecific binding of perpetrator can improve the accuracy of CYP3A4 induction predictions. Of the seven compounds used in this evaluation, significant parent loss and nonspecific binding were observed for rifampicin (29.3-38.3%), pioglitazone (64.3-78.6%), and rosiglitazone (57.1-75.5%). As a result, the free measured EC50 values (EC50u) of pioglitazone, rosiglitazone, and rifampicin were significantly lower than the nominal EC50 values. In general, the accuracy of the induction predictions, using multiple static models, improved when corrections were made for measured medium concentrations, medium protein binding, and nonspecific binding of the perpetrator, as evidenced by 18-29% reductions in the root mean square error. The relative induction score model performed better than the basic static and mechanistic static models, resulting in lower prediction error and no false-positive or false-negative predictions. However, even when the EC50u value was used, the induction prediction for bosentan, which is a substrate of organic anion transporter proteins, was overpredicted by approximately 2-fold. Accounting for the ratio of unbound intracellular concentrations to unbound medium concentrations (Kpuu,in vitro) (0.5-7.5) and the predicted multiple-dose Kpuu,in vivo (0.6) for bosentan resulted in induction predictions within 35% of the observed interaction.

Although polypharmacy is a particular challenge in daily rheumatological practice, clinical research on the effects of hydroxychloroquine (HCQ), a commonly used drug for patients with rheumatic diseases, is sparse on cytochrome P450 (CYP)-mediated metabolism. We have shown that pre-treatment with pantoprazole does not alter HCQ absorption in healthy volunteers. In this paper, we report the effects of a single 400 mg dose of HCQ on specific CYP3A and CYP2D6 substrates in healthy volunteers.

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 [2,3,4]. 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 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.

The primary objective of this trial was to evaluate the effect of the proton-pump inhibitor pantoprazole on the absorption of HCQ; the results of the primary endpoint have been published previously [11].

First, CYP3A and CYP2D6 baseline activities were evaluated with microdosed midazolam (30 g; Dormicum V 5 mg/5 ml, Cheplepharm Arzneimittel GmbH, Greifswald, Germany, in 100 ml water) and yohimbine (50 g; Yohimbinum hydrochloricum D4, Deutsche Homopathie-Union-Arzneimittel GmbH and Co. KG, Karlsruhe, Germany). The drugs were administered 3 h after a standardized meal. The timing of the meal was the same on trial day 6 when HCQ was administered with a meal prior to the microdosed drugs. Starting after the baseline assessments, participants were 1:1 randomized either to a 9-day course of oral pantoprazole 40 mg (Pantoprazol HEXAL, Holzkirchen, Germany) once daily (to reach a steady state) or to control (without pantoprazole) for the evaluation of the primary objective of the trial. At day 6, a single oral dose of 400 mg HCQ (two tablets of Quensyl 200 mg; Sanofi-Aventis, Frankfurt, Germany) was administered to all participants with a standardized meal. Pantoprazole intake on this day took place 1 h before the meal (Fig. 1). A total of 3 h after the intake of HCQ (i.e. 4 h after pantoprazole), participants received the microdosed oral probe drugs. Plasma samples (lithium heparin) were collected for 6 h (pre-dose and 0.5, 1, 2, 2.5, 3, 4, and 6 h after administration). CYP phenotyping using microdosed midazolam [14, 16, 17] and yohimbine [15] has been published previously.

Trial design. Adapted from the trial protocol published in [13]. At baseline, cytochrome P450 (CYP) 3A and CYP2D6 activity were phenotyped with a microdose of midazolam and yohimbine. Thereafter, one group was started on a 9-day course of once daily (qd) pantoprazole, while the other group served as a control group. On the 6th day, a single dose of hydroxychloroquine 400 mg was administered, after which the CYP3A and CYP2D6 phenotyping was repeated. Cmax plasma peak concentration

Genotyping of CYP2D6 was performed using a single-base primer extension method as described previously [15]. CYP2D6 activity was scored on the basis of the sum of the allele activity scores assigned by PharmGKB ( ) and CPIC ( ) as follows: 0 poor metabolizer; 0.5 and 1 intermediate metabolizer; and 1.25, 1.5 and 2 normal metabolizer.

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.

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.

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].

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.

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