BACKGROUND AND PURPOSE:Neonatal hypoxic-ischemic encephalopathy (HIE) remains a leading cause of mortality and long-term morbidities, and therapeutic hypothermia (TH), while the standard of care, is not universally effective. The objective of the study was to use T2* values as a surrogate marker of myelination to characterize early myelination after HIE and explore whether sildenafil, used as an adjunct to TH, is associated with improved myelination. MATERIAL AND METHODS:This prospective neuroimaging study included neonates with HIE treated with TH; a subset of neonates with MRI-confirmed brain injury received sildenafil in addition to TH. Myelination was assessed using a T2* mapping. T2* values were measured in eight brain regions at three time-points (days 2, 10, and 30). T2* values were compared among neonates without brain injury treated with TH alone (NoBI+TH), neonates with brain injury treated with TH alone (BI+TH), and neonates with brain injury treated with TH plus sildenafil (BI+TH/S). RESULTS:A total of 167 MRI scans were obtained from 77 neonates: 32 NoBI+TH neonates, 25 BI+TH neonates, and 20 BI+TH/S neonates. On day 2, neonates with brain injury exhibited significantly higher T2* values in the thalamus, posterior limb of the internal capsule [PLIC], anterior white matter [AWM], and anterior corpus callosum [ACC] than the NoBI+TH group. By day 10, elevated T2* values persisted in AWM and ACC in both brain injury groups, and also in PLIC in the BI+TH group. At day 30, T2* values in the BI+TH group remained persistently elevated across these regions, with additional increases in the thalamus, optic radiations [OR], and posterior corpus callosum [PCC]. In contrast, the BI+TH/S group demonstrated significantly lower T2* values in thalamus, PLIC, AWM, OR, ACC, and PCC compared to BI+TH; in several regions (thalamus, PLIC, AWM), T2* values in the BI+TH/S group were no longer different from NoBI+TH. CONCLUSIONS:T2* values were increased after HIE despite TH, consistent with a possible disruption of early myelination. Sildenafil as an adjunct to TH was associated with lower T2* values over the first month of life, suggesting a potential neurorestorative effect on myelination.

OBJECTIVE:To evaluate the safety and tolerability of higher doses of sildenafil in neonates with hypoxic-ischemic encephalopathy (HIE) and brain injury. STUDY DESIGN:A phase 1b open-label dose-finding clinical trial in neonates with moderate-severe HIE and confirmed brain injury on a day-2 magnetic resonance imaging during therapeutic hypothermia (TH). Enteral sildenafil was administered every 12 hours (q12 h) for 7 days. All participants received an initial dose 2.0 mg/kg, and a second dose of 2.5 mg/kg. Starting from the third dose, group 1 received 2.5 mg/kg q12 h and group 2 received 3.0 mg/kg q12 h. Primary outcome was incidence of dose-limiting toxicities. Secondary outcomes explored day-30 neuroimaging and 18-month neurodevelopment. RESULTS:Among the 30 neonates born between October 2019 and December 2021, 20 displayed day-2 brain injury and 13 received sildenafil (8 in group 1; 5 in group 2). In group 1, 25% (2/8) experienced transient hypotension after the first dose, linked to antiseizure medications. No significant hypotension occurred in group 2 when sildenafil was administered separately. At the 3.0 mg/kg/dose, steady-state sildenafil concentrations persisted beyond TH. Death or significant 18-month neurodevelopmental impairment occurred in 50% (4/8) of group 1 and 60% (3/5) of group 2. Among the survivors, partial recovery of brain injury was seen in 80% (4/5) of group 1 and 75% (3/4) of group 2; cerebral palsy developed in 0% (0/5) and 50% (2/4), respectively. CONCLUSIONS:Enteral sildenafil up to 3.0 mg/kg q12 h was safe and well tolerated in a small single-center cohort of neonates with HIE treated with TH. Phase 2 trials are needed to assess multicenter feasibility and efficacy. TRIAL REGISTRATION:ClinicalTrials.gov NCT04169191.

For the toxicokinetic evaluation of a lipid conjugate of the cell-penetrating peptide cyclic nonaarginine, we aimed to develop a highly sensitive plasma quantification. Due to the amphiphilic properties and high number of basic amino acids, plasma bioanalysis with LC-MS/MS instruments is difficult. Challenges in particular include chromatographic characteristics, minimal extraction recovery, and resistance against collision-induced dissociation. We developed a quantification of a nonextractable CPP-lipid conjugate by eliminating the lipid part by phospholipase D digestion, enabling the efficient extraction of a surrogate peptide for analysis. Chromatographic separation was feasible only with trifluoroacetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by postcolumn addition of aqueous ammonia. Efficient extraction of the surrogate peptide fragment was achieved by protein precipitation with TFA. This enabled the highly sensitive quantification of the CPP-lipid conjugate in plasma in the low picomolar range (lower limit of quantification of 0.1 ng/mL; 34 pM). The assay was validated according to the pertinent recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the determination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. Applicability was further demonstrated by validation of the assay for human plasma. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates by using lipase digestion to generate extractable surrogate fragments. Additionally, TFA-based UPLC-MS/MS analysis with desolvation of TFA adducts by postcolumn addition of ammonia is feasible for the regulated, highly sensitive quantification of arginine-rich peptides.

PurposePregabalin, gabapentin, and carbamazepine, a potent inducer of cytochrome P450 (CYP) 3A4 and P-glycoprotein, are frequently used antiepileptic drugs that are often administered together with factor Xa inhibitors (FXaI). We aimed to investigate whether potentially clinically relevant drug-drug interactions occur with these combinations.MethodsIn an open-label fixed-sequence trial in 36 healthy volunteers, we evaluated the pharmacokinetics of 60 mg edoxaban and of a microdosed FXaI cocktail (25 µg apixaban, 50 µg edoxaban, and 25 µg rivaroxaban) before and during treatment with carbamazepine (12 evaluable volunteers, individually dosed to therapeutic concentrations), gabapentin (11 volunteers, titrated to 3 × 400 mg/d), and pregabalin (12 volunteers, titrated to 2 × 300 mg/d). The antiepileptics were dosed to steady-state and the CYP3A activity was evaluated by assessing the pharmacokinetics of microdosed midazolam (30 µg).ResultsCarbamazepine reduced the area under the plasma concentration-time curve (AUC∞) of 60 mg edoxaban by a factor of 0.48 (geometric mean ratio (GMR) with 90% CI (0.41–0.56); p < 0.0001) and Cmax by a factor of 0.47 (0.34–0.66) and reduced the exposure of the edoxaban metabolite M-4 to a similar extent. Carbamazepine also decreased the exposure (AUC∞) of microdosed apixaban, edoxaban, and rivaroxaban by a factor of 0.66, 0.59, and 0.56, respectively. Gabapentin and pregabalin did neither affect the exposure of 60 mg edoxaban nor the exposure of any microdosed FXaI.ConclusionCarbamazepine decreased FXaI exposure to a clinically relevant extent and dose adjustment may be required to maintain an adequate anticoagulant effect, whereas gabapentin and pregabalin do not require dose adjustment of FXaI.

Background/Objectives: Bempedoic acid (BA) is a novel cholesterol-lowering agent with proven positive effects on cardiovascular endpoints. Because it is an inhibitor of the hepatic transporters OATP1B1 and OATP1B3, two uptake transporters regulating the intrahepatic availability of statins, it increases the systemic exposure of co-administered statins. This interaction could raise the risk of myopathy. We hypothesized that the drug interaction between BA and statins could be mitigated by staggered administration. Methods: This was a single-centre, open-label, randomized, two-arm, cross-over, phase I drug interaction trial in healthy volunteers (EudraCT-No: 2022-001096-13). The primary objective was to evaluate the OATP1B1 inhibitory effect of BA on exposure to pravastatin after simultaneous administration versus different schedules of staggered administration. A secondary objective was to evaluate the impact of SLCO1B1 genotypes (*1, *5, *15, *37) on pravastatin exposure. Pravastatin was administered in single oral doses of 40 mg at six visits. After a baseline visit with pravastatin alone, BA was dosed to steady state at the approved oral dose of 180 mg. Outcome measures were the area under the plasma concentration–time curve, extrapolated to infinity (AUC∞) and Cmax of pravastatin, 3α-hydroxy-pravastatin (pravastatin 3-iso), and pravastatin lactone, and their geometric mean ratios (GMRs) of different schedules of administration. Log-transformed AUC∞ and Cmax were compared with one-way ANOVA with a 90% confidence interval (CI). Results: Fourteen participants completed all visits. At BA steady state, the GMRs of pravastatin AUC∞ and Cmax were 1.80 (90% CI 1.31–2.46) and 1.95 (90% CI 1.40–2.72), respectively, compared to baseline. There was no significant difference in pravastatin exposure between simultaneous vs. staggered administration. There was no statistically significant difference in pravastatin 3-iso or pravastatin lactone between different administration modes. For the AUC∞ of pravastatin and pravastatin 3-iso, haplotype was a significant source of variation (63% and 20%, respectively), while the type of administration (simultaneous vs. staggered) had no significant impact. Conclusions: The increase in pravastatin exposure with concomitant intake of BA was larger than expected. There was no significant difference between simultaneous vs. staggered administration of pravastatin and BA, possibly due to a population that was heterogenous in SLCO1B1 haplotypes.

Lipid-based drug delivery systems can be surface-modified by lipid conjugates of the substance in question. The most important modifications include arginine-rich cell-penetrating peptides (CPP). A toxicokinetic evaluation of these lipid conjugates is im-portant during preclinical and clinical development of drug delivery formulations. Due to their amphiphilic properties and high number of basic amino acid residues, lipid conjugates of CPP exhibit difficult characteristics in regard to their plasma bioanalysis with LC-MS/MS instruments. These especially include challenging chromatography with extensive carry-over and minimal extrac-tion recovery, and, due to large numbers of basic amino acids and the resulting immobility of protons, resistance against collision-induced dissociation. We developed a surrogate quantification of a CPP-lipid conjugate relying on elimination of the lipid part by phospholipase D digestion. Chromatographic separation was only feasible with trifluoro acetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by post-column addition of aqueous ammonia. Efficient extraction of the surrogate peptide fragment was achieved by protein precipitation with TFA. This enabled the highly sensitive quantification of the CPP-lipid conjugate in plasma in the low picomolar range (lower limit of quantification of 0.1 ng/mL; 34 pM). The assay was validated ac-cording to the pertinent recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the deter-mination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates and especially the TFA-based UPLC-MS/MS analysis with post-column desolvation of TFA adducts by ammonia is a feasible approach for the highly sensitive quantification of arginine-rich peptides and other related substances with challenging chromatographic characteristics.

Medical care can fail for various reasons: diseases can remain undetected and their severity misjudged, therapies can be incorrectly dosed or ineffective, and therapies can trigger new conditions or adverse drug reactions (ADR). To manage the complexity of changing patient circumstances, data-driven techniques play an increasingly important role in monitoring patient safety and treatment success. Therefore, clinical prediction models need to consider longitudinal factors (“Prescribing Monitoring”) to ensure clinically meaningful results and avoid misclassification in the dynamic health situation of the individual patient. We have conducted a scoping review (OSF registration: https://doi.org/10.17605/OSF.IO/P93TZ ) on prediction models for ADR to collect potential use cases for Prescribing Monitoring. This review identified 2435 relevant studies in English that were published in MEDLINE or EMBASE. Two reviewers screened the records for inclusion, with a third reviewer making the final decision in the event of discrepancies. In order to derive recommendations on the way towards a Prescribing Monitoring system, the following elements were extracted and interpreted: the prediction models used, selection of candidate predictors, use of longitudinal factors, and model performance. A total of 56 studies were included after the screening process. We identified the main areas of current research in ADR prediction, all covering clinically important outcomes. We identified Prescribing Monitoring use cases based on their potential to (i) make individual predictions considering specific patient characteristics, (ii) make longitudinal predictions in a near time frame, and (iii) make dynamic predictions by updating predictions with previous risk predictions and newly available data. As a further aside, we use hyperkalaemia as an example to discuss the framework for developing Prescribing Monitoring in an electronic health record (EHR). This scoping review provides an overview of the use of time-varying effects and longitudinal variables in current prediction model research. For application to clinical cases, prediction models should be developed, validated and implemented on this basis, so that time-dependent information can enable continuous monitoring of individual patients.

Adequate secondary prevention in survivors of intracerebral hemorrhage (ICH) who also have atrial fibrillation (AF) is a long-standing clinical dilemma because these patients are at increased risk of recurrent ICH as well as of ischemic stroke. The efficacy and safety of oral anticoagulation, the standard preventive medication for ischemic stroke patients with AF, in ICH patients with AF are uncertain. PRESTIGE-AF is an international, phase 3b, multi-center, randomized, open, blinded end-point assessment (PROBE) clinical trial that compared the efficacy and safety of direct oral anticoagulants (DOACs) with no DOAC (either no antithrombotic treatment or any antiplatelet drug). Randomization occurred in a 1:1 ratio and stratification was based on ICH location and sex. The two co-primary binary endpoints included ischemic stroke and recurrent ICH which will be analyzed hierarchically according to the intention-to-treat principle. Secondary efficacy endpoints encompassed all-stroke and systemic embolism, all-cause and cardiovascular mortality, major adverse cardiac events, and net clinical benefit. Secondary safety endpoints included any major hemorrhage and intracranial hemorrhage. All outcome events were adjudicated by an independent committee. Results of PRESTIGE-AF are expected to support risk-adjusted secondary prevention in ICH survivors with AF and to inform clinical guideline recommendations.

Medication history taking at hospital admission is still prone to errors. Despite numerous quality improvement initiatives, new strategies to improve medication history taking are still sought and evaluated. Unfortunately, the gold standard research methodology for evaluation is resource-intensive, as it requires each patient to complete two medication history interviews. Therefore, a new study design and quality parameter were developed. We aimed to pilot our newly developed study design and quality parameter in a study on medication history taking by telephone. In this prospective interventional study, patients with scheduled admissions had their medication histories taken either by telephone before admission (intervention) by a pharmacist or in-person by physicians or medical interns upon admission (control). Following the newly developed design, we compared a patients’ new medication histories to the respective pre-visit medication lists available in the medical records to calculate the new endpoint: the difference in the number of updates per patient. Further, we surveyed patients and staff on their satisfaction. We enrolled 76 intervention and 75 control patients. In the intervention group, a mean of 4.93 (± 4.45, 0–18) updates were found vs. 3.40 (± 3.75, 0–21) in the control group. Accordingly, the incident rate of number of updates per patient was 1.34 times higher in the intervention group (p = 0.044). The distribution of the types of updates was similar with the most common type of update being newly initiated medicines in both groups. Medication history taking by telephone took 15.7 ± 9.8 min (mean ± SD), including preparation, interview, and documentation. Survey results showed that intervention patients felt positive about the telephone interviews. Both groups were open to other digital approaches, e.g., online platforms. The new study design proved feasible to evaluate medication history taking by telephone with comprehensible results. The telephone approach delivered more updates compared to standard care, however, the proposed endpoint needs to be validated against the gold standard before widespread application Patient acceptance for this and other digital approaches was high in both groups.

Introduction Real-world effectiveness of a new treatment is relevant information for patients, healthcare professionals and payers, especially when patients encountered in routine clinical care differ significantly from those recruited in the randomised controlled trials (RCTs) that led to approval. However, obtaining effect estimates can be challenging when a new drug has only recently been marketed and real-world data (RWD) are not yet available. For new breast cancer (BC) therapies, we illustrate how RCT inferences can be transported to a target population and how a synthetic population can be generated to mimic a target population for which no RWD is yet available.Methods and analysis In our framework, we defined the data-generating process for the RCT population and the real-world (target) population with confounders, effect-modulating covariates and survival times as outcomes. First, we conducted generalisability and transportability (G&T) analyses to transport the RCT results to the simulated target population, applying the inverse probability of sampling weighting and outcome model-based estimator approach. We then used Synthea to generate a synthetic target population based on German BC survival rates and combined both approaches into a coherent strategy.Results Effect estimates (HRs with 95% CIs) transported from the RCT to our defined target population closely matched the expected real-world effect (RCT: 0.68 (0.65; 0.71); real-world: 0.75 (0.71; 0.79); transported from RCT: 0.76 (0.71; 0.81)). BC survival rates were very similar between observed and synthetic data (prediction error in absolute survival rates: 1.62%).Conclusion Combining G&T with synthetic data may inform decision-making in situations where RWD are not (yet) available.

To investigate the effect of amiodarone on apixaban pharmacokinetics in cardiac surgery patients with postoperative atrial fibrillation. Apixaban concentrations of postoperative cardiac surgery patients with or without amiodarone therapy were quantified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in clinical routine. A population pharmacokinetic model was built using nonlinear mixed effects modeling in NONMEM® 7.5 using first-order conditional estimation with interaction. The impact of amiodarone and creatinine clearance (CrCL) on apixaban exposure under various dosing regimens was analyzed using Simulx® (Lixoft). A total of 33 patients with 76 apixaban concentrations were included. A one-compartment model best described the pharmacokinetics of apixaban with a clearance (CL/F) of 3.05 L/h, apparent volume of distribution (Vd/F) of 23.7 L, and an absorption rate constant (ka) of 0.652/h. Interindividual variability (IIV) was observed in CL/F but not in Vd/F and ka. The covariates amiodarone and CrCL were independently associated with apixaban CL/F. Under concomitant amiodarone therapy, simulations predicted an increase of 44–49

Background: Findings on the effectiveness of medication reviews led by community pharmacists (CPs) are often inconclusive. It has been hypothesized that studies are not sufficiently standardized, and thus, it is difficult to draw conclusions. Objective(s): To examine differences in the way CP-led medication review studies are set up. This was accomplished by investigating (1) patient selection criteria, (2) components of the medication review interventions, (3) types of outcomes, and (4) measurement instruments used. Methods: A systematic literature search of randomized controlled trials of CP-led medication reviews was carried out in PubMed and Cochrane Library. Information on patient selection, intervention components, and outcome measurements was extracted, and frequencies were analyzed. Where possible, outcomes were mapped to the Core Outcome Set (COS) for medication review studies. Finally, a network analysis was conducted to explore the influence of individual factors on outcome effects. Results: In total, 30 articles (26 studies) were included. Most articles had a drug class-specific or disease-specific patient selection criterion (n = 19). Half of the articles included patients aged >= 60 years (n = 15), and in 40% (n = 12/30) patients taking 4 drugs or more. In 24 of 30 articles, a medication review was comprised with additional interventions, such as distribution of educational material and training or follow-up visits. About 40 different outcomes were extracted. Within specific outcomes, the measurement instruments varied, and COS was rarely represented. Conclusion: The revealed differences in patient selection, intervention delivery, and outcome assessment highlight the need for more standardization in research on CP-led medication reviews. While intervention delivery should be more precisely described to capture potential differences between interventions, outcome assessment should be standardized in terms of outcome selection by application of the COS, and with regard to the selected core outcome measurement instruments to enable comparison of the results.

AbstractThe dose dependence of the effect of enzyme inducers and the effect of the combined administration of two inducers that exert their effect via the same induction pathway (pregnane X receptor) have not been well studied. Using oral midazolam microdoses (30 μg), we have investigated CYP3A4 induction by St. John's wort (SJW) in 11 healthy volunteers using low (300 mg/day containing 7.48 mg hyperforin), therapeutic (900 mg/day), and supratherapeutic doses of SJW (1800 mg/day) for 14 days. SJW was then co‐administered with rifampin (600 mg/day) for a further 7 days to evaluate the effect of the combined administration of two inducers. In addition, intravenous midazolam microdoses (10 μg) were administered before SJW, at SJW 1800 mg/day, and during administration of the two inducers to assess the hepatic contribution to total induction (semi‐simultaneous administration). Administration of SJW increased oral midazolam clearance 1.96‐fold (300 mg/day), 3.86‐fold (900 mg/day), and 5.62‐fold (1800 mg/day), and 17.5‐fold after the addition of rifampin. Concurrently, the clearance of intravenous midazolam increased 2.05‐fold (1800 mg/day) and 2.93‐fold (SJW + rifampin). These results show that rifampin significantly enhances the induction of the highest SJW doses both hepatically and overall and suggest that these metabolic effects occur predominantly in the gut. These findings also suggest that in drug interactions involving strong and moderate enzyme inducers, the perpetrator effects of the strong inducer are decisive for the 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. In the trial, participants were randomized into two groups (HCQ plus a 9-day course of pantoprazole, or HCQ only). As a secondary endpoint, the effects of a single oral dose of HCQ on the exposure of the oral microdosed CYP3A probe drug midazolam (30 μg) and the oral microdosed CYP2D6 probe drug yohimbine (50 μg) were studied in 23 healthy volunteers (EudraCT no. 2020-001470-30, registered 31 March 2020). The exposure of the probe drugs after intake of HCQ compared with baseline values was quantified by the partial area under the plasma concentration–time curve 0–6 h after administration (AUC0–6 h) for yohimbine and the partial AUC2–4 h for midazolam. Under HCQ, yohimbine AUC0–6 h was unchanged, independent of CYP2D6 genotypes and pantoprazole exposure. Midazolam AUC2–4 h was 25

Background:Event analysis is a promising approach to estimate the acceptance of medication alerts issued by computerized physician order entry (CPOE) systems with an integrated clinical decision support system (CDSS), particularly when alerts cannot be interactively confirmed in the CPOE-CDSS due to its system architecture. Medication documentation is then reviewed for documented evidence of alert acceptance, which can be a time-consuming process, especially when performed manually. Objective:We present a new automated event analysis approach, which was applied to a large data set generated in a CPOE-CDSS with passive, noninterruptive alerts. Methods:Medication and alert data generated over 3.5 months within the CPOE-CDSS at Heidelberg University Hospital were divided into 24-hour time intervals in which the alert display was correlated with associated prescription changes. Alerts were considered "persistent" if they were displayed in every consecutive 24-hour time interval due to a respective active prescription until patient discharge and were considered "absent" if they were no longer displayed during continuous prescriptions in the subsequent interval. Results:Overall, 1670 patient cases with 11,428 alerts were analyzed. Alerts were displayed for a median of 3 (IQR 1-7) consecutive 24-hour time intervals, with the shortest alerts displayed for drug-allergy interactions and the longest alerts displayed for potentially inappropriate medication for the elderly (PIM). Among the total 11,428 alerts, 56.1% (n=6413) became absent, most commonly among alerts for drug-drug interactions (1915/2366, 80.9%) and least commonly among PIM alerts (199/499, 39.9%). Conclusions:This new approach to estimate alert acceptance based on event analysis can be flexibly adapted to the automated evaluation of passive, noninterruptive alerts. This enables large data sets of longitudinal patient cases to be processed, allows for the derivation of the ratios of persistent and absent alerts, and facilitates the comparison and prospective monitoring of these alerts.

Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug–drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity–liquid chromatography–mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01–10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin’s weaker induction-related drug–drug interaction risk in vivo.