In vivo CYP3A4 activity does not predict the magnitude of interaction between itraconazole and tacrolimus from an extended release formulation

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1 Received: 12 April 2018 Accepted: 3 July 2018 DOI: /bcpt ORIGINAL ARTICLE In vivo CYP3A4 activity does not predict the magnitude of interaction between itraconazole and tacrolimus from an extended release formulation Thomas Vanhove 1,2 Pieter Annaert 3 Noël Knops 4 Henriëtte de Loor 1,2 Jan de Hoon 5,6 Dirk R J Kuypers 1,2 1 Department of Microbiology and Immunology, KU Leuven University of Leuven, Leuven, Belgium 2 Department of Nephrology and Renal Transplantation, University Hospitals Leuven, Leuven, Belgium 3 Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven University of Leuven, Leuven, Belgium 4 Department of Pediatric Nephrology and Solid Organ Transplantation, University Hospitals Leuven, Leuven, Belgium 5 Department of Pharmaceutical and Pharmacological Sciences, Clinical Pharmacology and Pharmacotherapy, KU Leuven, Leuven, Belgium 6 Department of Pharmaceutical and Pharmacological Sciences, Center for Clinical Pharmacology, University Hospitals Leuven, KU Leuven, Leuven, Belgium Correspondene: Dirk R. J. Kuypers, Department of Nephrology and Renal Transplantation, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium (dirk.kuypers@uzleuven.be). Abstract The magnitude of interaction between the CYP3A4 substrate tacrolimus and various CYP3A4 inhibitors is highly unpredictable. We investigated whether an individual's baseline in vivo CYP3A4 activity, assessed using the oral midazolam (MDZ) probe, could be used to predict the magnitude of drug drug interaction between tacrolimus and the potent CYP3A4 inhibitor itraconazole. In a prospective single arm open label study, 16 healthy volunteers were administered single doses of MDZ and tacrolimus before and after a 4 day course of itraconazole. Itraconazole treatment resulted in a 9.0 fold decrease in MDZ apparent oral clearance (CL/F) and a 3.3 fold decrease in tacrolimus CL/F (P < for each). MDZ CL/F and tacrolimus CL/F were positively correlated both at baseline (r = 0.582, P = 0.018) and after itraconazole (r = 0.811, P < 0.001). Furthermore, baseline MDZ CL/F was positively correlated to the fold change in MDZ CL/F resulting from CYP3A4 inhibition (r = 0.759, P = 0.001). However, no predictors of change in tacrolimus CL/F resulting from CYP3A4 inhibition were identified, including baseline MDZ CL/F (P = 0.453), baseline tacrolimus CL/F (P = 0.759) and fold change in MDZ CL/F between both phases (P = 0.274). In conclusion, baseline oral MDZ clearance does not predict the magnitude of interaction between tacrolimus and itraconazole. 1 INTRODUCTION Tacrolimus, a calcineurin inhibitor used extensively in solid organ transplantation, is characterized by high pharmacokinetic variability and a narrow therapeutic window, necessitating therapeutic drug monitoring (TDM). Between and within subject variability in the expression and activity of cytochrome P450 (CYP) isoenzymes 3A4 and 3A5, present in gut and liver, are major determinants of variability in tacrolimus exposure. CYP3A5 activity is only meaningful in individuals possessing at least one functional CYP3A5*1 allele (referred to as CYP3A5 expressors ) but is severely reduced in carriers of two loss of function alleles (CYP3A5*3, *6 and *7, referred to as CYP3A5 non - expressors ). 1 As a result, tacrolimus oral clearance (CL/F) is about 2.5 fold higher in CYP3A5 expressors compared to non expressors. 2 CYP3A4 activity, which can be quantified using the midazolam (MDZ) drug probe, also varies widely between individuals and is a key determinant of tacrolimus CL/F and dose requirements. 3 Studies with MDZ (which, in vivo, does not seem to reflect CYP3A5 activity 2 ) have demonstrated that intraindividual CYP3A Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society) wileyonlinelibrary.com/journal/bcpt Basic Clin Pharmacol Toxicol. 2019;124:50 55.

2 VANHOVE ET AL. 51 activity is subject to time related changes resulting from factors such as intestinal pathology, food constituents and concomitant administration of drugs that induce CYP3A4 (such as corticosteroids 4 ) or inhibit CYP3A4. 5 Several moderate to potent CYP3A4 inhibitors (e.g. azole antifungals, certain calcium channel blockers and protease inhibitors) are regularly prescribed to tacrolimus treated transplant recipients, resulting in significant drug drug interactions (DDIs). To avoid tacrolimus overexposure and toxicity after the initiation of a CYP3A4 inhibitor, the dose of tacrolimus will generally be pre emptively reduced and TDM intensity increased with further dose adjustments until the new steady state tacrolimus dose requirement becomes apparent. However, a major challenge is the fact that the magnitude of interaction between tacrolimus and any given CYP3A4 inhibitor is highly unpredictable. For example, after voriconazole initiation, the change in tacrolimus dose corrected exposure varies more than 15 fold between patients and this variability is largely unexplained. 6 CYP3A4 inhibitors seem to have less of an effect on CYP3A5 expressors 7 due to the CYP3A5 enzyme being intrinsically resistant, 8,9 but in clinical practice, the effect of CYP3A5 genotype is quite limited and unpredictable. 6 There remains, therefore, an unmet clinical need to identify the factors that determine the magnitude of CYP3A4 related DDIs, with the ultimate goal of predicting the victim drug's optimal dose adjustment before the interaction occurs. We hypothesize that an individual's baseline CYP3A4 activity is one such determinant of DDI magnitude. It has, for example, been demonstrated that CYP3A4 is induced more by dexamethasone in patients with low baseline CYP3A4 activity, 10 and that prior administration of a CYP3A4 inhibitor blunts the effect of a second CYP3A4 inhibitor. 6 The aim of this study, therefore, was to assess whether combined intestinal and hepatic CYP3A4 activity, assessed using the oral MDZ drug probe, can predict the magnitude of interaction between tacrolimus and the potent CYP3A4 inhibitor itraconazole. 2 METHODS 2.1 Study population This was a single centre prospective single arm open label study. Sixteen healthy adult non smoking men (aged 18 28) with no relevant medical history were recruited. None of the subjects had taken any CYP3A4/5 interfering medication for at least 1 month prior to the start of and during the study. All subjects abstained from alcohol, grapefruit or pomelo from 7 days prior to the start of the study until the end of the study. On days 1 and 6, subjects were administered a single oral dose of 3 mg tacrolimus (Advagraf, Astellas Pharma Europe, Staines, UK) and 2 mg of midazolam (2 ml of a 1 mg/ml solution of Dormicum; Roche, Basel, Switzerland) with 250 ml of water in the morning after an overnight fast. Two 4 ml blood samples were collected in ethylenediaminetetraacetic acidcontaining tubes before and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hour after drug administration. One whole blood sample was stored at 80 C pending tacrolimus quantification. The other sample was centrifuged for 10 minutes at 1860 g and 4 C, after which plasma was stored at 80 C pending MDZ quantification. On days 2 to 5, 200 mg of itraconazole (two tablets of 100 mg Sporanox; Janssen Cilag, Beerse, Belgium) was administered twice daily with 250 ml of water, approximately 2 hour before breakfast and dinner. The study was performed according to the most recent version Declaration of Helsinki, registered at clinicaltrials.gov (NCT ), and approved by the ethics committee of the University Hospitals Leuven (S58603) and the Belgian Federal Agency for Medicines and Health Products (EudraCT , All participants provided written informed consent. 2.2 Analytical methods Plasma concentrations of MDZ were measured using a highperformance LC MS/MS method, as previously described. 11 Whole blood concentrations of tacrolimus were measured using an ultraperformance LC MS/MS method, adapted from a previously described method. 3 In short, the calibration curve of tacrolimus, which covered a linear range from to 72 ng/ml, was prepared by spiking different tacrolimus solutions (Milli Q water/ acetonitrile (50/50)) on blank human whole blood. Calibration curves were constructed using peak area ratios of analyte to internal standard using 1/X weighted linear regression. For sample preparation, 20 μl Internal Standard (20 ng/ml tacrolimus 13C d4), 50 μl solution of Milli Q water/acetonitrile (50/50) and 50 μl zinc sulphate (0.1 M) were added to 50 μl whole blood. After vortex mixing for 10 seconds, 200 μl acetonitrile was added. Subsequently, the samples were vortex mixed and centrifuged. The supernatant was then transferred into a 96 well 2 ml collection plate, 100 μl Milli Q water and 200 μl of Milli Q water/ acetonitrile (50/50) were added to each sample and 50 μl was injected onto an UPLC (Acquity H Class,Waters, Zellik, Belgium). Chromatographic separation was performed on an Acquity BEHC18 column ( mm 1.7 μm particle size; Waters, Zellik, Belgium). The mobile phase, delivered at a flow rate of 0.5 ml/min at 55 C, was a gradient of Milli Q water and acetonitrile. The detection was performed with a Xevo TQS tandem mass spectrometer (Waters, Zellik, Belgium). The ammonium adducts of tacrolimus and the internal standard were detected with

3 52 VANHOVE ET AL. multiple reaction monitoring in electrospray positive ionisation mode. Ion transitions were m/z > and > , respectively, for tacrolimus and the internal standard. The limit of detection and the lower limit of quantification were and ng/ml, respectively. The total, within run, between run and between day precision (n = 10) were all below 4% coefficient of variation. The recovery of tacrolimus in whole blood was 104%. 2.3 Genotyping Genomic DNA was isolated from whole blood samples using a salting out procedure. 12 The quantity and quality of genomic DNA were verified with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Participants were genotyped for the following single nucleotide polymorphisms (SNPs): CYP3A5*1 (rs776746) and CYP3A4*22 (rs ) using a previously published PCR restriction fragment length polymorphism method Pharmacokinetic and statistical analysis Data are presented as mean ± SD except when stated otherwise. Normality was tested using the Shapiro Wilk test. AUC, Cl/F, Cmax values were not normally distributed and Ln transformed for analysis. Differences between means were assessed using t test for normally distributed continuous data (ANOVA in case of more than two categories of the predictor variable) and using Mann Whitney U test for ordinal data. The following variables were considered as possible predictors of tacrolimus AUC 0-24 in a multivariable regression analysis: CYP3A5 genotype, MDZ AUC 0-24, haematocrit, weight and age. A two sided P value < 0.05 was considered statistically significant. All reported R² values are semipartial. Statistical analyses were performed using IBM SPSS Statistics version 22 (IBM, NY, USA). Calculation of non compartmental analysis (NCA) pharmacokinetic parameters and figure generation was performed using GraphPad Prism version 6 (San Diego, CA, USA). 3 RESULTS 3.1 Genotype and pharmacokinetic parameters Of the 16 study subjects, four (25%) were CYP3A5 expressors, of whom one was homozygous CYP3A5*1/*1. One subject (a CYP3A5 non expressor) carried the CYP3A4*22 allele. No adverse events occurred. Noncompartmental pharmacokinetic parameters of tacrolimus and MDZ before and during CYP3A4 inhibition are shown in Table 1; concentration time profiles are presented in Figure 1. The decrease in CL/F resulting from itraconazole treatment averaged ninefold (range ) for MDZ and 3.3 fold (range ) for tacrolimus (P < for each). 3.2 Relationship between MDZ and tacrolimus clearance MDZ CL/F and tacrolimus CL/F were positively correlated both baseline (pearson r = 0.582, P = 0.018) and after itraconazole administration (r = 0.811, P < 0.001). Baseline MDZ CL/F was strongly related to the fold change in MDZ CL/F resulting from itraconazole administration (r = 0.759, P = 0.001), but not to fold change in tacrolimus CL/F (r = 0.202, P = 0.453). Baseline tacrolimus CL/ F was also not correlated to the fold change in tacrolimus CL/F (r = 0.174, P = 0.519). These correlations are plotted in Figure 2. In multivariable linear regression, the only independent predictors of baseline tacrolimus CL/F were TABLE 1 Pharmacokinetic parameters of midazolam and tacrolimus before and after itraconazole treatment (n = 16) Variable Baseline After itraconazole Difference (%) P Midazolam AUC 0-24 (h ng/ml) 32.9 ± ± % <0.001 CL/F (ml/min) ± ± % <0.001 T max (h) a 0.5 [ ] 0.5 [ ] C max (ng/ml) 13.5 ± ± % <0.001 Tacrolimus AUC 0-24 (h ng/ml) 50.6 ± ± % <0.001 CL/F (L/h) 69.7 ± ± % <0.001 T max (h) a 2.0 [ ] 3.0 [ ] <0.001 C max (ng/ml) 5.5 ± ± % <0.001 AUC, area under the concentration time curve; CL/F, oral clearance; C max, maximum blood concentration; T max, time to reach maximum blood concentration. a Median [interquartile range].

4 VANHOVE ET AL. 53 FIGURE 1 Concentration time profiles of midazolam (A) and tacrolimus (B) before and after itraconazole administration CYP3A5 genotype (B = ± 10.77, R² = 0.36, P = 0.007) and baseline MDZ CL/F (B = 0.04 ± 0.02, R² = 0.20, P = 0.016; overall R² = 0.60). Independent predictors of tacrolimus CL/F during itraconazole inhibition were also CYP3A5 genotype (B = 9.33 ± 2.46, R² = 0.13, P = 0.008) and MDZ CL/F during inhibition (B = 0.25 ± 0.04, R² = 0.63, P < 0.001; overall R² = 0.76). No predictors of the fold change in tacrolimus CL/F before and after itraconazole administration were identified. Specifically, fold change in MDZ CL/F and change in haematocrit did not predict change in tacrolimus CL/F (r = 0.041, P = and r = 0.291, P = 0.274, respectively). 4 DISCUSSION This is the first study to assess whether quantification of in vivo CYP3A4 activity using the MDZ drug probe could be used to predict the magnitude of interaction between a CYP3A4 substrate and a CYP3A4 inhibitor. In 16 healthy volunteers, baseline MDZ CL/F was a strong predictor of the decrease in MDZ CL/F resulting from treatment with itraconazole, indicating that pharmacological inhibition decreases CYP3A4 activity less in subjects with a constitutionally low enzyme activity. This is biologically plausible. Orally administered CYP3A4 inhibitors generally have a larger effect on pre systemic clearance (i.e. bioavailability) than on systemic clearance Baseline bioavailability is therefore likely to be a major determinant of the potential magnitude of CYP3A4 related interactions: if bioavailability is 10% in a patient, it can theoretically increase tenfold to 100%, but if bioavailability is 50% (e.g. in subjects with very low intestinal/hepatic CYP3A4 activity [such as CYP3A4*22 carriers]), it can only increase twofold. 5 In spite of this, MDZ CL/F was not predictive of the magnitude of interaction between itraconazole and tacrolimus. The exact reasons for the lack of an association between MDZ CL/F and change in tacrolimus CL/F remain speculative, but may be related to fundamental differences in the metabolism of MDZ and tacrolimus. Firstly, the fact that itraconazole is a dual inhibitor of CYP3A4 and P glycoprotein (P gp) 17 is likely to be relevant. Tacrolimus, but not midazolam, is a combined substrate for CYP3A4 and P gp, 18 which may contribute to a discrepancy in the change in MDZ CL/F and tacrolimus CL/F resulting from itraconazole administration. However, the differential effects of CYP3A4 and P gp inhibition on tacrolimus disposition cannot be formally assessed due to the lack of a validated in vivo probe for P gp. 19 Additonally, itraconazole was not coadministered with tacrolimus on day 6 to minimize its inhibitory effect on P gp activity. Secondly, MDZ, a Biopharmaceutical Classification System (BCS) class I compound (high solubility, high permeability) is rapidly absorbed in the proximal intestine. Tacrolimus, a BCS class II compound (low solubility, high permeability), was administered as extended release formulation (which has become standard of care in our renal transplant patients) and likely has a large absorptive window over a substantial part of the intestine. Because CYP3A4 content in enterocytes decreases from proximal to distal intestine, tacrolimus absorption is at least comparable in the distal compared with the proximal intestine. 23 If distal absorption of tacrolimus is less dependent on CYP3A4, this may blunt the effect of pharmacological CYP3A4 inhibition on the overall disposition of the drug. That may also explain why the average decrease in CL/F was much greater for MDZ (ninefold) than for tacrolimus (3.3 fold), and why the change in MDZ CL/F was not predictive of the change in tacrolimus CL/F. CYP3A5 genotype had no clear effect on DDI magnitude, although this study was not powered to assess that

5 54 VANHOVE ET AL. FIGURE 2 Correlations between midazolam oral clearance (MDZ CL/F) and tacrolimus (Tac) CL/F during the baseline (A) and itraconazole (B) phases; between baseline MDZ CL/F and fold change in MDZ CL/F (C) or change in tacrolimus CL/F (D); between baseline tacrolimus CL/ F and change in tacrolimus CL/F (E) relationship (only four subjects were CYP3A5 expressors). Clinical evidence suggests that CYP3A5 expressors are intrinsically resistant to both CYP3A4 inhibition and induction, 7,24 but a large recent analysis of solid organ recipients indicates that, at least with regard to the potent CYP3A4 inhibitors voriconazole and posaconazole, this effect is limited and inconsistent. 6 It is noteworthy that the range in DDI magnitudes observed in the current study (the change in tacrolimus CL/F varied between 1.6 and 4.4 fold) was not as wide as that observed in clinical practice (where it can range from 1.0 to almost 20 fold). 6,25 This is likely to be at least partly related to the fact that, in the current study, tacrolimus was administered to healthy volunteers who were free from comorbid conditions such as anaemia, intestinal malabsorption and diarrhoea. A limitation of the current study is that subjects were administered a single dose of tacrolimus and, consequently, were not in pharmacokinetic steady state. Although it seems unlikely that CYP3A4 phenotyping using MDZ would have an added value in predicting the magnitude of CYP3A4 related DDIs in renal recipients in tacrolimus steady state conditions, the current study cannot exclude this possibility. It must be noted that although MDZ and tacrolimus are both substrates for CYP3A4, a significant interaction between them under the conditions of the current experiment is unlikely. In vitro data indicate that MDZ non competitively inhibits tacrolimus metabolism by hepatic microsomes with a Ki of around 2 3 μg/ml, 26 while the plasma concentrations of MDZ observed in the current cohort are in the range of 1 30 ng/ml, that is 1000 fold lower. Similarly, studies indicate that tacrolimus is not a significant CYP3A4 inhibitor in vivo. 27 In conclusion, baseline CYP3A4 activity, assessed using the oral MDZ probe, does not predict the magnitude of interaction between tacrolimus and itraconazole. With current knowledge, it remains impossible to predict an individual's optimal tacrolimus dose reduction needed upon initiation of a potent CYP3A4 inhibitor. ACKNOWLEDGEMENTS We would like to thank our study nurses J. De Vis, V. Verbeek and H. Wielandt. We also thank M. Dekens and J. Paulissen for their technical assistance. CONFLICT OF INTEREST The authors declare that they have no conflict of interest.

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