BIOPHARMACEUTICS & DRUG DISPOSITION
`Biopharm. Drug Dispos. 27: 421–426 (2006)
`Published online in Wiley InterScience
`(www.interscience.wiley.com) DOI: 10.1002/bdd.524
`
`Everolimus Drug Interactions: Application of a Classification
`System for Clinical Decision Making
`
`John M. Kovarik*, Doris Beyer and Robert L. Schmouder
`Novartis Pharmaceuticals, Basel, Switzerland and East Hanover, NJ, USA
`
`ABSTRACT: Introduction. More than half of all drugs used in medical practice are metabolized by
`cytochrome CYP3A. Coadministration of drugs that share this elimination pathway may lead to
`pharmacokinetic drug interactions. Efforts are underway by clinical, drug development and
`regulatory scientists to classify CYP3A-related drug interactions with the ultimate goal of
`improving guidance for clinical intervention. The CYP3A inhibitory classification system ranks
`inhibitors according to the fold-increase in area-under-the-curve (AUC) of a probe substrate as:
`strong (55-fold), moderate (>2.0- to 4.9-fold), or weak (42.0-fold). This classification system was
`applied to characterize everolimus as a CYP3A substrate.
`Methods. Five open-label crossover drug interaction studies were performed in 12–16 healthy
`subjects each. Subjects received a single 2 mg dose of everolimus alone and again during single- or
`multiple-dose treatment with the probe inhibitors ketoconazole, erythromycin, verapamil,
`cyclosporine and atorvastatin.
`Results. The fold-increase in everolimus AUC was: 15.0 with the strong inhibitor ketoconazole;
`4.4, 3.5 and 2.7 with the moderate inhibitors erythromycin, verapamil and cyclosporine; and no
`change with the weak inhibitor atorvastatin. Subjects with low baseline AUCs when everolimus was
`given alone tended to have AUC increases of a higher magnitude (more potent interaction) in the
`presence of an inhibitor.
`Conclusions. Strong CYP3A inhibitors should be avoided when possible during everolimus
`treatment as compensatory everolimus dose reductions could be difficult to manage. Everolimus
`therapeutic drug monitoring should be used to guide individualized dose adjustments when
`moderate CYP3A inhibitors are added to or withdrawn from the regimen. Routine everolimus
`therapeutic drug monitoring should be sufficient to determine whether dose adjustments are
`needed when weak CYP3A inhibitors are coadministered. This rational and systematic approach to
`drug interactions on everolimus yielded clinically useful, structured guidelines for dose
`adjustment. Copyright # 2006 John Wiley & Sons, Ltd.
`
`Key words: drug interactions; enzyme inhibition; everolimus
`
`Introduction
`
`A large proportion of drugs used in clinical
`medicine}up to 60% by some estimates}
`utilize the cytochrome P450 (CYP) 3A pathway
`for biotransformation [1]. Consequently,
`the
`
`*Correspondence to: Novartis Pharma AG, Building WSJ 103.426,
`4002 Basel, Switzerland. E-mail: john.kovarik@Novartis.com
`
`potential for pharmacokinetic drug interactions
`when coadministering agents that share this
`pathway is clearly of clinical relevance. It is
`understandable that drug development scientists
`and regulatory authorities share the desire to
`harmonize approaches to assess drug interac-
`tions and to develop a CYP3A classification
`system to communicate risk to health care
`providers and patients. While several regulatory
`
`Copyright # 2006 John Wiley & Sons, Ltd.
`
`Received 14 October 2005
`Revised 10 March 2006
`Accepted 28 June 2006
`
`Ex. 1049-0001
`
`

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`422
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`J.M. KOVARIK ET AL.
`
`agencies worldwide have issued general drug
`interaction guidances for industry [2–4],
`the
`specifics of study design and methodology have
`not been addressed in a comprehensive manner.
`The drug metabolism and clinical pharmacol-
`ogy technical working groups of the Pharmaceu-
`tical Research and Manufacturers of America
`have issued a consensus statement to begin to
`address some of these issues and to define a drug
`interaction data package that can be expected by
`regulatory authorities in the submission dossier
`of a new drug [5].
`In addition to defining
`currently preferred and alternate probe CYP
`substrates to use in clinical drug interaction
`studies,
`they also cite a CYP3A inhibitory
`classification system to allow ranking the magni-
`tude of
`interactions via this pathway. Using
`midazolam as a prototype CYP3A substrate, this
`system classifies the inhibitory potential of a new
`drug as strong if the plasma area-under-the-
`curve (AUC) of midazolam increases 55-fold,
`moderate if the increase is >2.0- to 4.9-fold, and
`weak if the increase is 42.0-fold [5]. This system
`could also be used to characterize a new drug as
`a substrate of CYP3A, placing it in reference to
`preferred probe inhibitors in the three classes.
`With the exception of midazolam, which was
`used to define the system,
`there are to our
`knowledge no examples in the scientific literature
`applying this classification system in a consistent
`manner during the development of a new drug.
`We present our attempt to do so with everolimus,
`a macrolide immunosuppressant, that acts as a
`proliferation signal
`inhibitor on T-lymphocytes.
`
`Everolimus is used to prevent acute rejection
`episodes after kidney and heart transplantation.
`It is primarily biotransformed via CYP3A with
`metabolites eliminated in the bile and is also a
`substrate of P-glycoprotein [6]. A series of clinical
`drug interaction studies were performed during
`the development of everolimus demonstrating that
`it fits into this classification system as a CYP3A
`substrate. The classification system provided a
`useful structure to rank the potential for interac-
`tions from coadministered drugs on everolimus
`and to recommend everolimus dose adjustments.
`
`Methods
`
`Five open-label crossover drug interaction stu-
`dies were performed in healthy subjects using
`the strong CYP3A inhibitor ketoconazole [7]; the
`moderate inhibitors erythromycin [8] verapamil
`[9] and cyclosporine microemulsion [10]; and the
`weak inhibitor atorvastatin [11]. All study proto-
`cols were approved by medical ethics committees
`and all subjects gave written informed consent to
`participate.
`In these studies the reference treatment was a
`2 mg single dose of everolimus (Certican, Novar-
`tis Pharmaceuticals) administered after an over-
`night fast of at least 10 h. The test treatment
`consisted of single-dose everolimus administered
`simultaneously with the inhibitor either under
`single-dose conditions or after steady state was
`reached during multiple-dose administration of
`the inhibitor as itemized in Table 1. Blood
`
`Table 1. Clinical drug interaction studies of CYP3A inhibitors on everolimus
`
`Inhibitor
`class
`
`Number of
`subjects
`
`Test regimen
`
`Strong
`
`Moderate
`
`Moderate
`
`Moderate
`
`Weak
`
`12
`
`16
`
`16
`
`12
`
`12
`
`Ketoconazole 200 mg b.i.d.  8
`days, Everolimus on day 4
`Erythromycin 500 mg t.i.d.  9
`days, Everolimus on day 5
`Verapamil 80 mg t.i.d.  6 days,
`Everolimus on day 2
`Cyclosporine 175 mg single dose,
`Everolimus on day 1
`Atorvastatin 20 mg single dose,
`Everolimus on day 1
`
`Everolimus
`AUC alone
`(mg.h/l)
`
`Everolimus AUC
`with inhibitor
`(mg.h/l)
`
`Fold-increase in
`everolimus
`AUCa
`
`90  23
`
`1324  232
`
`15.0 (13.6–16.6)
`
`116  37
`
`115  45
`
`74  26
`
`120  37
`
`524  225
`
`392  142
`
`193  47
`
`118  46
`
`4.4 (3.5–5.4)
`
`3.5 (3.1–3.9)
`
`2.7 (2.2–3.2)
`
`1.0 (0.8–1.2)
`
`Ref
`
`[7]
`
`[8]
`
`[9]
`
`[10]
`
`[11]
`
`aFold-increase is point estimate and 90% confidence interval.
`AUC, area under the concentration-time curve; b.i.d., twice daily; t.i.d., thrice daily.
`
`Copyright # 2006 John Wiley & Sons, Ltd.
`
`Biopharm. Drug Dispos. 27: 421–426 (2006)
`DOI: 10.1002/bdd
`
`Ex. 1049-0002
`
`

`
`EVEROLIMUS DRUG INTERACTIONS
`
`423
`
`samples were obtained before each everolimus
`dose and then at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24,
`36, 48, 72, 96 and 120 h postdose. Everolimus
`whole blood concentrations were determined by
`a validated liquid chromatography method with
`mass spectrometric detection as previously de-
`scribed [12]. The assay limit of quantification was
`0.3 ng/ml. The area under the concentration-time
`curve extrapolated to infinity (AUC) was derived
`by noncompartmental methods. The AUCs were
`log-transformed and compared between treat-
`ments by conventional bioequivalence testing to
`yield the test/reference ratio of the geometric
`means and 90% confidence interval. The ratio
`served as the estimate of the magnitude of the
`drug interaction on everolimus expressed as the
`fold-increase in everolimus exposure. The inter-
`subject variability in the magnitude of
`the
`interaction was derived as the standard deviation
`divided by the mean of these AUC-ratios (AUC
`with inhibitor/AUC alone). Relationships be-
`tween the reference everolimus AUC and the
`fold-increase in the test AUC were explored by
`graphical evaluation and fitting a descriptive log
`function through the data.
`
`Results
`
`Interaction magnitude
`
`As summarized in Table 1 the population
`average fold-increase in everolimus AUC in the
`presence of strong, moderate and weak CYP3A
`inhibitors fell within the bounds of the classifica-
`tion system [5] in all cases. A related rapamycin
`macrolide, sirolimus, reported similar fold-in-
`creases in sirolimus AUC of 10.9 in the presence
`of ketoconazole, 4.2 in the presence of erythro-
`mycin, 3.3 in the presence of cyclosporine
`microemulsion, 2.2 in the presence of verapamil,
`and no relevant change in the presence of
`atorvastatin [13]. The fold-increases in everoli-
`mus and sirolimus AUC were similar to that of
`the prototype CYP3A substrate, midazolam, in
`the presence of
`these inhibitors. Published
`studies reported mean fold-increases in midazo-
`lam AUC in the presence of multiple-dose
`ketoconazole of 8.7, 11 and 16 [14–16], in the
`presence of multiple-dose erythromycin of 4.4
`
`Copyright # 2006 John Wiley & Sons, Ltd.
`
`and 3.8 [17–18] and in the presence of multiple-
`dose verapamil of 2.9 [19]. The lack of change in
`the AUC of atorvastatin}a recommended and
`sensitive probe substrate of CYP3A}in the
`presence of everolimus [11] indicates that ever-
`olimus does not inhibit CYP3A to a clinically
`relevant extent.
`
`Interaction variability
`
`Figure 1 depicts the individual fold-increases in
`everolimus AUC. The associated intersubject
`variability was generally moderate to high at
`20% in the presence of ketoconazole, 57% for
`erythromycin, 28% for verapamil, 34% for
`cyclosporine microemulsion and 36% for ator-
`vastatin. While all subjects exhibited a strong
`interaction magnitude (55-fold increase) with
`ketoconazole and a weak interaction magnitude
`(52.0-fold) with atorvastatin,
`some subjects
`receiving moderate inhibitors had fold-increases
`which transgressed the categorical boundaries.
`Specifically, for cyclosporine microemulsion 3 of
`12 subjects (25%) had weak interactions between
`1.5- to 1.9-fold. For erythromycin 6 of 16 subjects
`(38%) had a strong interaction ranging from
`5.1- to 12.6-fold and for verapamil 1 of 16 subjects
`(6%) had a strong interaction of 6.3-fold.
`
`Exposure-response associations
`
`The pharmacokinetic data were explored to look
`for predictive or explanatory factors for the
`
`Magnitude: Wk Mod
`
`Strong
`
`Ketoconazole
`
`Erythromycin
`
`Verapanil
`
`Cyclosporine
`
`Atorvastatin
`
`0
`
`2
`
`4
`
`6
`8 10 12 14 16 18 20 22 24
`Fold-increase in everolimus AUC
`
`Figure 1. Fold-increase in everolimus AUC in the presence of
`various CYP3A inhibitors against a categorical background
`for weak inhibition (Wk, 42.0-fold increase), moderate
`inhibition (Mod, 2.0- to 4.9-fold increase) and strong inhibition
`(Strong, 55-fold increase). Shown are the individual fold-
`increases (open circles), the population average (vertical bar),
`and the 90% confidence interval (horizontal bar)
`
`Biopharm. Drug Dispos. 27: 421–426 (2006)
`DOI: 10.1002/bdd
`
`Ex. 1049-0003
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`

`
`J.M. KOVARIK ET AL.
`
`by monitoring predose blood levels along with
`the clinical condition of the patient [6]. Given the
`small influence of weak CYP3A inhibitors on
`everolimus pharmacokinetics,
`it is anticipated
`that routine therapeutic drug monitoring would
`indicate whether an individual patient needs an
`everolimus dose adjustment when a weak
`CYP3A inhibitor is added to or removed from
`the regimen. At the other extreme, the large
`influence of strong CYP3A inhibitors on ever-
`olimus exposure would make compensatory
`dose adjustments difficult to achieve given the
`tablet strengths available. Hence, coadministra-
`tion of strong CYP3A inhibitors with everolimus
`is not recommended.
`Addition or removal of moderate CYP3A
`inhibitors to an everolimus regimen should be
`accompanied by therapeutic drug monitoring to
`guide compensatory everolimus dose adjust-
`ments. The study data shown in Figure 1 indicate
`that most everolimus AUC increases were in the
`range of 2- to 4-fold and this can serve as a
`general
`indicator of what
`to expect when
`coadministering a moderate CYP3A inhibitor.
`However, some increases in everolimus exposure
`may extend into the strong interaction range.
`Figure 2 suggests that patients who may be more
`susceptible to a drug interaction from a CYP3A
`inhibitor appear to be those with low baseline
`everolimus exposure. Such patients would likely
`require a higher everolimus dose than the
`population average dose in order to maintain
`everolimus blood levels in the therapeutic range
`in the absence of CYP3A inhibitors. Clinicians
`treating such patients should be more alert for
`drug interactions when adding a moderate
`CYP3A inhibitor to the regimen until everolimus
`therapeutic drug monitoring results are available
`from the analytical
`laboratory to confirm the
`actual magnitude of the interaction.
`
`Discussion
`
`This overview of the everolimus drug interaction
`program during drug development demonstrates
`that
`the influence of
`five different CYP3A
`inhibitors on everolimus fits into the proposed
`CYP3A inhibitory classification system proposed
`
`Biopharm. Drug Dispos. 27: 421–426 (2006)
`DOI: 10.1002/bdd
`
`Ketoconazole
`
`Erythromycin
`
`0
`
`25
`
`Verapamil
`75 100 125 150 175 200 225 250
`50
`Baseline everolimus AUC (µg.h/L)
`
`424
`
`24
`
`21
`
`18
`
`15
`
`12
`
`0369
`
`Fold-increase in everolimus AUC
`
`Figure 2. Relationship between everolimus AUC in the absence
`of coadministered drugs (baseline AUC) and the fold-increase
`in everolimus AUC in the presence of CYP3A inhibitors
`ketoconazole (filled triangles), erythromycin (open squares)
`and verapamil (filled circles). Shown is the best fit of a
`for ketoconazole, y ¼
`logarithmic trendline to the data:
`9 lnðxÞ þ 55 ðr2 ¼ 0:617Þ; for erythromycin, y ¼ 1:6 lnðxÞ þ
`11:3 ðr2 ¼ 0:423Þ; for verapamil,y ¼ 4:5 lnðxÞ þ 26 ðr2 ¼ 0:275Þ
`
`magnitude of the interaction on everolimus or its
`intersubject variability. The blood or plasma level
`of the inhibitor during the test treatment was in
`no case predictive of
`the magnitude of
`the
`interaction on everolimus [7–11]. The baseline
`everolimus AUC, however, was inversely related
`to the fold-increase in everolimus AUC in the
`presence of the inhibitor as shown in Figure 2 for
`ketoconazole, erythromycin, and verapamil. In
`each case a linear regression did not adequately
`account for the curvature in these relationships;
`hence,
`they were described with logarithmic
`trendlines as specified in the figure legend. In
`general, the lower the baseline everolimus AUC,
`the greater the interaction magnitude in the
`presence of the inhibitor. There was no apparent
`relationship for everolimus with cyclosporine for
`which the scatterplot appeared flat. Since ator-
`vastatin essentially did not change the ever-
`olimus AUC, no relationship was observed for
`this weak inhibitor.
`
`Clinical implications
`
`Everolimus is marketed at dose strengths of 0.25,
`0.5, 0.75 and 1 mg tablets and 0.1 and 0.25 mg
`dispersible tablets [20].
`In organ transplant
`medicine, everolimus dosing is individualized
`
`Copyright # 2006 John Wiley & Sons, Ltd.
`
`Ex. 1049-0004
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`

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`EVEROLIMUS DRUG INTERACTIONS
`
`425
`
`by the Pharmaceutical Research and Manufac-
`turer’s Association [5]. Such a classification
`system begins to address the desire expressed
`by industry sponsors to develop a ranking
`system to allow better assessment and compar-
`ison of different drugs in their drug interaction
`potential and to communicate risk to health care
`providers and patients [5].
`With regard to comparing drug interaction
`potential among drugs, the two macrolide im-
`munosuppressants, everolimus and sirolimus,
`differ in chemical structure and biotransforma-
`tion pathways [21–22] which in theory could
`yield differences in their susceptibility to meta-
`bolic drug interactions. Nonetheless, comparing
`the everolimus
`results presented here with
`published data from sirolimus suggests that they
`share a generally similar risk for CYP3A inhibi-
`tion drug interactions as interpreted against the
`background of this classification system.
`This classification system also serves as a
`framework to communicate drug interaction risk
`to clinicians. The risk of increased drug exposure
`when coadministering strong CYP3A inhibitors
`led to the recommendation to avoid these come-
`dications in the product labels of both everolimus
`[20] and sirolimus [13]. Coadministration of
`moderate CYP3A inhibitors, however, appears to
`be associated with a generally lower magnitude in
`the fold-increase of everolimus AUC; however, the
`healthy subject data indicate that some individuals
`are more susceptible to CYP3A inhibition interac-
`tions resulting in larger increases in exposure. The
`data further suggest that these individuals tend to
`have lower everolimus exposure in the absence of
`comedications as shown in Figure 2. This pattern
`has been observed in other drug interaction
`studies with CYP3A substrates [23–25].
`It
`is
`speculated that individuals with higher expression
`of CYP3A (manifested by lower exposure when
`the drug is given alone) have more enzyme to
`inhibit and thereby elicit a stronger drug interac-
`tion in the presence of a CYP3A inhibitor [24–26].
`In other words, intersubject variability in enzyme
`levels is likely a strong contributor to variability in
`the magnitude of drug interactions. In light of this
`underlying variability, moderate CYP3A inhibitors
`may generally be used with everolimus provided
`that
`it
`is accompanied by therapeutic drug
`monitoring to quantify the increase in everolimus
`
`Copyright # 2006 John Wiley & Sons, Ltd.
`
`exposure after adding the moderate inhibitor and
`to individually adjust
`the everolimus dose to
`compensate for the interaction [20].
`the CYP3A
`An additional application of
`inhibitor classification system in risk manage-
`ment is as a qualitative guide to drug interactions
`not specifically tested by the sponsor. This is
`explicitly recognized in the European Agency for
`the Evaluation of Medicinal Products drug
`interaction guidance which states:
`‘It seems
`in vivo studies with strong
`reasonable that
`inducers/inhibitors may be used to extrapolate
`qualitatively to other inducers/inhibitors of the
`same enzyme’ [4]. In the case of everolimus, the
`summary of product characteristics extends the
`recommendation to avoid strong CYP3A inhibi-
`tors based on ketoconazole data to include
`itraconazole, voriconazole, clarithromycin and
`ritonivir [20]. Similar qualitative extensions are
`made for moderate CYP3A inhibitors [20].
`While the CYP3A inhibitor classification sys-
`tem has many desirable traits, some investigators
`cite points for caution as well [27]. For example,
`when using the system to classify a new drug as
`an inhibitor with midazolam as the probe
`substrate, the data might not be extrapolatable
`to other substrates. The classification results may
`be study design-specific if the magnitude of the
`interaction depends on the drug doses studied,
`administration under single-dose or multiple-
`dose conditions, or relative timing of coadminis-
`tration (simultaneously versus delayed). Further-
`more,
`the potency of an individual drug
`interaction may be understated if multiple ‘mild’
`or ‘moderate’ inhibitors are coadministered in a
`patient’s drug regimen leading to a stronger
`combined interaction or if the victim drug is a
`substrate of multiple CYP enzymes or transpor-
`ters inhibited by the perpetrator drug.
`
`Conclusions
`
`Mindful of the caveats mentioned above, drug
`development scientists, regulatory reviewers and
`clinicians can use this CYP3A inhibitory classifi-
`cation system as a tool to design drug interaction
`programs,
`to manage regulatory risk and to
`guide pharmacotherapy in the patient care
`setting. The example of everolimus demonstrates
`
`Biopharm. Drug Dispos. 27: 421–426 (2006)
`DOI: 10.1002/bdd
`
`Ex. 1049-0005
`
`

`
`426
`
`J.M. KOVARIK ET AL.
`
`that applying a rational and systematic approach
`to drug interactions on everolimus yielded
`clinically useful, structured guidelines for dose
`adjustment.
`
`Acknowledgements
`
`The clinical studies described herein were spon-
`sored by Novartis Pharmaceuticals.
`
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