DRUG INTERACTIONS/DISPOSITION

J Clin Pharmacol 2008;48:1323-1338 1323

This study investigated the potential pharmacokineticinteraction between the direct renin inhibitor aliskirenand modulators of P-glycoprotein and cytochrome P4503A4 (CYP3A4). Aliskiren stimulated in vitro P-glycopro-tein ATPase activity in recombinant baculovirus-infected Sf9 cells with high affinity (Km 2.1 μmol/L) andwas transported by organic anion-transporting peptideOATP2B1-expressing HEK293 cells with moderate affin-ity (Km 72 μmol/L). Three open-label, multiple-dosestudies in healthy subjects investigated the pharmaco-kinetic interactions between aliskiren 300 mg and digoxin0.25 mg (n = 22), atorvastatin 80 mg (n = 21), or keto-conazole 200 mg bid (n = 21). Coadministration withaliskiren resulted in changes of <30% in AUCτ and Cmax,ss

of digoxin, atorvastatin, o-hydroxy-atorvastatin, and ρ-hydroxy-atorvastatin, indicating no clinically signifi-cant interaction with P-glycoprotein or CYP3A4substrates. Aliskiren AUCτ was significantly increased bycoadministration with atorvastatin (by 47%, P < .001) or

ketoconazole (by 76%, P < .001) through mechanismsmost likely involving transporters such as P-glycopro-tein and organic anion-transporting peptide and possi-bly through metabolic pathways such as CYP3A4 in thegut wall. These results indicate that aliskiren is a sub-strate for but not an inhibitor of P-glycoprotein. On thebasis of the small changes in exposure to digoxin andatorvastatin and the <2-fold increase in exposure toaliskiren during coadministration with atorvastatinand ketoconazole, the authors conclude that the poten-tial for clinically relevant drug interactions betweenaliskiren and these substrates and/or inhibitors of P-glycoprotein/CPY3A4/OATP is low.

Keywords: Cytochrome P450; direct renin inhibitor; druginteraction; P-glycoprotein

Journal of Clinical Pharmacology, 2008;48:1323-1338© 2008 the American College of Clinical Pharmacology

Pharmacokinetics of the Oral DirectRenin Inhibitor Aliskiren in Combination With

Digoxin, Atorvastatin, and Ketoconazole inHealthy Subjects: The Role of P-Glycoprotein

in the Disposition of Aliskiren

Sujata Vaidyanathan, PhD, Gian Camenisch, PhD, Helmut Schuetz, PhD,Christine Reynolds, Ching-Ming Yeh, PhD, Marie-Noelle Bizot, PhD,

Hans Armin Dieterich, MD, Dan Howard, PhD, and William P. Dole, MD

P-glycoprotein (multidrug-resistant protein 1, MDR1)is an efflux transporter protein that is a member

of the adenosine triphosphate (ATP)–dependent bind-ing cassette (ABC) superfamily of transmembraneproteins. P-glycoprotein is expressed in the smallintestine, kidney, liver, and blood-brain barrier andinfluences intestinal absorption, renal secretion, andbiliary excretion of many drug substrates, includingdigoxin, verapamil, and some HMG-CoA reductaseinhibitors (statins).1 Clinically significant interactions

From Novartis Pharmaceuticals Corporation, East Hanover, New Jersey(Dr Vaidyanathan, Ms Reynolds, Dr Yeh, Dr Howard); Novartis PharmaAG, Basel, Switzerland (Dr Camenisch, Dr Schuetz, Dr Dieterich); NovartisPharma SAS, Rueil-Malmaison, France (Dr Bizot); and Novartis Institutesfor Biomedical Research, Cambridge, Massachusetts (Dr Dole). Submittedfor publication October 25, 2007; revised version accepted July 6, 2008.Address for correspondence: William P. Dole, MD, Novartis Institutes forBiomedical Research, 400 Technology Square, Building 605-820, Cambridge,MA 02139; e-mail: bill.dole@novartis.com.DOI:10.1177/0091270008323258

1324 • J Clin Pharmacol 2008;48:1323-1338

in the gastrointestinal tract and kidney have beenobserved between P-glycoprotein inhibitors anddrugs that are substrates for P-glycoprotein, althoughthe clinical significance of effects on absorptionremains uncertain. Well-characterized examplesinclude the interaction of azole antifungal agents(eg, ketoconazole and itraconazole) with P-glycopro-tein substrates such as digoxin and statins.2-4 Activehepatic uptake is a key process in hepatic elimina-tion of drugs, and several members of the organicanion-transporting peptide (OATP) family arelocated on the liver sinusoidal membranes inhumans,4 have broad substrate specificity, and playa key role in hepatobiliary excretion of drugs.OATP1B1 and OATP1B3 are expressed exclusivelyin the liver, whereas OATP2B1 is more widely dis-tributed in tissues.4 Many drugs that are substratesor inhibitors of P-glycoprotein are also substrates orinhibitors of cytochrome P450 isoenzyme 3A4(CYP3A4),5 and so determination of the potential forinteraction of new drugs with both P-glycoproteinand CYP3A4 is of considerable clinical importance.

Aliskiren is the first in a new class of orallyeffective direct renin inhibitors to be approved for thetreatment of hypertension at once-daily doses of 150mg and 300 mg. New antihypertensive treatmentoptions are clearly required, as rates of blood pressure(BP) control among patients with hypertension remainlow.6 Direct inhibition of renin is an attractiveapproach to antihypertensive therapy because chronicactivation of the renin system is associated withhypertension and associated target organ damage.7

Aliskiren is a potent inhibitor of human renin in vitro(IC50 = 0.6 nM),8,9 and clinical studies have shown thatonce-daily treatment with aliskiren provides similarantihypertensive effects to the angiotensin receptorblockers (ARBs), losartan and irbesartan,10,11 and supe-rior antihypertensive effects to the angiotensin-converting enzyme (ACE) inhibitor ramipril.12 Aliskirenis rapidly absorbed following oral administration inhealthy subjects, and maximum plasma concentra-tions are reached within 1 to 3 hours after administra-tion. Aliskiren exhibits a mean elimination half-life ofabout 40 hours (34-41 hours),13-15 and following once-daily dosing with aliskiren, steady state is reachedafter 7 to 8 days. The absolute bioavailability of a single75-mg oral dose of aliskiren (relative to an intravenousdose) determined in 9 humans was 2.6%.14

Aliskiren is unlikely to affect the pharmacokinet-ics of coadministered drugs through interactions atthe level of CYP450 isoenzymes, as studies on the invitro activity of specific CYP450 isoenzymes inhuman liver microsomes have shown no significant

inhibition by aliskiren.16 Indeed, coadministrationof aliskiren had no effect on the pharmacokinetics ofthe CYP3A4 substrates lovastatin and amlodipine inhealthy volunteers.17,18 In vitro studies indicate thataliskiren is a substrate for CYP3A4.14 Incubation ofaliskiren with a series of 22 recombinant humanCYP isoenzymes in human liver microsomesshowed that CYP3A4 is the major isoenzymeresponsible for the oxidative metabolism of aliskirenwith Km 24.3 ± 2.2 μmol/L (data on file); ketocona-zole almost completely inhibited metabolism ofaliskiren in human liver microsomes. However,apparent intrinsic hepatic clearance of the totalmetabolite formulation of aliskiren was low (41.3μL/mg/min). Consistent with the in vitro findings,elimination of aliskiren in vivo predominantlyinvolves biliary/fecal excretion of the drug in theunchanged form. Thus, elimination of either an oralor intravenous dose was predominantly biliary/fecalin rats (up to 90%) and marmosets (up to 78%). Inhealthy humans, 81% of a 300-mg oral dose ofaliskiren was eliminated unchanged in the feces,19

whereas the renal excretion of unchanged aliskirenin humans, after an intravenous dose of 20 mg, was7.5% of the dose20; oxidized metabolites in excretaaccounted for approximately 1.4% of an adminis-tered radioactive dose.19

Inhibitors of P-glycoprotein may modify the phar-macokinetics of aliskiren, as in vitro and in vivo ani-mal studies indicate that P-glycoprotein may play amajor role in the absorption/elimination of aliskiren.Thus: (1) binding studies in the human colonic ade-nocarcinoma cell line Caco-2, an established in vitromodel system of intestinal transport21 (predomi-nantly P-glycoprotein22), showed that aliskiren is ahigh-affinity (Km 3 μmol/L), moderate-capacity (Jmax29 × 10–5 nmol/min⋅cm2) substrate for P-glycopro-tein-mediated transport, which was inhibited byPSC833 (an inhibitor of MDR123); (2) studies in bileduct-cannulated rats demonstrated biliary clearanceof intravenously administered 14C-aliskiren that wasinhibited by intravenous PSC833 but not by intra-venous probenecid (an inhibitor of MRP24,24); (3)aliskiren Cmax and AUC following oral administra-tion to rats were significantly increased by coadmin-istration with PSC833, possibly because of an effect ofPSC833 on the uptake of aliskiren into hepatocytes(T. Faller, personal communication, [2004]); and (4)studies with OATP1B1-transfected Chinese hamsterovary (CHO) cells and OATP1B3-transfected Madin-Darby canine kidney (MDCK) cells showed thataliskiren is not transported by these hepatic trans-porters (data on file).

VAIDYANATHAN ET AL

The aim of the present study was to investigatethe potential for drug interaction between aliskirenand substrates/inhibitors of P-glycoprotein. First, anin vitro study was conducted to assess whetheraliskiren is a substrate of P-glycoprotein, using anestablished assay for the activatory effects of drugson ABC transporter ATPase activity. In addition,uptake of aliskiren by the hepatic transporterOATP2B1 was investigated in vitro. Second, 3 sepa-rate clinical studies in healthy subjects assessed thepharmacokinetics and safety of aliskiren alone or incombination with the cardiac glycoside digoxin(which is a substrate for, but does not inhibit, P-glycoprotein), the HMG-CoA reductase inhibitoratorvastatin (which is a substrate and inhibitor ofP-glycoprotein, and also a substrate for other trans-porters such as OATP1B), and the antifungal agentketoconazole (which inhibits P-glycoprotein but isnot transported by it and inhibits CYP3A4).25-27

METHODS

Preclinical Studies

ATPase Activity of ABC Transporters inMembrane Vesicle Preparations

ATPase activity of human MDR1, the humanmultidrug-resistant associated protein 2 (MRP2),and the human breast cancer resistant protein (MXR)was determined in membrane vesicles purified fromrecombinant baculovirus-infected Sf9 cells (forMDR1 and MRP2, SOLVO Biotechnology, Budapest,Hungary) and from a selected mammalian cell line(for MXR, SOLVO Biotechnology), as reported previ-ously.28,29 Membrane vesicles were maintained at–80°C before use in ATPase experiments.

Transporter-mediated efflux was determined asdescribed previously.28,29 Briefly, for activation studiesmembrane suspension in the presence and absenceof aliskiren 0.04, 0.13, 0.40, 1.21, 3.63, 10.89, 32.67,and 98.00 μmol/L, and Na3VO4 60 mmol/L (withglutathione [GSH] 2 mmol/L added for MRP2 exper-iments) was pipetted into a 96-well plate and pre-incubated for 5 minutes at 37°C.

The ATPase reaction was started by addition of 25mmol/L Mg-ATP solution, followed by incubation at37°C (20 minutes for MDR1 and MXR, 30 minutesfor MRP2), and stopped by adding sodium dodecylsulfate (SDS; 5%). Ammonium molybdate/zinc acetatecolorimetric detection reagent was then added, fol-lowed by incubation for a further 25 minutes at37°C. All studies were performed in triplicate.

Optical density was measured at 730 nm, and theamount of free phosphate (Pi) liberated (nmol/well)was calculated using a previously determined phos-phate standard curve. Na3VO4-sensitive ATPaseactivity was calculated as the amount of Pi liberatedper milligram membrane protein per minute, sub-tracting blank values obtained by performing theassay in the absence of Na3VO4.

Verapamil, probenecid, and sulfasalazine wereobtained from Sigma (St. Louis, Missouri); aliskirenwas provided by Novartis (Basel, Switzerland).A 5-mmol/L stock solution in dimethyl sulfoxide(DMSO) was prepared for each compound; additionof the stock solution or serial dilutions to the assaymix produced a final DMSO concentration of 2% ofthe total volume (v/v). All solutions were main-tained at pH 7.4. All other reagents were of analyti-cal grade.

Kinetics of ATPase Activity

Specific ATPase activity for an active transportprocess exhibiting Michaelis-Menten kinetics wascalculated using the following equation30,31:

ActATP = ActATP, base +ActATP, max . C0 / Km,

1 + C0 / Km

where ActATP (nmol Pi/mg/min) is the transporter-dependent ATP consumption, ActATP,base (nmolPi/mg/min) is the baseline transporter-dependentATP consumption, ActATP,max (nmol Pi/mg/min) isthe maximal transporter-dependent ATP consump-tion, and Km (μM) is the Michaelis-Menten con-stant for the substrate, which reflects the intrinsicaffinity for the transporter system involved in thetransport process. All statistical analyses were per-formed using Microsoft Excel (Microsoft, Redmond,Washington).

Assessment of the Involvement of OATP2B1 inthe Hepatic Uptake of Aliskiren

OATP2B1-expressing human embryonic kidneyHEK293 cells, (Prof Y. Sugiyama, Graduate School ofPharmaceutical Sciences, University of Tokyo, Tokyo,Japan) were cultured for 3 days in a culture mediumcontaining Dulbecco’s modified Eagle’s medium(DMEM; Gibco-BRL, Carlsbad, California) low glu-cose (1 g/L) supplemented with 10% fetal bovine serum(FBS; Sigma-Aldrich) and 1% penicillin/streptomycinat 37°C with 5% CO2. Cells were seeded into 12-wellplates at a density of 1.5 × 105 cells per well with1 mL of culture medium for 48 hours, which was then

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DRUG INTERACTIONS/DISPOSITION 1325

1326 • J Clin Pharmacol 2008;48:1323-1338

replaced with culture medium supplemented with 5mmol/L sodium-butyrate and incubated for 24 hoursto induce the expression of the OATP2B1 trans-porter system. After washing and preincubating thecells with culture medium, uptake studies were ini-tiated at 37°C by adding Krebs-Henseleit buffer con-taining [14C]aliskiren at concentrations between 0.03and 500 μmol/L.19 After incubation for 4 minutes,the incubation buffer was removed and replaced byice-cold buffer. The cells were washed 3 times with1 mL of ice-cold Krebs-Henseleit buffer, solubilizedin 500 μL of 0.2 mol/L sodium hydroxide, and incu-bated overnight at room temperature. Aliquots (500 μL)were transferred into scintillation vials, and 250 μLof 0.4 mol/L hydrochloric acid was added. Countingwas performed using a Tri-Carb 2500 TR (CanberraPackard, Meriden, Connecticut). All studies wereperformed in triplicate.

Drug uptake (µL/min/mg protein) was determinedfrom the specific activity of the samples and the con-trols in the incubation medium, the radioactivity inthe cells after incubation, and the protein content ofthe cells in the test wells. The binding constant Kmwas determined by linear regression from the slopeand intercept of an Eadie-Scatchard plot.

Clinical Studies

All subjects provided written informed consent priorto study participation. The study protocols wereapproved by the Independent Investigational ReviewBoard (Plantation, Florida), and all 3 studies wereperformed in compliance with good clinical practiceand adhered to the principles of the Declaration ofHelsinki of the World Medical Association.

Aliskiren/Digoxin Study

Subjects underwent a baseline evaluation on day –1,followed by 2 open-label treatment periods. Inperiod 1 (days 1-7), subjects received once-dailytreatment with aliskiren 300 mg for 7 days, fol-lowed by a washout period of 10 days (Figure 1a). Inperiod 2, subjects received once-daily treatmentwith digoxin 0.25 mg for 9 days (days 18-26), fol-lowed by coadministration of aliskiren 300 mg oncedaily for 7 days (days 27-33). Subjects were confinedto the clinic on days 1 to 7 and days 17 to 33.

Aliskiren/Atorvastatin Study

After a screening period of up to 21 days, subjectsunderwent a baseline evaluation on day –1, followed

VAIDYANATHAN ET AL

(a)

(b)

(c)

Aliskiren/digoxin study

Aliskiren/atorvastatin study

Aliskiren/ketoconazole study1101987654321yaD

Treatment Aliskiren 300 mg Aliskiren 300 mg + ketoconazole 200 mg twice dailyAliskiren drug levelsKetoconazole drug levels

Time after dosing (h) 0 0.25 0.5 1 1.5 2 3 4 6 8 10 12 16 24Aliskiren determination

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33Treatment tuohsaWgm 003 neriksilA Aliskiren 300 mg + digoxin 0.25 mgDigoxin 0.25 mgAliskiren drug levelsDigoxin drug levels

Time after dosing (h) 0 0.25 0.5 1 1.5 2 3 4 6 8 10 12 16 24Aliskiren determinationDigoxin determination

Time after dosing (h) 0 0.25 0.5 1 1.5 2 3 4 6 8 10 12 16 24Atorvastatin determinationAliskiren determination

91817161514131211101987654321DayTreatment Atorvastatin 80 mg Atorvastatin 80 mg + aliskiren 300 mgAliskiren 300 mgWashoutAtorvastatin drug levelsο-hydroxy-atorvastatin levelsρ-hydroxy-atorvastatin levelsAliskiren drug levels

Figure 1. Study design and blood sampling schedules for studies investigating the interaction of aliskiren with (a) digoxin, (b) atorvas-tatin, and (c) ketoconazole. Filled circles indicate predose blood sample only; triangles indicate blood sampling throughout the24-hour postdose interval according to the schedules shown.

by 2 open-label treatment periods. In period 1 (days1-4), subjects received once-daily treatment with ator-vastatin 80 mg for 4 days, followed by a washoutperiod of 4 days (Figure 1b). In period 2, subjectsreceived once-daily treatment with aliskiren 300mg for 7 days (days 9-15), followed by coadministra-tion of atorvastatin 80 mg once daily for 4 days(days 16-19). Subjects were confined to the clinic ondays 1 to 4 and days 9 to 20.

Aliskiren/Ketoconazole Study

After a screening period of up to 21 days, subjectsunderwent a baseline evaluation on day –1, fol-lowed by a single treatment period (Figure 1c).Subjects received once-daily treatment with aliskiren300 mg for 7 days (days 1-7), followed by coadmin-istration of aliskiren with ketoconazole 200 mgtwice daily for 4 days (days 8-11). On the days whenpharmacokinetic assessments were performed (day7 and day 11), subjects continued to fast for 4 hoursafter drug administration. Subjects were confined tothe clinic throughout the study (12 days).

Study Objectives and Dose Selection

The primary objective of these studies was toinvestigate the effect of coadministration of aliskirenwith digoxin, atorvastatin, or ketoconazole on thepharmacokinetics of these drugs in healthy subjects.A secondary objective was to assess the safety andtolerability of coadministration of aliskiren withdigoxin, atorvastatin, and ketoconazole. The 300-mgdose of aliskiren used in the present study was cho-sen because this is the highest clinical dose antici-pated for use in the treatment of hypertension. The0.25-mg daily dose of digoxin is the standard start-ing dose of the drug for patients younger than 70years of age with normal renal function,32 whereasatorvastatin was used at the highest licensed dosefor the treatment of hypercholesterolemia.33 The200-mg twice-daily dose of ketoconazole is the high-est recommended daily dose for treating fungalinfections in clinical practice and is the dosage com-monly used in drug interaction studies involvingP-glycoprotein inhibition.34 Aliskiren, digoxin, ator-vastatin, and the morning dose of ketoconazole wereadministered orally in tablet form with 240 mL ofwater in the morning, after an overnight fast of atleast 10 hours. The evening dose of ketoconazole200 mg was administered alone after a 12-hourinterval, and no fluid intake apart from that given fordrug administration was allowed from 1 hourbefore until 1 hour after dosing.

Clinical Study Populations

Inclusion criteria for all 3 clinical studies specifiedmale or female subjects ages 18 to 45 years and ingood health, as determined from medical history,physical examination, vital signs (supine systolic BP90-140 mm Hg, diastolic BP 50-90 mm Hg andpulse rate 40-90 bpm with no evidence of posturalhypotension), electrocardiogram (ECG), and labora-tory tests. Subjects had a body weight of at least 50kg and within –20% to +20% of normal for theirheight and frame size according to the MetropolitanLife Insurance tables.

Exclusion criteria for participants in all 3 clinicalstudies included: smoking (use of tobacco productin the previous 3 months); clinically significant ECGabnormalities or abnormal laboratory values; ahistory of autonomic dysfunction, renovascular dis-ease, or acute or chronic bronchospastic disease;clinically significant drug allergy, atopic allergy,known hypersensitivity to drugs similar to the studydrugs or immunocompromise, and any conditionthat might significantly alter the absorption, distrib-ution, metabolism, or excretion of study drugs.Subjects were excluded if they had used any pre-scription or over-the-counter medication (exceptacetaminophen) during the 2 weeks prior to dosing.

Subjects were not permitted to engage in strenu-ous physical exercise for 7 days before dosing untilafter the study completion evaluation or to take alco-hol for 72 hours before dosing until after the studycompletion evaluation. Intake of xanthine-containingfood or beverages was discontinued 48 hours beforedosing and was not permitted while subjects wereadmitted. In the aliskiren/digoxin study, intake offruit juices was prohibited during the study.

Pharmacokinetic Assessments

In the aliskiren/digoxin and aliskiren/atorvastatinstudies, blood samples for analysis of drug concen-trations (aliskiren, atorvastatin, and atorvastatinmetabolites o-hydroxy-atorvastatin and ρ-hydroxy-atorvastatin) were collected predose on the final 3days of each treatment period and at frequent inter-vals for up to 24 hours postdose on the final day ofeach treatment period. Samples for analysis ofdigoxin trough concentrations were taken for the last7 days of treatment with digoxin alone and the last 6days of coadministration of aliskiren and digoxin;digoxin was discontinued if the trough digoxin con-centration was >2.0 ng/mL. In the aliskiren/keto-conazole study, blood samples for analysis of aliskirenconcentrations were taken over 24 hours postdose

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DRUG INTERACTIONS/DISPOSITION 1327

1328 • J Clin Pharmacol 2008;48:1323-1338

(0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, and 24 hours) onthe last day of treatment with aliskiren alone orcoadministered with ketoconazole. Treatment peri-ods and blood sampling schedules for the final dayof each treatment period in the 3 studies are shownin Figure 1a-c. Inspection of mean trough plasmaconcentrations of aliskiren, digoxin, and atorvas-tatin indicated that steady-state conditions had beenreached at the time when pharmacokinetic para-meters were assessed (data not shown).

All blood samples were collected by directvenipuncture or by an indwelling cannula insertedin a forearm vein. Samples were either collected intoa lithium heparinized tube (for aliskiren, digoxin, oratorvastatin analysis) or EDTA tube (for ketocona-zole trough concentration determination) and frozenat –20°C until analysis was performed.

Plasma concentrations of aliskiren were deter-mined by a liquid chromatography/tandem massspectrometry (LC/MS/MS) method. The assay con-sisted of a solid-phase extraction on Oasis MCX car-tridges using an automated system followed byreversed-phase high-performance liquid chromatog-raphy (HPLC) on a Metachem MetaSil basic columnusing gradient elution with 10 mM aqueous ammo-nium acetate/acetonitrile. Detection was performedin MS/MS using electrospray ionization (ESI). Themasses for aliskiren were precursor ion m/z 552 andproduct ion m/z 436. A derivative of aliskiren (gem-dimethyl d6-aliskiren) was used as an internal stan-dard. Within-study assay validation across the 3studies showed an assay precision (coefficient ofvariation [CV]) of 2.9% to 10.7% (aliskiren/digoxinstudy), 6.5% to 21.7% (aliskiren/atorvastatin study),and 3.2% to 6.2% (aliskiren/ketoconazole study);across the 3 studies, bias was –1.6% to 11.0%. Thelower limit of quantification for this assay wasapproximately 0.5 ng/mL; samples with concentra-tions below this level were assumed to be 0 ng/mL.

Plasma concentrations of digoxin were deter-mined by solid-phase extraction on an Oasis HLB30-mg 96-well plate followed by reverse-phase liq-uid chromatography (isocratic elution with 5 mMammonium acetate/acetonitrile 60:40 v/v) with tan-dem mass spectrometric detection. The masses fordigoxin were precursor ion m/z 798.6 and production m/z 651.5. A derivative of digoxin (digoxin-d3)was used as an internal standard. Within-studyassay validation showed an assay precision (CV) of4.6% to 8.4% and a bias of –8.3% to 0.6%. Thelower limit of quantification was 0.2 ng/mL.

Plasma concentrations of atorvastatin and its act-ive metabolites o-hydroxy-atorvastatin and ρ-hydroxy-atorvastatin were determined by liquid phase extraction

followed by liquid chromatography (gradient elutionwith methanol-water 70:30, 1 mM ammonium formate[pH 3.00] followed by methanol followed by methanol-water-ammonium formate) with tandem mass spectro-metric detection in turbo ion spray (TIS) positive ionmode. The masses for atorvastatin were precursor ionm/z 559.2, product ion m/z 440.2; for o-hydroxy-atorvastatin, were precursor ion m/z 575.3, productionm/z 466.2; and for ρ-hydroxy-atorvastatin, were precur-sor ion m/z 575.3, product ion m/z 440.2. The d5derivatives of atorvastatin, o-hydroxy-atorvastatin, andρ-hydroxy-atorvastatin were used as internal standards.Within-study assay validation showed assay precisions(CV) of 0.7% to 4.3% (atorvastatin), 1.0% to 5.6% (o-hydroxy-atorvastatin), and 1.2% to 7.9% (ρ-hydroxy-atorvastatin), and assay bias of –7.5% to –5.6% (atorvas-tatin), –7.2% to –5.5% (o-hydroxy-atorvastatin), and–9.5% to 5.5% (ρ-hydroxy-atorvastatin). The lowerlimit of quantification for atorvastatin and the 2 activemetabolites was 0.25 ng/mL.

Plasma concentrations of ketoconazole weredetermined by reverse-phase HPLC. Briefly, thisconsisted of liquid/liquid extraction (tert-butylmethylether, pH 12) followed by HPLC at 40°C onan Inertsil column using isocratic elution with ace-tonitrile/water/diethylamine (500:500:0.5, v/v/v)with pH adjusted to 8 with 85% (v/v) orthophos-phoric acid, and ultraviolet (UV) detection at 254nm with clotrimazole as internal standard. Within-study assay validation showed an assay precision(CV) of 2.7% to 6.2% and a bias of –3.3% to 7.5%.The lower limit of quantification was 10 ng/mL.

Safety and Tolerability Assessments

Safety and tolerability assessments included the reg-ular monitoring and recording of all adverse events(AEs), and concomitant medications or significantnondrug therapies. Evaluations of routine bloodchemistry, blood counts with white cell differentialand urine analyses, as well as a physical examina-tion, ECGs, and monitoring of vital signs were per-formed at regular intervals in each study.

Statistical Analyses

Pharmacokinetic parameters (AUCτ, Cmax,ss, Cmin,ss,tmax,ss, and CL/F) were determined at steady state bynoncompartmental methods using WinNonlinEnterprise (Version 4.1, Pharsight Corp, MountainView, California). For sample size estimation, theintrasubject CV for aliskiren AUC and Cmax wasassumed to be between 0.50 and 0.70. In all 3 stud-ies, a sample size of 18 completed subjects (assum-ing a CV of 0.60) therefore provided 80% power thatthe 90% confidence interval (CI) for ratios of

VAIDYANATHAN ET AL

aliskiren pharmacokinetic parameters (for coadmin-istration compared with aliskiren alone) would havean upper CI within 80% and a lower CI within 44%of the true mean ratio.

Log-transformed pharmacokinetic parametersAUCτ and Cmax,ss were compared between treatmentsby a linear mixed effect model, with treatment as afixed factor and subject as a random factor. Pointestimate and corresponding 90% CI for the ratio ofgeometric means (coadministration vs administra-tion alone) were used to assess drug-drug interac-tions. The P values, ratio of geometric means, and90% CI for the ratio of geometric means were deter-mined from an analysis of variance (ANOVA) modelusing the log-transformed values with subject andtreatment as factors (subject was treated as a randomvariable and treatment as a fixed variable). A 90% CIcontained fully within the bioequivalence intervalfrom 0.80 to 1.25 was taken as evidence of a lack ofclinically significant drug interaction.

RESULTS

In Vitro Studies

In vitro measurements of Na3VO4-sensitive ATPaseactivity in membrane vesicles showed that aliskirenproduced complete stimulation of P-glycoprotein(MDR1) ATPase activity, equivalent to that observedwith the stimulating agent verapamil. The effect ofaliskiren was concentration-dependent and reachedits maximum between 1 and 10 μmol/L (Figure 2a).The stimulatory effect of aliskiren on MDR1 activitywas fitted to a Michaelis-Menten equation with a Kmof 2.1 ± 0.5 μmol/L (mean ± SEM), indicating rela-tively high affinity of aliskiren for MDR1. In theinhibition assay (performed in the presence of vera-pamil 40 μmol/L), aliskiren had no inhibitory effecton MDR1 ATPase activity (data on file). Aliskirendid not stimulate ATPase activity of either MXR orMRP2 (Figure 2b,c).

Incubation of aliskiren in vitro with OATP2B1-expressing HEK293 cells indicated the involvementof this solute carrier in human hepatic uptake. Thelinear concentration plot of aliskiren uptake showednonlinear saturation kinetics (Figure 2d) and an esti-mated Km of 72 μmol/L (by Eadie-Scatchard transfor-mation). Therefore, aliskiren has moderate affinityfor the human hepatic uptake transporter systemOATP2B1. Inhibition experiments indicated thataliskiren caused only moderate inhibition ofOATP2B1 at high concentrations, exhibiting an IC50of 95 μmol/L (data on file).

Clinical Studies

Patient Characteristics

A total of 22 subjects were enrolled in thealiskiren/digoxin study; 19 completed the study.One discontinuation was due to an AE (clinicallysignificant ECG abnormality observed with digoxinadministration), 1 subject was discontinued on day6 due to noncompliance with clinic rules, and 1subject withdrew consent (non-AE-related reasons).Of 21 subjects enrolled in the aliskiren/atorvastatinstudy, 20 completed the study; the 1 discontinuationwas due to a protocol violation (elevated serum coti-nine levels found at the baseline visit). Of 21subjects enrolled in the aliskiren/ketoconazolestudy, 20 completed the study; the 1 discontinuationwas due to administrative reasons.

A higher proportion of male subjects were enrolledin the aliskiren/digoxin study compared with thealiskiren/atorvastatin and aliskiren/ketoconazole stud-ies, and this is reflected in the higher mean baselineheight and weight observed (Table I). The predomi-nant race was black in the aliskiren/digoxin studyand Caucasian in the other 2 studies.

Pharmacokinetic Analyses

Aliskiren/digoxin study. Coadministration ofaliskiren with digoxin had no significant effect onaliskiren AUCτ (ratio of geometric means, 1.02 [90%CI: 0.93, 1.13]; P = .706) or Cmax,ss (0.98 [90% CI: 0.80,1.19]; P = .830; Figure 3) or on median tmax,ss.Aliskiren CL/F was also unchanged by coadminis-tration with digoxin (Table II).

Coadministration of aliskiren with digoxindecreased digoxin AUCτ by 15% (ratio of geometricmeans, 0.85 [90% CI: 0.75, 0.97]; P = .049) but had noeffect on digoxin Cmax,ss (geometric mean ratio 0.91[90% CI: 0.84, 0.99]; P = .081; Figure 4) or mediantmax,ss (Table III).

Aliskiren/atorvastatin study. Coadministration ofaliskiren with atorvastatin significantly increasedaliskiren AUCτ by 47% (ratio of geometric means,1.47 [90% CI: 1.29, 1.67]; P < .001) and Cmax,ss by50% (geometric mean ratio 1.50 [90% CI: 1.22,1.85]; P = .003; Figure 3). Although there was nochange in median tmax,ss (Table II), the mean valuefor tmax,ss tended to be smaller during coadministra-tion of aliskiren with atorvastatin (2.16 ± 1.25hours) compared with aliskiren alone (2.43 ± 1.66hours; P = NS). The arithmetic mean AUC in thefirst 2 hours after administration (AUC0-2 h) ofaliskiren was 62% higher when coadministered

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1330 • J Clin Pharmacol 2008;48:1323-1338

with atorvastatin (578 ± 259 vs 356 ± 170 ng⋅h/mLfor aliskiren alone). Coadministration of aliskirenwith atorvastatin decreased aliskiren CL/F by 34%(arithmetic mean ratio; Table II), but the semi-logarithmic plasma concentration-time plots showedno obvious change in the terminal elimination ofaliskiren when coadministered with atorvastatin(Figure 3b, inset).

Coadministration of aliskiren with atorvastatinreduced atorvastatin Cmax,ss by 23% (ratio of geomet-ric means, 0.77 [90% CI: 0.67, 0.88]; P = .003; Figure4, Table III), with no effect on atorvastatin AUCτ(geometric mean ratio 0.91 [90% CI: 0.84, 1.00];P = .093) or median tmax,ss (Table III). AUCτ for the activeatorvastatin metabolite o-hydroxy-atorvastatin wasalso reduced by 11% (geometric mean ratio 0.89

VAIDYANATHAN ET AL

Figure 2. Interaction of aliskiren with MDR1, MXR, and MRP2 ATPase activity in membrane vesicles and uptake of aliskiren by trans-porter OATP2B1-expressing cells. (a-c) Na3VO4-sensitive ATPase activity in membrane vesicles purified from recombinant baculovirus-infected Sf9 cells is shown. Symbols show ATPase activity at baseline (open circles) and the effect of aliskiren at the concentrationsindicated (filled circles) determined alone (activation): (a) concentration dependency of MDR1 ATPase activity, following treatment withthe active control verapamil 50 μmol/L (open triangle); (b) concentration dependency of MXR ATPase activity, following treatment withthe active control sulfasalazine 10 μmol/L (open triangle); and (c) concentration dependency of MRP2 ATPase activity in the presenceof GSH, following treatment with the active control probenecid 1 mmol/L (open triangle). (d) Uptake of aliskiren by OATP2B1-expressingHEK293 cells is shown. MDR1, multidrug-resistant protein 1; MXR, breast cancer resistant protein; MRP2, multidrug-resistant protein;Pi, inorganic phosphate; HEK, human embryonic kidney. Values are shown as mean ± SD of experiments performed in triplicate.

[90% CI: 0.82, 0.98]; P = .037) and Cmax,ss by 22%(geometric mean ratio 0.78 [90% CI: 0.67, 0.91];P = .010; Figure 3) with no change in median tmax,ss(Table III). Coadministration of aliskiren had no sig-nificant effect on ρ-hydroxy-atorvastatin AUCτ (geo-metric mean ratio 0.91 [90% CI: 0.82, 1.01]; P = .119)or Cmax,ss (geometric mean ratio 0.90 [90% CI: 0.75,1.08]; P = .320; Figure 4), although median tmax,ss wasincreased from 2.0 hours (atorvastatin alone) to 4.0hours (atorvastatin/aliskiren; Table III).

Aliskiren/ketoconazole study. Coadministration ofaliskiren with ketoconazole significantly increasedaliskiren AUCτ by 76% (ratio of geometric means,1.76 [90% CI: 1.64, 1.89]; P < .001) and Cmax,ss by 81%(geometric mean ratio 1.81 [90% CI: 1.57, 2.09];P < .001; Figure 3). Aliskiren Cmin,ss was also increasedby 64% (arithmetic mean ratio), and tmax,ss wasdecreased from 2.0 hours (aliskiren alone) to 1.0

hour (aliskiren/ketoconazole; Table II). Arithmeticmean AUC in the first 2 hours after administration(AUC0-2 h) of aliskiren was 127% higher when coad-ministered with ketoconazole (889 ± 464 vs 392 ±289 ng⋅h/mL for aliskiren alone). Coadministrationwith ketoconazole decreased aliskiren CL/F by 43%(arithmetic mean ratio) compared with aliskirenadministered alone (Table II), but semi-logarithmicplasma concentration-time plots showed no obviouschange in the terminal elimination of aliskiren whencoadministered with ketoconazole (Figure 3c, inset).

Safety and Tolerability

Once-daily oral treatment with aliskiren 300 mgwas well tolerated when administered alone or incombination with digoxin, atorvastatin, or ketocona-zole. All AEs reported across the 3 studies were mildor moderate in severity, with the exception of 1

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Table II Pharmacokinetic Parameters for Aliskiren Administered Alone and in Combination WithDigoxin, Atorvastatin, or Ketoconazole

Aliskiren/Digoxin Study Aliskiren/Atorvastatin Study Aliskiren/Ketoconazole Study

Aliskiren Aliskiren 300 mg ++ Aliskiren Aliskiren 300 mg ++ Aliskiren Aliskiren 300 mg ++300 mg Digoxin 0.25 mg 300 mg Atorvastatin 80 mg 300 mg Ketoconazole 200 mg

Parameter (n == 21) (n == 19) (n == 20) (n == 20) (n == 21) (n == 20)

Cmax,ss, ng/mL 246 240 392 588 369 675tmax,ss, h 1.0 (0.6-6.0) 2.0 (0.6-6.0) 2.03 (0.50-6.00) 2.00 (0.50-6.02) 2.0 (0.5-4.1) 1.0 (0.5-4.0)AUCτ, ng⋅h/mL 1605 1593 2180 3201 2106 3750Cmin,ss, ng/mL 23.7 24.4 ND ND 30.0 48.0CL/F, L/h 194.7 ± 57.0 196.4 ± 58.7 149.3 ± 63.3 98.4 ± 32.9 162.4 ± 85.4 92.4 ± 51.7

Values are presented as geometric mean except for CL/F values, which are presented as arithmetic mean ± SD, and tmax values, which are presented asthe median (range). ND, not determined.

Table I Subject Baseline and Demographic Characteristics

Aliskiren/Digoxin Aliskiren/Atorvastatin Aliskiren/Ketoconazole Parameter Study (n == 22) Study (n == 21) Study (n == 21)

Age, y 30.2 ± 6.0 27.1 ± 8.9 27.6 ± 6.8Range 19-38 18-45 18-41

Gender, male/female 16/6 6/15 11/10Race, n (%)

Caucasian 6 (27.3) 16 (76.2) 13 (61.9)Black 11 (50.0) 5 (23.8) 2 (9.5)Other 5 (22.7) 0 6 (28.6)

Weight, kg 80.4 ± 13.3 67.4 ± 10.8 69.4 ± 8.0Height, cm 178 ± 11 168 ± 11 171 ± 9BMI, kg/m2 25.5 ± 4.0 23.8 ± 2.8 23.8 ± 3.0

Values are presented as mean ± SD unless otherwise stated. BMI, body mass index.

1332 • J Clin Pharmacol 2008;48:1323-1338

severe AE in the aliskiren/atorvastatin study (a caseof abdominal pain in a subject receiving aliskirenand atorvastatin in combination). There was only 1discontinuation due to an AE across the 3 studies; 1subject in the aliskiren/digoxin study was discontin-ued due to clinically significant ECG abnormalities(ST-T wave abnormalities likely due to the effect ofdigoxin and prolonged QTc) starting on day 26 (aftertreatment with digoxin alone). This subject hadshown clinically insignificant ECG abnormalities atscreening (but not at baseline), and was discontinuedfrom the study on day 28. Follow-up ECG findingsshowed clinically insignificant ST-T wave changes.

Most patients had at least one laboratory value orvital sign measurement outside the normal range ineach of the 3 studies, but no trends were noted. Asexpected, most subjects experienced small decreasesin BP during treatment with aliskiren. The only clini-cally significant ECG abnormality observed was anisolated case of a short PR interval in a subject receiv-ing digoxin alone; subsequent ECG tracings in thissubject showed no clinically significant abnormali-ties. Overall, there was no evidence of ECG changeswith aliskiren administered alone or coadministeredwith digoxin, atorvastatin, or ketoconazole.

The most frequently observed AEs were mild andincluded headache, dizziness, nausea, abdominalpain, and diarrhea. There were no notable differ-ences in either the incidence or type of AEsobserved during coadministration of aliskiren withdigoxin or atorvastatin compared with these agentsadministered alone (Table IV). Six subjects reportedAEs during coadministration of aliskiren with keto-conazole, compared with only 2 subjects reportingAEs with aliskiren alone; this was due to 3 mildcases of headache with aliskiren/ketoconazole.

DISCUSSION

P-glycoprotein (MDR1) is a drug transporter proteinfound in the kidney, liver, intestine, and blood–brainbarrier that is involved in the elimination of a rangeof commonly used drugs, and many drug–druginteractions involve the induction or inhibition of P-glycoprotein-mediated transport.4 The OATPs alsohave a key role in hepatobiliary excretion of drugs,particularly in mediating hepatic uptake.4 OATP1B1and OATP1B3 are located exclusively in the liver,whereas OATP2B1 is more widely distributed in tis-sues.4 In vitro and in vivo animal studies have indi-cated that the oral direct renin inhibitor aliskirenmay be a substrate for intestinal and biliary efflux byP-glycoprotein. The aims of the present study were

VAIDYANATHAN ET AL

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Figure 3. Plasma concentration-time profiles for aliskiren fol-lowing once-daily administration of aliskiren 300 mg alone or incombination with (a) digoxin 0.25 mg once daily, (b) atorvastatin80 mg once daily, or (c) ketoconazole 200 mg twice daily inhealthy subjects. Figure shows plasma concentrations of aliskirenat steady state; inset graphs show semi-logarithmic plots ofconcentration-time profiles. Data are presented as mean ± SEM.

to assess whether aliskiren alters in vitro ATPaseactivity in membrane vesicles containing MDR1 pro-tein (ie, is a substrate for P-glycoprotein transport) oris transported in vitro by cells expressing OATP2B1.

Another aim was to investigate the potential forpharmacokinetic interaction in healthy subjects betweenaliskiren and digoxin (P-glycoprotein substrate but notan inhibitor), atorvastatin (P-glycoprotein substrate

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Table III Pharmacokinetic Parameters for Digoxin and Atorvastatin (and Atorvastatin Active Metabolites)Administered Alone and in Combination With Aliskiren

Digoxin Atorvastatin o-Hydroxy-Atorvastatin ρρ-Hydroxy-Atorvastatin

Digoxin Atorvastatin Atorvastatin Atorvastatin 0.25 mg ++ 80 mg ++ 80 mg ++ 80 mg ++

Digoxin Aliskiren Atorvastatin Aliskiren Atorvastatin Aliskiren Atorvastatin Aliskiren 0.25 mg 300 mg 80 mg 300 mg 80 mg 300 mg 80 mg 300 mg

Parameter (n == 20) (n == 19) (n == 21) (n == 20) (n == 21) (n == 20) (n == 21) (n == 20)

Cmax,ss, ng/mL 1.43 1.31 43.0 33.7 40.9 32.1 3.6 3.3tmax,ss, h 1.0 1.0 0.5 0.6 1.5 1.0 2.0 4.0

(0.5-3.0) (0.5-8.0) (0.5-2.0) (0.5-4.0) (0.5-4.0) (0.5-6.0) (0.5-6.1) (1.6-10.0)AUCτ, ng⋅h/mL 14.89 12.74 162 148 234 210 37 34

Values are presented as geometric mean except for tmax values, which are presented as the median (range).

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ο-hydroxy-atorvastatin (atorvastatin 80 mg; n = 21)ο-hydroxy-atorvastatin (atorvastatin 80 mg + aliskiren 300 mg; n = 20)ρ-hydroxy-atorvastatin (atorvastatin 80 mg; n = 21)ρ-hydroxy-atorvastatin (atorvastatin 80 mg + aliskiren 300 mg; n = 20)

4 8 12 16 20 24

0Time (h)

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0Time (h)

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Figure 4. Plasma concentration-time profiles for (a) digoxin, (b) atorvastatin, and (c) atorvastatin metabolites (o-hydroxy-atorvastatinand ρ-hydroxy-atorvastatin) following once-daily administration of digoxin 0.25 mg or atorvastatin 80 mg alone or in combination withaliskiren 300 mg once daily in healthy subjects. Figure shows plasma concentrations at steady state. Data are presented as mean ± SEM.

1334 • J Clin Pharmacol 2008;48:1323-1338

and inhibitor and a substrate for other transporterssuch as OATP1B),35 and ketoconazole (CYP3A4and P-glycoprotein inhibitor but not transported byP-glycoprotein).25,26,36,37

The major findings of this study are as follows: (1)aliskiren is a substrate for P-glycoprotein andOATP2B1-mediated transport in vitro; (2) in healthysubjects, coadministration with aliskiren had noclinically important effects on the pharmacokineticsof digoxin (15% decrease in digoxin AUC) or ofatorvastatin (Cmax,ss decreased by 23%, but the geo-metric mean ratios for AUC were within the range0.80-1.25 for atorvastatin, o-hydroxy-atorvastatin,and ρ-hydroxy-atorvastatin); (3) aliskiren CL/F wasreduced, and aliskiren AUC and Cmax significantlyincreased by coadministration with (a) the P-glycoprotein/CYP3A4/OATP substrate atorvastatin(aliskiren CL/F reduced by 34%; AUCτ and Cmax,ssincreased by 47% and 50%, respectively) and (b)the P-glycoprotein/CPY3A4 inhibitor ketoconazole(aliskiren CL/F reduced by 43%; AUCτ and Cmax,ssincreased by 76% and 81%, respectively); and (4)aliskiren was well tolerated alone or when coadmin-istered with digoxin, atorvastatin, or ketoconazole.

Measurement of Na3VO4-sensitive ATPase activityof ABC transporters expressed in isolated membranevesicles is a well-established in vitro assay for theidentification of P-glycoprotein substrates.28 Ourobservation that aliskiren stimulates in vitro ATPaseactivity of P-glycoprotein with a Km of 2.1 μmol/Lindicates that aliskiren is a relatively high-affinitysubstrate for P-glycoprotein transport. By compari-son, the calcium channel blocker nifedipine exhibits

a Km of 10 μmol/L in this assay, whereas verapamilhas a Km of 0.5 μmol/L.28 The maximum stimulationof P-glycoprotein ATPase activity achieved withaliskiren was similar to that observed with vera-pamil. However, aliskiren did not inhibit verapamil-stimulated P-glycoprotein ATPase activity. Thisfinding suggests that aliskiren is not an inhibitor ofP-glycoprotein, although it should be noted that theATPase assay is not specifically designed to distin-guish P-glycoprotein substrates from inhibitors.26

Aliskiren had no notable interaction with the ABCtransporters MXR or MRP2. In vitro investigations ofthe affinity of aliskiren for OATP2B1 expressed inHEK293 cells38 showed that aliskiren is a substratefor OATP2B1-mediated transport in vitro (Km = 72μmol/L). Aliskiren also acted as an inhibitor ofOATP2B1 transport, but only at concentrationsunlikely to be observed in vivo (IC50 = 95 μmol/L).Previous in vitro experiments have demonstratedthat aliskiren is a substrate for metabolism byCYP3A4 but is not an inhibitor of the in vitro activ-ity of cDNA-expressed CYP3A4 in microsomalpreparations.16 Based on these findings, aliskirenwould not be expected to alter significantly the invivo pharmacokinetics of substrates for P-glycopro-tein, OATP2B1, or CYP3A4, and the results of thepresent study would appear to confirm this. Thus,aliskiren had no major effect on exposure to digoxin(P-glycoprotein substrate).3,4,39

In human subjects, aliskiren had no clinically rel-evant effect on the AUCτ of atorvastatin (P-glycoprotein,OATP1B1,35 OATP2B1,40 and CYP3A4 substrate) orof o-hydroxy-atorvastatin or ρ-hydroxy-atorvastatin

VAIDYANATHAN ET AL

Table IV Safety and Tolerability of Study Treatments

Aliskiren/Digoxin Aliskiren/Atorvastatin Aliskiren/Ketoconazole Study Study Study

Aliskiren Aliskiren Aliskiren 300 mg ++ 300 mg ++ 300 mg ++

Aliskiren Digoxin Digoxin Atorvastatin Aliskiren Atorvastatin Aliskiren Ketoconazole 300 mg 0.25 mg 0.25 mg 80 mg 300 mg 80 mg 300 mg 200 mg

AE Category (n == 22) (n == 21) (n == 20) (n == 21) (n == 20) (n == 20) (n == 21) (n == 21)

Any AE 8 (36.4) 14 (66.7) 10 (50.0) 6 (28.6) 10 (50.0) 7 (35.0) 2 (9.5) 6 (28.6)Discontinuation 0 0 1 (5.0) 0 0 0 0 0

due to AEHeadache 2 (9.1) 4 (19.0) 2 (10.0) 1 (4.8) 1 (5.0) 1 (5.0) 0 3 (14.3)Dizziness 3 (13.6) 2 (9.5) 1 (5.0) 0 4 (20.0) 2 (10.0) 0 0Nausea 0 2 (9.5) 1 (5.0) 0 3 (15.0) 2 (10.0) 1 (4.8) 2 (9.5)Abdominal pain 1 (4.5) 0 1 (5.0) 0 3 (15.0) 3 (15.0) 0 0Diarrhea 1 (4.5) 1 (4.8) 2 (10.0) 0 1 (5.0) 2 (10.0) 0 0

Table shows all individual adverse events (AEs) occurring in more than 1 patient in more than 1 treatment period across the 3 studies. Results are pre-sented as the number (%) of patients reporting at least 1 AE in the respective category.

(both derived from CYP3A4 metabolism and sub-strates for P-glycoprotein).35,41 However, theobserved 23% reduction in atorvastatin Cmax duringcoadministration with aliskiren may indicatecompetition between aliskiren and atorvastatin atthe level of P-glycoprotein or OATP2B1. Furtherstudies are required to investigate the interactionbetween aliskiren and atorvastatin at the level of P-glycoprotein and OATP2B1.

Aliskiren CL/F was reduced and exposure signifi-cantly increased by coadministration with eitheratorvastatin or ketoconazole, both of which inhibitthe efflux transporter, P-glycoprotein, and CYP3A4.Both atorvastatin and ketoconazole caused a signifi-cant increase in the aliskiren AUC between time 0and 2 hours (tmax), suggesting increased absorption ofaliskiren. Calculation of the elimination half-life (t1/2)of aliskiren at steady state was not possible due to thesmall number of time points available for the elimi-nation phase (which begins at >12 hours postdose inmost patients). However, semi-logarithmic plasmaconcentration-time plots indicated that the terminalphase elimination of aliskiren appeared unchangedby either atorvastatin or ketoconazole. Taken togetherwith the in vitro and in vivo animal studiesdescribed previously, these observations are consis-tent with an effect of atorvastatin and ketoconazole toinhibit P-glycoprotein-mediated intestinal and bil-iary efflux and/or CYP3A4-mediated metabolism ofaliskiren (thus increasing absorption of aliskiren andbioavailability F). The proposed lack of a role for P-glycoprotein in the terminal phase elimination ofaliskiren is supported by the observation thataliskiren plasma concentrations at steady state fol-lowing administration of aliskiren 300 mg alonewere no higher than 0.7 μmol/L, below the Km ofaliskiren for P-glycoprotein in vitro. The lack ofeffect of digoxin on aliskiren pharmacokinetics isalso consistent with this interpretation because atclinically relevant concentrations, digoxin is a sub-strate, but not an inhibitor, of P-glycoprotein25,42 andhas little interaction in vitro with OATP2B1.40

Aliskiren is a substrate for both P-glycoproteinand CYP3A4. The lack of effect of cimetidine (a lesspotent inhibitor of CYP3A4) and lovastatin (whichinteracts with CYP3A4 and is an inhibitor of P-glycoprotein) on the pharmacokinetics of aliskirensuggests that metabolism by CYP3A4 is unlikely toplay a major role in the in vivo elimination ofaliskiren.18 Unchanged aliskiren accounted for 81%of the radioactivity in plasma following oral admin-istration of radioactive-labeled aliskiren in healthysubjects,19 and exposure to metabolites is very low;

the 2 major oxidized metabolites of aliskirenaccounted for only 3% and 1%, respectively, of theradioactivity in the plasma at tmax. However, there isinsufficient evidence to determine precisely the rela-tive importance in vivo of P-glycoprotein- orOATP2B1-mediated intestinal efflux and biliaryclearance, as well as gut wall CYP3A4 metabolism inthe disposition of aliskiren. Indeed, enzyme kineticdata on the in vitro intrinsic clearance of aliskirenshow similar clearance of aliskiren in human intes-tine and liver microsomes (data on file). There is con-vincing evidence of a role for P-glycoprotein, aspreclinical studies have clearly demonstrated P-glycoprotein-mediated intestinal efflux and biliaryclearance of aliskiren. Moreover, the aliskiren plasmaconcentration-time profile in many human subjectsin the present study exhibited a distinctive double-peak characteristic of possible enterohepatic recircu-lation, which would be consistent with the biliaryelimination of aliskiren demonstrated in rats. Thefinding that aliskiren AUC was significantlyincreased by 29% (P < .05) by coadministration withthe calcium channel blocker amlodipine in healthysubjects17 is also evidence in favor of elimination ofaliskiren by P-glycoprotein rather than CYP3A4,because amlodipine is an effective inhibitor of P-gly-coprotein (IC50 22 μmol/L)43 but a weak inhibitor ofCYP3A4.44 On the other hand, a previous study inhealthy subjects estimated that oral administration ofan 80-mg dose of atorvastatin with about 240 mL ofwater would produce an intestinal luminal concen-tration of approximately 560 μmol/L,41 which wouldbe sufficient to inhibit not only intestinal P-glycopro-tein activity (atorvastatin IC50 15 μmol/L) but alsogut wall CYP3A4 activity (atorvastatin IC50 <10μmol/L for CYP3A4).45 Moreover, ketoconazoleis a much more potent inhibitor of CYP3A4 (IC500.02-0.05 μmol/L) than of P-glycoprotein (IC50 3μmol/L).23 It is therefore possible that the effects ofatorvastatin and ketoconazole on aliskiren pharma-cokinetics may be explained by inhibition of gut wallCYP3A4 activity, although it should be noted thatthere is considerable interindividual variability inthe intestinal expression of CYP450 isoenzymes.46

Further studies are required to identify the relativeimportance of P-glycoprotein and CYP3A4 activity inthe disposition of aliskiren. Interaction with a lowerdose of aliskiren (150 mg once daily) could define themagnitude of the interaction with competitiveinhibitors such as ketoconazole. The inhibition ofCYP3A4 by ketoconazole is dose dependent, and 400mg ketoconazole once daily is the optimal regimen toachieve maximum inhibition of CYP3A4.47

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Aliskiren treatment was well tolerated in healthysubjects, whether administered alone or coadminis-tered with digoxin, atorvastatin, or ketoconazole.The most common reported adverse experiencesduring treatment with aliskiren across the 3 studieswere headache and dizziness—AEs that might beanticipated following administration of an anti-hypertensive agent to normotensive healthy subjects.Overall, there was no evidence that coadministra-tion of aliskiren with digoxin, atorvastatin, or keto-conazole was associated with safety or tolerabilityissues. All 3 studies employed a multiple-dosedesign, which reflects the dosage regimen that islikely to be used with aliskiren in clinical practice,and measurement of trough plasma concentrationsof aliskiren confirmed that comparisons of pharma-cokinetic parameters and safety were made understeady-state conditions. Aliskiren AUCτ (1671 ng⋅h/mL)and Cmax (mean Cmax 261 ng/mL) in the aliskiren/digoxin study were slightly lower than in the other2 studies but were within the range of values observedin previous multiple-dose studies of aliskiren300 mg (mean AUCτ 1110-2135 ng⋅h/mL; Cmax 198-342 ng/mL).13,48

In large-scale clinical trials, aliskiren at once-daily doses of up to 300 mg exhibits a safety profilecomparable to that of placebo.11,15 Since aliskirenexhibits linear pharmacokinetics up to 600 mg, theexposure expected during coadministration ofaliskiren 300 mg with atorvastatin 80 mg oncedaily or ketoconazole 200 mg twice daily shouldremain below that observed at a dose of 600 mg.Moreover, as the adverse effects associated with the600-mg dose of aliskiren were gastrointestinal andpossibly related to local drug concentrations,49 theincrease in plasma concentrations of aliskiren fol-lowing coadministration of aliskiren 300 mg withatorvastatin or ketoconazole would not be expectedto lead to an increase in adverse events. The presentstudy in healthy subjects suggests that modificationof the starting dosage of aliskiren is unlikely to berequired when aliskiren is administered in combina-tion with digoxin, atorvastatin, or ketoconazole.Caution must be exercised in extrapolating thesefindings to other patient groups; however, previouspharmacokinetic studies have shown that initialdose adjustment of aliskiren is not required based onage, gender, race, the presence of renal or hepaticimpairment, or type 2 diabetes.14

In summary, the preclinical results of the presentstudy show that the direct renin inhibitor aliskiren isa substrate for but not an inhibitor of P-glycoproteinactivity and that OATP2B1 is the likely hepatic

uptake transporter of aliskiren. Moreover, the lack ofany clinically significant effect of aliskiren on thepharmacokinetics of either digoxin or atorvastatin inhealthy subjects indicates that aliskiren is not aninhibitor of P-glycoprotein–OATP-mediated trans-port or of metabolism by CYP3A4 in vivo. Exposureto aliskiren was significantly increased by coadmin-istration of the maximum recommended dosage ofatorvastatin and ketoconazole (both P-glycoproteininhibitors), suggesting that P-glycoprotein, OATP2B1,and/or CYP3A4 play a role in aliskiren disposition,most likely at the level of the intestine. Based on thisstudy and on the safety profile of aliskiren fromlarge-scale clinical trials, adjustment of the startingdose of aliskiren should not be required duringcoadministration with digoxin, atorvastatin, orketoconazole.

The authors thank Dr Ann Taylor and Dr Richard White forwriting and editorial support.

Financial disclosure: This study was supported by NovartisPharma AG. All authors are employees of Novartis and are eligi-ble for Novartis stock and stock options.

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