Pharmacokinetics for once-daily versus twice-daily tacrolimus formulations in de novo liver...

ORIGINAL ARTICLE

Pharmacokinetics for Once-Daily Versus Twice-Daily Tacrolimus Formulations in De Novo LiverTransplantation: A Randomized, Open-Label TrialLutz Fischer,1 Pavel Trunecka,2 Bruno Gridelli,3 Andre Roy,4 Alessandro Vitale,5 Andres Valdivieso,6

Evaristo Varo,7 Daniel Seehofer,8 Stephen Lynch,9 Didier Samuel,10 Bo-Goran Ericzon,11

Karim Boudjema,12 Carmen Karpf,13 and Nasrullah Undre14

1Department of Hepatobiliary Surgery and Transplantation, Universitat Krankenhaus Eppendorf, Hamburg,Germany; 2Department of Hepatogastroenterology, IKEM, Prague, Czech Republic; 3Chirurgia Generale IIICentro Trapianto Fegato Pediatrico, Ospedali Riunitit di Bergamo, Bergamo, Italy; 4Centre Hospitalier del’Universite de Montreal (CHUM) Hospital St. Luc, Montreal, Canada; 5Unita di Chirurgia Oncologica,Istituto Oncologico Veneto, Istituto di Ricovero e Cura a Carattere Scientifico, Padova, Italy; 6Servicio deCirugıa General y Digestivo, Unidad de Trasplante Hepatico, Hospital de Cruces, Universidad del PaısVasco, Bilbao, Spain; 7Servicia Cirugıa General y Digestivo, Unidad de Trasplante Hepatico, Hospital Xeralde Galicia, Santiago de Compostela, Spain; 8Klinik fur Allgemein - Viszeral - und Transplantationschirurgie,Charite Campus-irchow Klinikum, Berlin, Germany; 9Queensland Liver Transplant Service, PrincessAlexandra Hospital, Brisbane, Australia; 10Centre Hepato Bilaire, Hospital Paul Brousse, Villejuif, France;11Department of Transplantation Surgery, Huddinge Hospital, Stockholm, Sweden; 12Service de ChirurgieHepato-biliaire et Digestive, Hopital Pontchaillou, Universite de Rennes 1, Rennes, France; 13Astellas GmbH,Munich, Germany (formerly Fujisawa, Germany at the time of study); and 14Astellas Pharma Europe, Ltd.,Staines, UK (formerly Fujisawa, Germany at the time of study)

Tacrolimus, a cornerstone immunosuppressant, is available as a twice-daily formulation (tacrolimus bid). A once-daily pro-longed-release formulation (tacrolimus qd) has been developed. This 6-week, randomized, phase 2, multicenter, open-label,prospective trial in primary liver transplant recipients investigated and compared the pharmacokinetics (PK) of tacrolimus forqd and bid formulations. All patients received tacrolimus-based immunosuppression (tacrolimus qd, n ¼ 67; bid, n ¼ 62).PK data were available for 77 patients (tacrolimus qd, n ¼ 45; bid, n ¼ 32). Tacrolimus area under the curve (AUC) from 0to 24 hours (AUC0-24) at equivalent doses was approximately 50% lower for tacrolimus qd than for bid on day 1 (146 versus264 ng�h/mL, respectively), but by day 14 was comparable between treatments (324 and 287 ng�h/mL, respectively) withhigher tacrolimus qd doses. There was a strong correlation between AUC0-24 and concentration at 24 hours for tacrolimusqd and bid (r ¼ 0.92 and r ¼ 0.76, respectively). Furthermore, the relationship between these 2 parameters (ie, the slope ofthe line) was also similar for the 2 formulations. Efficacy endpoints were comparable for both formulations at 6 weeks with

Abbreviations: AE, adverse event; AUC0-24, area under the curve from 0 to 24 hours; AZA, azathioprine; bid, twice daily; BPAR,biopsy-proven acute rejection; C24, concentration at 24 hours; Cmax, maximum concentration; Cmin, minimum blood concentra-tion; MMF, mycophenolate mofetil; NEC, not elsewhere classified; PK, pharmacokinetics; qd, once daily; SD, standard deviation;Tmax, time to maximum concentration.

This study and statistical analyses, and the editorial and project management services of Acumed involved in the preparation ofthis manuscript, were supported by Astellas Pharma Europe, Ltd., Staines, UK.

L. Fischer, B. Gridelli, A. Roy, A. Vitale, A. Valdivieso, E. Varo, D. Seehofer, B. Ericzon, and K. Boudjema have declared noconflicts of interest. P. Trunecka has acted as study investigator and advisory board member for Astellas and has receivedspeaker’s fees. S. Lynch has received honoraria for chairing and speaking at international meetings for Astellas and Janssen-Cilag, Ltd. D. Samuel has acted as a consultant and also received research grants from Astellas and Novartis and acted as aconsultant for Roche. C. Karpf was an employee of Astellas at the time of the study, and N. Undre is currently an employee ofAstellas.

Address reprint requests to Nasrullah Undre, Ph.D., Astellas Pharma Europe, Ltd., Lovett House, Lovett Road, Staines, Middlesex, TW18 3AZ,UK. Telephone: þ44 1784 419 591; FAX: þ44 1784 419 527; E-mail: [email protected]

DOI 10.1002/lt.22211View this article online at wileyonlinelibrary.com.LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

LIVER TRANSPLANTATION 17:167-177, 2011

VC 2011 American Association for the Study of Liver Diseases.

no marked differences in incidence, nature, or severity of adverse events between treatments (although the study was notpowered to draw efficacy conclusions). These results suggest that targeting the same trough levels will achieve similar totalAUC over 24 hours for both tacrolimus qd and tacrolimus bid in de novo liver transplant recipients. Liver Transpl 17:167-177, 2011. VC 2011 AASLD.

Received February 8, 2010; accepted October 6, 2010.

Prograf (Astellas Pharma Europe, Ltd., Staines, UK) isan immediate-release formulation of the immunosup-pressive agent tacrolimus, administered twice-daily(bid) for the prevention and treatment of allograftrejection in liver, kidney, and heart transplantation;hereafter, this formulation will be referred to as tacro-limus bid. A prolonged-release formulation of tacroli-mus (Advagraf; Astellas Pharma Europe, Ltd., Staines,UK) has been developed to provide once-daily (qd)dosing while ensuring similar efficacy and safety tothe standard bid formulation; hereafter, it will bereferred to as tacrolimus qd. Tacrolimus is a medica-tion exhibiting a narrow therapeutic index1,2 and theoral bioavailability is highly variable between individ-uals.3 Systemic exposure to tacrolimus (area underthe curve [AUC]) is a significant variable for efficacyand safety, and therefore, therapy is optimized on anindividual patient basis by monitoring trough levelsas surrogate markers of exposure (AUC).3 Therefore,the aim in the development of tacrolimus qd was toapply the same drug-level monitoring concept as isestablished for tacrolimus bid.

The AUC from 0 to 24 hours after dosing (AUC0-24)of tacrolimus qd has previously been shown to beapproximately 10% lower in comparison to that of thebid formulation in stable liver transplant patientsconverted on a 1:1 (milligram:milligram) total dailydose basis (measured at steady-state 14 days afterconversion).4 Furthermore, follow-up data in stableliver transplant patients converted from tacrolimusbid to tacrolimus qd demonstrate good efficacy andsafety profiles at 2 years.5 However, the pharmacoki-netics (PK) of tacrolimus qd in de novo–transplantedpatients has not been investigated previously. The pri-mary objective of this study was to evaluate and com-pare PK parameters of tacrolimus qd and tacrolimusbid in de novo liver transplant recipients. Assess-ments were made following the first dose and understeady-state conditions in patients undergoing pri-mary liver transplantation.

PATIENTS AND METHODS

Study Design and Procedures

This was a 6-week, phase 2, multicenter, open-label,prospective, 1:1 randomized trial comparing the PK oftacrolimus qd and tacrolimus bid in patients under-going primary liver transplantation. Patient enroll-ment began in January 2003 and the last evaluationwas performed in January 2004. Patients were allo-cated to treatment groups, locally at each center,using sealed randomization envelopes provided by the

Data Operations Department of Astellas Pharma, Ltd.Randomization was performed preoperatively on a 1:1(tacrolimus qd:tacrolimus bid) basis stratified by cen-ter, with each center being allocated a unique sequenceof patient numbers. As this was an open-label study,no blinding was necessary. Visits were scheduled atdays 0, 1, 3, 7, 11, 14 and weeks 4 and 6. At the endof the 6-week study period, patients in the tacrolimusqd group had the option to continue treatment withtacrolimus qd as part of a long-term extension study.Patients treated with tacrolimus bid were not followedafter 6 weeks. Also reported here are 1-year follow-upresults from the tacrolimus qd group.

Patients

Eligible patients were aged between 18 and 65 years,undergoing primary whole or split liver transplanta-tion, had provided written informed consent, hadreceived a first dose of tacrolimus and corticosteroidswithin 6-12 hours (but not later than 18 hours,depending on time of surgery) of skin closure, andwere expected to be maintained on tacrolimus andcorticosteroid treatment throughout the study.

Patients were excluded if they had malignancy(except hepatic cell carcinoma fulfilling Milan criteria)or history of malignancy within the last 5 years, hadsystemic infections requiring treatment, had serumcreatinine >175 lmol/L, required induction therapy,had received an ABO-incompatible graft, had previ-ously received or were receiving an organ transplantother than liver, or were likely to be nonadherent tothe study protocol. Pregnant women and nursingmothers were also excluded.

Patients could be withdrawn due to withdrawal ofconsent, intolerable adverse event, retransplantation,noncompliance with study protocol, loss to follow-up,pregnancy or initiation of prohibited concomitantmedication known to affect the pharmacokinetics oftacrolimus (ketoconazole, fluconazole, itraconazole,clotrimazole, nifedipine, nicardipine, erythromycin,clarithromycin, josamycin, danazol, ethinyl estradiol,omeprazole, calcium antagonists such as diltiazem,nefazodone, phenobarbital, phenytoin, rifampicin).

The study was conducted at 21 centers: 16 inEurope, 3 in Canada, and 2 in Australia. The studywas conducted in accordance with the Declaration ofHelsinki, fifth revision (2000).

Study Treatment

The first dose of tacrolimus qd was administeredwithin 6-12 hours (but not later than 18 hours,

168 FISCHER ET AL. LIVER TRANSPLANTATION, February 2011

depending on time of surgery) after skin closure, inthe morning following transplantation. The recom-mended first oral daily dose of tacrolimus for bothgroups was to be in the range of 0.10-0.15 mg/kg,with tacrolimus qd given as 1 dose in the morning.The first dose of tacrolimus bid was administeredorally within 6-12 hours (but not later than 18 hours,depending on time of surgery) after skin closure, inthe morning following transplantation. The seconddose of tacrolimus bid was taken in the evening of theday following transplantation. Patients unable toswallow a capsule could receive the contents of thetacrolimus bid capsules via nasogastric tube. It wasnot permitted to administer the dose of tacrolimus qdby nasogastric tube. Doses of tacrolimus qd weretaken orally in the morning, whereas tacrolimus bidwas taken as 2 daily oral doses, on an empty stomachor at least 1 hour before or 2-3 hours after a meal.Capsules were to be swallowed intact, with fluid (pref-erably water). Doses of both tacrolimus qd and tacro-limus bid were adjusted on an individual patient basisaccording to clinical signs aided by whole-blood tacro-limus trough level monitoring. Whole-blood sampleswere taken daily during the first 2 weeks, at weeks 4and 6, and whenever clinically indicated. Concentra-tions of tacrolimus were determined locally usingmicroparticle enzyme immunoassay (MEIA; IMx) orenzyme multiplied immunoassay technique. The rec-ommended target tacrolimus whole-blood trough levelrange was 10-20 ng/mL for all patients.

Patients received an intravenous bolus (500-1000 mg)of methylprednisolone (or equivalent) prior to reperfu-sion, with subsequent doses given as oral prednisone

(or equivalent) at a dose of 15-20 mg/day during thefirst month and tapered doses thereafter. The use ofantibodies for induction therapy was not permitted, buttheir use for the treatment of acute rejection wasallowed.

Pharmacokinetic Profiles and Assay

PK profiles were obtained following the initial dose oftacrolimus qd or tacrolimus bid (day 1 posttransplan-tation), and twice under steady-state conditions (onday 14 and at 6 weeks 6 7 days after transplantation)(Fig. 1). For both tacrolimus qd and tacrolimus bid,the first blood sample was taken in the morningbefore the first dose of study treatment was adminis-tered (0) and at 0.5, 1, 2, 3, 4, 6, 8, 12, 12.5, 13, 14,15, 16, 18, 20, and 24 hours after administration.The assays were based on serial whole-blood samplescollected in ethylene diamine tetraacetic acid tubesand frozen at �20�C (�4�F) within 2 hours of collec-tion, then stored until shipment to a central labora-tory (AAI Deutschland GmbH & Co. KG) for analysis.Tacrolimus concentrations were determined usingvalidated high-performance liquid chromatographytandem mass spectroscopy assay methods (lower limitof quantification ¼ 0.1 ng/mL).6 Whole-blood calibra-tion standards, quality control samples and studysamples were thawed and 1-mL aliquots taken. Inter-nal standard (20 lL, 50 ng/mL) was added and mixedbriefly. Aliquots were extracted using protein precipi-tation and solid phase extraction using C18 200 mg/3-mL extraction cartridges. Elutes were evaporated todryness under a stream of nitrogen at 40�C, and

Figure 1. Study design and schedule of PK profiles.

LIVER TRANSPLANTATION, Vol. 17, No. 2, 2011 FISCHER ET AL. 169

residues redissolved in a 50:50 mix (vol/vol) of aceto-nitrile and water, mixed and centrifuged, before beingsubmitted for assay.

As an additional aid for monitoring exposure, lim-ited sampling AUC monitoring was performed on days1, 3, 7, 11, 14, and as clinically indicated thereafter(for example, if signs of toxicity or lack of efficacywere observed within the recommended target ranges,and the clinician felt that a limited AUC samplingwould be a helpful aid in optimizing dose). Blood sam-ples were collected at 0 (predose) and at 2, 4, 8, and12 hours postdose for both formulations, with anadditional sample at 24 hours postdose for tacrolimusqd. Tacrolimus levels were assayed at the investiga-tional site and the resulting data were sent to anexternal panel (Covance Clinical Research Unit, Ltd.,Leeds, UK) to estimate the AUC using the linear trape-zoidal method. The AUC values were immediatelymade available to the investigational site to aid in ex-posure estimation; the decision to make any doseadjustments was the responsibility of the site investi-gator. All procedures were performed in compliancewith the principles of Good Laboratory Practice.

Objectives and Endpoints

The objective of this study was to evaluate and com-pare PK parameters following first administration andunder steady-state conditions in de novo liver trans-plant recipients treated with tacrolimus qd and tacro-limus bid.

The primary endpoint was a comparison of AUC0-24

for tacrolimus qd and tacrolimus bid on day 1, day 14,and week 6 after transplantation. Secondary endpointsincluded the comparison between tacrolimus qd andtacrolimus bid of the maximum concentration of tacro-limus (Cmax), time to maximum concentration (Tmax),trough levels (minimum blood concentration [Cmin] at12 hours for tacrolimus bid and at 24 hours for tacroli-mus bid and tacrolimus qd), correlation between Cmin

and AUC0-24, incidence of and time to biopsy-provenacute rejection (BPAR) events at 6 weeks, overall fre-quency of acute rejection episodes at 6 weeks, severityof BPAR at 6 weeks, graft and patient survival at 6weeks, and incidence of adverse events (AEs) at 6weeks.

All acute rejection episodes were classified asbiopsy-proven or suspected acute rejection episodes(further classified as spontaneously resolving, cortico-steroid-sensitive or corticosteroid-resistant acuterejections). The incidence of graft loss (defined asretransplantation or death) and duration of patientsurvival were also recorded. In addition, AEs andabnormal laboratory measurements for hematologyand biochemistry (performed at baseline, at the timeof PK profiling and at week 4) were recorded for bothtreatment groups. Clinical laboratory parameters(including red blood cell count, hemoglobin, sodium,potassium, creatinine, total bilirubin, and albumin)were also measured at baseline, at the same time asPK profile sampling, and at weeks 4 and 6.

Statistical Analyses

From previous early-phase studies with tacrolimusqd,8 a steady-state AUC0-24 ratio of 0.9 and a stand-ard deviation (SD) of 0.35 for each treatment arm wasassumed in this study and it was estimated that 32patients with 3 complete PK profiles each would beadequate to obtain well-founded information on theprimary parameter and its ratio. Taking into consider-ation potential dropout rates, it was planned to enrollapproximately 40 patients per treatment arm.

PK analyses were conducted in the PK EvaluableSet (patients who received all doses of study medica-tion, provided 3 complete evaluable PK profiles, andhad no major protocol violations). Comparison of PKparameters for tacrolimus qd and tacrolimus bid(AUC0-24, Cmax, and Cmin) was performed using a2-sided 90% confidence interval for the ratio ofmeans, with an acceptance interval of 80%-125% forsimilarity between any 2 parameters. Descriptivestatistics were calculated for Tmax. The correlationbetween Cmin and AUC0-24 was assessed for bothtacrolimus qd and tacrolimus bid.

PK parameters were calculated from the complete24-hour concentration–time profiles of tacrolimus inwhole-blood following administration of tacrolimus qdor tacrolimus bid. AUC0-24 was estimated using thelinear trapezoidal method. Cmax, Tmax, and minimumblood concentration at 12 or 24 hours were obtaineddirectly from the 24-hour concentration–time profiles.

Efficacy and safety analyses were conducted for theFull Analysis Set (patients who received at least 1dose of study medication). Incidence of acute rejectionand BPAR were assessed using a chi-square test. Theincidence of, and time to, first acute rejection and tofirst BPAR episode was calculated using Kaplan-Meiersurvival estimates. The difference between treatmentswas assessed using the Wilcoxon Gehan test. Differ-ences between treatments in the severity of BPAR epi-sodes were assessed using the Wilcoxon rank-sumtest. Patient and graft survival were analyzed usingKaplan-Meier estimates (Wilcoxon Gehan test).

RESULTS

Study Population

A total of 133 patients were randomized to tacrolimusqd (n ¼ 69) or tacrolimus bid (n ¼ 64) treatment. Ofthese patients, 4 did not receive study medication.Therefore, 129 (tacrolimus qd, n ¼ 67; tacrolimus bid,n ¼ 62) treated patients were included in the efficacyand safety analyses (Full Analysis Set). Evaluable PKdata (from patients with 3 evaluable PK profiles withoutany major PK relevant protocol violations) were avail-able for 77 patients (tacrolimus qd, n ¼ 45; tacrolimusbid, n ¼ 32). Patient disposition (Fig. 2) was generallycomparable between the tacrolimus qd and tacrolimusbid groups; however, slightly more patients were with-drawn following administration of tacrolimus bid.

Baseline demographics and disease characteristicsfor all randomized and treated patients (Full Analysis


Set; Table 1) were similar for patients who receivedtacrolimus qd or tacrolimus bid. Patient demographicsfor the PK Evaluable Set were also similar for the 2treatment groups (Table 1), with the exception of meanpatient age, which was higher for the tacrolimus bidtreatment group (52.7 years, range 35-65 years) com-pared with the tacrolimus qd treatment group (48.6years, range 26-64 years, P ¼ 0.044; Student t test).

Donor characteristics and surgical details werecomparable for the tacrolimus qd and tacrolimus bidtreatment groups (Table 1).

Drug Administration and Exposure

In the Full Analysis Set, all patients received mainte-nance corticosteroid therapy. Median daily starting dosewas 750 mg/day in both treatment arms (with rangesof 20-1500 mg for tacrolimus qd and 25-1500 mg fortacrolimus bid). Maintenance daily doses were graduallytapered to a median of 15 mg/day in both treatmentarms (with ranges of 10-25 mg for tacrolimus qd and5-20 mg for tacrolimus bid) by week 6.

Other immunosuppressive medication, generallyadministered as prophylaxis, was received by 16/67(23.9%) patients in the tacrolimus qd group and17/62 (27.4%) patients in the tacrolimus bid group.In the tacrolimus qd group, 1 (1.5%) patient received

mycophenolate mofetil (MMF), 14 (20.9%) receivedazathioprine (AZA), and 1 (1.5%) received both MMFand AZA. In the tacrolimus bid group, 4 (6.5%)patients received MMF, 12 (19.4%) received AZA, 1(1.6%) received both MMF and AZA and 1 (1.6%)received sirolimus. Of these patients, 2 patients in thetacrolimus qd group and 4 patients in the tacrolimusbid group had MMF or AZA added at the time of treat-ment of a rejection episode.

The mean total daily doses of both tacrolimus qd andtacrolimus bid increased during the early posttrans-plant period (Fig. 3A). Apart from the first dose (day 1),the mean tacrolimus qd dose (0.117 mg/kg on day 1 to0.226 mg/kg at week 6) was higher than the corre-sponding tacrolimus bid dose (0.110-0.162 mg/kg).Trough levels were similar for patients treated withtacrolimus qd and tacrolimus bid (Fig. 3B).

During the study, patients could receive concomi-tant medication (Table 2).

Pharmacokinetics

On day 1, mean AUC0-24 of tacrolimus at equivalentdoses was approximately 50% lower for tacrolimus qdthan for tacrolimus bid (146 versus 264 ng�h/mL,respectively; Table 3 and Fig. 4). The AUC for tacrolimusqd on day 1 was outside the 90% confidence interval

Figure 2. Patient disposition.


(80-125), and therefore was not equivalent to AUC withtacrolimus bid. However, AUC0-24 for tacrolimus qd wascomparable with that of tacrolimus bid by day 14 (324versus 287 ng�h/mL, respectively) and at week 6 (364 ver-sus 301 ng�h/mL; respectively) although the mean daily

doses of tacrolimus qd were higher after day 1 (Fig. 3A),ie, in order to achieve similar tacrolimus exposure, higherdoses of tacrolimus qd were required. Furthermore, byday 4, trough levels were similar for both formulations(Fig. 3B). There was a strong correlation between AUC0-24

TABLE 1. Baseline Demographics and Disease Characteristics

Characteristics Tacrolimus qd (n ¼ 67) Tacrolimus bid (n ¼ 62)

Full Analysis SetMale patients (n [%]) 49 (73.1) 45 (72.6)Age (years), mean (range) 49.4 (24-65) 52.4 (27-68)Weight (kg), mean (range) 78.0 (40-127) 77.5 (48-142)Race, n (%)Caucasian 65 (97.0) 61 (98.4)Black 1 (1.5) 0Asian 0 1 (1.6)Other 1 (1.5) 0

Viral status, n (%)Hepatitis B surface antigen–positive 6 (9.0) 5 (8.1)Anti-hepatitis C–positive 21 (31.3) 19 (30.6)Existing glucose metabolism disorder 19 (28.4) 21 (33.9)Primary diagnosis, n (%)Cirrhosis* 48 (71.6) 47 (75.8)Primary biliary 2 (3.0) 2 (3.2)Secondary biliary 1 (1.5) 1 (1.6)Posthepatitic 19 (28.4) 19 (30.6)Hepatitis B 3 (4.5) 4 (6.5)Hepatitis B delta 2 (3.0) 1 (1.6)Hepatitis C 14 (20.9) 15 (24.2)Autoimmune 3 (4.5) 2 (3.2)Alcoholic 23 (34.3) 17 (27.4)Cryptogenic 3 (4.5) 4 (6.5)Other 2 (3.0) 3 (4.8)

Carcinoma† 10 (14.9) 6 (9.7)Budd–Chiari 2 (3.0) 0Metabolic disease 2 (3.0) 1 (1.6)Sclerosing cholangitis 3 (4.5) 6 (9.7)Other 2 (3.0) 2 (3.2)

PK Evaluable Set Tacrolimus qd (n ¼ 45) Tacrolimus bid (n ¼ 32)Male patients, n (%) 34 (75.6) 24 (75.0)Age (years), mean (range) 48.6 (26-64) 52.7 (35-65)‡

Weight (kg), mean (range) 78.2 (40-127) 78.9 (48-142)Caucasian 45 (100) 32 (100)Viral statusHepatitis B–positive 5 (11.1) 4 (12.5)Anti-hepatitis C–positive 13 (28.9) 8 (25.0)

Existing glucose metabolism disorder 13 (28.9) 6 (18.8)

Donor Characteristics Tacrolimus qd (n ¼ 67) Tacrolimus bid (n ¼ 62)Male donor, n (%) 43 (64.2) 35 (56.5)Donor age (years), mean (range) 41.2 (6-78) 45.2 (15-81)Partial liver transplant, n (%) 7 (10.4) 8 (12.9)Assessment of liver, n (%)Very good/good 61 (91.0) 56 (90.3)Fair/poor/not recorded 6 (9.0) 6 (9.7)

Total ischemia time (hours), mean (range) 8.2 (2.2-16.3)§ 8.2 (2.0-16.0)||

*Multiple reports possible†All hepatocellular carcinoma‡P ¼ 0.044; all other demographic parameters were not significantly different§n ¼ 63||n ¼ 58.


and C24 for both tacrolimus qd and tacrolimus bid (r ¼0.92 and r¼ 0.76, respectively; Fig. 5).

Tmax occurred later for tacrolimus qd comparedwith tacrolimus bid (day 1: 5.0 versus 2.9 hours; day14: 2.8 versus 1.9 hours; week 6: 2.8 versus 1.8hours, respectively), reflecting the prolonged-releasecharacteristics of the qd tacrolimus formulation.

Secondary Endpoints

Rejection and Survival Rates

The overall frequency of BPAR in the Full Analysis Setwas comparable for tacrolimus qd and tacrolimus bid(26.9 and 27.4%, respectively). There were no markeddifferences in the classification or histological severityof the acute rejections reported.

One patient receiving tacrolimus qd (1.5%) and3 patients receiving tacrolimus bid (4.8%) experiencedgraft loss during the study in the Full Analysis Set. Inthe tacrolimus qd group, this loss was as a result ofdeath due to cardiac arrest (caused by pulmonaryhypertension) whereas in the tacrolimus bid group,2 graft losses were the result of retransplantation(graft dysfunction and graft ischemia) and 1 due todeath caused by pneumonia. One patient in eachgroup experienced graft loss after withdrawal from thestudy (due to graft dysfunction in the tacrolimus qdgroup and portal vein thrombosis in the tacrolimusbid group).

The estimated graft survival rates with tacrolimusqd and tacrolimus bid at week 6 were similar at 96.9and 93.3%, respectively. There was no graft loss

Figure 3. Summary of (A) dailytacrolimus doses and (B) troughconcentrations (Full AnalysisSet).


beyond week 1 for tacrolimus qd–treated patients andbeyond week 3 for tacrolimus bid–treated patients.

Estimated patient survival rates at week 6 weresimilar for tacrolimus qd and tacrolimus bid (98.4and 98.1%, respectively).

Adverse Events

AEs reported during the study were consistent withthe well-described safety profile of tacrolimus bid.7

There were no marked differences in the incidence,nature, or severity of AEs between tacrolimus qd andtacrolimus bid (Fig. 6). The most frequently reportedAEs (Medical Dictionary for Regulatory Activities high-level term) causally related to the study drug in thetacrolimus qd and tacrolimus bid groups were: renalfailure and impairment (32.8% versus 29.0%), diabe-tes mellitus (17.9% versus 24.2%), vascular hyperten-sive disorders not elsewhere classified (14.5% versus16.4%), and hyperglycemic conditions not elsewhereclassified (9.0% versus 16.1%). Renal failure andimpairment was the most frequently reported seriousAE and was reported in 6.0% of tacrolimus qd-treatedpatients and 6.5% of tacrolimus bid–treated patients(P ¼ not significant).

Hepatic function was comparable between tacroli-mus qd–treated and tacrolimus bid–treated patientsthroughout the study. At week 6, aspartate amino-transferase levels were 34.9 6 53.6 and 37.7 650.2 U/L, respectively, and alanine aminotransferaselevels were 68.4 6 138.8 and 77.8 6 138.3 U/L,respectively. Renal function, as determined by serumcreatinine levels and creatinine clearance, was compara-ble for tacrolimus qd and tacrolimus bid during the study(Table 4). At week 6, serum creatinine levels were 99.5 6

TABLE 2. Concomitant Medications Administered

During the Study* (Full Analysis Set)

Full Analysis Set

Tacrolimus

qd (N ¼ 67)

Tacrolimus

bid (N ¼ 62)

n (%) n (%)

Furosemide 56 (83.6) 49 (79.0)Ranitidine 43 (64.2) 37 (59.7)Bactrim 36 (53.7) 37 (59.7)Insulin 34 (50.7) 32 (51.6)Ursodeoxycholic acid 31 (46.3) 28 (45.2)Heparin-fraction 29 (43.3) 27 (43.5)Heparin 26 (38.8) 29 (46.8)Albumin human 27 (40.3) 24 (38.7)Metronidazole 27 (40.3) 24 (38.7)Morphine 28 (41.8) 22 (35.5)Nystatin 27 (40.3) 23 (37.1)Acetylcysteine 28 (41.8) 21 (33.9)Aciclovir 29 (43.3) 19 (30.6)

*Received by at least 40% of patients in either group.

TABLE 3. Comparison of PK Parameters of Tacrolimus Administered as Tacrolimus qd and

Tacrolimus bid (PK Evaluable Set)

PK Parameters Tacrolimus qd (n ¼ 45) Tacrolimus bid (n ¼ 32)

Day 1* Mean (SD)Cmax (ng/mL) 10.59 (6.26) 19.75 (8.48)C24 (ng/mL) 4.21 (3.31) 8.98 (5.90)Tmax (hours)† 5.0 (4.0) 2.9 (1.8)AUC0-24 (ng�h/mL) 145.97 (103.03) 263.82 (153.36)ln(AUC0-24) ratio (90% confidence interval)

tacrolimus qd:bid50.3% (39.0-65.0)

Day 14‡ Mean (SD)Cmax (ng/mL) 25.65 (11.61) 25.07 (12.13)C24 (ng/mL) 8.82 (3.18) 8.53 (2.85)Tmax (hours) 2.8 (2.5) 1.9 (1.5)AUC0-24 (ng�h/mL) 324.19 (119.07) 286.99 (88.03)ln(AUC0-24) ratio (90% confidence interval)


Week 6§ Mean (SD)Cmax (ng/mL) 29.20 (10.28) 28.52 (11.51)C24 (ng/mL) 9.96 (3.53) 9.70 (3.21)Tmax (hours) 2.8 (1.7) 1.8 (1.7)AUC0-24 (ng�h/mL) 364.28 (110.52) 301.10 (60.76)ln(AUC0-24) ratio (90% confidence interval)


*Mean total daily dose on day 1: tacrolimus qd ¼ 0.118 mg/kg; tacrolimus bid ¼ 0.112 mg/kg.†Tacrolimus bid: Relative to preceding morning or evening dose.‡Mean total daily dose on day 14: tacrolimus qd ¼ 0.221 mg/kg; tacrolimus bid ¼ 0.176 mg/kg.§Mean total daily dose at week 6: tacrolimus qd ¼ 0.209 mg/kg; tacrolimus bid ¼ 0.165 mg/kg.Log-normalized AUC used for comparison between formulations.


30.7 and 99.2 6 48.8 lmol/L and creatinine clearancerates were 83.3 6 31.7 and 85.5 6 25.7 mL/minute fortacrolimus qd and tacrolimus bid, respectively. Therewere no clinically significant findings for other laboratoryvariables, body weight, blood pressure or pulse rate.

One-Year Follow-Up

Patients from the tacrolimus qd group had the optionto enter long-term follow-up at the end of the study.Patients must have been fully informed and musthave given written informed consent to demonstratecomprehension of the purpose and risks of the studyto participate. Patients were excluded from the follow-up phase if they were pregnant, nursing or not using

adequate contraception during the study. Of the 67patients in the Full Analysis Set who had received atleast 1 dose of tacrolimus qd, 47 (70.1%) patientschose to enter the long-term extension phase and con-tinue treatment with tacrolimus qd.

At entry to follow-up the mean tacrolimus qd dose(SD) was 0.168 (0.105) mg/kg. At 1 year after enteringthe follow-up phase, mean tacrolimus qd dose (SD)was 0.096 (0.073) mg/kg. Safety and efficacy findingswere consistent with the well-documented character-istics of tacrolimus bid1,8,9 with Kaplan-Meier esti-mated rates of both patient and graft survival of95.7% at 1-year follow-up. There were 2 graft losses,which were both due to patient deaths (acute respira-tory failure and abnormal hepatic function). Overall,

Figure 4. Mean AUC0-24 (SD) fortacrolimus (PK Evaluable Set).

Figure 5. Correlation betweenAUC0-24 and Cmin for tacrolimusadministered as tacrolimus qdand tacrolimus bid (PK EvaluableSet). Lines of best fit: tacrolimusqd: y ¼ 34.401x; tacrolimus bid:y ¼ 29.583x.


the Kaplan-Meier estimated rate of patients free fromBPAR at 1-year follow-up was 95.6%. At 1-year fol-low-up, mean (SD) serum creatinine and creatinineclearance rates were 101.0 lmol/L (20.5) and 86.3mL/minute (27.1), respectively.

DISCUSSION

This is the first study to characterize the PK of tacroli-mus in de novo liver transplant recipients. The pri-mary objective of this study was to compare PKparameters of tacrolimus qd and bid following livertransplantation. Systemic exposure (AUC0-24) on day 1was 50% lower for tacrolimus qd than for tacrolimusbid at equivalent doses. Following dose adjustments,the systemic exposure to tacrolimus as measured bywhole-blood trough level was similar by day 4 for bothformulations. In order to achieve similar tacrolimusexposure, higher doses of tacrolimus qd were required.

The reasons for differences in the extent of absorptionbetween the 2 tacrolimus formulations during the im-mediate posttransplant period are not yet fully under-stood. In order to achieve similar exposure to tacroli-mus bid, the initial dose of tacrolimus qd would needto be higher. A higher starting dose of tacrolimus qdhas been investigated in subsequent studies.

Because tacrolimus is a narrow therapeutic indexmedication, doses are optimized using whole-bloodtrough level monitoring as a surrogate marker ofexposure (AUC). Thus it is critical to ensure that therelationship and correlation between AUC and troughlevels is similar between any 2 formulations. Thisstudy has demonstrated a good correlation betweenwhole-blood tacrolimus trough levels and AUC0-24, anda similar slope of the regression line, in de novo livertransplant patients, for both tacrolimus qd and tacroli-mus bid. This indicates that, for drug-level monitoring,the same recommended target levels can be applied for

Figure 6. Most frequentlyreported AEs assessed by theinvestigator as causally relatedto study medication (MedicalDictionary for Regulatory Activitieshigh-level term). Full Analysis Set.No differences between tacrolimusqd and tacrolimus bid wereassociated with P < 0.05 (Fisher’sexact test). Renal failure wasdefined as any event reportedby the investigator as: renalinsufficiency, oliguria, acute renalfailure, acute renal impairment nototherwise specified, anuria, andincrease in serum creatinine.

TABLE 4. Renal Function

n Tacrolimus qd (n ¼ 67) n Tacrolimus bid (n ¼ 62)

Serum creatinine (lmol/L) Mean 6 SDDay 0 67 78.9 6 21.7 62 82.0 6 26.6Day 1 65 93.0 6 33.0 62 96.1 6 32.9Day 14 58 98.2 6 40.6 51 97.0 6 46.0Week 4 53 98.9 6 26.8 43 99.8 6 51.8Week 6* 51 99.5 6 30.7 42 99.2 6 48.8

Creatinine clearance (mL/minute) Mean 6 SDDay 0 67 111.7 6 39.4 62 106.4 6 38.6Day 1 65 100.3 6 45.0 62 93.2 6 36.2Day 14 58 90.2 6 34.3 51 93.5 6 38.1Week 4 53 84.2 6 27.7 43 86.4 6 28.4Week 6* 51 83.3 6 31.7 42 85.5 6 25.7

Full Analysis Set. Normal ranges: Serum creatinine < 133 lmol/L; Creatinine clearance 91 to 130 mL/minute.*Completers only.Comparisons between formulations at each time point did not reach statistical significance.


both formulations to ensure similar exposure (AUC).This observation is also supported by data from a pre-vious PK study in stable liver transplant patients con-verted from tacrolimus bid to tacrolimus qd.6

Although the study was not powered to show differ-ences in efficacy and safety profiles, these were com-parable for tacrolimus qd and bid during the 6-weekstudy period, with patient survival rates over 98% andgraft survival rates over 93% for both formulations.BPAR rates were also similar for tacrolimus qd andtacrolimus bid at 6 weeks. The overall safety profile oftacrolimus qd was comparable to that of tacrolimusbid in this 6-week study. Although some types of AEconsidered to be tacrolimus-related were higher in thetacrolimus qd arm (eg, infection, renal failure andimpairment, headache, and abnormal liver function),other AEs were higher in the tacrolimus bid arm (eg, di-abetes mellitus, hyperglycemia, tremor, nausea). There-fore, it is not possible to draw definite conclusions fromthe comparison between the 2 formulations from this6-week study with relatively small patient numbers.The higher incidence of infection in the tacrolimus qdgroup was mainly a result of procedure-related infec-tions (eg, wound and catheter-related infection); impor-tantly, the overall incidence and pattern of infection, aswell as the incidence of serious or dangerous infections,was similar between groups. The reasons for thegreater incidence of headaches and anemia amongtacrolimus qd recipients is unclear, particularly withthe small sample size of the current study.

For tacrolimus qd patients who were entered into thelong-term follow-up, efficacy and safety profiles wereconsistent with the well-described characteristics oftacrolimus bid after 1 year,1 with patient and graft sur-vival rates in excess of 95% at 1-year follow-up. Renalfunction was similar between tacrolimus bid and qd for-mulations at the end of the 6-week study and remainedstable with tacrolimus qd after 1 year of follow-up.

The strengths of the study design are that thisstudy was adequately powered to compare PK param-eters, and validated assay methods10 were used todetermine tacrolimus concentrations. Notably, the PKEvaluable Set only included patients who completedall PK evaluations and had no other protocol viola-tions. A potential limitation associated with the studymay be that, due to the 6-week study duration andrelatively low patient numbers, the study was notdesigned to show differences in efficacy and safetybetween tacrolimus qd and tacrolimus bid. These pa-rameters will be investigated in further phase 3 trialsthat are currently underway.

The higher mean total daily doses with tacrolimusqd versus tacrolimus bid achieved similar exposure asmeasured by trough levels and as determined by theobserved AUC at week 2 and week 6.

In conclusion, this study provides evidence thattacrolimus qd can be administered to de novo livertransplant recipients; furthermore, the equally goodcorrelation and similar slope of the line for tacrolimustrough levels and AUC for both formulations suggeststhat targeting the same trough level should achieve

similar total AUC over the 24-hour period for bothtacrolimus qd and tacrolimus bid.

ACKNOWLEDGMENTSTacrolimus Modified Release Liver Study Group:L. Fischer (Germany); P. Trunecka (Czech Republic);B. Gridelli (Italy); M. Colledan (Italy); A. Roy (Canada);D. D’Amico (Italy); A. Valdivieso (Spain); E. Varo(Spain); J. Langrehr (Germany); S. Lynch (Australia);D. Samuel (France); B.-G. Ericzon (Sweden); K. Boudjema(France); G. McCaughan (Australia); R. Margreiter (Aus-tria); J. Tchervenko (Canada); C. Scudamore (Canada);C. Margarit (Spain); G.P. Pageaux (France); J. O’Grady(UK); L. Backman (Sweden); M. Meurisse (Belgium).The design and results of this study have been pre-sented previously in an abstract at the AmericanTransplant Congress 2005: 6th Annual Joint Meetingof the American Society of Transplant Surgeons andthe American Society of Transplantation; May 21-25,2005; Seattle, WA, USA.The authors thank Caroline Anthony, a professionalmedical writer with Acumed, for her assistance indrafting and revising the manuscript.

REFERENCES

1. Scott LJ, McKeage K, Keam SJ, Plosker GL. Tacrolimus:a further update of its use in the management of organtransplantation. Drugs 2003;63:1247-1297.

2. Staatz C, Tett S. Clinical pharmacokinetics and pharma-codynamics of tacrolimus in solid organ transplantation.Clin Pharmacokinet 2004;43:623-653.

3. Wallemacq P, Armstrong VW, Brunet M, Haufroid V, HoltDW, Johnston A, et al. Opportunities to optimize tacroli-mus therapy in solid organ transplantation: report of theEuropean Consensus Conference. Ther Drug Monit 2009;31:139-152.

4. Florman S, Alloway R, Kalayoglu M, Lake K, Bak T, KleinA, et al. Conversion of stable liver transplant recipientsfrom a twice-daily Prograf-based regimen to a once-dailymodified release tacrolimus-based regimen. TransplantProc 2005;37:1211-1213.

5. Florman S, Alloway R, Kalayoglu M, Punch J, Bak T,Melancon J, et al. Once-daily tacrolimus extendedrelease formulation: experience at 2 years postconver-sion from a Prograf-based regimen in stable liver trans-plant recipients. Transplantation 2007;83:1639-1642.

6. Alak AM, Moy S, Cook M, Lizak P, Niggebiugge A,Menard S, Chilton A. An HPLC/MS/MS assay for tacroli-mus in patient blood samples. Correlation with results ofan ELISA assay. J Pharm Biomed Anal 1997;16:7-13.

7. First MR, Fitzsimmons WE. New drugs to improve transplantoutcomes. Transplantation 2004;77(9 Suppl):S88-S92.

8. O’Grady JG, Hardy P, Burroughs AK, Elbourne D; the UKand Ireland Liver Transplant Study Group. Randomizedcontrolled trial of tacrolimus versus microemulsifiedcyclosporin (TMC) in liver transplantation: poststudy sur-veillance to 3 years. Am J Transplant 2007;7:137-141.

9. Jonas S, Neuhaus R, Junge G, Klupp J, Theruvat T,Langrehr JM, et al. Primary immunosuppression withtacrolimus after liver transplantation: 12-years follow-up. Int Immunopharmacol 2005;5:125-128.

10. Jusko WJ, Thomson AW, Fung J, McMaster P, Wong SH,Zylber-Katz E, et al. Consensus document: therapeuticmonitoring of tacrolimus (FK-506). Ther Drug Monit1995;17:606-614.


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