PHARMACOKINETICS AND DISPOSITION

Population pharmacokinetics of plasma-derived factor IXin adult patients with haemophilia B: implicationsfor dosing in prophylaxis

Sven Björkman & Victor Åhlén

Received: 14 November 2011 /Accepted: 3 January 2012 /Published online: 27 January 2012# Springer-Verlag 2012

AbstractPurpose Knowledge of the pharmacokinetics (PK) ofplasma-derived factor IX (FIX) is still inadequate, withconflicting findings on its elimination half-life and as yet noanalysis of the variance in PK between and within individuals.The aim of the study was thus to characterize the PK ofplasma-derived FIX, including estimates of variance betweenand within patients, in adult patients and to predict the varia-tion between individuals in dose requirement to produce atarget trough level during regular prophylaxis.Methods Plasma FIX versus time data were compiled fromfour published and one unpublished PK study involving atotal of 26 adult patients with severe haemophilia B. Thenumber of PK assessments per patient varied between oneand eight, yielding in total 893 measured FIX levels from 80study occasions. A population PK model was developed todescribe the whole dataset. Parameter values from the modelwere used to calculate the dose requirement to maintain atrough level of 1% of normal FIX activity in each patient.Results The disposition of FIX was well described by athree-compartment PK model. The median eliminationhalf-life was 31 h, and the variation between individualswas modest both in PK and in dose requirement duringtwice-weekly prophylaxis.Conclusion With twice weekly dosing, the need for PK-based dose tailoring of FIX in adult patients appears to belimited. However, monitoring FIX levels should be consid-ered in children, in patients who do not respond satisfacto-rily to standard dosing, and if treatment is switched fromplasma-derived to recombinant FIX.

Keywords Factor IX . Haemophilia B . Pharmacokinetics .

Dosing . Prophylaxis

Introduction

Haemophilia B is a rare disease characterized by congenitaldeficiency of coagulation factor IX (FIX). Treatment orprevention of bleeding events in patients with haemophiliaB requires that an adequate plasma level of coagulationfactor is obtained by infusion of a FIX concentrate. In thecases of spontaneous bleedings, trauma or surgery, FIXprocoagulant activity (FIX:C) levels of between 0.30 and1.0 IU/mL are aimed for, depending on the site and severityof the bleeding [1, 2]. Prophylactic treatment, on the otherhand, theoretically requires the long-term maintenance of a“protective” FIX:C plasma level. Since a constant level ofFIX:C cannot be obtained with intermittent administration,the target is an adequate minimum (i.e. trough or pre-dose)level. This was originally suggested as 0.01 IU/mL, but theactual requirement may vary between patients [3–5]. Irre-spective of the aims and type of treatment, the achievedplasma level and therapeutic response depend on the doseand on the pharmacokinetics (PK) of FIX:C in the patient.Thus, rational dosing requires a knowledge of the PK ofFIX, preferably in the individual, but at least in the popula-tion of patients under consideration [4–7].

Plasma-derived and recombinant FIX differ in PK prop-erties and thus in dose requirements for both treatment andprophylaxis. The PK of recombinant FIX is well docu-mented according to required procedures for a new drug,and the findings are generally consistent between studies[8]. The PK of plasma-derived FIX, however, has beeninvestigated for five decades in small studies of varyingdesigns and study protocols, in particular as regards the

S. Björkman (*) :V. ÅhlénDepartment of Pharmaceutical Biosciences, Uppsala University,Box 591, SE-751 24, Uppsala, Swedene-mail: sven.bjorkman@farmbio.uu.se

Eur J Clin Pharmacol (2012) 68:969–977DOI 10.1007/s00228-012-1211-z

number of blood samples taken and total time of sampling[6, 7]. It has been shown that adequate blood sampling for atleast 56 and most preferably 72 h is required to yield reliableestimates of the PK of plasma-derived FIX [6, 9] (Europeanregulatory guidelines [10] stipulate sampling up to 50 h,with an optional 72-h sample). To date, eight studies withsampling for at least 72 h have been reported in the literature[9, 11–17]. Compilation of these findings reveals a fragmen-tary description of the PK of FIX [8], with conflicting valuesreported for even such a basic parameter as elimination half-life. It is also important to estimate the magnitude of vari-ance in PK, both between individuals and, if possible,between study occasions within the same individual. Suchdata are available for coagulation factor VIII (FVIII)[18–20] but not for FIX.

Consequently, the first aim of this study was to charac-terize the PK of plasma-derived FIX, including estimates ofvariance between and within patients, in a reasonably largesample of adult patients by means of a population PKmodel. The second aim was to predict variation betweenindividuals in dose requirement to produce a 0.01 IU/mLtrough level of FIX:C during regular prophylaxis, using theestimates from this model. For these purposes, we collectedFIX:C versus time data from 80 study occasions in 26patients with severe haemophilia B. Multi-dose FIX:C lev-els during prophylaxis were then simulated as previouslydescribed [21, 22].

Methods

Patients

Data on patient characteristics, dosing and FIX:C levelsversus time were compiled for 12 subjects in the Malmö(Sweden) cohort of prophylaxis patients. These subjects hadparticipated in four original studies, performed between1990 and 2004, which had all been separately approved bythe Ethics Committee of Lund University [9, 12, 21, 23].Data from another 14 patients were provided by Bio Prod-ucts Laboratory Ltd. (Elstree, UK; www.bpl.co.uk) from anunpublished study performed between 1997 and 1999 on thehigh-purity FIX preparation Replenine-VF. In all studies,inclusion criteria were biochemically severe disease (definedin Malmö as baseline FIX:C <0.01 IU/mL and in theReplenine-VF study as <0.02 IU/mL), informed consent andno indication of circulating FIX antibodies (inhibitors). Thepatients were ambulant and had no apparent bleedings at thestudy occasions. All samples were analysed using the one-stage clotting assay [24]. The within-day coefficient of varia-tion (CV) of this assay in Malmö was 11% at 0.04 IU/mL,5.4% at 0.48 IU/mL and 4.9% at 1.0 IU/mL [9]. At BioProducts, the within-day CV was generally around 2%, and

the between-day CV was 5.7 % at 0.55 IU/mL and 3.9% at0.94 IU/mL (Roger Luddington, personal communication).

The first Malmö data were from a cross-over comparisonof the three high-purity FIX preparations Preconativ, Nano-tiv (both, at the time, from Kabi Pharmacia, Stockholm,Sweden) and Mononine (from Armour, Eastbourne, UK),yielding fifteen 72-h datasets from five patients [12]. Theblood sampling times were at 0, 5, 10, 15, 20, 30 and 45 minand 1, 2, 3, 6, 9, 12, 24, 32, 48 and 72 h after the end of the10-min infusion. These patients and one more patient thenparticipated in a 104-h blood sampling study in which twodoses of Nanotiv were given 1 month apart, generating 12datasets [9]. The sampling times were 0, 5 and 20 min and 1,3, 6, 9, 24, 30, 34, 48, 56, 72, 80, 96, 100 and 104 h after theend of the 15-min infusion. The third study [21] exploredtailored dosing of FIX in these six and another two patients,about 4 years later. Complete 72-h profiles (sampling times0 and 5 min and 1, 6, 9, 24, 31, 48, 56 and 72 h after the 5-min infusion) were obtained in the two new patients. Fouradditional samples were later obtained from each patient, at48, 60, 72 and 76 h after the latest FIX infusion duringprophylactic treatment. Another 3–4 years later, five of thesepatients had single blood samples taken during a follow-upstudy [23], in which the PK of FIX:C was also estimated infour new patients by limited sampling (3 blood samples, at10 min, 45 and 50 h after infusion).

In the Replenine-VF study, the test preparation was infusedtwice, with a 12-week interval, in all 14 patients. Seven of thesepatients had received a comparator preparation (Replenine: 4patients; Alphanine: 1 patient; BeneFIX: 2 patients) 1 weekprior to the first dose of Replenine-VF. The data obtained withReplenine and Alphanine were also included in the analysis(but not the BeneFIX data since this is recombinant FIX). Thedose used for all preparations was 71–90 IU/kg. Blood sampleswere taken before infusion and at 10 and 30 min and 1, 3, 6, 9,12, 24, 30, 36, 50 and 56 h after the end of infusion. Exact timeswere recorded and used in the data evaluation. The study wasperformed in accordance with present European regulatoryguidelines [10].

The total number of study occasions was thus 80, ofwhich 60 yielded full 56- to 72- or 104-h PK datasets andthe remaining 20 sparse data (1–4 FIX:C values). The totalnumber of plasma FIX:C values was 893 (Fig. 1). The ageof the patients, as recorded on each occasion, ranged from16 to 65 (median 39) years and their body weight (BW)from 47 to 115 (median 70) kg. The number of studyoccasions per patient was: 1 (n05 patients), 2 (n010), 3(n05), 4 (n01), 6 (n01), 7 (n02) and 8 (n02).

Data analysis

The baseline level of FIX:C, which was available from theclinical or study records of the patient, was subtracted from

970 Eur J Clin Pharmacol (2012) 68:969–977

all measured values. In a few cases, pre-infusion values ofup to 0.1 IU/mL were found as residues from previousdoses. This extra background was subtracted assumingfirst-order elimination of previously present FIX [9].

The PK model was built using non-linear mixed effectsmodelling by means of the software NONMEM, ver. 6(ICON Development Solutions, Ellicott City, MD). Theprocedure was similar to that used in an earlier study onFVIII [19]. The model was built in a stepwise fashion. First,the structural (compartmental PK) model was developed inconjunction with the residual error model. Inter-individualvariance (IIV) was added and, since most patients hadreceived FIX several times, inter-occasion variance (IOV)was also included [25]. Finally, age and FIX concentratepreparation were tested as covariates (BW was includedalready in the structural model, as will be described below).

The model-fitting was performed using subroutinesADVAN11 TRANS1 and the first-order conditional estima-tion method (FOCE) with interaction. The statistical pack-age R (The R Foundation for Statistical Computing, Vienna,Austria) and Xpose ver. 4 [26] were used for data setcheckout, model exploration and diagnostics. The abilityof each successive model to describe the data was evaluatedusing graphical evaluation, the objective function value(OFV) and the precision (percentage relative standard error;%RSE) of parameter estimates. The main tool for selectionbetween hierarchical models was the likelihood ratio test,i.e. the difference in OFV between models. If the modelsdiffer by one parameter, a difference in OFV of >3.84 issignificant at the 5% level. The corresponding value forp00.01 is 6.63.

The structural models tested were the standard two- andthree-compartment PK models. The two-compartment modelparameters were elimination and inter-compartmental clearance

(CL and Q2) and volumes of the central and second compart-ment (V1 and V2). For the three-compartment model, a secondinter-compartmental clearance (Q3) and a third compartmentvolume (V3) were added. The residual error was described by acombined additive and proportional error model, in whicheither of these terms could be deleted if it was found to benon-significant. Exponential models were applied to accountfor IIV and IOV in the PK parameters according to:

Parameter valueik ¼ Typical value� expηiþkik ð1ÞThe subscripts i and k denote individual and occasion,

respectively, and the typical value is the mean value of theparameter in the population. ηi is the random effect account-ing for individual deviation from the typical value (i.e. IIV),and κik is the random effect accounting for the IOV. ηi andκik are assumed to be symmetrically distributed with a meanof 0 and an estimated variance of ω2 and π2, respectively.

Allometric scaling based on BW was applied to the PKparameters [19, 27], as described by the equation:

Typical value ¼ θ1 � BW

Median BW

� �θ2

ð2Þ

In this expression, θ1 is the parameter value for a subjectwith a BW corresponding to the median BW of the popula-tion, and θ2 is the allometric exponent. Initially, θ2 wasfixed to 0.75 for the clearance parameters (CL, Q2, Q3)and to 1.0 for the volume parameters (V1, V2, V3).

Following the building, evaluation and choice of the finalmodel, individual values for CL, Q2, Q3, V1, V2, V3 andVss (V1 + V2 + V3) were obtained for all patients and allstudy occasions as empirical Bayes estimates (EBE), i.e. byBayesian estimation using the population model parametersin conjunction with the FIX:C level data from each individ-ual [28]. Mean residence time (MRT) and all half-lives (t½α, β and γ) were calculated by standard methods [29].Finally, in vivo recovery [6] was calculated as the inverseof V1 (expressed as dL/kg).

Shrinkage of the EBE distribution, i.e. η-shrinkage (Shrη)was estimated as [30, 31]:

Shrη ¼ 1� SDðηEBEÞw

ð3Þ

In this expression, SD(ηEBE) is the standard deviation ofthe EBEs of η, and ω is the population model estimate ofthe standard deviation in η. Shrinking of the distribution ofindividual weighted residuals (IWRES) towards zero, i.e. ε-shrinkage (Shrε), was calculated as [30, 31]:

Shr" ¼ 1� SDðIWRESÞ ð4Þwhere IWRES are the individual weighted residuals. Thefinal diagnostic test was a visual predictive check (VPC),i.e. creating a large set of simulated datasets by means of the

Time after dose (hours)

FIX

:C (

IU/m

L)

0.5

1.0

1.5

0 20 40 60 80 100

Fig. 1 Observed plasma-derived factor IX procoagulant activity (FIX:C) levels as function of time after infusion, together with a visualpredictive check (VPC) of model-predicted FIX:C levels. Solid linesMedian and 5–95th percentile of the observed data from all patients,dashed lines 5–95th percentile around the median and the 5–95thpercentiles of the model predictions

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model and comparing the distribution of the simulatedconcentration (level) versus time data with the real data[32].

The dose of FIX needed to maintain a 0.01 IU/mLtrough level (Cmin) of exogenous FIX:C (i.e. above thepatient’s own baseline level) during regular prophylactictreatment was calculated as previously described [21, 22]for each patient. In brief, FIX:C versus time curves weresimulated for 2 weeks of treatment from the tri-exponential PK functions, assuming administration twicea week (“Monday and Thursday” [8]) of the dose used inthe individual PK study. Trough levels obtained on days10 and 14 (corresponding to a treatment time exceedingby fourfold the longest elimination half-life found at anystudy occasion) were used to calculate the adjusted doseneeded to obtain 0.01 IU/mL.

Results

The model development is outlined in Table 1. Based on thesignificant (P<0.01) decrease in OFV, a three-compartmentmodel described the data better than a two-compartmentmodel. Incorporation of IOV for CL and V1 as well as IIVfor all volume parameters further improved the model. Asfor covariates, BW was accounted for by the allometricscaling. Estimation of the value of the exponent for CL(instead of fixing it at 0.75) improved the model significant-ly, while estimation of the exponent for V1 (instead of fixingit at 1.0), alone or simultaneously with the CL exponent, didnot. Age was not a significant separate covariate for CL orV1. There was no significant systematic difference in CLbetween preparations investigated in Malmö and those inthe Bio Products Laboratory study. However, within theMalmö data, the CL of Mononine was significantly lowerthan that of the other preparations.

Thus, the final population PK model for FIX:C was athree-compartment model with a combined additive andproportional residual error model. There was good agree-ment between model predictions and observations of FIX:Clevels (Figs. 1, 2). The η-shrinkage values were 15% for CL,5% for V1, 12% for V2 and 25% for V3, while the ε-shrinkage was −34%. The primary typical parameter esti-mates, θ, (in an individual with a BW of 70 kg) are pre-sented in Table 2. Their precision was high for the centraland third compartment parameters (CL, V1, Q3 and V3; %RSE ≤10%) and lower but still acceptable for the secondcompartment parameters (Q2 and V2: %RSE ≤35%). Thedistribution of FIX could be described as comparatively fast(Q202.0 L/h) to a shallow (0.81 L) second compartment,with an additional slow (Q300.17 L/h) distribution to adeeper (2.9 L) third compartment. Inter-occasion, orwithin-individual variance (IOV) was lower than inter-individual variance (IIV) in CL and V1.

Table 3 shows a summary of individual (empirical Bayes)estimates (EBE) of the FIX:C disposition. On some studyoccasions where three or less FIX:C values had beenobtained the estimates proved to be poorly supported bythe data. The table is therefore based on 68 study occasions,at which at least four blood samples were taken in 22 of thepatients and depicts variance in PK both within (IOV) andbetween (IIV) patients as well as the probable influence ofthe differences in sampling schedules (from 17 post-infusionsamples over 104 h [9] down to 4 samples in the timeinterval 48–72 h after dose [21]). Still, the ratio of the 90thand 10th percentile of the values was only 1.8 for CL (1.7for CL per kilogram BW), 1.6 for Vss (2.2 for Vss perkilogram BW), 1.7 for MRT and 2.0 for the eliminationhalf-life. It exceeded 3 only for V2, V3 and the first distri-bution half-life.

Figure 3 shows the calculated dosing needed to produce a0.01 IU/mL (1%) trough level in the same 22 patients. These

Table 1 Model building stepsthat resulted in statistically sig-nificant decreases of the objec-tive function value

NOP, Number of estimatedparameters (θ, η and κ; for def-inition see Data analysis) in themodel; OFV, objective functionvalue; IIV, inter-individual vari-ance; CL clearance; V1, V2, V3,volume of the central, secondand third compartment,respectively

Model Description of model NOP OFV

Structural model:

1 Two-compartment with IIV on V1 and CL 8 −4523

2 Three-compartment with IIV on V1 and CL 10 −4543

Inter-occasion variance (added to model 2):

3 For V1 11 −4630

4 For CL 11 −4879

5 For both V1 and CL 12 −5032

Further IIV (added to model 5):

6 For V2 13 −5108

7 For V2 and V3 14 −5148

Covariates (added to model 7):

8 Estimation of allometric exponent for CL 15 −5156

9 Preparation0Mononine, as covariate on CL (added to model 8) 16 −5161

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doses were calculated based on the combined PK informa-tion obtained in each patient, i.e. the averages of calcula-tions from the separate study occasions are presented. Thevariation between the patients was small and unrelated toBW, except that the patient with BW093 kg stands out as

having a high dose requirement. The highest CL (474 mL/h)and shortest elimination half-life (15 h) shown in Table 2were found in this patient (at the other study occasion thesevalues were 350 mL/h and 19 h, i.e. still high and lowcompared to the medians).

Discussion

The primary reason for performing this study was the cleardiscrepancies in reported PK of plasma-derived FIX [6–8].For example, according to a recent summary [8], studieswith adequate blood sampling carried out on between sixand 25 adult subjects showed mean elimination half-livesranging between 18 and 43 h. This issue is of clinicalinterest since uncertainty in the estimated half-life seriouslyinfluences the calculation of trough levels and dose require-ments to maintain a pre-determined level during prophylaxis[5]. The prevalence of haemophilia B is estimated at ap-proximately 1 in 30 000 males [33]. Of these, only 30–40%have severe disease [34] and would be eligible for a PKstudy. Consequently, with 26 patients and 80 infusions, ourstudy is, to the best of our knowledge, the largest PK studyon plasma-derived FIX, with adequate blood sampling,reported to date and the first to use population PK method-ology. It is also the first one in which FIX has been found tofollow three-compartment PK. Distinguishing three-compartment from two-compartment PK with the conven-tional separate curve-fitting on FIX:C data from each pa-tient, as in previous studies [9, 11–17], may be virtuallyimpossible, since a few data points deviating from the two-compartment curve are not sufficient to reject the simplermodel in favour of the more complex one. Adequatestatistical power is more easily obtained with a popula-tion model—in our case with a disposition pattern madeup from 893 data points.

Differences in study methodology and difficulties to fit amodel to individual FIX:C curves may also explain part ofthe variation in reported PK parameter values. EliminationCL (easily determined from the area under the curve) rangedbetween 3.8 and 6.3 mL/h per kilogram in six studies [9, 12,13, 15–17]. Together with the results from the present study,these values confirm that the CL of plasma-derived FIX issubstantially lower than the 7.5–9.1 mL/h per kilogramnormally reported for recombinant FIX [8]. Vss has notbeen calculated in any previous study, with the exceptionof the two studies [9, 12] whose data are included in thepresent model. In the detailed analysis [9] of FIX PK basedon 104 h of sampling, the mean values of V1, V2 and Vsswere 82, 65 and 147 mL/kg. When the two-compartmentpopulation PK model (Table 1, model 1) was applied to thepresent data, typical V1, V2 and Vss were 90, 51, and141 mL/kg. In the three-compartment model, the typical

Individual predicted level (IU/mL)

Obs

erve

d le

vel (

IU/m

L)

0.1

1

0.1 1

Fig. 2 Goodness of fit. Observed and individually model-predictedFIX:C levels plotted against each other. Line Line of identity

Table 2 Parameter estimates (θ, η and κ) for the final model

Parameter Mean %RSE

Structural model parameters:

Clearance (CL: L/h)a 0.29 3.7

Volume of central compartment (V1: L)a 5.71 3.6

Distribution clearance to compartment 2 (Q2: L/h)a 1.99 35

Volume of compartment 2 (V2: L)a 0.81 19

Distribution clearance to compartment 3 (Q3: L/h)a 0.17 6.7

Volume of compartment 3 (V3: L)a 2.89 10

Inter-individual variability parameters:

Clearance (%CV) 23 64b

Volume of central compartment (%CV) 19 37b

Volume of compartment 2 (%CV) 63 30b

Volume of compartment 3 (%CV) 78 81b

Inter-occasion variability parameters:

Clearance (%CV) 15 36b

Volume of central compartment (%CV) 12 24b

Residual variability parameters:

Additive residual error (IU/mL) 0.0037 10

Proportional residual error (%CV) 9.2 11

Covariate parameters:

Allometric exponent of clearance 1.26 16

CL (% difference for preparation Mononine) -16 47

%RSE, Relative standard error; %CV, percentage coefficient ofvariationa Typical value for an individual with a median body weight, i.e. 70 kgb The %RSE for corresponding variance or covariance term

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values of V1, V2, V3 and Vss were 82, 12, 41, and 136 mL/kg. The three-compartment model thus approximately di-vided up the V2 of the two-compartment model into a smallV2 and a larger V3, with the V1 and Vss remaining practi-cally unchanged. The previous [9] “physiological” interpre-tation of V2 as one extravascular compartment could thus bemodified to V2 and V3 representing extravascular compart-ments characterized by fast and slow exchange of FIX,respectively, with the general circulation. The eliminationhalf-life of plasma-derived FIX has previously been foundto average 29–34 h [9, 11, 12, 14, 16, 17]; however, meanvalues of 18 [13] or 43 h [15] have also been reported after acomplete 72 h of blood sampling. Our study establishes that

the average elimination half-life of plasma-derived FIX isapproximately 30 h, thereby confirming that the averagehalf-life is longer for plasma-derived FIX than the 18–24h generally found [8] for recombinant FIX.

Incremental in vivo recovery, a parameter traditionallyused in coagulation factor PK and ubiquitously [1–20, 22]estimated or discussed, was also calculated. It represents theFIX:C level observed immediately after infusion in relation-ship to the administered dose, that is, recovery equals C0

(IU/dL) divided by dose (IU/kg). Recovery is normallymeasured by taking a blood sample soon after the end ofinfusion (precise sampling time varies between studies), butapplying this concept in a PK model means calculatingdistribution of the dose in V1. Thus a “model-predicted”recovery becomes the inverse of V1 in IU/dL. The recoveryestimated here agrees with previously reported mean valuesranging between 1.0 and 1.7 IU/dL per IU/kg [7–9, 13–17].

BW and age were tested as covariates in the model. Theformer was accounted for by allometric relationships. Ini-tially, the allometric exponent of CL estimates (CL, Q2 andQ3) was set to 0.75 [19, 27]. This is similar to using bodysurface area (BSA) as a proportionality factor since BSA isapproximately related to BW to the power of 0.7, or 2/3[35]. For volume terms, the default allometric exponent is1.0, i.e. assuming direct proportionality between volumes ofdistribution and BW. The allometric exponent of CL wasestimated at 1.26 instead of 0.75, while the remaining oneswere kept at their default values. Since all subjects were adultswith mostly normal body weights, success at finding biologi-cally meaningful empirical values for allometric exponents

Table 3 Individual pharmaco-kinetic parameter estimates from68 study occasions in 22 patients

Parameter Unit Medianvalue

10th and 90thPercentiles

Totalrange

Clearance (CL) mL/h 259 192–351 156–474

mL/h per kg 4.16 3.01–5.13 1.94–6.12

Volume of central compartment (V1) L 5.48 4.12–6.65 3.37–8.30

mL/kg 82 57–104 48–124

Incremental in vivo recovery IU/dL / IU/kg 1.23 0.97–1.77 0.81–2.10

Volume of compartment 2 (V2) L 0.84 0.36–1.23 0.34–1.76

mL/kg 12 4.7–22 4.6–27

Distribution clearance tocompartment 2 (Q2)

L/h 1.91 1.55–2.20 1.48–2.53

Volume of compartment 3 (V3) L 3.22 1.69–4.54 0.47–5.20

mL/kg 48 22–87 6.5– 97

Distribution clearance to compartment3 (Q3)

L/h 0.16 0.13–0.19 0.13–0.22

Volume of distribution atsteady state (Vss)

L 9.02 7.51–11.6 4.95–13.7

mL/kg 148 97–214 69– 245

First distribution half-life (t½ α) Hours 0.26 0.11–0.40 0.11–0.51

Second distribution half-life (t½ β) Hours 7.0 4.4–9.2 1.7–10.7

Elimination half-life (t½ γ) Hours 31 22–43 15–52

Mean residence time (MRT) Hours 35 27–46 20–55

Fig. 3 Dose requirement of FIX to maintain a 0.01 IU/mL trough levelof exogenous FIX:C. The weekly dose is divided into the “Mondaydose” (solid bars) and “Thursday dose” (diagonally striped bars)

974 Eur J Clin Pharmacol (2012) 68:969–977

cannot be expected. However, since the allometric exponentfor CL was estimated with a 16% RSE and an improvement of8.1 in OFV, it was retained in the model. Age could not beidentified as a significant covariate, which was expected sinceno data were available from children. The model is thereforevalid only for adult patients. This has limited practical meaningsince recombinant FIX (with different PK) is preferred toplasma-derived FIX in patients who have not previously beenexposed to plasma-derived products, which is normally thecase for children newly diagnosed with haemophilia.

The only other significant covariate was a 16% lower CLfor Mononine, which approximately agrees with previous[12] findings from a conventional PK comparison betweenNanotiv, Preconativ and Mononine. This may be related todifferences in true potency (i.e. of actual dose) of the threepreparations in the study. The finding of no significantdifference in CL between preparations investigated inMalmö and in the Replenine-VF study suggests that therewere no important between-site differences in the laboratoryassays.

Model diagnostic plots (in particular, the observed vs.individual predicted level, Fig. 2) have been shown tobecome misleading when either η- or ε-shrinkage reachesaround 20–30%. In all, the obtained shrinkage values wereregarded as acceptable. A negative ε-shrinkage means thatthe standard deviation of the individual weighted residualsis >1, which can be found when, as in the present case, thedataset is characterized by rich data from a small number ofsubjects [31]. Thus, all diagnostic plots (Figs. 1, 2) testify toa satisfactory fit of the final model to the data.

The variance in PK of FIX between the patients was low,with an IIV of only 23% CV for CL and 19% CV for V1.Incorporation of inter-occasion variance (IOV) for CL or V1improved the model significantly; however, the IOV waseven lower than the IIV. These estimates are based on allavailable data, which were used to build the model. How-ever, obtaining individual EBEs could be difficult for studyoccasions where only sparse data were collected. Thus,individual estimates were calculated for 68 study occasionsin 22 patients (not after selective deletion of “bad” estimatesbut by general exclusion of all occasions with <4 bloodsamplings). The variance was modest for these estimatesas well (Table 3), in particular for key PK parameters suchas CL, V1, incremental in vivo recovery and eliminationhalf-life. This implies that PK determination in the individ-ual gives better information than application of the meanpopulation PK.

The precise relationship of FIX effect, that is the preven-tion of bleeding, to FIX:C level is not known. Thereby, theconcept of keeping a satisfactory minimum level of exoge-nous FIX:C by means of repeated infusions is generallyapplied. Setting a target coagulation factor level of0.01 IU/dL is a convenient way to illustrate how to maintain

a “protective” plasma level but does not imply that it is inreality the critical value for all patients [5, 8, 36]. There wasonly a modest variation in average dose requirement to givethis trough level—approximately twofold for 21 of the 22patients (Fig. 3). This observation supports a previous find-ing [21] that infusion every third day in eight adult patientsrequired doses ranging only between 750 and 1500 IU (13–21 IU/kg) to produce a trough level of 0.014–0.017 IU/mL.The calculated doses (both here and previously) were low incomparison to an originally recommended [3] dosage of 25–40 IU/kg twice weekly. However, they were estimated foradult patients, and higher doses per kilogram are possiblyneeded in children. The calculated doses are in fair agree-ment with the 9 or 18 IU/kg twice weekly successfully usedin an early prophylaxis study [37].

Infusion of plasma-derived FIX twice a week means thatthe intervals between infusions are approximately two- tothreefold the median elimination half-life. Since recombi-nant FIX has a typical half-life of 18–24 h [8], the compa-rable dosing scheme would be every other day. In this case,the variation in calculated dose was four- to fivefold (5–23 IU/kg) [22]. Considerably higher variation (approximate-ly tenfold ranges) has been estimated for FVIII in adolescentto adult patients [5, 36, 38]. This is at least in part explainedby the fact that dosing intervals for FVIII are longer on thehalf-life timescale. Two days correspond to fourfold a typ-ical 12-h half-life and three days to sixfold. The longer thedosing interval the greater is the impact of individual vari-ation. Consequently, with twice-weekly dosing, thereappears to be less need for PK tailoring in adult haemophiliaB patients than during normal prophylactic dosing of FVIIIin haemophilia A.

Clinical implications

Characterizing the disposition of plasma-derived FIX andascertaining that intra-individual variance is lower thaninter-individual variance are prerequisites for PK-based cal-culation of appropriate dosing during the prophylactic treat-ment of haemophilia B. The present study achieved thesegoals, but only for adult patients. However, at least in theaffluent part of the world, recombinant FIX is normallypreferred to plasma-derived FIX for the treatment of chil-dren. The study also confirms the difference in PK betweenthe two types of FIX. Twice-weekly dosing, with 15 IU/kgfor the 3-day dosing interval and 30 IU/kg for the 4-dayinterval, rounded upwards to the nearest available vial size,would be adequate to maintain a 0.01 IU/mL trough level inmost adult patients. Whether this is sufficient also to preventbleeding has, however, to be investigated for each patient.The variation between individuals was modest both in termsof PK and in dose requirement to achieve the 0.01 IU/mL

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trough level. Thus, with this dosing there appears to bea limited need for routine PK dose tailoring in adultpatients. However, monitoring of FIX:C levels shouldbe considered in children, in patients who do not re-spond satisfactorily to standard dosing, and in patientswhose treatment is switched from plasma-derived torecombinant FIX.

Acknowledgements The authors wish to thank Bio Products Labo-ratory for making these data available, Rukhsana Shaikh-Zaidi forproviding additional information whenever needed and Peter Collinsfor helping us to find data and to get in touch with the company, and forcritical reading of the manuscript. The original researchers in theReplenine-VF clinical study were Dr. P. Collins (Cardiff), Dr. C. Raper(Hull), Dr. V. Mitchell (Leicester), Dr. D. Prangnell (Lincoln), Dr. T.Nokes (Basingstoke), Professor C.A. Lee (London) and Dr. T. Baglin(Cambridge). The authors also wish to thank Anna Folkesson, SivJönsson, Akash Khandelwal and Kajsa Harling for advice and technicalhelp in various stages of the modeling.

Conflict of interest None.

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