Keywords
factor VIII - hemophilia A - pharmacokinetic - observational study - population PK
Introduction
Regular replacement therapy (“prophylaxis”) with clotting factor concentrates has
been demonstrated to reduce the frequency of joint bleeds (hemarthroses) in individuals
with moderate/severe hemophilia A and B, resulting in the prevention of disabling
and painful joint damage and improved health outcomes.[1]
There is evidence that the risk of spontaneous joint bleeding in persons with hemophilia
A increases with the duration of time spent with factor VIII activity (FVIII:C) levels
below 1 international unit (IU)/dL (1%).[2] As such, the recommendation has been to maintain FVIII:C levels >1% for persons
with severe hemophilia. Factor levels (peaks and troughs) in persons with severe hemophilia
A receiving prophylaxis are primarily influenced by the individual's pharmacokinetic
(PK) profile together with the frequency of FVIII infusions. In clinical practice,
FVIII doses are typically calculated based on body weight without adjustment for individual
PK profiles between individuals with severe hemophilia A.[3] This may result in vastly different levels of protection between individuals when
given similar doses on a per weight basis; those with a “better” PK profile, as evidenced
by a slower clearance (Cl) of infused FVIII and a longer terminal half-life (t
1/2), may be well protected while those with a “worse” PK profile, as evidenced by a
faster Cl of infused FVIII and a shorter t
1/2, may be inadequately protected. To optimize treatment, there has been an increased
interest in individualized PK-based dosing.
Traditional PK assessment for persons with moderate/severe hemophilia A, however,
requires a long washout period (minimum 72 hours) and a total of 5 to 11 blood samples
drawn at specific time points following an infusion of FVIII with the subject in a
nonbleeding state.[4]
[5] This is demanding for both patients/families and health care professionals alike.[6]
In 1977, Sheiner et al proposed the population PK (PPK) model (also known as nonlinear
mixed effects analysis) that has recently become a popular approach used by PK experts.[7]
[8]
[9] FVIII PPK models have been developed using individual PK measurements from a defined
population of persons with hemophilia A and covariates such as age, weight (or lean
body mass/fat-free mass), and von Willebrand factor antigen (VWF:Ag) levels.[10] Bayesian forecasting, a method of inference using Bayes' theorem to update probabilities
as information becomes available, is used to generate PK parameters using PPK models.
Most importantly, these models allow PK analysis to be accomplished without mandatory
washouts and with sparse blood sampling (2–4 timed blood samples) from the subject
under study.[11]
[12]
The principal aim of this study was to compare two PK protocols for persons with severe
hemophilia A receiving ADVATE: (1) a more traditional, 6-sampling time point PK protocol
with a minimum 72-hour washout and (2) a more practical, single clinic visit “reverse”
2-sampling time point PK protocol with no-washout. The latter was achieved during
a single 3-hour outpatient hemophilia clinic visit, with the first sample drawn upon
arrival to the clinic, 24 hours following infusion of a regular prophylaxis dose in
the patient's home. Immediately after this sample is drawn, the patient receives an
administration of FVIII (in clinic), and then 3 hours postinfusion a drawing of the
second sample is done.
The following PK parameters were determined: Cl; t
1/2; volume of distribution at steady state (V
ss); and time to FVIII:C of 1% above baseline (tt1%). Our working hypothesis was that
a “user-friendly” (single outpatient hemophilia clinic visit, sparse blood sampling),
no-washout PK protocol would yield PK profiles that would be comparable to the more
demanding (multiple clinic visits, more frequent blood samples) washout PK protocol.
If confirmed, this could set the stage for more widespread use of individualized,
PK-guided prophylaxis in persons with hemophilia.[13] This study also afforded us the opportunity to reinforce the previously described
influence of blood group and VWF:Ag levels on PK parameters, and to confirm the large
interindividual variability that exists in PK parameters. The results of our international
collaborative PK study are detailed in this communication.
Methods
Study Design
Seven hemophilia treatment centers participated in this international, multicenter,
prospective sequential design study: The Hospital for Sick Children (Toronto, Canada);
St. Paul's Hospital (Vancouver, Canada); University Hospital Brno (Brno, Czech Republic);
University Hospital Motol (Prague, Czech Republic); Royal Children's Hospital (Melbourne,
Australia); Royal Prince Alfred Hospital (Sydney, Australia); and The Children's Hospital
at Westmead (Sydney, Australia).
In this observational study, all participants were required to complete a 6- and a
2-point PK protocol (see below) within 6 months of enrollment. Depending on participant
preference, PK blood samples were taken from a peripheral vein or an indwelling peripheral
or central venous access device (e.g., a port-a-catheter) following the recommended
protocol for blood discards (1–2 mL for an indwelling peripheral cannula, 5–7 mL for
a port-a-catheter).
Blood samples were collected into 3.2% sodium citrate tubes and processed locally
within 3 hours of collection, centrifuging at 2,500 × g for 15 minutes at room temperature
and aliquoting the resulting platelet-poor plasma into cryovials for storage at −20°C
for a maximum of 48 hours before transferring to −80°C. Frozen samples were shipped
on dry ice to the central reference coagulation laboratory (Department of Pathology
and Molecular Medicine, Queen's University, Kingston, Canada) for analysis.
6-Sampling Time Point PK Protocol
Participants were required to undergo a minimum washout of 72 hours to ensure a low
baseline measure of FVIII:C. They were infused intravenously with approximately 50 IU/kg
body weight rounded to the closest full vial size of a standard half-life (SHL) recombinant
full-length FVIII concentrate (rFVIII; ADVATE; Baxalta US Inc., a Takeda company,
Lexington, Massachusetts, United States) in clinic. Blood samples were taken at 6
time points: < 30 minutes preinfusion; and 1 hour ± 5 minutes, 3 hours ± 15 minutes,
9 hours ± 1 hour, 24 hours ± 2 hours, and 48 hours ± 2 hours postinfusion ([Fig. 1]). This protocol, requiring a washout and six samples, was designed as a reference
protocol for the more practical two-sample single-clinic visit protocol without a
washout (see the following).
Fig. 1 Flowchart of the 6-point and 2-point pharmacokinetic (PK) protocols.
Reverse 2-Sampling Time Point PK Protocol
This protocol consisted of two blood samples collected during a single-clinic visit
with no washout. The first blood sample was drawn in clinic 24 hours ± 2 hours after
the participant infused their regular prophylactic dose of rFVIII (ADVATE) at home
(15–50 IU/kg); then, following an infusion of approximately 25 IU/kg rFVIII (ADVATE;
rounded to the nearest full vial size) administered at the time of the first blood
draw in clinic, a postinfusion sample was taken at 3 hours ± 15 minutes ([Fig. 1]). As such, the 2-sampling point PK protocol could be accomplished in one 3-hour
outpatient clinic visit. This timeframe was chosen as a routine clinic visit that
includes medical/nursing, physiotherapy, and psychosocial assessments and possible
other blood work takes, in our experience, approximately 3 hours to complete.
Participant Selection Criteria
Both adults and children were included in this study, with no minimum or maximum age
cut-offs. Inclusion criteria included a confirmed diagnosis of severe hemophilia A,
defined in this study as a baseline FVIII:C level of <2%; participant receiving ADVATE
for prophylaxis; and body weight ≥12 kg and ≤120 kg. Exclusion criteria included presence
of an inhibitor to FVIII (≥0.6 BU/mL using the Nijmegen modification of the Bethesda
assay); a history of recent events that might affect FVIII half-life (e.g., infection,
significant bleed, surgery or an invasive procedure) within 2 weeks of blood sampling;
human immunodeficiency virus (HIV) positivity with a CD4 count <200 cells/µL; or significant
hepatic dysfunction defined as alanine aminotransferase or aspartate aminotransferase
levels >5× the upper limit of normal. The last two criteria were used to ensure that
participants had no laboratory evidence of clinically severe hepatitis or HIV-associated
immunodeficiency, comorbidities that might impact on the PK handling of FVIII. Inhibitor
status for participants was determined according to the two most recent, consecutive
inhibitor levels on record prior to study enrolment.
Written informed consent was obtained from each participant and/or their parents/legal
guardians (as appropriate); local research ethics boards approved the study. The study
was registered on ClinicalTrials.gov (NCT02750085).
Laboratory Assays
Plasma samples were assayed in a central reference coagulation laboratory (Kingston,
Canada) using one-stage and chromogenic FVIII:C assays. The Siemens BCS XP system
(Marburg, Germany) was used for both assays, and all reagents used were from Siemens.
Standard human plasma (ORKL17) was used to generate standard curves. FVIII-deficient
plasma (OTXW17), Owren's veronal buffer (B4237–23), CaCl2 (ORHO37), and Actin FS (B4218–100; activating material ellagic acid) were used for
the one-stage assays. The FVIII chromogenic assay kit (B4238–40) containing reagents
FX, FIX, substrate, substrate buffer, and Owren's veronal buffer were used for the
chromogenic FVIII:C assay. One-stage FVIII:C assays were also performed on plasma
samples in the routine clinical coagulation laboratories of study sites as per their
local protocols.
VWF:Ag levels were measured by enzyme-linked immunosorbent assay (ELISA) using polyclonal
rabbit anti-human VWF-coating antibody (Ab) (A0082; Dako, Glostrup, Denmark), polyclonal
rabbit anti-human VWF/HRP detecting Ab (P0026; Dako), and Cryocheck Normal Reference
Plasma (CCNRP-05; Precision BioLogics, Dartmouth, Canada). VWF propeptide (VWF:pp)
levels were measured by ELISA using Ab pair anti-human VWF:pp MW1939 (Antibody Chain,
Utrecht, Netherlands). The reference plasma used for the VWF:pp assay was Cryocheck
Normal Reference Plasma. FVIII non-neutralizing Ab levels were measured by ELISA.
ADVATE was used as a coating material, human anti-human FVIII monoclonal Ab EL-14
as reference Ab (kindly provided by Jan Voorberg, Sanquin, University of Amsterdam,
the Netherlands), and goat anti-human IgG-HRP as detecting Ab (2040–05; Southern Biotech,
Birmingham, United States).
Pharmacokinetic Parameters and Analysis
The PK parameters reported herein were chosen in accordance with the guidelines from
the FVIII and FIX Subcommittee of the Scientific and Standardization Committee of
the International Society on Thrombosis and Haemostasis (ISTH).[4] They include Cl (mL/h/kg); t
1/2 (hours); V
ss (dL/kg); and tt1% (hours). t
1/2 is defined as the time required for the FVIII:C level to decrease by 50% after the
initial phase of distribution is completed, and thus reflects drug elimination.[14]
PK parameters from the 6-point PK were calculated using Phoenix WinNonlin 7.0 (Certara
USA, Inc.; Princeton, United States), considered the gold standard for analyzing individual
PK data, and ADVATE myPKFiT v. 3.1 (hereafter referred to as myPKFiT; Baxalta U.S.
Inc., a Takeda company, Lexington, Massachusetts, United States). PK parameters from
the 2-point PK were calculated only using the myPKFiT dosing tool.
PK analysis using WinNonlin is based on a two-compartment model with single bolus
input using first-order output and micro-constants as primary parameters. The myPKFiT
dosing tool was built from the ADVATE pre-licensure PK data using 184 full PK datasets
(10 postinfusion samples) from 100 adults/adolescents with hemophilia A (10–65 years
of age) and 52 reduced sample PK datasets (four postinfusion samples) from 52 boys
with hemophilia A (1–6 years of age).[15]
[16] The myPKFiT Web-based dosing tool is based on a Bayesian algorithm that assumes
a two-compartment model for ADVATE as best fit for data and uses four basic parameters
(Cl; volume of distribution [V1]; volume of the second compartment [V2]; and intercompartmental
Cl [Q]). The model demonstrates good agreement between model predictions and observations
of FVIII levels. Interindividual variance, as well as the covariates of age and body
weight, are incorporated into the model. The model and its development are detailed
in Björkman et al.[11]
Statistical Analyses
Statistical analyses were conducted using MedCalc Statistical Software version 18.10.2
(MedCalc Software; Ostend, Belgium). Intraclass correlations (ICCs) were used to assess
the agreement of myPKFiT PK parameters obtained with the 6-point and 2-point PK protocols.
The agreement of PK parameters of the 6-point protocol between WinNonlin and myPKFiT
was also calculated using ICCs. The ICC values are interpreted as: >0.8, almost perfect;
0.6 to 0.8, substantial; 0.4 to 0.6, moderate; 0.2 to 0.4, fair; 0.01 to 0.2, slight;
and <0.01, poor agreement.[17]
[18] Pearson correlation coefficients were used to determine the extent of linear correlation
between PK parameters and (1) VWF:Ag; (2) VWF:pp; (3) VWF:pp/Ag ratio; and (4) age.
Pearson correlation coefficient values are interpreted as: 1, perfect; 0.9 to 0.8,
very strong; 0.7 to 0.6, moderate; 0.5 to 0.3, fair; 0.2 to 0.1, poor; and 0, no agreement.
A negative value means that the variables are inversely related.[19]
Paired t-tests were used to compare FVIII:C levels from the one-stage and chromogenic FVIII:C
assays, and one-way analysis of variance (ANOVA) was used for comparing VWF:Ag levels
in O versus non-O blood group subjects. Results from paired t-tests and one-way ANOVA are presented as means ± standard deviations. Postinfusion
FVIII:C levels were corrected using the Björkman formula, FVIII = FVIII * (1 − [baseline/C
max]).[20] Descriptive statistics, i.e., means, medians, standard deviations, and confidence
intervals, were used to summarize data, and p-values are presented where appropriate. A p-value <0.05 was considered to be statistically significant. A sample size of 35 patients
was deemed necessary to achieve 80% power to detect an ICC of 0.8 under the alternative
hypothesis, when the ICC under the null hypothesis is 0.5 using an F-test with a significance
level of 0.01.
Results
Thirty-nine males (median age: 11 years; range: 2–69 years) with severe hemophilia
A (FVIII:C < 1%, n = 31; FVIII:C: 1 to <2%, n = 8) participated in this study. Twenty-nine of the 39 study subjects were of age
18 years or less at the time of study. Characteristics of the study group are summarized
in [Table 1].
Table 1
Characteristics of the study cohort
|
Pediatric
|
Adult (>18 y)
|
Total[a]
|
|
(<12 y)
|
(12–18 y)
|
|
Sample size (n)
|
20
|
9
|
10
|
39
|
|
Age (y)
|
6 (2–11)
|
14 (13–17)
|
28.5 (22–69)
|
11 (2–69)
|
|
Weight (kg)
|
23.6 (13.5–53.0)
|
70.4 (43.8–77.6)
|
83.5 (74.0–105.0)
|
48.4 (13.5–105.0)
|
|
Blood group (%)
|
A (35.0%)
|
A (33.3%)
|
A (50.0%)
|
A (38.5%)
|
|
B (20.0%)
|
B (0%)
|
B (0%)
|
B (10.3%)
|
|
AB (0%)
|
AB (11.1%)
|
AB (20.0%)
|
AB (7.7%)
|
|
O (40.0%)
|
O (44.5%)
|
O (20.0%)
|
O (35.9%)
|
|
Unknown (5.0%)
|
Unknown (11.1%)
|
Unknown (10.0%)
|
Unknown (7.7%)
|
|
VWF:Ag[b] (IU/mL)
|
0.8 (0.4–1.3)
|
1.0 (0.5–1.7)
|
1.0 (0.5–1.6)
|
0.8 (0.4–1.7)
|
|
VWF:pp[b] (IU/mL)
|
1.1 (0.6–3.5)
|
1.1 (0.8–1.7)
|
1.1 (0.8–2.3)
|
1.1 (0.6–3.5)
|
|
VWF:pp/VWF:Ag[b]
|
1.4 (0.7–4.5)
|
1.0 (0.6–2.7)
|
1.0 (0.9–2.1)
|
1.2 (0.6–4.5)
|
Abbreviations: VWF:Ag, von Willebrand factor antigen; VWF:pp, von Willebrand factor
propeptide.
Note: Values shown are medians with ranges shown in parenthesis.
a All cases were negative for non-neutralizing FVIII binding antibodies (IgG) except
for four subjects who had low-level positive results.
b Levels were measured after a 72-hour washout. The total sample size for these variables
is 35. No samples were available for analysis for four patients, all from the same
hemophilia treatment center.
PK parameters obtained with the WinNonlin program (two-compartment model) and the
myPKFiT dosing tool were compared using both one-stage and chromogenic FVIII:C levels
from the washout, 6-point PK protocol. An almost perfect agreement was observed when
comparing Cl (0.95 and 0.94, respectively) and V
ss (0.81 and 0.81, respectively) using one-stage and chromogenic FVIII:C results.
Comparison between the 6-Point and 2-Point PK Protocols
The PK parameters of Cl and t
1/2, calculated using the myPKFiT dosing tool for the (washout) 6-point and the (no washout)
single-clinic visit 2-point PK protocols using both one-stage and chromogenic FVIII:C
levels (determined in the central laboratory), are shown in [Fig. 2] as spaghetti plots connecting single subjects (all participants, those with O blood
group, and those with non-O blood group). For all participants, the agreement between
the protocols for Cl was substantial and moderate using the one-stage and chromogenic
FVIII:C levels, respectively, and for t
1/2, it was almost perfect using both assays ([Table 2]).
Fig. 2 Spaghetti plots showing, in the same subjects, the PK parameters of (A) clearance and (B) terminal half-life generated using the myPKFiT dosing tool and factor VIII:C levels
from the washout, 6-sampling time point and the no-washout, single-clinic visit 2
time-point PK protocols. a: All participants (n = 35); b: subjects with O blood group (n = 12); c: subjects with non-O blood group (n = 20). PK, pharmacokinetic.
Table 2
Agreements between the washout, 6-sampling time point, and the no-washout, single-clinic
visit 2 time-point PK protocols for the PK parameters of clearance and terminal half-life
generated using the ADVATE myPKFiT dosing tool and factor VIII:C levels[a]
|
PK parameter
|
One-stage assay
|
Chromogenic assay
|
|
ICC (95% CI)
|
ICC (95% CI)
|
|
All participants (n = 35)
|
Cl (mL/h/kg)
|
0.73 (0.52, 0.85)
|
0.54 (0.25, 0.74)
|
|
t
1/2 (h)
|
0.84 (0.70, 0.91)
|
0.80 (0.63, 0.89)
|
|
O blood group (n = 12)
|
Cl (mL/h/kg)
|
0.52 (−0.04, 0.84)
|
0.35 (−0.25, 0.76)
|
|
t
1/2 (h)
|
0.37 (−0.23, 0.76)
|
0.37 (−0.23, 0.77)
|
|
Non-O blood group (n = 20)
|
Cl (mL/h/kg)
|
0.83 (0.63, 0.93)
|
0.50 (0.08, 0.76)
|
|
t
1/2 (h)
|
0.93 (0.84, 0.97)
|
0.85 (0.66, 0.94)
|
Abbreviations: Cl, clearance; PK, pharmacokinetic; t
1/2, terminal half-life.
Note: Data for 35/39 subjects were available for analysis: the ADVATE myPKFiT application
was unable to generate PK parameters for the 2-point PKs of four subjects (two subjects
had doses <10 IU/kg which is outside the allowable range of 10–100 IU/kg for the dosing
tool, one subject had technical issues with the FVIII:C samples and one subject did
not have adequate documentation of the FVIII infusion prior to the clinic visit for
the 2-sampling time point PK study). Blood group status was not available for three
subjects.
a Factor VIII:C determinations done in central laboratory.
For both Cl and t
1/2, there was an approximate three- to five-fold difference between the lowest and highest
values obtained with the one-stage and chromogenic FVIII:C assays for both the 6-
and 2-point PK protocols ([Fig. 3]).
Fig. 3 Box and whisker plots of (A) clearance (Cl) and (B) terminal half-life (t
1/2) for one-stage and chromogenic FVIII:C assays (n = 39 for 6-point PK protocol; n = 35 for 2-point PK protocol). The maximum and minimum values are given beside the
plots, with the fold difference in the values as indicated.
Results for the PK parameter of V
ss and the agreement between the 6- and 2-point PK protocols are provided in the [Supplementary Material] ([Supplementary Fig. 1] and [Supplementary Table 1], available in the online version). For all participants, the agreement between the
two PK protocols for V
ss was moderate using FVIII:C levels determined with both the one-stage and chromogenic
FVIII assays.
The degree of agreement between the 6- and 2-point PK protocols for the parameters
of Cl and t
1/2 was greater for non-O blood group subjects than for O blood group subjects ([Table 2], [Supplementary Table 1], available in the online version), perhaps reflecting the difference in Cl of FVIII
and therefore circulating FVIII levels at different time points between O and non-O
blood group subjects, or the smaller number of O blood group subjects. The degree
of agreement between the two protocols for V
ss was moderate for both O and non-O blood group subjects.
The agreements between the 6- and 2-point protocols for PK parameters generated using
the myPKFiT dosing tool and the FVIII:C levels measured in the local coagulation laboratories
of the study sites ([Supplementary Table 2], available in the online version) are very similar to those measured in the central
laboratory ([Table 2] and [Supplementary Table 1], available in the online version).
Comparison between One-Stage and Chromogenic FVIII:C Assays
PK parameters calculated using the myPKFiT dosing tool were used to compare one-stage
and chromogenic FVIII:C levels for both protocols (6- and 2-point). An almost perfect
agreement was observed on both protocols when comparing Cl (0.84 and 0.91, respectively)
and t
1/2 (0.91 and 0.93, respectively). V
ss showed a substantial agreement (0.60 and 0.71, respectively). The frequency distributions
of Cl and t
1/2 calculated using the myPKFiT dosing tool from the 6-point and 2-point PK protocols
(one-stage and chromogenic) are presented in [Fig. 3(A, B)].
One-stage and chromogenic FVIII:C levels were compared for each sampling time point
using data from the washout, 6-point PK protocol. FVIII:C levels with the chromogenic
assay were significantly higher at early time points, 1 and 3 hours (by 17.8 and 9.0%,
respectively), but significantly lower at later time points, pre- and at 48 hours
postinfusion (by 22.1 and 18.4%, respectively) in comparison to the one-stage assay.
The mean difference between the one-stage and chromogenic assay FVIII:C levels for
the 9- and 24-hour time points was not statistically significantly different. These
results are presented graphically in [Supplementary Fig. 2] (available in the online version).
PK Parameters among Blood Groups
PK parameters calculated with the myPKFiT dosing tool using both one-stage and chromogenic
FVIII:C levels from the washout, 6-point PK protocol were compared between subjects
who were O blood group and those who were non-O blood group (A, B, or AB). Subjects
who were O blood group had significantly higher Cl values than subjects who were non-O
blood group for both the one stage (4.76 ± 1.06 vs. 3.80 ± 1.33 mL/h/kg, respectively;
p = 0.03) and chromogenic (4.95 ± 1.25 vs. 3.67 ± 1.16 mL/h/kg, respectively; p = 0.004) FVIII:C assays. Reflecting these differences in Cl of infused FVIII, subjects
who were O blood group had significantly lower t
1/2 values than subjects who were non-O blood group for both the one-stage (9.9 ± 1.8
vs. 12.1 ± 2.97 hours, respectively; p = 0.02) and chromogenic (8.8 ± 1.6 vs. 11.0 ± 2.6 hours, respectively; p = 0.01) FVIII:C assays. Subjects who were O blood group had a shorter tt1% when compared
with subjects who were non-O blood group for both the one-stage (60.4 ± 12.6 vs. 74.1 ± 20.0 hours,
respectively; p = 0.03) and chromogenic (54.4 ± 11.8 vs. 68.7 ± 17.9 hours respectively; p = 0.01) FVIII:C assays. There were no statistically significant differences between
subjects who were O blood group and non-O blood group for V
ss derived from both FVIII:C assays.
PK Parameters and VWF:Ag and VWF:pp Levels
PK parameters calculated with the myPKFiT dosing tool using both the one-stage and
chromogenic FVIII:C levels from the washout, 6-point PK protocol were compared with
VWF:Ag levels. There were very strong negative correlations between VWF:Ag levels
and Cl of infused FVIII derived from both the one-stage assay (r = −0.74, p < 0.0001) and the chromogenic assay (r = −0.76, p < 0.0001). Similarly, there were strong positive correlations between VWF:Ag levels
and t
1/2 derived with both the one-stage assay (r = 0.69, p < 0.0001) and the chromogenic assay (r = 0.76, p < 0.0001). There was also a very strong positive correlation between VWF:Ag levels
and tt1% with both the one-stage assay (r = 0.72, p < 0.0001) and the chromogenic assay FVIII:C levels (r = 0.77, p < 0.0001). There was a moderate negative correlation between VWF:Ag and V
ss derived from the one-stage assay (r = −0.53, p = 0.001) and a strong negative correlation with the chromogenic assay (r = −0.64, p < 0.0001). There were significant differences between VWF:Ag levels in subjects who
were O blood group and non-O blood group (p = 0.002), with the O group subjects (0.72 ± 0.16 IU/dL) having lower VWF:Ag levels
compared with the non-O blood group subjects (1.08 ± 0.37 IU/dL). There was also a
significant difference between VWF:pp/VWF:Ag ratios in subjects who were O blood group
and those who were non-O blood group (1.63 ± 0.55 and 1.19 ± 0.54, respectively, p = 0.031). This finding is consistent with higher Cl of VWF in O blood group patients.
The 6-point PK parameters Cl, t
1/2, tt1%, and V
ss calculated with myPKFiT for both the one-stage and chromogenic FVIII:C levels were
compared with VWF:pp levels and VWF:pp/VWF:Ag levels. No statistically significant
correlations were found between the PK parameters and VWF:pp levels or VWF:pp/VWF:Ag
ratios.
PK Parameters and Age
PK parameters calculated with the myPKFiT dosing tool using both the one-stage and
chromogenic assay FVIII:C levels were compared with age. There were fair negative
correlations between age and Cl derived from the one-stage (r = −0.50, p = 0.001) and chromogenic assays (r = −0.49, p = 0.002) and between age and V
ss derived from the one-stage (r = −0.39, p = 0.014) and chromogenic assays (r = −0.57, p = 0.0001). Reflecting these results, there were also moderate positive correlations
between age and t
1/2 derived from the one-stage (r = 0.63, p < 0.0001) and the chromogenic FVIII:C levels (r = 0.67, p < 0.0001), as well as between age and tt1% derived from the one-stage (r = 0.58, p = 0.0001) and chromogenic assays (r = 0.62, p < 0.0001).
Discussion
Several recent reports have documented the potential benefits of individualized prophylaxis
regimens based on measured PK profiles of subjects with hemophilia A.[7]
[21]
[22]
[23] A challenge to the implementation of PK-directed prophylaxis regimens in the past
was that traditional PK studies in individuals with hemophilia were demanding as they
required a long washout to achieve trough levels below the limit of quantitation of
FVIII using conventional one-stage or chromogenic FVIII:C assays, followed by multiple
postinfusion blood samples. Consequently, such PK studies were impractical from a
clinical perspective.[6] More recently, the development of PPK dosing tools have, with Bayesian forecasting,
facilitated measurement of PK profiles without an obligatory washout and with sparse
blood sampling. In a recent communication from the Scientific Standardization Committee
of the ISTH, Iorio and colleagues recommended two to three sampling time points at
least 12 hours apart, e.g., 4 to 8, 16 to 28, and 40 to 60 hours following infusion
of a SHL FVIII concentrate; for extended half-life (EHL) FVIII concentrates, an additional
sample obtained at 60 to 84 hours was recommended. The authors also stressed that
the most informative sample is the one taken at 24 hours.[12] Although this is a marked improvement over the traditional washout, multiple blood
sampling PK protocol advocated by the ISTH several years ago, this modified, no-washout
PPK protocol still requires blood sampling at several outpatient clinic visits. This
will either increase the number of nonroutine clinic visits or increase the time until
PK results will be available (if blood samples are collected only during routine outpatient
hemophilia comprehensive care clinic visits, generally every 6 to 12 months).
To the best of our knowledge, this is the first study to compare a no-washout, sparse,
reverse sampling, single-clinic visit PK protocol (2-point PK protocol) to a washout,
multiple clinic visit protocol (6-point PK protocol) as recommended in the past by
the ISTH.[4] The times of the blood draws in this study were chosen based on the myPKFiT User
Manual, which states that a PK profile can be generated when the recommended minimum
sampling conditions, one at 3 to 4 hours and the second at 24 to 32 hours postinfusion,
are met[7]
[24]; this also complies with the recent ISTH guidance published by Iorio and colleagues.[12] Most importantly, we chose this 2-sampling time point protocol, in which the samples
are taken in a reverse order of time, i.e., 24-hour sample first, followed by the
3-hour sample, based on the fact that sampling can be performed during a routine comprehensive
care hemophilia clinic visit, which in our experience, if it includes medical, nursing,
and physiotherapist reviews, generally lasts approximately 3 hours. Thus, this “reverse”
sampling PK protocol is convenient for patients and their families, and practical
for caregivers. We reasoned that if the results of our study yielded good comparability
between a no-washout, single-clinic visit 2-point PK protocol and a more traditional
washout, multiple clinic visit, 6-sampling time point PK protocol, the uptake of PK
profiling of subjects with hemophilia A receiving prophylaxis with ADVATE using the
myPKFiT PPK dosing tool at routine comprehensive care hemophilia clinic visits could
be greatly enhanced.
Indeed, the results of this study are encouraging. We demonstrated, using data from
the washout, 6-point PK protocol with FVIII:C levels measured using either the one-stage
or chromogenic assays, that results obtained with the two-compartment PPK myPKFiT
dosing tool and with the gold standard individualized PK program WinNonlin are comparable.
We then determined that there was moderate to almost perfect agreement for all PK
parameters, Cl, t
1/2, and V
ss, obtained with the washout, 6-point PK protocol and the no-washout, single out-patient
hemophilia clinic visit, 2-point PK protocol using the one-stage and chromogenic assays
done in the central laboratory. Results for Cl and t
1/2, the two PK parameters of greatest importance with regard to dosing for prophylaxis
regimens, are highlighted in this study. Of note and of importance from a practical
perspective, very similar results were obtained using the one-stage FVIII:C results
reported by the local coagulation laboratories. Overall, we interpret these findings
as positive from a clinical viewpoint, suggesting that the no-washout, single-clinic
visit 2-point PK protocol analyzed with the myPKFiT PPK dosing tool generally yields
a very good approximation of the PK profile for ADVATE infused into subjects with
severe hemophilia A.
When the FVIII:C levels for each time point were compared between assays, the chromogenic
assay showed higher levels at 1 and 3 hours postinfusion and lower levels for the
pre- and 48-hour postinfusion samples. These findings are consistent with results
reported by Morfini and colleagues,[25] where it was shown that when FVIII:C levels are above 25 IU/dL, the chromogenic
assay yields higher FVIII:C levels than does the one-stage assay and lower FVIII:C
levels than the one-stage assay when FVIII:C levels are below 25 IU/dL. These differences
did not impact in any significant way on the PK parameters of Cl or t
1/2, as detailed in this report.
Additional important findings from this study relate to the importance of baseline
VWF:Ag levels on Cl of infused ADVATE which was significantly higher in subjects with
lower VWF:Ag levels, resulting in lower t
1/2 values in these subjects. In parallel, Cl of infused ADVATE was significantly higher
in subjects who were O blood group as compared with non-O blood group subjects, reflecting
the well-known fact that levels of VWF:Ag, the binding partner for FVIII in the circulation,
are substantially lower in blood O group subjects as compared with non-O blood group
subjects.[26]
[27]
[28] In fact, in a recent publication, Lunghi et al showed that there is a higher intercompartment
Cl, as well as transfer rates between central and peripheral compartments, in O blood
group subjects when compared with non-O blood group subjects.[28] These differences between O and non-O blood group subjects might have had an impact
on the agreement between the 6- and the 2-point PK protocols, since our results showed
that the non-O group had a greater level of agreement than the O group.
This association between blood groups and VWF:Ag on both Cl and t
1/2 of infused SHL and EHL FVIII concentrates has been documented in several studies[26]
[27]
[29]
[30] and reflects the fact that clearance of infused FVIII is driven by clearance of
its binding partner, VWF:Ag. The importance of these associations should not be underestimated.
The approximate three- to five-fold difference in Cl and t
1/2 between inhibitor-negative subjects with severe hemophilia A observed in this study,
and also reported by others,[25]
[31]
[32] exceeds the approximate 1.5-fold difference in Cl and t
1/2 observed between EHL and SHL FVIII concentrates,[33] and should be taken into consideration when recommending individual prophylaxis
regimens for subjects with severe hemophilia A. This study also showed a negative
correlation between V
ss and VWF:Ag, which could be due to the fact that VWF retains FVIII inside the plasma
compartment; this may reduce FVIII in the extravascular spaces, creating a decrease
in V
ss.
Another relationship of note relates to the effect of age on Cl and t
1/2 of infused FVIII. In this study, as in some other reported studies,[20]
[34]
[35] Cl of infused FVIII (e.g., ADVATE) was higher in younger subjects. As subjects age,
there is a change in body size and fluid volumes that impacts on the V
ss and therefore would decrease Cl and increase t
1/2 of FVIII.[36] We believe that this effect is most marked for very young boys with hemophilia,
since a recent study comparing PK parameters of two EHL FVIII concentrates in inhibitor-negative
adolescent males with hemophilia A failed to document an age effect on Cl and t
1/2.[26] It appears likely, therefore, that PK profiles will not change substantially after
adolescence and do not need to be repeated at regular intervals in such age groups
(adolescents and adults). It is prudent, however, to consider repeat PK testing when
switches are made from one FVIII concentrate to another and at an interval of every
few years in young boys with hemophilia A <12 years of age, and especially <6 years
of age.
This study has some limitations. First is the number of samples collected for PK analysis.
Some patients might not robustly fit a two-compartment model based on six samples;
however, the collection of more blood samples has ethical and physiological limitations
in pediatric patients that would make it challenging to obtain more blood samples.
Therefore, the 6-point PK protocol based on the ISTH guidelines was used as a reference,
rather than a traditional PK study based on more blood samples.[4]
[5] Second is the premise that there is negligible inter-occasion variability in our
2-sample reverse sampling strategy as it requires two different infusions. The fact
that the infusion doses varied within the 2-point PK protocol precluded our ability
to directly compare the tt1% between the 6- and 2-point PK protocols and this comparison
is therefore not made in this study. Third, this study depends on patients/families
taking the correct dose of factor and reporting the exact time of infusions. This
is particularly important as we were indeed not able to perform the 2-sample PK protocol
on three subjects due to incorrect doses (outside the myPKFiT ranges), or due to inadequate
documentation of prior infusions. Fourth, the product-specific myPKFiT tool for ADVATE,
only, was used in the determination of PK parameters in this study; in future studies,
other PPK programs that are not product-specific, such as the WAPPS-Hemo (Web-based
Application for the Population Pharmacokinetic Service Hemophilia),[37] could be utilized. Fourth, as WinNonlin requires more than five samples to be able
to perform a PK profile, it was not possible to compare the results from the 2-point
PK from the myPKFiT dosing tool with those from the WinNonlin program. Finally, and
most importantly, our study excluded subjects with several a priori determined comorbidities
(e.g., presence of a recent bleed, laboratory evidence of clinically significant active
hepatitis) that potentially could impact the clearance of infused FVIII; this represents
a “best case” baseline scenario and it is likely that in a “real-world” setting, observed
PK profiles may, in some cases, reflect presence of comorbidities, recognized or even
unrecognized. However, it is very important that baseline PK profiles are obtained
with subjects in a nonbleeding state and free of active systemic infection; when such
studies are performed as part of a switch from one FVIII concentrate to another, the
PK study should only be performed after the subject has received a reasonable number
of infusions of the new FVIII concentrate and not at the time of the first infusion.
Conclusion
In summary, this study, conducted in 39 inhibitor-negative subjects with severe hemophilia
A (median age of 11 years, and with 71.8% of cases ≤18 years of age), demonstrated
that a no-washout, single (3 hour) outpatient hemophilia clinic visit, 2-sampling
time point PK protocol analyzed with the ADVATE myPKFiT PPK dosing tool generates
PK parameters, Cl, t
1/2, and V
ss, with sufficient accuracy compared with a washout, 6-sampling time point, 3 clinic
visit PK protocol. Thus, a 2-sampling time point PK protocol is very attractive for
routine clinical use associated with comprehensive care hemophilia assessment visits.
Results from the initial 2-sampling time point PK study, performed with subjects in
a nonbleeding state on prophylaxis after at least a few infusions of the FVIII concentrate
under study, can be used to guide design of individual prophylaxis regimens. Additionally,
of importance, the results can inform to the timing of future PK sampling time points
at times convenient for subjects with hemophilia and their families and caregivers
leading to more robust estimates of PK profiles. Although there remains controversy
amongst health care providers involved with the assessment and care of persons with
hemophilia regarding the clinical value of PK studies, we believe that PPK programs
such as the ADVATE myPKFiT dosing tool and others (e.g., the WAPPS/Hemo Program[37]) are important tools for optimization of prophylaxis regimens at the individual
patient level with the goal of preventing long-term hemophilic arthropathy.
What is known about this topic?
-
Traditional pharmacokinetic (PK) assessments are demanding for persons with hemophilia
A. They require a washout period and multiple blood sampling time points over a 72-hour
period following infusion of a standard half-life FVIII clotting factor concentrate
such as ADVATE.
-
Population PK (PPK) models have been developed for persons with hemophilia A.
What does this paper add?
-
PPK parameters were obtained with a traditional washout, 6-sampling time point PPK
and a more practical, no-washout, reverse 2-sampling time point, single-visit PPK
protocol in persons with severe hemophilia A using the ADVATE myPKFiT dosing tool
and were compared.
-
The “user-friendly” 2-point PPK protocol was found to generate PK parameters with
sufficient accuracy. Thus, 2-point PPK results can be used to guide prophylaxis regimens
in persons with severe hemophilia A receiving ADVATE.
