Pharmacokinetics/Pharmacodynamics
Pharmacokinetics and Pharmacodynamics of
a Human Monoclonal Anti-FGF23 Antibody
(KRN23) in the First Multiple Ascending-Dose
Trial Treating Adults With X-Linked
Hypophosphatemia
The Journal of Clinical Pharmacology
2016, 56(2) 176–185
© 2015 The Authors. The Journal of
Clinical Pharmacology Published by
Wiley Periodicals, Inc. on behalf
of American College of Clinical
Pharmacology
DOI: 10.1002/jcph.570
Xiaoping Zhang, PhD1, Erik A. Imel, MD2, Mary D. Ruppe, MD3,
Thomas J. Weber, MD4, Mark A. Klausner, MD1, Takahiro Ito, MSc1,
Maria Vergeire, MD1, Jeffrey Humphrey, MD1, Francis H. Glorieux, MD, PhD5,
Anthony A. Portale, MD6, Karl Insogna, MD7, Thomas O. Carpenter, MD7,
and Munro Peacock, MD2
Abstract
In X-linked hypophosphatemia (XLH), serum fibroblast growth factor 23 (FGF23) is increased and results in reduced renal maximum threshold
for phosphate reabsorption (TmP), reduced serum inorganic phosphorus (Pi), and inappropriately low normal serum 1,25 dihydroxyvitamin D
(1,25[OH]2D) concentration, with subsequent development of rickets or osteomalacia. KRN23 is a recombinant human IgG1 monoclonal
antibody that binds to FGF23 and blocks its activity. Up to 4 doses of KRN23 were administered subcutaneously every 28 days to 28 adults with
XLH. Mean standard deviation KRN23 doses administered were 0.05, 0.10 0.01, 0.28 0.06, and 0.48 0.16 mg/kg. The mean time to reach
maximum serum KRN23 levels was 7.0 to 8.5 days. The mean KRN23 half-life was 16.4 days. The mean area under the concentration–time curve
(AUCn) for each dosing interval increased proportionally with increases in KRN23 dose. The mean intersubject variability in AUCn ranged from
30% to 37%. The area under the effect concentration–time curve (AUECn) for change from baseline in TmP per glomerular filtration rate, serum
Pi, 1,25(OH)2D, and bone markers for each dosing interval increased linearly with increases in KRN23 AUCn. Linear correlation between serum
KRN23 concentrations and increase in serum Pi support KRN23 dose adjustments based on predose serum Pi concentration.
Keywords
X-linked hypophosphatemia (XLH), fibroblast growth factor 23 (FGF23), human anti-FGF23 antibody (KRN23), serum phosphorus, pharmacokinetics
Loss-of-function mutations in PHEX cause X-linked
hypophosphatemia (XLH), the most common heritable
form of rickets and osteomalacia. A consequence of the
PHEX mutation is increased expression of FGF23 in
bone.1,2 FGF23 is a hormone that reduces the abundance of
sodium-phosphate cotransporters in the apical membrane of
renal proximal tubular cells, resulting in reduced renal
tubular phosphate reabsorption (TmP) and serum phosphorus concentration (measured as inorganic phosphorus,
Pi).3–5 FGF23 also decreases renal 1a-hydroxylase activity,
resulting in reduced blood levels of 1,25-dihydroxyvitamin
D (1,25[OH]2D). Thus hypophosphatemia and inappropriately low normal serum 1,25(OH)2D levels for the level of
serum Pi represent 2 characteristic biochemical consequences of increased FGF23 observed in XLH.6,7
In children with XLH, impaired bone mineralization
results in clinical and radiographic features of rickets,
including lower-extremity bowing, widening of growth
plates, and short stature.7,8 In adults, impaired mineralization
results in osteomalacia and pseudofractures.8 The presence
1
Kyowa Hakko Kirin Pharma Inc., Princeton, NJ, USA
Indiana University School of Medicine, Indianapolis, IN, USA
3
The Methodist Hospital, Houston, TX, USA
4
Duke University Medical Center, Durham, NC, USA
5
Shriners Hospital for Children, Montreal, Quebec, Canada
6
University of California, San Francisco, CA, USA
7
Yale Center for X-Linked Hypophosphatemia, Yale University
School of Medicine, New Haven, CT, USA
2
This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial and no modifications
or adaptations are made.
Submitted for publication 29 March 2015; accepted 9 June 2015.
Corresponding Author:
Xiaoping Zhang, PhD, Kyowa Hakko Kirin Pharma Inc., 212 Carnegie
Center, Suite 101, Princeton, NJ 08540 Email: [email protected]
Clinicaltrials.gov identifier: NCT01340482
2Zhang et al
of
Using
a reduced
a streptavidin-alkaline
mineralization rate
phosphatase
in active conjugate
osteomalacia
to
results
catalyzein the
increased
chemiluminescent
serum and urine
substrate,
concentrations
the resulting
of
9
Current therapy,
biochemical
chemiluminescent
markers
light
of bone
intensity
turnover.
was measured,
and the
typically
with oral
calcitriolwere
and calculated.
phosphate supplements,
concentrations
of KRN23
The calibratargets
the biochemical
FGF23
excess
tion range
was 50 to consequences
3000 ng/mL. ofThe
lower
limitand
of
theaccuracy
underlying
defect
improves
bone was
mineralization,
quantification
50 ng/mL.10,11
The but
assay
(relative
in
renal
reabsorption
is notofcorrected,
andtheclinical
error)
andPiprecision
(coefficient
variation for
mean)
responses
inacceptance
bone are criteria
highly variable
among
patients.
were within
(< 20%)
for a majority
Current
treatment
daily
doses of
medicaof quality
controlrequires
samplesmultiple
(94.6%).
A small
amount
of
tion
andcontrol
has serious
potential
including
quality
samples
(5.4%) complications
showed relative
errors
hypercalciuria,
nephrocalcinosis,
nephrolibetween 20.7%hypercalcemia,
and 26.7%. Ten
individual lots of
pooled
Thus,
safer The
and
thiasis,
parathyroid
hyperplasia.
human and
serum
were spiked
with 50 12,13
ng/mL
KRN23.
14
more
efficacious
needed.
relative
error of therapies
detectionare
ranged
from
-11.4% to 7.2%,
KRN23
a recombinant
human immunoglobulin
(Ig)
whereas
allisthe
unspiked individual
lots yielded concenmonoclonal
antibody
that
binds
intact
FGF23
(and
G
trations
below
the
low
limit
of
quantification
(50
ng/mL),
1
FGF23
fragments)
at the
N-terminal
domain. An
confirming
the selectivity
of the
assay.
antimurine FGF23 antibody binding to FGF23 blocked
biologic
activity
of FGF23Antibodies
in mice.12 In hypophosphaDetection
of Anti-KRN23
temic
mice, injection
of were
antibody
to
Serum Phex-deficient
samples for anti-KRN23
antibodies
obtained
murine
FGF23
increased
both
serum
Pi and
before each
dose and
on day 36
after
the fourth
dose 1,25
or at
D concentrations
bywere
increasing
(OH)
early 2withdrawal.
Samples
assayedrenal
using expression
a validated
of
type IIa sodium-phosphate
and renal
electrochemiluminescent
(ECL)cotransporters
method. A sequential
1a-hydroxylase
activity,
respectively.
After assay,
serial
screening algorithm
employed
an initial 15
screening
injections
in these
mice, defective
mineralization,
abnora subsequent
immunodepletion
assay,
and a neutralizing
mal
development,
andCalifornia,
delayed skeletal
assaycartilage
at Kyowa
Hakko Kirin
Inc (Lagrowth
Jolla,
were
improved,
confirming
that increased
FGF23
underCalifornia).
Testing
only proceeded
to the
subsequent
lies
thestep
murine
boneresult
disease
suggests that
assay
if themetabolic
earlier assay
wasand
positive.
inhibition
of FGF23
be useful
for treating
The bridging
ECLactivity
format may
for both
the screening
and
rickets
andconfirmation
osteomalaciaassays
in humans
specificity
beginswith
withXLH.
a streptavidinRecently,
a phase by
1 randomized,
placebo-controlled,
coated
plate supplied
Meso Scale Discovery
(MSD). In
double-blind
trial control
of KRN23
in adults with
XLH
the screening assay,
and bioanalytical
samples
are
demonstrated
that
a single
intravenous
or subcutaneous
first diluted with
diluent
buffer
on a 96-well,
round-bottom
dose
of KRN23plate.
resulted
in prolonged
inhibition
FGF23
polypropylene
Then,
diluted samples
are of
incubated
Serum
Pi, containing
TmP per glomerular
filtrate
rate
activity.
with a 16,17
Master
Mix
biotinylated
KRN23
D
increased
in
a
dose(TmP/GFR),
and serum
(capture antibody)
and 1,25(OH)
ruthenylated-KRN23
(detection
2
dependent
manner, and
the half-life
antibody). Following
incubation
with oftheKRN23
labeledgiven
drug
subcutaneously
to 19 days.
Thepolypropylene
greater than
mixture, sampleswas
are 13
transferred
from the
4-week
of the streptavidin-coated
Pi effect suggested that
subcutaneplate toduration
the blocked,
MSD
plate in
ous
injection
of KRN23
everytemperature
4 weeks might
be an
duplicate
and incubated
at room
with shaking.
effective
treatmentunbound
to increase
serumisPiwashed
in XLHaway,
patients.
After incubation,
material
and
Subsequently
a phase
1/2 dose-escalation
studytoofthe
repeated
MSD Read Buffer
(ECL
substrate) is added
wells.
subcutaneous
doses
of KRN23
at 28-day intervals
in adults
The ECL signal
intensity
is immediately
read using
an
with
was
undertaken
to assess
thesignal
efficacy
and which
safety
MSDXLH
Sector
Imager
measuring
MSD
counts,
18
We report
here the
objective
of
of
KRN23.
is used
to evaluate
the presence
of secondary
anti-KRN23
antibodies
the
was to evaluate
the pharmacokinetics
and study,
assess which
the specificity
of any antibodies
relative to
(PK),
pharmacodynamics
(PD), and PK/PD
KRN23.
The specificity confirmation
assayrelationships
follows the
of
serum
KRN23
andscreening
the biochemical
variables.
same
format
as the
assay, except
that prior to
incubation with the labeled drug mixture, controls and
bioanalytical samples are first diluted with buffer containMethods
ing immunodepletive antigen (KRN23). Screening assay
Subjects
cut point was 1.88 for signal ratio.
The study was approved by the relevant local institutional
review
boards (IRBs):
Human Research Protection Program,
Pharmacodynamic
Assessments
at
Yalesamples
University
of Medicine;assessments
Human Subjects
Blood
for School
pharmacodynamic
were
Office
at Indiana
University
IRB;
Copernicus
at
collected
at screening,
before
each
dose, on Group
days 3,IRB
7, 12,
1773
The Journal of Clinical Pharmacology / Vol XX No XX 2015
Research
Triangle
Park in
North
Carolina;
forlast
the
18, and 26
after each
dose,
and
on dayCommittee
36 after the
Protection
Human
Subjects at measurements
the University of
Texas
dose (finalofvisit).
Biochemical
included
Health
Science
Center
Houston; Committee
on Human
D (25[OH]D),
serum Pi,
1,25(OH)
2D, 25-hydroxyvitamin
Research
at calcium,
the University
of Californiahormone
San Francisco;
creatinine,
and parathyroid
(PTH).and
Pi,
IRB
at McGill
University.
study in
was
in
calcium,
and creatinine
wereThe
measured
theconducted
correspondaccordance
with the
International
Conference
onon
Harmoniing 2-hour urine
collected
after an
8-hour fast
the day
zation
E6 first
Good
Clinical
guidelines
the
before the
injection
andPractice
on days 3,
7, 12, andand
26 after
Declaration
of and
Helsinki.
All36subjects
provided
written
each injection
on day
after the
last injection.
informed
to screening.
Adults (18
of
Calcium, consent
Pi, and prior
creatinine
were measured
in a years
24-hour
age)
a clinical
XLH,
serum
urine with
collected
on the diagnosis
day beforeof the
first intact
injection,
on
19
FGF23
pg/mL,
days 3, 12,30and
26 after
each injection,
on day
36 after
TmP/GFR
< 2.0and
mg/dL,
creatinine
the last injection.
TmP/GFR
was calculated
usingand
an
clearance
60 mL/min
(Cockcroft-Gault
equation),
established
based
on glomerular
filtration
serum
calciumequation
< 10.8 mg/dL
at screening
were eligible.
Key
Blood
and
rate calculated
creatinineorclearance.
exclusion
criteriafrom
were pregnancy
lactation;20
history
within
samples
measurement
of bone
turnover
markers
3urine
months
beforeforscreening
of major
surgery,
or receipt
of a
were collected
priorortoa each
dose and atvaccine.
the final
visit.
monoclonal
antibody
live (attenuated)
Vitamin
Bone
formation
markers included
bone alkaline
D
or vitamin
D analogues,
calcium serum
or phosphate
supplephosphatase
(BALP),
procollagen
type
1, N propeptide
ments,
or aluminum
hydroxide
were not
permitted
for at least
(P1NP),
and screening
osteocalcin.
Bone resorption
10
days before
and throughout
the study.markers
included serum carboxy-terminal cross-linked telopeptide
of type
1 collagen
(CTx), and urine amino-terminal
Study
Design
and Treatment
cross-linked
type 1 collagen
The
dose of telopeptide
KRN23 wasofcalculated
using (NTx).
the subject’s
Serum
FGF23
was
measured
at baseline
using an
weight
on intact
the dosing
day.
Subjects
received
a subcutaneELISA
(Kainos
All using
other
ous
doseassay
of KRN23
everyLaboratories,
28 days, for upJapan).
to 4 doses,
variablesofin0.05,
serum
1,25[OH]
abiochemical
stepwise escalation
0.1,(25[OH]D,
0.3, and 0.6
mg/kg. For
2D,
PTH,subject,
BALP, the
CTx,
osteocalcin,
Pi, calcium,
creatieach
dose
was increased
based on and
the fasting
nine) and
in urine samples
(Pi,
creatinine,
and
serum
Pi concentration
on day
26,calcium,
as follows:
if the serum
NTx)
were26analyzed
Quest
Diagnostics
using
Pi
on day
was (1) by
2.5
mg/dL,
the dose Inc.
increased
automated
1standardized
dose level; (2)
>2.5 butmethodologies.
3.5 mg/dL, the previous dose
was repeated; (3) >3.5 mg/dL, the dose was withheld,
Pharmacokinetic
and Statistical
Analysis
and
Pi was retested
28 days later.
If the repeat was
Pharmacokinetic
parameters
using
(1)
2.5 mg/dL, (PK)
the most
recent were
dose estimated
was repeated;
serum
concentration–time
for allwas
accumulated
(2)
>2.5
but 3.5 mg/dL, data
the dose
reduced 1 doses
dose
from (3)
day>3.5
1 tomg/dL,
the last
visit on and
dayserum
120.
level;
the follow-up
dose was withheld
A reevaluated
noncompartmental
method
employed
to
Pi
another analysis
28 days later
at thewas
discretion
of the
21
These
included
area
the
derive PK parameters.
investigator
and the sponsor.
Dosing
could
be under
modified
serum concentration–time
curve including
from zero to
last measured
based
on safety observations,
adverse
events
concentration
(AUClastparameters.
), area under the serum concentraand
safety laboratory
tion–time curve from zero to infinity (AUCinf), apparent
volume of distribution
Investigational
Product (V/F), apparent clearance (CL/F),
and terminal
elimination half-life
the fourth
dosing
KRN23
was manufactured,
labeled, after
and packaged
in single).
Pharmacokinetic
parameters
estimated
for
interval
use
vials (tby
Kyowa
Hakko
Kirin
Co.,
Ltd
(Tokyo,
Japan).
1/2
each 28-day dosing interval included area under the serum
concentration–time
curve (AUCn), maximum serum
Pharmacokinetic
Assessments
), predoseof KRN23
serum concentration
concentration
concentration
(Cmax,n
Blood
samples for
measurement
(Cmin,n
), and time
to each
peak dose,
serumonconcentration
),
were
obtained
before
days 3, 7, 12,(T
18,
and
max,n
where
denotes
the number
of doses
received
(1, 2,dose
3, and
26
aftern each
dosing
and on day
36 after
the fourth
or
4) early
up to the
measuredSerum
dosing KRN23
interval. concentrations
Descriptive statistics
at
withdrawal.
were
were usedatfor
serumHakko
KRN23
concentration
data(La
and
PK
measured
Kyowa
Kirin
California, Inc
Jolla,
parameters. All
PKaparameters
derivedenzyme-linked
using Phoenix
California)
using
validatedwere
sandwich
WinNonlin (version
6.3;(ELISA;
Pharsightdata
Company,
Mountain
immunosorbant
assay
on file),
which
View, California).
employs
an anti-KRN23 mouse monoclonal antibody as
underantibody
the effectand
concentration–time
for the
the Area
capture
a biotinylated curve
anti-KRN23
change fromantibody
baseline prior
the first dose
for TmP,
monoclonal
as thetodetection
antibody
(bothserum
were
D
were
calculated
for
day
1
to
day
120
of
Pi, and 1,25(OH)
supplied
from Kyowa
Hakko
Kirin
Co.,
Ltd,
Japan).
2
178
Zhang et al
Using a streptavidin-alkaline phosphatase conjugate to
catalyze the chemiluminescent substrate, the resulting
chemiluminescent light intensity was measured, and the
concentrations of KRN23 were calculated. The calibration range was 50 to 3000 ng/mL. The lower limit of
quantification was 50 ng/mL. The assay accuracy (relative
error) and precision (coefficient of variation for the mean)
were within acceptance criteria (< 20%) for a majority
of quality control samples (94.6%). A small amount of
quality control samples (5.4%) showed relative errors
between 20.7% and 26.7%. Ten individual lots of pooled
human serum were spiked with 50 ng/mL KRN23. The
relative error of detection ranged from -11.4% to 7.2%,
whereas all the unspiked individual lots yielded concentrations below the low limit of quantification (50 ng/mL),
confirming the selectivity of the assay.
Detection of Anti-KRN23 Antibodies
Serum samples for anti-KRN23 antibodies were obtained
before each dose and on day 36 after the fourth dose or at
early withdrawal. Samples were assayed using a validated
electrochemiluminescent (ECL) method. A sequential
screening algorithm employed an initial screening assay,
a subsequent immunodepletion assay, and a neutralizing
assay at Kyowa Hakko Kirin California, Inc (La Jolla,
California). Testing only proceeded to the subsequent
assay step if the earlier assay result was positive.
The bridging ECL format for both the screening and
specificity confirmation assays begins with a streptavidincoated plate supplied by Meso Scale Discovery (MSD). In
the screening assay, control and bioanalytical samples are
first diluted with diluent buffer on a 96-well, round-bottom
polypropylene plate. Then, diluted samples are incubated
with a Master Mix containing biotinylated KRN23
(capture antibody) and ruthenylated-KRN23 (detection
antibody). Following incubation with the labeled drug
mixture, samples are transferred from the polypropylene
plate to the blocked, streptavidin-coated MSD plate in
duplicate and incubated at room temperature with shaking.
After incubation, unbound material is washed away, and
MSD Read Buffer (ECL substrate) is added to the wells.
The ECL signal intensity is immediately read using an
MSD Sector Imager measuring MSD signal counts, which
is used to evaluate the presence of anti-KRN23 antibodies
and assess the specificity of any antibodies relative to
KRN23. The specificity confirmation assay follows the
same format as the screening assay, except that prior to
incubation with the labeled drug mixture, controls and
bioanalytical samples are first diluted with buffer containing immunodepletive antigen (KRN23). Screening assay
cut point was 1.88 for signal ratio.
Pharmacodynamic Assessments
Blood samples for pharmacodynamic assessments were
collected at screening, before each dose, on days 3, 7, 12,
The Journal of Clinical Pharmacology / Vol 56 No 2 20163
18, and 26 after each dose, and on day 36 after the last
dose (final visit). Biochemical measurements included
serum Pi, 1,25(OH)2D, 25-hydroxyvitamin D (25[OH]D),
creatinine, calcium, and parathyroid hormone (PTH). Pi,
calcium, and creatinine were measured in the corresponding 2-hour urine collected after an 8-hour fast on the day
before the first injection and on days 3, 7, 12, and 26 after
each injection and on day 36 after the last injection.
Calcium, Pi, and creatinine were measured in a 24-hour
urine collected on the day before the first injection, on
days 3, 12, and 26 after each injection, and on day 36 after
the last injection. TmP/GFR was calculated using an
established equation based on glomerular filtration
rate calculated from creatinine clearance.20 Blood and
urine samples for measurement of bone turnover markers
were collected prior to each dose and at the final visit.
Bone formation markers included serum bone alkaline
phosphatase (BALP), procollagen type 1, N propeptide
(P1NP), and osteocalcin. Bone resorption markers
included serum carboxy-terminal cross-linked telopeptide
of type 1 collagen (CTx), and urine amino-terminal
cross-linked telopeptide of type 1 collagen (NTx).
Serum intact FGF23 was measured at baseline using an
ELISA assay (Kainos Laboratories, Japan). All other
biochemical variables in serum (25[OH]D, 1,25[OH]2D,
PTH, BALP, CTx, osteocalcin, Pi, calcium, and creatinine) and in urine samples (Pi, calcium, creatinine, and
NTx) were analyzed by Quest Diagnostics Inc. using
standardized automated methodologies.
Pharmacokinetic and Statistical Analysis
Pharmacokinetic (PK) parameters were estimated using
serum concentration–time data for all accumulated doses
from day 1 to the last follow-up visit on day 120.
A noncompartmental analysis method was employed to
derive PK parameters.21 These included area under the
serum concentration–time curve from zero to last measured
concentration (AUClast), area under the serum concentration–time curve from zero to infinity (AUCinf), apparent
volume of distribution (V/F), apparent clearance (CL/F),
and terminal elimination half-life after the fourth dosing
interval (t1/2). Pharmacokinetic parameters estimated for
each 28-day dosing interval included area under the serum
concentration–time curve (AUCn), maximum serum
concentration (Cmax,n), predose serum concentration
(Cmin,n), and time to peak serum concentration (Tmax,n),
where n denotes the number of doses received (1, 2, 3, and
4) up to the measured dosing interval. Descriptive statistics
were used for serum KRN23 concentration data and PK
parameters. All PK parameters were derived using Phoenix
WinNonlin (version 6.3; Pharsight Company, Mountain
View, California).
Area under the effect concentration–time curve for the
change from baseline prior to the first dose for TmP, serum
Pi, and 1,25(OH)2D were calculated for day 1 to day 120 of
4Zhang et al
treatment
Using a streptavidin-alkaline
or up to the last measured
phosphatase
time beforeconjugate
withdrawal
to
) andchemiluminescent
for each of the 4 dosing
intervals
(AUEC
catalyzelastthe
substrate,
the (AUEC
resulting
1,
,
AUEC
,
and
AUEC
).
Pharmacodynamic
(PD)
AUEC
chemiluminescent
light
intensity
was
measured,
and
the
2
3
4
variables
were estimated
softwareThe
(version
9;
concentrations
of KRN23using
wereSAS
calculated.
calibraSAS
Institute
Inc.,
Carolina).
tion range
was
50North
to 3000
ng/mL. The lower limit of
Scatterplotswas
were
toassay
explore
PK/PD
correquantification
50 generated
ng/mL. The
accuracy
(relative
lations
using
AUCn(coefficient
for PK measures
andfor
AUEC
error) and
precision
of variation
the mean)
n for
PD
Pearson correlation
coefficients
were
weremeasures.
within acceptance
criteria (< 20%) for a(R)
majority
derived
from
linearsamples
regression
as appropriate.
of quality
control
(94.6%).
A small amount of
During
the study
conduct,
TmP/GFR
was manually
quality
control
samples
(5.4%)
showed relative
errors
derived
nomogram
developed
bylots
Walton
and
between from
20.7%a and
26.7%. Ten
individual
of pooled
24
In thewere
analysis,
was calculated
Bijvoet.
human serum
spikedTmP/GFR
with 50 ng/mL
KRN23.from
The
20
the
following
equation
: ranged from -11.4% to 7.2%,
relative
error of
detection
whereas all the unspiked individual lots yielded concenTmP=GFRðmg=dLÞ
TRPx½serum
phosphorous
trations
below the low¼limit
of quantification
(50 ng/mL),
ð1Þ
confirming
the
selectivity
of
the
assay.
ðmg=dLÞ�ðif TRP <¼ 0:86Þ;
Detection
of Anti-KRN23
Antibodies
TmP=GFRðmg=dLÞ
¼ 0:3xTRP=ð1
� 0:8xTRPÞ
Serum samples for anti-KRN23 antibodies were obtained
x½serum phosphorousðmg=dLÞ�ðif TRP > 0:86Þ;
before each dose and on day 36 after the fourth dose or at
ð2Þ
early withdrawal. Samples were assayed using a validated
electrochemiluminescent
method.(TRP)
A sequential
where
tubular reabsorption(ECL)
of phosphate
is 1-(2screening
employed
an initial screening
assay,
hour
urinealgorithm
phosphorus
[mg/dL])/(serum
phosphorus
[mg/dL])
� (serum
creatinine assay,
[mg/dL])/(2-hour
urine
a subsequent
immunodepletion
and a neutralizing
assay at Kyowa
Hakko Kirin California, Inc (La Jolla,
creatinine
[mg/dL]).
California).
Testing
onlyderived
proceeded
to 2the
subsequent
The TmP/GFR
values
from the
methods
were
compared,
only
the calculated
results
are reported.
assay step but
if the
earlier
assay result
was positive.
The bridging ECL format for both the screening and
specificity confirmation assays begins with a streptavidinResults
coated plate supplied by Meso Scale Discovery (MSD). In
the screening assay,
control and
bioanalytical(Table
samples
Demographics
and Baseline
Characteristics
1) are
first
diluted
with
diluent
buffer
on
a
96-well,
round-bottom
KRN23 was administered to 28 subjects. Twenty-six
polypropylene
plate.
Then, diluted
are withdrew
incubated
subjects
(92.9%)
received
4 doses;samples
1 subject
with
a
Master
Mix
containing
biotinylated
KRN23
after 3 doses because of injection-site urticaria.
One
(capture
antibody)
and
ruthenylated-KRN23
(detection
subject withdrew after 1 dose because of the presence of
antibody). toFollowing
incubation nephrocalcinosis
with the labeled (this
drug
moderate
severe preexisting
mixture,
samples
are
transferred
from
the
polypropylene
subject did not provide PK samples). The number of
plate to who
the blocked,
streptavidin-coated
plate in
subjects
contributed
to the analysis wasMSD
27. Eighteen
duplicate
and
incubated
at
room
temperature
with
shaking.
subjects (64%) were female, and 26 (93%) were white;
After incubation,
washed
and
mean
body weightunbound
was 75.6material
kg, andismean
ageaway,
was 41.5
MSD
Read
Buffer
(ECL
substrate)
is
added
to
the
wells.
years. Mean TmP/GF (1.6 � 0.4 mg/dL) and serum
The(1.9
ECL
signal
intensity
immediately
read reference
using an
Pi
� 0.3
mg/dL)
were isbelow
the normal
MSD
Sector
Imager
measuring
MSD
signal
counts,
which
range. The mean 1,25(OH)2D level (36.6 � 14.3 pg/mL)
is
used
to
evaluate
the
presence
of
anti-KRN23
antibodies
was within the normal reference range, and only 2
and assess
thedecreased
specificity
of any
relative
to
subjects
were
below
the antibodies
reference range
. The
KRN23.
The
specificity
confirmation
assay
follows
the
mean 25(OH)D was 25.0 � 9.1 ng/mL. Mean PTH was
same format
screening
assay, serum
except FGF23
that prior
to
increased
at as
74 the
pg/mL,
and mean
was
incubation
with
the
labeled
drug
mixture,
controls
and
increased at 91 pg/mL. Mean serum calcium, fasting
bioanalytical
samples /creatinine
are first diluted
contain2-hour
urine calcium
ratio,with
andbuffer
24-hour
urine
ing
immunodepletive
antigen
(KRN23).
Screening
assay
calcium excretion were in the normal range. No subject
cut point
was 1.88 forMean
signal values
ratio. for both formation
was
hypercalciuric.
markers (BALP, osteocalcin, PINP) and resorption
Pharmacodynamic
markers
(NTx, CTx)Assessments
were in the normal reference ranges,
Blood
samples
for pharmacodynamic
assessments were
although some subjects
had increased levels.
collected at screening, before each dose, on days 3, 7, 12,
1793
The Journal of Clinical Pharmacology / Vol XX No XX 2015
18, and Doses
26 after each dose, and on day 36 after the last
KRN23
dosesubjects
(final received
visit). Biochemical
measurements
All
an initial dose
of 0.05 mg/kg.included
For the
D,
25-hydroxyvitamin
D
(25[OH]D),
serum Pi,
1,25(OH)
second
dose,
26 (96.3%)
received
0.1
mg/kg,
and
1
remained
2
creatinine,
calcium,
and parathyroid
hormone
(PTH).
Pi,
at
0.05 mg/kg
(1 withdrew).
For the third
dose, 25
subjects
calcium,received
and creatinine
were1measured
correspond(92.6%)
0.3 mg/kg,
continuedinatthe
0.05,
and 1 at
ingmg/kg.
2-hourFor
urine
after
8-hour(61.5%)
fast onreceived
the day
0.1
thecollected
fourth dose,
16 an
subjects
before
the first
injection and
on mg/kg,
days 3, 7,
and 26 from
after
0.6
mg/kg,
8 continued
at 0.3
1 12,
increased
eachtoinjection
day 0.1
36toafter
the last
injection.
0.05
0.1 mg/kg,and
andon
1 from
0.3 mg/kg
(1 withdrew).
Calcium,
and creatinine
measuredThe
in mean
a 24-hour
No
subjectPi,
required
dose delaywere
or reduction.
total
urine collected
on the day increased
before thefrom
firstthe
injection,
on
KRN23
doses administered
first to the
days 3, dose:
12, and0.05
26 after
each
injection,
on�day
36 after
fourth
� 0.0,
0.10
� 0.01,and
0.28
0.06,
and
the last
injection.
TmP/GFR
was calculated
0.48
� 0.16
mg/kg for
dosing intervals
1, 2, 3,using
and an
4,
established equation based on glomerular filtration
respectively.
rate calculated from creatinine clearance.20 Blood and
urine samples forResults
measurement of bone turnover markers
Pharmacokinetic
were collected prior to each dose and at the final visit.
The mean KRN23 PK exposure (AUCn) increased from
Bone
formation
serum
bone
alkaline
174
� 63
after themarkers
first doseincluded
to 2610 �
974 mg
� h/mL
after
phosphatase
(BALP),
procollagen
type
1,
N
propeptide
the fourth dose (Table 2). The AUClast for the 4 dosing
(P1NP), and osteocalcin. Bone resorption markers
intervals was 4340 � 1320 mg � h/mL. The extrapolated
included
serum carboxy-terminal
cross-linked telopeptide
part
for AUC
inf (5240 � 1880 mg � h/mL) compared with
of
type
1
collagen
(CTx),
and
urineT amino-terminal
AUClast was less than 25%. The mean
max values were
cross-linked telopeptide of type 1 collagen (NTx).
similar (7.0 to 8.5 days) across the 4 dosing intervals. The
Serummean
intact FGF23
was measured
an
terminal
t1/2 estimated
after at
thebaseline
fourth using
dosing
ELISA
assay
(Kainos
Laboratories,
Japan).
All
other
interval was 16.4 days. Mean serum KRN23 concentrabiochemical variables in serum (25[OH]D, 1,25[OH]2D,
tion increased as mean dose increased (Figure 1A). The
PTH,
osteocalcin,
Pi, calcium,
and creatimean BALP,
KRN23CTx,
serum
PK exposures
(log-transformed
nine)
and
in
urine
samples
(Pi,
calcium,
creatinine,
log
Cmax,n, Cmin,n, and AUCn) increased with increased and
NTx) were analyzed by Quest Diagnostics Inc. using
dose in a proportional manner, with correlation coefstandardized
automated
methodologies.
ficients
(R) of
0.998, 0.993,
and 0.998, respectively
(Figure 1B shows AUCn). The mean intersubject
Pharmacokinetic and Statistical Analysis
variability in AUCn ranged from 30% to 37%. The
Pharmacokinetic
parameters
CL/F
was 0.186(PK)
� 0.078
mL/h/kg,were
and estimated
the V/F using
was
serum
concentration–time
data
for
all
accumulated
doses
98.3 � 34.6 mL/kg.
from day 1 to the last follow-up visit on day 120.
A noncompartmental
analysis method was
employed to
Pharmacokinetic
and Pharmacodynamic
Relationships
21
These
included
area
under the
derive
PK
parameters.
Serum Phosphorus. Mean serum Pi concentrations
serum concentration–time curve from zero to last measured
increased to a maximum on day 7, returned to near
concentration
(AUC
), area
concentrabaseline
by day
28 last
after
eachunder
dose,the
andserum
increased
with
),
apparent
tion–time
curve
from
zero
to
infinity
(AUC
successive doses of KRN23 (Figure 2A). inf
AUEC
n for
volume of distribution (V/F), apparent clearance (CL/F),
serum Pi was linearly correlated with AUCn for serum
and terminal
elimination
half-life
KRN23
(R ¼ 0.812;
Figure
2B). after the fourth dosing
).
Pharmacokinetic
parameterstoestimated
for
interval
(t
1/2
TmP/GFR.
TmP/GFR values increased
a maximum
each 28-day dosing interval included area under the serum
on day 7 after each dose, returned to near baseline by day
concentration–time
curvesuccessive
(AUCn), doses
maximum
serum
28,
and increased with
of KRN23
),
predose
serum
concentration
concentration
(C
max,n
(Figure 2C). AUEC
n for TmP/GFR was linearly correlated
(Cmin,n), and time to peak serum concentration (Tmax,n),
with
serum KRN23AUCn (R ¼ 0.822; Figure 2D).
where
n denotes
the number of doses received (1, 2, 3, and
1,25(OH)
2D. Serum 1,25(OH)2D concentrations in4)
up
to
the
measured
interval.
statistics
creased to a maximumdosing
by day
3 afterDescriptive
each dose and
then
were used for serum KRN23 concentration data and PK
returned to near baseline values by day 28 (Figure 2E).
parameters.
All PK parameters were derived using Phoenix
AUEC
n for serum 1,25(OH)2D were linearly correlated
WinNonlin
(version AUC
6.3; Pharsight
Company, Mountain
with serum KRN23
n (R ¼ 0.666; Figure 2F).
View,
California).
Bone Markers. The AUECn for serum bone formation
Area under
the effect
concentration–time
curve forwith
the
markers
increased
as KRN23
AUCn increased
change
from
baseline
prior
to
the
first
dose
for
TmP,
serum
moderate correlation. The correlation coefficients (R)
Pi, and 1,25(OH)2D were calculated for day 1 to day 120 of
180
Zhang et al
The Journal of Clinical Pharmacology / Vol 56 No 2 20165
Table 1. Subject Demographics and Disease Characteristics at Baseline
Characteristica
Demographics
Age (years)
Sex (male/female), n
Race (white/other), n
Weight (kg)
Height (cm)
Serum FGF23 (pg/mL), median (range)
Serum Pi (mg/dL)
TmP/GFR (mg/dL)
Serum 1,25(OH)2D (pg/mL)b
Serum 25(OH)D, ng/mL
Serum total calcium (mg/dL)
Serum PTH (pg/mL), median (range)
BALP (mg/L)
KRN23
Reference Range
n ¼ 27
41.5 � 14.0 (19–66)
9/18
26/1
75.6 � 19.9 (51.3–121.9)
150.1 � 12.5 (121.9–170.2)
NA
NA
NA
NA
NA
91 (36–3520)
1.9 � 0.3 (1.2–2.8)
1.6 � 0.4 (0.8–2.3)
36.6 � 14.3 (10–62)
25.0 � 9.1 (12–44)
9.1 � 0.4 (8.5–10.2)
74 (38–143)
28.3 � 12.8 (8.2–52.4)
P1NP (ng/mL)
64.1 � 30.7 (11–123)
Osteocalcin (ng/mL)
29.3 � 17.7 (6.5–95.4)
NTx/creatinine ratio (nmol BCE/mmol)
41.8 � 20.3 (12–112)
CTx (pg/mL)
24-Hour urine calcium (mg/24 hours), median (range)
24-Hour urine creatinine (g/24 hours), median (range)
2-Hour calcium/creatinine ratio (mg/g creatinine), median (range)
24-Hour urine phosphate (mg), median (range)
752 � 389 (214, 1899)
67 (11–253)
1.13 (0.54–3.01)
36 (7–192)
2244 (1087–4785)
8–5422
2.5–4.523
2.5–4.224
15.9–55.625
32–10025
8.5–10.323
10–6523
M: 3.7–20.9
F: 2.9–14.5 (premenopausal)
F: 3.8–22.6 (postmenopausal)23
M: 22–87 (18þ years)22
F: 19–83 (premenopausal)23
F: 16–96 (postmenopausal)23
M: 8.0–37023
F: 7.0–38.0 (pre- and postmenopausal
without osteoporosis)23
M: 12–99 (18–29 years)23
M: 9–60 (30–59 years)23
M: not established (60þ years)23
F: 4–64 (premenopausal, 18þ years)23
M: 87–11200 (18–129 years)23
M: 70–1780 (30–139 years)23
M: 60–1700 (40–149 years)23
M: 87–1345 (50–168 years)23
M: not established (69þ years)23
F: 64–1640 (18–129 years)23
F: 60–1650 (30–139 years)232
F: 40–1465 (40–149 years)23
F: not established (50þ years)232
M: 50–1300 F: 50–125023
0.63–12.5023
M: 10–124023 F: 10–132023
M: 1103–14 90323 F: 521–13 67723
a
Data are presented from baseline and are expressed as mean � standard deviation (range) except where noted.
n ¼ 24 for serum 1,25(OH)2D.
1,25(OH)2D, 1,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D; BALP, bone alkaline phosphatase; F, female; FGF23, fibroblast growth factor 23; PTH,
parathyroid hormone; M, male; NA, not applicable; Pi, serum phosphorus (inorganic); TmP/GFR, renal tubular maximum reabsorption rate of phosphate to
glomerular filtration rate.
b
were 0.373 for BALP (Figure 3A), 0.484 for P1NP
(Figure 3B), and 0.537 for osteocalcin (Figure 3C). The
AUECn for the bone resorption markers increased as
KRN23 AUCn increased. The moderate correlations are
shown by the R values of 0.446 for CTx (Figure 3D) and
0.310 for NTx/creatinine (Figure 3E).
Other Biochemistry Parameters. No meaningful effect of
KRN23 treatment was observed on AUECn for other PD
variables (Figure 4): serum calcium (R ¼ 0. 238, Figure 4A),
PTH (R ¼ 0.118, Figure 4B), fasting 2-hour calcium/
creatinine ratio (R ¼ 0.058, Figure 4C), or 24-hour urine
calcium (R ¼ 0.146, Figure 4D). There were also no
meaningful PK/PD correlations observed for the following
PD variables: serum 25(OH)D (R ¼ 0.160), creatinine
(R ¼ 0.351), ionized calcium (R ¼ 0.099); and calcium
(R ¼ 0.146), creatinine (R ¼ 0.149), and calcium/creatinine
ratio (R ¼ 0.291) in 24-hour urine samples (Supplementary
Figure S1).
6Zhang et al
Table
Pharmacokinetic Parameters
Using2.a KRN23
streptavidin-alkaline
phosphatase
1813
The Journal of Clinical Pharmacology / Vol XX No XX 2015
18, and 26 after each dose, and on day 36 after the last
conjugate to
measurements4 included
catalyze
the
chemiluminescent
substrate,
the
resulting
Dosing Interval
1
2 dose (final visit). Biochemical
3
chemiluminescent
light intensity 27was measured, and the 27 serum Pi, 1,25(OH)2D,2725-hydroxyvitamin D (25[OH]D),
Number
of subjects
26
8.50were
� 2.90 (2.98,
14.9) 34 The 7.04
� 1.71 (3.95, creatinine,
11.9) 24
7.45 � 3.83
19.9) 27
7.00 � 3.22 (2.87–15.0)
Tconcentrations
max,n (days)
calcium,
and(1.96,
parathyroid
hormone
(PTH). 46Pi,
of KRN23
calculated.
calibraCmax,n (ng/mL)
386 � 145 (193, 688) 38
947 � 307 (356, 1710) 32
2490 � 843 (421, 3950) 34
4750 � 1800 (1030, 8110) 38
calcium,
and
creatinine
were
measured
in
the
correspondtion
range
was
50
to
3000
ng/mL.
The
lower
limit
of
Cmin,n (ng/mL)
147 � 53.4 (60.7, 253) 36
364 � 130 (119, 662) 36
1080 � 587 (104, 2560) 54
1470 � 827 (166, 2890) 56
ing302-hour urine
after35an 8-hour
on 4280)
the day
quantification
was 50 ng/mL.
assay
(relative
AUC
174 � The
63 (71.6,
316) accuracy
36
430 � 127 (156, 685)
1220collected
� 432 (228, 2350)
2610 � fast
974 (572,
37
n (mg � h/mL)
Data
fromand
all 4 precision
dosing intervals
(n ¼ 27)
before the first injection and on days 3, 7, 12, and 26 after
error)
(coefficient
of variation for the mean)
AUC
� h/mL)acceptance criteria
AUCinf(<
(mg
� h/mL)
CL/F (mL/h/kg)
t1/2 (days)
last (mg
each injection andV/Fon(mL/kg)
day 36 after the last
injection.
were
within
20%) for a majority
4340 � 1320 (1340–6450) 30
5240 � 1880 (1570, 8990) 36
0.186 � 0.0780 (0.0835, 0.472) 42
98.3 � 34.6 (51.2, 21.2) 35
16.4 � 5.83 (5.58, 29.5) 35
Calcium, Pi, and creatinine were measured in a 24-hour
of quality control samples (94.6%). A small amount of
urine collected on the day before the first injection, on
quality control samples (5.4%) showed relative errors
Mean � standard deviation (min, max) CV% is reported.
days 3, 12, and 26 after each injection, and on day 36 after
between 20.7% and 26.7%. Ten individual lots of pooled
the last injection. TmP/GFR was calculated using an
human serum were spiked with 50 ng/mL KRN23. The
established equation based on glomerular filtration
relative error of detection ranged from -11.4% to 7.2%,
Anti-KRN23-Antibodies
values were similar across the 4 dosing intervals
and ranged
rate calculated from creatinine clearance.20 Blood and
whereas all the unspiked individual lots yielded concenNo positive anti-KRN23 antibodies were detected after
from 7.0 to 8.5 days, indicating that rate of absorption of
urine samples for measurement of bone turnover markers
trations below the low limit of quantification (50 ng/mL),
KRN23 treatment in any subject from screening assay
KRN23 after subcutaneous administration was consistent
were collected prior to each dose and at the final visit.
confirming the selectivity of the assay.
testing. Therefore, no further confirmative assay was
during every 28-day dosing regimen. Because of intraBone formation markers included serum bone alkaline
needed.
subject dose escalation of KRN23 in this study and a mean
phosphatase (BALP), procollagen type 1, N propeptide
Detection of Anti-KRN23 Antibodies
KRN23 t1/2 of 16.4 days, steady-state conditions were not
(P1NP), and osteocalcin. Bone resorption markers
Serum samples for anti-KRN23 antibodies were obtained
Baseline FGF23 and Serum Pi Response
reached for any dose between 0.1 and 1.0 mg/kg.
included serum carboxy-terminal cross-linked telopeptide
before each dose and on day 36 after the fourth dose or at
No apparent correlation between overall serum area under
Administration of a fixed KRN23 dose every 4 weeks for
of type 1 collagen (CTx), and urine amino-terminal
early withdrawal. Samples were assayed using a validated
the effect concentration–time curve (AUEClast) for serum
at least 5 times of the KRN23 t1/2 (ie, approximately 82
cross-linked telopeptide of type 1 collagen (NTx).
electrochemiluminescent (ECL) method. A sequential
Pi and baseline intact FGF23 levels was observed
days) would be necessary to reach KRN23 steady-state
Serum intact FGF23 was measured at baseline using an
screening algorithm employed an initial screening assay,
(R ¼ 0.0943).
serum concentrations.
ELISA assay (Kainos Laboratories, Japan). All other
a subsequent immunodepletion assay, and a neutralizing
Unlike a standard multiple ascending-dose study
biochemical variables in serum (25[OH]D, 1,25[OH]2D,
assay at Kyowa Hakko Kirin California, Inc (La Jolla,
design for a novel monoclonal antibody therapy
PTH, BALP, CTx, osteocalcin, Pi, calcium, and creatiCalifornia). Testing only proceeded to the subsequent
Discussion
(parallel-dose groups),27 the present study employed
nine) and in urine samples (Pi, calcium, creatinine, and
assay step if the earlier assay result was positive.
KRN23 given subcutaneously in 4 increasing doses every
an intradose-escalation scheme. The design was based
NTx) were analyzed by Quest Diagnostics Inc. using
The bridging ECL format for both the screening and
28 days to adults with XLH, increased TmP, and serum Pi
on the practical reason that the prevalence of XLH is
standardized automated methodologies.
specificity confirmation assays begins with a streptavidinand 1,25(OH)2D concentrations in a KRN23 PK-dependent
only 1:20 000.6 For the intradose-escalation design, the
coated plate supplied by Meso Scale Discovery (MSD). In
manner. The PK exposure to KRN23, as measured by Cmax,
dose–response relationship cannot be analyzed directly.
Pharmacokinetic and Statistical Analysis
the screening assay, control and bioanalytical samples are
We therefore used PK exposure (AUCn) and PD
n, Cmin,n, and AUCn values, increased in a dose-proportional
Pharmacokinetic (PK) parameters were estimated using
first diluted with diluent buffer on a 96-well, round-bottom
manner. The V/F for a typical subject weighing 70 kg was
response (AUECn) for the assessment of PK/PD
serum concentration–time data for all accumulated doses
polypropylene plate. Then, diluted samples are incubated
estimated to be 6.9 L, which is slightly larger than the
relationships.
from day 1 to the last follow-up visit on day 120.
with a Master Mix 26
containing biotinylated KRN23
plasma volume (3–5 L ). The mean t1/2 for KRN23
This study demonstrated that increases in TmP/GFR,
A noncompartmental analysis method was employed to
(capture antibody) and ruthenylated-KRN23 (detection
(16.4 days) after multiple-dose KRN23 administration was
D (AUEC ) were linearly
serum Pi, and serum 1,25(OH)
derive PK parameters.21 These2 included narea under the
antibody). Following incubation with the labeled drug
similar to the mean t1/2 (17.6, 15.1, 13.4, and 18.7 days) after
correlated with increases in KRN23 PK exposure (AUCn)
serum concentration–time curve from zero to last measured
mixture, samples are transferred from the polypropylene
single subcutaneous administration of KRN23 at doses of
following multiple-dose administration of KRN23 in
concentration (AUClast), area under the serum concentraplate to the blocked, streptavidin-coated MSD plate in
0.1, 0.3, 0.6, and 1.0 mg/kg, respectively, indicating that
adults with XLH. TmP/GFR
and serum Pi concentrations
tion–time curve from zero to infinity (AUCinf), apparent
duplicate and incubated at room temperature with shaking.
elimination of KRN23 is not dose dependent. Mean Tmax
reached maximum values as KRN23 concentrations at
volume of distribution (V/F), apparent clearance (CL/F),
After incubation, unbound material is washed away, and
and terminal elimination half-life after the fourth dosing
MSD Read Buffer (ECL substrate) is added to the wells.
interval (t1/2). Pharmacokinetic parameters estimated for
The ECL signal intensity is immediately read using an
each 28-day dosing interval included area under the serum
MSD Sector Imager measuring MSD signal counts, which
concentration–time curve (AUCn), maximum serum
is used to evaluate the presence of anti-KRN23 antibodies
concentration (Cmax,n), predose serum concentration
and assess the specificity of any antibodies relative to
(Cmin,n), and time to peak serum concentration (Tmax,n),
KRN23. The specificity confirmation assay follows the
where n denotes the number of doses received (1, 2, 3, and
same format as the screening assay, except that prior to
4) up to the measured dosing interval. Descriptive statistics
incubation with the labeled drug mixture, controls and
were used for serum KRN23 concentration data and PK
bioanalytical samples are first diluted with buffer containparameters. All PK parameters were derived using Phoenix
ing immunodepletive antigen (KRN23). Screening assay
WinNonlin (version 6.3; Pharsight Company, Mountain
cut point was 1.88 for signal ratio.
View, California).
Area under the effect concentration–time curve for the
Pharmacodynamic
Assessments
Figure 1. Mean serum KRN23 concentration over time (A) and dose proportionality between mean log KRN23 AUCn and mean log dose during the 4
change
baseline
prior
to the
dose
TmP,
Blood
samples
for
pharmacodynamic
assessments
were
dosing intervals (B). Error bars represent standard deviation (SD). The mean � SD dose from
was 0.05
� 0.0, 0.10
� 0.01,
0.28first
� 0.06,
andfor
0.48
� 0.16serum
mg/kg
for
dosing intervals
1, 2, 3, and
4, respectively.
N ¼on
27 for
dosing
and n1,25(OH)
¼ 26 for dosing
interval
4.
D
were
calculated
for
day
1
to
day 120 of
collected
at screening,
before
each dose,
days
3, 7,intervals
12, 1 toPi,3 and
2
182
Zhang et al
The Journal of Clinical Pharmacology / Vol 56 No 2 20167
Figure 2. Pharmacodynamic (PD) profiles over 4 dosing intervals (left) and relationship of pharmacokinetic (PK) and PD parameters (right). (A and B)
Serum Pi; (C and D) TmP/GFR; (E and F) 1,25(OH)2D. AUCn, area under the KRN23 serum concentration–time curve; AUECn, area under the effect
concentration–time curve for PD change from baseline during interval n (n ¼ 1, 2, 3, or 4); R, Pearson correlation coefficient.
about the same time after each dose, and their levels
increased and decreased in parallel. The direct relationship between serum KRN23 concentration and serum
Pi increase reflects the dose-dependent blocking action of
the antibody on renal phosphate reabsorption and supports
dose adjustment of KRN23 based on peak and trough
serum Pi concentrations.
In the present study, the body weight–based dose was
employed instead of a fixed dose, which is usually
used for subcutaneous route of administration.28 As the
study design was an intradose escalation, the body
weight–based dosing strategy was employed to minimize
variability.
In the present analysis, TmP/GFR values were both
manually read using a nomogram24 and calculated using
an equation.20 The values derived from 2 methods
resulted in a linear correlation with an R of 0.996.
Thus, the equation20 that is easier to perform than manual
reading may be used in future clinical trials.
The increased serum 1,25(OH)2D levels observed in
the study might have been expected to increase
intestinal calcium absorption (not measured) and
potentially increase serum calcium concentration and
urine calcium excretion. However, serum calcium
and urine calcium did not change, and there were no
correlations between serum KRN23 concentrations and
serum and urine calcium changes. This might suggest
that the increased absorbed calcium was deposited in
bone, a strong likelihood in the setting of osteomalacia
in XLH patients.
8Zhang et al
Using a streptavidin-alkaline phosphatase conjugate to
catalyze the chemiluminescent substrate, the resulting
chemiluminescent light intensity was measured, and the
concentrations of KRN23 were calculated. The calibration range was 50 to 3000 ng/mL. The lower limit of
quantification was 50 ng/mL. The assay accuracy (relative
error) and precision (coefficient of variation for the mean)
were within acceptance criteria (< 20%) for a majority
of quality control samples (94.6%). A small amount of
quality control samples (5.4%) showed relative errors
between 20.7% and 26.7%. Ten individual lots of pooled
human serum were spiked with 50 ng/mL KRN23. The
relative error of detection ranged from -11.4% to 7.2%,
whereas all the unspiked individual lots yielded concentrations below the low limit of quantification (50 ng/mL),
confirming the selectivity of the assay.
1833
The Journal of Clinical Pharmacology / Vol XX No XX 2015
18, and 26 after each dose, and on day 36 after the last
dose (final visit). Biochemical measurements included
serum Pi, 1,25(OH)2D, 25-hydroxyvitamin D (25[OH]D),
creatinine, calcium, and parathyroid hormone (PTH). Pi,
calcium, and creatinine were measured in the corresponding 2-hour urine collected after an 8-hour fast on the day
before the first injection and on days 3, 7, 12, and 26 after
each injection and on day 36 after the last injection.
Calcium, Pi, and creatinine were measured in a 24-hour
urine collected on the day before the first injection, on
days 3, 12, and 26 after each injection, and on day 36 after
the last injection. TmP/GFR was calculated using an
established equation based on glomerular filtration
rate calculated from creatinine clearance.20 Blood and
urine samples for measurement of bone turnover markers
were collected prior to each dose and at the final visit.
Bone formation markers included serum bone alkaline
phosphatase (BALP), procollagen type 1, N propeptide
(P1NP), and osteocalcin. Bone resorption markers
included serum carboxy-terminal cross-linked telopeptide
of type 1 collagen (CTx), and urine amino-terminal
cross-linked telopeptide of type 1 collagen (NTx).
Serum intact FGF23 was measured at baseline using an
ELISA assay (Kainos Laboratories, Japan). All other
biochemical variables in serum (25[OH]D, 1,25[OH]2D,
PTH, BALP, CTx, osteocalcin, Pi, calcium, and creatinine) and in urine samples (Pi, calcium, creatinine, and
NTx) were analyzed by Quest Diagnostics Inc. using
standardized automated methodologies.
Detection of Anti-KRN23 Antibodies
Serum samples for anti-KRN23 antibodies were obtained
before each dose and on day 36 after the fourth dose or at
early withdrawal. Samples were assayed using a validated
electrochemiluminescent (ECL) method. A sequential
screening algorithm employed an initial screening assay,
a subsequent immunodepletion assay, and a neutralizing
assay at Kyowa Hakko Kirin California, Inc (La Jolla,
California). Testing only proceeded to the subsequent
assay step if the earlier assay result was positive.
The bridging ECL format for both the screening and
specificity confirmation assays begins with a streptavidincoated plate supplied by Meso Scale Discovery (MSD). In
Pharmacokinetic and Statistical Analysis
the screening assay, control and bioanalytical samples are
Pharmacokinetic (PK) parameters were estimated using
first diluted with diluent buffer on a 96-well, round-bottom
serum concentration–time data for all accumulated doses
polypropylene plate. Then, diluted samples are incubated
from day 1 to the last follow-up visit on day 120.
with a Master Mix containing biotinylated KRN23
A noncompartmental analysis method was employed to
(capture antibody) and ruthenylated-KRN23 (detection
Figure 3. Relationship of pharmacokinetic and bone marker change from baseline. (A) BALP; (B) P1NP;
(C) Osteocalcin; (D) CTx; (E) NTx/
included area under the
derive PK parameters.21 These
antibody). Following incubation with the labeled drug
creatinine. AUCn, area under the KRN23 serum concentration–time curve; AUECn, area under the effect concentration–time curve for bone marker
serum
concentration–time
curve
from
zero to last measured
mixture,
samples
are
transferred
from
the
polypropylene
change from baseline during interval n (n ¼ 1, 2, 3, or 4); R, Pearson correlation coefficient.
concentration (AUClast), area under the serum concentraplate to the blocked, streptavidin-coated MSD plate in
tion–time curve from zero to infinity (AUCinf), apparent
duplicate and incubated at room temperature with shaking.
volume of distribution
(V/F),paradoxical
apparent clearance
After
is2washed
and
osteomalacia.
An initial
rise in(CL/F),
both
Theincubation,
increases unbound
in serum material
1,25(OH)
D may away,
also have
and terminal
after the fourth
dosing
MSDexpected
Read Buffer
(ECL substrate)
is added
the levels
wells.
formation
andelimination
resorptionhalf-life
bone biomarkers
was seen
in
been
to decrease
the increased
serumtoPTH
).
Pharmacokinetic
parameters
estimated
for
interval (t
The ECLfrom
signal
intensityhyperparathyroidism
is immediately readpresent
using an
patients
with
hypophosphatemic
osteomalacia
on
startresulting
secondary
in
1/2
each treatment
28-day dosing
included
area under
the serum
MSD Sector
Imager measuring
signalin
counts,
ing
withinterval
the active
vitamin
D analogue,
many
of the patients.
However,MSD
no change
serumwhich
PTH
concentration–time
curve D(AUC
),
maximum
serum
is used
to evaluate
the presence
anti-KRN23
antibodies
1-alpha
hydroxy vitamin
,
and
phosphate
supplewas
observed,
possibly
becauseofresolution
of secondary
n
3
predose returning
serum concentration
concentration
(Cmax,n
and assess the specificity
any antibodies
to
ments
with the
bone), markers
to normal
hyperparathyroidism
takes of
considerably
more relative
time than
(Cmin,n), and
time after
to peak
serummonths
concentration
(Tmax,n),
KRN23.
The specificity
confirmation assay follows the
reference
range
several
of successful
that
evaluated
in this study.
9
where n denotes
number ofinitial
dosesincrease
receivedis(1,generally
2, 3, and
same
as theinscreening
assay,
except that
to
The the
paradoxical
treatment.
Theformat
increase
both bone
formation
andprior
bone
4) up to the measured
interval. stimulation
Descriptive statistics
incubation with
the labeled
drug moderate
mixture, controls
and
considered
to be duedosing
to osteoblast
of bone
resorption
markers
and the
correlation
were
used
for
serum
KRN23
concentration
data
and PK
bioanalytical
samples
are AUEC
first diluted
with
buffer
containturnover.
Thus,
the
rise
in
biomarkers
with
KRN23
and
serum
KRN23
between
their
serum
n
parameters.
All
PK
parameters
were
derived
using
Phoenix
ing immunodepletive
antigen
(KRN23).
Screening
assay
is
of
great
interest
because
it
implies
an
indirect
suggests
that
the
increase
in
serum
phosphate
and
1,25
AUC
n
WinNonlin
(version
6.3;
Pharsight
Company,
Mountain
cut point
1.88 intervention
for signal ratio.
effect
of was
KRN23
on bone turnover. As a
(OH)
D
was
starting
to
heal
the
osteomalacia.
2
View,
California).
group, the patients had normal levels of bone biomarkOverall
PK/PD results after multiple doses of KRN23
Area underadministration
the effect concentration–time
curve forrelathe
Pharmacodynamic
Assessments
ers
at baseline, although
several subjects had clearly
subcutaneous
confirmed the PK/PD
change from
baseline
prior
to the firstKRN23
dose forsubcutaneous
TmP, serum
Blood samples
pharmacodynamic
assessments
were
increased
levels,for
probably
indicating varying
degrees
of
tionships
observed
after
single-dose
Pi, and 1,25(OH)2D were calculated for day 1 to day 120 of
collected at screening, before each dose, on days 3, 7, 12,
184
Zhang et al
The Journal of Clinical Pharmacology / Vol 56 No 2 20169
Figure 4. Pharmacodynamic (PD) profiles over 4 dosing intervals (left) and relationship of PK and PD parameters (right). (A and B) Serum
calcium; (C and D) serum PTH; (E and F) 2-hour urine calcium/creatinine ratio; (G and H) 24-hour urine calcium. AUCn: area under the KRN23
serum concentration–time curve; AUECn, area under the effect concentration–time curve for PD change from baseline during interval n (n ¼ 1, 2,
3, or 4); R, Pearson correlation coefficient.
administration,15,16 and further support the mechanism of
action for KRN23 to block FGF23 activity.
In conclusion, the effects of KRN23 administered
subcutaneously at 28-day intervals on serum Pi, TmP/
GFR, and serum 1,25(OH)2D levels were sustained after
each dose. There was also stimulation of bone turnover.
PK dose proportionality and the linear PK/PD relationship
between serum KRN23 and Pi concentrations support
adjusting the KRN23 dose based on serum Pi levels.
KRN23 is a promising treatment for XLH patients.
Acknowledgments
We are grateful to the dedicated patients who participated in the
study and to the subinvestigators, clinical study coordinators,
and research nursing staff at the 6 study sites (Indiana
University, Indianapolis, Indiana; The Methodist Hospital,
Houston, Texas; Duke University Medical Center, Durham,
North Carolina; Shriners Hospital for Children, Montreal,
Quebec, Canada; the University of California, San Francisco,
San Francisco, California; and Yale University School of
Medicine, New Haven, Connecticut). We thank Val Barra and
Yamamoto Katsuhiko for analysis of serum KRN23 and FGF23
concentrations (Kyowa Hakko Kirin California, Inc, La Jolla,
California), Kavita Gumbhir-Shah (PharmaMed Resources
LLC, New Jersey) for conducting PK/PD data analysis, Shirley
McKernan for editing and formatting of the manuscript (Kyowa
Hakko Kirin Pharma, Inc.).
Declaration of Conflicting Interests
The data in this article were obtained from the KRN23-INT001study funded by Kyowa Hakko Kirin Pharma Inc. Drs. T.O.
Carpenter and M.D. Ruppe received consulting fees (other than
10
Zhang et al
advisory
Using a board
streptavidin-alkaline
or board of directors)
phosphatase
and research
conjugate
grants.
to
Drs.
Imel, the
Ruppe,
Weber, Insogna, Portale,
Glorieux,
Peacock,
catalyze
chemiluminescent
substrate,
the resulting
and
Carpenter received
research
grantswas
during
the study conduct.
chemiluminescent
light
intensity
measured,
and the
Drs.
Zhang, Klausner,
Vergeire,
and calculated.
Humphrey and
Mr.calibraIto are
concentrations
of KRN23
were
The
employees
Kyowa
Kirin
Pharma
Inc.lower
Drs. E.A.
Imel,
tion rangeofwas
50 Hakko
to 3000
ng/mL.
The
limit
of
T.O.
Carpenter,was
M.50Peacock,
F.H.assay
Glorieux,
T.J.(relative
Weber,
quantification
ng/mL. The
accuracy
A.A.
and K. (coefficient
Insogna have
grants
error)Portale,
and precision
of received
variationresearch
for the mean)
and/or
consulting
fees (other
than (<
advisory
board
board of
were within
acceptance
criteria
20%)
for or
a majority
directors)
Ultragenyx
Pharmaceuticals,
for amount
subsequent
of qualityfrom
control
samples
(94.6%). A Inc.
small
of
work
related
to KRN23.
quality
control
samples (5.4%) showed relative errors
between 20.7% and 26.7%. Ten individual lots of pooled
human serum were spiked with 50 ng/mL KRN23. The
Funding
relative error of detection ranged from -11.4% to 7.2%,
This study was funded by Kyowa Hakko Kirin Pharma Inc.
whereas all the unspiked individual lots yielded concentrations below the low limit of quantification (50 ng/mL),
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2009;73(24):2061–2070.
andAlzheimer
terminaldisease.
elimination
half-life
after the fourth dosing
28.
Kavanaugh
McInnes I, Mease P, et
al. Golimumab,
a new human
parameters
estimated
for
interval
(t1/2).A,Pharmacokinetic
necrosis factor a antibody, administered every four weeks as
eachtumor
28-day
dosing interval included area under the serum
a subcutaneous injection in psoriatic arthritis: twenty-four–week
concentration–time
curveof (AUC
serum
n), maximum
efficacy and safety results
a randomized,
placebo-controlled
),
predose
serum
concentration
concentration
(C
max,n 2009;60(4):976–986.
study. Arthritis Rheum.
(Cmin,n), and time to peak serum concentration (Tmax,n),
where n denotes the number of doses received (1, 2, 3, and
Supporting
Information
4) up to the measured
dosing interval. Descriptive statistics
were
used
for
serum
concentration
data and
PK
Additional supportingKRN23
information
may be found
in the
parameters.
All
PK
parameters
were
derived
using
Phoenix
online version of this article at the publisher’s web-site.
WinNonlin (version 6.3; Pharsight Company, Mountain
View, California).
Area under the effect concentration–time curve for the
change from baseline prior to the first dose for TmP, serum
Pi, and 1,25(OH)2D were calculated for day 1 to day 120 of