SUPPLEMENTAL MATERIAL
Pharmacokinetics, Biotransformation, and Excretion of
[14C]Etelcalcetide (AMG 416) Following a Single
Microtracer Intravenous Dose in Patients With Chronic
Kidney Disease On Hemodialysis
Raju Subramanian, Xiaochun Zhu, M. Benjamin Hock, Bethlyn J. Sloey, Benjamin Wu,
Sarah F. Wilson, Ogo Egbuna, J. Greg Slatter, Jim Xiao, Gary L. Skiles
Amgen Inc., Thousand Oaks, California, USA
Address correspondence to: Raju Subramanian
Amgen Inc.
One Amgen Center Drive
Thousand Oaks, CA 91320
Phone: 805-447-6301
Fax: 805-447-1010
Email: [email protected]
Target journal: Clinical Pharmacokinetics
Subramanian et al 1
SUPPLEMENTAL METHODS
Sample Collection and Processing
Blood samples were collected into potassium-EDTA for [14C]etelcalcetide-derived
radioactivity (referred to as the “total C-14”) analysis, etelcalcetide and total M11 (TM11)
bioanalysis, and biotransformation product profiling according to the schedule described.
An aliquot of whole blood from each time-point was saved for carbon-14 (C-14)
analysis. The remainder was processed to plasma by centrifuging the whole blood at
1500×g for 15 minutes at 4°C. An aliquot of plasma was set aside for C-14 analysis. The
remaining plasma was acidified with citric acid (20mg/mL) to prepare acidified aliquots
for bioanalysis and biotransformation product profiling. The prepared non-acidified and
acidified plasma was stored at −70°C.
The discharged urine from each patient was collected in 24-hour intervals during the in-
clinic period (pre- and postdose; study days 1 to 11). The weight of the total urine in
each 24-hour period was recorded and aliquots of this pooled urine were stored at
−70°C for further C-14 analysis and biotransformation product profiling.
Total dialysate was collected for C-14 analysis and radioprofiling at each hemodialysis
session for (1) for each patient during the in-clinic period (study days 1 to 11) and
outpatient days (study days 14 to 39), and (2) for select patients (n=4) at the six time-
points in the extended pharmacokinetic collection period. The dialysate from each
hemodialysis session was collected in collection barrels in approximately 1-hour
intervals and their weight was recorded for each interval. After thorough mixing, a fixed
percentage of aliquots from each interval were combined and aliquots of this pooled
dialysate were stored at −70°C for C-14 analysis and biotransformation product profiling.
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The feces from each patient was collected during the in-clinic period (pre- and postdose;
study days 1 to 11). The total weight of each discharge was recorded at the clinic and
stored at −70°C until shipment to Covance (Madison, WI). At Covance, the feces
samples were combined by patient at 24-hour intervals, combined with appropriate
volume of ethanol/water (1:1 v/v) mixture and homogenized. A sub-sample of each
homogenized sample was freeze-dried, and then ground in a pestle and mortar; these
were used for C-14 analysis. The freeze-dried samples were stored at room
temperature. Aliquots of homogenized feces were stored at −70°C for biotransformation
product profiling.
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Bioanalysis of Etelcalcetide and Total M11
Plasma concentrations of etelcalcetide and TM11 were determined by liquid
chromatography-tandem mass spectrometry (LC-MS/MS) method. Etelcalcetide
concentrations were measured in acidified plasma by an electrospray ionization (ESI)
positive-ion mode LC-MS/MS assay at Alturas Analytics (Moscow, ID). The validated
method used solid phase extraction; the MS response was linear over the range of
0.2─100 ng/mL (lower limit of quantification [LLOQ], 0.2 ng/mL). Stable isotope-labeled
etelcalcetide was used as the internal standard. Sample extracts were injected into a
high-performance liquid chromatography (HPLC)-MS/MS triple quadrupole mass
spectrometer (Sciex API5500 or 6500; Sciex, Farmingham, MA). A Discovery HS-C18,
3-µm HPLC column (2.1×50 mm; Supelco, Bellefonte, PA) was used to separate
etelcalcetide and the internal standard from interfering compounds. The peak area of
the product ion of etelcalcetide was measured against the peak area of the product ion
of the internal standard.
TM11 is the sulfhydryl (reduced) form of the D-amino acid peptide backbone in
etelcalcetide [5]. TM11 was liberated by chemical reduction of all etelcalcetide-related
mixed disulfides with an intact D-amino acid backbone present in plasma. Reduction
was performed with tris(2-carboxyethyl)phosphine (TCEP), a disulfide bond reducing
agent. TM11 concentrations were measured by ESI positive-ion mode LC-MS/MS at
Amgen Inc. Acidified plasma was reduced with TCEP followed by protein precipitation
with heptafluorobutyric acid in 50:50 acetonitrile:methanol. The MS response was linear
over the TM11 concentration range of 50─2000 ng/mL (LLOQ, 50 ng/mL); stable
isotope-labeled etelcalcetide was used as the internal standard. Sample extracts were
separated by reversed-phase LC using a gradient elution, followed by LC-MS/MS (Sciex
API 4000).
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Accelerator Mass Spectrometry
Sample Preparation
The C-14 content in whole blood, plasma, urine, and fecal samples was determined by
accelerator mass spectrometry (AMS). All samples underwent a graphitization
procedure before C-14 detection by AMS. Carbon carrier (sodium benzoate) was added
as necessary during sample preparation to achieve approximately 2 mg of carbon.
Urine samples were processed with a 10-fold dilution and a 100-µL aliquot volume;
select plasma samples that were too concentrated for direct AMS analysis were
processed with a 10-fold dilution; blood (20-µL aliquots) and lyophilized feces (3- to 4-mg
aliquots) were directly analyzed by AMS.
C-14 detection by AMS
The prepared cathodes were placed in the ion source of a single-stage AMS (National
Electrostatics Corp., Middleton, WI) using a Cs+ ion beam, and the 14C:12C ratio of all
sources of carbon in the sample was determined. The prepared quartz sample tubes
were heat-sealed under vacuum and then heated for 2 hours at 900°C to oxidize all
carbon in the sample to CO2. The CO2 was then cryogenically transferred and sealed
into an evacuated glass tube containing TiH2 and Zn (reducing agent), with cobalt
powder as catalyst. Samples were heated for 4 hours at 500°C, then for 6 hours at
550°C, reducing CO2 to solid carbon (graphite). The resultant graphite was pressed into
aluminum cathodes. When possible, background 14C:12C ratios were measured from
predose sample and subtracted from the ratios determined for postdose sample. The
lower limit of quantification for C-14 values obtained following AMS analysis was
dependent on the carbon content and dilution factor of the samples. The limits of
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quantification, in ng equivalents of etelcalcetide/(mL or g), were approximately as
follows: plasma, 0.65; whole blood, 1.32; vomit, 0.57; dialysate, 0.009; urine, 0.28; and
feces, 5.77.
Biotransformation Product Profiling
Sample Preparation
For each biomatrix, samples from all patients were pooled to obtain a single sample
representative of all patients in the study. For urine, the weights of individual aliquots
included in the pool (study day 1 to 10) were proportional to the weight of sample
collected in each study day from each patient. A grand pool representing all patients
was then prepared by combining the individual patient pool in proportion to the total
weight of urine, and then acidified with FA (final 1% v/v). For dialysate, the weights of
individual aliquots included in the pool were proportional to the weight of sample
collected in study day 4 from each patient and this pool was acidified with formic acid
(final 1% v/v). For plasma, volume aliquots of acidified plasma at each time-point were
pooled proportional to the time interval to approximate an AUC pool from 0 to start of
first dialysis session postdose (approximately 68 hours) and another AUC pool from end
of first dialysis session (approximately 72 hours) to the start of the second dialysis
session postdose (approximately 116 hours). Equal volumes of this AUC pool were then
combined to prepare one representative sample, one for AUC0-68hr and another for AUC72-
116hr, across all patients.
Urine: The pooled urine sample was concentrated by a solid phase extraction (SPE)
procedure. The SPE column (Oasis WCX 6cc, 150 mg, 30 μm, Waters Corp., Milford,
MA) was conditioned with methanol (3 mL) and 5 mM ammonium acetate (3 mL),
respectively. Pooled human urine (4809 μL) was then loaded onto the column. The
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column was then washed with 5 mM ammonium acetate (4 mL) and acetonitrile/water
(1:4 v/v; 4 mL) sequentially. The column content was finally eluted down with
methanol/trifluoroacetic acid (v/v=98/2; 8 mL), dried by nitrogen gas overnight at 23°C.
The residue was reconstituted into 300 μL of 0.1% formic acid and analyzed by liquid
chromatography–high-resolution mass spectrometry (LC-HRMS). Recovery of the
eluted radioactivity from the SPE was approximately 84%. For tris(2-carboxyethyl
phosphine (TCEP) reduction, pooled urine was first incubated with TCEP (final 10 mM)
at 37°C for 1.5 hours and then prepared by SPE as described above.
Plasma: Area under the plasma concentration-time curve (AUC) pooled plasma was
subjected to a protein precipitation procedure. The supernatants obtained following
protein precipitation were concentrated to enable LC-HRMS profiling. Pooled samples
(3 mL) were treated with 3 mL of acetonitrile/methanol (1:1 v/v) and vortex-mixed for
approximately 1 minute. The resulting suspension was centrifuged for 20 minutes at
1500×g. The supernatant was transferred to a 15-mL Falcon tube. The extraction
procedure was repeated once more, and the combined supernatants were dried down to
~300 μL in a vacuum centrifuge (SpeedVac; Savant SPD2010; Thermo Fisher Scientific,
Waltham, MA). The concentrated supernatant was analyzed by LC-HRMS. For TCEP
reduction, 1 mL of pooled plasma was first diluted with 2 mL of 0.1% aqueous
trifluoroacetic acid and then incubated with TCEP (final 10 mM) at 37°C for 1.5 hours.
The reduced plasma was processed the same as above.
For sample preparation of SAPC for LC-HRMS analysis, AUC pooled human plasma (1
mL) was centrifuged at 20,817×g for 5 minutes; the top layer was transferred to a new
centrifuge tube, diluted with 0.5 mL of 20 mM pH 7.1 sodium phosphate buffer (Buffer
A), and vortex-mixed. This diluted plasma was loaded onto a buffer-exchange column
(Econo-Pac 10 DG column; Bio-Rad, Hercules, CA). The column was then eluted five
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times (1 mL each time) with Buffer A. Five 1-mL fractions (labeled fractions 1–5) were
collected. Fractions 2 to 4 were loaded onto an albumin affinity gel column (Affi-Gel®
column, 153-7301; Bio-Rad). The affinity gel column was then washed with 25 mL of
Buffer A to remove nonalbumin proteins and small molecules. Subsequently, 20 mL of
NaSCN phosphate buffer (1.5 M NaSCN, 20 mM sodium phosphate in water, pH 7.1)
was used to elute the serum albumin protein. The eluting fractions (20 mL) were loaded
onto a Vivaspin 20 tube (molecular weight cutoff, 10,000 Da; Sartorius Stedim Biotech
GmbH; Goettingen, Germany) and centrifuged at 2250×g (20°C) to remove the salts.
After being washed with water (20 mL each time) three times, the remaining desalted
solution (~2.5 mL) was transferred to a 4-mL vial and lyophilized using a ModulyoD
Freeze Dryer (Thermo Fisher Scientific Inc.; San Jose, CA). An aliquot of dried human
serum albumin sample (0.33 mg) was dissolved into 495 μL of buffered solution (pH 5.5)
containing urea (4M), ammonium acetate (50 mM), and hydroxylamine (20 mM) and
digested via addition of 16.5 μL of Lys-C (1 μg/μL) with a substrate:enzyme ratio of 1:20
(w/w). The digestion solution was gently vortex-mixed and incubated at 37°C for 24
hours. The digestion reaction was stopped by addition of 5.1 μL of formic acid (1% v/v
final solution). The digest was then analyzed by LC-HRMS
Dialysate : The pooled dialysate sample (49.7 mL) was dried down to approximately 5
mL in a SpeedVac at 35°C. The concentrate was processed by the urine SPE method,
and the reconstituted solution (300 μL) was analyzed by LC-HRMS. Recovery of the
eluted radioactivity from the SPE was approximately 91%. For TCEP reduction, the
pooled dialysate (49.7 mL) was incubated with TCEP (final 10 mM) at 37°C for 1.5
hours. The TCEP-treated dialysate was processed the same as above.
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LC─Fraction Collection+AMS and LC–High-Resolution MS
Radioprofiles by LC─fraction collection followed by off-line AMS (LC-FC+AMS) and
biotransformation product identification by LC–high-resolution mass spectrometry
(HRMS) were performed on pooled samples of plasma, urine, and dialysate with and
without TCEP reduction at Xceleron and Amgen Inc., respectively. Samples from all
patients were pooled to obtain a representative sample, which was acidified with formic
acid (1% v/v). Formation of serum albumin peptide conjugate (SAPC) was confirmed by
LC-HRMS analysis of the lysyl endopeptidase (Lys-C; Wako Chemicals USA, Inc.,
Richmond, VA) digest of SAPC in pooled human plasma.
LC-HRMS analysis of plasma, urine, dialysate, and SAPC in plasma was performed as
previously described [13]. For C-14 biotransformation profiling by LC-FC+AMS, samples
were prepared as follows: pooled urine was centrifuged for 10 minutes at 3210×g at 4°C
and supernatants were used for LC-FC+AMS. Pooled plasma was diluted eight-fold with
aqueous formic acid (0.2%, v/v) and centrifuged for 10 minutes at 3210×g at 4°C; the top
layer was used for LC-FC+AMS; direct injection of diluted plasma was performed to
enable detection of protein conjugates. Pooled dialysate (5 mL) was dried to
approximately 0.5 mL in a vacuum centrifuge (SpeedVac) at ambient temperature, and
the concentrate was used for LC-FC+AMS. TCEP-reduced samples of urine, plasma,
and dialysate were generated as described for LC-HRMS.
LC-FC+AMS analyses used the same LC components and methods as described for
LC-HRMS, with smaller injection volumes (urine and dialysate, 80 µL; plasma, 20 µL);
the flow post column was set completely for fraction collection. Fifteen-second fractions
were collected from each LC run (single injection runs for urine and dialysate; five
injections for plasma, with fractions pooled from each run before AMS sample
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processing). Fractions were pooled based on LC-HRMS chromatograms to optimize
peak resolution and prepared for graphitization and C-14 determination by AMS.
LC-HRMS Analysis of Human Serum Albumin Digest by Lys-C
The LC-HRMS system was an Agilent 1200 (Agilent Technologies, Santa Clara, CA) LC
system with a binary pump, photodiode array detector, a degasser, a refrigerated
autosampler, and a column heater and in-line positive ionization electrospray ionization
(ESI) HRMS (LTQ Velos Orbitrap; Thermo Fisher, Waltham, MA). Data were acquired
using Xcalibur software (version 2.1).
Samples (50 µL) were injected onto a Vydac 218MS52 column (250×2.1 mm; Grace;
Deerfield, IL) maintained at 40°C and eluted with a gradient method that used mobile
phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) as follows:
0 to 2 minutes, 3% B, 0.2 mL/min; 2 to 45 minutes, 3% to 40% B, 0.2 mL/min; 45 to 46
minutes, 40% to 80% B, 0.2 to 0.3 mL/min; 46 to 58 minutes, 80% B, 0.3 mL/min; 58 to
60 minutes, 80% to 3% B, 0.3 to 0.2 mL/min; 60 to 65 minutes, 3% B, 0.2 mL/min.
MS analysis of the Lys-C digests was performed using positive ionization ESI HRMS
(LTQ Velos Orbitrap; Thermo Fischer Scientific, Inc.) in-line using full-scan mode (mass-
to-charge ratio [m/z] 200–1000) at a resolution of 30,000, followed by collision induced
dissociation MS/MS scan event with a resolution of 7500 at 35% collision energy.
MS/MS scan was implemented to either the most abundant ion observed in the full-scan
spectrum or predefined m/z list.
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Pharmacokinetic Analysis
Apparent terminal half-life (t½,z) was calculated as t½,z=ln(2)/λz, where λz was the first-
order terminal rate constant estimated via linear regression of the terminal log-linear
decay of the concentration-time profile from the time points acquired immediately before
the start of hemodialysis sessions.
The ratio of the total (TM11) AUC to the total C-14 AUC was calculated as
AUCTM11/AUC[14C]etelcalcetide; the ratio of etelcalcetide AUC to the total C-14 AUC was
calculated as AUCetelcalcetide/AUC[14C]etelcalcetide.
Nonhemodialysis pharmacokinetics parameters analyzed included maximum plasma
concentration (Cmax); time to Cmax (tmax); the last observed plasma concentration (Clast);
time to Clast (tlast); the area under the plasma concentration-time curve (AUC) from time 0
on study day 1 (predose) to start of hemodialysis on study day 4 (3 days postdose;
AUC3d) and from time 0 on study day 1 (predose) to study day 11 (10 days postdose;
AUC10d), estimated using the linear trapezoidal method; apparent terminal half-life (t½,z;
the ratio of TM11 AUC to the total C-14 AUC; and the ratio of etelcalcetide AUC to the
total C-14 AUC.
Additional pharmacokinetics parameters from the hemodialysis period based on
individual plasma etelcalcetide pharmacokinetics samples included hemodialysis
clearance (CLHD) and the hemodialysis extraction ratio (EHD) using the following formulas:
CLHD = EHD × QP
EHD = (AUCA,HD – AUCV,HD)/AUCA,HD
where AUCA,HD is the area under the arterial plasma concentration-time curve
obtained during hemodialysis session on study day 4; AUCV,HD is the area under the
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venous plasma concentration-time curve obtained during hemodialysis session on
study day 4; and QP is the plasma flow rate through the dialyzer, calculated using the
following equation:
QP = QB × (1 − HCT)
where QB is the blood flow rate through the dialyzer during hemodialysis on study
day 4 and HCT is the hematocrit count obtained on study day −1.
Anti-Etelcalcetide Antibody Analysis
A validated dual-flow cell immunoassay was used to screen for and confirm the
presence of anti-etelcalcetide and anti-SAPC antibodies. For both screening and
confirmatory assays, etelcalcetide and SAPC were separately immobilized onto
carboxymethyl-dextran coated sensor chip flow cell surfaces in a Biacore™ 3000
instrument (GE Healthcare Bio-Sciences, Pittsburgh, PA). In the screening assay, serum
specimens were injected over both surfaces to initially detect etelcalcetide-reactive
antibodies. Confirmation that the sample binding was due to etelcalcetide-reactive
antibodies was demonstrated by injecting a solution of goat anti-human (IgA+IgG+IgM)
antibody after each human specimen injection. A specimen was considered positive if
both the screening and confirmatory reactivity was greater than the assay cut point on
either surface. Sensitivities for anti-etelcalcetide antibody detection on etelcalcetide and
SAPC surfaces were 1289 and 244 ng/mL, with lower limits of reliable detection of 2000
and 500 ng/mL, respectively.
Subramanian et al 12
SUPPLEMENTAL TABLE
Supplemental Table 1. Summary of Cumulative Total C-14 Excretion Ratios in Urine and Feces on Nonsampled Days
Patient
1 2 3 4 5 6
Cumulative Excretion (% of Administered C-14 Dose)
CED, SD4 to SD11a 17.63 16.49 11.66 10.27 14.61 16.55
CED, SD14 to SD176a 44.11 47.75 38.66 NCb NCb 45.35
CEU, SD1 to SD10a 0.34 0.18 2.44 4.26 3.47 0.07
CEF, SD1 to SD10a 1.07 1.07 1.19 0.29 1.10 1.34
Ratio of Cumulative Excretion (Study Days 1 to 11)
CEU, SD1 to SD10/CED, SD4 to SD11 0.02 0.01 0.21 0.42 0.24 0.00
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Patient
1 2 3 4 5 6
CEF, SD1 to SD10/CED, SD4 to SD11 0.06 0.06 0.10 0.03 0.08 0.08
CE=cumulative excretion in respective matrices (U=urine; F=feces; D=dialysate); SD=study day.
aSee Materials and Methods section 2.8.
bExcretion during extended pharmacokinetics collection period not collected; hence, value not computed.
Subramanian et al 14
SUPPLEMENTAL FIGURE LEGENDS
Supplemental Fig. 1 Radioactivity excretion rate in dialysate following
administration of a single intravenous dose of
[14C]etelcalcetide (10 mg; 26.3 kBq) to patients with CKD on
hemodialysis (n=6). CKD=chronic kidney disease
Supplemental Fig. 2 HPLC radiochromatogram of pooled urine (0–240 hours)
following a single intravenous dose of [14C]etelcalcetide (10
mg; 26.3 kBq) to patients with CKD on hemodialysis.
CKD=chronic kidney disease; dpm=disintegrations per minute;
HPLC=high-performance liquid chromatography
Supplemental Fig. 3 HPLC radiochromatogram of TCEP treated plasma, dialysate
and urine obtained following a single intravenous dose of
[14C]etelcalcetide (10 mg; 26.3 kBq) to patients with CKD on
hemodialysis. (a) AUC pooled plasma (0–68 hours), (b) pooled
dialysate from the first hemodialysis session, (c) pooled urine
(0–240 hours). CKD=chronic kidney disease; HPLC=high-
performance liquid chromatography; TCEP=tris(2-carboxyethyl
phosphine); TM11=total M11
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Supplemental Fig. 1
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Supplemental Fig. 2
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Supplemental Fig. 3
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