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Absorption, Metabolism, and Excretion of GBT440, a Novel Hemoglobin S (HbS) Polymerization
Inhibitor for the Treatment of Sickle Cell Disease (SCD), in Healthy Male Subjects
Peter M. Rademacher, PhD; Athiwat Hutchaleelaha, PhD; Carla Washington, PhD; Joshua Lehrer-Graiwer, MD; MPhil, FACC
Global Blood Therapeutics, South San Francisco, CA
Structures of metabolites were proposed by interpretation of
their mass spectral fragmentation patterns and comparisons
with reference standards, if available
Figure 1. Proposed pharmacokinetic model of GBT440
RBC
Clinical study design
●●
The pharmacokinetics, mass balance, and metabolite profile of
[14C]GBT440 were evaluated in 7 healthy male subjects
Hb
I
440 cpx
To evaluate the disposition kinetics of GBT440 at steadystate concentrations, a loading/maintenance dose schema was used
–– Each subject received an oral loading dose of 2000 mg GBT440
on day 1 followed by oral maintenance doses of 400 mg once
daily from days 2 to 4
PO
–– Once the target steady-state was achieved, a single [14C]GBT440
400-mg dose (approximately 100 μCi) was administered orally
on day 5
–– Blood, plasma, urine, and feces were collected serially to day 27
or when subjects met the discharge criteria
Analysis of blood and plasma GBT440
concentrations
●●
Blood and plasma samples were analyzed for GBT440
concentrations using liquid chromatography-tandem
mass spectrometry
–– The analytical range was 50 to 100,000 ng/mL for blood and 10
to 20,000 ng/mL for plasma samples
●●
GBT440 concentration in red blood cells (RBCs) was calculated
from the whole blood and plasma concentrations and
hematocrit values
Determination of total radioactivity
●●
●●
●●
●●
Total radioactivity (TRA) levels in whole blood, plasma, urine, and
fecal samples were measured using liquid scintillation counting (LSC)
Whole blood and homogenized feces samples were submitted
to combustion analysis before LSC
Plasma and urine samples were analyzed directly by LSC
TRA in RBCs was derived from the whole blood and plasma
TRA data and hematocrit values
Metabolite identification in plasma, whole blood,
urine, and feces
●●
The [ C]-containing material was extracted from blood, plasma,
and fecal homogenates using 0.5% acetic acid in acetonitrile
methanol (1:1, vol/vol), and the extracts were concentrated
14
●●
Urine samples were centrifuged and concentrated
●●
The blood, plasma, and fecal extracts and urine concentrates
were subjected to liquid chromatography separation and
fraction collection
©2016 Global Blood Therapeutics
Protein
I
440
complex
Tmax (h)
Cmax (µg/mL)
AUC(0-∞) (µg●h/mL)
t1/2 (h)
RBC/Plasma
Blood/Plasma
440
GBT440 was well-absorbed
●●
In this study, a 98.0% average recovery of TRA in urine and
feces over the course of the study was achieved
●●
440
Tissue
I
440 cpx
440
●●
After oral administration, GBT440 is absorbed from the gut and enters the bloodstream. GBT440 has high
specific binding toward hemoglobin leading to high RBC:plasma concentration ratio. Metabolism of free
drug is a driving force of drug elimination. The half-life of GBT440 is much shorter than the half-life of the
RBC, which supports the hypothesis that the binding of GBT440 to hemoglobin is a reversible process.
Pharmacokinetics (Figure 2; Table 1)
After oral administration, GBT440 reached maximum
concentration (Cmax) in plasma and whole blood with median
time to maximum concentration (Tmax) values of 2 hours
GBT440 rapidly partitions into the RBC with a high specificity
with blood/plasma ratio of ~32:1, which corresponded to an
RBC/plasma ratio of ~71:1
After reaching Cmax, GBT440 concentrations decreased in a
monophasic manner, with the terminal elimination phase
for GBT440 in plasma, whole blood, and RBCs appearing to
decrease in a parallel manner, with geometric mean half-life
(t1/2) values of 67 hours in whole blood and 66 hours in RBCs
Figure 2. M
ean (±SD) blood, plasma, and RBC
concentration–time profiles of GBT440 In
humans after oral dosing of 400 mg
(100 µCi) [14C] GBT440 at steady state
1000000
–– GBT440 was eliminated predominately in feces
●●
–– Four metabolites were identified, each accounting
for 5.62%, 2.66%, 1.66% and <6% of the dose in the 0-216-hour
human feces
Although GBT440 has high specific binding to hemoglobin,
it was completely excreted from the body, with a t1/2 of
approximately 3 days in healthy subjects
●●
Because the t1/2 of GBT440 was much shorter than
RBC lifespan (~120 days), this supports the hypothesis
that the binding between GBT440 to hemoglobin is a
reversible process
●●
After an oral administration, approximately one-third of
the dose was excreted as the unchanged drug into the feces
(unabsorbed and/or via biliary excretion)
●●
Two-thirds of the administered dose was metabolized and
excreted into urine and feces
●●
The major metabolic pathway was via phase 1 and phase
2 metabolism
●●
Because GBT440 was not excreted directly into the urine,
the pharmacokinetics are unlikely to be affected in patients
with renal disorders
Figure 4. Representative radio-chromatograms of pooled
blood (A), plasma (B), urine (C), and feces (D)
GBT440
100
80
60
40
20
0
0.00
M5/M6/M20
10.0
●●
100
20
Urine
Feces
Total (Urine + Feces)
48
96
144
192
240
288
336
384
432
480
528
Whole blood
Plasma
–– In plasma, unchanged GBT440 was the prominent circulating
radioactive component, accounting for 48.8% of the TRA
10
1
96
192
288
Time on Day 5 (h)
384
480
40.00
50.00
60.00
M9/M19
60
50
40
30
20
10
0
0.00
M15/M17
70.00 Mn
–– There was 1 major phase 2 metabolite (M2,
GBT440 O-dealkylation-sulfation), accounting for 16.8% of
the TRA
GBT440
M16
M11
M12
M13
M2
10.0
20.00
30.00
40.00
M5/M6
50.00
60.00
Feces
(63%)
70.00 Mn
GBT440
(unabsorbed)
(33%)
Metabolites
(30%)
GBT440 Dose
C
M12/M14
1200
1000
800
600
400
200
0
0.00
M21
M1
10.0
M19
M10
Urine
(35%)
M9
M11
M15
M7
M19
20.00
30.00
M18
–– Two potential active metabolites (M5, M6) were identified but
only accounted for 2.5% of the dose in whole blood
1000
30.00
B
–– In whole blood, most of the TRA was unchanged GBT440
(97.5%), whereas 3 metabolites accounted for the remaining
TRA (2.5%)
10000
20.00
40
Metabolite identification in plasma, whole blood,
urine, and feces (Figures 4 and 5)
RBC
CONCLUSIONS
–– Unchanged GBT440 was the most abundant
radioactive component, accounting for 33.3% of the
administered dose
Time (Hours Postdose)
●●
Phase 2 metabolism:
Glucuronide conjugation,
sulfate conjugation and
methylation
Feces
60
0
Blood
1
80
0
Plasma
100000
–– GBT440 glucuronidation and reductionglucuronidation products, which are phase 2 metabolites,
were the most abundant metabolites in urine, accounting for a
combined 9.22% of dose
A
100
RESULTS AND DISCUSSION
●●
Most of the administered radioactivity (88.2%) was recovered
by 144 hours postdose (day 7)
Figure 3. Mass balance study
PO, by mouth; RBC, red blood cell
●●
GBT440 was eliminated primarily in feces (62.6% of the total
radioactive dose), with urinary excretion accounting for 35.4%
of the total radioactive dose
Phase 1 metabolism:
Hydroxylation, oxidative
dealkylation and reduction
–– Unchanged GBT440 accounted for 0.08% of the
administered dose, and the rest were metabolites
●●
●●
Figure 5. Proposed Metabolic Pathways of GBT440
–– An average of 34.3% of the dose was recovered in the
urine samples
AUC(0-∞), area under the curve from time 0 extrapolated to infinite time; Cmax, maximum serum concentration;
CV, coefficient of variation; RBC, red blood cell; t1/2, half-life
Tissues/organs
Elimination
●●
Blood
2.0 (0-6)
133 (15.5)
10,400 (16.6)
66.3 (8.7)
–
32.1 (20.1)
Mass balance study (Figure 3)
Plasma
GBT440 Concentration (ng/mL)
●●
RBC
6.0 (0-6)
290 (19.4)
22,700 (18.9)
65.8 (9.0)
70.5 (16.6)
–
ARO (DRM)
METHODS
●●
Metabolite characterization was accomplished by liquid
chromatography-tandem mass spectrometry in conjunction with an
appropriate radioactivity monitor to facilitate metabolite peak detection
–– Urine was a relatively minor excretion route for GBT440
in humans
ARO (DRM)
The goal of this open-label study is to determine
the absorption, metabolism, and elimination routes of GBT440
●●
Urine
ARO (DRM)
●●
GBT440 is an oral, once-daily therapy that modulates
hemoglobin affinity for oxygen, thereby inhibiting hemoglobin
polymerization in sickle cell disease
Table 1. Mean (%CV) pharmacokinetic parameters of
GBT440 in healthy male subjects after oral dosing of
400 mg (100 µCi) [14C] GBT440 at steady state
●●
40.00
50.00
M5/M6
60.00
Metabolites
(35%)
GBT440
70.00 Mn
M12
M13
Disclosure
All authors are employed by and have equity ownership of Global Blood Therapeutics.
D
ARO (DRM)
●●
[14C]GBT440 and [14C]-related materials were detected by
solid scintillation counting using a Packard TopCount® NXTtm
Microplate Counter
Cumulative Percent of Administered Dose
INTRODUCTION
●●
GBT440
350
300
250
200
150
100
50
0
0.00
M4
M5/M6
M8
10.0
20.00
30.00
40.00
50.00
60.00
70.00 Mn
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PRESENTED AT the 58th annual meeting of the American Society of Hematology (ASH); December 3‑6, 2016; San Diego, CA