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Using Accelerator Mass Spectrometry to
Explain the Pharmacokinetics of
VismodegibCornelis E.C.A. Hop
Topics to be Addressed
• Why AMS?
• AMS for mass balance studies with
vismodegib
• AMS for absolute bioavailability studies with
vismodegib
Vismodegib - Approved by the FDA for the
Treatment of BCC in January 2012
MW = 421
pKa = 2.8
clogP = 3.9
Human PK Predictions
• Predicted human PK:• Low clearance: < 1 ml/min/kg
• Lower volume of distribution: < 1 ml/min/kg
• Long half life
• Incomplete absorption:• Rat F = 53%; Dog F = 33 %
• Based on BDC rat and dog data, Fa is 42% and 31%, respectively
F = Fa * Fg * Fh
• Minimal turnover in microsomes
and hepatocytes for all species
except cyno; Human Clhep = 0.5
mL/min/kg
• Low clearance in rat and dog,
but moderate in cyno
Clinical PK of VismodegibPhase I Cancer Patient Study
A B
C (A) Increase in exposure after single 150 or
270 mg dose with sustained [plasma]
(B) Steady state reached quickly;
unexpected with 10-14 day half-life
(C) Steady state concentration independent
of dose
(D) Particle size effect on day 1 that was
not present at steady state (data not
shown)
Graham et al., Clin Cancer Res 2011, 17, 2512
5
EOP1 FDA Comments
• According to 21 CFR 320.25, the bioavailability (fraction
absorbed by IV/PO routes) of GDC-0449 should be
assessed.
• Absolute bioavailability study
• Address the absorption, distribution, metabolism and
excretion (ADME study) of GDC-0449 in humans and if
needed characterize the effect of renal and or hepatic
impairment on the PK of GDC-0449.
• Mass balance study
… But How?
• Traditional mass balance study:• How ethical is it to have healthy volunteers exposed
to 50 – 100 mCi for several months?• Can you keep them in the clinic that long?
• The amount excreted each day is likely very small and may not be detectable with liquid scintillation counting
• Poor mass balance?
• Can metabolites in plasma, feces and urine be detected?
Mass Balance Studies for Compounds with Long T1/2
• Poor mass balance is likely for vismodegibRoffey et al., Drug Metab Rev 2007, 39, 17
vismodegib
… But How?
• Traditional mass balance study:• How ethical is it to have healthy volunteers exposed
to 50 – 100 mCi for several months?• Can you keep them in the clinic that long?
• The amount excreted each day is likely very small and may not be detectable with liquid scintillation counting
• Poor mass balance
• Can metabolites in plasma, feces and urine be detected?
• Study is likely to fail
• 14C tracer study with accelerator mass spectrometry detection!
AMS allows quantitative separation of 12C, 13C
and 14C from a sample containing a very low
level 14C-tagged drug or biopharmaceutical
Accelerator Mass Spectrometer
Flow Diagram of AMS Procedures
for Biomedical Samples
Analyse by AMS
Administer nCi 14C-drug Collect blood etc
CombustGraphitise
Types of AMS Studies in Drug Development
Phase 0
Human micro-dosing study Drug candidate selection using human micro-dosing data
Investment decisions
Phase I/II
IV PK, Human Absolute Bioavailability study Intravenous tracer + concomitant oral dose
Mass balance and nano-tracing human metabolism study Low dose of 14C incorporated into the therapeutic oral dose of drug
Mass balance and metabolite profiling – As an alternative to conventional Human Radiolabel Study?
– Addresses MIST guidelines
To answer specific ADME questions
To investigate/mitigate metabolism liabilities & retention issues
Mass balance and metabolite profiling
Microtracer Absolute Bioavailability Studies with
NCEs Intended for Regulatory Submission
13
Courtesy of Xceleron & Graham Lappin
AMS Mass Balance Study Design
• Amount of radioactivity greatly reduced: 1,000 nCi
• It is feasible to send volunteers home after two
weeks
Time (days)
Cp/D
ose
150 mg PO on day 1 (unlabeled)
+ 1,000 nCi/10 µg 14C PO
Cold
Hot
How to ensure acceptable mass
balance?
Continuous sampling until day 14
and interim sampling on days 21,
28, 35, 42, 49, and 56 and
interpolation
Dose as homogenous suspension
Vismodegib Mass Balance Results
Total Recovery = 86.6%Confined D1-D14
Feces Recovery
= 82.2%
Urine Recovery = 4.4%
• Renal impairment study not needed - widens exclusion criteria for ongoing trials
• Need for hepatic impairment study dependent on metabolite profiling
Vismodegib Predominant in Plasma
Time (hr)
0 200 400 600 800 1000 1200 1400
Pla
sm
a C
once
ntr
atio
n (
ng o
r n
g e
qu
iv p
er
mL
)
0.1
1
10
100
1000
10000
Plasma GDC-0449 "cold" (ng/mL)
Plasma total radioactivity "hot" (ng equiv/mL)
GDC-0449 is major drug-species in plasma - no significant circulating metabolites
Graham et al., Drug Metab Dispos 2011, 39, 1460
Metabolic Profiling in Feces
• Mainly vismodegib in circulation
• Mainly excreted in feces as parent compound
(including un-absorbed material) and oxidative
metabolites
GDC-0449
M3dp
m/g
fec
es h
om
og
en
ate
GDC-0449
M3dp
m/g
fec
es h
om
og
en
ate
feces 0-72 hrs
GDC-0449
M3
M1
M13
M18
feces 72-312 hrs
Graham et al., Drug Metab Dispos 2011, 39, 1460
Metabolism of Vismodegib
Oxidative metabolism
and unique pyridine ring
opening
Graham et al., Drug Metab Dispos 2011, 39, 1460
AMS Absolute Bioavailability Study Design
• Dose cold vismodegib orally and 14C labeled
vismodegib intravenously at the Tmax
• No concerns about linearity of iv PK because hot and
cold present at the same time
• Single dose and steady state (one week dosing)
Time (days)
Cp/d
ose
150 mg PO
on day 1
500 nCi/10 µg 14C IV dose
on day 1 at Tmax = 2 hr
Time (days)
Cp/d
ose
150 mg PO qd
for 7 days
500 nCi/10 µg 14C
IV dose on day 7
Vismodegib Absolute Bioavailability ResultsDose Normalized GDC-0449 Concentration vs. Time
Time (hours)
0 200 400 600 800 1000 1200 1400
Dose n
orm
alized G
DC
-0449 P
lasm
a C
oncentr
ation
(ng/m
L/d
ose)
0.01
0.1
1
10
100
1000
Oral Mean
IV Mean
Dose Normalized GDC-0449 Concentration vs. Time
Time (hours)
0 200 400 600 800 1000 1200 1400
Dose n
orm
alized G
DC
-0449 P
lasm
a C
oncentr
ation
(ng/m
L/d
ose)
0.01
0.1
1
10
100
1000
Oral Mean
IV Mean
• Slow IV CL consistent with rate limited systemic elimination (not “flip-flop”)
• Absolute bioavailability determined to be 31.8 + 4.6%
• Does F and/or CL change with daily dosing?
• Total clearance increased close to two-fold
• Absolute bioavailability decreased to 7.4 + 2.5%
dose-normalized
data
Graham et al., Br J Clin.Pharmacol 2012, 73, 788
T1/2
(d)
CL
(mL/h)
Vss
(L)
Single
dose13.0 43.4 16.4
Multiple
dose10.3 78.5 26.8
Summary of Vismodegib IV PK
0.01 mL/min/kg
+81%
No evidence for CYP induction in human hepatocytes
Graham et al., Br J Clin.Pharmacol 2012, 73, 788
Plasma Protein Binding
• Binding to both HSA and AAG, but AAG binding can be saturated• The higher concentration at steady state results in a larger free
fraction
Graham et al., Br J Clin.Pharmacol 2012, 73, 788
T1/2
(d)
CL
(mL/h)
CLu
(mL/h)
Vss
(L)
Vss,u
(L)
Single
dose13.0 43.4 13,152 16.4 4,970
Multiple
dose10.3 78.5 9,937 26.8 3,392
Summary of Vismodegib IV PK
• The intrinsic clearance and volume of distribution does
not change significantly from single to multiple dose
Graham et al., Br J Clin.Pharmacol 2012, 73, 788
T1/2
(d)
CL
(mL/h)
CLu
(mL/h)
Vss
(L)
Vss,u
(L)
Oral
AUC
(mM.hr)
F
(%)
Single
dose13.0 43.4 13,152 16.4 4,970 2,850 31.8
Multiple
dose10.3 78.5 9,937 26.8 3,392 356 7.4
Summary of Vismodegib IV & Oral PK
• The total oral exposure is reduced by close to eight-fold
due to non-sink absorption conditions
-88% -77%
Graham et al., Br J Clin.Pharmacol 2012, 73, 788
Summary of Vismodegib Human
Pharmacokinetics• Vismodegib is absorbed slowly after a single oral dose mainly due to
poor solubility
• Terminal elimination half-life is very long after both oral and IV administration due to very slow elimination
• The CL is incredibly low, 0.01 mL/min/kg
• Metabolism is via oxidation and pyridine ring opening
• The Vss of 0.23 L/kg is low, but is still indicative of distribution out of the plasma space.
• Mean bioavailability is moderate, 31.8%, with little inter-individual variability; micromolar plasma concentrations were achieved with a single 150 mg oral dose
• At steady state, the absorption and clearance are reduced, but the intrinsic clearance has not changed
• All of this is driven by very slow clearance, non-sink absorption and non-linear plasma protein binding
• AMS enabled these studies and absolute bioavailability and mass balance studies using AMS are now a standard part of drug development
We Can Rationalize the Clinical PK of Vismodegib!Phase I Cancer Patient Study
A B
C (A) Increase in exposure after single 150 or
270 mg dose with sustained [plasma]
(B) Steady state reached quickly;
unexpected with 10-14 day half-life
(C) Steady state concentration independent
of dose
(D) Particle size effect on day 1 that was
not present at steady state (data not
shown)
Graham et al., Clin Cancer Res 2011, 17, 2512
Acknowledgements
• DMPK, Clinical
Pharmacology and
Clinical Operations
colleagues and others
on the HH team
• PRA and Xceleron and
all healthy volunteers
History of Accelerator Mass Spectrometry
• Highly specialised nuclear physics instrument
originally developed for carbon dating in the 1970’s
• It uses very high energies to separate rare isotopes
which are then measured with high precision
• Willard Frank Libby won the Nobel Prize for Chemistry
for his method to use carbon-14 for age determination
in archaeology, geology, geophysics
• Approximately 160 AMS instruments worldwide
• First biological application described in 1989 (LLNL)
• Xceleron first company in the world to use AMS for
biomedical research
AMS
Atoms separated by differences 12C,13C and 14C atoms
individually countedin mass, charge and energy
-decay of 14C atom Detected by LSC as photons
of light in photomultiplier tube
LSC
0.012% of 14C decays per annum; 1 billion 14C atoms ≡ 1 dpm
Key
Sample containing 12C 13C and 14C atoms
Principle of LSC vs AMS Detection
1000 14C atoms required for valid measurement
1,000,000 more sensitive than LSC
100,000 more sensitive than MS
Xceleron’s 5 MV AMS
0.01
0.1
1
0 500 1000 1500 2000 2500
Time (h)
Lo
g p
erc
en
t o
f to
tal
ex
cre
ted
pe
r 2
4 h
pe
rio
d
Up to 168 h all
excreta collected
After 168 h, collections over
selected 24 h periods
AUC0- = 90.5 % recovery
Subjects were administered 3 mg/1 μCi of drug X orally
Interpolation to Ensure Adequate Radioactivity
Recovery for Long Half-Life Drugs