importance of metabolism in mass balance study: is bextra
TRANSCRIPT
Importance of metabolism in mass balance study: Is Bextra’s skin reaction associated with its reactive metabolites?
Jeff Zhang (张继跃)
Novartis China (诺华中国)
DMPK, Nanjing, June 25, 2016
Outline
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Important of mass balance and metabolite profiling using
radiolabeling materials
Study design for mass balance study and regulatory
guidelines
Case study for mass balance and metabolite profiling for
Bextra (Valdecoxib) and detection of potential reactive
metabolites
Mass balance and metabolite profiling studies, why do we do them?
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Investigation of the basic pharmacokinetics and ADME to aid the extrapolation of safety and efficacy data to humans.
Major outcome for mass balance study
• Estimation of rate and extent of absorption (total and parent)
• Excretion and mass balance
• Tissue distribution
• Elimination behavior
• Determination of clearance mechanism
Mass balance and metabolite profiling studies, why do we do them?
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Major outcome for metabolite profiling
• Metabolic pattern and ID
• Estimation of first pass effect
• Estimation of extent of metabolism
• Evaluation of species difference in metabolism
• Major circulating metabolites and human specific metabolites for liability of safety testing of drug metabolite (MIST)
• Reactive and long-live metabolites
Mass balance studies, when do we do them?
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Rat ADME is conducted normally with Phase I, mouse ideally before the carcinogenicity study, rabbit later (if at all) before reprotox, human before Phase III
Industry practice – varies, many companies complete ADME in toxicology species before Phase I, others wait as long as possible.
FDA guidance: carcinogenicity study protocol:
Safety testing of drug metabolite (MIST) and mass balance data required by the regulatory agencies prior to initiation of Phase III, achieving adequate mass balance (% recovery) in the human [14C]-AMDE study is critical.
Considerations in the choice of radioisotope
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ADME studies are nearly always done with radiolabel.
• Almost all pharmaceutical studies with small molecules are done with 14C and 3H.
• Other labels can be considered (125I, 35S).
Position of radiolabel in molecule
• If portions of the molecule are metabolically cleaved from the label, this portion of the molecule can no longer be followed.
• If the molecule is cleaved into two roughly equal pieces, more than one label position may be needed to fully elucidate the metabolism.
• It is preferable to study the different label positions separately (14C and 3H dual labelling).
Dose and ADME process
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Dose with radiolabeled drug (20-100 uCi)
Collect blood, urine and feces (bile) from predose and postdose at different time points
Measure the total excreted in urine, bile and feces
Profile plasma, urine, bile and fecal samples
Identify metabolites
Sample collection
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Urine and feces are collected at timed intervals postdose. Cages are rinsed at the end of the study, sometimes during. Carcasses are collected. Rinsing agent should both dissolve drug and free caked material from cage. Pure organic solvents not recommended with metal cages.
CO2 and organic volatiles can be collected, usually not necessary for pharmaceuticals. Typically only done with rodents but large animals are possible.
Make sure CO2 and volatiles collection are validated. Typical trapping solutions NaOH, ethanolamine, Carbosorb.
Plasma is collected for total radioactivity PK and metabolite ID.
Results of mass balance and metabolic profiling
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Criteria :
• Rodents >95% ideally, can live with >90%.
• Nonrodents >95% ideally, can live with high eighties.
To my knowledge, ADME study has never been rejected by a regulator for poor mass balance.
Total radioactivity pharmacokinetics.
Samples for metabolite profiling
Major circulating metabolites
Case study : Story of Bextra (Valdecoxib)
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A non-steroidal anti-inflammatory drug (NSAID) used in the treatment of osteoarthritis, rheumatoid arthritis, and painful menstruation and menstrual symptoms. It is a selective cyclooxygenase-2 inhibitor
Approved by US FDA in Nov. 2001 and available by prescription in tablet form until 2005 when the FDA requested that Pfizer withdraw Bextra from the market. FDA cited "potential increased risk for serious cardiovascular (CV) adverse events," an "increased risk of serious skin reactions"
In September 2009, Pfizer paid a $2.3 billion for settlement with civil and criminal fine
Valdecoxib
SC-65872
Mass balance and metabolite profiling studies for Valdecoxib
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Mouse, rat (Tox), dog (Tox), monkey ADME were done before Phase II
Human and rabbit (Reprotox) ADME were completed before Phase III
Cumulative percentage of total radioactive dose excreted in urine and feces
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Species Dose
(mg/kg)
Collection
Interval (days)
Urine Feces Total
(Urine+Feces)
Rat 2.5 2 33 58 91
Dog
Mouse 5 7 42 55 97
Rabbit 5 2 40 60 100
Cyno Monkey
Human 50 mg 8 75 17 92
Recovered > 90 % cross species with majority excreted in feces for animals, but in urine for human.
Rat ADME for Valdecoxib
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A single 2.5 mg/kg oral dose of [14C]SC 65872 to 8 male and 8 female rats (a specific activity of 63 µCi/mg)
Suspension formulation as 0.5% methylcellulose and 0.1% Tween 80 aqueous solution.
Collected blood at 1, 2, 5, 8, 12, 24 hrs and bile at 6 hr postdose
Collected urine and feces at 1 day prior to dosing and 1 and 2 days postdose
Collected tissues at 2 and 168 hrs
Tissues distribution in rats
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Well distributed in liver, kidney and lung after 2 hrs
Minor dose remained in liver, skin, muscle and fat after 168 hrs
168 hrs 2 hrs
% of dose radioactivity excreted in urine and feces
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Mean total 90.7% of dose recovered with 32.8% in urine and 57.9% in feces in 2 days
0
20
40
60
80
100
0 6 12 18 24 30 36 42 48
Urine +
Feces
Feces
Urine
Female Rats
0
20
40
60
80
100
0 6 12 18 24 30 36 42 48
Postdose Time (hours)
Male Rats
LC-MS-NMR systems for metabolic profiling and metID
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HPLC Auto
Sampler
HPLC
Column
Splitter
MS
UV PC
PC Fraction
Collector
Radioactive
Detector NMR PC
LC-MS/MS and NMR Systems
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HPLC : Two Shimadzu LC-10 A Pumps
MS: Finnigan Quantum (TSQ)
Finnigan LCQ-Deca
PE Sciex Q-Trap
PE Sciex Q-Star Paulsor (Q-TOF)
-- Electrospray and APCI interfaces
-- Precursor ion scan, neutral loss scan, data-dependent scan
-- Metabolite ID software
-- Stable isotope-labeled compounds for isotopic dilution technique
NMR: Bruker DRX-600
-- 1H and 19F
HPLRC profiles in rat plasma and RBC
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M1 as a major metabolite circulating in rat plasma and RBC
HPLRC profiles in rat urine and feces
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0
13000
26000
39000
52000
65000
0 5 10 15 20 25
0
16000
32000
48000
64000
80000
0 5 10 15 20 25
SC-65872M7
M12 & M14
M1-GM6
M3-GM15
M13
M10M8
M3 M9
M1
SC-65872
M7
M12 & M14
M1-GM6M3-G
M15 M13
M10
M8
M3 M9
M1
Female Rat Urine, 0-24 hours
Male Rat Urine, 0-24 hours
0
1000
2000
3000
4000
5000
0 5 10 15 20 25
Female Rat Urine, 24-48 hours
Male Rat Urine, 24-48 hours
0
2000
4000
6000
8000
10000
0 5 10 15 20 25
Retention Time (minutes)
SC-65872M7
M12 & M14
M1-GM6
M3-G
M15
M13M10
M8M3
M9
M1
SC-65872M7
M12 & M14
M1-GM6
M3-G M15 M13
M10M8
M3 M9
M1
0
1000
2000
3000
4000
5000
0 5 10 15 20 25
0
600
1200
1800
2400
3000
0 5 10 15 20 25
SC-65872
M12/M14
M1-G
M6
M3-G M15 M13
M3
M1
M7M8
SC-65872
M12/M14
M1-GM6
M3-G
M15M13M3
M1
M7M8
Female Rat Feces, 0-24 hours
Male Rat Feces, 0-24 hours
0
700
1400
2100
2800
3500
0 5 10 15 20 25
0
300
600
900
1200
1500
0 5 10 15 20 25
Retention Time (minutes)
SC-65872M12/M14
M1-G
M6M3-G
M15 M13
M3
M1M7M8
SC-65872M12/M14
M1-GM6M3-G
M15 M13
M3
M1M7
M8
Female Rat Feces, 24-48 hours
Male Rat Feces, 24-48 hours
Time % Dose Percentage of dose radioactivity excreted as identified metabolite from both urine and feces
Sex h Profiled M6 M3-G1 M12 M14 M1-G M3 M15 M7 M8 M11 M1 M13 SC-65872
M 0-48 59.9 1.76 3.36 4.31 1.50 1.71 0.562 2.66 1.32 1.65 1.56 7.74 4.30 7.61
F 0-48 68.9 2.86 3.07 6.86 2.37 1.16 0.116 2.07 1.98 4.56 2.45 14.6 1.96 7.81
MF 0-48 64.4 2.31 3.21 5.59 1.93 1.44 0.339 2.37 1.65 3.11 2.00 11.2 3.13 7.71
HPLRC profiles in rat bile
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Major metabolites in rats bile as glucuronides of oxidative drug
0
600
1200
1800
2400
3000
3600
0 5 10 15 20 25 30
0
800
1600
2400
3200
4000
4800
0 5 10 15 20 25 30
0
600
1200
1800
2400
3000
3600
0 5 10 15 20 25 30
Retention Time (minute)
M1
SC-65872
SC-65872 & SC-66905 Standards
Male Rat 2, 0-6 hr Bile
Female Rat 2, 0-6 hr Bile
M10-G
M1
M5-G
M1-G
SC-65872M8
M9
M15M2-G
M9-G
?M15-G
M3-G1
M10-G M1
M5-G
M1-G
SC-65872M8
M9
M15M2-G
M9-G?
M15-G
M3-G1
Proposed biotransformation pathway of Valdecoxib in rats
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No GSH-adducts detected in rats
Ketone metabolites M11 and M13 observed
Mouse ADME study: unusual metabolites observed in mouse RBC
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Two unusual metabolites observed mouse plasma samples in late time points
0.5 hr
0
3000
6000
9000
12000
0 5 10 15 20 25 30TIme (min)
[14C
]DP
M
M1
37.9%
SC-65872
59.1%
M21
1.28%
6 hr
0
1750
3500
5250
7000
0 5 10 15 20 25 30TIme (min)
[14C
]DP
M
M1
17.4%
M20
46.5%
M11
11.5%SC-65872
1.23%
M21
5.48%
Plasma RBC
Accurate mass measurement of M20 and M21
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Metabolites M20 M21
Accurate Mass 391.0420 407.0372
Elemental Composition C17H15N2O5S2 C17H15N2O6S2
Difference from Valdecoxib CH2SO2 CH2SO3
Difference between M20 & M21 M20 + O
(Hydroxylation)
J. Y. Zhang, et al., Drug Metab. Dispos., 31, 491-501 (2003)
Characterization of M20
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Methyl sulfate metabolite confirmed by MS/MS and NMR
M20 Daughter ions of 391
79
118
390205172
312
0
25
50
75
100
50 100 150 200 250 300 350 400 450
m/z
158144
251233
270
SH2N
O O
O
N
SO O
312
79118
144
0
25
50
75
100
50 100 150 200 250 300 350
m/z
206144
SC-65872 Daughter ions of 313118
80
172 233192
SH2N
O O
O
N
233
80118
144
Proton NMR MS/MS
79
312
MS/MS spectra of M21 and M21-G
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Additional methyl sulfate metabolites detected and confirmed
These unusual metabolites difficult to be identified without radioactive monitoring
Proposed biotransformation pathway of Valdecoxib in mice
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Potential to form reactive epoxide followed by GSH-adduct formation
Ketone metabolites might lead
to form Schiff bases with protein
Human ADME for Valdecoxib
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Dose human subjects with 50 mg,100 uCi
Collect blood at 1, 1.5, 4, 8, 12, 16, 24, 48 and 72 hrs postdose
Collect urine and feces at 1 day prior to dosing and 1, 2, 3, 4, 5, 6 and 8 days postdose
Percentages of Dose Excreted in Urine and Feces
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Mainly excreted via renal clearance
Collection % of dose excreted
Time (day) Urine Feces U + F
0-1 58.3 11.6 69.9
1-2 11.8 4.52 16.3
2-3 3.07 1.30 4.37
3-4 1.41 0.23 1.64
0-4 74.6 17.7 92.3
Plasma profiling of 14C-Valdecoxib in human
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Minor metabolites circulating in
plasma
Exposure of M1 was less than
10% P in humans
No MIST issue
Urine and fecal profiling of 14C-Valdecoxib in human
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Application of enzymatic and basic hydrolyses for Phase II metabolites
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Beta-Glucuronidase
• Ether-linked glucuronide conjugates
• Acyl glucuronide conjugates (C-1 only)
Basic hydrolysis (pH > 10)
• Acyl glucuronide conjugates
• N-linked glucuronide conjugates
HPLRC and LC-MS profiles in human urine
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0
1000
2000
3000
0 5 10 15 20 25
0
1000
2000
3000
0 5 10 15 20 25
0
1000
2000
3000
0 5 10 15 20 25
Time (minutes)
P
M1
M2-G
M1-G
M3-G
P-G
M5-G
PPPP
P
M2
M1
M5
P-G
M3
M4
P
M1
M2-G
M1-G
M3-G M4
M5-G
(a) Human urine
(b) Human urine with
-glucuronidase
(c) Human urine with
base, pH 10
HPLRC LC-MS
P+O+G
P+2O+G
P+G
MS/MS spectra of M1-G and M2-G
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M1-G and M2-G identified as O-glucuronide
0
25
50
75
100
50 100 150 200 250 300 350 400 450 500 550
m/z
113
443
196
85
329
313
M1-GDaughter ions of 505
75
299256
247504
0
25
50
75
100
50 100 150 200 250 300 350 400 450 500 550
m/z
113
118
73
85
329
313
M2-GDaughter ions of 505
59
298
286
234
SH2N
O O
O
N
O OCOOH
OH
OHHO
329
313
299
S
HN
O O
O
N
O
O
OHHOOC
HOHO
329
313
234298
Characterization of P-G by MS/MS and NMR
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P-G identified as the sulfonamide-N-glucuronide
HPLRC Profiles of [14C]SC-65872 after Incubations with HLM and CYPs
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CYP2D6 and CYP3A4 are responsible for oxidative metabolism of Valdecoxib
Biotransformation Pathway of Valdecoxib in Human
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No GSH-adduct and ketone metabolites observed in human ADME
M13 ?
Potential reactive metabolites and idiosyncratic toxicity
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~8 % 14C dose unaccounted after 4 days, which might remain in body
The epoxide intermediate might lead to protein adducts as GSH adduct observed in mice, dogs and rabbits (data didn’t show)
Ketone metabolites (M11, M13) might lead to form Schiff bases with protein
M11 M13
Summary: mass balance and metabolite profiling in radiolabel
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Advantage
• Widely adapted and acceptable practices for most programs
• Able to accomplish most MIST tasks
• Provide complete human ADME data
• Guide to identify unusual and long-live metabolites
• Acquire metabolism and disposition data in FIH
Limitation
• ADME requires a lot of resources, most of which will be wasted since large percent of clinical candidates failed in development
Acknowledgements
My former colleagues at
G.D. Searle & Co