Importance of metabolism in mass balance study: Is Bextra ...

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

| Presentation Title | Presenter Name | Date | Subject | Business Use Only 2

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?


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?


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?


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


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


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


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


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)


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


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


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


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


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


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


HPLC Auto

Sampler

HPLC

Column

Splitter

MS

UV PC

PC Fraction

Collector

Radioactive

Detector NMR PC

LC-MS/MS and NMR Systems


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


M1 as a major metabolite circulating in rat plasma and RBC

HPLRC profiles in rat urine and feces


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


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


No GSH-adducts detected in rats

Ketone metabolites M11 and M13 observed

Mouse ADME study: unusual metabolites observed in mouse RBC


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


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


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


Additional methyl sulfate metabolites detected and confirmed

These unusual metabolites difficult to be identified without radioactive monitoring

Proposed biotransformation pathway of Valdecoxib in mice


Potential to form reactive epoxide followed by GSH-adduct formation

Ketone metabolites might lead

to form Schiff bases with protein

Human ADME for Valdecoxib


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


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


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


Application of enzymatic and basic hydrolyses for Phase II metabolites


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


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


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


P-G identified as the sulfonamide-N-glucuronide

HPLRC Profiles of [14C]SC-65872 after Incubations with HLM and CYPs


CYP2D6 and CYP3A4 are responsible for oxidative metabolism of Valdecoxib

Biotransformation Pathway of Valdecoxib in Human


No GSH-adduct and ketone metabolites observed in human ADME

M13 ?

Potential reactive metabolites and idiosyncratic toxicity


~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


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

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