6.Applications of Accelerator Mass Spectrometry (AMS… 06zdl .pdf · Applications of Accelerator...

Donglu Zhang

Applications of Accelerator Mass

Spectrometry (AMS) in Clinical Studies

Donglu ZhangDrug Metabolism and PharmacokineticsGenentech, One DNA WaySouth San Francisco, CA 94080

At 2015 Nanjing International DMPK SymposiumAt 2015 Nanjing International DMPK SymposiumJune 26, 2015June 26, 2015

Outline

Introduction to AMS Basic AMS Concepts, AMS validation

Drivers for AMS

Microtracer, microdosing vs normal dosing

AMS study logistics

Potential DMPK-related AMS Applications- Case Potential DMPK-related AMS Applications- Case

Studies Phase 0 and 1 studies and animal studies for MIST and

Early human PK

Absorption/Bioavailability: Saxagliptin

Mass Balance/Bioavailability: Vismodegib

Metabolic profiles/Mass Balance: Ixabepilone

Limitations and future perspectives

Contributors

Sophia Xu

Nilgun Comezoglu

Van Ly

Lisa Christopher

Griff Humphreys

Cyrus Khojasteh

Harvey Wong

Shuguang Ma

Qin Yue

Cornelis E.C.A. Hop

BMS Genentech

Griff Humphreys

Mark Arnold

Vitalea

Accium

Xceleron

Accelerator Mass Spectrometry (AMS)

Accelerator mass spectrometry (AMS) is a technique for measuringlong-lived radionuclide that occur naturally in environment(-decay half life of 14C is 5730 year, 2.3x10-10 DPM/14C).

AMS uses a particle accelerator in conjunction with ion sources, largemagnets, and detectors to separate out interferences and countsingle rare isotope atoms in the presence of a large number of stableatoms (14C:12C ratio = 10-12).

AMS technique is used for a wide variety of radiocarbon dating and AMS technique is used for a wide variety of radiocarbon dating andtracing applications in the geological and planetary sciences,archaeology, and biomedicine.

AMS differs from other forms of mass spectrometry in that itaccelerates ions to very high kinetic energies before mass analysis.AMS can accurately analyze elemental and isotopic compositionswith high sensitivity.

AMS Applications in Medical Research

AMS as an analytical tool

Very sensitive and accurate technique for measuring 14C, 13C and12C etc.

Traditionally used for radiocarbon dating, introduced toPharmaceticals in the last decade.

Quantification based on measurement of 14C/ C (defined as fMC,fraction modern carbon) ratio in a sample.

It is an atom counter detector and typically linear over 4 orders ofmagnitude.

5

It is an atom counter detector and typically linear over 4 orders ofmagnitude.

LC-AMS can typically achieve LLOQ of 10 amol of 14C (dependingon specific activity, sample loaded onto HPLC column, molecularweight, etc.).

Clinical studies that can utilize AMS

Phase 0 and phase I studies

Absolute bioavailability

Metabolic profiling

Mass balance

AMS-Scheme

3. Filtermolecule destruction


2. Filtermass analysis

(low-energy end)


(low-energy end)

massspectrometer

charge exchange


(high-energy end)


(high-energy end)

massspectrometer

(Stripping process)




(low-energy end)


(low-energy end)

massspectrometer

charge exchange


(high-energy end)


(high-energy end)

massspectrometer

(Stripping process)

veca

rbo

nio

nb

ea

m

-ve ions converted to +ve ions

Pre-accelerated and passedthrough a spherical

electrostatic analyzer

Again accelerated usingelectrostatic quadrapole

triplet and analyzing magnet

12C4+ and 13C4+ weremeasured in faradays cup

5. Filterparticle identification


electrostaticdeflector

1.Filternegative ion formation


13C / 12C14C / 12C

13CH

-

12CH2-

14C-

argonstrippergasnegativeions positiveions

14C +

13C +

12C +

H+

10-9

1

1

:

:

7Li2-

12C

13C



electrostaticdeflector



13C / 12C14C / 12C

13CH

-

12CH2-

14C-

argonstrippergasnegativeions positiveions

14C +

13C +

12C +

H+

10-9

1

1

:

:

7Li2-

12C

13C

-ve

carb

on

ion

be

am

Ion source: Multi CathodeSource of –ve ions with

Cs sputtering

measured in faradays cup

14C4+ measured usingelectrostatic deflector

Courtesy of Vitalea Sciences

Accelerator mass spectrometer at Lawrence Livermore National Laboratory

Typical Sample Processing: Key Steps

Adding carbon carrier: Liquid paraffinPlasma sample (Human plasma contains ~4% of carbon, no need to add external

carbon).

Combustion (Oxidation) In the combustion stage, Carbon dioxide(CO2 ) is produced from the pretreated

samples. Samples are combusted at 850℃ for 2 h with CuO and Ag.

Graphitization (reduction)After combustion, the Carbon dioxide is purified by passing it through two LiquidAfter combustion, the Carbon dioxide is purified by passing it through two Liquid

nitrogen/alcohol trap to remove water. The CO2 is collected in a storage and reactedwith H2 for the graphitization in a heat sealed graphitization tube under vacuum.Placed in a furnace at 500℃ for 3-4 h, slow cooling to ambient temperature.

Packing cathodes with graphiteAfter the graphitization process, the cathode is packed with graphite. Place the

cathodes AMS sample wheel and the sample wheel stored under vacuum.

AMS analysis Insert the sample wheel contained graphite packed cathodes into the ion source of

AMS instrument.

-decay of 14C atom Detected by LSC as photonsof light in photomultiplier tube

LSC

0.012% of 14C decays per annum; 1 billion 14C atoms ≡ 1 dpm

AMS vs LSC Detection

AMS

Atoms separated by differences 12C,13C and 14C atomsindividually countedin mass, charge and energy

Sample containing 12C 13C and 14C atoms

1000 14C atoms required for valid measurement

Source: GNE

Sensitivity of AMS vs. LC-MS/MS: Driver for AMS

250 nCi (10 µg)

LC

-AM

S

Sensitivity dependenton structure, mobile

phase and extract“cleanliness”

pg/mL

ng/mL

>500 pg/mL

LC

-MS

/MS

10Source: Xceleron

14C-analyte component

20 fg/mL

200 fg/mLL

C

fg/mL

pg/mL1-50 pg/mL

LC-MS operating at maximumperformance, system clean andoptimized tuning parameters.Maintaining LLOQ challenging

Pharmacological Dose (mg)Macrotracer (50-100 uCi):Traditional ADME study

Tra

ce

rA

cti

vit

y

Clinical Studies Using Radiotracer

Pharmacological Dose (mg)Microtracer (1 uCi):

Phase I Study

Microdose Dose (<100 ugor 1/100 pharmacological

dose)Microtracer (1 uCi):

Phase 0 Study

Total Drug Dose

Tra

ce

rA

cti

vit

y

No Toxicity Concern of IV Microdose

Single dose 1000 x human microdose in rats with 8 days observation

IV or intended human route of administration (n=10)

biochemistry on days 2 and 8

animals sacrificed on day 8

gross necropsy, limited histopathology

Abbreviatd genotoxicity (optional)

Comparative in vitro metabolism; microsomes or hepatocytes

Minimal Tox Package for Phase 0 Microdose

Comparative in vitro metabolism; microsomes or hepatocytes

in vitro hERG assay

Food and Drug Administration, US Department of Health and Human Services, Guidance forIndustry Investigators and Reviewers. Exploratory IND Studies. January 2006.

EMEA, Position Paper on Non-clinical Safety Studies to Support Clinical Trials with a SingleMicrodose. Position paper CPMP/SWP/2599, 23 June 2004.

Microdose and Analytical Sensitivity Requirements

Microdose:

1/100th of efficacious dose and ≤100 µg

Fully characterized pharmacokinetics (PK) Fully characterizedelimination phase including Cmin

Volume of Distribution (VD):

The VD is a reflection of how widely a drug will distribute throughoutthe body

14

the body

The volume of distribution is given by the following equation:

Low VD drug and a good mass spec response compound: Sensitivityon LC-MS/MS may be sufficient

High VD drug: There is need for sensitive PK assay

Low VD drug but a poor mass spec response compound: There isneed for sensitive PK assay

Radiotracer and Radiospecific Activity of Carbon-14

Theoretical radio SA: 0.0624 Ci/mmol (2.31 TBq/mol) per atom, half life 5730years.

The radio SA will be 62.4 mCi/mmol if one carbon atom is labeled with 14C in amolecule; The radio SA will be 187.2 mCi/mmol (3 x 62.4 mCi/mmol) if 3 carbonatoms are labeled with 14C in a molecule.

For a molecule with MW of 500 Da, the radio SA will be 124.8 uCi/mg (62400uCi/500 mg) if one carbon is labeled.

For a regular dose of 10 mg (MW 500 Da) with a tracer of 200 nCi radioactivity, For a regular dose of 10 mg (MW 500 Da) with a tracer of 200 nCi radioactivity,the radio SA is 0.02 uCi/mg (200 nCi/10 mg), approximately 0.016%(0.02/124.8*100) of the molecules are labeled with 14C – A radiotracer.

For a microdose of 100 ug (MW 500 Da) with a tracer of 200 nCi radioactivity,the radio SA is 2 uCi/mg (200 nCi/100 ug), approximately 1.6% (2/124.8*100) ofthe molecules are labeled with 14C – A radiotracer.

For any dose (MW of 500 Da) and a radio SA of 5 uCi/mg, approximately 4%(5/124.8*100) of the molecules are labeled with 14C – A radiotracer.

For an ADC (150,000 Da) with DAR of 2 and radio SA of 0.83 uCi/mg, 100% ofmolecules are labeled with two 14C – not a radiotracer.

General Limitations and Sensitivity in Radioactive Studies

There may be a maximum of radiospecific activity possible when a

radiolabeled compound is synthesized.

A limit on the amount of 14C that can be incorporated into themolecule, radiolytic instability and size of the molecule (150,000 Dafor ADCs) etc.

The amount of radioactivity that can be administered to humans islimited by regulation.

16

limited by regulation.

Poor absorption or low bioavailability will limit the amount ofradioactivity getting into the biological samples.

For certain sample preparations such as protein, DNA,incorporation of radioactivity may be very low.

Low Risk:

14C-dose ≤ 1 μCi (0.037 MBq): radiation burden < 0.1 mSv

≤ 0.1 mSv is ICRP-62 category I (minimal burden)

No need for dosimetry calculation and supporting animal studies

Other regulations

No license is required if total amount is low (< 10 MBq, 1 μCi=37

Radiation Safety Aspects With Microtracer

No license is required if total amount is low (< 10 MBq, 1 μCi=37kBq).

To avoid contamination: dosing of radiotracer in facilities with lowradioactivity background, not typical mass balance facilities.

Dosing of subjects under supervision of medical investigator who islicensed to use radiation.

Microtracer can be considered as excipient although a CoAincluding specific radioactivity is needed.

Combined Microdose with Radiotracer to Shorten the Timeline

Source: PRA

AMS Procedure Flow Diagram

Administer nCi 14C-drug Collect blood etc Combust

Analyse by AMS Graphitize

Microdosing/tracer label studies + AMS: Study Timeline

1week

Dosesubjects

IEC FinalDocs

SubmitIEC

IECApproval

ScreeningStarts

3 weeks2 weeks

Recruitment

4 weeks

Startwriting

Requires FastSponsor Review

drug product partIND/IMPD

setup pharmaceutical analysis,

14C synthesis

Clinic Execution‘x’ weeks

2 weeks

LPLV DB Lock

1st DraftCSR

6 Weeks

writingprotocol

This may be extendeddepending on AMS data

setup pharmaceutical analysis,API + 14C-compound

parent vs metabolite LCtransfer-> AMS lab

Microdosing and AMS: Some Caveats

Samples converted to graphite before AMS

AMS only gives total 14C content, will not distinguish co-eluting metabolites bymass.

No metabolite structural information.

Off-line HPLC separation of parent compound and metabolites: critical.

Excess of cold parent drug spiked in samples as internal standard forextraction recovery (based on UV-peak).extraction recovery (based on UV-peak).

HPLC separation by fractionation

Need certainty on full separation of parent from metabolites

Which may affect number of assays and timelines of AMS work

Requires communication between AMS provider and DMPK/bioanalysis group.

Not all AMS labs have a MS detector to identify drug-related componentsalthough HPLC methods can be derived from nonclinical metabolism studies.

Drug Discovery and DevelopmentMicrodose/tracer and Developing MIST Strategy

In Vitro and In VivoMetabolism Studies

In vitro metabolite profiles-species comparison

PrePre--DevelopmentDevelopment Phase 0Phase 0 Phase 1Phase 1

Short-term Tox Studies

IND-Enabling Studies

Plasma samples frommultiple dosed animals

FIH Studies (SAD/MAD)

Plasma samples from singleand multiple dosed subjects

Microtracer in SAD or MAD

Major human in vitro metabolites

(microsomes/hepatocytes)

Idea (qualitative) of in vivocoverage of major human in vitrometabolites from preclinicalstudies

multiple dosed animals

Metabolite profiles aftermultiple doses - forcomparison with single dosestudy, other species, and invitro met profile

Animal ADME

Microdose/microtracer inhumans

Microtracer in SAD or MADsubjects

Compound SelectedFor Development

IND Filing

Examples of Microdose

Data from two trials: EUMAPP and CREAM and other literature data.

21 out of 26 drugs displayed linear pharmacokinetics (within 2x exposureextrapolated from microdose to pharmacological doses) includingfenofenadine, a P-gp and OATP substrate.

Where there is no-linearity with PO, IV scaled well.

Examples of not scalable: Propafenone, dose-dependent 1st pass metabolismby CYP2D6; warfarin, target-mediated disposition at low dose.by CYP2D6; warfarin, target-mediated disposition at low dose.

DDI at microdoses: caffeine (CYP1A2), tolbutamide (CYP2C9), fenofenadine(p-gp), midazolam (CYP3A4) by pharmacological doses of fluvoxamine andketoconazole.

Not widely used the industry because 1) cost effective only if poor PK at amicrodose, 2) the saved time will delay development for a compound withgood microdose PK

Lippin G (2010) Bioanalysis, 2, 509Liu et al (2011) Drug Metab Dispos 39, 1840.

Addressing MISTMethods to Obtain AUC

Traditional approach: (Method 1)Quantitation by LC/MS/MS of parent and metabolites

Synthetic standards needed for parent and metabolites

All samples analyzed (up to 24 samples for 1 dose, 3 animals/dose, 8 time points)

AUC determined by Trapezoidal rule (WinNonLin)

Pooling method - individual animal: (Method 2)Samples from individual animals pooled based on time and volume (Hamilton et al)Samples from individual animals pooled based on time and volume (Hamilton et al)

One sample per study animal number (Up to 3 samples, 3 animals)

Quantitation by LC/MS/MS. [AUC0-t = Cpool * time]

Pooling method - per dose: (Method 3)Samples from all animals in same dose group pooled based on time and volume

One sample per dosage (1 sample)

Quantitation by LC/MS/MS. [AUC0-t = Cpool * time]

Suitable for quantitation by AMS

Hamilton et al., Clin Pharmacol Ther 1981, 29, 408-413.

Example Using Microtracer for ADME in FIH

10 mg50 mg

+

100 nCi

100 mg200 mg

10 mg25 mg

Part B

Multiple dose SafetyMultiple dose Safety

Part A

Single dose SafetySingle dose Safety

25 mg25 mg

TimelineTimeline

AmendmentAmendment

+

40 nCi

on Day 4

Introduction to Absolute Bioavailability Study of Saxagliptin

Onglyza™ (saxagliptin, BMS-477118) is an oral DPP4 inhibitor, approved for

the treatment for Type 2 diabetes. A clinical absolute BA study was required to

support regulatory submissions.

Prior experience with LC-MS/MS method development indicated the potential

for poor extraction recovery at concentrations below 0.5 ng/mL. Based on

human ADME data after oral administration, several metabolites were alsohuman ADME data after oral administration, several metabolites were also

identified in plasma.

A pilot microtracer study of [14C]saxagliptin in dogs was initiated prior to the

human study to provide samples for method development and to assess AMS

methodology. The method was successfully validated and was applied to the

quantification of [14C]saxagliptin in human plasma from an absolute BA study.

Arnold ME, LaCreta F, Bioanalysis 2012, 4:1831-1834.

Absolute Bioavailability (BA): Why and What

Required by some regulatory agencies as part of thedrug registration process.

Used to describe the actual amount of drugabsorbed from a non-intravenous (IV) formulation.

Expressed as the area under curve (AUC) of a drugformulation relative to that of an IV dosing of theformulation relative to that of an IV dosing of thedrug.

27

Traditional Approach of Absolute BA Studies

Two-way cross-over study design with a washout period

Need to develop intravenous (IV) formulation which mayhave potential drug solubility issue

Mandatory pre-clinical toxicity testing to ensure adequatesafety

Day-to-day variability in pharmacokinetics (PK) due toseparate administrations

Costly and time-consuming

Usually employs traditional bioanalytical techniques (LC-MS/MS)

28

Advantages of Using Microdose for Absolute BA Studies

The most expedient path to generate human BA data is toco-administer the oral therapeutic dose and an IV [14C]-microdose of drugOral: LC-MS/MS

IV: Accelerator Mass Spectrometer (AMS)

Single period, IV microdose (< 100 µg) at Tmax of oral doseSingle period, IV microdose (< 100 µg) at Tmax of oral dose

No need for cross-over dosing

No day-to-day variation

Reduced effort in IV formulation

No need for toxicology studies

Saves costs and resources of largerclinical study

29

Specific Challenges for Saxagliptin

Sample preparation

During LC-MS/MS method development, poor extraction recovery wasobserved at low concentrations (<0.5 ng/mL), presumably due to non-specific binding and/or tight binding of the drug to plasma DPP4.

The LC-MS/MS assay used a detergent and 45-min heated sonication toovercome extraction losses.

High sensitivity is needed to capture the concentration of [14C]saxagliptin at

NH2N

O

OH

NC

*

High sensitivity is needed to capture the concentration of [ C]saxagliptin atCmin.

Peak Isolation

Chromatographic separation of saxagliptin from its metabolites is critical.

Reference standards for many of the minor metabolites were not available.

Xu Xet. al. J. of Chromatography B., 201230

[14C]Saxagliptin Oral Microtracer in Dogs-Study Design

3 male beagle dogs, oral administration of [14C]saxagliptin

Share all human metabolites

Dose selection - 0.071 mg/kg (3.4 nCi/kg)

14C dose selected to mimic IV dose (~200 nCi) in humans

The mass dose chosen to mimic the 5 mg human oral dose

Sample Collection

31

Sample Collection

Collect samples from 3 dogs over 24 h post-dose and pool by time points

Split and submit portions to 2 AMS laboratories and an LC-MS/MS lab

Study Objectives

Determine the LLOQ (sensitivity) of the 14C AMS assay

Compare values obtained between labs and techniques (AMS vs. LC-MS/MS)

Xu X, et al., Bianalysis 2012, 6, 497-504.

Data from Dog Microtracer Study

10

100

Saxa

glip

tinC

onc

(ng/m

L)

20

25

30

Saxa

glip

tinC

onc

(ng/m

L)

LC-MS/MS Assay

Vitalea AMS Assay

Accium AMS Assay

0.01

0.1

1

0 5 10 15 20 25

Saxa

glip

tinC

onc

(ng/m

L)

Time (h)

0

5

10

15

0 5 10 15 20 25

Saxa

glip

tinC

onc

(ng/m

L)

Time (h)

32

Microdose Study Design for Absolute BA Determination

24 h blood sampling forsaxagliptin/[14C]saxagliptin

plasma PK

8 healthy male subjects

~40 µg (240 nCi) [14C]saxagliptin single dose IV infusion (15 min)administered at 1 hr (Tmax) after the oral dose of 5 mg

Parent drug concentration measuredusing two analytical methods

AUC for oral dose by LC-MS/MS AUC for IV dose By UPLC-AMS

33

Dose Normalized Mean Plasma Concentration-Time Profile

IV dose: 15 min infusion of [14C]Saxa (40 ug,240 nCi) @ 1 h after oral dose by LC-AMS

Abs BA = 50%

Oral dose; 5 mg Saxaby LC-MS/MS

Abs BA = 50%90% CI, N=8

34

Cmin

Boulton DW et al., Br J Clin Pharm 2013, 75, 763-768.

Summary and Conclusions

Microdose approach for human absolute BA studies is efficient. AMS is

the technology of choice to meet the needed assay sensitivity.

Sample preparation and chromatographic separation are the major

challenges in an LC-AMS measurement.

Drug concentrations in plasma measured by 2 AMS labs were

comparable and were similar to values obtained by LC-MS/MS. Acomparable and were similar to values obtained by LC-MS/MS. A

technical validation is sufficient for an LC-AMS quantification.

Data from the human absolute BA study generated under AMS were

included in regulatory filings and resulted in the drug approval.

35

Introduction to Vismodegib Mass Balance Study

A B

36

• Steady state reached quickly

• Unexpected with a10-14 day half-life

Graham et al., Clin Cancer Res 2011, 17, 2512.

MW = 421pKa = 2.8

clogP = 3.9

Combined Mass balance Study withAbsolute BA Determination of Vismodegib

• Mass Balance Study

• Understanding of major routes of vismodegib elimination

• Quantification of parent and metabolites in plasma, urine and feces

• Absolute Bioavailability Study• Absolute Bioavailability Study

• Understanding vismodegib absorption and distribution after asingle dose and at steady state

• Fulfills regulatory requirement for product registration

Specific Challenges for vismodegib and Opportunity

• If traditional mass balance study:

• How ethical is it to have healthy volunteers exposed to 50 – 100uCi for several months?

• The amount excreted each day is likely very small and may not bedetectable with liquid scintillation counting

• Study is likely to fail• Study is likely to fail

• 14C tracer study with accelerator mass spectrometry

AMS Mass Balance Study Design

• Amount of radioactivity: 1,000 nCi

• It is feasible to send volunteers home after two weeks (lowlevel of radioactivity in urine and feces)

• Continuous sampling until day 14

Time (days)

Cp/D

ose

150 mg PO on day 1 (unlabeled)+ 1,000 nCi/10 µg 14C PO

Cold

Hot

• Continuous sampling until day 14and interim sampling on days 21,28, 35, 42, 49, and 56 andinterpolation to ensure acceptablemass balance?

Graham et al., Drug Metab Dispos 2011, 39, 1460.

0.1

1L

og

pe

rce

nt

of

tota

le

xc

rete

dp

er

24

hp

eri

od

Up to 168 h allexcreta collected

AUC0- = 90.5 % recovery

Interpolation to Ensure Adequate Radioactivity Recoveryfor a Long Half-Life Drug

0.01

0 500 1000 1500 2000 2500

Time (h)

Lo

gp

erc

en

to

fto

tal

ex

cre

ted

pe

r2

4h

pe

rio

d

After 168 h, collections overselected 24 h periods

Subjects were administered 3 mg/1 μCi of drug®

Vismodegib Mass Balance Results

Total Recovery = 86.6%Confined D1-D14

Feces Recovery

= 82.2%

Urine Recovery = 4.4%

Graham et al., Drug Metab Dispos 2011, 39, 1460.

Summary of Vismodegib Mass Balance

• 14C Tracer study with accelerator mass spectrometry wassuccessfully used to determine mass balance andabsolute bioavailability of Vismodegib in one study.

• Interim sampling for long period of sample collections anddata interpolation was demonstrated as a good strategyto determine mass balance for a drug with long terminalto determine mass balance for a drug with long terminalelimination half-life.

Ixabepilone - BMS-247550

A semi-synthetic analog of the natural product epothilone B

S

N HN

O

O O

OH

OH

*

*

*

**

* Denotes the position of 14C label

A semi-synthetic analog of the natural product epothilone B

and marked as an anticancer drug

Preclinical ADME study showed degradation and complex

metabolic profiles

Radiolytic instability at 1-10 uCi/mg but stable at 1-2 nCi/mg

Eight patients were each dosed with 70 mg and 80 nCi

at 1.14 nCi/mg

Plasma, urine, and feces were collected

Comezoglu et al, Drug Metab Pharmacokint 2007, 26, 511.

Metabolic Pathways of Ixabepilone

S

N

HO

O

OH

OH

O

N

BMS-249798(known degradant)

S

N

OHO

N

HOH

O

S

N HN

HO

O O

OH

OH

HO


S

N

O

OH

-2H

S

N

HO

O

OH

OH

O

N


S

N

OHO

N

HOH

O

S

N HN

HO

O O

OH

OH

HO


S

N

O

OH

-2H

OOHBMS-567637(known degradant)

S

N HN

O O

OH

OH

Ixabepilone (BMS-247550)

S

N HN

O

O O

OH

OH

OH

M8

S

N HN

O

O O

OH

OH

OH

M41

N HN

O OOH

-2H

M19

S

N HN

O

O O

OH

OH

OH-2H

M16

OOHBMS-567637(known degradant)

S

N HN

O O

OH

OH

Ixabepilone (BMS-247550)

S

N HN

O

O O

OH

OH

OH

M8

S

N HN

O

O O

OH

OH

OH

M41

N HN

O OOH

-2H

M19

S

N HN

O

O O

OH

OH

OH-2H

M16

0 . 0 0 0 0

0 . 0 0 5 0

0 . 0 1 0 0

0 . 0 1 5 0

0 . 0 2 0 0

0 . 0 2 5 0

0 . 0 3 0 0

0 . 0 3 5 0

0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

8 - 9

1 1

1 7

1 9

2 4

2 7

2 8

3 1

3 2

3 3

3 4

1 5

2 0 - 2 1

2 3

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3 6

3 7

2 6

0 . 0 0 0 0

0 . 0 0 5 0

0 . 0 1 0 0

0 . 0 1 5 0

0 . 0 2 0 0

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0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

8 - 9

1 1

1 7

1 9

2 4

2 7

2 8

3 1

3 2

3 3

3 4

1 5

2 0 - 2 1

2 3

2 9

3 6

3 7

2 6

0 . 0 1 0 0

0 . 0 1 2 0

3 10 . 0 1 0 0

0 . 0 1 2 0

3 1

APlasma 4 hr

B

0 . 0 0 0 0

0 . 0 0 5 0

0 . 0 1 0 0

0 . 0 1 5 0

0 . 0 2 0 0

0 . 0 2 5 0

0 . 0 3 0 0

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0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

8 - 9

1 1

1 7

1 9

2 4

2 7

2 8

3 1

3 2

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0 . 0 0 0 0

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0 . 0 3 0 0

0 . 0 3 5 0

0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

8 - 9

1 1

1 7

1 9

2 4

2 7

2 8

3 1

3 2

3 3

3 4

1 5

2 0 - 2 1

2 3

2 9

3 6

3 7

2 6

0 . 0 1 0 0

0 . 0 1 2 0

3 10 . 0 1 0 0

0 . 0 1 2 0

3 1

APlasma 4 hr

B

Radioactivity Profiles of Ixabepilone in Human Plasma

0 . 0 0 0 0

0 . 0 0 2 0

0 . 0 0 4 0

0 . 0 0 6 0

0 . 0 0 8 0

0 . 0 1 0 0

0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

5

7 - 8

1 0

1 3

1 5

2 2

2 5

2 7

2 9 - 3 0

3 1

3 2

3 4

3 5

2 43 3

3 6

3 7

0 . 0 0 0 0

0 . 0 0 2 0

0 . 0 0 4 0

0 . 0 0 6 0

0 . 0 0 8 0

0 . 0 1 0 0

0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

5

7 - 8

1 0

1 3

1 5

2 2

2 5

2 7

2 9 - 3 0

3 1

3 2

3 4

3 5

2 43 3

3 6

3 7

Plasma, 8 hr

0

0 . 0 0 1

0 . 0 0 2

0 . 0 0 3

0 . 0 0 4

0 . 0 0 5

0 . 0 0 6

0 . 0 0 7

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

T i m e [ m i n ]

dp

mp

er

fracti

on

CPlasma, 8 hrRepeat Analysis

0 . 0 0 0 0

0 . 0 0 2 0

0 . 0 0 4 0

0 . 0 0 6 0

0 . 0 0 8 0

0 . 0 1 0 0

0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

5

7 - 8

1 0

1 3

1 5

2 2

2 5

2 7

2 9 - 3 0

3 1

3 2

3 4

3 5

2 43 3

3 6

3 7

0 . 0 0 0 0

0 . 0 0 2 0

0 . 0 0 4 0

0 . 0 0 6 0

0 . 0 0 8 0

0 . 0 1 0 0

0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 4 5 . 0 5 0 . 0

T i m e ( m i n )

dp

mp

er

fracti

on

B M S - 2 4 9 7 9 8

B M S - 3 2 6 4 1 2

B M S - 2 4 7 5 5 0

5

7 - 8

1 0

1 3

1 5

2 2

2 5

2 7

2 9 - 3 0

3 1

3 2

3 4

3 5

2 43 3

3 6

3 7

Plasma, 8 hr

0

0 . 0 0 1

0 . 0 0 2

0 . 0 0 3

0 . 0 0 4

0 . 0 0 5

0 . 0 0 6

0 . 0 0 7

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

T i m e [ m i n ]

dp

mp

er

fracti

on

CPlasma, 8 hrRepeat Analysis

0

0.05

0.1

0.15

0.2

0.25

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fracti

on

3 5

8-9

1012

13 BMS-249798

17

18-19

23

26

27

BMS-247550

31

32

7 11

20-21

24

29-30

35

0

0.05

0.1

0.15

0.2

0.25

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fracti

on

3 5

8-9

1012

13 BMS-249798

17

18-19

23

26

27

BMS-247550

31

32

7 11

20-21

24

29-30

35

AUrine

0

0.05

0.1

0.15

0.2

0.25

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fracti

on

3 5

8-9

1012

13 BMS-249798

17

18-19

23

26

27

BMS-247550

31

32

7 11

20-21

24

29-30

35

0

0.05

0.1

0.15

0.2

0.25

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fracti

on

3 5

8-9

1012

13 BMS-249798

17

18-19

23

26

27

BMS-247550

31

32

7 11

20-21

24

29-30

35

AUrine

Mass Balance:

Radioactivity Profiles of Ixabepilone in Human Urine/Feces

Time (min)Time (min)

0.0000

0.0050

0.0100

0.0150

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fra

cti

on

BMS-326412

BMS-247550

4

6

8

10

12

13-15

16

17-19

20

22

24-25

2628-30

32

33

35

7

9

11

23

31

36

0.0000

0.0050

0.0100

0.0150

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fra

cti

on

BMS-326412

BMS-247550

4

6

8

10

12

13-15

16

17-19

20

22

24-25

2628-30

32

33

35

7

9

11

23

31

36

4

6

8

10

12

13-15

16

17-19

20

22

24-25

2628-30

32

33

35

7

9

11

23

31

36

BFeces

Time (min)Time (min)

0.0000

0.0050

0.0100

0.0150

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fra

cti

on

BMS-326412

BMS-247550

4

6

8

10

12

13-15

16

17-19

20

22

24-25

2628-30

32

33

35

7

9

11

23

31

36

0.0000

0.0050

0.0100

0.0150

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Time (min)

dp

mp

er

fra

cti

on

BMS-326412

BMS-247550

4

6

8

10

12

13-15

16

17-19

20

22

24-25

2628-30

32

33

35

7

9

11

23

31

36

4

6

8

10

12

13-15

16

17-19

20

22

24-25

2628-30

32

33

35

7

9

11

23

31

36

BFeces

Mass Balance:25.1% of dose in urine52.7% of dose in fecesDuring 0-168 h collections

Summary of Ixabepilone Human ADME

• 14C Ixabepilone undergoes radiolysis degradation.

• 14C microtracer in a pharmacological dose with AMS wassuccessfully used to determine mass balance andmetabolite profiles of ixabepilone in human ADME study.

• Although chemical degradation was prominent forixabepilone, AMS analysis of plasma shows that theixabepilone, AMS analysis of plasma shows that theparent was the major circulating component in humancirculation.

• Fraction collection, AMS analysis, and comparison withanimal and in vitro metabolites showed that manyoxidative metabolites and degradation products in humanurine and feces.

Technical Limitations for AMS Studies in ADME

No Structural Information

Efficient Extraction Requirement

In the absence of an internal standard, reliable and high extractionrecovery of the low level of [14C] material is critical to achieve theneeded sensitivity and reliability in derived concentrations from theAMS analysis.

Peak Isolation Peak Isolation

AMS cannot distinguish co-eluting metabolites by mass, thuschromatographic separation of parent from its metabolites is critical.

The AMS labs do not have easy access to a benchtop MS detectorfor identification of drug-related components

Reference standards for many of the minor metabolites are often notavailable

Factors Impacting Sensitivity in AMS

In theory, only 1,000 atoms are need to achieve statistical limits ofdetection which translates to a zeptomole detection limit (10-21) forAMS

In practice, the limit of detection for AMS depends on the s/n ratio,which is further dependent upon: The amount of 14C in the isotopically enriched drug

Endogenous background from extraction

49

Quality of the signal/noise above background

Ways of to reduce endogenous carbon in samples Removal of endogenous proteins via selective extraction

Remove co-eluted metabolites/interferences

Contamination must be controlled at the site of sample collectionand sample analysis as background level of radioactivity willsignificantly impact sensitivity (small sample size can easily causecontamination, usually 0.1% of conventional sample size) Elevated 14C baseline can have a significant impact on accuracy/ sensitivity

of LOQ measurement

LC-MS/MS

Integrated separation/detectiondoesn’t require offline fractioncollection

Can simultaneously detect parentand metabolites

High throughput

AMS

Accelerates ions to extraordinarilyhigh kinetic energies before massanalysis

Derives extraordinary sensitivityfrom direct counting of the amount ofionized atoms (e.g., 14C) in sample

AMS vs. LC-MS/MS

High throughput

Can reaching sub pg/mL level, butrequires extraordinary sample cleanup efforts

Cost and time savings

Improving sensitivity

Wide linear range (>104)

No need for IS

Low throughput and long dataturnaround

Costly and limited vendors (Vitalea,Accium, etc.)

50

Path to Successful Microdose/Microtracer Studies

• Understand project needs and timelines: PK/massbalance/metabolite profiles

• Important elements to consider: analytical sensitivityrequirement, AMS vs LC-MS/MS, separation frommetabolite and parent, no Structural Information fromAMS, complementary to regular LC-MS/MS, need

51

AMS, complementary to regular LC-MS/MS, needefficient extract recovery

• Plan early: Model pharmacokinetics of IV dose: VD

and Cmin, Cmax ratio of tracer to cold in circulation

• Team collaboration: clinical Pharmacology,metabolism, radiosynthesis and bioanalytical

AMS Limitations and Future Perspectives

• Microtracers leveraged with AMS sensitivity offer unique clinical testing

• i.v. Absolute Bioavailability (efficient)

• Low 14C mass balance (radiolysis or dosimetry limited)

• PK/Dermato/Occular (sensitivity, dosimetry limited)

• Pediatrics (single blood drop PK)

• Instrument and total bioanalysis can meet needs ofregulated bioanalysis

• Low voltage instruments are becoming available

Single-dropliquid analysis

replacing graphite

• Analysis costs have dropped but graphite process cumbersomeand not automatable – major hindrance for Pharma(low capacity, relatively expensive)

• However – Direct CO2 interfaces are ready for commercialization

• EA-CO2 (ETH Zurich)

• LC/AMS (LLNL) Order of magnitude leap in throughput

• Simpler Laser based systems (cavity ring down) are approachingAMS sensitivity

• Real-time CO2 analysis

• Inexpensive benchtop instruments (200K)

• Technological renaissance

• Next wave: ADCs, Biotherapeutics

Cavity ring-downLaser System

Slide courtesy of LLNL/Stephen R. Dueker

Backup Slides

Readings

Vogel JS and Love AH, Quantitating isotopic molecular labels with accelerator mass spectrometry P402-422. InBiological Mass Spectrometry in Methods in Enzymology vol 402.

Garner RC et al., A validation study comparing accelerator MS and liquid scintillation counting for analysis of 14C-labeled drug in plasma, urine, and faecal extracts. J Pharmaceut Biomed Anal 24: 197-2009.

Comezglu SN et al (2007) Biotransformation profiling of 14C ixabepilone in human plasma, urine and fecessamples using accelerator mass spectrometry (AMS) Drug Metab Pharmacokinetics 26:511-522.

Graham RA et al (2011) A single dose mass balance study of the Hedgehog pathway inhibitor vismodegib (GDC-0449) in humans using accelerator mass spectrometry. Drug Metab Dispos 39: 1460-7.

Graham RA et al (2012) single and multiple dose intravenous and oral PK of the Hedgehog pathway inhibitorvisodegib in healthy femle subjects. Br J Clin Pharm 74: 788-796.visodegib in healthy femle subjects. Br J Clin Pharm 74: 788-796.

Boulton DW et al (2013) simultaneous oral therapeutic and intravenous 14C-microdose to determine the absoluteoral bioavailability of saxagliptin and dapagliflozin. Br J Clin Pharm 75:763-768.

Lappin G (2012) Microdosing: current and the future 2, 509-517.

Dingley KH et al. (1998) Attomole detection of 3H in biological samples using accelerator mass spectrometry:Application in low‐dose, dual‐isotope tracer studies in conjunction with C‐14 accelerator mass spectrometry.Chem Res Toxicol 11, 1217–1222.

Dueker SR et al. (2000) Long‐term kinetic study of beta‐carotene, using accelerator mass spectrometry in anadult volunteer. J Lipid Res 41, 1790–1800.

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