Achieving Regulatory Approval: Overcoming Bioanalytical Challenges in an Onglyza Micro-tracer Absolute Bioavailability Study with Accelerator Mass Spectrometry

Xiaohui (Sophia) Xu, Lisa J. Christopher, Stephen Dueker, Pete Lohstroh, Vikram Roongta, Laura Cojocaru, Chi Fung (Anther) Keung, Kai (Kevin) Cao, Samuel Bonacorsi, W. Griffith Humphreys, Bruce Stouffer and Mark Arnold
Research & Development, Bristol-Myers Squibb, Lawrenceville, NJ Introduction
Onglyza (saxagliptin, BMS-477118) is an oral, small molecule DPP4 inhibitor, indicated for the treatment for Type II diabetes. A clinical absolute bioavailability (BA) study was required to fully understand the disposition of saxagliptin.

Xiaohui (Sophia) Xu Rt. 206 & Province Line Road Lawrenceville, NJ 08543-4000 Phone: 609-252-7271 E-mail: xiaohui.xu@bms.com

Accelerator Mass Spectrometer (AMS)

AMS is very sensitive and accurate technique for measuring 14C, 13C and 12C. AMS traces low abundance carbon-14 for high specificity, and detects them for high sensitivity. The quantified 14C/C isotope ratio is a property of the isolate and is independent of ionization or detection efficiencies. Again accelerated using electrostatic quadrapole triplet and analyzing magnet
4. Filter 4. Filter mass analysis mass analysis (high-energy end) (high-energy end) mass spectrometer mass spectrometer

Optimizing Extraction Efficiency

Utilized surrogate approach ([13C]saxagliptin and measured by LC-MS/MS instead of [14C]) to evaluate recovery. It was efficient and cost effective. Remove steps that had the potential to conflict with AMS procedures Add 200-fold excess of unlabeled saxagliptin to the plasma instead of CHAPS. The unlabeled saxagliptin prevents nonspecific binding of [13CD2]saxagliptin and displace it from DPP4 binding sites in plasma Treatment with CHAPS surfactant 45 min sonication
Recovery at incubation conditions vs. freshly prepared saxagliptin

Representative UV Chromatograms from Standard and Samples

[14C]saxaliptin
From Standard Curve

Incurred Sample Reanalysis (ISR) Data

500.0 Concentration (pg/mL) 400.0 300.0 200.0 100.0 0.0 0 5 10 Sam ple Num ber ISR Data Original data 15 20 % Difference 40 % Difference 20 0 -20 -40

The clinical study design included concurrent IV administration of a [14C]saxagliptin microdose (40 g, 240 nCi) at the projected Tmax of a 5 mg therapeutic oral dose. The analytical plan involved analyzing plasma concentrations of derived from the IV route of administration by AMS (accelerator mass spectrometry) and unlabeled saxagliptin from the oral dose with a validated LC-MS/MS method. [14C]saxagliptin
2. Filter 2. Filter mass analysis mass analysis (low-energy end) (low-energy end)

-ve ions converted to +ve ions

charge exchange

-ve carbon ion beam

The human ADME study showed that saxagliptin and 5-hydroxysaxagliptin (an active metabolite) were the major drug-related components in the circulation along with several minor metabolites.

Pre-accelerated and passed through a sperical electrostatic analyzer

Recovery Percentage (%)

LC-MS/MS method development indicated the potential for poor extraction recovery at low saxagliptin concentrations (i.e. <0.5 ng/mL).

110

90

3. Filter 3. Filter molecule destruction molecule destruction (Stripping process)

12 C

70

[14C]saxaliptin
From extracted sample of subject 7 @1.25 hr
0 5 10 15 20 25 30 Incubation tim e at room tem perature (hr) CHAPS and Saxa No CHAPS w ith saxa No saxa w ith CHAPS No CHAPS no saxa

Chromatographic separation of saxagliptin from its metabolites was required for AMS analysis of [14C]saxagliptin

and 13C5+ were measured in faradays cup

12C4+

After overcoming the analytical challenges, the method was successfully validated and was applied to the quantification of [14C]saxagliptin in human plasma from an absolute BA study.

13 C

/ 14 C /
negat eons v i i ar onst per g p gas i r 1 : 9 10: 1 13C H

12 C 12 C

Kinetic equilibration was established at both 24 hr incubation at room temperature or 4 hr incubation at 37C in the presence of excess unlabeled saxagliptin

50

%Difference= (ISR data-Mean)*100%/Mean

13 C

Incubation time optimization at room temperature

Dose Normalized Mean Plasma Drug Concentration-Time Profile Following Oral and IV Administration to Human Subjects (n=8) Validation Curve, Sample Analysis Curve and QC Summary
Human Validation Curve Plot
80.00 Measured STD Valiue (fMC) 80.00

Absolute BA Study
An absolute BA study compares the exposure of the active parent drug in systemic circulation following non-intravenous administration with the exposure of the same drug following intravenous (IV) administration.

Ion source: Multi Cathode Source of ve ions with Cs sputtering

Extraction Recovery of [14C]Saxagliptin from Human Plasma

posi eons t i v i 14C + 13C + 14C 12C 2H 7Li 2 12C + H+

electrostatic 14C4+ measured

deflector elecrostatic deflector

using

100

100

IV dose: 15 min infusion of [14C]Saxagliptin (40 ug, 240 nCi) @ 1 h after oral dose Measured by LC-AMS Abs BA = 50% 90% CI: 48 - 53% %CV: 6.6%

37 C Incubation

Traditional approach: Two-way cross-over study design with a washout period Mandatory toxicology studies in non-clinical species LC-MS/MS measurement used for both IV and non-IV portion Expedient approach: Single interval study design utilizing an IV microdose co-administered with an non-IV therapeutic dose Toxicology studies in non-clinical species may not be required* Plasma concentrations from non-IV dose measured by LC-MS/MS Plasma concentrations from IV route measured by AMS *No supporting toxicology studies required provided that the IV micro-tracer dose utilized was less than 1/100th of the therapeutic oral dose in humans (and <100 g) and NOAEL in animals

60

60

60.00

General AMS Process for Absolute BA Measurement

Quantification based on measurement of 14C/C ratio in a sample Data are often reported as Modern with Modern being equal to 98 amol 14C per mg carbon or 0.01356 DPM/mg C Calculations: fMC (fraction modern carbon, ratio of 14C/C) DPM/mL pg/mL Add unlabeled compound in excess
1 .4 1 .2

Weighting 1/Y2, Linear Regression Validation

60.00

Measured QC Valiue (fMC)

1.Filter 1.Filter negative ion formation negative ion formation

5. Filter 5. Filter particle identification particle identification

80

80

40

40

20

20

0 0 6 12 18 24

fMC (amol 14C/mg C)

1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 6 .6 6 .8 7 .0 7 .2 7.4 7 .6 7.8 8 .0 8.2 8 .4

Fraction collection, sample preparation, and AMS accuracy Are the source of error UV Absorbance Fractions corresponding to analyte AMS analysis
T im e p o s t in je c t io n

Time (Hr)
Percentage of [14C]saxagliptin remained in protein pellet after extraction

Test samples were prepared by adding [14C]saxagliptin at a concentration of 1.1 ng/mL to human plasma, followed by equilibration for 2 h at 37C & freezing overnight. To evaluate extraction recovery, the samples were thawed, and unlabeled saxaglitpin (1.8 ug/mL) was added. Samples were then incubated for 24 hr at room temperature or for 8 hr at 37C. At various times, the carbon-14 remaining in the protein pellet after protein precipitation was determined by AMS. At room temperature, only the 24 hr time point had a recovery > 90%. Whereas, at elevated temperature (37C), the >90% extraction was achieved within 6 h and displayed a competitive (sigmoidal) release profile.

Room Temperature Incubation

40.00

40.00

Curve 1: Y=4.934X+0.2167 Curve 2: Y=4.988X+0.2751

20.00

20.00

0.00 0.000

4.000

8.000

12.000

0.00 16.000

QC 1 QC 2 Linear (STD 2) Linear (STD 1)

Oral dose; 5 mg Saxa Measured by LC-MS/MS

In both validation and sample analysis: 5 points STD curve in duplicates 3 levels of QCs in 4 replicates No failed QC or STD points in either validation or sample analysis runs Two month storage stability at -20C was established

Nominal Concentration (DPM/mL)

Human Sample Analysis Curve Plot

90.0 80.0 Measured Value (fMC ) 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 0.0 4.0 8.0 12.0 16.0 Nominal Concentration (DPM/mL)

Sample

Extract

Absolute BA Study Design in Expedient Microdose Approach

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

Chromatographic separation

Revised Chromatographic Conditions

Biotransformation ADME Method (70 min)
4 8 0

Weighting 1/Y2, Linear Regression

QC 1 QC 2 QC 3 Linear (STD 2) Linear (STD 3)

Collect parent drug as fractions

B) Human

BMS-510849 (M2)

Main Sources of Procedural losses Add 12C carrier tributyrin Graphitization Oxidation/Reduction CuO/Zn + TiH

3 8 0

2 8 0

Saxa M46 M13 M1 M3 D1 M45

1 8 0

24 hr blood sampling for saxagliptin/ [14C]saxagliptin plasma PK parameter estimation

AMS is non-selective and measures only and 12C Chromatographic separation is required to collect the portion only containing saxagliptin, which is critical for selectivity, sensitivity and reproducibility Pooled samples from a recent clinical study were run using a longer run time LC-MS method (70 min) to identify metabolites. The rnu time was optimized <15 min, by following the LC-MS MRM transitions of parents drug Mobile Phase B B B B B B

14C, 13C

Sample analysis

Linear (STD 1)

Curve 1: Y=4.7750X+0.2207 Curve 2: Y=4.4660X+0.2437 Curve 3: Y=4.6160X+0.2306

Conclusions
Extraction recovery was optimized/maximized: Minimized non-specific binding and DDP4 specific binding at low concentrations. Metabolite separation from saxagliptin was assured during method development by monitoring components with LC-MS/MS: Minimized background contamination/interference. The use of standard curve and QCs may compensate for background 14C in the system and possibly correct for concentration dependent-non linearities in recovery. The raw AMS measurement, 14C/C ratio, expressed as fMC (fraction Modern Carbon) and known DPM/mL of [14C]saxagliptin standards, were used to construct a weighted (1/y2) leastsquares, linear regression model and used to predict the concentration of unknowns. A technique-appropriate validation was conducted that fully demonstrated the accuracy, precision, stability, specificity and recovery of the AMS method across the concentration range of 0.025 to 15.0 DPM/mL (equivalent to 1.91 to 1144 pg/mL). The absolute bioavailability of saxagliptin was at ~50%. Pharmacokinetic results indicated good agreement in the terminal phase half-life values between the IV and oral routes demonstrating that the IV radioactive microdose provides a sound approach to determine absolute bioavailability. Data from the saxagliptin absolute BA study were included in regulatory filings which results in drug approval.

CPM

Summary of QC data for the analysis of [14C]saxaglitpin by AMS Quality Controls Nominal Values Mean (dpm/mL) N %CV %Accuracy Mean (dpm/mL) N %CV %Accuracy Mean (dpm/mL) N %CV %Accuracy 0.075 DPM/mL 0.0775 4 1.4 103.4 0.0683 4 2.0 91.0 0.0726 4 2.1 96.8 0.750 DPM/mL Run 1 0.6859 4 1.0 91.5 Run 2 0.7333 4 4.0 97.8 Run 3 0.7142 4 8.8 95.2 10.0 DPM/mL 9.5177 4 4.5 95.2 10.7034 4 2.1 107.0 10.3755 4 1.4 103.8

Challenges
Extraction Recovery During development of an LC-MS/MS assay for saxagliptin, poor extraction recovery was observed at concentrations below (<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 utilized CHAPS and 45-min heated sonication to overcome extraction losses. However, use of CHAPS may interfere with AMS analysis as it may give high carbon background. In the absence of an internal standard, reliable and high extraction recovery of the critical to achieve the needed sensitivity and reliability in derived concentrations from the AMS analysis. Peak Isolation Chromatographic separation of saxaglitpin from its metabolites is critical, since AMS cannot distinguish co-eluting metabolites by mass as it only counts various isotopes of carbon AMS labs do not have bench-top MS detectors for identification of drug-related components & their retention times (use UV detection) Reference standards for many of the minor metabolites were not available [14C]saxagliptin is

8 0

-2 0

1 0

1 5

2 0

2 5

Time (minutes)

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

Time (min) 0.01 10.00 10.02 11.00 11.01 12.50

% 10.0 50.0 95.0 95.0 10.0 10.0

Parent drug concentration measured using two analytical methods

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

M2

Optimized Method (12 min) All metabolites identified, better separated

P

M2

saxagliptin

Chromatographic Condition
M3 &M46 M13 & M1
1 116 2 232 3 347 4 462

D1

M45
5 577 6 693 Time, min 7 808 8 923 9 1038 10 1154 11 1269

Analytical Column: Acuity, 2.1 x 100 mm, 5 m Flow: 0.3 mL/min MP (A): 10mM NH4OAc (aq) MP (B): 10mM NH4OAc in MeOH

Acknowledgements
The authors would like to thank the contributions from Brad Maxwell, David Boulton, Frank LaCreta, Ramaswamy Iyer, Dennis Garner and Pathanjala Kadiyala, all current BMS employees. The authors would also like to thank Tandem Labs, West Trenton, NJ and Vitalea Sciences, Sacramento, CA for their excellent support in sample analysis. The authors would also like to thank Quotient Clinical, UK for performing the clinical study.

Time (minutes)

Presented at the 12th Annual Land OLakes Bioanalytical Conference: Agency and Industry Perspectives on Biomarkers, Bloodspots and Beyond Merrimac, WI July 11-15, 2011

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