The Use of Dried Blood Spot Samples for the Quantitative Bioanalysis of Drugs in PreClinical

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The Use of Dried Blood Spot Samples for the
Quantitative Bioanalysis of Drugs in PreClinical
Clinical Studies

• Neil Spooner
• PCD DMPK, GlaxoSmithKline, Ware, UK
• (Neil.Spooner_at_gsk.com)

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Background
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What are Dried Blood Spots? (DBS)

• Technique has been around for gt40 years
• Easy way of collecting, shipping storing blood
  samples
• Widely used in new born screening, therapeutic
  drug monitoring for trials in remote areas,
  e.g. anti-malarials
• Blood from animal/human spotted onto collection
  card
• Lyses cells (anti microbial)
• Denatures proteins
• Cards air dried stored/shipped desiccated at
  room temperature
• Discs punched out of the DBS for quantitative
  analysis
• Validated LC-MS/MS bioanalytical methods

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Blood Spot Card

• Currently use 2 types of Whatman paper
• FTA
• FTA Elute
• Both contain additives (designed for nucleic acid
  analysis)
• Untreated (Guthrie / 903) paper
• Uniformity issues
• Lower recoveries than treated papers

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Why are GSK Interested in DBS?

• Originally came from the need to handle small
  volume blood / plasma samples (lt100 µL) for
  quantitative bioanalysis
• Paediatrics
• Juvenile tox
• Other benefits of small blood volumes and DBS
  soon realised

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Advantages for Pre-Clinical

• Ethical benefits of reduced volumes (typically 10
  - 20µL per spot)
• Refinement
• Elimination / reduction of rodent warming
• Reduction
• Serial sampling from one rodent rather than
  composite bleeds from several
• Reduction / removal of satellite rodents
• Data quality
• Serial vs composite
• TK from central study animals
• Enables juvenile studies
• Costs
• Animal numbers
• Procedures
• Test substance

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Current Practice - Rats

• Typically 6 blood samples of between 200 to
  250µL (total volume up to 1500µL) per TK occasion
• Approximately 10 minutes warming prior to
  sampling
• Typical short term toxicology study dose group
  structure, males and females

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Proposed Practice - Rats

• Reduced blood volume requirement from 1500 to
  480µL
• No prior warming
• Collect toxicokinetic samples from toxicology
  animals
• Remove requirement for satellites on short term
  toxicology studies

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Advantages for Clinical

• Reduced volume enables paediatric studies
• Simplified blood sampling (finger prick)
• Findings from GSK pilot clinical study
• All staff (14) found blood spotting to be easy
• 10 out of 11 subjects preferred finger prick to
  venous cannula
• 2/3 finger pricks per timepoint (if required)
• gt6 samples per session / visit
• Improved recruitment
• Ideal for Phase II/III in developing countries

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Other Advantages

• Simplified process better quality
• ? Centrifugation
• ? Sub-aliquotting
• ? Freezing
• ? Defrosting
• Reduced costs
• Shipping non-hazardous (inactivates HIV Hep
  B)
• Storage
• Potential for greater compound/metabolite
  stability

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Regulatory Perspective

• Blood v plasma
• Blood data has already supported regulatory
  filings

ICH S3A says .The quantification of systemic
exposure provides an assessment of the burden on
the test species and assists in the
interpretation of similarities and differences in
toxicity across species, dose groups and sexes.
The exposure might be represented by plasma
(serum or blood) concentrations or the AUCs of
parent compound and/or metabolite(s). .The
choice of analyte and the matrix to be assayed
(biological fluids or tissue) should be stated
and possible interference by endogenous
components in each type of sample (from each
species) should be investigated. Plasma, serum or
whole blood are normally the matrices of choice
for toxicokinetic studies
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Validating the Technology
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Validating the Technology (1)

• Initially 10 structurally diverse compounds were
  validated
• Precision and accuracy
• On card stability
• Assay ruggedness to pipetting error
• 12 additional compounds then tested in rats
• Direct comparison of conventional technique
  (bloodwater 11 v/v) vs blood spot using in-vivo
  samples

Bloodwater DBS
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Validating the Technology (2)

• Pilot in vivo Safety Assessment studies started
  in January 2007
• nonGLP 7 day studies
• Plasma primary data
• DBS and blood/water samples also taken
• All pre-clinical Sites involved

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GSKs Strategy

• World Wide agreement and implementation
• DBS adopted as preferred technique for assessment
  of TK of all new oral compounds selected as
  candidates, where a bioanalytical method has been
  validated
• Once we commit to a matrix at pre-candidate
  selection that matrix will not change throughout
  the life of the compound, including Clinical
  phases
• Switch to DBS for late stage compounds only
  considered for particular circumstances, e.g.
  paediatrics, drugs of the developing world
• gt50 compounds have DBS methods developed
• 9 compounds have reached phase 1 pre-clin GLP
  studies
• Clinical pilot study completed
• FTiH studies initiated (rapid turnaround)
• Large scale Clinical studies 1Q09
• Investigating pre-clinical microsampling
  strategies

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Process Details
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Blood Spotting

• Aliquot 15µL blood per spot
• 3 spots per sample plus spare
• Using a pipette or capillary
• Do NOT allow tip to touch card surface!
• Dry for approx 2 hours at room temperature
• Ship store in sealable bags containing desiccant

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Sample Prep Analysis

• Analytical sample obtained by punching small
  circular disc (typically 3 mm) from centre of DBS
• Manually
• Automation BSD1000
• Extract disc in organic solvent (typically
  methanol) containing internal standard
• Quantitation by validated LC-MS/MS assay
• Sensitivity

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Assay Validation

• Precision, accuracy linearity
• Whole blood stability at 37C for 4 hours
• On card stability
• Assay robustness to pipetting error (10 - 20µL)
• Dilution with control matrix extract
• Total Blanks
• Blanks
• Processed sample stability

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Acetaminophen in Human DBS Assay Performance
Data
n6 replicates
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Acetaminophen in Human DBS On Card Stability

• Human DBS on FTA paper stored at room temperature
  for 113 days

n6 replicates
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Acetaminophen in Human DBS Assay Robustness to
Pipetting Error
n6 replicates
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Acetaminophen in Human DBS 10-fold Sample
Dilution

• 10,000 ng/mL sample extracted from card then
  diluted 10-fold using extract from blank DBS
  samples (n6)

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Acetaminophen in Human DBS Influence of
Spotting Device
n6 replicates
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Acetaminophen in Human DBS Influence of Blood
Temperature
n6 replicates
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Metabolite Stability

• Qualitative assessment of the glucuronide
  sulfate metabolites of acetaminophen
• Individual samples as DBS (FTA cards) and whole
  blood
• 6 24 hours at room temperature
• No detectable formation of acetaminophen
• Compound investigated in US showed frozen plasma
  instability possibly due to esterase activity
• DBS on card stability for at least 1 week

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Areas for Further Investigation

• Microsampling procedures
• Further reductions in blood volumes
• Alternative papers and additives
• Reduced variability
• Reduced suppression
• Enhanced ionisation
• Improvements in handling
• Drying
• Shipping
• Storage
• Automation
• Direct elution / desorption
• Immunoassay
• Already demonstrated for mAbs

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Publications

• M. Barfield, N. Spooner, R. Lad, S. Parry, S.
  Fowles (2008) J. Chromatogr. B 870 32-37
• Application of dried blood spots combined with
  HPLC-MS/MS for the quantification of
  acetominophen in toxicology studies
• Y. Ramakrishnan, S. Shabbir, S. Miller, J.
  Houlden, J. Shadare, O. Dewit, M. Barfield, N.
  Spooner (2008) Proceedings of the British
  Pharmacological Society In Press
• A study to evaluate dried blood spot technology
  as a potential sample collection technique
  following oral administration of paracetamol in
  healthy human volunteers
• N. Spooner, R. Lad, M. Barfield (2009) Anal.
  Chem. In Press
• Dried blood spots as a novel sample collection
  technique for the determination of
  pharmacokinetics in clinical studies
  considerations for the validation of a
  quantitative bioanalytical method

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Acknowledgements

• Matthew Barfield (PCD DMPK, GSK)
• Rakesh Lad (PCD DMPK, GSK)
• Susan Fowles (PCD DMPK, GSK)
• Cerys Lovatt (SA, GSK)
• Wesley Dopson (SA, GSK)
• Yujay Ramakrishnan (Addenbrookes CCI, GSK)
• Odile Dewit (Addenbrookes CCI, GSK)

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Contacts
GSK Whatman, now part of GE Healthcare
Neil Spooner GSK - UK Neil.Spooner_at_gsk.com 44 1920 882550 Christopher Evans GSK - USA Christopher.2.Evans_at_gsk.com 1 610 270 7184 For Europe Claire Dupont Claire.dupont_at_ge.com 33 6 8393 9070 For the US Jim Robbins Jim.Robbins_at_ge.com 1 973 979 1372