Biomarkers in drug discovery and development · aptuit.com aptuit.com. Biomarkers are used in pre- clinical and clinical drug development as . indicators of likely response to drug:

Margit Wissenbach,PhD

Dir Business Development Scandinavia

Biomarkers in drug discoveryand development

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Biomarkers are used in pre-clinical and clinical drug development as indicators of likely response to drug: therefore, they enjoy utility both as predictors of possible EFFICACY and

also as warning signs of potential TOXICITY

Efficacy biomarkers

rapid confirmation that the drug has hit its

targetIndicator of

pharmacological activity

Safety biomarkers

measurable indicators of toxicological and adverse side effects

of drug activitycells

proteins

genes

+

Quality assessment of biological drug activity Translational approach to clinic development

Reduced attrition

Biomarkers

Applications for biomarkers

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Single biomarker approach (ELISA, EIA, RIA, IRMA);

Disease-driven biomarker panels (Luminex® and Meso Scale Discovery® platforms):

acute stress markers: corticosterone and ACTH - potential indicators of specific compound activity or side effects;

metabolic markers/adipokines: prolactin, insulin, amylin, glucagon, GLP-1, glucose, leptin, resistin, adiponectin, MCP-1, ghrelin - to assess metabolic liability displayed by some compounds;

inflammatory markers: Cytokines/Chemokines (e.g. IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GRO-KC, MIP-1α, GM-CSF, IFN-γ & TNF-α) to test for pro- or anti-inflammatory activity;

hypothalamic neuroendocrine markers: prolactin, TSH, LH, GH, FSH; neuroplasticity panel: serum BDNF, VEGF, cAMP; sex hormones: testosterone, progesterone, estradiol.

Quantification in complex biological samples (e.g. serum, plasma, urine, saliva, brain tissue) from preclinical species and in clinical samples

Use of biomarkers in support of disease hypothesis

Regulatory requirements for biomarker validation

Little regulatory guidance on biomarker assay validation is available: In 2003 the American Association of Pharmaceutical Scientists participated in

a Biomarker Method Validation Workshop to address the validation challenges of biomarker assays in support of drug development Biomarker method development and validation is based on a “fit-for-

purpose” approach.

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Cardiac Troponin I (cTnI):

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represents a biomarker of cardiac toxicity in the clinic

can be used as reverse translational biomarker since animal studies have demonstrated that it’s a marker of the same diseases that occur in humans

has been successfully applied as marker of cardiac injury in different preclinical animal species

is measured in preclinical by automated chemiluminescent immunoassay on the Siemens ADVIA Centaur CP

courtesy by jolisfukyu.tokai-sc.jaea.go.jp

Aminoterminal pro-brain natriuretic peptide (NT-proBNP):

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courtesy by Roche Diagnostics

is primarily secreted from the cardiac ventricular myocytes in response to cardiac stress

used in the clinic for the diagnosis of left ventricular systolic and diastolic dysfunction

has been evaluated in preclinical studies in the dog

is measured in preclinical by ELISA

Biomarkers in a 26-week dog study

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CTnI was essential in the characterization of the onset of the lesions and its slow evolution;

excellent correlation between cTnI and developing pathology was observed with cTnIelevations detectable long before light microscopy changes;

transmission electron microscopy analysis was capable to detect ultrastructural changes in the heart which were concomitant to cTnI elevations

NT-proBNP determination in conjunction with echocardiography and morphometry acted as an early marker of heart hypertrophy anticipating echocardiography changes as well morphometry

cTnI and NT-proBNP are reliable, sensitive and non-invasive markers of drug-induced cardiac changes in the dog


CARDIAC MARKERS NT-proBrain Natriuretic Peptide Serum ELISA Atrial Natriuretic Peptide Serum ELISA Cardiac Troponine I Serum MSD or Centaur CP

Immunoassay Cardiac Troponin T Serum MSD Fatty acid binding protein 3 Serum MSD Myosin light chain 3 Serum MSD CK-MB Serum ELISA AST, myoglobin, aldolase, CK and LDH isoemzymes Serum Clinical Chemistry

Analyzer HORMONES Prolactin Serum Immulite 2000

Immunoassay Growth hormone Serum Immulite 2000

Immunoassay Insulin Serum Immulite 2000

Immunoassay IGF-1 (Insulin-like growth factor 1) Serum ELISA INFLAMMATION/IMMUNITY IL-1beta, TNF-alpha, IFN-gamma, IL-2, IL-6, IL-12/23 (p40)

Plasma Luminex

IFN-gamma Splenocytes ELISpot Intracellular cytokines Whole

blood/tissues Flow Cytometry

Histamine Serum ELISA T cell activation markers (CD25,CD69, HLA-DR) Whole blood,

PBMC Flow cytometry

T/B/NK cells Immunophenotyping Whole blood Flow cytometry LIVER INJURY MARKERS Arginase 1 Arginase 1 Arginase 1 αGST αGST αGST

CARDIAC MARKERS

NT-proBrain Natriuretic Peptide

Serum

ELISA

Atrial Natriuretic Peptide

Serum

ELISA

Cardiac Troponine I

Serum

MSD or Centaur CP Immunoassay

Cardiac Troponin T

Serum

MSD

Fatty acid binding protein 3

Serum

MSD

Myosin light chain 3

Serum

MSD

CK-MB

Serum

ELISA

AST, myoglobin, aldolase, CK and LDH isoemzymes

Serum

Clinical Chemistry Analyzer

HORMONES

Prolactin

Serum

Immulite 2000 Immunoassay

Growth hormone

Serum


Insulin

Serum


IGF-1 (Insulin-like growth factor 1)

Serum

ELISA

INFLAMMATION/IMMUNITY

IL-1beta, TNF-alpha, IFN-gamma, IL-2, IL-6, IL-12/23 (p40)

Plasma

Luminex

IFN-gamma

Splenocytes

ELISpot

Intracellular cytokines

Whole blood/tissues

Flow Cytometry

Histamine

Serum

ELISA

T cell activation markers (CD25,CD69, HLA-DR)

Whole blood, PBMC

Flow cytometry

T/B/NK cells Immunophenotyping

Whole blood

Flow cytometry

LIVER INJURY MARKERS

Arginase 1

Arginase 1

Arginase 1

αGST

αGST

αGST


ONCOLOGY AlPhafetoprotein (AFP) Serum ELISA Caspase3 Tumor section Immunohistochemistry Carbohydrate antigen-19-9 (CA-19-9) Serum ELISA Carbohydrate antigen-15-3 (CA-15-3) Serum ELISA Carbohydrate antigen-125 (CA-125) Serum ELISA Carcinogenic Embryonic Antigen (CEA) Serum Chemiluminescent

Immunoassay CD31 Tumor section Immunohistochemistry CYFRA 21-1 (Cytokeratin) Serum Monoclonal based assay Galectin-3 Serum ELISA HER2 Expression Serum ELISA Ki67 Tumor section Immunohistochemistry PCNA Tumor section Immunohistochemistry p-histone-3 Tumor section Immunohistochemistry Prostate Specific Antigen (PSA) Blood/plasma Chemiluminescent

Immunoassay Putresceine TPA (Cytokeratin) Serum Monoclonal based assay TPS (Cytokeratin) Serum Monoclonal based assay TRAIL/TRAIL Rs Blood/plasma TUNEL Tumor section Immunohistochemistry VEGF, VEGF-C, VEGF-D, Tie-2, Flt-1, PIGF and bFGF Serum MSD P53 Serum MSD OXIDATIVE STRESS MARKERS GSH-GSSG ratio Muscle and liver Fluorimetric Assay Nitrotyrosine Muscleand liver ELISA Reactive Oxygen Species (ROS) Plasma, muscle

and liver Fluorimetric Assay

ONCOLOGY

AlPhafetoprotein (AFP)

Serum

ELISA

Caspase3

Tumor section

Immunohistochemistry

Carbohydrate antigen-19-9 (CA-19-9)

Serum

ELISA

Carbohydrate antigen-15-3 (CA-15-3)

Serum

ELISA

Carbohydrate antigen-125 (CA-125)

Serum

ELISA

Carcinogenic Embryonic Antigen (CEA)

Serum

Chemiluminescent Immunoassay

CD31

Tumor section


CYFRA 21-1 (Cytokeratin)

Serum

Monoclonal based assay

Galectin-3

Serum

ELISA

HER2 Expression

Serum

ELISA

Ki67

Tumor section


PCNA

Tumor section


p-histone-3

Tumor section


Prostate Specific Antigen (PSA)

Blood/plasma

Chemiluminescent Immunoassay

Putresceine

TPA (Cytokeratin)

Serum


TPS (Cytokeratin)

Serum


TRAIL/TRAIL Rs

Blood/plasma

TUNEL

Tumor section


VEGF, VEGF-C, VEGF-D, Tie-2, Flt-1, PIGF and bFGF

Serum

MSD

P53

Serum

MSD

OXIDATIVE STRESS MARKERS

GSH-GSSG ratio

Muscle and liver

Fluorimetric Assay

Nitrotyrosine

Muscleand liver

ELISA

Reactive Oxygen Species (ROS)

Plasma, muscle and liver

Fluorimetric Assay


RENAL MARKERS Albumine Serum and urine MSD Lipocalin -2 Serum and urine MSD KIM-! Serum and urine MSD Osteopontin Serum and urine MSD SEX HORMONES Progesterone Serum ELISA Testosterone Serum ELISA Dihydrotestosterone Serum ELISA STRESS MARKERS Corticosterone Serum/plasma RIA THYROID MARKERS Triiodothyronine (T3) Serum Centaur CP Immunoassay

or ELISA Thyroxine (T4) Serum Centaur CP Immunoassay

or ELISA Thyroid stimulating hormone (TSH) Serum Centaur CP Immunoassay

or ELISA or Immulite 2000 Immunoassay

RENAL MARKERS

Albumine

Serum and urine

MSD

Lipocalin -2

Serum and urine

MSD

KIM-!

Serum and urine

MSD

Osteopontin

Serum and urine

MSD

SEX HORMONES

Progesterone

Serum

ELISA

Testosterone

Serum

ELISA

Dihydrotestosterone

Serum

ELISA

STRESS MARKERS

Corticosterone

Serum/plasma

RIA

THYROID MARKERS

Triiodothyronine (T3)

Serum

Centaur CP Immunoassay or ELISA

Thyroxine (T4)

Serum

Centaur CP Immunoassay or ELISA

Thyroid stimulating hormone (TSH)

Serum

Centaur CP Immunoassay or ELISA or Immulite 2000 Immunoassay

InstrumentationTECHNOLOGIES

LuminexECL

ELISA

Flow Cytometry

ELIspot

MATRIX

Rodents blood and derivativesPrimate blood and derivativesHuman blood and derivatives

Primary cells

Gene expression

Biomarkers in drug discovery�and development Aptuit: A Truly Integrated R&D Offering BiomarkersApplications for biomarkersUse of biomarkers in support of disease hypothesis�Regulatory requirements for biomarker validationCardiac Troponin I (cTnI):Aminoterminal pro-brain natriuretic peptide (NT-proBNP):Biomarkers in a 26-week dog study Dias nummer 10Dias nummer 11Dias nummer 12Instrumentation

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Biomarkers in drug discovery and development · aptuit.com aptuit.com. Biomarkers are used in pre- clinical and clinical drug development as . indicators of likely response to drug: