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1 - 04/04/2016
Ghrelin Immunoassay & Translational Medicine
Stéphane Denépoux, PhD 11th Annual Biomarkers Congress, Manchester UK 25th & 26th of February, 2016
2 - 04/04/2016
Whatever your molecule…
Bertin Pharma proposes a unique BioAnalysis integrated platform throughout the drug development process thanks to a long track-record in assay methods completed in compliant environments.
Biomarker assay application > soluble protein > membrane protein > cell surface antigens
Drug analysis expertise NCEs peptide > Protein > Therapeutic drug antibodies
Immunogenicity assessment (ADA) > Screening test > Confirmatory assay > Neutralising assay
Bertin Pharma handles chemical entities, biologics, peptides and proteins thanks its highly experimented staff
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Sample preparation expertise > Appropriate sampling > Macromolecules Stabilization in Biofluids > Validated extraction methods (incl. immunoaffinity)
Ligand binding assays & flow cytometry > Singulex or Multiplex > Various detection system
LC-MS/MS MRM powered by MS2Plex > Highly specific > High sensitivity (fmol/μg of total protein) > Simultaneous quantification > state-of-the art validated assays
Bertin Pharma designs in collaboration with you the best scenario to assay your active compounds
Our customized solutions
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Whatever your Development Stage
Drug discovery & translational medecine
Non-clinical assay
Clinical trials & central lab operations
Bertin Pharma is your outsourcing partner whatever the phase you are…
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Our main Features...
A solid track record > sustainable background in set-up & validation methods for NCEs, biologics & biomarkers > recognized biomarker kit trademarks
Multi-disciplinary teams supported by senior management > pharmacological or toxicological team > expert in various analytical methods: Biochemical methods, Hyphenated methods, etc
Up-to-date means, design & quality > fit-to-purpose development assay > update & validation of biomarker assays > two GLP site facilities
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Bertin Pharma Capacities in Translational Medicine
LC-MS/MS Quantification of DMPK & Oncology Biomarkers
How Biomarkers evolve with Drug Development Phases
Presentation of The Ghrelin ImmunoAssay Case Study
Bertin Pharma : Biomarkers & Translational Medicine
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human CYP, UGT & Drug Transporters MDR-1 BCRP BSEP MRP1, MRP2, MRP3 NTCP OCT1, OCT2
Multiplex assays Hepato OATP1B1, OATP1B3, MDR-1, BCRP Kidney OCT2, OAT1, OAT3, MDR-1 BBB MDR-1, BCRP
Oncology Related Biomarkers FAK2, FGFR4, FGFR2, MET, CD33, ERBB3, EGFR, ERBB2,
IGFR, FLT3, VEGFR1, ERBB4, CD37, FGFR1, RET, PDGFRβ, FAK1, C-KIT, PDGFRα, FGRFR3, VEGFR2
Others human, mouse or monkey proteins assays to be developed upon request
DMPK & Oncology Biomarkers Currently Available
OAT1, OAT2, OAT3 OATP1B1, OATP1B3 CYP 1A1,1A2, 3A4, 3A5, 2B6, CYP 2C8, 2C9, 2C19, 2D6, 2E1 UGT 1A1, 1A3, 1A6, 1A9, 2B7, 2B15
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ADME applications: in vitro /ex vivo model validation
in vitro Model Validation
in vitro / in vivo Correlations
Induction / Inhibition Monitoring
Clinical Applications & PBPK
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Drug Development Phases ask for different Biomarkers
Screening Animal Studies First in Human Studies Larger Clinical Trials
Biomarkers need to be adapted to the Drug Development Stages
The Ghrelin Case
Biomarkers & Translational Medicine Issues
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Resolving unexpected ImmunoAssay challenges
Dealing with Species Specific issues
GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor screening
Anorexia & renutrition compound screening strategies
Contribution to constitutional anorexia syndrome description
Ghrelin Immunoassay & Translational medicine
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28 Amino Acid Hormone discovered at the same time by Kojima in Japan & Tomasetto in France
Produced by Endocrine Stomac Cells & by Hypothalamus
Present as an Acylated and an UnAcylated form Ghrelin O-acyltransferase (GOAT) is responsible for
Acylation. The Acylated form stimulates GH-Secretion
Receptors
Actylated Ghrelin : Increases apetite & adipose tissue
generation Stimulates neuroendocrine (GH secretion),
metabolic (insulin inhibition) & extra-endocrine pathways.
Ghrelin : the GH-releasing Hormone
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Resolving unexpected ImmunoAssay challenges
Dealing with species specific issues
GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor screening
Anorexia & renutrition compound screening strategies
Contribution to constitutional anorexia syndrome description
Ghrelin Immunoassay & Translational medicine
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Specific Quantitation of Acylated and UnAcylated Ghrelin is essential to :
Understand its Biological Fonctions Understanding of its implication in Nutrition Disorders (Cachexy, Mental Anorexia, Obesity)
> & for Growth Hormone Secretion and Mobility Disorders To support Ghrelin analogs Screenings such as GOAT inhibitors
Analytical Method Setting up for:
Screening on Cellular Models Screening on Animal Models
> (rodents & non rodents) Clinical Trials Support
Ghrelin: 2 forms
Blood basal level Variability between individuals Depending on physiological conditions:
> for instance feeding behaviour, … Ghrelin: Assessment of blood level
Concentration measured blood sample
Setting-up the Ghrelin ImmunoAssay Kit: Allowing Ghrelin to become a nutrition research biomarker
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Setting up of the Ghrelin ImmunoAssay
Peptide are unstable in biological matrix like blood, plasma or tissue
Other By-product residues (enzymatic, chemical or physical degradation)
Preproghrelin (117 AA)
Desacyl ghrelin (28 AA)
BuChEAPT-1
Blood
Stomach
Acyl ghrelin (28 AA+ acylation)
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Ghrelin assay: How
Physical or chemical treatment Without protection, half time of ghrelin is around few minutes
Storage at very low temperature (-80°C for instance) Use Protease inhibitor
Wide range of products today available More or less specific Some of them are toxic
Protease inhibitor : Sample treatment have to be compatible with the ImmunoAssay:
No interference with assay (to be checked for each solution)
Preproghrelin (117 AA)
Desacyl ghrelin (28 AA)
BuChEAPT-1
Blood
Stomach
Acyl ghrelin (28 AA+ acylation)
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Ghrelin assay: one step format
Sandwich immunoassay in one step for the immunological reaction:
Peptide (AG or UAG)
Immunological Reaction Wash
Detection Ellman reagent
Detection antibody
Capture antibody
One Step Ghrelin EIA kit: One step assay (immunological reaction in one step) Use of PHMB (p-Hydroxymercuribenzoate) as Protease Inhibitor Evaluation of Protease inhibition activity in vitro in plasma Validation for rat/mouse, and human plasma samples
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Ghrelin Easy Sampling ImmunoAssays
Easy Sampling Ghrelin ImmunoAssay (AG and UAG) EIA kits validated: Human, Rat, Mouse, Dog, Pig (undervalidation)
Validation items: Calibration curve: accuracy, CV Intra-, inter-run precision and accuracy: 5 levels of QC including LLOQ Parallelism, Specificity, Dilution linearity Stability: short term, freeze and thaw stability, long term stability
Peptide (AG or UAG)
First Immunological
Reaction Wash
Detection Ellman reagent
Detection antibody
Capture antibody
Wash
Second
Immunological Reaction
First Step Second Step Remove AEBSF
19 - 04/04/2016
Resolving unexpected ImmunoAssay challenges
GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor screening
Dealing with Species Specific Issues
Anorexia & renutrition compound screening strategies
Contribution to constitutional anorexia syndrome description
Ghrelin Immunoassay & Translational medicine
20 - 04/04/2016
Main Objectives & Obstacles Acylated Ghrelin Quantification in an in vitro Sample Miniaturization / Automation associated with high
performances with less than 1 % false positives
Primary Evaluated Parameters Choice of 384 wells trays Choice of Automated Systems Operating Conditions Set Up
Milestones
Spotting a 25% Inhibition Threshold : 188 pg/mL <1% False Positives > 10 000 Screened compounds
Ghrelin Monitoring : in early Screening Phases
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Main Objectives & Obstacles Quantification of AG and UAG in PreClinical Species (Mice,
Rats, Pigs, Dogs) Establishing Robust Quantification Methods on a very large
Dilution scale (LLOQ: 10 pg/mL to 20 pg/mL) Limiting UnAcylation during Sampling and Conservation
Primary Evaluated Parameters Operating Procedures / Sample Management Adapting Antibody Couples for each Species Optimization of Sample PreTreatment Conditions
Milestones LLOQ : pg/mL for mice/rat/dog, 10 pg/mL for minipigs Available Assays : 4 mice/rat, 2 dogs, 2 pigs
Ghrelin Monitoring : in animal Studies
Congrès Int d’endocrinol., 2014 Erasmus University, Alizé Pharma & Bertin Pharma
Peptides 32 (2011) 2274–2282 UMR 894 Inserm/Université Paris Descartes
22 - 04/04/2016
Resolving unexpected ImmunoAssay challenges
Dealing with species specific issues
GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor screening
Anorexia & Renutrition Screening Strategies
Contribution to the Constitutional Anorexia Syndrome Description
Ghrelin Immunoassay & Translational medicine
23 - 04/04/2016
Main Objectives & Obstacles Sensitivity, Sampling Procedures Availability of human Samples for Analytical Validations
Primary Evaluated Parameters
Operating Procedures / Sample Management Optimization of Sample PreTreatment Conditions Assay Validation following Guidelines
Milestones 4 ImmunAssay Available Clinical Trials sponsored by Alizé Pharma:
> Phase II in Prader-Willi Syndrome (childhood obesity) > Phase Ib in type 2 Diabetes > PreClinical Trials in Ischemia / Reperfusion injury
Ghrelin Monitoring : in Clinical Trials
Impossible d’afficher l’image.
Congrès Intl de neuroendocrinol., 2014 UMR 894 Inserm/Université Paris Descartes
Bertin Pharma
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J. Epelbaum, D. Grousselles, M.-T. Bluet-Pajot (Inserm U. 549) Ghrelin physiology & ImmunoAssay Set Up
M. Kojima (Nat Cardiovascular Center Res. Institute, Osaka)
Ghrelin co-founder & Patent owner
C. Tomasetto (CNRS/Inserm U.184/ULP/ICBMG) Ghrelin co-founder & mAb owner
Atomic Energy Research Center :
AChE as an ImmunoAssay uniquer marker for EIA
P. Vitaux & P. Brune (Bertin Pharma) ImmunoAssay Kit Production Department
Our International Scientific Network
25 - 04/04/2016
Chemical entities
Let’s Discuss your Projects on :
Biologics mAbs, Antisense & Fusion Proteins
Advanced Therapy Medicinal Products Cell, Gene & Tissue Therapies
Vaccines Medical Devices
Cosmetics Nutraceuticals
Thank you for your attention!
For additional details, we remain at your entire service
Take a chance to visit our website:
www.bertinpharma.com
11th Annual Biomarkers Congress, Manchester UK
February 25th & 26th, 2016
Stéphane Denépoux, PhD [email protected]
Tel: +33 1 39 30 60 68