ghrelin immunoassay & translational medicine

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Ghrelin Immunoassay & Translational Medicine

Stéphane Denépoux, PhD 11th Annual Biomarkers Congress, Manchester UK 25th & 26th of February, 2016

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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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LC-MS/MS Quantification : w/ Pr Tetsuya Terasaki

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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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Dealing with species specific issues





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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

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Dealing with Species Specific Issues




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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

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Dealing with species specific issues


Anorexia & Renutrition Screening Strategies

Contribution to the Constitutional Anorexia Syndrome Description


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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

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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

ghrelin immunoassay & translational medicine

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