Biomakers of osteoarthritis

(1)

Yves Henrotin, PhD

University of Liège

(2)

Osteoathritis: an ageing-related disease

2 0 10 20 30 40 50 60 70 15-24 25-34 35-44 45-54 55-64 65+ Age (années) P ré va le n ce (%) Hommes Femmes

A 65 ans, 30% des sujets ont une arthrose invalidante

X-Rays prevalence

(3)

Osteoarthritis an «old disease»

Local mechanical disease

Cartilage degradation Overloading

Overuse

Joint instability Malignancy

(4)

Adysregulation of chondrocyte

metabolism

MMP-3 ADAMTS4 ADAMTS5 AGG COL2 Catabolism Anabolism Catabolism Anabolism MMP-13 MMP-3 ADAMTS4 ADAMTS5 AGG COL2 Healthy _{osteoarthritis} NO PGE2 IL-6 COX2 iNOS IL-1b

(5)

OA affects the whole joint and

surrounding tissue

Muscle Atrophy ↓Strength ↓Neuromuscular control Ligament Laxity Capsule Retraction Fat pad/ Tendons Inflammation Synovial membrane Inflammation Cartilage/ Meniscus Degradation Subchondral bone Sclerosis Edema (BML)

(6)

OA diagnosis : symptoms

and standard radiography

Osteophytes

Joint space narrowing Bone sclerosis Attrition Geodes Pain Stiffness Swelling Cracks Deformity Malalingment X-ray _Symptoms

These signs and symptoms occur in the late stage of the disease

(7)

Radiographic and clinical signs are preceeded by a silent molecular phase

(D Patra & L Sandell, J Knee Surg, 2011)

Pre-OA

initiation Silentmolecular phase Pre-radiographic phase Radiographic Joint replacement Genetic

biomarkers Biochemicalbiomarkers MRIImaging X-ray imaging Joint death

Genetic

predisposition Cartilage and joint tissue metabolic changes Structural changes in bone , cartilage, and other soft tissues Structural changes in bone, JSN and pain End-stage disease

(8)

Drug discovery is protracted, risky and

costly

Nothing new to offer at the

patients and the OA research community

(9)

Clinical trials end-point



Symptoms modification (3 to 6 months)

Pain

Physical function

Patient global assessment



Structure modification (1 to 3 years)

Imaging outcomes

(10)

The main limitations of JSN

 Indirect measure of the alteration in

articular cartilage.

 Fails to measure a dynamic process

 Confounded by the presence of

meniscal lesions and extrusion.

 Changes overtime are small, and occur

in only a subset (progressors) of patients.

 Poorly reproducible (full extension).

 Poorly correlated with joint function and

(11)

Why do we need of biological

markers?



To enrich our understanding of OA

pathogenesis



To detect early OA



To discriminate progressor and non

progressor



To monitor progression of OA and

efficacy of treatment



To surrogate clinical end-point



To decrease the length and cost of

(12)

Definition - Classification

Genomic/proteomic substances Imaging, Questionnaire, VAS RNA, DNA Metabolites Proteins

X-ray MRI Ultrasound

Fragments Peptides

Soluble or « wet » biomarkers « Dry » biomarkers

A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or

pharmacologic responses to a therapeutic intervention. »

Biomarkers Definitions Working Group I. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther 2001; 69: 89-95.

(13)

OA Biomarker candidates

lymphatics Blood ~5 liters urines kidney Bone cartilage Synovium bone sPINP uCTX-I Cartilage sColl2-1 sFibulin3 sCOMP sYKL40 sC2C uCTX-II PIIANP CPII

Synovium & inflammation sColl2-1NO2 sPIIINP sHA sCRP ultrasensible Metalloproteases sMMP1(collagenase) sMMP3 (stromelysin Cytokines, MPO Agrec-1

(14)

The long and winding road…

Discovery Assay dev Exploration & proof-of-concept Clinical qualification CE kit production Assay validation Regulatory & market adoption Clinical surrogacy Companio n/diagnosti c tool

(15)

Process of soluble biomarkers

development

SURROGATE Qualification Validation Discovery Brain/OMICS technics Analytical performance of assay Assessing clinical value

(16)

(17)

 The most abundant protein in cartilage

 Relatively specific of hyaline cartilage

 Makes up only 1% of all collagens

 Collagen breakdown is a critical event

in the pathology of osteoarthritis

(18)

Post-translational modifications

Post-Translational

Modifications _{Modified Protein}

Modified Protein Modified Protein Modified Protein Modified Protein Modified Protein Modified Protein Altered Physiological Function Disease Protein Aging Comorbidities i.e diabetes Inflammation

i.e oxidative stress

 Phosphorylation  Methionine Oxidation  Deamidation*  Glycosylation  Ubiquitination  Nitration*  Chlorination  …

(19)

Coll2-1NO2: a joint inflammation

related biomarkers

Deberg et al. O&C 2008

Coll2-1 HRGYPGLDG NH2 Coll2-1NO₂ HRGY(NO2)PGLDG NO + O₂. ONOO-+

HEALTHY CARTILAGE DAMAGED CARTILAGE

COLL2-1NO₂

FIBRILLATION ZONE CELL CLUSTER ZONE

Cleavage site of MMP-1, MMP-8 and MMP-13 of type II Collagen molecule

(20)

20 CONT ROL OA RA 0 100 200 300 400 500 C o ll 2 -1 c o n c e n tr a ti o n , nM

COLL 2-1 IN OA AND RA

Serum levels

*** * CONT ROL OA RA 0 100 200 300 400 500 C o ll 2 -1 c o n c e n tr a ti o n , nM

Deberg M et al. New serum biochemical markers (Coll2-1 and Coll2-1NO2) for studying oxidative-related

(21)

CONT ROL OA RA 0.00 0.25 0.50 0.75 1.00 C o ll 2 -1 N O 2 c o n c e n tr a ti o n , n M

Diagnosis:

COLL 2-1NO

₂

discriminates

OA and RA patients

***

* ***

(22)

22 CONTROL OA RA 0.00 0.25 0.50 0.75 1.5 2.0 C o ll 2 -1 N O2 /C o ll 2 -1 , %

COLL 2-1 NO

₂

/ COLL 2-1 RATIO

Serum levels

* _**

(23)

Página 8 de 32

Blanco FJ et al. Nature Rheumatology Review, 2010

Diagnostic: Coll2-1NO2 discriminates Mitochondrial Haplogroups

Haplogroups J

Lower probability to develop OA Lower X-ray OA severity

(24)

MM P-1 (x1 0) MM P-3 (x1 0) MM P-1 3 (x1 00) Col l2-1 Col l2-1 NO 2 (x1 000) Col l2 ra tio (x1 000) MPO C2C CPI I C2C :CPI I (x1 000) Cat hepsi n K (x10) HA YKL-4 0 CO MP (1/1 0) Haplogroup J Haplogroup H * * * * * * MM P-1 (x1 0) MM P-3 (x1 0) MM P-1 3 (x1 00) Col l2-1 Col l2-1 NO 2 (x1 000) Col l2 ra tio (x1 000) MPO C2C CPI I C2C :CPI I (x1 000) Cat hepsi n K (x10) HA YKL-4 0 CO MP (1/1 0) Haplogroup J Haplogroup H * * * * * * Figure 1 Rego-Perez I. et al ARD, 2010 *

Diagnosis: OA patients with haplogroup J have lower levels of Coll2-1NO2

Coll2 -1 Coll2 -1 N O2

(25)

Proteomic analysis: classical workflow of protein identification Spot extraction and in-gel digestion Cy3 Cy5

Cy3 + Cy5 _Mass

Spectrometry

With/Without

Liquid chromatography Separation

(26)

Increased (9)

B-actin

a1-microglobulin Fibulin-3 fragments Apoptosis inducing factor-2

Decreased (9)

Serpinsb1 et b3

Mannan binding lectin serum proteases-2 Kinnogen 1 Spot extraction and in-gel digestion Cy3 Cy5 Cy3 +Cy5

Urinary proteome

Henrotin et al. Arthritis Rheum 2012

OA/N > 1.5

(27)

Fib3-1: TCQDINECETTNECR

Fib3-2: CVCPVSNAMCR

Immunization of rabbits Antiserum production

Specific immunoassays of Fib3-1 and Fib3-2 development and

validation in human serum

Fib 3-2:ROC curve

0 ₂₀ ₄₀ ₆₀ ₈₀ 100 0 20 40 60 80 100 AUC = 0.846 100 - Specificity (%) S e n s it iv it y ( % )

Fib3-1: ROC curve

0 ₂₀ ₄₀ ₆₀ ₈₀ 100 0 20 40 60 80 100 AUC = 0.759 100% - Specificity% S e n s it iv it y ( % ) Sensibility Specificity Fib3-1 (cut-off: 71.1 pM) 78.5% 68.4% Fib3-2 (cut-off: 163.7 pM) 75.0% 86.4% CT OA 0 50 100 150 200 250 300 350 *** Fi b3 -2 (p M ) CT OA 0 25 50 75 100 125 150 *** Fi b3 -1 (p M )

Fibulin-3 fragments (Fib3-1 and Fib3-2): potential diagnostic biomarkers

(28)

BIOVISCO study: Study design

Open-label, observational prospective study

D-15

D1 _D7 D14

D30 D60 D90

HylanGF-20

sHA, 1, sColl2-1NO2, s C2C, sCOMP,

sCS-846, sCPII, CTX-II, Fib3-1, Fib3-2

Henrotin Y et al. Journal of Orthopaedic Research

(29)

BIOVISCO study

An open label observational prospective study

Conrozier et al, J Orthp Res, 2012; Henrotin et al, J Orthp Res,2013.

D1

(after the last injection)

90 days

(after the last injection)

p-Value D1 vs D90 sColl2-1 (nM) 140.34(882.44-285.32) 128.41 (85.6-241.34) 0.05* sColl2-1NO2 (nM) 0.400 (0.050-1.010) 0.370 (0.14-0.870) 0.025* uCTX-II (ng/nmolcreat) 392.7 (90.0-816.4) 306.0 (90-1123.9) 0.02* sPIICP (ng/ml) 817.9 (131.4-1848.6) 874.8.3 (326.4-1435.0) 0.41 sC2C (ng/ml) 223.6 (99.4-329) 209.5 (135.9-291.7) 0.11 sCOMP (U/L) 10.9 (6.0-20.2) 10.5 (6.0-20.0) 0.82 sCS846 (ng/ml) 99.8 (45.9-172.3) 102.2 (53.0-190) 0.38 sHA (ng/ml) 34.1 (15.4-211) 33.3 (9.5-230.1) 0.38

 45 patients with unilateral symptomatic tibiofemoral and/or patellofemoral OA

 3-weekly intraarticular injection of hyalan G20 (Synvisc®)  Follow-up D1, D30 and D90 after the last injection

(30)

(31)

Critical needs!

 Drugs that can impact the disease

progression

 Large cohorts representative of the general

population and designed for the qualification of the biomarkers

 A sensitive imaging gold standard detecting

early structural changing in joint tissues

 The inclusion of biomarkers as secondary

(32)

 New technologies adapted to a personalized management

 Combination of biomarkers in multiplex tests

 Agregate score including clinical, imaging

and biological parameters

 Companion biomarkers for drugs

(Theranostic)

(33)

Thank you for your attention !

Team

International collaborations:

F Blanco (La coruna, Spain) T Conrozier (CHU Lyon, France) V Kraus (Duke University, USA) L Punzi (University of Padova, Italy)

A Mobasheri (University of Notttingham, UK) J Monfort (Hospital del mare (Spain)

P Richette (Lariboisiere, France) J Runhaar (Erasmus MC, Rotterdam)