E. Demoncheaux, PhD

From 10 thousand to 1: How Science Helped Find Just The Right Drug Target

In Exhaled Breath

eBiomarkers

Need Biomarkers for…

Patient Identification / Outcome Predictors

Therapeutic Category / Mechanism of Action

New Indications

Definition of the Measurements

• A Biomarker is a measurement purported to reflect a disease status or the mechanism of action of a drug

• A Surrogate is a measurement that reflects the activity of the fundamental mechanism of the disease, i.e. a substitute for the disease

• An Endpoint is a measurement that is recognized by the Regulatory Authorities as a specific and final expression of the disease in patients

Biomarker MeasurementsCharacterisation of Biological Systems

• Validation of assays in human samples – Blood– Urine– Tissues

• Development of sampling from the lungs– Sputum– Condensates

– Exhaled breath

From Cells to Human Body

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Analysis of Exhaled Gases

• Non-invasive • Commercial nutritional monitoring (information

on digestive processing)• Monitoring of exposure • Monitoring of treatment

– Compliance– Pharmacogenomics

• Monitoring of disease

From Hippocrates to Dr McCoy

William Harvey (1578-1657)

Molecular Profile of the Breath

• Product of composition inspiratory air and volatile molecules in blood

• Bulk matrix is mixture of:– Nitrogen– Oxygen– Carbon dioxide– Water vapour– Inert gases

• Remainder of breath (<0.000001%) is mixture of thousands of different compounds

Breath and Disease

• Water vapour as non-invasive monitor of mortality

• Distinctive breath odours for:– Uncontrolled diabetes– Liver disease– Renal failure

– Dental disease

– Can be confounded by diet (garlic, onions, fish, ethanol, etc.)

• Conducting airways– Trachea– Bronchi

– Bronchioles

• Respiratory airways– Respiratory bronchioles– Alveolar ducts

– Alveoli

Anatomy

• Dichotomous branching• ~23 generations

Branching Structure of Airways

• Alveoli• Alveolar ducts• Pores of Kohn

• Liquid lining layer

Pulmonary Microstructure

• Capillaries surround alveoli

• Sheet flow of blood

Pulmonary Microcirculation

Collection of Exhaled Breath and Breath Condensate

From Kharitonov et al. 2001

From Tsoukias et al. 2001

Biomarker Assays

Analysis of the content of exhaled breath condensate (nitrates, chemokines, cytokines, etc.)

Biomarkers Discovery

• Proteomics

• Protein chip

• mRNA profiling for secreted proteins

• ELISAs, RIAs, Westerns

• MS, LC/MS

• Imaging techniques (MRI, MALDI)

• Chemiluminescence

Nitric Oxide

• 1859 Amyl Nitrate discovered• 1867 Brunton: Amyl Nitrate

lowers BP

• 1879 Glycerine Trinitrate• 1900+ Nitrites and Nitrates• 1980 Furchgott: EDRF (rabbit

endothelium)• 1988 EDRF is NO (identity

confirmed by Pr Moncada using chemiluminescence)

Nitric Oxide: Biosynthetic Pathway

• Catalytic oxidation of L-arginine• Stoichiometric synthesis of NO and L-citrulline

• NO is poorly soluble• Partition mostly to the gas phase and is excreted in the breath

Nitric Oxide in the Lung

How Do We Measure Nitric Oxide in the breath ?

• Gas sample drawn in a chemiluminescent analyser at a fixed flow rate

• Reaction of ozone with NO under high vacuum = chemiluminescent signal

• Limit of detection of 1 ppb (40 x 10-12 mol/l at 20ºC)

Exhaled NO Levels Are Decreased in Infants with Cystic Fibrosis

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Cystic Fibrosis Controls(n = 5) (n = 11)

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Elphick et al. 2001

Increased NO Decreased NO

ExhaledAsthma Pulmonary HypertensionAllergen challenge

Cystic fibrosis

Upper respiratory tract infectionCOPD

Lower respiratory tract infectionBronchiectasisCirrhotic patientsCirrhotic patients withhepatopulmonary syndrome

NasalAllergic rhinitis Kartagener's syndrome

Cystic FibrosisPrimary ciliary dyskinesia

Diseases Affecting Exhaled and Nasal Nitric Oxide

Pulmonary Hypertension

• PH characterised by a thickening of the wall of small pulmonary arteries

• Pulmonary blood vessels are involved in all forms of the disorder such as – Pulmonary arterial hypertension – Hypoxic pulmonary hypertension (PH

and Chronic Obstructive Pulmonary Disease, COPD)

© Merck Pharmaceutical, Inc., 2000

Chronic Pulmonary Hypertension

• Expression of eNOS is decreased in some pulmonary vessels (Mason et al. 1998)

• Exhaled nitric oxide is decreased (Cremona et al. 1994)

• Whole body production of nitric oxide is decreased in primary pulmonary hypertension (Demoncheaux et al. 2005)

Nitric Oxide Is an Important Constituent of Cigarette Smoke

• Countries with high NO yield cigarettes have low incidence of COPD, Lung Cancer and Ischaemic heart disease

• Nitric oxide yields of cigarette depends on its nitrate content

• Wide variation in NO dose per puff

Borland and Higenbottam 1987,1988,1989

Inhaled Nitric Oxide: Clinical Applications

• Pulmonary Hypertension

• Hypoxic respiratory failure (FDA approval in 1999)

Selective pulmonary vasodilatation with inhaled nitric oxide in PPH

Pepke-Zabaet al. 1991

From BiomarkersDiscovery (eNO) to Drug Discovery (iNO)

From Hippocrates to Pr Higenbottam

“Measure all that is measurable and make those things measurable which have hitherto not been measured”

Galileo Galilei

From Hippocrates to Dr McCoy