Metabolomics & Pharmacometabonomics

FATIMATUZZAHRA’ ABD AZIZ, PhD

Outline

◎What is metabolomics?
◎Why metabolomics?
◎Metabolomics platforms
◎Metabolomics work flow
◎Application of metabolomics
◎Challenges in metabolomics
What is metabolomics

◎Metabolites: bio-active small molecules

involved in biochemical network.
◎Metabolome: represents the collection of
all metabolites in a given biological sample.
◎Metabolomics is the systematic
identification and quantitation of the
metabolites.
Metabolomics & Pharmacometabonomics

• Metabolomics:
• A descriptive study of all the
metabolites (metabolome) in a
cellular system
• Metabonomics:
•Interactions between metabolic
property of an organism and the
biological and genetic changes
• Metabolomics approach not only may
able to diagnose diseases but also
phenotype them

•Figure Source: American Society for Pharmacotherapy & Therapeutics

http://www.ascpt.org/My-ASCPT/Special-Interest-Groups/Pharmacometabolomics-Interest-Group
•TAClayton, JC Lindon, O Cloarec, H Antti, C Charuel, G Hanton, J Provost, J Net, D Baker, RJ Walley, JR Everett, JK Nicholson.
Pharmaco-metabonomic phenotyping and personalized drug treatment. Nature April 2006; 440(20)
• Ian D. Wilson1 . Drugs, bugs, and personalized medicine: Pharmacometabonomics enters the ring. PNAS August 2009; 106(34):14187–
14188
 Pharmacometabonomics
• Pharmacometabonomics
•The prediction of a drug or xenobiotic intervention outcome
in an individual based on a mathematical model of Pre-
intervention metabolite signatures
•i.e. Prediction of:
•Efficacy
•Toxicity

• One of the major factors influencing patient’s response to any

medication is drug Pharmacokinetic (ADME).

•Differences in the balance of Pharmacokinetic leading to

detoxification vs. toxicity are the difference between a treatment
being Safe and Effective or causing an adverse drug reaction

•TAClayton, JC Lindon, O Cloarec, H Antti, C Charuel, G Hanton, J Provost, J Net, D Baker, RJ Walley, JR Everett, JK Nicholson. Pharmaco-
metabonomic phenotyping and personalized drug treatment. Nature April 2006; 440(20)
• Ian D. Wilson1 . Drugs, bugs, and personalized medicine: Pharmacometabonomics enters the ring. PNAS August 2009; 106(34):14187–14188
 Pharmacometabonomics
• Pharmacometabonomic approach can amounts to:

•Response-targeted Pre-dose phenotyping, might provide

the basis of a future population-screening tool for selecting
individuals according to their suitability for treatment with
particular drugs, drug classes or drug doses

•Potentially avoiding Adverse drug reactions by Pre-dose

phenotyping and drugs and dose levels could be targeted
more effectively according to the metabolic and other
characteristics of each individual

•TAClayton, JC Lindon, O Cloarec, H Antti, C Charuel, G Hanton, J Provost, J Net, D Baker, RJ Walley, JR Everett, JK Nicholson. Pharmaco-
metabonomic phenotyping and personalized drug treatment. Nature April 2006; 440(20)
• Ian D. Wilson1 . Drugs, bugs, and personalized medicine: Pharmacometabonomics enters the ring. PNAS August 2009; 106(34):14187–14188
 Pharmacometabonomics

Urine – Pre-dose Post-dose

99 healthy male NMR analysis Acetaminophen 1g NMR analysis
volunteers

• High predose urinary levels of p-cresol sulfate had low postdose

urinary ratios of acetaminophen sulfate to acetaminophen glucuronide

• p-cresol known to be produced from protein-derived tyrosine in

reactions involving gut bacteria

• In individuals with high bacterially mediated p-cresol generation,

competitive O-sulfonation of p-cresol reduces the effective systemic
capacity to sulfonate acetaminophen

• Gut bacteria might influence both drug-induced responses and disease

development
•TA Clayton, D Baker, JC Lindon, JR Everettc, and JK Nicholson. Pharmacometabonomic identification of a significant host-
microbiome metabolic interaction affecting human drug metabolism. PNAS August 2009; 106(34):14728–14733
 History
Metabolomic
Burt, T., & Nandal, S. (2016). Pharmacometabolomics in Early-Phase Clinical Development. Clinical and translational science, 9, 128-138.
Outline

◎What is metabolomics?
◎Why metabolomics?
◎Metabolomics platforms
◎Metabolomics work flow
◎Application of metabolomics
◎Challenges in metabolomics
Why metabolomics?

◎End point of cellular regulation

◎Dynamic & time sensitive
◎Sensitivity
◎High throughput
◎Suitable platform for system biology
◎Integrated studies:
◉metabolomics + GWAS
◉Metabolomics + transcriptomics
◎Gate to personalised medicine
Advantages of Metabolomics approach

•Affected by both Genes and Environment

• Closer to Phenotype

• Simple and Non-invasive

• Relatively Ease of Analysis

• Potential to identify Novel biochemical pathways

Outline

◎What is metabolomics?
◎Why metabolomics?
◎Metabolomics platforms
◎Metabolomics work flow
◎Application of metabolomics
◎Challenges in metabolomics
Comparison of NMR vs MS for Metabonomics
Analytical Considerations NMR MS
Sensitivity
Reproducibility – w/in lab
Reproducibility – across labs
Quantitation
Sample Prep Requirements
Sample Analysis Automation
Versatility
Selectivity
Non-selectivity

Taken from D.G. Robertson, Toxicological Sciences, 85, 809, 2005

Outline

◎What is metabolomics?
◎Why metabolomics?
◎Metabolomics platforms
◎Metabolomics work flow
◎Application of metabolomics
◎Challenges in metabolomics
Metabolomics work flow

• Sample preparation
Analytical • Data acquisition
experiment

• Data pre processing

• Data processing
Data
analysis • Model validation

• Screening of marker metabolites

• Metabolites Identification
Biomarker
discovery • Confirmation of biomarker
 Profiling (Untargeted)

Data Reduction
25
PC2
20
15
10 ANIT
Data Collection 5
0
-5
-10
-15
Control
-20 PAP PC1
-25
-30 -20 -10 0 10
Sample Prep

Metabolite Identification
 Quantitative (Targeted)

Sample Prep
25
PC2
20
15
Biological Interpretation 10 ANIT
5
0
-5
-10
-15
Control
-20 PAP PC1
-25
-30 -20 -10 0 10

Data Reduction

Metabolite Identification & Quantification

Analytical experiment

blood

Feces urine

Type of
samples
Prostate
saliva
secretion

Bronchial
Ascitic fluid
washes
Analytical experiment
◎Sample preparation : collection & handling

•Require careful handling, special requirements for diet,

physical activity, current drug use.

• due to high susceptibility of metabolic pathways to

exogenous environment, maintaing low temperature
and consistent sample extraction is essential.

•Preservative: sodium azide

•For biofluids, standard sample volume : ~ 1mL

 Human/animal factors:
Gender, age, genetic, hormone differences
Environmental factors:
Dietary inputs, stress, drug use, disease,
allostatic load, pollutant exposure

Sample Personnel handling, centrifuge speed &

Collection time, type of tubes

Type of preservative and standard

Sample
use, sample preparation technique,
Storage
volume, pH

Temperature,
duration of Sample
storage Preparation
Instrumental
variation Postacquisition,
processing,
normalization
Analysis: procedure, scaling
NMR/MS

Data
Processing
Data Analysis
Pharmacometabonomic Involves Many Samples

Looking for subtle differences in many spectra requires some data

reduction/simplification
Topspin 3.5 pl 5 (Bruker® BioSpin, Germany)
AMIX 3.0 (Bruker BioSpin, Germany)
The Need for Multivariate Statistical Analysis
◎There are 10s, 100s or even 1000s of samples
◎Each spectrum (MS or NMR) can contain hundreds or
thousands of signals
◎Metabolic perturbations may be very subtle and effect a
small number of the observed metabolites
◎How do we find the needle metabolites in the biofluid
haystack
SIMCA-P+ 14.0 (Umetrics, Sweden)
Outline

◎What is metabolomics?
◎Why metabolomics?
◎Metabolomics platforms
◎Metabolomics work flow
◎Application of metabolomics
◎Challenges in metabolomics
Application of metabolomics

◎Personalised medicine
◎Nutrigenomic
◎Agricultural
◎Functional genomics
◎Toxicology
◎Biomarker discovery
Metabolomics study in USM

Warfarin

Aspirin alcohol

Clopidogrel Gentamicin
Metabolomics Disease Diagnosis
Metabolic fingerprint
Diagnosis of Alcohol Dependence

Hamza Mohamed Amin Mostafa, Arwa Mohamed Amin Mostafa, Nor Hayati Arif, Teh Chin Hoe, Vikneswaran
a/l Murugaiyah, & Ibrahim, B. (2014). METABOLOMIC ANALYSIS OF BLOOD AND URINE TO IDENTIFY
ALCOHOL-DEPENDENCE BIOMARKERS. The Medical Journal of Penang Hospital(Supplement 2014).
(Poster Abstract)
 Metabolomics
Metabolomics Disease Diagnosis
Disease Diagnosis
Scatter Plot
Diagnosis of Alcohol Dependence
The OPLS-DA model of 3 groups each12 subjects of AD, social drinkers
and healthy control groups was done.

Hamza Mohamed Amin Mostafa, Arwa Mohamed Amin Mostafa, Nor Hayati Arif, Teh Chin Hoe, Vikneswaran a/l
Murugaiyah, & Ibrahim, B. (2014). METABOLOMIC ANALYSIS OF BLOOD AND URINE TO IDENTIFY ALCOHOL-
DEPENDENCE BIOMARKERS. The Medical Journal of Penang Hospital(Supplement 2014). (Poster Abstract)
 Metabolomics
Disease
Metabolomics Diagnosis
Disease Diagnosis
Accuracy, Sensitivity & Specificity
Diagnosis of Alcohol Dependence
OPLS-DA model

Type of Analysis Specificity Sensitivity Accuracy

OPLS-DA in 81.82% 90.91% 57.58%

(AD vs Social drinkers
vs Healthy control)
OPLS-DA in 90.91% 90.91% 90.91%
(AD vs Social
drinkers+Healthy
control)

Hamza Mohamed Amin Mostafa, Arwa Mohamed Amin Mostafa, Nor Hayati Arif, Teh Chin Hoe, Vikneswaran a/l
Murugaiyah, & Ibrahim, B. (2014). METABOLOMIC ANALYSIS OF BLOOD AND URINE TO IDENTIFY ALCOHOL-
DEPENDENCE BIOMARKERS. The Medical Journal of Penang Hospital(Supplement 2014). (Poster Abstract)
Prediction of drug toxicity: Gentamicin

1. Trimethylamine-N-oxide
2. Methanol
3. Deoxyinosine
4. Xylose
5. 2-phosphoglycerate
6. Glucose-6 –phosphate
7. lactate
Pre-dose serum
Fingerprinting models Identification models

Model

Summary of all fingerprinting model of pre-dose serum for number of pre-dose metabolites with VIP
more than 1, score plot model value, accuracy and permutation values.
Prediction of drug toxicity: Gentamicin

Pre-urine metabolites
1.4-pyridoxic acid
2.2-oxoglutarate
3.Citrate
4.Betaine
5.Hippuric acid
6.allantoins
7.urea
Pre-dose urine
Fingerprinting models Identification models

Model

Summary of all fingerprinting model of pre-dose serum for number of pre-dose metabolites with VIP
more than 1, score plot model value, accuracy and permutation values.
Outline

◎What is metabolomics?
◎Why metabolomics?
◎Metabolomics platforms
◎Metabolomics work flow
◎Application of metabolomics
◎Challenges in metabolomics
Challenges
 Challenges
1) Patient
• Demand?
• Safety
• Privacy- data shared
• Peace of mind?
• Employers discrimination?
• Ethnic discrimination?

•Personalized Medicine Coalition www.personalizedmedicinecoalition.org/sciencepolicy/personalmed-101_overview.php.

•Lester. DS. Will Personalized Medicine Help in 'Transforming' the Business of Healthcare? Personalized Medicine. 2009;6(5):555-565.
 Challenges
2) Clinician
• New knowledge
• Cost

3) Industries
• Pharmaceuticals, diagnostics, biotechnology
• New business model – reduced market size,
profit?
• Disease prevention doesn’t pay?
•Personalized Medicine Coalition www.personalizedmedicinecoalition.org/sciencepolicy/personalmed-101_overview.php.
•Lester. DS. Will Personalized Medicine Help in 'Transforming' the Business of Healthcare? Personalized Medicine. 2009;6(5):555-565.
 Challenges
4) Regulations
• Patient safety and privacy,
• High quality and clinical utility
• Genetic Information Nondiscrimination Act
of 2005 (GINA) – In USA

5) Insurance
• Genetically susceptible patient –
coverage?
• Forced Patient to go for genetic testing?
Thank you