Ms. Suruchi Ramkumar Sharma
Under the guidance of:
Dr. (Mrs.) Vaishali Dixit
M.E.T Institute of Pharmacy, Bandra (W)
The US FDA defines a biomarker as a
objectively measured & evaluated as an
normal biologic, pathogenic or
pharmacologic responses to therapeutic
• It can be specific cells, molecules or genes,
gene products, enzymes or hormones.
• Helps in early diagnosis, disease prevention,
drug target identification, drug response etc.
• serum LDL for cholesterol
• blood pressure for stroke
• C-reactive protein (CRP) for inflammation
Disease – related
Drug – related
Risk indicator / predictive
Indicate effectiveness of a
drug in a specific patient
Diagnostic & prognostic
How the patient’s body will
Biomarkers validated by genetic and
molecular biology methods can be classified
into three types:
Type 0 - Natural history markers
Type 1 - Drug activity markers
Type 2 - Surrogate markers
ASSAY TECHNIQUES 11
In Toxicology & Clinical trials -
• In vivo monitoring
• Early detection of metabolic changes
• Detection of organ-specific effects
• Establishment of “NO EFFECT” level
• Determination of toxic mechanism
Important issues to remember:
• Cell types differ in susceptibility to toxic agents
• One organ – many cell types
• Cellular injury vs. organ function impairment
• Oxygen concentration gradients
• Metabolizing enzymes (e.g., Cytochrome P450)
Localization of damage:
Centrilobular (zone 3)
• Most hepatotoxicants (CCl4, APAP)
• Less oxygen + high P450 conc.
Periportal (zone 1)
• Phosphorus, aflatoxin, allyl alcohol
• High oxygen + highest dose at site
Midzonal (zone 2) –
• Massive necrosis - iproniazid, MAOI
Serum Indicators Urine Indicators
Blood Urea Nitrogen
Blood Creatinine Color/turbidity
Urinary protein –
tubular (low MW) or
glomerular (high MW)
Volume Urinary glucose –
no elevation of blood
Osmolality Urinary brush border
(ALP, AST, GGT)
Develop new biomarkers that predict toxicity
in the preclinical development of NCEs early
in the drug development process and are
translatable to the clinic.
To reduce the time and cost associated with
drug discovery and approval.
Metabolic profiling methods used are based
on metabolomics and/ or metabonomics.
Metabolomics is especially important in the
early ADME/Tox stage of drug testing.
Acetaminophen (APAP) is responsible for 50%
of all drug-induced acute liver failure.
• High-throughput LC/MS-based metabolomic
assays rapidly investigate APAP and its
metabolites excretion profile in urine from
• Biomarker : Changes in the SAMe
concentrations inversely proportional with
urinary APAP–NAC concentration.
NMR-based approach to investigate toxicity
induced by Bay41-4109, an anti-hepatitis B
• Biomarker : Fatty acid metabolism disorder
and mitochondrial dysfunction.
GC/MS was used to study carbon
tetrachloride-induced acute liver injury in
• Biomarker : Elevated levels of maleate and
several fatty acids in the liver.
UPLC/MS to investigate toxicity by CCl4 and α-
• Biomarker : Changes in bile acids.
Integration of OMICS:
• Metabolomics + transcriptomics liver
samples of two mouse strains regulated
glutamate and glutamine networks
diabetes and obesity.
• Metabolomic + proteomics liver samples
of CD1 mice acute hepatotoxicity induced
by valproic acid altered glucose levels.
NMR and HPLC-TOF/MS approach for
Cyclosporin A in a rodent model.
• Biomarker : Elevated levels of urinary
glucose, acetate, trimethylamine, succinate
and reduced levels of urinary TMAO +
decreased levels of kynurenic acid,
xanthurenic acid, citric acid and riboflavin.
• Biomarker : Increased levels of glucose,
hydroxybutyrate, creatine, creatinine, TMAO
and decreased concentration of glutathione.
Gentamicin in SD rats,
• Biomarker : Increased urinary level of
glucose and decreased level of TMAO by NMR
with decreases in xanthurenic and kynurenic
acids and changes in sulfation patterns by
• Biomarker : UPLC/MS showed increase in
urinary levels of 6-hydroxymelatonin.
Cisplatin in mice,
• Biomarker : Altered urinary levels of glucose,
amino acids and Krebs cycle intermediates
that preceded changes in serum creatinine.
Chronic toxicity testing of nephrotoxicants:
gentamicin, cisplatin and tobramycin in SD
rats, non targetted analysis after 1, 5 and 28
days dosing caused Aminoaciduria (marker
of kidney damage) due to decreased renal
Used in the drug development process
• Early drug development studies
e.g. used in phase I study for establishing
doses and dosing regimen for future phase II
• Safety studies
e.g. APAP induced hepatotoxicity study
• Proof of concept studies
• Molecular profiling
A new face in diagnostics, therapeutics &
Potential to encourage innovation, improve
efficiency, save costs.
Potential to encourage innovation, improve
efficiency, save costs, and gain research
organizations a valuable advantage.
The ultimate promise of a future towards
• Biomarkers in toxicology, Timbrell J. A.,
Volume 129, Issue 1, Pages 1-12, 7 August
• Metabolomics approaches for discovering
biomarkers of drug-induced hepatotoxicity
and nephrotoxicity, Beger R. D., Volume 243,
Issue 2, Pages 154–166, March 2010.
• Biomarkers of Toxicity, NTP Workshop,
Workshop report, September 20-21, 2006.
• Clinical Biomarkers in drug discovery and
development, Richard Frank and Richard
Hargreaves, Volume 2, Pages 566-580, July
• Biomarkers and surrogate endpoints, Clinical
research and applications, G. D. Downing,
• Biomarkers in Drug Development – A CRO
Perspective, John Allison & Steve Brooks,
pages 15-19, 2004.