Presented by:

Ms. Suruchi Ramkumar Sharma

F.Y. M.Pharm

Under the guidance of:

Dr. (Mrs.) Vaishali Dixit

M.E.T Institute of Pharmacy, Bandra (W)

12-Mar-2012

Definition:

The US FDA defines a biomarker as a characteristic i.e.

objectively measured & evaluated as an indicator of

normal biologic, pathogenic or pharmacologic responses to therapeutic

intervention.2

• 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.

Examples• serum LDL for cholesterol • blood pressure for stroke• C-reactive protein (CRP) for inflammation

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Disease – related Biomarkers

Drug – related Biomarkers

Risk indicator / predictive biomarkers

Indicate effectiveness of a drug in a specific

patient

Diagnostic & prognostic biomarker

How the patient’s body will process it

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Based on Characteristics

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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

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Discovery of

Molecular Biomarkers

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

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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) concentration gradients

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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) –• Beryllium• Massive necrosis - iproniazid, MAOI

Liver Lobule

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Cholestatic injury Cytotoxic injury Altered hepatic function

Alkaline Phosphatase [AP, ALP]

Aspartate aminotransferase [AST]

Creatine phosphokinase [CPK]

5’‐Nucleotidase [5-NT]

Lactate Dehydrogenase [LDH]

Choline Esterase [ChE](acetylcholine esterase and butyrylcholine esterase)

γ - Glutamyl Transpeptidase [GGT]

Alanine aminotransferase [ALT]

Total Serum Bile Acids

Ornithine carbamyl transferase [OCT]

Decreased dye clearance •Sulfobromophthalein•Indocyanine green

Plasma Bilirubin Alanine aminotransferase [SDH]

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Serum Indicators Urine Indicators

Blood Urea Nitrogen (BUN)

Physical characteristics

Chemical Characteristics

Blood Creatinine Color/turbidity (RBC’s, bilirubin)

Urinary protein –tubular (low MW) or glomerular (high MW)

Volume Urinary glucose –no elevation of blood glucose but glucosuria (tubular)

Osmolality Urinary brush border enzymes (ALP, AST, GGT)

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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.

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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 rats.

• Biomarker : Changes in the SAMe concentrations inversely proportional with urinary APAP–NAC concentration.20

NMR-based approach to investigate toxicity induced by Bay41-4109, an anti-hepatitis B virus compound.

• Biomarker : Fatty acid metabolism disorder and mitochondrial dysfunction.

GC/MS was used to study carbon tetrachloride-induced acute liver injury in mice.

• Biomarker : Elevated levels of maleate and several fatty acids in the liver.

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UPLC/MS to investigate toxicity by CCl4 and α-napthylisothiocyanate.

• 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.

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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.

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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 MS. Glucosuria.

• Biomarker : UPLC/MS showed increase in urinary levels of 6-hydroxymelatonin.

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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 reabsorption. 25

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 studies.

• Safety studies e.g. APAP induced hepatotoxicity study• Proof of concept studies• Molecular profiling

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Clinical biomarker in Lung Cancer: Volumetric growth analysis of lesions in high resolution CT.

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A new face in diagnostics, therapeutics & drug development.

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 personalized healthcare.

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• Biomarkers in toxicology, Timbrell J. A., Volume 129, Issue 1, Pages 1-12, 7 August 1998.

• 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.

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• Clinical Biomarkers in drug discovery and development, Richard Frank and Richard Hargreaves, Volume 2, Pages 566-580, July 2003.

• Biomarkers and surrogate endpoints, Clinical research and applications, G. D. Downing, Elsevier (2000).

• Biomarkers in Drug Development – A CRO Perspective, John Allison & Steve Brooks, pages 15-19, 2004.

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DISCUSSION