Green - Biochemical

Principles of
Clinical Pharmacology
Biochemical
Mechanisms for Drug
Toxicity
Dr. Ashley Beasley Green, PhD

Technical Project Lead
Biomolecular Measurement Division
Material Measurement Laboratory
National Institute of Standards and Technology
Biochemical Mechanisms
of Drug Toxicity
Ashley Beasley Green, PhD

Biomolecular Measurement Division
Material Measurement Laboratory (MML)
National Institute of Standards and Technology (NIST)
NIH Principles of Clinical Pharmacology

Presentation Outline
• Adverse Drug Reaction (ADR)
• Prevalence and Economic Impact
• Mechanisms of ADRs
• Intrinsic vs. Idiosyncratic Reactions
• Risk Assessment of ADRs
Impact of drug-induced reactions?
• Adverse Drug Reaction (ADR):
“a response to a drug which is noxious and unintended and which occurs at
doses normally used in man for prophylaxis, diagnosis, or therapy of disease or
for modification of physiological function” WHO Technical Report 498 [1972]
Economic Impact:
Inpatient Outpatient
Adverse Drug Events
(ADEs) contribute an
additional $3.5 billion
to U.S. health care • 2 million stays • Most • > 3.5 million
costs (2006 USD) annually common post- office visits
• Increased discharge • ~1 million ED
length of stay complication visits
(~1.7-4.6 days)
U.S. Department of Health and Human Services, Office of Disease Prevention and Health Promotion. (2014). National Action Plan for Adverse Drug Event
Prevention. Washington, DC
The Fate of Drugs
Non-Toxic
Drug Excretion
Metabolites
Principles of Clinical Pharmacology, ed. 3. ch.16, Fig. 16.4.

The Fate of Drugs
Non-Toxic
Drug Excretion
Metabolites
Bioactivation
Enzyme Reactive
Metabolites
Types of Adverse Drug Reactions (ADRs)

Type A (Intrinsic) Type B (Idiosyncratic)
Dose-dependent Not Dose-dependent
Predictable Unpredictable
Low incidence, rare
High incidence
(individual susceptibility?)
Low mortality High mortality
Directly destructive, Indirect (metabolic) Immunoallergic (hypersensitivity
and cholestatic reaction) and Metabolic (“host”)
Acetaminophen (APAP)
• Over-the-counter analgesic and antipyretic
• Function
• Inhibit Prostaglandin H2 Synthetase conversion of AA→PGH2
Cell Membrane Phospholipids

Phospholipase A2
Arachidonic Acid (AA)

Acetaminophen
Prostaglandin H2 Synthetase
(APAP)
Prostaglandin H 2 (PGH2)
Prostacyclin (PGI2) Thromboxane A2
Prostaglandin F2α (PG F2α) Prostaglandin E2 (PGE2)

• FDA Warning for Acetaminophen
Etiology of Acute Liver Failure (ALF) in the USA
US Acute Liver Failure Study Group (ALFSG), January 2017 (n= 2,436)
1200
Num ber of Patients
1000
800
600
400
200
0
http://www.utsouthwestern.edu/labs/acute-liver/
The alternative language states:

“Liver warning: This product contains
acetaminophen. Severe liver damage may occur
if you take • more than 4,000 mg of
acetaminophen in 24 hours • with other drugs
containing acetaminophen • 3 or more alcoholic
drinks every day while using this product.”
https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM310477.pdf
Acetaminophen
(APAP)
Metabolism via
Phase II Conjugation Metabolism via
Glucuronidation
Phase I Oxidation
P450s 2EI, 5%
55-60% 1A2, 2A4
Eliminated in Urine
Sulfation 10%
20-30%
Catechol Derivative
NAC
Glutathione N-acetylcysteine
(GSH) (NAC)
N-acetyl-p-
benzoquinone imine
(NAPQI)
• Half-Life (t1/2): 2.0-2.5 hour Glutathione

(GSH) Depletion
• With hepatic injury, APAP t1/2 increase Glutathione Conjugate Cysteine adduct
to >4 hour APAP APAP
• Daily dose of APAP:
• Adult (>12 yrs.): 4 g/day
• Children (<12 yrs.): 50-75 mg/kg/day
• Overdose of APAP:
• Adult (>12 yrs.): >7.5 g/day
• Children (<12 yrs.): >150 mg/kg/day Covalent cell binding Mercapturic acid
and injury adduct APAP
Hodgman M and Garrard AR. Crit Care Clin. 2012; 28, 499-516
Acetaminophen
(APAP)
Glucuronidation
P450s 2EI, 5%
55-60%
1A2, 2A4
Eliminated in Urine
Sulfation Reactive
20-30% 10%M etabolite
Catechol Derivative
NAC
Major Metabolites (~85%); (GSH) (NAC)
Inactive Form s (renal
elim ination) N-acetyl-p-
benzoquinone imine
(NAPQI)
Glutathione
(GSH) Depletion
Non-Toxic Glutathione Conjugate Cysteine adduct
Drug Excretion
Metabolites APAP APAP
Bioactivation
Enzyme Reactive Further Metabolism
Metabolites and/or Excretion
Covalent cell binding Mercapturic acid
and injury adduct APAP
Acetaminophen
(APAP) Drug
Non-Toxic
Excretion
Metabolites
uronidation
Bioactivation
P450s 2EI, 5% Enzyme Reactive
5-60% Metabolites
Bioactivation 1A2, 2A4 A
Eliminated in Urine
Sulfation Reactive GSH, etc.
20-30% 10%
M etabolite B
Catechol Derivative Macromolecular-Drug
NAC
Non-toxic Adducts
Excretion Oxidative Stress:
Product GSH Depletion
(GSH) ↓ATP ↓↓ ATP
(NAC) • Tissue Necrosis
N-acetyl-p- • Hypersensitivity Reaction
benzoquinone imine • Carcinogenesis
Apoptosis Necrosis • Teratogenesis
(NAPQI)
Oxidative Glutathione
Stress (GSH) Depletion
Glutathione Conjugate Cysteine adduct
APAP APAP
Increase
Oxidative ↑ M itochondrial
Permeability (MPT)
Stress
Hepatocellular
and injury Necrosis
adduct APAP
Acetaminophen Variations in APAP susceptibility
(APAP)
Alcohol • Alcohol consumption (increase metabolic activation)
uronidation Non-Toxic
Drug Excretion
5%
Metabolites
P450 2EI
5-60%
Bioactivation Eliminated in Urine
Sulfation Reactive Bioactivation
20-30% 10% Enzyme Reactive
M etabolite
Catechol Derivative Metabolites A
NAC
GSH, etc.
B
Glutathione Macromolecular-Drug
N-acetylcysteine
(GSH) Non-toxic
(NAC) Adducts
Excretion Oxidative Stress:
N-acetyl-p- Product GSH Depletion
benzoquinone imine
(NAPQI) ↓ATP ↓↓ ATP
• Tissue Necrosis
• Hypersensitivity Reaction
Oxidative Glutathione • Carcinogenesis
Apoptosis Necrosis •
Stress (GSH) Depletion Teratogenesis
Glutathione Conjugate Cysteine adduct

APAP APAP
Increase
Permeability (MPT)
Stress
Hepatocellular
and injury Necrosis
adduct APAP
Acetaminophen Variations in APAP susceptibility
(APAP)
• Inflammatory stress
*APAP-induced Hepatocellular Necrosis via Immune Response
uronidation
P450s 2EI, 5%
5-60%
Bioactivation 1A2, 2A4
Eliminated in Urine
Sulfation Reactive
20-30% 10%
M etabolite
Catechol Derivative
NAC
(GSH) (NAC)
N-acetyl-p-
benzoquinone imine
(NAPQI)
Oxidative Glutathione
Stress (GSH) Depletion Necrotic Hepatocytes
Krenkel O,Conjugate
Glutathione Mossanen JC and Tacke F. Hepatobiliary
Cysteine adduct Surg Nutr. 2014 Dec; 3(6): 331-43.
APAP APAP
Increase
Permeability (MPT)
Stress
Hepatocellular
and injury Necrosis
adduct APAP
Type B- Idiosyncratic DILI
• Drugs usually eliminated during preclinical testing
• Idiosyncratic DILI accounts for large percent of post-marketing restrictions by FDA
• Hepatotoxicity not related to pharmacological action of
drug
• Wide range in the severity of toxic effects
• Limited mechanistic understanding
• Theories for Toxicity
• Reactive Intermediate Hypothesis
• Mitochondrial Dysfunction Hypothesis
• Genetic Polymorphism Hypothesis
• Hapten Hypothesis
• The Danger Hypothesis
• Failure-to-Adapt Hypothesis
• Multiple Determinant Hypothesis
• Inflammatory Stress Hypothesis
Shaw PJ, Ganey PE, and Roth RA. Toxicol Sci. 2010 Nov; 118(1): 7-18
Idiosyncratic DILI
Theories for Mechanism of Action
Reactive Intermediate Hypothesis
Mitochondrial Dysfunction Hypothesis
Genetic Polymorphism Hypothesis
Genetic Polymorphisms
Bioactivation
Enzyme Reactive Macromolecular-Drug
Drug DILI
Metabolites Adducts
Increase
Oxidative
Stress ↑ M itochondrial
Permeability (MPT)
Hepatocellular
Necrosis
Roth R.A. and Ganey P.E. J Pharmacology and Experimental Therapeutics. 2010; 332(3), 692-697)
Shaw PJ, Ganey PE, and Roth RA. Toxicol Sci. 2010 Nov; 118(1): 7-18
Isoniazid
• First-line agent in the treatment of active or latent tuberculosis
• Mechanism of Action
• Bactericidal action: Block formation of mycolic acids
• Mycolic acid: essential component of mycobacterial cell wall synthesis
• Disruption of cell wall results in cell death
• Animal model used to elucidate biochemical mechanism
• ~20% of treated patients develop elevated liver enzymes and bilirubin (small percentage develop hepatitis)
Isoniazid N-acetylisoniazid
Theories of Toxicity
Reactive Intermediate Hypothesis
Mitochondrial Dysfunction Hypothesis
Amidase Amidase
Genetic Polymorphism Hypothesis*
Isonicotinic Acid
NAT2
NH2-NH2 CH3CO-NH2-NH2 NAT2 CH3CO-NH2-NH-COCH3
Hydrazine Aceytlhydrazine Diaceytlhydrazine
Amidase
CYP2E1 ↑ Mitochondrial
GSH Binding/ Permeability
Depletion (MPT)
Toxic ↑ Oxidative Hepatocellular
Metabolite Stress Apoptosis
Principles of Clinical Pharmacology,ed 3. ch.16, Fig. 16.6. NAT2: N-Acetyltransferase 2

Hapten Hypothesis
• Drug-induced hepatotoxic reaction mediated by an immune response
• Chemically reactive drug or reactive metabolite (hapten) binds endogenous protein that
initiates an immune response
Cho T and Uetrecht J. Chem Res Toxicol. 2017 Jan 17: 30(1): 295-314
Hapten Hypothesis
• Drug-induced hepatotoxic reaction mediated by an immune response
• Chemically reactive drug or reactive metabolite (hapten) binds endogenous protein that
initiates an immune response
• Macromolecule-drug adduct: neoantigen
• Example Drug: Halothane
• Volatile general anesthetic
• 2 types of hepatotoxicity of Halothane
Lipid Peroxidation
Halothane Type 1 2-chloro-1,1,1-
trifluoroethyl radical
Type 2 Trifluoroacetic acid (TFA)
Trifluoroacetyl-chloride
Liver Protein
Principles of Clinical Pharmacology,ed 3. ch.16, Fig. 16.7.

Hapten Hypothesis: Penicillin
• Adverse reaction (immune response) is mediated by IgE antibodies
that recognize the β-lactam-modified proteins
Thiazolidine ring
β-lactam ring
Major
Side Chain
Penicilloyl-Protein (antigen)
Penicillin
Common Adverse Drug Reactions Incidence: ≥1%

Typical lesions of chronic urticaria
Diarrhea, nausea, rash, neurotoxicity, urticaria and/or super
infection
Infrequent Adverse Drug Reactions Incidence: 0.1-1%

Fever, vomiting, erythema, dermatitis, angioedema, seizures,
and/or pseudo membranous colitis
True Anaphylaxis Incidence: 0.01%

http://w ww.worldallergy.org/professional/allergic_
Hypersensitivity with hypotension, angioedema, bronchospasm diseases_center/urticaria/urticariasynopsis.php
and urticaria
Bhattacharya S. J Adv Pharm Technol Res. 2010 Jan; 1(1): 11-17. Gonsales-Estrada A and Radojicic C. Cleve Clin J Med. 2015 May; 82(5): 295-300.
Hapten Hypothesis: Penicillin
• Adverse reaction (immune response) is mediated by IgE antibodies
that recognize the β-lactam-modified proteins
Thiazolidine ring
β-lactam ring
Major
Side Chain
Penicilloyl-Protein (antigen)
Penicillin
Common Adverse Drug Reactions Incidence: ≥1%

Clinical Evaluation:
Diarrhea, nausea, rash, neurotoxicity, urticaria and/or super
infection Skin Test
Negative Positive
Infrequent Adverse Drug Reactions Incidence: 0.1-1%
Fever, vomiting, erythema, dermatitis, angioedema, seizures, Not allergic or Avoid penicillin
and/or pseudo membranous colitis Graded-dose
Challenge required
True Anaphylaxis Incidence: 0.01%

Hypersensitivity with hypotension, angioedema, bronchospasm Desensitization
and urticaria Therapy
Bhattacharya S. J Adv Pharm Technol Res. 2010 Jan; 1(1): 11-17. Gonsales-Estrada A and Radojicic C. Cleve Clin J Med. 2015 May; 82(5): 295-300.
Inflammatory Stress Hypothesis
• Drug therapy coupled to inflammatory stress precipitates Type B adverse drug reaction
• Inflammatory stress induced by several factors:
• Infection, intestinal microbial disturbance, cell death, etc.
• Example Drug: Trovafloxacin (Trovan, Pfizer)

• Broad-spectrum fluoroquinolone antibiotic Trovafloxacin (TVX)
• ~140 severe hepatic reactions reported/14 resulted in liver failure
• Restrictions in 1999 and withdrawn in 2001--due to hepatotoxicity
• Mode of action for hepatotoxicity identified via animal model
• Mechanism of action (prevent bacterial growth):
• Bactericidal activity exerted by parent compound (no reactive metabolite)
• Inhibit bacterial DNA gyrase (Gram-negative) and Topoisomerase IV (Gram-positive) which
prevents replication of bacterial DNA during growth and reproduction
Bacterial DNA
B B Trovafloxacin B B B B
B B A A A A A A
A A DNA-DNA gyrase Complex Trovafloxacin stabilizes DNA-DNA Broken DNA strands are released,
forms (DNA strands broken but gyrase Complex (Broken DNA this results in cell death
Bacterial DNA strands cannot be released and
held together via DNA gyrase)
Gyrase DNA replication is blocked)
http://www.antibiotics-info.org/levofloxacin.html
Example: Trovafloxacin (TVX)
• Mode of action for hepatotoxicity: inflammation-drug interaction animal models
Mouse Model: TVX/LPS-Induced Liver Injury

TVX/LPS-induced Liver Injury Time-dependence of Liver Injury
Veh: Sterile Saline; LPS: Lipopolysaccharide; ALT: Alanine Transaminase
Shaw PJ, Hopfensperger MJ, Ganey PE, and Roth RA. Toxicol Sci. 2007; 100(1): 259-266
Mouse Model: TVX/LPS-Induced Liver Injury
Effect of TVX/LPS on TNFα Effect of TVX/LPS on Liver Injury
Veh: Sterile Saline; LPS: Lipopolysaccharide; ALT: Alanine Transaminase; Etan: Etanercept
Shaw PJ, Hopfensperger MJ, Ganey PE, and Roth RA. Toxicol Sci. 2007; 100(1): 259-266
Gene Expression after TVX/ LPS-Induced Liver Injury
• Global Gene Profiling:
•Genes involved in interferon signaling (IFN-γ) play a role in TVX/LPS co-exposure
•Early Marker of Hepatotoxicity
TVX/LPS
↑ 193 ↑ JAK/STAT and IFN
↓ 580 Signaling Pathways
↑ 116 ↑ 534
↓ 284 ↓ 230
↑ 114
TVX/Veh ↓ 105
Veh/LPS
↑ 197 ↑ 10 ↑ 364
↓ 168 ↓9 ↓ 173
Shaw PJ, et al. Toxicol Sci. 2009; 107(1): 270-280

How do we develop safe drugs?
• Integration of risk assessment strategies early in drug development phase
• Minimize risk of attrition during preclinical phase, clinical trials and post-market
• Early risk assessment during drug development phase
Total R&D cost: $2.6 billion on average (in 2013 USD) http://phrma-docs.phrma.org/sites/default/files/pdf/rd_brochure_022307.pdf
Adverse Outcome Pathway
Mode of Action Pathway
Toxicity Pathway
Adverse Chemical
Macro-
Cellular Tissue Organ Individual
Outcome molecular
Insult Response Response Response Response
Pathway Interaction
Formation of
Reactive
Metabolites
Reactive metabolite formation

combined with dose
Existing Risk Assessment Approaches
Reactive metabolite formation integrated with

measures of cellular response
Thompson RA, et al. Chem Res Toxicol. 2016 Apr 18; 29(4): 505-533.
Risk Assessment Approaches
• Existing Risk Assessment Approaches
• Formation of Reactive Metabolite
• Reactive metabolite formation combined with dose
• Reactive metabolite formation integrated with measures of cellular response
• Removal of structural motifs linked to reactive metabolite formation or metabolism-

mediated toxicity
• Elimination of candidates based on in silico assessment of structure motifs—no
synthesis required
• Minimizes risk of reactive metabolite formation — reduce chemical library of drug
candidates
*Elimination of phenyl- or thiophene-containing compounds---

prevent patient access to several safe and efficacious drugs
• Detection of Reactive Metabolite Formation

• Trapping Approach
▪ Dependent upon electrophilic nature of reactive metabolite
▪ Trapping Agents:
• GSH-trap soft electrophiles (epoxides, quinones, etc.)
• KCN-trap iminium ions
• Methoxylamine-trap aldehydes and α,β-unsaturated carbonyl compounds
▪ Detection: LC-MS
• Electrochemical Approach
• Time-dependent Inactivation (TDI) of drug metabolizing enzyme
(P450s)
Example of Trapping Approach
• Characterization of 1,3-Bisphosphoglycerate (1,3-BPG)
• Primary intermediate of glycolysis pathway
• Sensitive to hydrolysis or isomerization
• Naturally converted to 3-PG and 2,3-BPG (enzyme-mediated)
• Reported Value: combined level of 1,2-BPG and 2,3-BPG
Glucose Pyruvate
3-Phosphoglyceraldehyde (GAP) 3-Phosphoglycerate (3-PG)
2,3-Bisphosphoglcerate (2,3-BPG)
Chang JW, et al. Anal Chem. 2016 Jul 5; 88(13): 6658-6661.

• New approach: Chemical trapping coupled to MS analysis
• Hydroxylamine (NH2OH): react with electrophilic carbonyls under mild aqueous
conditions and yield a stable acid compatible with extraction and MS
Glucose Pyruvate
3-Phosphoglyceraldehyde (GAP) 3-Phosphoglycerate (3-PG)
2,3-Bisphosphoglcerate (2,3-BPG)

3PGha
Fragmentation Transition:
199.98→79



• Detection of Reactive Metabolite Formation

• Trapping Approach
▪ Dependent upon electrophilic nature of reactive metabolite
▪ Trapping Agents:
• GSH-trap soft electrophiles (epoxides, quinones, etc.)
• KCN-trap iminium ions
• Methoxylamine-trap aldehydes and α,β-unsaturated carbonyl compounds
▪ Detection: LC-MS
• Electrochemical Approach
• Time-dependent Inactivation (TDI) of drug metabolizing enzyme
(P450s)
Reactive Metabolite Assessment: used to eliminate drug candidates but not
quantitative; issue with incorporation for HTP drug screen
• Quantitative assessment of reactive metabolite

• Labeled trapping agent or drug molecule
• Provide level estimates of reactive metabolite that can form in humans
• Account for drug dose?
• Covalent binding assessment
• Drug-induced modification of protein
• Combine covalent binding and dose
Reactive Metabolite/Dose Assessment: Difficult to set cutoff values to

distinguish safe drugs---the mode of toxicity is complex
• Address the complexity of the toxicity mechanism

• Couple reactive metabolite formation with cellular response
• Multifactorial Approach
• Prediction tool used to distinguish between drugs with high and low
tendency to cause a Type B (idiosyncratic) adverse reaction
• Covalent Binding (CVB) Burden coupled to In vitro Panel
▪ CVB Burden: Quantitative estimate of ability to covalently bind protein
▪ In vitro Panel: Profile the biological effects of drugs
Risk Assessment: Multifactorial
Multifactorial Approach 36 drugs: 27 severe or marked
idiosyncratic ADR concern and 9
Integrated In Vitro with low concern
Hazard Matrix
No Hazard
Distinguished between safe and unsafe drugs with

high sensitivity (100%) and specificity (78%)
Thompson RA, et al. Chem Res Toxicol. 2012 Aug 20: 25(8): 1616-1632.
• Integration of risk assessment strategy early in drug development phase
Adverse Outcome Pathway
Mode of Action Pathway
Toxicity Pathway
Macro-
Chemical Cellular Tissue Organ Individual
molecular
Insult Response Response Response Response
Interaction
Formation of
Reactive
Metabolites
Reactive metabolite formation

combined with dose Existing Risk Assessment Approaches
unable to determine metabolic or
immune activation
Reactive metabolite formation integrated with
measures of cellular response
Microphysiological Models:
Spheroids, Bioreactors, Media Flow
• New Risk Assessment Approach: Microphysiological Models
• Risk of sensitization and severity of clinical symptom
• State of immune system of patient
• Dose
• Frequency, route and duration of exposure
• Sex
• Immunogenetic predisposition
• 3D Spheroid Hepatic Cultures
• Enhanced liver phenotype
• Metabolic activity
• Stability in culture not attainable with conventional two-dimensional hepatic models
• Coculture
• Bioreactors
• Media Flow
• Removal of metabolites and bile
• Concentration gradients
Inflammatory Stress Hypothesis
What if we can use an in vitro model
to determine inflammation-mediated
drug toxicity?
• Example Drug: Trovafloxacin (Trovan, Pfizer) Trovafloxacin (TVX)
• Broad-spectrum fluoroquinolone antibiotic

• ~140 severe hepatic reactions reported/14 resulted in liver failure
• Restrictions in 1999 and withdrawn in 2001--due to hepatotoxicity
• Mode of action for hepatotoxicity identified via animal model
• Mechanism of action (prevent bacterial growth):
• Bactericidal activity exerted by parent compound (no reactive metabolite)
• Inhibit bacterial DNA gyrase (Gram-negative) and Topoisomerase IV (Gram-positive) which
prevents replication of bacterial DNA during growth and reproduction
Bacterial DNA
B B Trovafloxacin B B B B
B B A A A A A A
A A DNA-DNA gyrase Complex Trovafloxacin stabilizes DNA-DNA Broken DNA strands are released,
forms (DNA strands broken but gyrase Complex (Broken DNA this results in cell death
Bacterial DNA strands cannot be released and
held together via DNA gyrase)
Gyrase DNA replication is blocked)
http://www.antibiotics-info.org/levofloxacin.html
Coculture: Trovafloxacin
• Human Liver Microtissue Model
• 3D liver microtissue
• 96-well format: HTP screening
• Primary human hepatocytes in combination with non-parenchymal cells (Kupffer and endothelial cells)
3D InsightTM Human Liver Microtissue
Stability: ATP Content
Functionality: Albumin Secretion
Messner S, et al. Arch Toxicol. 2013 Jan; 87 (1): 209-213.

Coculture: Trovafloxacin
• Human Liver Microtissue Model
• 3D liver microtissue
• 96-well format: HTP screening
• Primary human hepatocytes in combination with non-parenchymal cells (Kupffer and endothelial cells)
• LPS addition: elevation of interleukin-6 (IL-6) secretion (responsive macrophages) and shift in
hepatotoxic threshold of trovafloxacin (TVX)
LPS-induced Inflammation Inflammation-induced Toxicity of TVX
Messner S, et al. Arch Toxicol. 2013 Jan; 87 (1): 209-213.

• New Risk Assessment Approach: Microphysiological Models
• Risk of sensitization and severity of clinical symptom
• State of immune system of patient
• Dose
• Frequency, route and duration of exposure
• Sex
• Immunogenetic predisposition
• 3D Spheroid Hepatic Cultures
• Enhanced liver phenotype
• Metabolic activity
• Stability in culture not attainable with conventional two-dimensional hepatic models
• Coculture
• Bioreactors
• Media Flow
• Removal of metabolites and bile
• Concentration gradients
• New Risk Assessment Approach: Computational Models
• Integration of risk assessment strategy early in drug development phase
Thank you!
Dr. Ashley Beasley Green

ashley.beasley@nist.gov
Course Directors
Dr. Lisa M. Cordes Dr. William Douglas Figg, Sr

PharmD, BCACP, BCOP PharmD, MBA

Green - Biochemical

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Principles of

Dr. Ashley Beasley Green, PhD

Ashley Beasley Green, PhD

NIH Principles of Clinical Pharmacology

Principles of Clinical Pharmacology, ed. 3. ch.16, Fig. 16.4.

Types of Adverse Drug Reactions (ADRs)

Cell Membrane Phospholipids

Arachidonic Acid (AA)

Prostacyclin (PGI2) Thromboxane A2

Prostaglandin F2α (PG F2α) Prostaglandin E2 (PGE2)

The alternative language states:

• Half-Life (t1/2): 2.0-2.5 hour Glutathione

Glutathione Conjugate Cysteine adduct

Principles of Clinical Pharmacology,ed 3. ch.16, Fig. 16.6. NAT2: N-Acetyltransferase 2

Type 2 Trifluoroacetic acid (TFA)

Principles of Clinical Pharmacology,ed 3. ch.16, Fig. 16.7.

Common Adverse Drug Reactions Incidence: ≥1%

Infrequent Adverse Drug Reactions Incidence: 0.1-1%

True Anaphylaxis Incidence: 0.01%

Common Adverse Drug Reactions Incidence: ≥1%

True Anaphylaxis Incidence: 0.01%

• Example Drug: Trovafloxacin (Trovan, Pfizer)

Mouse Model: TVX/LPS-Induced Liver Injury

Veh: Sterile Saline; LPS: Lipopolysaccharide; ALT: Alanine Transaminase

Mouse Model: TVX/LPS-Induced Liver Injury

Effect of TVX/LPS on TNFα Effect of TVX/LPS on Liver Injury

Shaw PJ, et al. Toxicol Sci. 2009; 107(1): 270-280

Adverse Outcome Pathway

Mode of Action Pathway

Reactive metabolite formation

Reactive metabolite formation integrated with

• Removal of structural motifs linked to reactive metabolite formation or metabolism-

*Elimination of phenyl- or thiophene-containing compounds---

• Detection of Reactive Metabolite Formation

3-Phosphoglyceraldehyde (GAP) 3-Phosphoglycerate (3-PG)

Chang JW, et al. Anal Chem. 2016 Jul 5; 88(13): 6658-6661.

3-Phosphoglyceraldehyde (GAP) 3-Phosphoglycerate (3-PG)

Chang JW, et al. Anal Chem. 2016 Jul 5; 88(13): 6658-6661.

Chang JW, et al. Anal Chem. 2016 Jul 5; 88(13): 6658-6661.

Chang JW, et al. Anal Chem. 2016 Jul 5; 88(13): 6658-6661.

Chang JW, et al. Anal Chem. 2016 Jul 5; 88(13): 6658-6661.

• Detection of Reactive Metabolite Formation

• Quantitative assessment of reactive metabolite

Reactive Metabolite/Dose Assessment: Difficult to set cutoff values to

• Address the complexity of the toxicity mechanism

Distinguished between safe and unsafe drugs with

Adverse Outcome Pathway

Mode of Action Pathway

Reactive metabolite formation

• Example Drug: Trovafloxacin (Trovan, Pfizer) Trovafloxacin (TVX)

• Broad-spectrum fluoroquinolone antibiotic

3D InsightTM Human Liver Microtissue

Stability: ATP Content

Functionality: Albumin Secretion

Messner S, et al. Arch Toxicol. 2013 Jan; 87 (1): 209-213.

LPS-induced Inflammation Inflammation-induced Toxicity of TVX

Messner S, et al. Arch Toxicol. 2013 Jan; 87 (1): 209-213.

• New Risk Assessment Approach: Computational Models

Dr. Ashley Beasley Green

Dr. Lisa M. Cordes Dr. William Douglas Figg, Sr

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