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PCS – The Integrated Drug Development Company
27th AGAH ANNUAL MEETING 2018Target organs in early medicines development – predictability and prevention of adverse reactions26. - 27. April 2018, Munich
Safety assessment of hepatic findings in non-clinical studiesDr. med. vet. Stephanie PlassmannBoard Certified Specialist in Pharmacology and Toxicology
Key objectives of preclinicalsafety programme
PreClinical approach
Liver toxicity
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Outline
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Key objectives of preclinicalsafety programme
PreClinical approach
Liver toxicity
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Outline
Identify initial safe starting dose and subsequent dose escalation schemes in
humans
Identify potential target organ toxicity incl. dose dependence, relation to exposure and where appropriate, reversibility
Identify safety parameters for clinical monitoring
ICH M3(R2): Key objectives
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Key objectives of preclinicalsafety programme
PreClinical approach
Liver toxicity
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Outline
Principal aims of testing (1)
Characterise dose‐response over time frame studied
Establish NOAEL (No Observed Adverse Effect Level)
Establish MTD (Maximum Tolerated Dose)
Identify target organs of toxicity
Identify parameters for clinical monitoring for potential adverse effects
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Principal aims of testing (2)
Potentially characterise reversibility of effects observed
Provide information on systemic (and tissue) exposures
Establish multiples of exposure/safety ratios
Provide basis for dose selection in subsequent preclinical studies in the species studied
To identify initial safe starting dose and dose range for subsequent human trials (in context with other studies)
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Choice of preclinical species - PD
Pharmacological relevance
Responsive to the primaryPD of the substance
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Choice of preclinical species - DMPK
Pharmacokineticrelevance
Similarity to humanswith regard to the PK
profile incl. biotransformation
Ensure exposure to mainhuman metabolite(s)
Consider separate studies with metabolite,
if not possible withparent compound
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Organ toxicity
General toxicity studies in
rodent and non‐rodent
Identify
Characterise
Assess human risk
Determine Clinical
monitoring parameters
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Principal approach
High dose selection
The dose selection needs to
be justified
Case by case
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High dose selection concepts
Maximum tolerated dose
Maximum feasible dose
Limit dose
Exposure multiples
Saturation of
absorption
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Maximum tolerated dose
Most robust
Ascertains highest possible systemic / tissue exposures
To identify all potential target organs of toxicity
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General toxicology
Key elemen
ts
Selection of relevant species
Dose range finding studies (non‐GLP)
Pivotal GLP studies
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Dose range finding (DRF) studies
ObjectivesNot uncommon to see mortality at doses > MTD
Identify Maximum tolerated dose (MTD) for main
studies
Particularly for CNS, CVS or other drugs targeting vital functions for
which the prevailing findings are dominated by
exaggerated pharmacological
effects
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Main 4-week studies to support phase I
GLP (Good laboratory practice)
Typically 4 groups of animals
1 control
3 treatment groups
10 animals/sex/group(rodent)
3‐4 animals/sex/group (non‐rodent)
Recovery is optional (depending on substance
class etc.)
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Treatment duration pivotal studies
• 1 day up to 6 months (rodent) to 9 months (non‐rodent)
• Duration of treatment in chronic toxicity studies see ICH S4
Varies from acute to chronic
studies
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Endpoints
In‐life (routine)
•Mortality, clinical signs, post‐dose observations, food consumption, body weight, clinical biochemistry, coagulation, haematology, ophthalmoscopy, ECG and blood pressure (non‐rodent), urinalysis
• Toxicokinetics
Necropsy and post‐mortem
•Macroscopic examination
•Organ weights
• Sampling of a full list of tissues (EMA guidance, Annex I)
•Histopathological evaluation of nearly all tissues and organs
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Key objectives of preclinicalsafety programme
PreClinical approach
Liver toxicity
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Outline
Organ evaluation from toxicity studies
Each organ will be evaluated histopathologically
• Routine stain: HE
• Additional staining optional (case by case)
• Other methods optional to follow‐up (exceptional, e.g. EM)
Rodents
• control and high dose initially
• target organs in mid and low dose (and recovery, if applicable)
Non‐rodents: all groups a priori due to lower power of study
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Organ evaluation from toxicity studies
Pathologist will diagnose findings for each individual animal
Determination of incidence for each finding in each group
Determination of severity of selected findings
Grading (minimal, slight, moderate, marked, severe)
Assessment of treatment‐relation of observations will be based on incidence and severity of findings in treated groups compared to controls
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Interpretation of liver findings
May be present in one or more testing species
• Rodent
• Non‐rodent
Changes may be evident as
• Clinical pathology alterations
• Organ weight increases
• Histopathological alterations
• Any of the above alone or in combination
Any findings in other organ systems?
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Clinical pathology alterations potentially indicative for impairment of the liver
Elevated serum
enzymes
Elevated serum
enzymes
Possible liver injury (AST,
ALT)
Biliary tract (gamma‐GT, Alkaline
phosphatase)
Bilirubin Bilirubin
Extrahepatic, hepatic or post‐hepatic increases
Alterations in protein levels e.g. albumin
Alterations in protein levels e.g. albumin
Associated with
functionality
Coagulation parametersCoagulation parameters
Many of these could also be
related to impairment of other organ systems
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Examples of morphological alterations at the histological level
Hepatocyte hypertrophy
Hepatocyte hyperplasia
Vacuolation
Lipid deposition
Cell degeneration
Inflammation
Necrosis
Hepatobiliary changes
Pre‐neoplastic changes
Neoplastic changes
Etc.
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Interpretation of findings
Overall finding considered test‐item related?
Not only from toxicology but
also pharmacology, DMPK etc.
Evaluate all results
Integrate information
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Observation in relation to
• Exacerbation of background lesion?
• Age related?
• Juvenile studies
• Old animals (carcinogenicity studies)
• Species?
• Strain?
Mechanistic considerations
• Acute?
• Subacute – subchronic?
• Chronic?
Treatment duration
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Liver metabolism?
Major organ involved in DMPK
Biotransformation of xenobiotic compounds
•CYP P450 oxidases: Phase I
• Conjugation: Phase II enzymes
•Create hydrophilic molecules which can be excreted
• Lipophilic compounds may be strong inducers of liver enzymes
• Involvement of transporters (efflux etc.)
Subject to stimulation of metabolic processes
•Morphologically visible at histopathological level if of sufficient degree
•May result in organ weight increases
•Organ weight increases may be associated with histopathological changes
Liver can be a primary target organ of toxicity or secondary due to
biotransformation
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Zonation
Phase I enzymes
Predominantly centrilobular localisation
Most constitutively expressed CYP450 and other phase I
enzymes
Phase II enzymes
Mostly periportal
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Interpretation of type of finding for human risk assessment
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Hepatocyte hypertrophy
Most common histological change associated with enzyme induction in animals
Often adaptive
Often non‐adverse
Due to stimulation of drug metabolism
However may proceed to (severe) toxicity at higher doses or upon prolonged treatment
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Liver hypertrophy (mouse) (1)
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Source: Courtesy of NIH Nonneoplastic Lesion Atlas: https://ntp.niehs.nih.gov/nnl/hepatobiliary/liver/
Liver hypertrophy (mouse) (2)
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Source: Courtesy of NIH Nonneoplastic Lesion Atlas: https://ntp.niehs.nih.gov/nnl/hepatobiliary/liver/
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Possible adverse consequences of hepatocyticstimulation
Degeneration
Necrosis
Proliferation
Hepatobiliary injury as result of increased formation of reactive intermediates
Oxidative injury
Formation of adducts covalently binding cellular molecules
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Hepatocyte necrosis and degeneration (mouse)
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Source: Courtesy of NIH Nonneoplastic Lesion Atlas: https://ntp.niehs.nih.gov/nnl/hepatobiliary/liver/
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Ennulat D. et.al. (2010)Toxicological Sciences 116(2), 397–412
Diagnostic Performance of Traditional Hepatobiliary Biomarkers of Drug‐
Induced Liver Injury in the Rat
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Diagnostic value of clinical pathology
Non‐invasive monitoring of liver disease in patients
Relationship between clinical pathology markers and liver morphology often unclear
ALT the most commonly used marker
May fail to identify liver disease of less marked degree
ALT and other hepatobiliary markers may lack relationship to severity or prognosis of the disease
Similarly, discrepancies between liver histopathology and hepatobiliary markers possible
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Diagnostic value of clinical pathology
Marked changes can be seen without histopathological correlate or vice versa
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Potential causes of discrepancies
Differences in timing of onset
Early indication
Short half‐lives
DepletionOther
pathophysiology such as restraint
Transporter function
pharmacologially influenced
Metabolic effects
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Potential causes of discrepancies
But most importantly: complexities
associated with DILI might confound interpertation
Requiring integrated assessment of combined
morphological findings rather than
discrete
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Summary Ennulat 2010 (Rat)
In the absence of hepatocellular necrosis or degeneration, manifestations of
Hypertrophy
Lipidosis
Hepatic inflammation
or hepatocellular cytoplasmic change
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Summary Ennulat 2010 (Rat)
Generally not associated with remarkable changes
in these traditional hepatobiliary markers Conclusion
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Conclusion Ennulat 2010 (Rat)
“In conclusion, the evaluation of the diagnostic utility of eight conventional serum biomarkers of hepatobiliary injury for prediction of drug‐induced liver injury in rat demonstrated the specificity and comparable diagnostic utility of ALT, AST, Tbili, and SBA for prediction of manifestations of hepatocellular necrosis/degeneration and biliary pathology. However, all eight conventional hepatobiliary markers evaluated had comparatively low diagnostic utility for manifestations of hepatocellular hypertrophy, lipidosis, cytoplasmic change, or inflammation.”
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Idiosyncratic liver toxicity
Not predicted from animal studies
Mechanism unknown
May be related to immune related pathology
May be related to differences in metabolism
Subject to individual sensitivity and hence unpredictable
Can lead to market withdrawal
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Summary and conclusion
Assess potential for liver toxicity in the overall context
Undertake integrated assessment
Critical appraisal of
results
Create robust
preclinical data
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5 Pillars of toxicological study conduct
• CAVEAT: High dose selection!
Robust study design and conduct
Senior toxicological expertise
High quality pathology (slide preparation)
Senior pathological expertise
Integrated interpretation
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Integrated Risk Assessment
Type of finding
Severity
Dose‐relation
Safety margin
Substance class
Indication
Mechanism of action
Chemical structure
Target population
DMPK
Predictivity for humans
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Thank you very much for your attention!
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PreClinical Safety (PCS) Consultants LtdNauenstrasse 63A
CH-4052 Basel
Switzerland
Website: www.pcsconsultants.com
Dr. med. vet. Stephanie PlassmannVeterinary SurgeonBoard Certified Specialist in Veterinary Pharmacology and ToxicologyEurotox Registered ToxicologistSenior Expert in Non-Clinical DevelopmentTel: +49 8133 908896
e-mail: stephanie.plassmann@pcsconsultants.com
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