plassmann agah-annual-2018 liver-safety fv1.0-for-website€¦ · (non‐rodent) •duration of...

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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

AGAH Annual Meeting 2018 2

Outline

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Liver toxicity


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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Liver toxicity


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)


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


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


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


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


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


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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Liver toxicity


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


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


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


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


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


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)


Source: Courtesy of NIH Nonneoplastic Lesion Atlas: https://ntp.niehs.nih.gov/nnl/hepatobiliary/liver/

Liver hypertrophy (mouse) (2)



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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


Hepatocyte necrosis and degeneration (mouse)



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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


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


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


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


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


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


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!


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: [email protected]


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