PRECLINICAL PHARMACOLOGY

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PRECLINICAL PHARMACOLOGY TOXICOLOGY IN CANCER
DRUG DEVELOPMENT

• Joseph E. Tomaszewski, Ph.D.
• Toxicology Pharmacology Branch
• DTP, DCTD, NCI

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PRECLINICAL TOXICOLOGY THE WHY AND HOW
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PRECLINICAL PHARMACOLOGY TOXICOLOGY WHY?

• Balance of
• Regulatory Issues
• Need for information
• Science
• Practical Considerations
• Preclinical Costs versus Patient Cost

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REASONS FOR TERMINATION OF DEVELOPMENT OF NCEs
Total Number of Drugs 198 (Excluding
Anti-Infectives, n77)
Lack of Efficacy
Commercial Reasons
5.0
46.0
Lack of Efficacy
30.0
ADRs in Man
Commercial Reasons
10.0
Misc
7.0
Misc
5.0
7.0
ADRs in Man
PK
Animal Toxicity
16.0
39.0
PK
11.0
Animal Toxicity
7.0
17.0
Ref Kennedy T, 1997. DDT, 10 436-444.
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FDA PRECLINICAL PHARMACOLOGY TOXICOLOGY
REQUIREMENTS
Ref DeGeorge, Cancer Chemother. Pharmacol., 41,
173-185, 1998.

• DRUGS
• Two Species - Rodent Non-rodent
• Clinical Route Schedule
• Pharmacokinetics - Optional

• BIOLOGICALS
• Most Relevant Species
• Clinical Route Schedule
• Biodistribution

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RECENT APPROACHES TO ONCOLOGY DRUG DEVELOPMENT
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REPRESENTATIVE SURFACE AREA TO WEIGHT RATIOS (km)
FOR VARIOUS SPECIES (Freireich, et al, Cancer
Chemotherapy Reports, 1966, 50 219-244)
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NCI STANDARDIZED PRECLINICAL TOXICOLOGY PROTOCOLS
FOR ANTI-NEOPLASTIC AGENTS (1980 - 1988)

• Mouse Lethality Studies
• Dog Toxicity Studies
• Rodent (Rat) Toxicity Studies

• Determine LD10 on Dx1 Dx5 Schedules
• Assess safety of 1/10 MELD10 Determine DLTs on
  Dx1 Dx5 Schedules.
• Assess safety of 1/10 MELD10 Determine DLTs on
  Dx1 Dx5 Schedules.

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NCI PRECLINICAL PHARM/TOX STUDIES (1988
Present) 1

• Agent-Directed Design
• Studies Guided by Pharmacokinetics/dynamics
  (PK/PD)
• Correlate PK/PD to Efficacy
• Correlate PK/PD to Safety Toxicity
• Incorporate In Vitro PK and Toxicity Data As
  Appropriate and Available

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NCI PRECLINICAL PHARM/TOX STUDIES (1988
present) 2

• Extrapolate PK and Toxicity Across Species
• Correlate PK/PD with Toxicity and Safety Across
  Species
• Ameliorate Toxicity by Change in Route and/or
  Schedule
• Compare Toxicity with Accepted Clinical Agents as
  Necessary

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ADVANTAGES OF AGENT-DIRECTED DRUG DEVELOPMENT

• Greater Scientific Basis for Development
• Permits Greater Flexibility
• Data Rich IND Submission to Support Phase I
• Preclinical Potential .. Less Expensive
• Permits PK/PD-Guided Dose Escalation in Phase I
• Optimal Schedule .. Greater Chance of Success?
• Patients .. Greater Chance of Effective Therapy?

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OBJECTIVES OF PRECLINICAL PHARMACOLOGY STUDIES
FOR ANTI-NEOPLASTIC DRUGS

• Development of Sensitive Analytical Methods
  (PK PD)
• Determine In Vitro Metabolism and Protein Binding
• Analog Evaluation - Determine Optimal Development
  Candidate
• Determine PK and PD in Various Species
• Identification and Analysis of In Vivo
  Metabolites
• Define Optimal Dose Schedule
• Correlate CP and/or AUC with Efficacy, Safety
  Toxicity and PD (Biomarkers)

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KEY PHARMACOLOGY CONTRIBUTIONS TO DRUG DEVELOPMENT

• 9-AC Suspension 72 Hr civ
• 17-AAG Parent Efficacy
• Active metabolite Efficacy
• Penclomedine High CP MAX Neurotoxicity
• CP lt Threshold No Neurotoxicity
• Isis 5320 High CP MAX BP?, APTT?, Bb?
• CP lt Threshold No Toxic Effects
• Halofuginone Tissue AUC Efficacy (?)
• gtgt Plasma AUC

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SELECTIVE RETENTION OF17-AAG IN TUMORS
Tumor
Plasma
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OBJECTIVES OF PRECLINICAL TOXICOLOGY STUDIES FOR
ANTI-NEOPLASTIC DRUGS

• DETERMINE IN APPROPRIATE ANIMAL MODELS
• The Maximum Tolerated Dose ( MTD )
• Dose Limiting Toxicities ( DLT )
• Schedule-Dependent Toxicity
• Reversibility of Adverse Effects
• A Safe Clinical Starting Dose

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RECENT TPB DRUG DEVELOPMENT EXAMPLE
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PYRROLOBENZODIAZEPINE(PBD, SJG-136, NSC 694501)
Energy-minimized SJG-136 cross-linked DNA adduct
View into minor groove
View down axis
A2780 Cells IC50 7.2 nM R-A2780 210 nM
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PYRROLOBENZODIAZEPINE (NSC 694501) MEASUREMENT
OF CROSSLINKING IN EX VIVO HUMAN LYMPHOCYTES
USING COMET ASSAY
Single Cell Gel Electrophoresis
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PYRROLOBENZODIAZEPINE (NSC 694501) TOXICITY
CONCERNS

• Potent DNA Minor Groove Cross-Linking Alkylating
  Agent Similar to Bizelesin Myelosuppression
  with Human Sensitivity Much Greater than Mouse
• Anthramycin-Like Compound Potential for Serious
  Cardiotoxicity

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SJG-136 BIZELESIN STRUCTURES
PYRROLOBENZODIAZEPINE
BIZELESIN
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BIZELESIN (NSC 615291)IN VITRO BONE MARROW
TOXICITY DATA
728X
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SJG-136, PBD (NSC 694501) IN VITRO BONE MARROW
TOXICITY DATA
3X
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SJG-136, PBD (NSC 694501) MYELOSUPPRESSION
SUMMARY

• In Vitro
• Bizelesin Human 728-fold gt Mouse
• Taxol Human 5-fold gt Mouse
• PBD Human 3-fold gt Mouse (IC90 826 nM)
• HTCFA PBD IC90 20-30 nM
• In Vivo
• Dose Limiting in Rats Dogs GI
• Based on In Vitro Bone Marrow and In Vivo MTDs,
  Projected Human MTD for PBD is within Mouse
  Efficacy Range (MED and MTD)
• PBD Has Greater Chance of Activity in Man

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SJG-136 ANTHRAMYCIN STRUCTURES
PYRROLOBENZODIAZEPINE
ANTHRAMYCIN
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SJG-136, PBD (NSC 694501) CARDIOTOXICITY
SUMMARY

• Anthramycin - Serious ECG changes leading to
  death
• PBD Serious GI BM Toxicity and Death
• No ECG Changes
• ? HR in Dogs at ?MTD
• No Heart Lesions
• cTnT Data Unchanged or Inconclusive
• PBD is Probably Non-Cardiotoxic

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SJG-136 CONCLUSIONS

• A Uniquely Designed Minor Groove Binder
• Highly Active in vitro in vivo
• Does Not Show Species Disparity in BM
• Does Not Appear to be Cardiotoxic
• DLTs (BM, GI) are Clinically Manageable
• MTD for Dog is 2X lower than Mouse MED,
• however Dog BM sensitivity is 2X gt Hu
• Projected Human MTD is within Range

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APPROACH TO TOXICOLOGY STUDIES - CONCLUSIONS

• Do Not Simply Use Standard Approach
• Do Not Think of Tox as Simply Checking a
  Regulatory Box ?
• Develop Preclinical Plan For Each Agent Using All
  Available Data
• In Vitro In Vivo Data Can Predict Human
  Sensitivity Toxicity
• Think Outside of the Box ??

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ACKNOWLEDGEMENTS

• TPB Contractors
• Pharmacology
• Mayo Foundation
• Ohio State University
• Southern Research
• Univ Alabama
• Univ Pittsburgh
• Univ Texas - MDA

• Toxicology
• Battelle
• IIT Research Inst.
• Southern Research
• SRI International
• Univ Illinois, Chicago
• Pathology Assoc.

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ACKNOWLEDGEMENTS

• TPB Staff
• PK Joseph M. Covey, Ph.D.
• PK Lee Jia, Ph.D.
• Tox Susan J. Donohue, Ph.D.
• Tox Elizabeth R. Glaze, Ph.D.
• Tox Karen M. Schweikart, Ph.D.
• Tox James O. Peggins, Ph.D.
• Former Tox Adaline C. Smith, Ph.D., DABT
• Sec Victoria Cordelli

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TPB CONTACT INFORMATION

• Phone No 301-496-8777
• Fax No 301-480-4836
• E-mail tpb_at_dtpax2.ncifcrf.gov
• Web Address http//dtp.nci.nih.gov/branches/tpb/i
  ndex.html

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Thank you,

• Are there any Questions?

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TPB TOX OMICS INITIATIVE Collection of
Frozen Samples During Toxicology and Pharmacology
Studies
NEW TPB INITIATIVES
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TYPICAL TOXICOLOGY STUDY DESIGN
Toxin-Treated Rat
Collect Target Organs Other Tissues Blood
Samples
Tissues
Plasma (Tissue)
Blood
Tissue Samples
DRUG ANALYSIS
REPOSITORY
HISTOPATHOLOGY
CLINICAL PATHOLOGY
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Tissue Collection for Tox Omics

• Brain, heart, kidney, liver, lung routinely
• Target tissues, eg, bone marrow, intestines
• Collection adjacent to histology sections, up to
  5 100mg samples
• Rapid collection and freezing, ie, liquid
  nitrogen
• Store in 4 mL cryotubes at approx. -70ºC

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Serum Samples for Tox Omics

• One or two 500 µL aliquots of serum will be
  collected per time point in non-rodent species.
  100 µL aliquots will be collected from rodents.
• Time points will depend on species and study
  design.
• At necropsy or unscheduled sacrifice, five 500 µL
  aliquots of serum will be collected from
  non-rodent species and five 200 µL aliquots will
  be collected from rodents.
• Serum will be rapidly frozen in 1 mL cryotubes
  and stored at approx. -70ºC.

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TOX OMICS STUDY DESIGN
Toxin-Treated Rat
Collect Target Organs Other Tissues Blood
Samples
RNA
(Blood)
Tissues, PBMCs, etc.
Target Organ Tissues
GENOMICS
All Samples
Serum
BIOMARKERS
PROTEOMICS
REPOSITORY
2D GEL
AND/ OR
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CURRENT TOXICOLOGY STUDY DESIGN
Tissues, BAL, PBMCs, etc.
BIOMARKERS
Toxin-Treated Rat
Collect Target Organs Other Tissues Blood
Samples
RNA
GENOMICS
Blood
D A T A B A S E
Tissues
HISTOPATHOLOGY
Plasma Tissue
All Samples
Blood
DRUG ANALYSIS
Serum
Target Organ Tissues
HEMATOLOGY CLIN CHEM
PROTEOMICS
REPOSITORY
Endpoints Timing are Agent-Specific