IND-enabling safety evaluation

(John E. Sagartz, Seventh Wave Laboratories)

Definitions and concepts:

  • TI – therapeutic index, ratio between the dose that produces toxic effects to the dose needed for therapeutic response (typically the 50% dose-response points are used); a higher ratio indicates a safer drug
  • NOEL – no observable effect level
  • NOAEL – no observable adverse effect level
  • LOEL – lowest observed effect level
  • MTD – maximum tolerated or minimally toxic dose
  • TEM – therapeutic exposure multiple; exposure at NOAEL divided by exposure at maximum human dose (exposure usually determined by AUC ratios)
  • STD10 – severely toxic dose in 10% of rodents (cancer indication)
  • HNSTD – highest non-severely toxic dose in non-rodents (cancer indication)
  • toxicokinetic parameters:
    • AUC = area under the plasma concentration vs. time curve
    • Cmax = maximum plasma concentration
    • Tmax = time to achieve maximum plasma concentration
    • T1/2 = elimination half-life
    • F = percent bioavailability

Nonclinical safety assessment is based on International conference on harmonization (ICH) guidelines

  • Harmonization of expectations between Europe, Japan, and the U.S. to improve the safety, efficacy, and quality of drugs
  • Necessary to evaluate:
    • Genetic toxicity (ICH S2)
    • Safety pharmacology (ICH S7A,B)
    • General toxicology with exposure assessment (ICH M3(R2), ICH S3)

Genetic toxicity (ICH S2)

  • Invitro and in vivo tests to evaluate the potential to induce mutations and chromosomal damage (e.g. bacterial mutations, cytogenetics, mammalian gene mutations)
  • The “standard battery” for genotoxicity:
    • Bacterial mutations – Ames assay (bacterial reverse mutation assay), identifies comparatively subtle effects on chromosomes (point mutations, substitutions, frame shifts) using tester strains of E. coli and Salmonella typhimurium; cost effective for exploratory programs
    • Cytogenetics – in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells (chromosomal aberrations assay)
    • Mammalian mutations – in vivo test for chromosomal damage using rodent hematopoietic cells

Safety pharmacology (ICH S7)

  • In vitro and in vivo studies conducted to determine whether the compound has any effects on the CNS, respiratory system, and cardiovascular system.
  • The “core battery” of tests:
    • Cardiovascular – ionic current assay (hERG) followed by in vivo QT assessment; in vivo studies in anesthetized or conscious telemetrized dogs or monkeys evaluate both electrophysiology and hemodynamics
    • Respiratory – can be included in some in vivo cardiovascular or general toxicology studies; usually single dose study in rats
    • Neurobehavioral – Functional Observational Battery or Irwin Assay to evaluate learning/memory, muscle grip strength, cranial nerve functions, and open field motility; usually single dose study in rats
  • Additional studies may be triggered by results from the general toxicology studies:
    • Immunotoxicology (NK and CMI parameters, immunization studies, etc.)
    • Renal toxicology
    • Gastrointestinal pharmacology (motility)

General toxicology (ICH M3(R2), ICH S3)

  • Requires two species, at least one of which is not a rodent
    • Generally: rats + either cynomolgus monkey or dog; pigs, rhesus monkeys, and ferrets are sometimes used as second species
  • No single study design or group of studies will fit all molecules – the program will need to be tailored to clinical plan, availability of API, PK/metabolism data, “formulatability”, etc.
    • In the U.S., the duration of preclinical studies supporting human trials is generally equal units of time.
    • Duration of dosing is guided by indication and anticipated duration of therapeutic use.
      • Acute toxicity
      • Sub-chronic (2, 4, 13 weeks of daily dosing – sequence dependent on requirements for clinical trial support)
      • Chronic (6 months in the rodent species, 9 months in the non-rodent species)
    • Dosing is via the intended route of human exposure

Dosing at the highest level will help explore toxicology early.

  • A low dose ideally is not associated with toxicity (establish TI).
  • A middle dose will establish NOEL/LOEL.

Endpoints for evaluation of toxicity:

  • Clinical observations – at least once daily, ideally conducted at/near Tmax (time to achieve maximum drug concentration in plasma)
  • Body weight and food consumption – daily weight measurements for short duration studies, twice weekly for 4 weeks or longer studies
    • very sensitive measure – best and easiest early measure of toxicology *cost effective for exploratory programs
  • Ophthalmic examination – conducted pre-study and during the last week
  • Electrocardiography (non-rodent) – conducted pre-study and during the last week
  • Clinical pathology – conducted pre-study or during the last week (non-rodent) or at/near termination (rodent); involves clinical chemistry, hematology, urinalysis, coagulation
  • Gross post-mortem evaluation – external examinations to include any in-life observations; evaluation of body cavity (thoracic, abdominal, pelvic, cranial)
  • Organ weights – both absolute and relative measurements (relative to brain weight, terminal body weight); typically includes adrenal gland, brain, epididymis, heart, kidney, liver, lung, ovary, pituitary, salivary gland, spleen, testis, thymus, thyroid/parathyroid
    • Second most sensitive measure after overall body weight
    • *Thymus weight is best choice for exploratory studies
  • Histopathology – microscopic examination of tissue sections (formalin-fixed, paraffin-embedded, H&E-stained); includes gross:micro correlation (see TABLE 4 for tissues examined)
    • Non-rodent – all tissues from all animals are examined
    • Rodent – initial comparison of high dose with control; “read down” of target tissues in low, mid, and recovery groups

Toxicokinetic studies (ICH S3)

  • Verify exposure to the test material in dosed groups and lack of exposure in control groups.
  • Measure various toxicokinetic parameters which are important in evaluating toxicology

Toxicology data interpretation

  • Multiple measures (NOEL, NOAEL, LOEL, MTD, TEM, STD10, HNSTD) and factors serve are important for human risk assessment, including:
    • Ability to identify NOAEL
    • Existence of biomarkers of injury
    • Reversibility of finding(s)
    • Likely relevance to humans
    • Known or postulated mechanism of toxicity

Determining the necessary exposure multiple (TEM)

  • There’s no set answer – it’s dependent on therapeutic indication, unmet medical need, nature of toxicity observed in animals, ability to monitor toxicity clinically, and reversibility of toxicity.
  • The therapeutic exposure window does not widen with increasing duration of dosing (stays the same or narrows).
  • For early short-duration studies, TEM should be as wide as possible.

Minimum requirements for IND application

  • Genetic toxicity standard battery
  • Safety pharmacology core battery
  • General toxicology with toxicokinetics (2 species, duration sufficient to cover duration of initial clinical studies)
  • All studies conducted according to Good Laboratory Practices (GLP)
  • See FIG. 2 for overall toxicology profile

Using binary inexpensive measures at an early stage will help predict further toxicology results later on (e.g. Ames assay, MTD).

  • For exploratory studies, estimated cost for acute rodent studies is on the order of $15-20K. Exploratory range finding studies in dogs/monkeys can be in the range of $40-50K.
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