Week 8

Question 1

Preclinical-Nonclinical Testing Objectives

Answer 1

• is the compound biologically active?
  Preclinical: Pharmacodynamics (in vitro; in vivo)
• is the compound safe?
  Nonclinical: Safety Pharmacology & Toxicity (in vitro; in vivo)
• what is the dose-response for the activity & safety?
  Nonclinical: Pharmaco/Toxicokinetic studies (“ADME”, in vitro; in vivo)

Question 2

Nonclinical (Toxicity) Testing

Answer 2

important part of preclinical evaluation: demonstrate safety of product prior to human clinical trials by testing
animal and in vitro models

• Requires Good Laboratory Practice (GLP) studies conducted to OECD Guidelines where available
• Application made by pharmaceutical company (“sponsor”) for registration of medicinal products
• Sent to Therapeutic Goods Administration (TGA), Commonwealth Department of Health, Canberra

Question 3

Drug data submitted by the Sponsor

Answer 3

• TGA Module 1 – Regional (Aust.) administrative information
• EU Module 2 – CTD summaries
• EU Module 3 – Quality (chemistry & quality control)
• EU Module 4 – Safety (non-clinical study reports)
  (pharmacology & toxicology data, “preclinical assessment”)
• EU Module 5 – clinical study reports

Question 4

Toxicology Written Summary contains

Answer 4

• Brief Summary
• Single-dose Toxicity
• Repeat-dose Toxicity
• Genotoxicity – in vitro, in vivo
• Carcinogenicity – long-term studies, short or medium-term studies
• Reproductive & Developmental Toxicity – fertility & early embryonic
  dev., embryo-fetal dev., pre- & post natal dev., offspring dosage
• Studies in Juvenile Animals
• Local tolerance – corrosion, irritation, sensitisation, phototoxicity
• Other Toxicity Studies – antigenicity, immunotoxicity, mechanistic
  studies, dependence, metabolites, impurities, other
• Discussion and Conclusions

Question 5

In Vivo Toxicity Testing lacks

Answer 5

extrapolation to human in vivo exposure is affected by their lack of ADME aspects

Is usually tested on one cell type only.

Question 6

Principles of In Vivo Toxicity Testing

Answer 6

Test compound administered to ID doses causing:
* no adverse effect
* major toxicity

Use two routes of administration:
* Route intended for human use (e.g. oral)
* Intravenous administration

Animals are closely monitored following dosing for the effects of the compound
Maximum possible PK & toxicological information is collected during the study

Question 7

Pharmacokinetics studies

Answer 7

Pharmacokinetics after single & repeated administration

Question 8

OECD Testing Guideline 417 (Toxicokinetics)

Answer 8

• Species – as per pharm/tox studies
• Number of animals – 4 males + 4 females
• Routes of administration – clinical use (oral); i.v. (to calc. “F”)
• Dose levels – single dose & 14 day repeat dose studies
  (incl. low “NEL”, 2 intermediate, high doses)
• Observations – ADME parameters (blood, urine, tissues, etc.)

Question 9

Distribution: Plasma drug levels

Answer 9

elimination half life
volume of distribution
plasma concentration
elimination rate constant
clearance
bioavailability

Question 10

Acute Toxicity study

Answer 10

Acute Toxicity study:
* Effects observed (usually in rodent & 2 nd species) after a single dose for a 14 day period
i.e. mortality, clinical signs (lethargy, body weight changes, etc.)
* Investigation of possible target organs by full autopsy
Timeline: Usually after successful Genotoxicity Testing
The information you want to get:
* Spectrum of toxicity
* Maximum Tolerated Dose (MTD)
* No (Observed) Adverse Effect Level, N(O)AEL
* Gender-based differences in reactions

Question 11

Repeat-dose toxicity studies

Answer 11

• Sub-acute (14 day), sub-chronic (3-6 months) & chronic (1-2 yrs) studies (usually oral dosage used for
  longer exposures)
• In 2 relevant species, both genders
• To determine target organs and dosage protocols for chronic studies

Measurements:
* food/water consumption, body weights
* any other observed abnormalities (e.g. neuronal)
* full autopsy and clinical chemistry on blood haematology & urinalysis
* major tissues examined histologically

Question 12

Clinical Chemistry Parameters

Answer 12

calcium, glucose, creatinine, total protein, cholestrol

Question 13

Haematology

Answer 13

haemoglobin, platelet count

Question 14

Urinalysis

Answer 14

pH
volume,
SG
glucose

Question 15

Genotoxicity studies

Answer 15

• In vitro non-mammalian cell system – e.g. gene mutation assays (Ames Test)
• In vitro mammalian cell system – e.g. chromosomal & DNA damage (unscheduled DNA Synthesis)
• In vivo mammalian system – e.g. chromosomal damage (clastogenic) assays & other genotoxic effects; transgenic mice

Question 16

Ames Test for Mutagenicity

Answer 16

bacterial mutagenicity assay

Salmonella His - mutants are mutated to wild type
- “reverse mutation”
* Rat liver fraction (S9) added to provide mammalian. CYP metabolism
* Quick screening assay used on all new compounds

Question 17

Chromosomal damage (clastogenic) assays

Answer 17

chromosomal aberrations, micronucleus assay
& sister chromatid exchange (SCE).

Question 18

Micronucleus Assay

Answer 18

Looks for potential to cause chromosome damage or spindle toxins.

Question 19

Carcinogenicity studies

Answer 19

Long-term studies: 2 yr Chronic toxicity
- given orally in feed at 3 doses:
* “ high” (“MTD” or 100x rec. human dose)
* “medium”
* “low” (no effect level, “NEL”)

Measurements:
* food/water consumption, body weights
* full autopsy/histopathology & clinical chem. at 0.5, 1, 1.5, 2yr
* type & incidence of tumours*
* any other observed abnormalities (e.g. neuronal)

Question 20

Stages of cancer

Answer 20

Initiation, transformation, promotion, progression.

Normal, preneoplastic, neoplastic, benign neoplasm, malignant neoplasm

Question 21

DART Testing

Answer 21

developmental and reproductive toxicology testing includes:
1. Fertility and general reproduction studies
2. Teratogenicity studies
3. Perinatal studies

Segment I: Male & Female Fertility and General Reproduction
* DONE IN ONE SPECIES ONLY (rat)
* M & F exposed to drug prior to mating for 10 or 2 weeks
(dosing from gametogenesis to implantation)
* F0 generation; exposed animals also mated with unexposed animals to determine sex-specific toxicity on reproductive performance
Measurements: fertility and fetal survival
Segment II: Embryo-Foetal Developmental Toxicity/Teratogenicity
* DONE IN TWO SPECIES (rat & rabbit)
* Effects of drug exposure on organogenesis in fetus during pregnancy (or extended dosing from implantation to just before parturition)
* In F0 dams and F1 offspring; includes 1st trimester (positive teratogen = aspirin)
Measurements: maternal condition; fertility, litter size, pup weight, resorption rate; fetal autopsy & histology for type & incidence of malformations (tissues & skeletal)

Segment III: Peri-/Post-Natal Development & Multi-generation
studies
* DONE IN ONE SPECIES ONLY (rat)
* For effects of exposure during the last trimester of pregnancy and
subsequently through to lactation & nursing (extended dosing to sexual maturation for juvenile tox study)
* In F0, F1 and F2 generations
Measurements: as for segment II: reduced birth weight, postnatal survival

Question 22

Local Tolerance Studies

Answer 22

• corrosion tests – in vitro tests have replaced many in vivo tests
• irritation tests – in vivo dermal & eye tests being replaced by in vitro tests
• sensitisation tests – irritant & allergic contact dermititis (immune mediated)
• phototoxicity – photoactivation by light exposure

Question 23

Sensitisation (allergy) Tests

Answer 23

Multiple exposures required for allergic response (measured as
erythema & oedema), due to 3 phases:
- Induction phase (sensitization occurs)
- Rest period (lymphocyte proliferation & distribution)
- Challenge phase (elicitation of respons

Question 24

Microarray analysis studies

Answer 24

Use of gene expression patterns to identify toxic potential
* Molecular biology technique: gene microarrays (‘gene chips’)
simultaneously measure changes in expression of 1000s of genes to screen for toxicity in target organs

Question 25

In Silico Prediction of Toxicity

Answer 25

In silico computer software tools rapidly screen new “lead” compounds for toxic potential early in drug development.

Question 26

What are the
advantages/disadvantages of the SCE test compared to the Ames test?

Answer 26

The advantage of the SCE test over the Ames test is the ability to identify physical exchange of DNA. SCE provides fast and easy in vitro results with results generally indicating mutagenic carcinogen. Ames test requires a higher concentration of compounds than SCE which has extremely sensitive detection.
The disadvantages of SCE tests can provide false negative or positive results depending on the metabolic activation, Ames test can also provide false results however it is more expensive and time consuming. Compounds that can induce double-strand DNA breaks can be undetected due to SCE’s insensitivity to compounds like some glycopeptides.