Preclinical-Nonclinical Testing Objectives
- 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)
Nonclinical (Toxicity) Testing
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
Drug data submitted by the Sponsor
- 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
Toxicology Written Summary contains
- 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
In Vivo Toxicity Testing lacks
extrapolation to human in vivo exposure is affected by their lack of ADME aspects
Is usually tested on one cell type only.
Principles of In Vivo Toxicity Testing
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
Pharmacokinetics studies
Pharmacokinetics after single & repeated administration
OECD Testing Guideline 417 (Toxicokinetics)
- 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.)
Distribution: Plasma drug levels
elimination half life
volume of distribution
plasma concentration
elimination rate constant
clearance
bioavailability
Acute Toxicity study
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
Repeat-dose toxicity studies
- 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
Clinical Chemistry Parameters
calcium, glucose, creatinine, total protein, cholestrol
Haematology
haemoglobin, platelet count
Urinalysis
pH
volume,
SG
glucose
Genotoxicity studies
- 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
Ames Test for Mutagenicity
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
Chromosomal damage (clastogenic) assays
chromosomal aberrations, micronucleus assay
& sister chromatid exchange (SCE).
Micronucleus Assay
Looks for potential to cause chromosome damage or spindle toxins.
Carcinogenicity studies
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)
Stages of cancer
Initiation, transformation, promotion, progression.
Normal, preneoplastic, neoplastic, benign neoplasm, malignant neoplasm
DART Testing
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
Local Tolerance Studies
- 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
Sensitisation (allergy) Tests
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
Microarray analysis studies
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
