Drug Development: Toxicology Flashcards
Standard in vivo toxicology screens
Single Dose acute studies
Dose-ranging studies
One-month repeat dose studies
Six-Month repeat dose studies
Single Dose acute studies
-Determine acute toxicity and find NTEL
- Animals dosed and adjusted depending on survival at 24h
- Repeated in small groups for ~15d to determine NTEL
Dose-ranging studies
- Determine toxicity from multiple doses and establish MTD
- Rodent/non-rodent groups dosed for 7-10d
- Clinical chemistry/haematology throughout test
One-month repeat dose studies
Support clinical stages and identify target organs/tissues
-Rodent/non-rodent groups dosed daily using proposed route
Detailed monitoring/terminal full autopsy
Recovery group
Six-month repeat dose studies
Similar to one month repeat dosing
Reqd for phase 2-3 clinical trials
Specialised in vivo tests
Oncogenicity studies
Reproductive studies
Oncogenicity studies- purposes
Drugs with intended long use
Drugs with slow excretion rates
If mutagenicity tests positive
Oncogenicity studies features
24 months in 2 rodent species administered by gavage (oral suspension)
Animals regularly examined for solid tumours/neoplasms
Types of reproductive toxicity tests
Fertility studies
Teratology studies
Post-natal development
Fertility studies features
Males- pregnant females sacrificed
- Position, resorption site, weight, sex of live foetuses noted
Females- as above; also observe oestrous cycles and abnormal weaning behaviour
Teratology studies features
For drugs where women of child-bearing age may be patients
- Dosed during organogenesis , sacrifice 1d pre-parturition
- Autopsy- effects on organ development
Post-natal development
Evaluate consequences on late uterine growth*, parturition, lactation
Female rats dosed day 6 of pregnancy to 21d post-parturition
Labour, delivery, maternal behaviour, litter size/survival, malformations, neonatal behaviour
Use of Zebrafish for in vivo tox
Roche
Quick, easy
Models cardiac function (QT prolongation- ICH guidelines)
CNS (same neurotransmitters, quantifiable learning, sleep, drug addiction, neurobehaviours)
GI (transparency- emetic liability to drugs, renal function)
Limitations: cannot do respiratory function, rapidly develop, BBB does not form until 10d, variation in drug uptake
In vitro toxicology assays
Ames Tet Mammalian Cell Mutation test (Mouse Lymphoma L5178Y cells) In vitro chromosome aberration In vitro micronucleus test In vivo comet assays
Ames test
Salmonella typhimurium grown
Ames test adv and disadv
+Cheap, fast, easy to interpet
+High sensitivity
- False negatives
- Not all carcinogens are mutagens
Mammalian Cell Mutation test
Mouse lymphoma L5178Y cells- are (Thymidine Kinase +/-)
Mutagens would remove functional copy
Cells grown in selective medium (TFT, toxic to DNA when incorporated by TK) or non-selective medium
Mutation frequency cloning efficacy in selective vs. non selective mediums
- Large growth indicates mutation
- Small growth indicates chromosome breakage
In vitro micronucleus test
Test substance administered at MTD in males and females; bone marrow is then harvested
- Bone marrow stained for presence of Micronucleated immature erythrocytes (mie)- product of chromosomal/spindle damage
- Mie values compared with vehicle control values
In vitro chromosome aberration
Blood lymphocytes incubated for 48h then test drug added; cells arrested in metaphase by colchicine
Number of cells with chromosome aberration (breakage) recorded
Comet assays
Animals treated with test drug at MTD for 24h
Hepatocytes lysed and electrophoresed
Extend of DNA liberated from comet head relates to extent of DNA damage
In silico models
CYP models
- SMARTCYP- online predictor of metabolism site
- Low signal to noise ratio in input data; easy to implement into chemical programming
-3D models- improved structual info and docking vs. 2D models- could reduce number of cells required for HTS
Tethered hepatocyte spheroids vs existing models
Conventional hepatocyte spheroids: cryopreserved hepatocytes in bilayer
- Analysis of CYP-mediated metabolism
- Expand during culture limiting O2 and drug access
Tethered spheroid model- attached to RGD/ galactose conjugated membranes
Comparable hepatocyte function level (albumin production) and urea excretory function
Example of application of in vitro tests
Sigma receptors- poorly understood, may mediate hallucinations convulsions
Haloperidol is an agonist of sigma receptors
Development of E-5842 potential new antipsychotic
- Ames test (negative result)
- Mouse lymphoma assay
- In vivo mouse lymphoma L5178Y cell assay
- Micronucleus test
- No increase in bone marrow toxicity - Significant chromosome aberrations at certain concentrations
Weight of evidence analysis based on results of these tests plus clinical conditions of use
Reproductive toxicity of Antipsychotics
2-4 Week Repeat Dosing with Sulpiride (D2 Antagonist)
Doses 1, 10, 100mg/kg
Also administered during pre-mating, mating and days 0-7 of gestation
Determine differential effects on follicle/ovarian histology
Dosing 1mg/kg for 2 or 4 weeks enough to induce adverse effects on fertility
