Drug Development: Toxicology

Question 1

Standard in vivo toxicology screens

Answer 1

Single Dose acute studies
Dose-ranging studies
One-month repeat dose studies
Six-Month repeat dose studies

Question 2

Single Dose acute studies

Answer 2

-Determine acute toxicity and find NTEL

• Animals dosed and adjusted depending on survival at 24h
• Repeated in small groups for ~15d to determine NTEL

Question 3

Dose-ranging studies

Answer 3

• Determine toxicity from multiple doses and establish MTD
• Rodent/non-rodent groups dosed for 7-10d
• Clinical chemistry/haematology throughout test

Question 4

One-month repeat dose studies

Answer 4

Support clinical stages and identify target organs/tissues

-Rodent/non-rodent groups dosed daily using proposed route
Detailed monitoring/terminal full autopsy
Recovery group

Question 5

Six-month repeat dose studies

Answer 5

Similar to one month repeat dosing

Reqd for phase 2-3 clinical trials

Question 6

Specialised in vivo tests

Answer 6

Oncogenicity studies

Reproductive studies

Question 7

Oncogenicity studies- purposes

Answer 7

Drugs with intended long use
Drugs with slow excretion rates
If mutagenicity tests positive

Question 8

Oncogenicity studies features

Answer 8

24 months in 2 rodent species administered by gavage (oral suspension)
Animals regularly examined for solid tumours/neoplasms

Question 9

Types of reproductive toxicity tests

Answer 9

Fertility studies
Teratology studies
Post-natal development

Question 10

Fertility studies features

Answer 10

Males- pregnant females sacrificed
- Position, resorption site, weight, sex of live foetuses noted

Females- as above; also observe oestrous cycles and abnormal weaning behaviour

Question 11

Teratology studies features

Answer 11

For drugs where women of child-bearing age may be patients

• Dosed during organogenesis , sacrifice 1d pre-parturition
• Autopsy- effects on organ development

Question 12

Post-natal development

Answer 12

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

Question 13

Use of Zebrafish for in vivo tox

Roche

Answer 13

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

Question 14

In vitro toxicology assays

Answer 14

Ames Tet
Mammalian Cell Mutation test (Mouse Lymphoma L5178Y cells)
In vitro chromosome aberration
In vitro micronucleus test
In vivo comet assays

Question 15

Ames test

Answer 15

Salmonella typhimurium grown

Question 16

Ames test adv and disadv

Answer 16

+Cheap, fast, easy to interpet
+High sensitivity

• False negatives
• Not all carcinogens are mutagens

Question 17

Mammalian Cell Mutation test

Answer 17

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

Question 18

In vitro micronucleus test

Answer 18

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

Question 19

In vitro chromosome aberration

Answer 19

Blood lymphocytes incubated for 48h then test drug added; cells arrested in metaphase by colchicine
Number of cells with chromosome aberration (breakage) recorded

Question 20

Comet assays

Answer 20

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

Question 21

In silico models

Answer 21

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

Question 22

Tethered hepatocyte spheroids vs existing models

Answer 22

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

Question 23

Example of application of in vitro tests

Answer 23

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

Question 24

Reproductive toxicity of Antipsychotics

Answer 24

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