Preclinical Testing Flashcards

1
Q

Data from lead optimisation

A

Preliminary CMC (Chemistry, Manufacture and Control)
*Formulation
*Stability of active ingredient
*Detailed physicochemical characterization
*Impurity analysis

Benchmark in vivo models
*In vivo models
*Validated disease models
*Models in other disease areas

ADME Profiling
*Optimized analytical method development
*Pharmaco-kinetic(PK) profile
*Oral bioavalibility
*Determine metabolism of drug

Preliminary Toxicology (non-GLP)
*Maximum tolerated dose(MTD)
*Repeat Dose toxicity (non-GLP)
*Preliminary Cardiovascular Safety

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2
Q

GMP

A

– Good Manufacturing Practice: is a quality system covering the
manufacture and testing of active pharmaceutical ingredients,
diagnostics, foods, pharmaceutical products, and medical
devices. Guidelines that outline the aspects of production and
testing can impact on the quality of a product

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3
Q

GLP

A

– Good laboratory practice: a system of management controls for
laboratories and research organizations to ensure the
consistency and reliability of results

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4
Q

GCLP

A

– Good clinical laboratory practice: a framework for a quality
system in analysis of clinical trial samples, ensuring GCLP
compliance overall of processes and results

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5
Q

Data from initial preclinical stage

A

Detail Preliminary CMC
ICH Stability
*ICH Impurity Analysis
*Develop prototype clinical formulation (pill, liquid, etc.)

Comprehensive ADME
*GLP PK profile
*GLP toxicokinetics (TK) profile
*Comprehensive determination of metabolites

GLP Toxicology Package
*Acute Tox study
*Subchronic repeat dose study
*Genotoxicity Battery
*Safety Pharmacology Core Battery

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6
Q

Chemical development

A

Chemical Development
* Improvement of the synthesis
– To reduce cost,
– To increase output, safety and quality (purity and consistency)
* Chemical development is non-GMP
* Chemical manufacturing (supplies for clinical trials) must be GMP

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7
Q

Salts

A

Salt and Formulation
* Find best salt for
– Stability
– Ease of formulation
* Find best formulation for chosen route of administration
– Tablet, capsule, solution, controlled release, protection in GI tract, etc.

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8
Q

Optimized analytical method development

A

Optimized analytical method development
Crucial to demonstrate exposure levels in toxicology studies – human starting doses are based on this data!

*Small molecules - HPLC/MS
-Can definitively show the molecular structure

*Biologics (proteins, antibodies, etc.) - ELISA
-Does not show structure
-Uses binding as an endpoint
-Does not demonstrate activity

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9
Q

Assay Validation

A

Assay needs to be validated for use in GLP studies and be performed GLP
-Extraction technique recovery
-Linearity of standard curve
-Intra- and inter-assay precision
-Bench top and freeze/thaw stability
-Sensitivity (lower limit of quantitation; LOQ)
-Establish Quality Control (QC) standards

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10
Q

Bioavailability

A

Bioavailability and PK (BAPK)
* Bioanalytical method development and validation
– use to quantify drug and/or metabolites usually in plasma
* Bioavailability – single dose, iv and intended route
* PK – rodent and non-rodent, drug availability by intended route, mean residence time, half-life
* Blood-brain barrier bioavailability – measure drug accumulation in brain, brain vs plasma levels

Interspecies scaling improves human PK predictions = better clinical trial dosage design

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11
Q

Metabolic Profile

A
  • Comparative metabolism
    – Important when interspecies differences are observed
    – Hepatic microsomes and cytosolic fractions from different species : human, mouse, rat, rabbit, dog, non-human primate, guinea pig, etc
    – Time course of loss of parent compound
    – Metabolite identification
  • Metabolic inhibition
    – Potential drug-drug interaction
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12
Q

Toxicology Evaluation

A

*Appropriate species – one rodent, one second species (dog, pig or monkey generally)
-Good exposure
-Metabolism similar to human – must cover all human metabolites
-Same pharmacologic activity as humans (same target binding, effect in disease models, pharmacologic effects)
*Exposures achieved in test species should be sufficient to cover multiples of the intended human dose/exposure in order to establish a safety margin
*Higher doses to evaluate possible toxicities that could occur
-FDA guidance to dose up to 1 g/kg, if possible
*Administer compound long enough to support intended clinical study
*Example Endpoints: body weight, clinical observations, serum chemistry, hematology, organ weights, histology, drug exposure (toxicokinetics)

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13
Q

Single-Dose (Acute) Toxicity (Non-GLP)

A

Single-Dose (Acute) Toxicity (Non-GLP)
* Determination of adverse effects within short time frame of single dose administration
* Animals observed for 14 days after dosing
* Minimal endpoints focused on clinical observation and may be non-terminal (e.g. dogs)
* Identifies single dose MTD

Range-finding
– Longer schedule of repeated dosing
– Establish dosage levels for subsequent toxicity studies

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14
Q

Acute and Repeated-Dose Toxicity (GLP)

A
  • Acute and repeated dose toxicity studies in 2 species (rodent and non-rodent) selected from non-GLP range-finding studies.
  • More comprehensive: (greater number/gender/species
  • More complete toxicology study.
  • Determination of adverse effects resulting from daily dosing to identify MTD and NOAEL (No Observed Adverse Effect Level)

Clinical Endpoints
-Survival
-Body weight
-Clinical signs
-Behavior (Eating, movement)

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15
Q

Genotoxicity/Mutagenicity Testing (GLP)

A
  • in vitro non-mammalian cell system – e.g. Ames Test – Salmonella typhimurium
  • in vitro mammalian cell system – e.g. CHO (Chinese Hamster Ovarian) cells -> Determine % chromosomal aberration across a range of drug concentrations
  • in vivo mammalian system –e.g. mouse micronucleus assay ->
    – Immature mice treated with mutagen for period of 2-4 weeks
    – RBCs observed under microscope for increased % of micronuclei
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16
Q

Assay pros and cons

A

In Vitro Assays
-easy to perform
-inexpensive
-fast results
-overly sensitive
- non-mammalian cells(Ames Test)
In Vivo Assays
-more accurate representation of human
-Requires a trained mouse researcher
-More expensive

17
Q

Carcinogenicity Testing - GLP

A

Carcinogenicity Testing - GLP
* Long term toxicity testing - ~lifetime exposure
* Usually in rats – 24 – 30 months
* Mouse or hamster may also be used
* 50 / gender / dose level and 100 / gender / control group
* Determine potential tumorigenic effects of drug

18
Q

Reproductive toxicology - GLP

A
  • Fertility and general reproductive performance – Rats
    – Dosing of males for 60-80 days prior to mating
    – Dosing of females for 14 days prior to mating and during gestation and lactation
  • Potential drug-induced embryotoxicity and teratogenicity
    -Rat/mouse and Rabbit
    -Escalating dosages
    -1 month of treatment in pregnant females during embryonic and fetal development.
  • Late fetal development, labour, delivery, lactation and newborn viability (Rat or Mouse)
    – pregnant females using escalting dose levels
    – Dosing from last gestation day (day 16-17) to end of weaning
    – If reproductive capacity of offspring is evaluated, study duration is 5-6 months
19
Q

Respiratory System – In Vivo

A

Whole Body Plethysmograph Chambers - Rats
*Indirect Volume Measurement: pressure change = inhaled gas expansion
*Measure Tidal Volume and Respiratory Rate before and after drug

20
Q

QT studies

A

Ion Channels and Cardiac Potential
-QT interval prolongation can result in Torsades de Pointes (TdP): heart arrhythmia – sudden cardiac arrest
-QT elongation is mediated by inhibition of hERG ion channel
-Withdrawn drugs due to TdP: Prenylamine, Lidoflazine, Terodiline, Astemizole, Grepafloxacin, Terfenadine….

21
Q

Cardiovacular Toxicity Studies – In Vitro

A
  • Recording of K+ current from hERG-expressing CHO cell line
  • Aims to generate full concentration-effect curve
  • Data plotted against measured drug concentration
22
Q

Irritation and sensitization testing - GLP

A

Irritation and sensitization testing - GLP
Other tests that may or may not be required:
*Rabbit eye test
*Skin tests- rabbit, guinea pig

23
Q

Translating Dosage from Animals to Humans

A
  • Maximum Recommended Starting Dose (MRSD) is converted from NOAEL in animal studies converted to HED divided by a safety factor of at least 10.
  • Human equivalent dose (HED) can be converted from animal dosage as a ratio of body weight, normalized by body surface area (BSA).
    This is Allometric scaling.
  • Isometric scaling (straight conversion based on body weight) may lead to overestimation of human dosage and/or underestimation of toxicity of a given dose.
  • Allometric scaling takes into account lower metabolism of larger animals (humans) compared to smaller animals (rodents, dogs, etc.).
  • Max Rubner (1883) demonstrated that while the ratio of blood volume to body weight decreases in larger animals, blood volume is constant to body surface area.
24
Q

Dosage Translation Calculation

A
  • USFDA suggest allometric scaling based on species K m when converting dosage from animal to human.
  • Km = body weight (kg) divided by BSA (m 2)
  • HED (mg/kg) = animal dose (mg/kg) × (animal K m /human Km )