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Lecture Contents -- Unit 5 Preclinical Studies Pharmacological profiling in vitro ex vivo in vivo Toxicology
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Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Dec 25, 2015

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Wilfrid Davis
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Page 1: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Lecture Contents -- Unit 5

• Preclinical Studies– Pharmacological profiling

• in vitro• ex vivo• in vivo

– Toxicology

Page 2: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

The Blurring Line Between Discovery, Pharmacology, and Preclinics

• Originally,– Discovery meant synthesis, screening, and in

vitro profiling,– Pharmacology meant activity and side effect

profiling in animals,– Preclinics meant dose finding in animals

• Current concepts involves an integrative approach with constant feedback

Page 3: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Why do Early-Stage Drugs Fail?

• Animal pharmacokinetic problems (33%)

• Animal toxicity (11%)

• Human toxicity (9%)

• Given the ethical implications and the high cost of animal experiments, good in vitro models for ADME testing are called for

Page 4: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Cell Culture can Provide Surrogate Models for Some Animal Experiments

Page 5: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Tissue Preparationsfor in vitro Liver Metabolism Studies

• S-9 subcellular fractions – contains liver enzymes in membrane-bound and soluble

form, but no nuclei or mitochondria

– capable of phase I and II metabolism

• Microsomal subcellular fractions– contains endoplasmatic reticulum vesicles

– capable of phase I but not phase II metabolism

• Hepatocytes• Liver slices

Page 6: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Ex vivo Receptor Imaging

Labeled vitamin Dreceptors on ratduodenum

Page 7: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Isolated Organ Receptor Models

• Guinea pig ileum– Muscarinic acetylcholine receptors (M3)– Histamine H1 and H3 receptors– Serotonine 5-HT3 receptors– Angiotensin AT2 receptors

• Rabbit jejunum– Alpha-2 adrenoceptors

• Rabbit aorta– alpha-1 adrenoceptors– Serotonine 5-HT2 receptors

Page 8: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.
Page 9: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

At some Point, in vivo Testing Becomes Unavoidable

Albino rat

Marmoset

Minipig

Page 10: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

ADME Profiling:A Major Objective for Pharmacology

• Absorption

• Disposition

• Metabolism

• Excretion

Page 11: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Some Important Acronyms

• NOEL = No Observed Effect Level

• MED = Minimally Effective Dose(LAD = Lowest Active Dose)

• ED50 = Effective Dose 50%

• NTEL = No Toxic Effect Level

• MTD = Maximal Tolerated Dose

• LD50 = Lethal Dose 50%

Page 12: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Of Limited Value: LD50 Testing

• LD50 results can vary widely between closely related species (e.g., mouse and rat)

• Predictive value limited to dose estimation in a particular animal strain, and for a particular route of administration

• Required by regulatory authorities

Page 13: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Behaviors Explored in Animal Testing

• Reward-seeking drug abuse potential

• Learning cognitive potential

• Conflict anti-anxiety potential

• Despair antidepressant potential

• Aggression

• Socialization

Page 14: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

The Irwin Test:The Simplest Behavioral Paradigm

• After administration of the compound, rats are observed for presence of pre-defined signs:

• Spontaneous motor activity• Body posture, Straub tail, muscle tone, gait• Tremor, writhing, convulsions• Fear, aggression• Reflexes (righting, pupil, ...)• Salivation, lacrimation, defecation

Page 15: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Behavioral Testing: Abuse Potential

Model for self-administrationof drugs

Page 16: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Behavioral Testing:General Activity Monitoring

An array of beams andphotocells detectsanimal movements

Page 17: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Cognitive Testing: Spacial Learning

Training to findsubmerged platformand recall the position

Page 18: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Cognitive Testing: Spacial Learning

Figure-8 maze

Radial-arm maze

Page 19: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Analgesia: Tail-Flick Testing

A photocell monitorsthe time lapsed until theanimal flicks its tailaway from a thermallight beam

Page 20: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Exertion and Metabolic Testing

Oxygen consumptionand blood metabolitestested in a treadmillsetup

Page 21: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Anxiolytics IncreasePunished Responding

Page 22: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Behavioral Despair:Forced Swimming and Tail Suspension

• When confronted with an aversive situation without the possibility to escape, rodents become immobile after a predictable period (model for despair)

• Antidepressants and stimulants will prolong the escape-directed behavior while minor tranquilizers and neuroleptics will shorten it identification of different psychotropic classes

Page 23: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Mutant Animals for Specific Investigations

Leptin administrationcan attenuate obesityin mice with a geneticdefect in the leptin gene(ob -/- knock-outs)

Page 24: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Microdialysis:Monitoring Metabolism in situ

• A probe with a semipermeable membrane, perfused with metabolically neutral solution, is inserted into the tissue

• Net composition changes upon passage reflect the concentrations of metabolites in the interstitial space (depending on flow rate and MW)

• Dialysis solution can be “spiked” with metabolically active compounds

• Continuous, real-time monitoring

Page 25: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Microdialysis:Monitoring Metabolism in situ

The perfusion fluid is pumpedfrom the microdialysis pump (1)through the catheter probe (2)into the microvial (3). The sample istransferred to the analyzer (4) which shows results on a display (5).

Microdialysis probefor CNS metabolism

Page 26: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

What is Toxicology?

• Derived from toxicon (gr.) = arrow

• Paracelsus (1493-1541): “Dosis facit venenum” (the right dose differentiates a poison from a remedy)

• Defined as the science of adverse effects on biological systems

• Modern toxicology requires an integrated approach

Page 27: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Issues in Toxicology

• In vitro mutagenicity and genotoxicity

• Acute toxicity (LD50)

• Subchronic and chronic toxicity

• Drug interaction toxicity

• Reproductive toxicity

• Developmental toxicity

• Ecotoxicity (environmental risk assessment)

Page 28: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Mutagenicity: The Ames Test

• Various mutant strains of Salmonella are exposed to the test compound– Histidine synthesis deficiency– rfa strain: increased permeability– uvrB strain: defect in DNA repair– plasmid factor strains

Page 29: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Mutagenicity:Looking for Polyploid Cells

Normal mitosisin healthy liver cell

Polyploid liver cellafter exposure to mutagen

Page 30: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

The Liver: Great Metabolizer, and First Target of Toxicity

Page 31: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

Developmental Toxicology

TeratogenicityEmbryotoxicity

Page 32: Lecture Contents -- Unit 5 Preclinical Studies –Pharmacological profiling in vitro ex vivo in vivo –Toxicology.

The Challenge After Pharm/Tox

„Shall we risk it?After all, it has cured rats, and primates have survived...“