M6 ADME Assays Notes

From 1991 to February, a study showed that 39% of failures in the preclinical phase (animal models) were due to poor pharmacokinetic properties.
By February, only 8% of failures were due to poor pharmacokinetic properties.
This significant reduction was attributed to the use of in vitro models to predict pharmacokinetic properties.

Pharmacokinetics: What the body does to the drug (absorption, distribution, metabolism, excretion).
Pharmacodynamics: What the drug does to the body (toxicity studies).

When a drug is taken orally, it's important to know:
- Where the drug goes.
- How long it takes to reach tissues.
- The concentration in the blood over time.
These parameters are difficult to know precisely, so approximations are used.

Clearance: How efficiently the body removes the drug from systemic circulation.
Volume of Distribution: The concentration of the drug in the plasma relative to the total amount of drug in the body.
Half-Life: The time it takes for drug concentration to reduce by 50%.
Bioavailability: The amount of drug that reaches systemic circulation compared to intravenous bolus injection.

To improve these parameters, it's essential to understand ADME:
- Absorption: How the drug is absorbed in the GI tract.
- Distribution: How the drug is distributed systemically in circulation, extracellular fluid, and tissues.
- Metabolism: How the drug is metabolized, primarily by the CYP450 enzyme system in the liver.
- Excretion: How the drug is excreted by the kidneys.
Alterations in the molecule's structure can affect these parameters.

Drug concentration in the blood increases when absorption is greater than excretion.
Maximum concentration ( $C_{max}$ ) is achieved when absorption equals excretion.
Drug concentration decreases when absorption is less than excretion.

Absorption, distribution, metabolism, and excretion can be assessed in vitro to predict a lead compound's suitability for in vivo studies.

Absorption is affected by the permeability of the lead molecule to cross barriers.
Simple model: Molecules are placed in chamber A, separated from chamber B by a membrane.
- The diffusion rate from A to B measures permeability (passive diffusion).
Alternative model: Replace the membrane with a monolayer of cells (e.g., Caco-2 cells).
- Caco-2 cells express transport proteins, allowing measurement of active transport.
- Transport activity can impact absorption, distribution, and excretion.

Metabolism involves phase one and phase two reactions.
- Phase one: oxidation, hydrolysis, reduction.
- Phase two: conjugation.
The goal is to increase the polarity of the lead compound for efficient excretion by the kidneys.
CYP450 enzymes are crucial in phase one metabolism.

Isolate hepatocytes or liver microsomes via differential centrifugation.
Incubate microsomes with and without the lead compound.
Also, incubate with specific CYP450 inhibitors.
Determine the drug's effect using CYP450 substrates, inhibitors, and inducers, along with the lead compound.
If the lead compound is metabolized in the presence of microsomes, CYP450 is involved.
If an inhibitor reduces metabolism, that specific CYP450 isoform is involved (e.g., CYP3A).