Week 3: Drug Transporters and Pharmacodynamics

Question 1

Drug Transporters

Answer 1

• Drug transporters have a specific directionality (either uptake or efflux mode)
• Drug transports in the epithelial cells of intestine, liver, kidney are localized to specific poles of the cell
• Enterocytes, hepatocyte, tubular cells each have one side facing the blood (basolateral membrane) and one side facing the lumen (apical membrane)
• For enterocytes, the apical membrane faces the gut lumen
• For hepatocytes, the apical membrane faces the bile canaliculi
• For kidneys, the apical membrane faces the tubular urine space

Question 2

Apical vs. Basolateral side

Answer 2

In the intestines:
APICAL (LUMINAL) SIDE: uptake transporters on the apical (luminal) side of enterocytes promote drug absorption into the body
APICAL SIDE: Efflux transporters on the apical membrane impede drug entry into the systemic circulation

In the liver:
BASOLATERAL SIDE: High activity of uptake transporters on the basolateral (blood) side, and
APICAL SIDE: Efflux transporters on the apical (bile) side of hepatocytes promotes excretion into bile and thus prevents high systemic levels of drugs

In the kidney:
BASOLATERAL SIDE: High activity of uptake transporters on the basolateral (blood) side, and
APICAL (URINE) SIDE: Efflux transporters on the apical (urine) side of tubular cells promotes excretion into urine and thus prevents high systemic levels of drugs

Question 3

Tissue Barriers

Answer 3

• True for blood-brain-barrier
• Not apical or basolateral
• Luminal (blood) vs. abluminal (tissue interstitium)
• High activity of uptake transporters on the blood side of capillary endothelial cells promotes tissue entry of xenobiotics
• High activity of efflux transporters on the blood side of capillary endothelial cells prevents xenobiotic entry into tissue
• Minimize central nervous system side effects (meds)

Question 4

Major Drug Transporter Families

Answer 4

Uptake transporters (Solute carrier, SLC)

Efflux transporters (ATP-binding cassette, ABC transporters)

Question 5

Uptake Transporters (Solute Carrier, SLC Transporters)

Answer 5

• Work as antiporters or use negative membrane potential to drive facilitated uptake
• Organic Anion Transporting Polypeptides (OATP) - transport anions, cations, or neutral molecules
• Organic Anion Transporters (OAT) - transport anions
• Organic Cation Transporters (OCT) - transport cations

Question 6

Efflux Transporters (ATP-Binding Cassette, ABC Transporters)

Answer 6

• Work as active transporters
• First found in cancer cells; most known for being able to reduce their intracellular accumulations of chemo/medications, lowering drug efficacies against tumours
• ABC transporters are found in normal body tissues
• -> P-glycoprotein (P-gp, MDR1)
• -> Multidrug resistance proteins (MRP)
• -> Breast Cancer Resistance Protein (BCRP)

Question 7

Drug Uptake Transporters

Answer 7

OATP: highly expressed in intestine, liver, BBB

• In intestine, promotes absorption
• In liver, update drug uptake for metabolism/excretion
• In BBB, not fully understood

OAT: liver and kidneys
-Promotes urinary elimination of drugs through renal secretions

OCT: liver and kidney
-Promote liver drug metabolism, urinary excretion, drug elimination

Question 8

Drug Efflux Transporters

Answer 8

• They are all similar and expressed in similar tissues
• Act to limit intestinal drug absorption, promote biliary and renal excretion, and prevent brain-rug distribution
• P-gp is expressed highly in the placenta (protects fetus from drugs in the mother’s circulation)
• BCRP is expressed highly in the mammary gland during lactation (why drugs can be found in high concentrations in milk)
• Regulate drug supply that dairy cows are treated with

Question 9

Renal Drug Transport Mechanisms

Answer 9

• Have significant impact on renal handling of medications
• A variety of uptake (blood side - OATs) and efflux (apical side - P-gp, MRP) transporters are expressed in proximal tubular epithelia.
• Inhibition of renal secretory transport is a cause for several drug-drug interactions
• Penicillin urinary excretion is inhibited by probenecid (blocks OATs)
• Penicillin is secreted by OAT transporters
• When inhibited, the renal elimination rate is decreased = longer half-life = longer dosing interval between doses
• Beneficial and purposeful drug interaction
• Digoxin urinary excretion is inhibited by quinidine (blocks P-gp; MDR1)
• Renal secretion of Digoxin is inhibited = higher concentrations of Digoxin = increased risk of CNS side effects

Question 10

Penicillin urinary excretion

Answer 10

Penicillin urinary excretion is inhibited by probenecid (blocks OATs)

Penicillin is secreted by OAT transporters

When inhibited, the renal elimination rate is decreased = longer half-life = longer dosing interval between doses

Beneficial and purposeful drug interaction

Question 11

Digoxin urinary excretion

Answer 11

Digoxin urinary excretion is inhibited by quinidine (blocks P-gp; MDR1)

Renal secretion of Digoxin is inhibited = higher concentrations of Digoxin = increased risk of CNS side effects

Question 12

P-gp (Efflux transporter)

Answer 12

• Cyclosporine (Pgp inhibitor) used with verapamil
• Without cyclosporine A, there is some verapamil in the brain (cool colours)
• In the presence of cyclosporine A, verapamil concentrations are great (warm colours)
• Cyclosporine A increases the brain concentration of verapamil
• P-gp is expressed on the lumenal (blood) cells
• Efflux reduced, more verapamil in the brain

Question 13

Pharmacodynamics

Answer 13

Quantitative description of the effect of a drug on the body.

Pharmacokinetics = what the body does to the drug

This is what effect the drug has on the body

Most (but not all) drugs exert their effects by binding to specialized macromolecules (i.e. receptors, enzymes)

Question 14

PK vs. PD

Answer 14

Closely related
Dose of drug → blood concentration
We want to be able to measure the conc of drug at its site of action but this is not feasible; blood is a surrogate for the site of action (look at what effect the drug has on the brain)
If there is not enough drug, you have to alter the dose (improve effect)

Question 15

Receptors

Answer 15

Detectors that detect the signal.

• In the simplest schematic, when receptors reside empty, they do not influence intracellular processes.
• Think of receptors as a power bars - many different slots for “drugs”
• When a drug is bound to a receptor, the receptor becomes activated and produces a biological response.
• Many drugs mimic endogenous ligands, some drugs block receptors.
• Signal transduction mediates response

*Note - a ligand is a molecule that binds to a receptor. A ligand can be a drug or an endogenous molecule

Question 16

Ligand

Answer 16

A ligand is a molecule that binds to a receptor. A ligand can be a drug or an endogenous molecule

Question 17

Drug and Receptor interactions

Answer 17

Rarely is it one type of bond that causes drug receptor binding, rather it is usually a combination of bonds.

Generally many weak bonds make up a drug-receptor interaction.

Ionic and covalent interactions are rare

Van der waals and hydrogen interactions are common

1. Van der waals interact with transient charges on other molecules (weak)
2. Hydrogen bonds are more positively polarized (stronger)
3. Ionic bonds occur when cations + anions (strong)
4. Covalent bonds share electrons (very strong)

Question 18

IMATINIB binding to Bcr-Abl Kinase

Answer 18

Specific shape (molecule) fits into the receptor’s binding pocket

Surrounding the drug = amino acids of the protein that help the drug interact (there must also be interactions with surrounding amino acids of the target)

Not just one bond that makes up the drug-enzyme interaction - many different ones

Question 19

Drug concentrations vs. receptor occupancy

Answer 19

Drug + Receptor <=> Drug Receptor Complex –> Biological effect

Kon = the rate of association (number of binding events per units time) and Koff = the rate of dissociation (the number of dissociation events per unit time).

There are several assumptions that we make when we use this model.
They include:
1. All receptors are equally accessible to drugs (almost always true).
2. Binding is reversible (almost always true).
3. Neither the drug or the receptor is altered by Kon Koff (not always true)
4. Receptors may undergo conformational change

Question 20

Law of Mass Action

Answer 20

The rate of a chemical reaction depends on the concentration of the reactants

Question 21

Equilibrium Dissociation Constant (Kd)

Answer 21

Kd is the equilibrium dissociation constant and represents the affinity of a drug-receptor pair.

• Smaller Kd = higher affinity
• Kd is calculated as Koff/Kon.
• Kd is most often influenced by its off rate (Koff) more than its on rate (Kon)
• Dissociation rate (Koff) determines how well the drug will stick to the receptor
• The Kd is expressed in units of concentration (i.e. nM or μM) and is the concentration of drug that occupies half the receptors at equilibrium.
• A small Kd means the receptor has a high affinity for the drug
• A large Kd means the receptor has a low affinity for a drug.
• The Kd represents the concentration at which 50% of the available receptors are occupied by the drug

Question 22

Fractional Receptor Occupancy

Answer 22

What fraction of a receptor is occupied by a drug.

• In order to look at this we must assume that the total number of receptors is constant.
• This is usually the case but there are some circumstances where the total number of receptors changes
• E.g. certain disease states and repeated exposure of certain drugs

[DR] / [Rt] = [D] / [D]+Kd

*Note: Rt = Total number of receptors

Question 23

Dose Response Curves

Answer 23

1. Graded - Shows the continuous relationship between dose and response.
2. Quantal - Shows the effect of various doses of a drug on the response of a population.
   - Used to describe “all or none” relationships (i.e. death, pregnant; you can’t be “kind of dead” or “kind of pregnant”)

Question 24

Graded Dose-Response Curves

Answer 24

• In many (but not all) situations, the magnitude of response is directly related to the amount of drug that occupies its receptor.
• Similarly, in most cases (but not all), the Emax occurs when all receptors are bound to drugs.
• In these cases the EC50 = Kd.
• The exception: spare receptors (more later)

E/Emax = [D]/[D]+Kd

Note: E= efficacy and Emax = maximal efficacy

Question 25

Potency

Answer 25

The amount of drug required to produce an effect of a given magnitude.

The EC50 is used to determine a drug’s potency.
–> The lower a drugs EC50, the more potent it is (e.g it takes a tiny bit of skunk spray to smell bad)

Question 26

Efficacy

Answer 26

The ability of a drug to elicit a response when it binds to its receptor.

A drug’s efficacy is dependent on the number of drug-receptor complexes.

Question 27

Potency vs. Efficacy

Answer 27

Drugs with a higher efficacy (B) are generally thought to be more therapeutically beneficial than drugs with high potency (A).

Question 28

Spare Receptor

Answer 28

Up until now we have assumed that 100% receptor occupancy is required to exert maximal effect (EC50 = Kd).
In some instances this is not the case!
Sometimes less than 100% occupancy results in maximal effect.
This discrepancy is described by the presence of “spare receptors”.

Less than 50% occupancy can cause 100% effect!!

• Spare receptors can be thought of as receptors in excess of those required to produce a full effect.
• There is nothing different about spare receptors!
• They are not hidden or different from other receptors.

Question 29

Spare Receptor

Answer 29

Up until now we have assumed that 100% receptor occupancy is required to exert maximal effect (EC50 = Kd).
In some instances this is not the case!
Sometimes less than 100% occupancy results in maximal effect.
This discrepancy is described by the presence of “spare receptors”.

Less than 50% occupancy can cause 100% effect!!

• Spare receptors can be thought of as receptors in excess of those required to produce a full effect.
• There is nothing different about spare receptors!
• They are not hidden or different from other receptors.

How can maximal efficacy be achieved with sub-maximal drug-receptor binding?
–> Amplification of intracellular signals!

Question 30

Quantal Dose-Response Curve

Answer 30

Plots the fraction of the population that responds to a given dose of drug.

• NOT graded, the patient either responds or they don’t (i.e. all or none).
• “Are you pain free, yes or no?” ← quantal vs. “Rate your pain from 1-10” ← graded

You can examine therapeutic effects, toxic effects and lethal effects using quantal dose-response curves.

• “Do you have liver toxicity from the tylenol?” ← examine toxic dose (TD50)
• Look at the lethal effect (LD50)
• Effective dose for 50% of the population (ED50)
• The doses that produce the response in 50% of the population are known as the ED50 (effective dose), TD50 (toxic dose) and LD50 (lethal dose)

We do not want toxic and therapeutic effects to overlap

Question 31

Therapeutic Index

Answer 31

An indicator of a drug’s relative safety.
It is calculated as the ratio of the doses of drugs that cause toxicity (or lethality) to the doses that produce therapeutic effects.

TD50 / ED50

The larger the therapeutic index, the safer the drug.
You want a low ED50 (very low dose of drug to produce an effect), high TD50 (need a very high dose to get a toxic effect)

Warfarin: The wanted effect dose overlaps with the unwanted effect
Some people may risk hemorrhage

Penicillin: The wanted effect dose never overlaps with the unwanted effect

When is it acceptable to use a drug with a small TI?
Cancer, heart episode (acceptable risks)