L5 - Measuring drug action Flashcards
Measure affinity: Radioligand binding
Label the drug with a radioactive isotope. H-3 or P-32
Can detect and measure amount of drug bound to tissue samples.
Homogenise brain and divide equally into two sets of test tubes. In first set, incubate samples with varying conc of radiolabelled drug.
In seconds set, incubate samples with varying conc of radiolabelled drug and a large excess of non labelled “cold” relaxin (drug). Filter and wash samples to leave behind only radiolabelled relaxin bound to brain.
Measure amount of radioactivity per sample.
To get a measure of specific relaxin binding, subtract non-specific binding (cold relaxin) from total relaxin. Shows Bmax which is max number of receptors per tissue and affinity (Kd) of radiolabelled drug. Kd is the conc required to occupy 50% of receptors.
Antagonist affinity:
The extent to which agonist curve is shifted by antagonist is a measure of the affinity of the antagonist for the receptor. Can measure shift by dose-ratio. Dose ratio is the ratio of the conc of agonist producing the same response in presence and absence of antagonist.
pA2:
It is the affinity of a reversible competitive antagonist. This is the negative log of conc of antagonist that is needed to double the agonist conc to produce the same response (dose ratio of 2).
High pA2 then the smaller conc of antagonist is needed for doubling effect. Higher the affinity of antagonist.
Constructing Schild Plot:
Construct log(agonist) vs response curve.
Add varying conc of antagonist.
Find the EC50 of agonist conc in presence of antagonist and absence of it ~ remember that it’s in log so must be converted.
Find dose ratio by doing presence of antagonist over absence.
Constructing Schild plot:
Plot log(dose ratio-1) vs. Log(antagonist conc).
Slope should be 1 if it is reversible competitive antagonist.
Extrapolate and see where cross on x axis. This is logKb.
PA2 = -logKb
Log(dose ratio-1)=log(antag)-logKb
