Principles of Pharmacology: Measurement of Drug Action at Receptors

Principles of Pharmacology – Measurement of Drug Action at Receptors

Radioligand Binding

  • Basic Principles of Radioligand Binding:

    • Radioligand binding assays are used to measure the affinity of drugs for receptors.
    • This method involves labeling a drug with a radioactive isotope to detect and measure the amount of drug bound to tissue samples.
    • Provides information on the number of specific binding sites (receptors) and the affinity of the drug for these sites.

  • Distinguishing Between Total, Non-Specific, and Specific Binding:

    • Total Binding: Represents the overall binding of the radioligand to the tissue, including both specific binding to receptors and non-specific binding to other proteins.
    • Non-Specific Binding: Binding of the radioligand to sites other than the target receptor. This is determined by incubating tissue samples with the radioligand in the presence of a large excess of non-radioactive (cold) ligand to saturate the receptors.
    • Specific Binding: Binding of the radioligand specifically to the target receptor. It is calculated by subtracting non-specific binding from total binding.
    • Specific Binding = Total Binding - Non-Specific Binding

  • Radioligand Binding Assay Steps:

    1. Homogenize a tissue sample (e.g., brain) and divide it into two sets of test tubes.
    2. Incubate the first set of tubes with varying concentrations of radiolabeled drug.
    3. Incubate the second set of tubes with the same concentrations of radiolabeled drug plus a large excess of non-radioactive drug.
    4. Filter and wash all samples to separate bound radioligand from free radioligand.
    5. Measure the amount of radioactivity (bound radioligand) in each sample.
    6. Plot the amount of radioactivity bound against the concentration of the labeled drug for both sets of tubes.

  • Saturation Plot:

    • A graph of specific binding versus the concentration of the radiolabeled drug.
    • Specific binding saturates as the receptor sites become fully occupied.
    • From this plot, the maximum number of receptors per tissue (Bmax) and the affinity (KD) of the radiolabeled drug can be determined.

  • Bmax and KD:

    • Bmax: The maximum number of binding sites (receptors) in the tissue.
    • KD: The equilibrium dissociation constant, representing the concentration of the drug required to occupy 50% of the receptors at equilibrium. A lower KD indicates higher affinity.

Reversible Competitive Antagonists

  • Reversible Competitive Antagonists:

    • Drugs that bind to the same receptor site as the agonist but do not activate the receptor.
    • Their effects can be overcome by increasing the concentration of the agonist (surmountable blockade).
    • Examples include pancuronium, cetirizine, and propranolol.

  • Effect on Agonist Concentration-Response Curve:

    • Reversible competitive antagonists produce a parallel shift to the right of the agonist log concentration vs. response curve.
    • The magnitude of the shift depends on the concentration and affinity of the antagonist.

  • Dose-Ratio:

    • The ratio of the concentration of agonist required to produce the same response in the presence and absence of the antagonist.
    • For a reversible competitive antagonist, the dose-ratio increases in direct proportion to the concentration of the antagonist.

  • pA2:

    • A measure of antagonist affinity.
    • Defined as the negative logarithm of the molar concentration of antagonist that produces a dose ratio of 2.
    • pA2 = -log[Antagonist Concentration] that produces a dose ratio of 2.
    • Example: If 6.3 x 10^{-9} M propranolol doubles the concentration of adrenaline needed to produce the same increase in heart rate, the pA2 for propranolol is 8.2.

Schild Analysis and Schild Plot

  • Schild Analysis:

    • A method used to determine the affinity of a reversible competitive antagonist and to verify that the antagonism is competitive.
    • Involves constructing log concentration-response curves for the agonist in the presence of several concentrations of the antagonist.

  • Schild Plot:

    • A plot of log (dose ratio - 1) versus log antagonist concentration.
    • For a reversible competitive antagonist, the Schild plot should be linear with a slope of 1.0.
    • The x-intercept of the Schild plot is equal to -log KB, where KB is the equilibrium dissociation constant for the antagonist.
    • For a reversible competitive antagonist, pA2 = -logKB = pKB.

  • Schild Equation:

    • log (dose ratio - 1) = log [antagonist concentration] - log KB

  • Steps in Producing a Schild Plot

    1. Construct log concentration-response curves for the agonist in the presence of several concentrations of antagonist
    2. From the curves read the logEC50 values for the agonist in the absence and presence of the different concentrations of antagonist
    3. Calculate the EC50 values for the agonist in the absence and presence of the different concentrations of antagonist
    4. Calculate the dose ratio for each concentration of antagonist
    5. Plot Log (dose ratio-1) versus Log [antagonist]
    6. The X-axis intercept occurs when Y = 0 i.e. when Log (DR-1) = 0. This occurs when the DR = 2, i.e. Log (2-1) = 0

  • Interpreting the Schild Plot:

    • A slope of 1.0 indicates that the antagonist is acting as a reversible competitive antagonist.
    • If the slope is significantly different from 1.0, the antagonism is likely not competitive.
    • The pA2 value obtained from the Schild plot is independent of the agonist used and is a measure of the antagonist's affinity for the receptor.

  • Importance of pA2:

    • Receptors are often characterized by the pA2 values calculated for different antagonists acting on them.
    • This provides a quantitative measure of the antagonist's affinity for the receptor.