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describe one pharmacodynamic and one pharmacokinetic in vitro assay. Make sure t
describe one pharmacodynamic and one pharmacokinetic in vitro assay. Make sure to address the principle of the assay, how it is performed, and what information it can provide.
Pharmacodynamic assay: Radioligand assay (RIA) systems
Background information provided from the module: Radioligand assay (RIA) systems: utilizing a radioisotope such as 3H, 14C, 33P, or 35S incorporated into the compound of interest or the endogenous agonist, such assays can provide information about affinity and mode of action between the drug and the target (both receptors and enzymes). 3H remains the most popular and easiest to incorporate radioisotope in the common laboratory. Upon binding with the target, the radioactivity is measured and the unbound drug is removed. The leftover radioactivity now is directly related to the amount of bound drug to the target and thus is quantifiable if the amount of target and the originally applied radioactivity are known. This type of assay remains popular due to its relative ease and wide applicability. But it has limitations in that it requires extensive clean-up steps to remove non-specific binding of the radioactive labeled agent to plastic or cellular membranes as well as handling radioactive waste. To avoid generation of radioactive waste, newer techniques utilize scintillation proximity assay (SPA) beads that require binding of the radioisotope material in order to emit light and scintillate. The addition of such beads therefore quenches the radioactive material and binds it so that no filtration and removal of non-specific binding waste is necessary. One has to be aware though that the reduction in radioactivity is now indicative of the drug-target interaction since the fraction that binds with the target will not scintillate. More information on the underlying kinetics of radioligand assays: https://www.giffordbioscience.com/radioligand-binding-assay/ Pharmacokinetic in vitro assay: Parallel Artificial Membrane Permeability Assay (PAMPA).
Background information provided in the module: Parallel Artificial Membrane Permeability Assay (PAMPA): similar to IAM, PAMPA can only measure passive diffusion but more accurately correlates to jejunal intestinal permeability of compounds. In this assay, the drug is diluted in a buffer (donor solution) and topped with an immiscible barrier of a lipid layer on a filter plate usually consisting of a phospholipid such as phophatidyl choline in dodecane or egg lecithin. On top of the layer added is a pH-adjusted buffer (acceptor solution). The system is allowed to incubate for a set period of time and samples are collected from the original drug solution, the donor, and the acceptor solution after incubation and the concentration of the drug are measured. The advantage of this system is that the lipid layer is consistent and the compound is not forced through it as is the case in IAM. The thickness of the lipid layer, the pH of the buffer solution, and the composition of the lipid layer all contribute to the permeability rate and need to be carefully adjusted.
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