Receptor binding assays specificity

AstraZeneca has filed a patent application on novel bis-aryl compounds as CB1/CB2 agonists that lack CNS penetration and thus avoid the unwanted side effects associated with activation of central CBi receptors [210]. Over 100 compounds are specifically claimed e.g. (321). Compounds were tested in receptor binding assays using human CBi and CB2 receptor preparations. Respective K values were in the ranges 50-5,000 and 15-2,800 nM, although no specific data were presented. 

The evaluations to determine relevant species for toxicity evaluation include various receptor binding assays and tissue cross-reactivity assessments, the later being routinely performed for monoclonal antibody based products (see Chapters 10 and 26). Species specificity can be determined using properly controlled in vitro cell-based response assays, cloning of receptors and ligands to determine compatibility, receptor-based response assays, immunoassays, genomic-based assays, and traditional biochemical-based assays as well as in vivo assessments with validated endpoints and markers for specificity. 

The Hill equation has only limited applicability as a model to predict the expression of agonism, because the parameters from this empirical equation depend on both drug-specific and system-related properties, complicating the extrapolation and prediction from in vitro to in vivo systems. With a fully mechanistic model, Zuideveld was better able to predict the in vivo affinity and efficacy of 5-HTia receptor agonists from in vitro receptor binding assay. 

Binding Characteristics. For ligand-receptor binding assays, we used an EDTA/Tris-citrate (1 mM, pH 7.4)-dialyzed mitochondrial fraction of housefly heads, the so-called 2 fraction, which was prepared by a modification of Squires s method (4). The final pellet was frozen at -25°C and then suspended in 50 mM Tris-citrate buffer (pH 7.4 at 3°C) for assays. 4-w-Propyl[2,3-3h]-2,6,7-trioxa-l-phosphabicyclo[2.2.2]— octane 1-oxide ([ HjPr-BP, 41 Ci/mmol) was used as a ligand for the reason described above. Radioreceptor assays were performed by filtration techniques with glass fiber filters. Specific [ HjPr-BP binding was taken as that displaced by excess unlabeled Pr-BP(10 yM). 

The compounds tetrandrine and fangchinoline inhibited platelet-activating factor (PAF)-induced human platelet aggregation. The ICjq of tetrandrine was 28.6 pM, whereas that of fangchinoline was 21.7 pM. Tetrandrine and fangchinoline also inhibited PAF-, thrombin- and arachi-donic acid-induced thromboxane B2 formation in human washed platelets. In the PAF-receptor binding assay, neither compound showed any inhibitory effects on the specific binding of PAF to its receptor (Kim et al. 1999). 

In any event, there is no standard tissue preparation that lends itself to use for all neurotransmitter and drug receptor binding assays. Each procedure is tailored to a particular receptor binding site in order to obtain the most robust assay, reliable and consistent kinetic constants, and receptor site specificity. 

The major drawback of radioreceptor assays relates to their specificity. While receptor binding assays are specific in that only a limited number... 

Exogenously administered substances are not the only agents which might interfere with a receptor binding assay. For example, opiate receptor binding as a radioreceptor assay for morphine would appear to be a relatively specific procedure since only opiates can interfere with the attachment to this receptor site. However, the discovery of endogenous opioid peptides makes the morphine assay less feasible with brain tissue because of the potential for these substances to be present in the tissue extracts. 

---