Analysis of the pharmacokinetic/pharmacodynamic relationship of a small molecule CXCR3 antagonist, NBI‐74330, using a murine CXCR3 internalization assay

Background and purpose: Pharmacokinetic/pharmacodynamic (PK/PD) models are necessary to relate the degree of drug exposure in vivo to target blockade and pharmacological efficacy. This manuscript describes a murine agonist‐induced CXCR3 receptor internalization assay and demonstrates its utility for PK/PD analyses. Experimental approach: Activated murine DO11.10 cells were incubated with agonist in the presence or absence of a CXCR3 antagonist and changes in surface CXCR3 expression were detected by flow cytometry. For PK/PD analysis, mice were dosed with a small molecule CXCR3 antagonist, NBI‐74330, (100 mg kg −1 ) orally or subcutaneously and plasma samples taken at specified timepoints for the CXCR3 internalization assay. Key results: Surface CXCR3 expression was specifically decreased in response to CXCL9, CXCL10 and CXCL11. CXCL11 was the most potent CXCR3 agonist in buffer (pA 50 =9.23±0.26) and the pA 50 for CXCL11 was unaltered in murine plasma (pA 50 =9.17±0.15). The affinity of a small molecule CXCR3 antagonist, NBI‐74330, was obtained in the absence or presence of plasma (buffer pA 2 value: 7.84±0.14; plasma pK B value 6.36±0.01). Administration of NBI‐74330 to mice resulted in the formation of an N‐oxide metabolite, also an antagonist of CXCR3. Both antagonists were detectable up to 7 h post oral dose and 24 h post subcutaneous dose. Measurement of CXCR3 internalization demonstrated significant antagonism of this response ex vivo , 24 h following subcutaneous administration of NBI‐74330. Conclusions and implications: The CXCR3 receptor internalization assay provides a robust method for determining agonist potency orders, antagonist affinity estimates and PK/PD analyses, which discriminate between dosing regimens for the CXCR3 antagonist NBI‐74330. British Journal of Pharmacology (2007) 152 , 1260–1271; doi: 10.1038/sj.bjp.0707519 ; published online 5 November 2007