Rapid solid-phase extraction method to quantify [¹¹C]-verapamil, and its [¹¹C]-metabolites, in human and macaque plasma

Abstract

Introduction

P-glycoprotein (P-gp), an efflux transporter, is a significant barrier to drug entry into the brain and the fetus. The positron emission tomography (PET) ligand, [¹¹C]-verapamil, has been used to measure in vivo P-gp activity at various tissue–blood barriers of humans and animals. Since verapamil is extensively metabolized in vivo, it is important to quantify the extent of verapamil metabolism in order to interpret such P-gp activity. Therefore, we developed a rapid solid-phase extraction (SPE) method to separate, and then quantify, verapamil and its radiolabeled metabolites in plasma.

Methods

Using high-performance liquid chromatography (HPLC), we established that the major identifiable circulating radioactive metabolite of [¹¹C]-verapamil in plasma of humans and the nonhuman primate, Macaca nemestrina, was [¹¹C]-D-617/717. Using sequential and differential pH elution on C₈ SPE cartridges, we developed a rapid method to separate [¹¹C]-verapamil and [¹¹C]-D-617/717. Recovery was measured by spiking the samples with the corresponding nonradioactive compounds and assaying these compounds by HPLC.

Results

Verapamil and D-617/717 recovery with the SPE method was >85%. When the method was applied to PET studies in humans and nonhuman primates, significant plasma concentration of D-617/717 and unknown polar metabolite(s) were observed. The SPE and the HPLC methods were not significantly different in the quantification of verapamil and D-617/717.

Conclusions

The SPE method simultaneously processes multiple samples in less than 5 min. Given the short half-life of [¹¹C], this method provides a valuable tool to rapidly determine the concentration of [¹¹C]-verapamil and its [¹¹C]-metabolites in human and nonhuman primate plasma.

Introduction

P-Glycoprotein (P-gp), a 170-kDa membrane ATP-binding cassette efflux transporter, is found in the luminal membrane of endothelial cells that form the blood–brain barrier (BBB), in the syncytiotrophoblasts of the placenta that form the blood–placental barrier (BPB), as well as in the cell membranes of other organs important in drug absorption and disposition [1], [2], [3]. At these sites, P-gp is predominantly responsible for restricting the entry of its substrate drugs or other xenobiotics into tissues (e.g., the brain and the fetus) or for drug absorption and elimination (e.g., the intestine, liver and kidneys) [1], [2], [3]. P-gp is encoded by MDR1 in humans and by mdr1a and mdr1b in rodents. Based on the mdr1a/b^(−/−) knockout mouse model, the function of P-gp at the BBB and BPB appears to be more important in drug disposition than in other organs [4], [5]. The brain distribution of verapamil, a P-gp substrate, in mdr1a/b^(−/−) mice is approximately 10-fold greater than that observed in the wild-type mice [4], [5], [6]. Likewise, the fetal distribution of the anti-HIV protease inhibitor, saquinavir, in the mdr1a/b^(−/−) fetuses is 20-fold greater than that in the wild-type fetuses [7]. Based on these data, it is widely presumed that P-gp activity at the human BBB and the BPB is important in restricting the distribution of drugs into the brain and fetus, respectively. However, it is not ethically possible to measure the distribution of drugs into human brain or fetal tissues in vivo. Unlike the mdr1a/b^(−/−) model, determining P-gp activity at the BBB or BPB in larger animals is difficult due to the cost of obtaining brain and fetal tissue samples at multiple time points. Thus, a highly sensitive imaging method is required that can noninvasively measure the brain (human) and fetal (large animals) tissue concentrations of drugs that are P-gp substrates.

An elegant and noninvasive method to examine P-gp activity at the BBB, BPB and other organs is positron emission tomography (PET) using [¹¹C]-verapamil as the P-gp substrate [8], [9], [10], [11]. Although verapamil is an excellent P-gp substrate (well established, approved for human IV administration and relatively safe), it suffers from the disadvantage of undergoing extensive hepatic metabolism by cytochrome P450 (CYP) enzymes (Fig. 1) [10], [12], [13], [14].

Multiple investigators have used high-performance liquid chromatography (HPLC) as a means of separating [¹¹C]-verapamil from its radiolabeled metabolites [15], [16], [17] with run times longer than 15 min. However, since [¹¹C] activity half-life is short (∼20 min), it is difficult to conduct rigorous HPLC quantification of the content of [¹¹C]-verapamil and its various metabolites in numerous blood or plasma samples obtained during a PET imaging study. Therefore, to rapidly quantify the content of [¹¹C]-verapamil and its metabolites circulating in the plasma, in both humans and nonhuman primates (Macaca nemestrina), we developed and validated a rapid solid-phase extraction (SPE) method to separate [¹¹C]-verapamil from its major circulating metabolites.