Simultaneous PET imaging of P-glycoprotein inhibition in multiple tissues in the pregnant nonhuman primate

Abstract

Studies in rodents indicate that the disruption of P-glycoprotein (P-gp) function increases drug distribution into the developing fetus and organs such as the brain. To simultaneously and serially evaluate the effect of P-gp activity and inhibition on the tissue distribution of drugs in a more representative animal model, we tested the feasibility of conducting whole-body PET of the pregnant nonhuman primate (Macaca nemestrina). We used (11)C-verapamil as the prototypic P-gp substrate and cyclosporine A (CsA) as the prototypic inhibitor.

Methods: Four pregnant macaques (gestational age, 145-159 d; gestational term, 172 d) were imaged after the intravenous administration of (11)C-verapamil (30-72 MBq/kg) before and during intravenous infusion of CsA (12 or 24 mg/kg/h, n = 2 each). The content of verapamil and its metabolites in plasma samples was determined using a rapid solid-phase extraction method. The plasma and tissue time-radioactivity concentration curves of (11)C were integrated over 0-9 min after each verapamil injection. The tissue or arterial plasma area under the time-concentration curve (AUC(tissue)/AUC(plasma)) served as a measure of the tissue distribution of (11)C radioactivity. CsA effect on (11)C radioactivity distribution was interpreted as P-gp inhibition. The change in the fetal liver AUC ratio served as a reporter of placental P-gp inhibition.

Results: CsA effect on tissue distribution of (11)C radioactivity (AUC ratios) did not increase with the mean blood concentration of CsA, indicating a near-maximal P-gp inhibition. CsA increased maternal brain and fetal liver distribution of (11)C radioactivity by 276% +/- 88% (P < 0.05) and 122% +/- 75% (P < 0.05), respectively. Changes in other measured tissues were not statistically significant.

Conclusion: These data demonstrate for the first time, to our knowledge, the feasibility of simultaneous, serial, noninvasive imaging of P-gp activity and inhibition in multiple maternal organs and the placenta in the nonhuman primate. Our findings, consistent with previous data in rodents, indicate that the activity of P-gp in the placenta and the blood-brain barrier is high and that the inhibition of P-gp facilitates drug distribution across these barriers.

FIGURE 1

A schematic as well details of the PET imaging protocol. As decribed in Methods, the protocol for one animal was modified as well as shortened.

FIGURE 2

Blood cyclosporine A (CsA) concentration-time profiles from the time of [¹¹C]-verapamil injection (t=0). The legend indicates animal number and CsA dose. The rectangle highlights the modest change in CsA blood concentration over 0-9 min., the focus of our data analysis. *Animal was studied for only 25 minutes.

FIGURE 3

Percent of [¹¹C]-radioactivity in plasma before and during cyclosporine A (CsA) infusion show that verapamil is rapidly metabolized in the pregnant macaque. At 9 minutes, CsA did not significantly affect the plasma radioactivity of verapamil and D-617/D-717, but decreased the radioactivity of the polar metabolites. At 40 minutes, less than 20% of the total radioactivity was verapamil in the presence or the absence of CsA. Data are expressed as mean ± SD (n=4). Data in the absence and presence of CsA (at a given time point) were compared using the Mann-Whitney test. * P<0.05.

FIGURE 4

Plasma (A) and tissues (B-L) [¹¹C]-radioactivity concentration-time profiles in a representative animal in absence (solid triangles) and presence (open triangles) of 12 mg/kg/hr cyclosporine A (CsA). In the presence of CsA, these profiles show greater distribution of radioactivity into the maternal brain (B) and the fetal liver (C), but lesser distribution into the maternal liver. In the maternal gallbladder, CsA initially increased the accumulation of [¹¹C]-radioactivity, but reduced it at later time points. CsA did not change the time-concentration profiles of radioactivity in the plasma or other tissues.

FIGURE 5

PET images of a pregnant M. nemestrina before (A) and during (B) the administration of 12 mg/kg/hr cyclosporine A (CsA). The PET scans (A and B) are SUV images summed over a period of 1 to 9 minutes following [¹¹C]-verapamil injection. The images A and B were scaled to the same pixel value. (C) Image produced by pixel-by pixel subtraction of scan A from scan B. The fetal liver was the reporter of [¹¹C]-radioactivity that crossed the placental barrier. Due to P-gp inhibition, CsA significantly increased the distribution of [¹¹C]-radioactivity into the maternal brain and fetal liver (yellow to red areas in B and C). In the maternal liver, CsA decreased the amount of [¹¹C]-radioactivity (darker area in C). (D) T2-weighted MRI of the same animal. The color scale reflects SUV as shown by the thermometer.

FIGURE 6

The effect of cyclosporine A (CsA) on the distribution (percent change in AUC_tissue/AUC_plasma) of [¹¹C]-radioactivity into maternal brain and fetal liver was large and significant. In contrast, its effect on the distribution of [¹¹C]-radioactivity into other tissues was insignificant or modest (gall bladder). Data are expressed as mean ± SD (n=4). * P<0.05.