Quantitative PET imaging of tumor integrin alphavbeta3 expression with 18F-FRGD2

Abstract

The development of noninvasive methods to visualize and quantify integrin alpha(v)beta(3) expression in vivo appears to be crucial for the success of antiangiogenic therapy based on integrin antagonism. Precise documentation of integrin receptor levels will allow appropriate selection of patients who will most likely benefit from an antiintegrin treatment regimen. Imaging can also be used to provide an optimal dosage and time course for treatment based on receptor occupancy studies. In addition, imaging integrin expression will be important to evaluate antiintegrin treatment efficacy and to develop new therapeutic drugs with favorable tumor targeting and in vivo kinetics. We labeled the dimeric RGD peptide E[c(RGDyK)](2) with (18)F and evaluated its tumor-targeting efficacy and pharmacokinetics of (18)F-FB-E[c(RGDyK)](2) ((18)F-FRGD2).

Methods: E[c(RGDyK)](2) was labeled with (18)F by conjugation coupling with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) under a slightly basic condition. The in vivo metabolic stability of (18)F-FRGD2 was determined. The diagnostic value after injection of (18)F-FRGD2 was evaluated in various xenograft models by dynamic microPET followed by ex vivo quantification of tumor integrin level.

Results: Starting with (18)F(-) Kryptofix 2.2.2./K(2)CO(3) solution, the total reaction time for (18)F-FRGD2, including final high-performance liquid chromatography purification, is about 200 +/- 20 min. Typical decay-corrected radiochemical yield is 23% +/- 2% (n = 20). (18)F-FRGD2 is metabolically stable. The binding potential extrapolated from graphical analysis of PET data and Logan plot correlates well with the receptor density measured by sodium dodecyl sulfate polyacrylamide electrophoresis and autoradiography in various xenograft models. The tumor-to-background ratio at 1 h after injection of (18)F-FRGD2 also gives a good linear relationship with the tumor tissue integrin level.

Conclusion: The dimeric RGD peptide tracer (18)F-FRGD2, with high integrin specificity and favorable excretion profile, may be translated into the clinic for imaging integrin alpha(v)beta(3) expression. The binding potential calculated from simplified tracer kinetic modeling such as the Logan plot appears to be an excellent indicator of tumor integrin density.

FIGURE 1

Schematic structure of ¹⁸F-FB–E[c(RGDyK)]₂ (¹⁸F-FRGD2).

FIGURE 2

Analysis of non–small cell lung cancer A549 tumor tissue integrin level by SDS-PAGE/autoradiography. NP-40–solubilized tumor tissue lysate (30 µg) was incubated with 1 × 10⁵ cpm of ¹²⁵I-echistatin for 2 h and increasing concentrations of echistatin. After separation on 0.6% SDS-PAGE, an autoradio-gram was obtained (A) and each radioactivity band was quantified by using a PhosphoImager system (B). Scatchard transformation of the sigmoid curve generated tissue receptor density (number of receptors/mg tissue) (C).

FIGURE 3

Representative HPLC profiles of the reference compound ¹⁸F-FRGD2, the soluble fractions of blood and urine samples, tumor, kidney, and liver homogenates collected 1 h after tracer injection. Dimeric RGD peptide tracer is metabolically stable in most organs and tissues.

FIGURE 4

Dynamic microPET study of U87MG tumor-bearing mouse over 60 min after injection of ¹⁸F-FRGD2 (3.7 MBq [100 µCi]), static scans at 2- and 3-h time points were also conducted to complete the tracer kinetic study. Decay-corrected, whole-body coronal images that contain the tumor are shown. (Reprinted with permission of BioTechniques to reproduce parts of Figure 6.)

FIGURE 5

(A) Time–activity curves derived from 60-min dynamic and 70-min, 120-min, and 180-min static microPET study. ROIs are shown as mean %ID/g ± SD (n = 3). (B) Comparison of tumor uptake in nude mice derived from 60-min dynamic microPET scans. (C) Logan plots derived from 60-min dynamic microPET data, which showed excellent linearity of normalized integrated (Int) tumor activity vs. normalized integrated muscle tissue activity effective for time >25 min. Slopes of fits represent DVRs. (Reprinted with permission of BioTechniques to reproduce parts of Figure 6.)

FIGURE 6

Correlation analysis is shown between tumor tissue receptor density (number of receptors/mg protein measured from SDS-PAGE/autoradiog-raphy using ¹²⁵I-echistatin as radioligand) vs. BP (calculated from Logan plot transformation of dynamic microPET data) (R² = 0.96) (A); tumor cell integrin expression (number of receptors/cell measured from whole-cell receptor-binding assay) vs. BP (R² = 0.69) (B); tumor tissue receptor density vs. tumor-to-background ratios (calculated from time–activity curves derived from dynamic microPET). Coefficient of determination R² is about 0.86, 0.87, and 0.98 at 5, 30, and 60 min after injection of ¹⁸F-FRGD2, respectively (C–E); tumor cell receptor density vs. tumor-to-background ratio at 60 min after injection of ¹⁸F-FRGD2. Coefficient of determination R² is 0.67 (F). Data derived from 6 tumor models (U87MG, C6, MDA-MB-435, PC-3, NCI-H1975, and A549) illustrate excellent linear relationship between tumor tissue receptor density vs. BP and tumor tissue receptor density vs. tumor-to-background ratio at 1 h after injection. (Reprinted with permission of BioTechniques to reproduce parts of Figure 6.)