64Cu-labeled phosphonium cations as PET radiotracers for tumor imaging

Abstract

Alteration in mitochondrial transmembrane potential (ΔΨ(m)) is an important characteristic of cancer. The observation that the enhanced negative mitochondrial potential is prevalent in tumor cell phenotype provides a conceptual basis for development of mitochondrion-targeting therapeutic drugs and molecular imaging probes. Since plasma and mitochondrial potentials are negative, many delocalized organic cations, such as rhodamine-123 and (3)H-tetraphenylphosphonium, are electrophoretically driven through these membranes, and able to localize in the energized mitochondria of tumor cells. Cationic radiotracers, such as (99m)Tc-Sestamibi and (99m)Tc-Tetrofosmin, have been clinically used for diagnosis of cancer by single photon emission computed tomography (SPECT) and noninvasive monitoring of the multidrug resistance (MDR) transport function in tumors of different origin. However, their diagnostic and prognostic values are often limited due to their insufficient tumor localization (low radiotracer tumor uptake) and high radioactivity accumulation in the chest and abdominal regions (low tumor selectivity). In contrast, the (64)Cu-labeled phosphonium cations represent a new class of PET (positron emission tomography) radiotracers with good tumor uptake and high tumor selectivity. This review article will focus on our recent experiences in evaluation of (64)Cu-labeled phosphonium cations as potential PET radiotracers. The main objective is to illustrate the impact of radiometal chelate on physical, chemical, and biological properties of (64)Cu radiotracers. It will also discuss some important issues related to their tumor selectivity and possible tumor localization mechanism.

Figure 1

Phosphonium cation conjugates for preparation of ⁶⁴Cu radiotracers. The phosphonium cation (PC) is used as the mitochondrion-targeting biomolecule to carry ⁶⁴Cu into the tumor cells where the negative mitochondrial potential is elevated as compared to normal cells. DOTA, DO3A, DO2A, NOTA and their derivatives are used as BFCs for ⁶⁴Cu chelation. Different linkers (L) are useful for modification of pharmacokinetics of ⁶⁴Cu radiotracers.

Figure 2

ORTEP drawings of In(3)⁺ (top) and Ga(3)⁺ (bottom). Crystallization water molecules and hydrogen atoms are omitted for the sake of clarity. The structure of Mn(3) is almost identical to that of In(3)⁺ despite their difference in the overall molecular charge.

Figure 3

Typical HPLC chromatograms of Cu(3), Mn(3), In(3)⁺ and Ga(3)⁺. The presence of a single peak suggests that they exist in solution as a single or “averaged” species. Obviously, Ga(3)⁺ (14.7 min) and In(3)⁺ (14.5 min) with the +1 overall molecular charge are more hydrophilic than Mn(3) (16.8 min) and Cu(3) (17.2 min) in their Zwitterion forms.

Figure 4

Comparison between the ⁶⁴Cu-labeled phosphonium cations and ^99mTc-Sestamibi with respect to their uptake in the glioma tumor, heart, liver and muscle, as well as their tumor/heart and tumor/lung ratios in the athymic nude mice bearing U87MG glioma xenografts.

Figure 5

Histograms to illustrate impacts of BFCs, overall charge and radiometal on the tumor uptake of ⁶⁴Cu and ¹¹¹In-labeled phosphonium cations in the athymic nude mice bearing U87MG human glioma xenografts.

Figure 6

Representative microPET images of the glioma-bearing mice administered with ~250 μCi of ⁶⁴Cu(2), ⁶⁴Cu(3) and ⁶⁴Cu(6) at 4 h p.i. Arrows indicate the presence of tumors.

Figure 7

Schematic presentation of the process for ⁶⁴Cu(6) to across the plasma and mitochondrial membranes. The main difference between tumor cells and normal cells is the mitochondrial potential. The difference in mitochondrial potential (Δψ_m) between carcinoma cells and epithelial cells is ~60 mV, which contributes to ~10-fold more accumulation of ⁶⁴Cu(6) in mitochondria according to the Nernst equation. The plasma potential (−30 – −60 mV) also pre-concentrates ⁶⁴Cu(6) in the plasma. The lower pH value (pH = 4.5 – 5.0) inside tumor cells makes it easier for the two acetate chelating arms to become dissociated from ⁶⁴Cu.

Figure 8

Schematic illustration to move a triphenylphosphonium cation across a lipophilic membrane. The electrostatic interaction between the triphenylphosphonium cation and positive charges outside the membrane is repulsive. This component of activation energy is due to the enthalpy input required to overcome charge repulsion and to remove salvation water molecules from the cation upon transfer into the membrane lipid core. In contrast, the hydrophobic interaction between the triphenylphosphonium cation and lipid core is attractive due to hydrophobicity of the lipophilic phosphonium cation and increased entropy (loss of water structure when moving a molecule into the lipid core).

Figure 9

Schematic presentation to illustrate the selectivity of cationic radiotracers based on their lipophilicity. ^99mTc-Sestamibi is lipophilic (log P = 1.1), and its membrane diffusion rate is so fast that it can readily localize in mitochondrion-rich, such as the heart, liver and kidneys. The ⁶⁴Cu-labeled phosphonium cations, such as ⁶⁴Cu(3), are very hydrophilic (log P = −1.5 – −2.7) due to the hydrophilic ⁶⁴Cu-DO3A chelate. The slow diffusion kinetics makes it difficult for the ⁶⁴Cu-labeled phosphonium cations to across plasma and mitochondrial membranes, thereby forcing them to localize in the tumor where mitochondrial potential is elevated (~60 mV). While the enhanced negative mitochondrial potential provides the thermodynamic driving force for the ⁶⁴Cu-labeled phosphonium cations to localize in energized mitochondria of tumor cells, the hydrophilicity offers a control of their cell-penetrating kinetics and tumor selectivity.

Figure 10

Schematic presentation to show capability of ⁶⁴Cu(DO3A-xy-ACR) to localize in glioma. ⁶⁴Cu(DO3A-xy-ACR) is the PET radiotracer for tumor imaging while Cu(DO3A-xy-ACR) is used as the fluorescent probe to demonstrate their mitochondrial localization. The results from this study provided strong indirect evidence to suggest that ⁶⁴Cu-labeled phosphonium cations are able to localize in the energized mitochondria of tumor cells.

Chart I

Synthesis of DO3A Conjugates.

Chart II

Synthesis of DO2A Conjugates.

Chart III

Synthesis of DOTA and NOTA Conjugates