Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye–Protein Interaction

Albertus W. Hensbergen; Tessa Buckle; Danny M. van Willigen; Margret Schottelius; Mick M. Welling; Felicia A. van der Wijk; Tobias Maurer; Henk G. van der Poel; Gabri van der Pluijm; Wytske M. van Weerden; Hans-Jürgen Wester; Fijs W.B. van Leeuwen

doi:10.2967/jnumed.119.233064

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FIGURE 1.
(A) Schematic overview of working hypothesis, exemplified by hybrid tracer inside PSMA binding site with interactions between dye and amphipathic funnel. (B) Selected hybrid tracers with functionalization introduced on indole with different lipophilicities. (C) Hybrid tracer based on control Cy5 dye. (D) Chemical structures of EuK and 2-mercaptoacetyl-seryl-seryl-seryl (mas₃) moieties. S1 and S1’ are the primary binding sites of the protein.
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FIGURE 2.
In vitro assessment of hybrid tracers. Shown is staining of extracellular expression of PSMA on C2-2B₄ cells after incubation with nuclear stain (Hoechst; in blue) and hybrid tracer (Cy5; in red), with half-maximal inhibitory concentrations (IC₅₀, measured with ¹²⁵I-EuK–I-BA [((S)-1-carboxy-5-(4-(iodo-benzamido)pentyl)carbamoyl)-l-glutamic acid] as competitive radioligand). (A) EuK-Cy5-mas₃. (B) EuK-(SO₃)Cy5-mas₃. (C) EuK-Cy5(SO₃)-mas₃. (D) EuK-(Ar)Cy5-mas₃. (E) EuK-Cy5(Ar)-mas₃. (F) Blocking (checkered) of PSMA with EuK–I-BA. Binding affinities are represented as mean ± SD.
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FIGURE 3.
In vivo SPECT imaging evaluation using BALB/c nude mice with orthotopically transplanted PC346C cells, each injected with a different PSMA hybrid tracer analog. All images are scaled relative to tumor. B = bladder; I = intestines; K = kidneys. Tumor is encircled in red.
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FIGURE 4.
White-light and ex vivo fluorescence imaging of tumor-containing prostate tissue (*), bladder (**), seminal vesicles (***), and abdominal adipose tissue (#) after injection of corresponding hybrid tracer.

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TABLE 1

Physical and Photophysical Characteristics of PSMA-Targeted Hybrid Tracers

Compound	λ_ex,max/λ_em,max in H₂O/PBS (nm)	Φ_F (% in PBS)	Brightness (×10⁴ M⁻¹ × cm⁻¹ in PBS)^†	Lipophilicity (logP_(o/w); n = 6)^*	PPB (n = 3)	Serum stability after 24 h (% remaining; n = 2)
EuK-Cy5-mas₃	644/663 (18)^‡	12	1.1	−2.13 ± 0.10	88.3 ± 1.6	Abs: 80 ± 6/Flu: 86 ± 5
EuK-(SO₃)Cy5-mas₃	648/664 (16)^‡	15	1.4	−2.86 ± 0.05	85.0 ± 2.3	Abs: 97 ± 2/Flu: 89 ± 6
EuK-Cy5(SO₃)-mas₃	648/664 (16)^‡	10	1.5	−2.70 ± 0.01	87.4 ± 2.0	Abs: 82 ± 7/Flu: 85 ± 1
EuK-(Ar)Cy5-mas₃	664/682 (18)^‡	9	0.3	−1.75 ± 0.10	90.7 ± 1.3	Abs: 85 ± 3/Flu: 79 ± 1
EuK-Cy5(Ar)-mas₃	663/680 (17)^‡	12	0.7	−1.84 ± 0.06	89.0 ± 2.5	Abs: 75 ± 0/Flu: 76 ± 0

↵† Calculated using molecular extinction coefficient of free fluorophore.
↵* Measured using ^99mTc-labeled hybrid tracers.
↵‡ Parenthetic numbers are Stokes shift.
λ_ex,max/λ_em,max = maximum λ_ex/λ_em; Φ_F = quantum yield; logP_(o/w) = the partition coefficient in octanol/water; PBS = phosphate-buffered saline; Abs = absorbance; Flu = fluorescence.