SUPPORTING INFORMATION
Albumin-Binding PSMA Ligands: Optimization of the Tissue Distribution Profile Martina Benešová 1,2, Christoph A. Umbricht 1, Roger Schibli1,2, Cristina Müller 1,2*
1. Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, 5232 Villigen-PSI, Switzerland 2. Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
E-Mail addresses:
[email protected];
[email protected];
[email protected];
[email protected]
* Correspondence to: PD Dr. Cristina Müller Center for Radiopharmaceutical Sciences ETH-PSI-USZ Paul Scherrer Institut 5232 Villigen-PSI Switzerland e-mail:
[email protected]
phone: +41-56-310 44 54; fax: +41-56-310 28 49
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1. Chemicals and Radionuclides All chemicals (analytical grade; ultrapure for the radiolabeling) and solvents (HPLC-grade; metal-free for the radiolabeling) were purchased commercially from abcr, Bachem, Fluka, CheMatech, Iris Biotech, Merck, and Sigma Aldrich. No-carrier-added
177
Lu ( 177LuCl3 in 0.04 M HCl) was provided by Isotope Technologies
Garching (ITG GmbH, Germany).
2. HPLC Purification of the PSMA Ligands Reversed-phase high performance liquid chromatography (RP-HPLC) of non-radiolabeled compounds was performed with a Merck-Hitachi system, equipped with a D-7000 interface, a L-7400 UV detector, and a L7100 pump. For analytical purposes a reversed-phase C18 column (5 µm, 4.6×150 mm, SunfireTM, Waters, USA) was used. Purification steps were carried out using a semi-preparative reversed-phase C18 column (5 µm, 10×150 mm, Sunfire TM, Waters, USA). In both cases, the mobile phase consisted of 0.1% trifluoroacetic acid in MilliQ water (A) and acetonitrile (B). For analytical runs, a linear gradient of 90% A and 10% B to 10% A and 90% B was used over a period of 10 min at a flow rate of 1 mL/min. Purification was performed using a linear gradient of 90% A and 10% B to 40% A and 60% B over a period of 15 min at a flow rate of 4 mL/min.
3. Radiolabeling and Quality Control The PSMA ligands were labeled with
177
Lu ( 177LuCl3 in 0.04 M HCl) in a mixture of sodium acetate (0.5 M,
pH 8) and HCl (0.05–0.1 M, pH 1) at pH 4.5. The reaction mixture was incubated for 10 min at 95°C. Quality control of the radioligands was performed using RP-HPLC (Merck Hitachi LaChrom L-7100 HPLC pump coupled with a L-7200 autosampler, a D-7000 interface and an HPLC Radioactivity Monitor LB 506 B from Berthold) equipped with a reversed-phase C18 column (5 µm, 150×4.6 mm; XterraTM, MS, Waters). The mobile phase consisted of MilliQ water containing 0.1% trifluoroacetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% B to 20% A and 80% B over a period of 15 min at a flow rate of 1 mL/min. Representative chromatograms of each radioligand are shown in Figure S1.
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Figure S1. Representative chromatograms of (A) 177
Lu-PSMA-ALB-07 and (D)
177
177
Lu-PSMA-ALB-02, (B)
177
Lu-PSMA-ALB-05 (C)
Lu-PSMA-617. The retention times are indicated as the average ± SD of at
least three independent runs.
4. Determination of n-Octanol/PBS Distribution Coefficients (LogD Values) The PSMA ligands were labeled at a specific activity of 50 MBq/nmol to determine their distribution coefficients (logD values) by a shake-flask method using liquid-liquid extraction followed by phase separation. Samples containing the radioligand (1.25 MBq, 25 pmol) in 25 µL phosphate buffered saline pH 7.4 (PBS) were added to each vial containing 1475 µL PBS and 1500 µL n-octanol. The vials were vortexed vigorously and then centrifuged at 2500 rpm for 6 min for phase separation. The concentration of radioactivity in a defined volume of each layer was measured in a γ-counter (Perkin Elmer, Wallac Wizard 1480). The distribution coefficients were expressed as the logarithm of the ratio of counts per minute (cpm) measured in the n-octanol phase to the cpm measured in the PBS phase.
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5. Filter Assay The PSMA ligands were labeled with
177
Lu at a specific activity of 50 MBq/nmol and incubated in either
human plasma, or in a solution of human serum albumin (HSA, 700 µM) or mouse plasma for 15 min at room temperature. As a control experiment, the radioligands were diluted in PBS (buffer solution without proteins). The free and plasma-bound fractions were separated using a centrifree ultrafiltration device (centrifugal filter units, Millipore; 30000 Da nominal molecular weight limit, methylcellulose micropartition membranes). The incubated solution was loaded onto the ultrafiltration device and centrifuged at 800 rfc for 40 min at 20 °C. Samples from the filtrate were measured in a γ-counter. The amount of plasma-bound compound was calculated based on the radioactivity measured in the filtrate (free fraction) relative to the corresponding loading solution (set to 100%) (Table S1). Table S1. In Vitro Data of 177Lu-PSMA-ALB-02, 177Lu-PSMA-ALB-05, 177Lu-PSMA-ALB-07 and 177
Lu-PSMA-617, respectively Protein-bound fraction tra Compound Code
[min] (n ≥ 3)
[% activity retained by the filter]
Log D
(n ≥3)
(n = 3) Human Plasma
HSA
Mouse plasma
PBS
177
Lu-PSMA-ALB-02
11.1 ± 0.1
–2.8 ± 0.09
95 ± 1.2
95 ± 1.7
87 ± 1.0
