An 86Y-Labeled Mirror-Image Oligonucleotide: Influence of Y-DOTA

Mar 18, 2008 - the ethylene bridge or via a chemical activation of a free carboxylic acid sidearm of DOTA. The positron-emitting radionuclide 86Y (t1/...
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Bioconjugate Chem. 2008, 19, 928–939

An 86Y-Labeled Mirror-Image Oligonucleotide: Influence of Y-DOTA Isomers on the Biodistribution in Rats† Joern Schlesinger,‡ Inge Koezle,‡ Ralf Bergmann,‡ Sergio Tamburini,§ Cristina Bolzati,§ Francesco Tisato,§ Bernhard Noll,‡ Sven Klussmann,| Stefan Vonhoff,| Frank Wuest,‡ Hans-Juergen Pietzsch,*,‡ and Joerg Steinbach‡ Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, Germany, ICIS-CNR, Corso Stati Uniti, 4, Padova, Italy, and NOXXON Pharma AG, Berlin, Germany. Received December 10, 2007; Revised Manuscript Received February 7, 2008

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide 86Y (t1/2 ) 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5′-aminohexyl UGA CUG ACU GAC-3′; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. 86Y radiolabeling of the DOTAL-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the 86Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)]- was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)]- isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)]- and conjugated to the L-RNA. The identity of the [86Y-DOTA]-L-RNA species was finally established by comparison of the radiometric (86Y) and UV–visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [86Y((S)-p-NH2-Bn-DOTA)]- complex isomers, while no significant differences were observed for the [86Y-DOTA]-L-RNA isomers. High renal excretions were found for the [86Y((S)-p-NH2-Bn-DOTA)]- complex isomers as well as for the L-RNA isomers.

INTRODUCTION The radiolabeling of oligonucleotides with radiometals (e.g. Ga, 111In, 99mTc, 86Y) for in ViVo imaging studies is usually performed via the bifunctional chelator (BFC)1 approach (1–4). BFCs should form highly stable complexes with the radiometal while enabling facile conjugation of the radiometal complex to the biomolecule under mild conditions. A frequently utilized chelator for trivalent metals, such as In3+, Ga3+, Y3+, and other ions of the lanthanide and actinide series, is 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetra68

†

Dedicated to Hartmut Spies in memoriam. * Author to whom correspondence should be addressed: H.-J. Pietzsch, Institute of Radiopharmacy, Forschungszentrum DresdenRossendorf, PF 51 01 19, 01314 Dresden, Germany; h.j.pietzsch@ fzd.de. ‡ Forschungszentrum Dresden-Rossendorf. § Corso Stati Uniti. | NOXXON Pharma AG. 1 Abbreviations: BIRD, bilinear rotation decoupling; BFC, bifunctional chelator; COSY, correlation spectroscopy; DOTA, 1,4,7,10tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; ESI-MS, electrospray ionization-mass spectrometry; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple-quantum coherence experiment; HPLC, high pressure liquid chromatography; i.d., inner diameter; L-ON, mirrorimage oligonucleotide; NMR, nuclear magnetic resonance; NOESY, nuclear overhause effect spectroscopy; p.i., post injection; PAGE, polyacrylamide gel electrophoresis; PET, positron emission tomography; Rf, retention factor; RT, retention time; (S)-p-NH2-Bn-DOTA, (S)-2(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid; (S)-p-SCN-Bn-DOTA, (S)-2-(4-isothiocyanatobenzyl)-1,4,7,10tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid; SAP, square antiprism; SDS, sodium dodecyl sulfate; SEC, size exclusion chromatography; SUV, standardized uptake value; TEAA, triethylammonium acetate; TLC, thin layer chromatography; TPPI, time-proportional phase incrementation; TSAP, twisted square antiprism.

acetic acid (DOTA). DOTA is known to form thermodynamically stable complexes with lanthanides and yttrium isotopes, and the corresponding radiometal complex show in many cases high kinetic inertness in ViVo (5). Examples of various DOTA derivatives used as BFCs are shown in Figure 1. The reactive functionality capable of being linked to the biomolecule can be introduced into the DOTA framework either at the ethylene bridge or via a chemical activation of a free carboxylic acid sidearm of DOTA. The positron-emitting radionuclide 86Y (t1/2 ) 14.7 h, β+ ) 33%, Eavg ) 664 keV) is an attractive isotope for molecular imaging purposes. The extensive use of the high-energy electronemitter 90Y (t1/2 ) 64.06 h, β- ) 72%, Eβ- ) 2.288 MeV), an excellent radionuclide for endoradiotherapy, makes the positron emitter 86Y an ideal radionuclide for dosimetry assessment of 90 Y-labeled radiotherapeutics (6). 86Y can be prepared on a small biomedical cyclotron employing the 86Sr(p,n)86Y nuclear reaction (7). The 14.7 h half-life of 86Y allows PET studies of up to two days. In the past decade, synthetic oligonucleotides have been suggested as molecular probes for the improved targeting of cancer using pretargeting methods (8, 9). In this connection, Hnatowich and co-workers have previously shown that complementary phosphorodiamidate morpholino oligomers (MORF/cMORF) are useful molecular recognition systems for targeting prostate tumor in nude mice in combination with a monoclonal antibody (10). Another promising class of synthetic oligonucleotides consists of mirror-image oligonucleotides (L-ON) (11). L-ON are constructed of an Lconfigured ribose or deoxyribose. As shown previously, L-ON display an extraordinary high metabolic stability in the biological environment due to the high resistance to enzymatic degradation (12). To explore the potential of complementary L-ON as recognition systems for pretargeting, the present work deals with basic radiopharmaceutical chemistry

10.1021/bc700453h CCC: $40.75  2008 American Chemical Society Published on Web 03/18/2008

86

Y-Labeled Mirror-Image Oligonucleotide

Figure 1. Bifunctional chelators based on the DOTA framework.

for labeling of an L-RNA as model system with the β+-emitter 86 Y and subsequent radiopharmacological evaluation. An important criterion for the use of radiolabeled L-ON as molecular probes for in ViVo imaging approaches is the radiochemical purity of the L-ON. The purpose of this work was to characterize precisely different isomers formed during the complexation of a DOTAfunctionalized L-RNA with Y(III). We report on the synthesis and NMR characterization of the nonradioactive [Y((S)-p-NH2Bn-DOTA)]- precursors isolated by HPLC and further analyzed by mass spectrometry, circular dichroism spectroscopy, and polarimetric measurements. The assessment of their solution structure allows one to determine the stereochemistry adopted by the pendant benzyl residue and to establish the identity with the compounds prepared at the “carrier-free” level with 86Y. The isolated [86Y((S)-p-NH2-Bn-DOTA)]- isomers were further characterized by a comparative biodistribution study in Wistar rats and metabolite analysis. To draw conclusions to the observed [86Y-DOTA]-L-RNA isomers, the amino groups of [Y(p-NH2-Bn-DOTA)]- were transferred into isothiocyanate functionalities and the isomers were attached to the L-RNA. The coupling products were analyzed via HPLC and MALDI-TOF analysis. Finally, the biodistribution profiles of the [86Y-DOTA]L-RNA isomers were examinated in rats, to characterize the influence of this chelate unit on the in ViVo behavior of the L-RNA conjugate.

EXPERIMENTAL SECTION Materials. L-RNA 1 [sequence: 5′-aminohexyl UGA CUG ACU GAC-3′, MW 3975] was obtained from NOXXON Pharma AG (Germany). (S)-p-SCN-Bn-DOTA 2 and (S)-p-NH2Bn-DOTA 4 were purchased from Macrocyclics (Dallas, TX, USA). 86Y was purchased as [86Y]YCl3 in 0.04 M HCl (500 µL) from QSA Global GmbH (Germany). Reactions and incubations were performed in 1.5 mL DNA low-binding tubes (Eppendorf, Germany) using a Thermomixer

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Comfort (Eppendorf, Germany). Lyophilization was carried out using an Alpha 2–4 LSC (Christ, Germany). L-RNA concentrations were determined via measurement of the UV absorbance at 260 nm using an Eppendorf (Germany) BIOPhotometer. Measurements of the pH-value in small volumes were performed using the pH sensor Biotrode (Metrohm, Germany). Chromatographic Purifications and Analysis. HPLC purification and analysis were performed with a LaChrom Instrument (Hitachi-Merck, Germany) system, coupled with the radioisotope detector Gabi (Raytest, Germany). The used HPLC columns were as follows: (A) XTerra MS C18 column (Waters, USA) 3.0 × 50 mm i.d., 2.5 µm; (B) XTerra MS C18 column (Waters, USA) 4.6 × 50 mm i.d., 2.5 µm; (C) PLRP-S RP18 column (Varian Inc., USA) 4.6 × 250 mm i.d., 5 µm; (D) Aqua C18 column (Phenomenex, USA) 4.6 × 250 mm i.d., 5 µm; and (E) Aqua C18 column (Phenomenex, USA) 10 × 250 mm i.d., 5 µm. System A(I): flow 0.35 mL/min, gradient of acetonitrile (5% at 0 min, 13.5% at 18 min, 20.5% at 24 min, and 50% from 24 to 35 min) in 50 mM triethylammonium acetate (TEAA). System A(II): flow 0.35 mL/min and isocratic 5% acetonitrile in 0.1 M ammonium acetate. System A(III): flow 0.4 mL/min, gradient of acetonitrile (5% at 0 min, 7.3% at 35 min, and 50% from 36 to 40 min) in 50 mM ammonium acetate. System A(IV): flow 0.35 mL/min, linear gradient of acetonitrile (5% at 0 min, 29% at 25 min) in 50 mM NH4OAc. System A(V): flow 0.3 mL/min and isocratic 2% acetonitrile. System B(I): flow 0.7 mL/min, gradient of acetonitrile (5% at 0 min, 7.7% from 41 to 44 min, 50% from 45 to 50 min) in 50 mM ammonium acetate. System B(II): flow 0.65 mL/min, gradient of acetonitrile (5% at 0 min, 50% from 20 to 25 min) in 0.1% TFA. System C(I): flow 1 mL/min, linear gradient of acetonitrile (5% at 0 min, 20% from 25 to 30 min) in 50 mM TEAA. System C(II): flow 1 mL/min, gradient of acetonitrile (5% at 0 min, 20% at 25 min, 40% at 30 min) in 50 mM TEAA. System C(III): flow 1 mL/min, linear gradient of acetonitrile (5% at 0 min, 11% at 25 min) in 50 mM TEAA. System D(I): flow 1 mL/min (E(I): flow 4 mL/min) and an isocratic elution with 5% acetonitrile in 50 mM triethylammonium hydrogencarbonate. Mass Spectrometry. Mass spectra were recorded on a Quattro Ultima ESI-MS (Micromass, UK) and on a Daltonics Autoflex II TOF/TOF50 (Bruker, USA). The MALDI-TOF matrix, 3-hydroxypicolinic acid, was purchased from Aldrich (USA). The L-RNA probes were desalted for MALDI-TOF measurements using C18 purification tips (ZipTip, Millipore, USA) as described elsewhere (13). NMR Studies. 1D and 2D spectra were recorded on an AMX300 spectrometer (Bruker, USA) equipped with direct and inverse broadband multinuclear probes and variable-temperature unit. 1H and 13C measurements were made at frequencies of 300.13 and 75.5 MHz, with complexes dissolved in D2O and referenced using TSP as internal standard. The phase-sensitive COSY spectra were acquired using 2K data points in F2 and 512 increments in F1 dimensions and processed to a 2K × 1K matrix after apodalization with a QSine bell function in both dimensions. The phase-sensitive (TPPI) NOESY spectra were recorded at 25 °C using a mixing time (80 and 140 ms at 25 °C and 250 ms at 55 °C for Y4b and 110 ms at 25 °C for Y4a) measured using the standard inversion–recovery pulse sequence and processed as the COSY spectra. HMQC spectra were recorded using BIRD preparation period and 2K × 1K data points and processed using a square sine bell function in both dimensions. HMBC experiments were recorded using the pulse sequence inv4lplrnd of the Bruker software and acquired with 512 increments in the F1 dimension and 4K data points in the F2 dimension. Four dummy scans and 250 scans were used for spectral recording.

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Circular Dichroism (CD) Spectroscopy and Polarimetric Measurements. CD measurements were carried out on a J-715 spectropolarimeter (Jasco GmbH, Germany) interfaced with a personal computer. The CD spectra were acquired and processed using the J-700 program for Windows. The experiments were carried out at room temperature using quartz cells (Hellma GmbH, Germany) with Suprasil windows and an optical path length of 0.02 cm. Spectra were recorded in the 190–300 nm wavelength range. The signal-to-noise ratio was improved by accumulating 8 scans. Polarimetric measurements were carried out on a 241 polarimeter (PerkinElmer LS, Finland) using an optical path length of 2 cm. Both CD and polarimetric measurements were performed using the deuterated aqueous solutions of Y4a and Y4b utilized for NMR studies. [Y((S)-p-NH2-Bn-DOTA)]-. (S)-p-NH2-Bn-DOTA 4 (25 mg, 40 µmol) in 1 mL ammonium acetate buffer (0.5 M, pH 6.8) was added to 87 µL of YCl3 (0.5 M), and the mixture (pH 6.1) was incubated for 30 min at 90 °C. A solution of DTPA (300 µL, 30 mM, pH 6.0) was added, and the reaction mixture was incubated for 10 min at 40 °C to complex unreacted Y. The resulting isomers Y4a and Y4b were fractionized by HPLC system E(I), concentrated by evaporation, and lyophilized. For ESI-MS measurements, Y4a and Y4b were dissolved in acetonitrile and methanol (1:4 v/v) and the spectra were collected in the negative ion mode. ESI-MS: Y4a, m/z 594.2 [M]-; Y4b, m/z 594.2 [M]-. HPLC: A(V), RT(Y4a) 2.1 min, RT(Y4b) 2.5 min; A(II), RT(Y4a) 4.4 min, RT(Y4b) 6.5 min; C(III), RT(Y4a), 13.7 min, RT(Y4b) 15.0 min; D(I), RT(Y4a) 12.9 min, RT(Y4b) 20.3 min. [Y((S)-p-SCN-Bn-DOTA)]-. The [Y((S)-p-NH2-BnDOTA)]- isomers Y4a and Y4b (8.3 mg, 14 µmol) in 1 mL water were added separately into 10 mL glass vials (Normag, Germany) equipped with a Teflon-coated magnetic stir bar and a rubber septum. CH2Cl2 (0.9 mL) and Cl2CS (0.1 mL) were added, and the mixtures were stirred vigorously at room temperature. A ninhydrine test was performed to follow the reactions as described elsewhere (14). After 3 h, the water layers were removed carefully and the [Y((S)-p-SCN-Bn-DOTA)]isomers Y2a and Y2b were transferred into new glass vials and washed twice with 1 mL CH2Cl2. Subsequently, the isomers were lyophilized and the compounds Y2a and Y2b were obtained as a pale yellow solid. The compounds Y2a and Y2b were dissolved in acetonitrile and water (1:4 v/v) and ESI-MS spectra of both isomers were collected in the positive ion mode. ESI-MS: Y2a, m/z 638.1 [M + 2H]+; Y2b, m/z 638.1 [M + 2H]+. HPLC: A(I), RT(Y2a) 32.7 min, RT(Y2b) 31.2 min, RT(2) 26.5 min; B(II): RT(Y2a) 13.8 min, RT(Y2b) 12.8 min, RT(2) 14.4 min. [Y-DOTA]-Hexanol. (S)-p-SCN-Bn-DOTA 4 (8.0 mg, 11.5 µmol) was conjugated to 6-amino-1-hexanol (Fluka, USA) (10.4 mg, 88.8 µmol) in water (251 µL, pH 8.4) by incubation for 4 h at 40 °C. HPLC analysis indicated the complete consumption of the DOTA precursor 4. Subsequently, 100 µL of the reaction mixture, 9.2 µL YCl3 (0.5 M in 0.04 M HCl), and 200 µL ammonium acetate buffer (0.5 M) were incubated for 20 min at 90 °C and pH 5.8. Additionally, the [Y((S)p-SCN-Bn-DOTA)]- isomers Y2a and Y2b (1.1 mg, 0.6 µmol) were conjugated separately to 6-amino-1-hexanol (2.4 mg, 0.6 µmol) by incubation in water for 1 h at 25 °C and pH 8.8. Product fractions of both reaction batches were separated via HPLC analyzed via ESI-MS. ESI-MS: Y6a, m/z 755.3 [M + 2H]+; Y6b, m/z 755.4 [M + 2H]+. HPLC: A(IV), RT(Y6a) 16.3 min, RT(Y6b) 17.0 min; C(II): RT(Y6a) 26.0 min, RT(Y6b) 26.8 min. DOTA-L-RNA. (S)-p-SCN-Bn-DOTA 2 (6.3 mg, 9.0 µmol) was conjugated to L-RNA 1 (2.4 mg, 0.6 µmol) in 0.7 M sodium

Schlesinger et al.

bicarbonate buffer (121 µL, pH 9.0) by incubation for 2 h at 40 °C. For the purification of DOTA-conjugate 3, 300 µL ethanol (abs.) and 1100 µL 70% ethanol were added, and the mixture was centrifuged for 20 min at –10 °C and 14 000 rpm. The supernatant with unreacted (S)-p-SCN-Bn-DOTA 2, and DOTA-thiourea 5 was removed and the pellet washed twice with 70% ethanol, centrifuged, and resuspended in 0.3 M sodium acetate for an additional precipitation with ethanol as described above. Subsequently, the pellet was resuspended in 100 µL water. DOTA-L-RNA 3 was purified via size exclusion chromatography (SEC) using a 25 × 490 mm column (P4 Biogel, Medium 90–180 µm, Bio-Rad Melville, USA) with 0.1 M ammonium acetate buffer (pH 7.0) as mobile phase at a flow rate of 17.5–18.4 mL/h. The UV absorbance was monitored at 260 nm. The identity of the DOTA-conjugate 3 was verified by mass spectrometry. MaldiTOF: 3, m/z 4521.4 [M + H]+. [Y-DOTA]-L-RNA. [Y((S)-p-SCN-Bn-DOTA)]- isomers Y2a and Y2b were conjugated separately to L-RNA 1 by incubation in borate buffer (12.5 mM, pH 9.6) for 3 h at 40 °C. Product fractions of both reaction batches were separated via HPLC and lyophilized. The separated HPLC fractions were analyzed via MALDI-TOF measurements. To confirm the identity of [86Y-DOTA]-L-RNA isomers Y3a and Y3b, the DOTA-modified L-RNA 3 was radiolabeled with carrier-added 86 Y using a molar ratio of 3 to YCl3 of 1:1. Both 86Y-complex isomers [86Y]3a and [86Y]3b were analyzed by HPLC and Maldi-TOF. MALDI-TOF: Y3a, m/z 4613 [M + 2H]+; Y3b, m/z 4612 [M + 2H]+. HPLC: A(III), RT(Y3a) 26.5 min, RT(Y3b) 29.4 min, RT([86Y]3a) 26.8 min, RT([86Y]3b) 29.8 min. [86Y((S)-p-NH2-Bn-DOTA)]-. (S)-p-NH2-Bn-DOTA 4 (66 µg, 0.1 µmol) in 450 µL ammonium acetate buffer (0.5 M, pH 7.0) was added to 100 µL of [86Y]YCl3 (21 MBq) and the mixture was incubated for 20 min at 90 °C. A solution of DTPA (10 µL, 30 mM, pH 6.0) was added, and the reaction mixture was incubated for 5 min at 40 °C to complex unreacted [86Y]Y3+. The [86Y((S)-p-NH2-Bn-DOTA)]- isomers [86Y]4a and [86Y]4b were separated by HPLC system A(II). Under these conditions, the [86Y(DTPA)]2- complex eluted with the void volume from the column. HPLC fractions containing [86Y]4a and [86Y]4b were concentrated at 90 °C under a dinitrogen stream, and the residues were dissolved in saline. HPLC fractions of the [86Y((S)-p-NH2-Bn-DOTA)]- isomers 86 [ Y]4a and [86Y]4b, containing TEAA as ion pair reagent, was used for estimation of the partition coefficient. A saturated n-octanol/water system was prepared in 1.5 mL tubes containing 400 µL buffer (25 mM disodium hydrogen phosphate and 0.09 M sodium chloride (pH 7.4)) and 400 µL n-octanol. The isomers [86Y]4a and [86Y]4b (30 µL, app. 250 kBq) were added separately, and the solutions were shaken vigorously for 5 min and incubated on a wave mixer for 30 min. The organic and aqueous phases were separated by centrifugation for 5 min at 5000 rpm. Subsequently, 300 µL of the n-octanol layer was transferred into a new tube, and the phase boundary was removed carefully by adding 200 µL to the waste. Afterward, the water layer (300 µL, pH 7.0) was transferred into a separate tube. The radioactivity of the organic and aqueous phases was measured on the gamma counter in five experiments, and the average was calculated. The partition coefficient log D7.0 was defined as the ratio of the amount of the radiotracer present in the organic layer to that present in the aqueous phase. Partition coefficient: log D7.0 ([86Y]4a), –2.0 ( 0.029; log D7.0 ([86Y]4b), -2.3 ( 0.032. HPLC: A(II), RT([86Y]4a) 5.0 min, RT([86Y]4b) 7.3 min; C(III): RT([86Y]4a) 14.4 min, RT([86Y]4b) 15.7 min.

86

Y-Labeled Mirror-Image Oligonucleotide

[86Y-DOTA]-L-RNA. Radiolabeling of the DOTA-modified 86 L-RNA 3 (95–160 µg, 21–36 nmol) with [ Y]YCl3 was performed in 300–350 µL ammonium acetate buffer (0.5 M, pH 7.0). After the addition of 50–100 µL of [86Y]YCl3 (13–45 MBq), the mixture was incubated for 30–60 min at 90 °C. A solution of DTPA (10 µL, 30 mM, pH 6.0) was added, and the reaction mixture was incubated for 10 min at 40 °C to complex unreacted [86Y]Y3+. The HPLC separation of the isomers [86Y]3a and [86Y]3b were performed on system B(I). Under these conditions, the [86Y(DTPA)]2- complex eluted with the void volume from the column. HPLC fractions containing the separated isomers [86Y]3a and [86Y]3b were concentrated in a stream of nitrogen at 40 °C for 10 min. HPLC: B(I), RT([86Y]3a) 29.7 min, RT([86Y]3b) 32.4 min; A(III), RT([86Y]3a) 27.5 min, RT([86Y]3b) 29.8 min. Determination of the Time-Dependent Radiochemical Yield of [86Y]3. The kinetics of 86Y-complexation of L-RNA 3 was followed by SDS-PAGE. In four attempts, L-RNA 3 (100 µg, 22 nmol) and [86Y]YCl3 (50 µL, 12–45 MBq) was incubated in ammonium acetate buffer (0.5 M, 300 µL, pH 7.0) at 90 °C for 60 min. At specific time points (0, 3, 9, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min), reaction aliquots (15 µL) were mixed with a solution of DTPA (10 µL, 15 mM, pH 6.0) and heated up to 40 °C for 10 min to complex free [86Y]Y3+ as [86Y(DTPA)]2- complex. SDS-PAGE was performed using a Multiphor II Electrophoresis (GE Healthcare, USA) system and SDS precast gels (GE Healthcare, USA), ExcelGel Gradient 8–18. A 10 bp DNA ladder (Invitrogen, USA) was applied as a molecular weight standard. After electrophoresis, the gel was exposed to a phosphor imaging plate for 2–4 min and scanned with a Fujix BAS 5000 (Fujifilm Europe, Germany). Subsequently, the gel was stained using ethidium bromide, and the DNA ladder was visualized and photographed under UV light. Radiochemical yields were calculated from the radioactivity area of the product peak related to the total radioactivity area using Tina 2.09 software (Raytest, Germany). Biodistribution. All animal experiments were performed in accordance with the German laws for the protection of animals and were approved by the regional council. The biodistribution profiles of the radiolabeled L-RNA isomers [86Y]3a and [86Y]3b and the [86Y((S)-p-NH2-Bn-DOTA)]- isomers [86Y]4a and [86Y]4b were studied in male Wistar rats (150 g body weight). For the time points 5 and 60 min, approximately 120 kBq aliquots of each compound were administered in 300 µL saline into the tail vein. For the longer examination period of 18 h, approximately 250 kBq aliquots of the L-RNA isomers [86Y]3a and [86Y]3b were administered. After the radiotracer application, four rats were anaesthetized for each radiotracer with ether at 5 min, 60 min, and 18 h, respectively. Blood was withdrawn by heart puncture, and the animals were euthanized. Organs and tissues were removed and weighed, and the radioactivity was measured in a calibrated well counter. The data were normalized to the amount of injected radioactivity and expressed as standardized uptake values (SUV). The SUV was calculated with following equation: SUV ) (organ activity/organ weight)/(total given activity/ rat body weight) The amount of radioactivity in urine was calculated as the total radioactivity of the injected dose subtracted by the values of all individual organs. Metabolite Analysis. The in Vitro stability analysis of the radiolabeled L-RNA [86Y]3b was performed via HPLC as described in detail in the Supporting Information. The ex ViVo autoradiography of the kidney sections containing the L-RNA [86Y]3b was detailed in the Supporting Information. [86Y((S)-p-NH2-Bn-DOTA)]-. The [86Y((S)-p-NH2-BnDOTA)]- somers [86Y]4a and [86Y]4b (2.3 MBq) were injected

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separately and intravenously into male Wistar rats. After sacrificing the rats 60 min p.i., the bladders were removed. Urine samples were collected by puncture of the bladders and methanol was added to the sample in a ratio of 1:2. The mixtures were shaken vigorously and centrifuged for 10 min at 13 000 rpm. The supernatants were transferred into a new vial, and the solutions were concentrated at 65 °C in a stream of nitrogen. TLC analyses were performed on Merck (Germany) RP18 plates with system (I): 0.1 M ammonium acetate in 5% acetonitrile (pH 7.0) and with system (II): methanol and 10% ammonium acetate (9:1 v/v) as the running buffer. The dried TLC plates were exposed to phosphor imaging plates for 10 min. The plates were scanned and the rates of radioactivity were calculated as described above. HPLC analyses of the prepared urine samples ([86Y]4a, 50 µL, 17 kBq; [86Y]4b, 100 µL, 90 kBq) were performed on system C(II). [86Y-DOTA]-L-RNA. The radiolabeled L-RNA [86Y]3 (2.3 MBq) isomers was injected intravenously into a male Wistar rat. Arterial blood samples of the rat were collected after injection of the radiolabeled compounds after 1, 3, 5, and 10 min p.i. The plasma of the blood samples was separated by centrifugation for 10 min at 13 500 rpm and 23 °C. Subsequently, the plasma samples were diluted in buffer (25 mM disodium hydrogen phosphate and 0.09 M sodium chloride (pH 7.4)) in a ratio of 1:1 and applied on a 20% PAGE. The plasma fraction of the blood probes obtained after 20 min p.i. contained insufficient radioactivity (95% for DOTA-L-RNA 3 were achieved as confirmed by HPLC chromatography. Only L-RNA 1 was detected as a minor (