MATERIALS AND METHODS Synthesis

Transcription

1 MATERIALS AND METHODS Synthesis Analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and Radio-HPLC were performed as described previously (1, 2). Preparative RP-HPLC was performed on the same system using a Multospher 100 RP 18-5 ( mm) column (CS GmbH, Langerwehe, Germany) applying various gradients of 0.1% (v/v) trifluoroacetic acid in H 2 O (solvent A) and 0.1% trifluoroacetic acid (v/v) in acetonitrile (solvent B) at a constant flow of 10 ml/min. Electrospray ionization-mass spectra (MS) were acquired on a Varian 500-MS IT mass spectrometer (Agilent Technologies, Santa Clara, USA). PSMA I&T (DOTAGA-(I-y)fk(Sub-KuE)) was synthesized according to a previously published protocol (2) by fragment condensation of the DOTAGA-coupled peptide linker (DOTAGA-iodo-D-Tyr-D-Phe-D- Lys-OH = DOTAGA-(I-y)fk) and the succinimidyl active ester of tert-butyl-protected Sub-KuE. After final acidic deprotection the product was purified by RP-HPLC (isocratic elution at 26% B). HPLC (25 to 55% in 15 min): t R = 11.5 min, K = 5.4. Calculated monoisotopic mass for PSMA I&T (C 63 H 92 IN 11 O 23 ): 1, found: m/z = 1,498.3 [M+H] +, 1,520.1 [M+Na] +. nat Ga-PSMA I&T: To a 2 mm solution of PSMA I&T in water an equal volume of 2 mm Ga(NO 3 ) 3 was added. After 30 min at 40 C, quantitative complex formation was confirmed by RP-HPLC and MS. HPLC (25 to 55% in 15 min): t R = 11.8 min, K = 5.6. Calculated monoisotopic mass for nat Ga-PSMA I&T (C 63 H 89 IN 11 O 23 Ga): 1,563.44/1, found: m/z = [M+2H] 2+, 1,564.5/1,566.3 [M+H] +. nat Lu-PSMA I&T: To a 2 mm solution of PSMA I&T was added LuCl 3 in water (2.5-fold molar excess using a 20 mm solution). The ph was adjusted to 5 using 0.1 M NH 4 OAc. After 30 min at 95 C, quantitative complex formation was determined by RP-HPLC and MS. HPLC (25 to 55% in 15 min): t R = 11.9 min, K = 5.6. Calculated monoisotopic mass for nat Lu-PSMA I&T (C 63 H 89 IN 11 O 23 Lu): 1, found: m/z = 1,669.9 [M+H] +, 1,691.9 [M+Na] +. Radiolabeling 68 Ga-labeling: For cell studies the 68 Ge/ 68 Ga-generator eluate (1 M HCl) was trapped on a strong cation exchange cartridge and eluted with aq. NaCl (5 M, 0.5 ml). 2-[4-(2-Hydroxyethyl)-1- piperazinyl]-ethanesulfonic acid (HEPES, 2.7 M aq., 140 µl) and PSMA I&T (3 nmol) were added and heated at 95 C (5 min, ph 4.5) (2, 3). After cooling, labeling efficiency and radiochemical purity were determined using Radio-thin layer chromatography (TLC) and Radio-HPLC (3, 4). For in vivo biodistribution and small animal PET studies, the 68 Ga-compounds were prepared in a GMP-compliant procedure using 5 nmol PSMA I&T in a fully automated synthesis module (GRP, Scintomics, Germany) similarly to the procedure described previously (4). The ethanol content, which is present in the final tracer preparation, was evaporated in vacuo. 177 Lu-labeling: For in vitro and animal studies, 0.91 nmol PSMA I&T was added to 24.5 MBq 177 Lu- LuCl 3 (120 GBq/µmole, IDB Radiopharmacy bv) in 0.05 M HCl. The ph was adjusted to ph 5 by the

2 addition of NH 4 OAc (0.1 M, 150 µl). After 30 min at 95 C the labeling efficiency was examined by Radio-TLC and Radio-HPLC. Radiolabeling for human application: PSMA-HBED-CC, as well as PSMA I&T were labeled with 68 Ga using an automated synthesis module (GRP, Scintomics, Germany) by adapting the procedure as described previously (3, 4). In brief, 25 µg of PSMA-HBED-CC or PSMA I&T were dissolved in HEPES buffer (1 M, 3 ml) for labeling with 68 Ga III and incubated at 95 C for 12 min, followed by a purification step using a Waters SepPak C18 light cartridge. 177 Lu-PSMA I&T for clinical use was prepared after incubation of µg PSMA I&T with 6-8 GBq of 177 LuCl 3 (ITG, Garching, Germany) at 90 C for 30 min in NaOAc (0.4 M, 800 µl, ph 5.5). To this buffer, 5-10 mg of gentisic acid was added to prevent radiolysis. The reaction solutions were diluted with saline and after sterile filtration a sample was taken for quality control (radio-hplc, radio-tlc, ph, limulus test, sterility testing, retention sample). Extrapolation to Humans The dose extrapolation to humans involved the scaling of the time-integrated activity coefficients and the subsequent calculation of the absorbed doses from the animal biodistribution data. The scaling was performed by two different methods. Method 1 was based on the assumption that the residence time for the same organ is the same in mice and humans (5, 6). Method 2 considered a relative mass scaling where the specific activity in a certain human organ is equal to the specific activity in the same mouse organ multiplied by the ratio of the body mass of human and mouse (6-8). Time-integrated activity coefficients ( residence times, RT) were calculated using the software solution NUKFIT as described by Kletting et al., choosing the optimal fit functions as proposed by the code (9). The dose calculation was performed for a selected group of organs using OLINDA/EXM V1.1 (10). Some special considerations for the RTs were used due to the differences between the organs/tissues used in OLINDA/EXM and the organs/tissues measured in our experiments: 1) Organ weights The weights of the human organs were extracted from OLINDA/EXM and, if not available, from ICRP 89 (7). Data for mice were taken from Bitar et al. (8) 2) Blood Total mouse blood volume is about 1.65 ml (8). The total activity RT mouse in the blood of a mouse is, therefore, the RT mouse for blood per ml of blood multiplied by In both methods we set the RT mouse equal to the RT human for blood in humans. 3) Bone Marrow For calculating the RT for the bone marrow we used the following approximation (11):

3 RT bone marrow = (RT blood /5300)*0.36*1500 4) Remainder of the Body The RT for the remainder of the body was set equal to the RT of blood 5) Stomach Content As during the experiments only the activities of the empty stomach were measured, we set the RT for the stomach equal to the RT of the stomach wall. 6) Heart MIRD divides the heart contribution as source organ into heart wall and heart content. The measurements were done on heart with contents. It was assumed that 10 % of the measured activity was associated to the heart wall and 90 % to the heart contents (6). 7) Bone The RT of the femur was multiplied by the ratio of total bone mass to the femur mass in mice in order to account the activity in the bone. Data for mice were taken from Bitar et al. (8) 8) Trabecular and Cortical Bone It was assumed that bone consisted of 33 % trabecular and 67 % cortical bone (6). 68 Ga-PSMA I&T PET imaging in patients Contrast enhanced CT was carried out after i.v. administration of nonionic iodinated contrast material (100 ml, 300 mg/ml (Ultravist 300; Bayer AG, Berlin, Germany) at 1 ml/s, 90 s delay). The further imaging/reconstruction parameters were: 120 kv, effective mas 44, gantry rotation time 0.6 s, reconstruction thickness 1.5 mm with an increment of 0.8 mm, reconstruction kernel B30f, matrix Immediately after the CT scan, whole-body PET scanning was performed from the base of the skull through the mid-thigh with a 3 min acquisition time per bed position (16.2 cm) in 3D. Reconstruction was conducted with an OSEM algorithm with 2 iterations/8 subsets and Gaussian-filtered to a transaxial resolution of 5 mm at full-width at half-maximum. Attenuation correction was performed using the contrast enhanced CT dataset.

4 RESULTS Internalization kinetics The cell binding and internalization kinetics of the PSMA-inhibitor 68 Ga-PSMA I&T in PSMAexpressing LNCaP cells for up to 1 h are summarized in Supplemental Figure 1. Supplemental Figure 1. Cell binding and internalization kinetics of 68 Ga-PSMA I&T at 37 C in LNCaP cells. Data are corrected for non-specific binding (10 µm PMPA) and expressed as mean ± SD.

5 Extrapolation to Humans Based on the biodistribution of 177 Lu-PSMA I&T in CD-1 mice (Supplemental Table 1), dosimetry estimates were calculated to humans. Supplemental Table 1. Biodistribution of MBq 177 Lu-PSMA I&T in CD-1 mice at 1, 6, 12, 24, 48 and 96 h p.i. (n = 5, respectively). Organ 1 h 6 h 12 h Blood ± ± ± Heart ± ± ± Lung ± ± ± Liver ± ± ± Pancreas ± ± ± Spleen ± ± ± Uterus ± ± ± Stomach ± ± ± Small intestine ± ± ± Large Intesine ± ± ± Adrenals ± ± ± Kidneys ± ± ± Muscle ± ± ± Bone ± ± ± Skin ± ± ± Brain ± ± ± Organ 24 h 48 h 96 h Blood ± ± ± Heart ± ± ± Lung ± ± ± Liver ± ± ± Pancreas ± ± ± Spleen ± ± ± Uterus ± ± ± Stomach ± ± ± Small intestine ± ± ± Large Intesine ± ± ± Adrenals ± ± ± Kidneys ± ± ± Muscle ± ± ± Bone ± ± ± Skin ± ± ± Brain ± ± ± Supplemental Table 2 provides a list of the time-integrated activity coefficients for a number of organs of relevance for dosimetry for both scaling methods. For both scaling methods, the highest values were observed for the kidney (8.3 h and 5.8 h). All other organs showed considerably lower residence times.

6 Supplemental Table 2. Time integrated activity coefficients for several organs scaled to humans. Residence Time Organ Method 1 Method 2 Heart contents Heart wall Lung Liver Kidneys Pancreas Spleen Uterus Stomach SI Contents Adrenals Muscle Brain Bone marrow Cortical bone Trabecular bone Remainder The corresponding absorbed doses, as calculated with OLINDA/EXM, for the two scaling methods are given in table 3. It contains information on the absorbed dose by beta particles (cols 2 and 5), photons (col 3 and 6) and the sum of both (col 4 and 7).

7 Supplemental Table 3. Dose Coefficients for penetrating and non-penetrating radiation based on both extrapolation methods. Method 1 mgy/mbq Method 2 mgy/mbq Target Organ Beta Photon Total Beta Photon Total Adrenals 5.21E E E E E E-02 Brain 4.78E E E E E E-04 Breasts 4.15E E E E E E-04 Gallbladder Wall 4.15E E E E E E-03 LLI Wall 4.15E E E E E E-04 Small Intestine 7.37E E E E E E-03 Stomach Wall 3.31E E E E E E-03 ULI Wall 4.15E E E E E E-03 Heart Wall 1.11E E E E E E-03 Kidneys 2.36E E E E E E+00 Liver 3.91E E E E E E-03 Lungs 7.13E E E E E E-02 Muscle 4.15E E E E E E-03 Ovaries 4.15E E E E E E-04 Pancreas 1.35E E E E E E-03 Red Marrow 5.23E E E E E E-03 Osteogenic Cells 3.53E E E E E E-03 Skin 4.15E E E E E E-04 Spleen 2.46E E E E E E-02 Testes 4.15E E E E E E-04 Thymus 4.15E E E E E E-04 Thyroid 4.15E E E E E E-04 Urinary Bladder Wall 4.15E E E E E E-04 Uterus 3.22E E E E E E-03 Total Body 1.01E E E E E E-03 Effective Dose (msv/mbq) 9.60E E-03

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