Development and Evaluation of an Albumin-Binding NODAGA-Folate

Transcription

1 Supporting Information (SI) 64 Cu- and 68 Ga-Based PET Imaging of Folate Receptor-Positive Tumors: Development and Evaluation of an Albumin-Binding NODAGA-Folate Renáta Farkas 1, Klaudia Siwowska 1, Simon Ametamey 2, Roger Schibli 1,2, Nicholas P. van der Meulen 1,3, Cristina Müller 1,2* 1 Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, Villigen-PSI, Switzerland 2 Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland 3 Laboratory of Radiochemistry, Paul Scherrer Institut, Villigen-PSI, Switzerland * Corresponding author: PD Dr. Cristina Müller Center for Radiopharmaceutical Sciences ETH-PSI-USZ Paul Scherrer Insitute 5232 Villigen-PSI Switzerland cristina.mueller@psi.ch phone: fax: Running Title: Albumin-binding 64 Cu-/ 68 Ga-NODAGA-folates for PET imaging 1

2 Organic Synthesis of NODAGA-AB-folate (rf42) General All reagents and solvents were purchased from Sigma-Aldrich Chemie GmbH (Germany), Merck (Germany) or Fluka (Switzerland). The protected amino-acids were obtained from Bachem, Switzerland. NODA-GA(t-Bu) 3 was purchased from CheMatech, France. Protected folic acid was kindly provided by Merck & Cie (Schaffhausen, Switzerland). High resolution mass spectrometry (HRMS) was performed on a Bruker's maxis (ESI-Qq-TOF-MS) spectrometer (Bruker Daltonik GmbH, Germany) and the results are given in m/z. HPLC was performed with a Merck-Hitachi system, which was equipped with a D-7000 interface, a L-7400 UV detector, and a L-7100 pump. For analytical purposes a reversed-phase C18 column (Sunfire TM, 5 µm, mm, Waters) was used. Purification steps were carried out using a semi-preparative reversed-phase C18 column (Sunfire TM, 5 µm, mm, Waters). Organic synthesis of the NODAGA-folate (rf42) Synthesis of compound 1. 4-(p-Iodophenyl)butyric acid (1045 mg, 3.60 mmol) was dissolved in DMF (5 ml). After addition of triethylamine (732 µl, 5.25 mmol), the reaction mixture was cooled down at 0 C. HBTU (1327 mg, 3.60 mmol) was added and the reaction mixture stirred for 30 min. A solution of Boc-Lys-OMe in DMF (6 ml) and triethylamine (1400 µl, mmol) was dropwise added and the reaction mixture was stirred for 1 h at 0 C. Subsequently, the product was precipitated by pouring the reaction mixture in ice/water mixture (30 ml). The white precipitation was filtered and purified by column chromatography with 1:3 hexane/ethyl acetate as an eluent to obtain the pure product 1. Yield: 1773 mg, 92%. HRMS calculated for C 22 H 34 IN 2 O 5 (M+H) , found Scheme 1: Synthesis of compound 1. 2

3 Synthesis of compound 3. Intermediate 1 (260 mg, mmol) was dissolved in a mixture of DCM (1 ml) and TFA (0.75 ml) for deprotection of the Boc group. The solution was stirred at rt for 20 min before removal of the solvent under reduced pressure. Crude product 2 (267 mg, mmol) was dissolved in a mixture of DMF (2 ml) and TFA (0.2 ml, mmol). It was dropwise added to a solution of Boc-Lys(Cbz)- OH (186 mg, mmol), dissolved in a mixture of DMF (2 ml) and TFA (0.102 ml, mmol) and activated with HBTU (124 mg, mmol). The mixture was stirred at rt for 1 h. The product was precipitated by pouring the reaction mixture in ice/water mixture (30 ml). The white precipitation was filtered and dried to afford compound 3. Yield: 380 mg, 85 %. HRMS calculated for C 36 H 52 IN 4 O 8 (M+H) , found Scheme 2: Synthesis of compound 3. 3

4 Synthesis of compound 5. Acidic deprotection of the Boc group of compound 3 (100 mg, mmol) was performed in a 1:4 mixture of TFA/DCM for 3 h at rt. Solvent was removed under reduced pressure. The crude compound 4 was directly used for the next reaction step. Protected folic acid (78 mg, mmol) was dissolved in DMF (2 ml) and triethylamine (0.026 ml, mmol) and HBTU (47.8 mg, mmol) was added. The mixture was stirred at rt for 20 min. A solution of compound 4 (102 mg, mmol) in DMF (2 ml) and triethylamine (0.044 ml, mmol) was dropwise added and stirred for 1 h. The product was precipitated by pouring the reaction mixture in ice/water mixture (40 ml). The beige precipitation was filtered and purified by column chromatography using 5% EtOH in DCM as eluent. The product was dried to obtain compound 5. Yield: 109 mg, 67%. HRMS calculated for C 57 H 68 F 3 IN 11 O 13 (M+H) , found Scheme 3: Synthesis of compound 5. 4

5 Synthesis of compound 6. The Cbz-protecting group of compound 5 (100 mg, mmol) was cleaved using thioanisole (8 equiv, 50 µl) dissolved in TFA (1 ml) within 6 h at rt. The solvent was removed under reduced pressure and the residue was recrystallized from cold diethyl-ether to obtain compound 6. Yield: 71 mg, 79%. HRMS calculated for C 49 H 62 F 3 IN 11 O 11 (M+H) , found Scheme 4: Synthesis of compound 6. 5

6 Synthesis of compound 7. Protected NODAGA chelator (20.85 mg, mmol) was dissolved in DMF (0.5 ml) and triethylamine (8.02 µl, mmol) and HBTU (14.54 mg, mmol) were added. The mixture was stirred for 20 min at rt. A DMF solution (0.5 ml) of compound 6 (49 mg, mmol) was added dropwise and stirred for 2 h. Thereafter, MilliQ water (5 ml) was added and the reaction mixture was extracted with ethyl acetate (3 x 3 ml). The combined organic layers were dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue was purified by column chromatography with 10% MeOH in DCM as eluent to obtain the pure compound 7. Yield: 54 mg, 83%. HRMS calculated for C 76 H 108 F 3 IN 14 NaO 18 (M+Na) , found Scheme 4: Synthesis of compound 7. 6

7 Synthesis of NODAGA-AB-folate (rf42) Deprotection of compound 7 (7 mg, 4.14 µmol) was achieved in two steps. Acid labile groups were cleaved using a mixture of TFA/TIPS/H 2 O (200 µl, 95 : 2.5 : 2.5) for 12 h at rt. After evaporation of the solvents 200 µl of an aqueous lithium hydroxide solution (1 M) was added and stirred for 4 h at rt. The solution was acidified with hydrochloric acid solution (1 M) resulting in precipitation of a yellow product. Purification was carried out using semi-preparative HPLC to afford the final product rf42 in 36% (2 mg) yield. The 1 H NMR and HRMS results are provided in the main manuscript. Scheme 5: Synthesis of NODAGA-AB-folate (rf42). 7

8 Preparation of 64 Cu- and 68 Ga-Labeled DOTA-Conjugate (cm10) Purpose. The previously developed and evaluated DOTA-folate conjugate (cm10) differs from the novel compound (rf42) solely with regard to the chelator. DOTA-folate (cm10) was used as a reference compound for comparison of the results. Fig. S1. Chemical structure of a previously developed DOTA-folate conjugate cm10. Results. Radiolabeling of cm10 with 64 Cu and 68 Ga, respectively, was carried out under the same conditions as for rf42. Quality control was performed by HPLC as reported in the main manuscript. 64 Cu-cm10 (t R = 9.7 min) and 68 Ga-cm10 (t R = 9.4 min) were stable (>95%) over a period of 4 halflives of the corresponding radionuclide when they were incubated in PBS ph 7.4. In Vitro Evaluation of 64 Cu-cm10 and 68 Ga-cm10 Purpose. In vitro experiments were performed with 64 Cu-cm10 and 68 Ga-cm10 in parallel to the evaluation of the radiolabeled rf42 conjugates ( 64 Cu-rf42 and 68 Ga-rf42) where it was appropriate and served as a reference for comparison. Results. The distribution coefficient (logd value) determined for 64 Cu-cm10 was ± 0.13 and 8

9 -3.94 ± 0.18 for 68 Ga-cm10. The fractions of the plasma-bound radiofolates are shown in Table S1. A significant difference (p < 0.05) of the protein-bound fractions of the folate radioconjugate was determined only between 68 Ga-rf42 and 64 Cu/ 68 Ga-cm10. Table S1. Results of ultrafiltration assay using samples of radiofolates after incubation in human plasma. The values represent the plasma bound fractions which were calculated as percentage of total added radioactivity. 64 Cu-rf42 68 Ga-rf42 64 Cu-cm10 68 Ga-cm10 Plasma protein bound fraction [% of totally added activity] 96.3 ± ± ± ± 0.1 Additional PET/CT Scans of 64 Cu- and 68 Ga-Labeled Folate Conjugates Purpose. PET/CT scans were performed with KB tumor-bearing mice which received the radiofolates only ( 64 Cu/ 68 Ga-rf42, 64 Cu/ 68 Ga-cm10) or the radiofolates with pre-injected folic acid (100 μg in 100 μl PBS ph 7.4) to demonstrate specific binding to FR-expressing tissues. Results In all cases the uptake in the KB tumor tissue (FR+) and in the kidneys (FR+) was significantly reduced in mice which received a pre-injection of folic acid (Fig. S2-S5). In these mice only background levels of accumulated radioactivity were observed in the heart, liver and intestines. As the radiofolates are circulating longer than folic acid which was used to block the receptors, the blockade was not complete anymore 2 h after injection and in some cases the tumors were therefore visible. Nevertheless, with these experiments it was confirmed that radifolates show FR-specific uptake. 9

10 Fig. S2. PET/CT images of KB tumor-bearing mice 2 h after injection of 64 Cu-rf42 only (A) and 64 Curf42 after a pre-injection of folic acid (B) shown as maximal intensity projections. During the in vivo PET (10 min) and CT (1.5 min) scan the mice were anesthetized with isoflurane/oxygen. (Tu = KB tumor xenograft, Ki = kidney, Bl = urinary bladder, BkG = background activity in the heart and intestines). Fig. S3. PET/CT images of KB tumor-bearing mice 2 h after injection of 64 Cu-cm10 only (A) and 64 Cu-cm10 after a pre-injection of folic acid (B) shown as maximal intensity projections. During the in vivo PET (10 min) and CT (1.5 min) scan the mice were anesthetized with isoflurane/oxygen. (Tu = KB tumor xenograft, Ki = kidney, Li = liver, Bl = urinary bladder). 10

11 Fig. S4. PET/CT images of KB tumor-bearing mice 2 h after injection of 68 Ga-rf42 only (A) and 68 Garf42 after a pre-injection of folic acid (B) shown as maximal intensity projections. During the in vivo PET (10 min) and CT (1.5 min) scan the mice were anesthetized with isoflurane/oxygen. (Tu = KB tumor xenograft, Ki = kidney, BkG = background activity in the heart an intestines). Fig. S5. PET/CT images of KB tumor-bearing mice 2 h after injection of 68 Ga-cm10 only (A) and 68 Ga-cm10 after a pre-injection of folic acid (B) shown as maximal intensity projections. During the in vivo PET (10 min) and CT (1.5 min) scan the mice were anesthetized with isoflurane/oxygen. (Tu = KB tumor xenograft, Ki = kidney, BkG = background activity in the heart an intestines). 11