Aim/Introduction: The use of an albumin binder has been shown to improve tumor uptake of prostate specific membrane antigen (PSMA)-targeting radiopharmaceuticals. We recently reported 177Lu-HTK01169, a 177Lu-PSMA-617 derivative that delivered 8.3-fold higher absorbed dose to LNCaP tumor xenografts than 177Lu-PSMA-617, at the expense of a lower tumor-to-kidney therapeutic index. We systematically investigated different linkers and albumin binders to maximize the therapeutic index of PSMA-binding radiopharmaceuticals. We aimed to develop novel radiopharmaceuticals that combine the effects of an improved linker with optimized albumin binders to maximize the tumor-to-kidney therapeutic index. Materials and Methods: Two novel PSMA-binding radiopharmaceuticals derived from the glutamate-ureido-lysine backbone, HTK03121 and HTK03123, were synthesized using a solid-phase approach. HTK03121 and HTK03123 were synthesized with a linker containing the lipophilic 3-(9-anthryl)-L-alanine and tranexamic acid, with the addition of albumin-binding motifs 4-(p-chlorophenyl)butanoyl]-Gly (HTK03121) and 4-(p-methoxyphenyl)butanoyl]-Gly (HTK03123). 177Lu labeling was conducted in acetate buffer (pH 4.5). SPECT/CT and biodistribution studies were performed in LNCaP tumor-bearing mice. Radiation dosimetry in mice was calculated using the OLINDA v.1.2. Results: SPECT/CT imaging showed that both 177Lu-HTK03121 and 177Lu-HTK03123 had very high and sustained tumor uptake and were excreted mainly via the renal pathway. The blood retention values (%ID/g) at 1, 4, 24, 72 and 120h were 22.6±5.35, 15.3±2.07, 4.33±0.88, 0.75±0.19 and 0.23±0.05, respectively for 177Lu-HTK03121 and 14.1±1.01, 7.30±1.39, 0.71±0.29, 0.16±0.01 and 0.09±0.06, respectively for 177Lu-HTK03123. Tumor uptake values of 177Lu-HTK03121 and 177Lu-HTK03123 all peaked at 24h (104±20.3 and 70.8±23.7 %ID/g, respectively), and remained stable up to 120h post-injection. For a 350-mg lesion size, the dose delivered to tumors were 13,316 and 13,498 mGy/MBq for 177Lu-HTK03121 and 177Lu-HTK03123 compared to 846 mGy/MBq for 177Lu-PSMA-617. Kidney doses were 15.8, 15.2 and 2.82 mGy/MBq for 177Lu-HTK03121, 177Lu-HTK03123 and 177Lu-PSMA-617, for tumor-to-kidney absorbed dose ratios of 843, 888 and 300 respectively. Thus, compared to 177Lu-PSMA-617, 177Lu-HTK03121 delivered 15.7-fold higher absorbed dose to tumor and a 2.8-fold improvement in the tumor-to-kidney dose ratio. 177Lu-HTK03123 delivered 16.0-fold higher absorbed dose to tumor, a 2.96-fold improvement in the tumor-to-kidney dose ratio. Conclusion: We report novel PSMA-targeting radiopharmaceuticals that showed, in a mouse model of prostate cancer, very high tumor uptake, 15.7- and 16.0-fold higher absorbed dose to tumors and superior tumor-to-kidney therapeutic ratios compared to 177Lu-PSMA-617. These compounds have the potential to improve treatment safety and efficacy using significantly lower quantities of 177Lu, and are promising candidates for clinical translation. References: Kuo H-T, et al. Molecular Pharmaceutics 2018;15:5183-5191.
Aim/Introduction: A number of albumin-binding radioligands, targeting the prostate-specific membrane antigen (PSMA), have been developed for radionuclide therapy of metastasized prostate cancer . 177Lu-PSMA-ALB-56 consists of a DOTA-functionalized PSMA ligand with a p-tolyl-based albumin-binding entity which resulted in high tumor accumulation and, hence, improved therapeutic efficacy compared to 177Lu-PSMA-617 . High blood activity levels would be limiting for therapy due to the risk of bone marrow toxicity. The aim of this study was, therefore, the development of a new class of PSMA ligands with a weak albumin binder and variable linker entities to enable fine-tuning of kinetic properties. Materials and Methods: Four glutamate-urea-lysine-based PSMA ligands were synthesized using solid-phase chemistry in analogy to the previously described synthesis of PSMA-ALB-56 . The p-tolyl-entity was replaced with an isobutyl-phenyl-propanoic acid-(Ibu)-based entity and connected either directly or via a negatively (Dα), neutral (N) or positively (DAB) charged linker to the ɛ-amino group of the lysine residue. 177Lu-labeling was performed under standard conditions and the new PSMA radioligands were tested using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor cells. Binding properties to plasma proteins were determined using an ultrafiltration assay. Biodistribution and SPECT/CT imaging studies were performed in PC-3 PIP/flu tumor-bearing Balb/c nude mice. Results: The PSMA ligands were obtained in moderate yields of 3-15% at high purity (>99%). 177Lu-labeling was achieved at up to 100 MBq/nmol with >96% radiochemical purity. PSMA-binding affinity (KD: 18-38 nM) and internalization into PSMA-positive PC-3 PIP tumor cells were in the same range for all radioligands, however, binding to proteins of human plasma was most pronounced for the 177Lu-Ibu-PSMA and 177Lu-Ibu-Dα-PSMA (95%) followed by 177Lu-Ibu-N-PSMA (93%) and 177Lu-Ibu-DAB-PSMA (89%). Similar observations were made in biodistribution studies where 177Lu-Dα-PSMA showed the most pronounced blood retention and highest tumor uptake. 177Lu-Ibu-DAB-PSMA revealed the best tissue distribution profile with high tumor uptake (65% IA/g at 4 h p.i. and 52% IA/g at 24 h p.i.) and most favorable tumor-to-blood dose ratios as compared to the other three candidates and 177Lu-PSMA-ALB-56. Conclusion: 177Lu-Ibu-DAB-PSMA was identified as the most promising candidate of this new class of albumin-binding radioligands. The favorable tissue distribution profile warrants in-depth investigations of the therapeutic potential of this new PSMA-targeting radioligand in tumor-bearing mice, which will be decisive for a potential future clinical translation. References:  Lau et al. Bioconjug Chem 2019, 30:487  Umbricht et al. Mol Pharm 2018; 15:2297
Aim/Introduction: The use of PSMA-targeted small molecules to image or treat late-stage prostate cancer is a clinically validated concept. Although the tissue distribution of small molecule ligands is typically rapid, the longer half-life of 64Cu (t1/2=12.7 h) may offer logistical advantages over 68Ga or 18F without compromising image resolution1. An additional benefit of 64Cu is that it forms a chemically identical theranostic pairing with 67Cu (t1/2=2.58 d, β-=100%), improving dosimetry and patient selection. Recently, we described novel trifunctional PSMA-targeting ligands with high tumor uptake and therapeutic index2, but the complexation of copper by DOTA is suboptimal. Sarcophagine chelators such as MeCOSar complex copper ions efficiently and with high stability3,4. We therefore aimed to develop a trifunctional sarcophagine ligand for PSMA-targeted theranostics with 64/67Cu. Materials and Methods: RPS-085 was synthesized by conjugation of a PSMA-targeting moiety, an Nε-(2-(4-iodophenyl)acetyl)lysine albumin binding group, and a bifunctionalized MeCOSar chelator. The IC50 of metal-free RPS-085 was determined by competition binding in LNCaP cells. Radiolabeling was performed at 25°C in 0.5 M NH4OAc, pH 5-6. 64Cu-RPS-085 was administered intravenously to male BALB/c mice bearing LNCaP xenograft tumors. The mice (n=4/time point) were sacrificed at 4, 24, and 96 h post injection (p.i.) for biodistribution.
Results: 64Cu-RPS-085 was radiolabeled in nearly quantitative yield in 20 min. The metal-free complex was a potent inhibitor of PSMA (IC50 = 29±2 nM). Accumulation of the tracer was primarily evident in tumor and kidneys. Activity in all other tissues, including blood, was less than 1 %ID/g. Tumor activity was 12.9±1.4 %ID/g at 4 h p.i., and remained at 9.8±1.3 %ID/g at 48 h p.i. By contrast, activity in the kidney peaked at 4 h (13.7±2.3 %ID/g) and cleared to 1.6±0.05 %ID/g at 48 h p.i. At 24 h p.i., the tumor-to-kidney ratio was 3.4±0.7, the tumor-to-blood ratio was 490±152, and the tumor-to-muscle ratio exceeded 10000.
Conclusion: 64Cu-RPS-085 combines rapid tissue distribution with prolonged retention in LNCaP xenograft tumors. Good contrast to background is evident by 4 h p.i., leading to an imaging window from 4-48 h p.i. Preliminary biodistribution studies indicate that tumor-to-background ratios increase with time, suggesting that the pharmacokinetic profile of 64Cu-RPS-085 would be suitable for PSMA-targeted radioligand therapy with 67Cu. References:  Conti M, Eriksson L. EJNMMI Phys. 2016;3:8.  Kelly J, et al. Eur J Nucl Med Mol Imaging. 2018;45:1841-1851.  Paterson BM, et al. Dalton Trans. 2014;43:1386-1396.  Gourni E, et al. Mol Pharm. 2015;12:2781-2790.
Aim/Introduction: Prostate cancer is the second commonly occurring malignance in men. The selection of an effective therapy form depends on the proper assessment of the disease progression. The prostate-specific membrane antigen (PSMA) is becoming increasingly recognized as a viable target for imaging and therapy of prostate and other types of cancer. In the present work we synthesized and investigated the series of HYNIC-PSMA conjugates with differentiated construction of the linker between the PSMA pharmacophore and HYNIC chelator. The aim of this study was to compare the biological properties in vitro and in vivo of the developed PSMA tracers. Materials and Methods: The HYNIC-PSMA conjugates (PSMA-T1,T2,T3 and T4) were synthesised using standard fmoc based solid support synthesis followed by HPLC purification. IC50 values of these compounds were determined by competitive binding assay on LNCaP cell membranes using radio-iodinated (131I)MIP1095 radioligand with known high affinity to PSMA (IC50=0.3). As the reference substances, the PSMA11 and PSMA617 were used. The HYNIC-PSMA inhibitors were labelled with 99mTc in the presence of tricine and EDDA co-ligands and SnCl2 as reducing agent. The binding affinity of 99mTc-HYNIC-PSMA tracers was evaluated carrying out the studies on LNCaP cell membranes (PSMA positive). The non-specific binding was determined using PC3 membranes known not to express PSMA. The preliminary biodistribution of 99mTc-HYNIC-PSMA tracers (T1,T2,T3 and T4) was conducted in normal Balb/c mice 4h piv. The selected 99mTc-PSMA-T4 tracer was further tested for pharmacokinetics in normal Wistar rats and Balb/c Nude mice with subcutaneously induced tumors using LNCaP cells. Results: The IC50 values of the three HYNIC-PSMA conjugates (T1,T3 and T4) were assessed at the level below 100nM, what means ca. 8 times lower value than obtained for PSMA11. The biodistribution study of 99mTc-HYNIC-PSMA conjugates showed that 99mTc-PSMA-T4 was characterized by the fastest elimination with urine and the significantly lowest accumulation in kidneys (37%ID/g 4h piv) - the critical organ for the labelled HYNIC-PSMA. The pharmacokinetic study in tumour-bearing mice showed very high accumulation of 99mTc-PSMA-T4 in tumors (20-30%ID/g), constant over 24h and impressive T/M ratios (230 and 550, 2 and 6h piv, respectively). Conclusion: In the conducted studies the 99mTc-PSMA-T4 was pointed out as the most promising candidate for SPECT imaging of the prostate cancer. The in vitro and in vivo studies showed a very high affinity of this tracer to PSMA, comparable to radiolabeled PSMA-617 inhibitor and much better than PSMA-11 used for PET diagnostics of prostate cancer. References:
Aim/Introduction: We prospectively studied prostate cancer patients with biochemical recurrence and no evidence for disease on conventional imaging with 18F DCFPyL PET/CT and multiparametric MRI of the pelvis. Materials and Methods: In this IRB approved trial, analysis was done on 30 prostate cancer patients with biochemical recurrence (average PSA 3.56 ng/mL, range 0.44-9.97 ng/mL) who underwent whole-body 18F-DCFPyL-PET/CT at 2 h p.i (299.9±15.5 MBq), and multiparametric pelvic MRI, 2 weeks apart. PET/CT and MR were independently read by nuclear medicine physicians and a radiologist, without knowledge of findings from either scan. Histologic biopsies were obtained from all. Results: Definitive prior therapy for the population included 14 who underwent radiation therapy, 8 prostatectomies and 8 with a combination of both. 18F DCFPyL detected a total of 91 lesions while MR detected 43. Only 6 lesions seen on 18F DCFPyL were outside the MRI field of view. In the prostate bed, 18F DCFPyL detected 21 lesions and MR detected 30, of which 12 were concordant. 18F DCFPyL detected 40 pelvic lymph nodes while MR reported 9 of which 4 were concordant. 18F DCFPyL detected 30 metastatic lesions consisting of soft tissue, bone and lymph nodes above the aortic bifurcation. MR detected 4 metastatic lesions in bone and a lymph node above the aortic bifurcation. A pubic bone lesion was concordant on both scans. Histologic validation was available from 69 biopsy specimens. MR and 18F DCFPyL were concordant with biopsy results in 42 sites. Histology was concordant only with MR in 8 lesions and with 18F DCFPyL alone in 17 lesions. One concordant MR and 18F DCFPyL lesion was false positive. There were no concordant false negatives. MR was false positive in 6 specimens while 18F DCFPyL was false positive in 2 sites. MR was false negative in 9 lesions and 18F DCFPyL was false negative in 8. 18F DCFPyL sensitivity 74%, specificity 92%, with MR sensitivity 70%, specificity 81%. Concordant MR/18F DCFPyL findings showed sensitivity 100% and specificity 97%. Conclusion: 18F DCFPyL and MR each had high sensitivity and specificity in biochemically recurrent prostate cancer patients with a slight advantage for 18F DCFPyL. Concordant findings with both modalities significantly increased sensitivity and specificity suggesting combined use would enhance clinical practice. References: none
Aim/Introduction: Inspired by the series of Radiohybrid PSMA inhibitors (rhPSMAs) recently developed in our group, we designed and evaluated another novel class of 18F-labeled PSMA ligands (siPSMAs) comprised of a Silicon Fluoride Acceptor (SiFA) moiety and non-chelator based pharmacokinetic modifiers. While the SiFA-moiety provides very fast and efficient radiofluorination, the modifier composition is decisive for the pharmacokinetic performance in vivo. Here we present the preclinical results of some selected siPSMAs differing in the modifier sequence with special regard to the most promising inhibitor siPSMA-14.
Materials and Methods: siPSMA ligands containing a urea-based binding motif linked by a spacer with a pharmacokinetic modifier in close proximity to a SiFA-moiety were synthesized via solid phase peptide synthesis. Direct radiofluorination of the SiFA-based PSMA inhibitors was achieved by isotopic exchange within 5 min at room temperature followed by fast tracer purification via solid phase extraction. For in vitro characterization, logP(o/w) values as well as binding affinities (IC50) on PSMA-expressing LNCaP cells were determined. Small animal μPET imaging and biodistribution studies were carried out on LNCaP tumor-bearing CB17-SCID mice. Results: Novel siPSMA ligands exhibit suitable lipophilicities (logP(o/w): -3.0 to -4.0) as well as high binding affinities. Biodistribution studies of 18F-siPSMA-11 and 18F-siPSMA-14 in mice showed high tumor uptake of up to 15 %ID/g at 1 h p.i. and low activity accumulation in non-target tissues and bones. Based on the high binding affinity, convenient lipophilicity and excellent biodistribution, siPSMA-14 was found to be the most promising candidate for first clinical studies in patients.
Conclusion: siPSMA inhibitors stand out for their simple and fast radiofluorination as well as for their good in vitro properties. Furthermore, tracer lipophilicity and thus excretion route and kinetics can easily be adjusted by the composition of the pharmacokinetic modifier leading to ligands with favorable in vivo distribution. The promising preclinical assessment of 18F-siPSMA-14 was confirmed in an encouraging proof-of-concept study in prostate cancer patients.
Aim/Introduction: Incomplete resection of prostate cancer (PCa) and its metastases may lead to disease recurrence and consequently poor patient outcome. To obtain complete resection of tumor tissue, prostate specific membrane antigen (PSMA) targeting multimodal ligands containing both a radiolabel and a photosensitizer may be used for intra-operative tumor detection, delineation, and tumor-targeted photodynamic therapy (tPDT). Previously we produced a selection of 12 multimodal PSMA ligands which demonstrated both radionuclide and fluorescence imaging potential for PCa. Here, we selected the multimodal ligand with the most favorable tumor targeting properties and evaluated its potential for PSMA tPDT.
Materials and Methods: The multimodal PSMA ligand PSMA-N064 consists of a PSMA-binding motif, a linker, the photosensitizer IRDye700DX and the chelator DOTAGA. In vitro, therapeutic efficacy of this ligand was evaluated using PSMA-transfected LS174T and PSMA-negative wild-type LS174T cells, which were incubated with PSMA-N064 followed by 100 J/cm2 NIR light irradiation (3-10 min). Additionally, PSMA-N064 (3 nmol/mouse) mediated tPDT, using 150 J/cm2 NIR light irradiation, was tested in BALB/c nude mice bearing PSMA+ LS174T-PSMA xenografts >30 mm3. Tumor growth and survival after tPDT (2h p.i.) was compared to control mice that received NIR-light irradiation or tracer injection only (n=5 mice per group).
Results: In vitro a dose-dependent PSMA-specific loss of cell viability was observed after 100 J/cm2 NIR irradiation, ranging from 29% ± 7.0% (3 nM) to 84% ± 2.6% (30 nM, P<0.001). Moreover, in a first in vivo feasibility study it was shown that PSMA-N064-mediated tPDT may lead to tumor growth delay and a prolonged median survival. In mice treated with tPDT the time interval until tumors reach a size of 500 mm3 (12.8 ± 1.1 days) was longer compared to only NIR-light irradiated (10 ± 4.9 days) or only tracer injected control mice (6.4 ± 1.7 days, P<0.05). In addition, tPDT prolonged survival (defined as tumor growth >1000 mm3, humane endpoint) in treated mice (16 days) compared to the two control groups (12 and 10 days, respectively).
Conclusion: Here, we demonstrated the feasibility of PSMA-targeted PDT using the newly developed PSMA-N064 multimodal ligand. In the future, this ligand will be used for intra-operative tumor detection and PSMA-tPDT. Use of tPDT during surgery can facilitate removal of unresectable tumor rest and positive surgical margins, potentially leading to improved surgical outcomes of PCa patients.
References: This work was supported by EKFS (2016-A64) and the Dutch Cancer Society (NKB-KWF 10443/2016-1).
Aim/Introduction: Comparison of the in vivo biodistribution and the treatment efficacy of 177Lu PSMA-R2 and 177Lu-PSMA-617 in mice with prostate cancer grafts. Materials and Methods: PSMA positive-PC3-PIP were subcutaneously implanted in athymic nude mice in the left flank. A single injection of 111MBq of 177Lu-PSMA-R2 or 177Lu-PSMA-617 or of saline was performed approximately two weeks later, concomitantly to randomization into groups of similar average tumor volumes expressed in mm3 . SPECT/CT imaging was performed in a subset of 12 mice (6 from 177Lu-PSMA-R2 group and 6 from 177Lu-PSMA-617 group). Acquisitions were performed 24h post injection of 177Lu-PSMA-R2 and 177Lu-PSMA-617. Tumor volumes were monitored daily and expressed either as absolute values in mm3 or as a volume relative to that measured the day of the 177Lu-PSMA- R2, 177Lu-PSMA-617 or saline administration. Tumor growth curves were compared using two-ways ANOVA from the day of first injection to day 14. Results: Absolute tumor volumes were significantly reduced in the 177Lu-PSMA-R2 and 177Lu-PSMA-617 groups vs control group (p<0.001). Similarly, tumor volumes from 177Lu-PSMA-R2 and 177Lu-PSMA-617 groups were significantly reduced in comparison to control group (p<0.001). No differences were observed between the tumor volumes of the treated groups. At later time points (14-36 days), the tumors of the control group started to reach the 1500mm3-limit volume as opposed to those of the treated groups. Control mice were therefore euthanized. Although 177Lu-PSMA-R2 and 177Lu-PSMA-617 were no longer administered, a tumor regression was observed in both groups. By day 36th, tumors were no longer detectable in 5 out of 10 tumors from 177Lu-PSMA-R2 group and 4 out of 10 tumors from 177Lu-PSMA- 617 group. Mean tumor volume was of 13.9±23.6 and 11.1±10.4 in 177Lu- PSMA-R2 and 177Lu-PSMA-617 groups, respectively. No statistical differences were found in tumor growth under 177Lu-PSMA-R2 or 177Lu-PSMA-617 treatment. SPECT/CT data acquired on 6 mice from each 177Lu-group showed strong 177Lu- PSMA-R2 and 177Lu-PSMA-617 uptakes at tumor level 24h p.i. with very low uptake elsewhere. VOI quantification confirmed visual observation. Interestingly, after 14 days of a single dose of 111 MBq of 177Lu-PSMA-R2 or 177Lu-PSMA-617, similar tumor growth was observed between 177Lu-PSMA-R2 and 177Lu-PSMA-617 treated mice despite this different tumor uptake at 24h. Conclusion: 177Lu-PSMA-R2 and 177Lu-PSMA-617 have a similar biodistribution in mice inoculated with PSMA positive-PC3-PIP tumor grafts. Similar significant reduction of tumor size was observed, despite the difference in clearance from tumor tissue. References: None