Polypeptide-Based Molecular Platform and Its Docetaxel/Sulfo-Cy5-Containing Conjugate for Targeted Delivery to Prostate Specific Membrane Antigen.

A strategy for stereoselective synthesis of molecular platform for targeted delivery of bimodal therapeutic or theranostic agents to the prostate-specific membrane antigen (PSMA) receptor was developed. The proposed platform contains a urea-based, PSMA-targeting Glu-Urea-Lys (EuK) fragment as a vector moiety and tripeptide linker with terminal amide and azide groups for subsequent addition of two different therapeutic and diagnostic agents. The optimal method for this molecular platform synthesis includes (a) solid-phase assembly of the polypeptide linker, (b) coupling of this linker with the vector fragment, (c) attachment of 3-aminopropylazide, and (d) amide and carboxylic groups deprotection. A bimodal theranostic conjugate of the proposed platform with a cytostatic drug (docetaxel) and a fluorescent label (Sulfo-Cy5) was synthesized to demonstrate its possible sequential conjugation with different functional molecules.

Prostate-specific membrane antigen PET-CT in patients with high-risk prostate cancer before curative-intent surgery or radiotherapy (proPSMA): a prospective, randomised, multicentre study.

BACKGROUND: Conventional imaging using CT and bone scan has insufficient sensitivity when staging men with high-risk localised prostate cancer. We aimed to investigate whether novel imaging using prostate-specific membrane antigen (PSMA) PET-CT might improve accuracy and affect management. METHODS: In this multicentre, two-arm, randomised study, we recruited men with biopsy-proven prostate cancer and high-risk features at ten hospitals in Australia. Patients were randomly assigned to conventional imaging with CT and bone scanning or gallium-68 PSMA-11 PET-CT. First-line imaging was done within 21 days following randomisation. Patients crossed over unless three or more distant metastases were identified. The primary outcome was accuracy of first-line imaging for identifying either pelvic nodal or distant-metastatic disease defined by the receiver-operating curve using a predefined reference-standard including histopathology, imaging, and biochemistry at 6-month follow-up. This trial is registered with the Australian New Zealand Clinical Trials Registry, ANZCTR12617000005358. FINDINGS: From March 22, 2017 to Nov 02, 2018, 339 men were assessed for eligibility and 302 men were randomly assigned. 152 (50%) men were randomly assigned to conventional imaging and 150 (50%) to PSMA PET-CT. Of 295 (98%) men with follow-up, 87 (30%) had pelvic nodal or distant metastatic disease. PSMA PET-CT had a 27% (95% CI 23-31) greater accuracy than that of conventional imaging (92% [88-95] vs 65% [60-69]; p<0·0001). We found a lower sensitivity (38% [24-52] vs 85% [74-96]) and specificity (91% [85-97] vs 98% [95-100]) for conventional imaging compared with PSMA PET-CT. Subgroup analyses also showed the superiority of PSMA PET-CT (area under the curve of the receiver operating characteristic curve 91% vs 59% [32% absolute difference; 28-35] for patients with pelvic nodal metastases, and 95% vs 74% [22% absolute difference; 18-26] for patients with distant metastases). First-line conventional imaging conferred management change less frequently (23 [15%] men [10-22] vs 41 [28%] men [21-36]; p=0·008) and had more equivocal findings (23% [17-31] vs 7% [4-13]) than PSMA PET-CT did. Radiation exposure was 10·9 mSv (95% CI 9·8-12·0) higher for conventional imaging than for PSMA PET-CT (19·2 mSv vs 8·4 mSv; p<0·001). We found high reporter agreement for PSMA PET-CT (κ=0·87 for nodal and κ=0·88 for distant metastases). In patients who underwent second-line image, management change occurred in seven (5%) of 136 patients following conventional imaging, and in 39 (27%) of 146 following PSMA PET-CT. INTERPRETATION: PSMA PET-CT is a suitable replacement for conventional imaging, providing superior accuracy, to the combined findings of CT and bone scanning. FUNDING: Movember and Prostate Cancer Foundation of Australia. VIDEO ABSTRACT.

Development of a new class of PSMA radioligands comprising ibuprofen as an albumin-binding entity.

Prostate-specific membrane antigen (PSMA)-targeted radioligands have been used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Recently, albumin-binding PSMA radioligands with enhanced blood circulation were developed to increase the tumor accumulation of activity. The present study aimed at the design, synthesis and preclinical evaluation of a novel class of PSMA-targeting radioligands equipped with ibuprofen as a weak albumin-binding entity in order to improve the pharmacokinetic properties. Methods: Four novel glutamate-urea-based PSMA ligands were synthesized with ibuprofen, conjugated via variable amino acid-based linker entities. The albumin-binding properties of the 177Lu-labeled PSMA ligands were tested in vitro using mouse and human plasma. Affinity of the radioligands to PSMA and cellular uptake and internalization was investigated using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor cells. The tissue distribution profile of the radioligands was assessed in biodistribution and imaging studies using PC-3 PIP/flu tumor-bearing nude mice. Results: The PSMA ligands were obtained in moderate yields at high purity (>99%). 177Lu-labeling of the ligands was achieved at up to 100 MBq/nmol with >96% radiochemical purity. In vitro assays confirmed high binding of all radioligands to mouse and human plasma proteins and specific uptake and internalization into PSMA-positive PC-3 PIP tumor cells. Biodistribution studies and SPECT/CT scans revealed high accumulation in PC-3 PIP tumors but negligible uptake in PC-3 flu tumor xenografts as well as rapid clearance of activity from background organs and tissues. 177Lu-Ibu-DAB-PSMA, in which ibuprofen was conjugated via a positively-charged diaminobutyric acid (DAB) entity, showed distinguished tumor uptake and the most favorable tumor-to-blood and tumor-to-kidney ratios. Conclusion: The high accumulation of activity in the tumor and fast clearance from background organs was a common favorable characteristic of PSMA radioligands modified with ibuprofen as albumin-binding entity. 177Lu-Ibu-DAB-PSMA emerged as the most promising candidate; hence, more detailed preclinical investigations with this radioligand are warranted in view of a clinical translation.

Diagnostic sensitivity of Tc-99m HYNIC PSMA SPECT/CT in prostate carcinoma: A comparative analysis with Ga-68 PSMA PET/CT.

BACKGROUND: Emerging data from published studies are demonstrating the superiority of Ga-68 PSMA PET/CT imaging in prostate cancer. However, the low yield of the Ge-68/Ga-68 from which Gallium-68 is obtained and fewer installed PET/CT systems compared to the SPECT imaging systems may limit its availability. We, therefore, evaluated in a head-to-head comparison, the diagnostic sensitivity of Ga-68 PSMA PET/CT and Tc-99m PSMA SPECT/CT in patients with prostate cancer. METHODS: A total of 14 patients with histologically confirmed prostate cancer were prospectively recruited to undergo Ga-68 PSMA PET/CT and Tc-99m HYNIC PSMA SPECT/CT. The mean age of patients was 67.21 ± 8.15 years and the median PSA level was 45.18 ng/mL (range = 1.51-687 ng/mL). SUVmax of all lesions and the size of lymph nodes with PSMA avidity on Ga-68 PSMA PET/CT were determined. Proportions of these lesions detected on Tc-99m HYNIC PSMA SPECT/CT read independent of PET/CT findings were determined. RESULTS: A total of 46 lesions were seen on Ga-68 PSMA PET/CT localized to the prostate (n = 10), lymph nodes (n = 24), and bones (n = 12). Of these, Tc-99m HYNIC PSMA SPECT/CT detected 36 lesions: Prostate = 10/10 (100%), lymph nodes = 15/24 (62.5%), and bones = 11/12 (91.7%) with an overall sensitivity of 78.3%. Lesions detected on Tc-99m HYNIC PSMA SPECT/CT were bigger in size (P < 0.001) and had higher SUVmax (P < 0.001) as measured on Ga-68 PSMA PET/CT compared to those lesions that were not detected. All lymph nodes greater than 10 mm in size were detected while only 28% of nodes less than 10 mm were detected by Tc-99m HYNIC PSMA SPECT/CT. In a univariate analysis, Lymph node size (P = 0.033) and the SUVmax of all lesions (P = 0.007) were significant predictors of lesion detection on Tc-99m HYNIC PSMA SPECT/CT. CONCLUSION: Tc-99m HYNIC PSMA may be a useful in imaging of prostate cancer although with a lower sensitivity for lesion detection compared to Ga-68 PSMA PET/CT. Its use is recommended when Ga-68 PSMA is not readily available, in planning radio-guided surgery or the patient is being considered for radio-ligand therapy with Lu-177 PSMA. It performs poorly in detecting small-sized lesions hence its use is not recommended in patients with small volume disease.

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

(68)Ga-HBED-CC-PSMA PET/CT Versus Histopathology in Primary Localized Prostate Cancer: A Voxel-Wise Comparison.

PURPOSE: We performed a voxel-wise comparison of (68)Ga-HBED-CC-PSMA PET/CT with prostate histopathology to evaluate the performance of (68)Ga-HBED-CC-PSMA for the detection and delineation of primary prostate cancer (PCa). METHODOLOGY: Nine patients with histopathological proven primary PCa underwent (68)Ga-HBED-CC-PSMA PET/CT followed by radical prostatectomy. Resected prostates were scanned by ex-vivo CT in a special localizer and histopathologically prepared. Histopathological information was matched to ex-vivo CT. PCa volume (PCa-histo) and non-PCa tissue in the prostate (NPCa-histo) were processed to obtain a PCa-model, which was adjusted to PET-resolution (histo-PET). Each histo-PET was coregistered to in-vivo PSMA-PET/CT data. RESULTS: Analysis of spatial overlap between histo-PET and PSMA PET revealed highly significant correlations (p < 10(-5)) in nine patients and moderate to high coefficients of determination (R²) from 42 to 82 % with an average of 60 ± 14 % in eight patients (in one patient R(2) = 7 %). Mean SUVmean in PCa-histo and NPCa-histo was 5.6 ± 6.1 and 3.3 ± 2.5 (p = 0.012). Voxel-wise receiver-operating characteristic (ROC) analyses comparing the prediction by PSMA-PET with the non-smoothed tumor distribution from histopathology yielded an average area under the curve of 0.83 ± 0.12. Absolute and relative SUV (normalized to SUVmax) thresholds for achieving at least 90 % sensitivity were 3.19 ± 3.35 and 0.28 ± 0.09, respectively. CONCLUSIONS: Voxel-wise analyses revealed good correlations of (68)Ga-HBED-CC-PSMA PET/CT and histopathology in eight out of nine patients. Thus, PSMA-PET allows a reliable detection and delineation of PCa as basis for PET-guided focal therapies.

Improved Synthesis and In Vitro Evaluation of an Aptamer Ribosomal Toxin Conjugate.

Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5' terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamer-toxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9.min-rGel conjugate demonstrated an IC50 of ∼60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t1/2 of ∼4 h, paving the way for future in vivo experiments.

PSMA ligand conjugated PCL-PEG polymeric micelles targeted to prostate cancer cells.

In this content, a small molecular ligand of prostate specific membrane antigen (SMLP) conjugated poly (caprolactone) (PCL)-b-poly (ethylene glycol) (PEG) copolymers with different block lengths were synthesized to construct a satisfactory drug delivery system. Four different docetaxel-loaded polymeric micelles (DTX-PMs) were prepared by dialysis with particle sizes less than 60 nm as characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Optimization of the prepared micelles was conducted based on short-term stability and drug-loading content. The results showed that optimized systems were able to remain stable over 7 days. Compared with Taxotere, DTX-PMs with the same ratio of hydrophilic/hydrophobic chain length displayed similar sustained release behaviors. The cytotoxicity of the optimized targeted DTX-PCL12K-PEG5K-SMLP micelles (DTX-PMs2) and non-targeted DTX-PCL12K-mPEG5K micelles (DTX-PMs1) were evaluated by MTT assays using prostate specific membrane antigen (PSMA) positive prostate adenocarcinoma cells (LNCaP). The results showed that the targeted micelles had a much lower IC50 than their non-targeted counterparts (48 h: 0.87 ± 0.27 vs 13.48 ± 1.03 µg/ml; 72 h: 0.02 ± 0.008 vs 1.35 ± 0.54 µg/ml). In vitro cellular uptake of PMs2 showed 5-fold higher fluorescence intensity than that of PMs1 after 4 h incubation. According to these results, the novel nano-sized drug delivery system based on DTX-PCL-PEG-SMLP offers great promise for the treatment of prostatic cancer.

Humoral immune response in prostate cancer patients after immunization with gene-based vaccines that encode for a protein that is proteasomally degraded.

Prostate-specific membrane antigen (PSMA), whose expression is upregulated in poorly differentiated, metastatic, and hormone refractory prostate cancer, could be targeted by gene-based vaccines. The aim of this study was to characterize the humoral immune response against PSMA in prostate carcinoma patients who have been vaccinated against PSMA with gene-based vaccines. Sera from prostate cancer patients who had been immunized repeatedly with plasmid DNA and a recombinant adenoviral vector, both carrying an expression cassette for human PSMA, and sera from healthy donors were tested for anti-PSMA antibodies by Western blot analysis and immunofluorescence. PSMA-producing LNCaP cells, recombinant PSMA protein, and a specific antibody against PSMA were used as positive controls. Specific anti-PSMA antibodies were detected by both Western blot and immunofluorescence in the sera of patients who had been vaccinated against PSMA with plasmid and recombinant adenoviral vectors. The specificity of the anti-PSMA antibodies was confirmed by preincubation and blocking experiments. Positive reactions were detected in 86% of the vaccinated prostate cancer patients. Anti-PSMA antibodies were not detected either in the patients' sera prior to vaccination or in the sera from healthy men and women. These data demonstrate that PSMA, a specific marker for prostate cancer, is a target for humoral immune response induced by gene-based PSMA vaccination. Detection of anti-PSMA antibodies by immunoblot analysis and by indirect immunofluorescence could be used to monitor the vaccination effect.