Design, Synthesis, and Biological Evaluation of Novel Nonsteroidal 18F-Labeled PET Probes Specifically Targeting Estrogen Receptor α.

The estrogen receptor α positive (ERα+) subtype represents nearly 70% of all breast cancers (BCs), which seriously threaten women's health. Positron emission computed tomography (PET) characterizes its superiority in detecting the recurrence and metastasis of BC. In this article, an array of novel PET probes ([18F]R-1, [18F]R-2, [18F]R-3, and [18F]R-4) targeting ERα based on the tetrahydropyridinyl indole scaffold were developed. Among them, [18F]R-3 and [18F]R-4 showed good target specificity toward ERα and could distinguish MCF-7 (ERα+) and MDA-MB-231 (ERα-) tumors efficiently. Especially, [18F]R-3 could differentiate the ERα positive/negative tumors successfully with a higher tumor-to-muscle uptake ratio (T/M) than that of [18F]R-4. The radioactivity of [18F]R-3 in the MCF-7 tumor was 5.24 ± 0.84%ID/mL and its T/M ratio was 2.49 ± 0.62 at 25 min postinjection, which might be the optimal imaging time point in PET scanning. On the contrary, [18F]R-3 did not accumulate in the MDA-MB-231 tumor at all. The autoradiography analysis of [18F]R-3 on the MCF-7 tumor-bearing mice model was consistent with the PET imaging results. [18F]R-3 exhibited the pharmacokinetic property of rapid distribution and slow clearance, making it suitable for use as a diagnostic PET probe. Overall, [18F]R-3 was capable of serving as a PET radiotracer to delineate the ERα+ tumor and was worthy of further exploitation.

Effect of dexmedetomidine infusion on postoperative sleep disturbances in women with breast cancer: A monocentric randomized-controlled double-blind trial.

BACKGROUND: Most women with breast cancer are prone to postoperative sleep disturbances (POSD). Little is known about the differences between sevoflurane and propofol combined with dexmedetomidine on POSD in the same context. We investigated the effect of intra-operative sevoflurane or propofol combined with intravenous dexmedetomidine on the incidence of POSD and postoperative sleep structures. METHODS: A monocentric, randomized-controlled, double-blind trial. Female patients undergoing radical surgery for breast cancer were randomly assigned to receive sevoflurane and placebo, sevoflurane and dexmedetomidine, propofol and placebo, or propofol and dexmedetomidine. Dexmedetomidine was administered at 1.0 µg kg-1 infusion 15 min before induction, then infused at 0.4 µg kg-1 h-1 until the surgical drain started to be placed. The primary outcome was the incidence of POSD within the postoperative first three days (defined as an Athens Insomnia Scale score ≥ 6 points on at least one day of postoperative first three days). The secondary outcome was the duration of sleep structures, collected from the Fitbit Charge 2® smart bracelet (Fitbit, Inc., San Francisco, CA, USA). RESULTS: There were 188 women analyzed with the modified intention-to-treat method. The incidences of POSD in the dexmedetomidine and placebo groups were similar (p = 0.649). In the sevoflurane sedation strategy, dexmedetomidine decreased nocturnal wakefulness on postoperative first day (p = 0.001). In the propofol sedation strategy, dexmedetomidine increased nocturnal deep sleep on postoperative first (p < 0.001) and third (p < 0.001) days. CONCLUSION: Intra-operative infusion of dexmedetomidine had no significant effect on POSD but decreased nocturnal wakefulness in the sevoflurane group and increased nocturnal deep sleep in the propofol group. TRIAL REGISTRATION: Registered at www.chictr.org.cn (ChiCTR2300070136).

Preparation and Bioevaluation of 18F-Labeled Small-Molecular Radiotracers via Sulfur(VI) Fluoride Exchange Chemistry for Imaging of Programmed Cell Death Protein Ligand 1 Expression in Tumors.

Nowadays, one of the most effective methods of tumor immunotherapy is blocking programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) immune checkpoints. However, there is still a significant challenge in selecting patients to benefit from immune checkpoint therapies. Positron emission tomography (PET), a noninvasive molecular imaging technique, offers a new approach to accurately detect PD-L1 expression and allows for a better prediction of response to PD-1/PD-L1 target immunotherapy. Here, we designed and synthesized a novel group of aryl fluorosulfate-containing small-molecule compounds (LGSu-1, LGSu-2, LGSu-3, and LGSu-4) based on the phenoxymethyl-biphenyl scaffold. After screening by the time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the most potent compound LGSu-1 (half maximal inhibitory concentration (IC50): 15.53 nM) and the low-affinity compound LGSu-2 (IC50: 189.70 nM) as a control were selected for 18F-radiolabeling by sulfur(VI) fluoride exchange chemistry (SuFEx) to use for PET imaging. [18F]LGSu-1 and [18F]LGSu-2 were prepared by a one-step radiofluorination reaction in over 85% radioconversion and nearly 30% radiochemical yield. In B16-F10 melanoma cell assays, [18F]LGSu-1 (5.00 ± 0.06%AD) showed higher cellular uptake than [18F]LGSu-2 (2.55 ± 0.04%AD), in which cell uptake could be significantly blocked by the nonradioactivity LGSu-1. In vivo experiments, micro-PET imaging of B16-F10 tumor-bearing mice and radiographic autoradiography of tumor sections showed that [18F]LGSu-1 was more effectively accumulated in the tumor due to the higher binding affinity with PD-L1. The above experimental results confirmed the potential of the small-molecule probe LGSu-1 as a targeting PD-L1 imaging tracer in tumor tissues.

Design, Synthesis, and Biological Evaluation of a Small-Molecule PET Agent for Imaging PD-L1 Expression.

Immunotherapy blocking programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) pathway has achieved great therapeutic effect in the clinic, but the overall response rate is not satisfactory. Early studies showed that response to treatment and overall survival could be positively related to PD-L1 expression in tumors. Therefore, accurate measurement of PD-L1 expression will help to screen cancer patients and improve the overall response rate. A small molecular positron emission tomography (PET) probe [18F]LP-F containing a biphenyl moiety was designed and synthesized for measurement of PD-L1 expression in tumors. The PET probe [18F]LP-F was obtained with a radiochemical yield of 12.72 ± 1.98%, a radiochemical purity of above 98% and molar activity of 18.8 GBq/µmol. [18F]LP-F had good stability in phosphate buffer saline (PBS) and mouse serum. In vitro assay indicated that [18F]LP-F showed moderate affinity to PD-L1. Micro-PET results showed that the tumor accumulation of [18F]LP-F in A375 tumor was inferior to that in A375-hPD-L1 tumor. All the results demonstrated that [18F]LP-F could specifically bind to PD-L1 and had a potential application in non-invasive evaluation of PD-L1 expression in tumors.

Stimuli-Responsive Macrocyclization Scaffold Allows In Situ Self-Assembly of Radioactive Tracers for Positron Emission Tomography Imaging of Enzyme Activity.

Target-enabled bioorthogonal reaction and self-assembly of a small-molecule probe into supramolecules have shown promise for molecular imaging. In this paper, we report a new stimuli-responsive bioorthogonal reaction scaffold (SF) for controlling in situ self-assembly by engineering the condensation reaction between 2-cyanobenzothiazole and cysteine. For probes with the SF scaffold, intramolecular cyclization took place soon after activation, which could efficiently outcompete free cysteine even at a low concentration and result in efficient aggregation in the target. Through integration with different enzyme-responsive substrates and an ammoniomethyl-trifluoroborate moiety (AmBF3), two radioactive positron emission tomography (PET) tracers, [18F]SF-DEVD and [18F]SF-Glu, were designed, which showed high stability under physiological conditions and could produce clear PET signal in tumors to detect enzyme activity (e.g., caspase-3, γ-glutamyltranspeptidase) timely and accurately. Our results demonstrated that the scaffold SF could serve as a general molecular scaffold in the development of smart PET tracers for noninvasive imaging of enzyme activity, which could contribute to tumor detection and treatment efficacy evaluation.

Development of a radiolabeled site-specific single-domain antibody positron emission tomography probe for monitoring PD-L1 expression in cancer.

Despite advances in immunotherapy for the treatment of cancers, not all patients can benefit from programmed cell death ligand 1 (PD-L1) immune checkpoint blockade therapy. Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level; hence, accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects. Therefore, based on the high affinity antibody Nb109, a new site-specifically radiolabeled tracer, 68Ga-NODA-cysteine, aspartic acid, and valine (CDV)-Nb109, was designed and synthesized to accurately monitor PD-L1 expression. The tracer 68Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95% and radiochemical purity of 97%. It showed high affinity for PD-L1 with a dissociation constant of 12.34 ± 1.65 nM. Both the cell uptake assay and positron emission tomography (PET) imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells. Meanwhile, dynamic PET imaging of a NCI-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression, allowing timely interventional immunotherapy. In conclusion, this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy.

Promising potential of a 18F-labelled small-molecular radiotracer to evaluate PD-L1 expression in tumors by PET imaging.

Programmed death ligand 1 (PD-L1) expression level is a reproducible biomarker for guiding stratification of patients to immunotherapy. However, the most widely used immunohistochemistry method is incompetent to fully understand the PD-L1 expression level in the whole body because of the highly complex PD-L1 expression in the tumor microenvironment. In this work, a novel small-molecular radiotracer [18F]LG-1 based on the biphenyl active structure was developed to evaluate PD-L1 expression in tumors. [18F]LG-1 was obtained by conjugating and radiolabeling with [18F]FDG with high radiochemical purity (>98.0%) and high molar activity (37.2 ± 2.9 MBq/nmol). In vitro experimental results showed that [18F]LG-1 could target PD-L1 in tumor cells and the cellular uptake in A375-hPD-L1 cells (PD-L1 + ) was clearly higher than that in A375 cells (PD-L1-). In vivo dynamic PET images of [18F]LG-1 provided clear visualization of A375-hPD-L1 tumor with high tumor-to-background contrast, and the tumor uptake was determined to be 3.98 ± 0.21 %ID/g at 60 min, which was 2.6-fold higher than that of A375 tumor. These results suggested that [18F]LG-1 had great potential as a promising PD-L1 radiotracer.

Immuno-PET imaging of 68Ga-labeled nanobody Nb109 for dynamic monitoring the PD-L1 expression in cancers.

The checkpoint blockade immunotherapy has become a potent treatment strategy for cancers, and programmed death ligand-1 (PD-L1) is a prominent checkpoint ligand that is highly expressed in some cancers. The identification of immune checkpoint marker PD-L1 is critical for improving the success of immunotherapy. Accordingly, the binding specificity and dynamic monitoring property of a non-blocking nanobody tracer 68Ga-NOTA-Nb109 to PD-L1 were assessed in this study. The endogenous expression level of PD-L1 in several cancer cells was measured by flow cytometry, Western blot, and cellular uptake assay. Sensitivity and specificity of 68Ga-NOTA-Nb109 in monitoring the expression of PD-L1 in vivo were evaluated by PET imaging of different tumor-bearing models (U87, high PD-L1 expression; HCT 116, medium PD-L1 expression; and NCI-H1299, low PD-L1 expression). In vivo PET imaging results agreed well with those detected in vitro. In addition, PET imaging of PD-L1 expression in U87 and NCI-H1299 xenografts using 18F-FDG was also performed for comparison. The maximum tumor-to-muscle uptake ratio of 68Ga-NOTA-Nb109 was more than twofold that of 18F-FDG in U87 xenograft. The change of PD-L1 expression in NCI-H1299 cells and xenografts induced by cisplatin (CDDP) was sensitively monitored by 68Ga-NOTA-Nb109. This study demonstrated the feasibility of tracer 68Ga-NOTA-Nb109 for specifically targeting endogenous PD-L1 and dynamic monitoring the change of PD-L1 expression, and could guide the immunotherapy and immunochemotherapy for refractory cancers.

A fluorine-18 labeled radiotracer for PET imaging of γ-glutamyltranspeptidase in living subjects.

The expression level of γ-glutamyltranspeptidase (GGT) in some malignant tumors is often abnormally high, while its expression is low in normal tissues. Therefore, GGT is considered as a key biomarker for cancer diagnosis. Several GGT-targeting fluorescence probes have been designed and prepared, but their clinical applications are limited due to their shallow tissue penetration. Considering the advantages of positron emission tomography (PET) such as high sensitivity and deep tissue penetration, we designed a novel PET imaging probe for targeted monitoring of the expression of GGT in living subjects, ([18F]γ-Glu-Cys-PPG(CBT)-AmBF3)2, hereinafter referred to as ([18F]GCPA)2. The non-radioactive probe (GCPA)2 was synthesized successfully and [18F]fluorinated rapidly via the isotope exchange method. The radiotracer ([18F]GCPA)2 could be obtained within 0.5 h with the radiochemical purity over 98% and the molar activity of 10.64 ± 0.89 GBq µmol-1. It showed significant difference in cellular uptake between GGT-positive HCT116 cells and GGT-negative L929 cells (2.90 ± 0.12% vs. 1.44 ± 0.15% at 4 h, respectively). In vivo PET imaging showed that ([18F]GCPA)2 could quickly reach the maximum uptake in tumor (4.66 ± 0.79% ID g-1) within 5 min and the tumor-to-muscle uptake ratio was higher than 2.25 ± 0.08 within 30 min. Moreover, the maximum tumor uptake of the control group co-injected with the non-radioactive probe (GCPA)2 or pre-treated with the inhibitor GGsTop decreased to 3.29 ± 0.24% ID g-1 and 2.78 ± 0.32% ID g-1 at 10 min, respectively. In vitro and in vivo results demonstrate that ([18F]GCPA)2 is a potential PET probe for sensitively and specifically detecting the expression level of GGT.

Pharmacological evaluation of imidazole-derived bisphosphonates on receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation and function.

Bisphosphonates (BPs) have been commonly used in the treatment of osteolytic bone lesions, such as osteoporosis and osteogenesis imperfecta. However, serious side-effects can occur during the therapy. To search for novel potent BPs with lower side-effects, a series of imidazole-containing BPs (zoledronic acid [ZOL]; ZOL derivatives by substitution of the hydrogen at the 2-position on the imidazole ring with a methyl [MIDP], ethyl [EIDP], n-propyl [PIDP], or n-butyl group [BIDP]) were developed and the effects on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation were investigated using the murine macrophage RAW 264.7 cells at the protein, gene, and morphological and functional levels. Influences of these BPs on the cell growth and proliferation of RAW 264.7 were also studied in order to determine cytotoxicity. The results showed that PIDP significantly inhibited the RANKL-induced osteoclast formation in a dose-dependent fashion without inducing cytotoxicity under the concentration of 12.5 µM. It exerted remarkable suppressive effects on the development of actin rings, the bone resorption, and the expressions of osteoclastogenesis-related gene and protein markers. The down-regulation of c-Jun N-terminal kinase (JNK), protein kinase B (Akt), and inhibitor of nuclear factor kappa-B (IκB) phosphorylation in the early signaling event and subsequent inhibition of the expression of c-Fos and nuclear factor of activated T cells (NFATc1) might be involved in these effects. All these results indicated that PIDP might be a promising drug to treat bone-related disorders.

Radiofluorinated Smart Probes for Noninvasive PET Imaging of Legumain Activity in Living Subjects.

Overexpression of legumain is closely associated with tumor proliferation, invasion, and metastasis. Because of its intrinsic properties, such as high sensitivity and resolution, positron emission tomography (PET) has become an effective imaging technique for early diagnosis, treatment response prediction, and monitoring. Herein, two legumain-targeting radiofluorinated smart probes (18F-2 and 18F-3) as well as a control probe (18F-1) were specifically designed for PET imaging of legumain activity in tumors. 18F-1, 18F-2, and 18F-3 were obtained with high radiochemical yield (RCY > 60%) and radiochemical purity (RCP > 99%) using a convenient "one-step" 18F-labeling method. The probes 18F-2 and 18F-3 exhibited high response to legumain activity and reductive environment and revealed comparable uptake in HCT116 cells (4.22% ± 0.14% and 4.64% ± 0.32% for 18F-2 and 18F-3, respectively; 8.46% ± 0.33% and 9.05% ± 0.24% for co-treatment of 18F-2 + 2 and 18F-3 + 3 at 1 h), while the control probe 18F-1 showed no response. PET imaging of tumor-bearing mice showed that the co-injection strategy (18F-2 + 2 and 18F-3 + 3) resulted in higher tumor uptake (3.57% ± 0.37% and 3.72% ± 0.19% ID/g at 10 min, respectively) than the single injection strategy (2.59% ± 0.19% and 2.60% ± 0.46% ID/g for 18F-2 and 18F-3, respectively). In addition, introduction of the trimeric histidine-glutamate (HEHEHE) tag to 18F-3 reduced the liver uptake by almost two-fold without any noticeable effect on the tumor uptake. All the results indicate that 18F-3 holds great potential applications in clinics for sensitive and specific PET imaging of legumain activity in tumors.

One-step radiosynthesis and initial evaluation of a small molecule PET tracer for PD-L1 imaging.

Programmed cell death protein-ligand 1 (PD-L1) is a crucial biomarker in immunotherapy and its expression level plays a key role in the guidance of anti-PD-L1 therapy. It had been reported that PD-L1 was quantified by noninvasive imaging with more developed radiotracers. In our study, a novel [18F]fluoride labeled small molecule inhibitor, [18F]LN was designed for positron emission tomography (PET) imaging in both PD-L1 transfected (A375-hPD-L1) and non-transfected (A375) melanoma-bearing mice. LN showed the specificity (IC50 = 50.39 ± 2.65 nM) to PD-L1 confirmed by competitive combination and cell flow cytometry (FACS) analysis. The radiotracer [18F]LN was obtained via 18F-19F isotope exchange from precursor LN. After radiosynthesis, [18F]LN was achieved with a high radiochemical purity (RCP) above 95% and got a favorable molar activity of 36.34 ± 5.73 GBq/µmol. [18F]LN displayed the moderate affinity (Kd = 65.27 ± 3.47 nM) to PD-L1 by specific binding assay. And it showed 1.3-fold higher uptake in A375-hPD-L1 cells than that in A375 cells. PET imaging revealed that [18F]LN could enter into PD-L1 expressing tumor site and visualize the outline of tumor. And tumor uptake (1.96 ± 0.27 %ID/g) reached the maximum at 15 min in the positive group, showed 2.2-fold higher than the negative (0.89 ± 0.31 %ID/g) or the blocked (1.07 ± 0.26 %ID/g) groups. Meanwhile, biodistribution could slightly distinguish the positive from the negative. The results indicated [18F]LN would become an efficient tool for evaluating PD-L1 expression with further optimization.

Radiopharmacological evaluation of a caspase-3 responsive probe with optimized pharmacokinetics for PET imaging of tumor apoptosis.

Early evaluation of the therapy efficiency can promote the development of anti-tumor drugs and optimization of the treatment method. Caspase-3 is a key biomarker for early apoptosis. Detection of caspase-3 activity is essential for quick assessment of the curative effect. We have reported a PET probe that could image drug-induced tumor apoptosis in vivo. However, high liver uptake limits its application. In order to optimize the pharmacokinetics of the previous probe, we introduced a hydrophilic peptide sequence to minimize liver uptake. The structure of the new probe was confirmed by mass spectrometry and nuclear magnetic resonance. This probe was able to cross the cell membrane freely and could be converted into a dimer through the condensation reaction of 2-cyano-6-aminobenzothiazole (CBT) and cysteine in response to intracellular activated caspase-3 and glutathione (GSH). The hydrophobic dimers further self-assembled into nanoparticles, which could enhance the probe aggregation in apoptotic tumor tissues. In vivo experiments showed that the tumor uptake of the new probe was higher than that of the previous probe, while the liver uptake of the new probe was significantly reduced. The new probe might be promising in imaging apoptotic tumors with suitable pharmacokinetics.

One-step 18F-fluorination of smart positron emission tomography tracer for sensing furin activity in tumors.

INTRODUCTION: Peptide analogues have attracted considerable attention in the field of developing novel positron emission tomography (PET) imaging agents due to their unique properties. Nevertheless, the complicated radiolabeling process and fast metabolism usually pose challenges to the clinical applications of peptide-based molecular probes. Herein a novel PET tracer containing a specific peptide sequence Arg-Val-Arg-Arg (RVRR), Acetyl-Arg-Val-Arg-Arg-Cys(StBu)-Gly(AMB[18F]F3)-CBT ([18F]1), was designed and radiosynthesized using a simple and convenient one-step 18F-fluorination procedure. The smart tracer can be activated by the protease furin and then undergoes an intermolecular cyclization reaction in tumor cells, leading to improved PET imaging efficiency of tumor. METHODS: The radiosynthesis of the target tracer [18F]1 and the control tracer [18F]1-ctrl was performed under facile conditions in pyridazine-HCl buffer (pH~2.5) at 80 °C within 30 min. The enzyme-controlled condensation was studied for non-radioactive compound 1 in the human breast cancer cell lysates (MDA-MB-468). The cellular uptake of [18F]1 and [18F]1-ctrl was studied and compared by measuring the activity in MDA-MB-468 cells using a γ-counter after incubation with 37 kBq of [18F]1 or [18F]1-ctrl, respectively. In vivo behavior of [18F]1 was examined through PET imaging of MDA-MB-468 tumor-bearing mice and compared with that of [18F]1-ctrl as well as that of [18F]1 co-injected with non-radioactive compound 1. RESULTS: The tracer [18F]1 was obtained with a high radiochemical yield (RCY) of 42.5 ± 1.47% and an excellent radiochemical purity (RCP > 99%). Under the activation of furin and GSH, the tracer suffered a condensation reaction to form dimers and then self-assembled into nanoparticles to produce enduring signal. The cellular uptake of [18F]1 and [18F]1-ctrl was determined to be 10.2 ± 0.37 and 1.19 ± 0.25%ID at 120 min, respectively. For in vivo PET imaging, [18F]1 exhibited the optimum tumor uptake of 2.39 ± 0.31%ID/g and the tumor-to-muscle uptake ratio of 2.93 ± 0.92 at 10 min post injection. Co-injection of [18F]1 and non-radioactive compound 1 produced a high tumor uptake ranging from 2.83 ± 0.23%ID/g to 3.40 ± 0.18%ID/g at 10 min and 60 min post injection, respectively. CONCLUSIONS: The one-step labeling method of tracer [18F]1 showed advantage in simplifying the radiolabeling process with high RCY, which could enable a real kit process for the synthesis of 18F-radiopharmaceuticals and was significant for the large-scale production of tracers for clinical applications. PET imaging results suggested that the tracer [18F]1 had good tumor uptake and the co-injection strategy of [18F]1 with 1 could enhance the imaging signal in tumor.

PET Imaging of Tumor PD-L1 Expression with a Highly Specific Nonblocking Single-Domain Antibody.

Although immunotherapy through programmed death 1/programmed death ligand 1 (PD-1/PD-L1) checkpoint blockade has shown impressive clinical outcomes, not all patients respond to it. Recent studies have demonstrated that the expression level of PD-L1 in tumors is one of the factors that correlate with PD-1/PD-L1 checkpoint blockade therapy. Herein, a 68Ga-labeled single-domain antibody tracer, 68Ga-NOTA-Nb109, was designed and developed for specific and noninvasive imaging of PD-L1 expression in a melanoma-bearing mouse model. Methods: The single-domain antibody Nb109 was labeled with the radionuclide 68Ga through a NOTA chelator. An in vitro binding assay was performed to assess the affinity and binding epitope of Nb109 to PD-L1. The clinical application value of 68Ga-NOTA-Nb109 was evaluated by a stability assay; by biodistribution and pharmacokinetics studies; and by PET imaging, autoradiography, and immunohistochemical staining studies on tumor-bearing models with differences in PD-L1 expression. Results:68Ga-NOTA-Nb109 was obtained with a radiochemical yield of more than 95% and radiochemical purity of more than 98% in 10 min. It showed a highly specific affinity for PD-L1, with an equilibrium dissociation constant of 2.9 × 10-9 M. A competitive binding assay indicated Nb109 to have a binding epitope different from that of PD-1 and PD-L1 antibody. All biodistribution, PET imaging, autoradiography, and immunohistochemical staining studies revealed that 68Ga-NOTA-Nb109 specifically accumulated in A375-hPD-L1 tumor, with a maximum uptake of 5.0% ± 0.35% injected dose/g at 1 h. Conclusion:68Ga-NOTA-Nb109 holds great potential for noninvasive PET imaging of the PD-L1 status in tumors and for timely evaluation of the effect of immune checkpoint targeting treatment.

Structure-based virtual screening and biological evaluation of novel non-bisphosphonate farnesyl pyrophosphate synthase inhibitors.

Farnesyl pyrophosphate synthase (FPPS) is known to participate in a variety of disease-related cell signaling pathway and bisphosphonates (BPs) are served as FPPS inhibitors. However, the high polarity of BPs often induces a series of side effects, limiting their applications. In the present study, novel non-BP FPPS inhibitors were discovered by in silico screening and experimental validation. From the structure-based virtual screening (SBVS) strategy combining molecular docking, pharmacophore and binding affinity prediction, 10 hits with novel scaffolds were filtered. The inhibition activity of hits against FPPS was identified and 7 hits showed comparable or higher inhibition activity than Zoledronate. The hit VS-4 with higher lipophilicity (XlogP = 1.81) and binding affinity (KD = 14.3 ± 2.63 µM) to FPPS was selected for further study on cancer cells with different FPPS expression level. Experimental results revealed that VS-4 could better target the FPPS high-expressing colon LoVo and HCT116 cancer cell lines with IC50 of 51.772 ± 0.473 and 43.553 ± 1.027 µM, respectively, whereas the IC50 value against FPPS low expressing MDA-MB-231 cells was >100 µM. The mechanism of VS-4 against colon cancer cells was investigated by flow cytometry and the results indicated that VS-4 induced cell apoptosis by increasing the intracellular reactive oxygen species (ROS) level. Taken together, the SBVS strategy could be used to discover promising non-BP FPPS inhibitors and the lead compound VS-4 might shed a light on designing more potent inhibitors as novel anticancer drugs.

Rational design of caspase-responsive smart molecular probe for positron emission tomography imaging of drug-induced apoptosis.

Purpose: Positron emission tomography (PET) imaging of apoptosis is very important for early evaluation of tumor therapeutic efficacy. A stimuli-responsive probe based on the peptide sequence Asp-Glu-Val-Asp (DEVD), [18F]DEVD-Cys(StBu)-PPG(CBT)-AmBF3 ([18F]1), for PET imaging of tumor apoptosis was designed and prepared. This study aimed to develop a novel smart probe using a convenient radiosynthesis method and to fully examine the sensitivity and specificity of the probe response to the tumor treatment. Methods: The radiolabelling precursor DEVD-Cys(StBu)-PPG(CBT)-AmBF3 (1) was synthesized through multistep reactions. The reduction together with caspase-controlled macrocyclization and self-assembly of 1 was characterized and validated in vitro. After [18F]fluorination in the buffer (pH= 2.5), the radiolabelling yield (RLY), radiochemical purity (RCP) and stability of the probe [18F]1 in PBS and mouse serum were investigated by radio-HPLC. The sensitivity and specificity of [18F]1 for detecting the drug-induced apoptosis was fully evaluated in vitro and in vivo. The effect of cold precursor 1 on the cell uptake and tumor imaging of [18F]1 was also assessed. The level of activated caspase-3 in Hela cells and tumors with or without apoptosis induction was analyzed and compared by western blotting and histological staining. Results: The whole radiosynthesis process of [18F]1 was around 25 min with RLY of 50%, RCP of over 99% and specific activity of 1.45 ± 0.4 Ci/µmol. The probe was very stable in both PBS and mouse serum within 4 h. It can be activated by caspase-3 and then undergo an intermolecular cyclization to form nanosized particles. The retained [18F]1 in DOX-treated HeLa cells was 2.2 folds of that in untreated cells. Within 1 h microPET imaging of the untreated Hela-bearing mice, the injection of [18F]1 resulted in the increase of the uptake ratio of tumor to muscle (T/M) only from 1.74 to 2.18, while in the DOX-treated Hela-bearing mice T/M increased from 1.88 to 10.52 and the co-injection of [18F]1 and 1 even led to the increase of T/M from 3.08 to 14.81. Conclusions: A caspase-responsive smart PET probe [18F]1 was designed and prepared in a kit-like manner. Co-injection of [18F]1 and 1 generated remarkably enhanced tumor uptake and signal-to-noise ratio in the tumor-bearing mice with drug-induced apoptosis, which correlated well with the expression level of activated caspase-3. This early readout of treatment response ensured that the probe [18F]1 could serve as a promising PET imaging probe for timely and noninvasive evaluation of tumor therapy.

A new GSH-responsive prodrug of 5-aminolevulinic acid for photodiagnosis and photodynamic therapy of tumors.

5-Aminolevulinic acid (5-ALA) and its two ester derivatives (5-ALA-OMe and 5-ALA-OHex) have been approved for photodiagnosis and photodynamic therapy (PDT) of tumors in the clinical. However, their pharmacological activities are limited by their instability under physiological conditions and lack of tumor selectivity. With the aim to overcome these shortcomings, a glutathione-responsive 5-ALA derivative (SA) was designed based on the fact that many types of tumor cells have higher intracellular glutathione level than normal cells. SA was synthesized by masking the 5-amion group of 5-ALA methyl ester (5-ALA-OMe) with a self-immolative disulfide linker. Compared with 5-ALA and 5-ALA-OMe, SA exhibited higher stability under physiological conditions, and it can efficiently release the parent compound 5-ALA-OMe in response to glutathione. In tumor cells, SA displayed excellent protoporphyrin IX (PpIX) production activity at low concentrations while 5-ALA and 5-ALA-OMe were ineffective at the same concentration. The SA-induced PpIX production was positively correlated with the intracellular glutathione level, and SA exhibited enhanced phototoxicity due to its excellent PpIX generation activity. This study indicates that modification of the amino group in 5-ALA derivatives with a self-immolative disulfide linker is an effective strategy to improve their chemical stability and pharmacological activities, and SA is a potential photosensitizer for photodiagnosis and PDT of tumors.

A protease-responsive fluorescent probe for sensitive imaging of legumain activity in living tumor cells.

Legumain, a lysosomal cysteine protease, is critical for pathological progression and has been found to play an important role in the occurrence and development of several cancers. However, its biological functions remain few recognized. To further understand the role of legumain activity in tumor progression, a legumain protease-responsive fluorescent probe was developed in the present study. The probe 1 was synthesized by conjugating an aminoluciferin fluorophore with an alanine-alanine-asparagine (AAN) peptide sequence. The successful synthesis of probe 1 was validated by NMR and MS spectra as well as HPLC analysis. The probe 1 was non-toxic and exhibited great stability in the physiological solutions. More importantly, compared with the aminoluciferin fluorophore, the peptide conjugation may dramatically increase the targeting specificity. Probe 1 was able to effectively detect the legumain activity in living HCT116 cells through fluorescence imaging. All these results implied that probe 1 could act as a promising fluorescent probe specialized for the monitoring of legumain activity in living cells.