Bis(Disulfide)-Bridged Somatostatin-14 Analogs and Their [111In]In-Radioligands: Synthesis and Preclinical Profile.

The overexpression of one or more somatostatin receptors (SST1-5R) in human tumors has provided an opportunity for diagnosis and therapy with somatostatin-like radionuclide carriers. The application of "pansomatostatin" analogs is expected to broaden the clinical indications and upgrade the diagnostic/therapeutic efficacy of currently applied SST2R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-c[Cys6-Phe7-DTrp8-Lys9-Thr10-Cys11]-Thr12-Ser13-Cys14]) and AT6S (DOTA-Ala1-Gly2-c[Cys3-Lys4-c[Cys5-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-Cys12]-Ser13-Cys14]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [111In]In-AT5S and [111In]In-AT6S were evaluated in a series of in vitro assays, while radioligand stability and biodistribution were studied in mice. The 8/12-mer bicyclic AT6S showed expanded affinity for all SST1-5R and agonistic properties at the SST2R, whereas AT5S lost all affinity to SST1-5R. Both [111In]In-AT5S and [111In]In-AT6S remained stable in the peripheral blood of mice, while [111In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST3R tumors in mice. In summary, high radioligand stability was acquired by the two disulfide bridges introduced into the SS14 motif, but only the 8/12-mer ring AT6S retained a pansomatostatin profile. In consequence, [111In]In-AT6S targeted SST2R-/SST3R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.

Side-Chain Modified [99mTc]Tc-DT1 Mimics: A Comparative Study in NTS1R-Positive Models.

Radiolabeled neurotensin analogs have been developed as candidates for theranostic use against neurotensin subtype 1 receptor (NTS1R)-expressing cancer. However, their fast degradation by two major peptidases, neprilysin (NEP) and angiotensin-converting enzyme (ACE), has hitherto limited clinical success. We have recently shown that palmitoylation at the ε-amine of Lys7 in [99mTc]Tc-[Lys7]DT1 (DT1, N4-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH, N4 = 6-(carboxy)-1,4,8,11-tetraazaundecane) led to the fully stabilized [99mTc]Tc-DT9 analog, displaying high uptake in human pancreatic cancer AsPC-1 xenografts but unfavorable pharmacokinetics in mice. Aiming to improve the in vivo stability of [99mTc]Tc-DT1 without compromising pharmacokinetics, we now introduce three new [99mTc]Tc-DT1 mimics, carrying different pendant groups at the ε-amine of Lys7: MPBA (4-(4-methylphenyl)butyric acid)-[99mTc]Tc-DT10; MPBA via a PEG4-linker-[99mTc]Tc-DT11; or a hydrophilic PEG6 chain-[99mTc]Tc-DT12. The impact of these modifications on receptor affinity and internalization was studied in NTS1R-positive cells. The effects on stability and AsPC-1 tumor uptake were assessed in mice without or during NEP/ACE inhibition. Unlike [99mTc]Tc-DT10, the longer-chain modified [99mTc]Tc-DT11 and [99mTc]Tc-DT12 were significantly stabilized in vivo, resulting in markedly improved tumor uptake compared to [99mTc]Tc-DT1. [99mTc]Tc-DT11 was found to achieve the highest AsPC-1 tumor values and good pharmacokinetics, either without or during NEP inhibition, qualifying for further validation in patients with NTS1R-positive tumors using SPECT/CT.

A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling.

Hyperinflammation plays an important role in severe and critical COVID-19. Using inconsistent criteria, many researchers define hyperinflammation as a form of very severe inflammation with cytokine storm. Therefore, COVID-19 patients are treated with anti-inflammatory drugs. These drugs appear to be less efficacious than expected and are sometimes accompanied by serious adverse effects. SARS-CoV-2 promotes cellular ATP release. Increased levels of extracellular ATP activate the purinergic receptors of the immune cells initiating the physiologic pro-inflammatory immune response. Persisting viral infection drives the ATP release even further leading to the activation of the P2X7 purinergic receptors (P2X7Rs) and a severe yet physiologic inflammation. Disease progression promotes prolonged vigorous activation of the P2X7R causing cell death and uncontrolled ATP release leading to cytokine storm and desensitisation of all other purinergic receptors of the immune cells. This results in immune paralysis with co-infections or secondary infections. We refer to this pathologic condition as hyperinflammation. The readily available and affordable P2X7R antagonist lidocaine can abrogate hyperinflammation and restore the normal immune function. The issue is that the half-maximal effective concentration for P2X7R inhibition of lidocaine is much higher than the maximal tolerable plasma concentration where adverse effects start to develop. To overcome this, we selectively inhibit the P2X7Rs of the immune cells of the lymphatic system inducing clonal expansion of Tregs in local lymph nodes. Subsequently, these Tregs migrate throughout the body exerting anti-inflammatory activities suppressing systemic and (distant) local hyperinflammation. We illustrate this with six critically ill COVID-19 patients treated with lidocaine.

177Lu-Dotatate plus long-acting octreotide versus high­dose long-acting octreotide in patients with midgut neuroendocrine tumours (NETTER-1): final overall survival and long-term safety results from an open-label, randomised, controlled, phase 3 trial.

BACKGROUND: The primary analysis of the phase 3 NETTER-1 trial showed significant improvement in progression-free survival with 177Lu-Dotatate plus long-acting octreotide versus high-dose long-acting octreotide alone in patients with advanced midgut neuroendocrine tumours. Here, we report the prespecified final analysis of overall survival and long-term safety results. METHODS: This open-label, randomised, phase 3 trial enrolled patients from 41 sites in eight countries across Europe and the USA. Patients were 18 years and older with locally advanced or metastatic, well differentiated, somatostatin receptor-positive midgut neuroendocrine tumours (Karnofsky performance status score ≥60) and disease progression on fixed-dose long-acting octreotide. Patients were randomly assigned (1:1) via an interactive web-based response system to intravenous 177Lu-Dotatate 7·4 GBq (200 mCi) every 8 weeks (four cycles) plus intramuscular long-acting octreotide 30 mg (177Lu-Dotatate group) or high-dose long-acting octreotide 60 mg every 4 weeks (control group). The primary endpoint of progression-free survival has been previously reported; here, we report the key secondary endpoint of overall survival in the intention-to-treat population. Final overall survival analysis was prespecified to occur either after 158 deaths or 5 years after the last patient was randomised, whichever occurred first. During long-term follow-up, adverse events of special interest were reported in the 177Lu-Dotatate group only. This trial is registered with ClinicalTrials.gov, NCT01578239. FINDINGS: From Sept 6, 2012, to Jan 14, 2016, 231 patients were enrolled and randomly assigned for treatment. The prespecified final analysis occurred 5 years after the last patient was randomly assigned (when 142 deaths had occurred); median follow-up was 76·3 months (range 0·4-95·0) in the 177Lu-Dotatate group and 76·5 months (0·1-92·3) in the control group. The secondary endpoint of overall survival was not met: median overall survival was 48·0 months (95% CI 37·4-55·2) in the 177Lu-Dotatate group and 36·3 months (25·9-51·7) in the control group (HR 0·84 [95% CI 0·60-1·17]; two-sided p=0·30). During long-term follow-up, treatment-related serious adverse events of grade 3 or worse were recorded in three (3%) of 111 patients in the 177Lu-Dotatate group, but no new treatment-related serious adverse events were reported after the safety analysis cutoff. Two (2%) of 111 patients given 177Lu-Dotatate developed myelodysplastic syndrome, one of whom died 33 months after randomisation (this person was the only the only reported 177Lu-Dotatate treatment-related death). No new cases of myelodysplastic syndrome or acute myeloid leukaemia were reported during long-term follow-up. INTERPRETATION: 177Lu-Dotatate treatment did not significantly improve median overall survival versus high-dose long-acting octreotide. Despite final overall survival not reaching statistical significance, the 11·7 month difference in median overall survival with 177Lu-Dotatate treatment versus high-dose long-acting octreotide alone might be considered clinically relevant. No new safety signals were reported during long-term follow-up. FUNDING: Advanced Accelerator Applications, a Novartis company.

PRRT neuroendocrine tumor response monitored using circulating transcript analysis: the NETest.

PURPOSE: Peptide receptor radionuclide therapy (PRRT) is effective for metastatic/inoperable neuroendocrine tumors (NETs). Imaging response assessment is usually efficient subsequent to treatment completion. Blood biomarkers such as PRRT Predictive Quotient (PPQ) and NETest are effective in real-time. PPQ predicts PRRT efficacy; NETest monitors disease. We prospectively evaluated: (1) NETest as a surrogate biomarker for RECIST; (2) the correlation of NETest levels with PPQ prediction. METHODS: Three independent 177Lu-PRRT-treated GEP-NET and lung cohorts (Meldola, Italy: n = 72; Bad-Berka, Germany: n = 44; Rotterdam, Netherlands: n = 41). Treatment response: RECIST1.1 (responder (stable, partial, and complete response) vs non-responder). Blood sampling: pre-PRRT, before each cycle and follow-up (2-12 months). PPQ (positive/negative) and NETest (0-100 score) by PCR. Stable < 40; progressive > 40). CgA (ELISA) as comparator. Samples de-identified, measurement and analyses blinded. Kaplan-Meier survival and standard statistics. RESULTS: One hundred twenty-two of the 157 were evaluable. RECIST stabilization or response in 67%; 33% progressed. NETest significantly (p < 0.0001) decreased in RECIST "responders" (- 47 ± 3%); in "non-responders," it remained increased (+ 79 ± 19%) (p < 0.0005). NETest monitoring accuracy was 98% (119/122). Follow-up levels > 40 (progressive) vs stable (< 40) significantly correlated with mPFS (not reached vs. 10 months; HR 0.04 (95%CI, 0.02-0.07). PPQ response prediction was accurate in 118 (97%) with a 99% accurate positive and 93% accurate negative prediction. NETest significantly (p < 0.0001) decreased in PPQ-predicted responders (- 46 ± 3%) and remained elevated or increased in PPQ-predicted non-responders (+ 75 ± 19%). Follow-up NETest categories stable vs progressive significantly correlated with PPQ prediction and mPFS (not reached vs. 10 months; HR 0.06 (95%CI, 0.03-0.12). CgA did not reflect PRRT treatment: in RECIST responders decrease in 38% and in non-responders 56% (p = NS). CONCLUSIONS: PPQ predicts PRRT response in 97%. NETest accurately monitors PRRT response and is an effective surrogate marker of PRRT radiological response. NETest decrease identified responders and correlated (> 97%) with the pretreatment PPQ response predictor. CgA was non-informative.

Optimizing the Profile of [99mTc]Tc-NT(7-13) Tracers in Pancreatic Cancer Models by Means of Protease Inhibitors.

BACKGROUND: The overexpression of neurotensin subtype 1 receptors (NTS1Rs) in human tumors may be elegantly exploited for directing neurotensin (NT)-based radionuclide carriers specifically to cancer sites for theranostic purposes. We have recently shown that [99mTc]Tc-DT1 ([99mTc]Tc-[N4-Gly7]NT(7-13)) and [99mTc]Tc-DT5 ([99mTc]Tc-[N4-ßAla7,Dab9]NT(7-13)) show notably improved uptake in human colon adenocarcinoma WiDr xenografts in mice treated with neprilysin (NEP) inhibitors and/or angiotensin-converting enzyme (ACE) inhibitors compared with untreated controls. Aiming toward translation of this promising approach in NTS1R-positive pancreatic ductal adenocarcinoma (PDAC) patients, we now report on the impact of registered NEP/ACE inhibitors on the performance of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 in pancreatic cancer models. METHODS: The cellular uptake of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 was tested in a panel of pancreatic cell lines, and their stability was assessed in mice treated or not treated with Entresto, lisinopril, or their combinations. Biodistribution was conducted in severe combined immunodeficiency (SCID) mice bearing pancreatic AsPC-1 xenografts. RESULTS: The Entresto + lisinopril combination maximized the metabolic stability of the fast-internalizing [99mTc]Tc-DT1 in mice, resulting in notably enhanced tumor uptake (7.05 ± 0.80% injected activity (IA)/g vs. 1.25 ± 0.80% IA/g in non-treated controls at 4 h post-injection; p < 0.0001). CONCLUSIONS: This study has shown the feasibility of optimizing the uptake of [99mTc]Tc-DT1 in pancreatic cancer models with the aid of clinically established NEP/ACE inhibitors, in favor of clinical translation prospects.

[99mTc]Tc-DB1 Mimics with Different-Length PEG Spacers: Preclinical Comparison in GRPR-Positive Models.

Background: The frequent overexpression of gastrin-releasing peptide receptors (GRPRs) in human cancers provides the rationale for delivering clinically useful radionuclides to tumor sites using peptide carriers. Radiolabeled GRPR antagonists, besides being safer for human use, have often shown higher tumor uptake and faster background clearance than agonists. We herein compared the biological profiles of the GRPR-antagonist-based radiotracers [99mTc]Tc-[N4-PEGx-DPhe6,Leu-NHEt13]BBN(6-13) (N4: 6-(carboxy)-1,4,8,11-tetraazaundecane; PEG: polyethyleneglycol): (i) [99mTc]Tc-DB7 (x = 2), (ii) [99mTc]Tc-DB13 (x = 3), and (iii) [99mTc]Tc-DB14 (x = 4), in GRPR-positive cells and animal models. The impact of in situ neprilysin (NEP)-inhibition on in vivo stability and tumor uptake was also assessed by treatment of mice with phosphoramidon (PA). Methods: The GRPR affinity of DB7/DB13/DB14 was determined in PC-3 cell membranes, and cell binding of the respective [99mTc]Tc-radioligands was assessed in PC-3 cells. Each of [99mTc]Tc-DB7, [99mTc]Tc-DB13, and [99mTc]Tc-DB14 was injected into mice without or with PA coinjection and 5 min blood samples were analyzed by HPLC. Biodistribution was conducted at 4 h postinjection (pi) in severe combined immunodeficiency disease (SCID) mice bearing PC-3 xenografts without or with PA coinjection. Results: DB7, -13, and -14 displayed single-digit nanomolar affinities for GRPR. The uptake rates of [99mTc]Tc-DB7, [99mTc]Tc-DB13, and [99mTc]Tc-DB14 in PC-3 cells was comparable and consistent with a radioantagonist profile. The radiotracers were found to be ≈70% intact in mouse blood and >94% intact after coinjection of PA. Treatment of mice with PA enhanced tumor uptake. Conclusions: The present study showed that increase of PEG-spacer length in the [99mTc]Tc-DB7-[99mTc]Tc-DB13-[99mTc]Tc-DB14 series had little effect on GRPR affinity, specific uptake in PC-3 cells, in vivo stability, or tumor uptake. A significant change in in vivo stability and tumor uptake was observed only after treatment of mice with PA, without compromising the favorably low background radioactivity levels.

Comparative evaluation of the new GRPR-antagonist 111 In-SB9 and 111 In-AMBA in prostate cancer models: Implications of in vivo stability.

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer, representing attractive targets for diagnosis and therapy with bombesin (BBN)-like radioligands. GRPR-antagonists have lately attracted much attention owing to inherent biosafety and favorable pharmacokinetics. We herein present the GRPR-antagonist SB9 structurally resembling the known BBN-based agonist AMBA (SB9 = [Leu13 NHEt-desMet14 ]AMBA). The profiles of 111 In-SB9 and 111 In-AMBA were directly compared in PC-3 cells and tumor-bearing mice. SB9 and AMBA displayed high GRPR affinities. 111 In-AMBA strongly internalized in PC-3 cells, while 111 In-SB9 remained bound on the cell surface showing a typical GRPR-radioantagonist profile. 111 In-SB9 was more stable than 111 In-AMBA, but coinjection of the neprilysin (NEP) inhibitor phosphoramidon (PA) stabilized both in vivo. The radioligands displayed high tumor uptake (20.23 ± 3.41 %ID/g and 18.53 ± 1.54 %ID/g, respectively, at 4 hours pi), but 111 In-SB9 washed faster from background. PA coinjection led to significant increase of tumor uptake, combined with better clearance for 111 In-SB9. In short, this study has revealed superior pharmacokinetics and higher stability for the GRPR-antagonist 111 In-SB9 vs the corresponding agonist 111 In-AMBA consolidating previous evidence that GRPR antagonists are preferable to agonists for tumor imaging and therapy. It has also demonstrated that further pharmacokinetic improvements were feasible by in situ metabolic radioligand stabilization using PA.

Comparing Gly11/dAla11-Replacement vs. the in-Situ Neprilysin-Inhibition Approach on the Tumor-targeting Efficacy of the 111In-SB3/111In-SB4 Radiotracer Pair.

Background: The GRPR-antagonist 68Ga-SB3 visualized prostate cancer lesions in animal models and in patients. Switching radiometal from 68Ga to 111In impaired tumor targeting in mice, but coinjection of the neprilysin (NEP)-inhibitor phosphoramidon (PA) stabilized 111In-SB3 in circulation and remarkably increased tumor uptake. We herein report on the biological profile of 111In-SB4: 111In-[dAla11]SB3. Methods: The biological responses of 111In-SB3/SB4 were compared in PC-3 cells and animal models. Results: Gly11/dAla11-replacement deteriorated GRPR-affinity (SB4 IC50: 10.7 ± 0.9 nM vs. SB3 IC50: 4.6 ± 0.3 nM) and uptake in PC-3 cells (111In-SB4: 1.3 ± 0.4% vs. 111In-SB3 16.2 ± 0.8% at 1 h). 111In-SB4 was more stable than 111In-SB3, but PA-coinjection stabilized both radiotracers in peripheral mice blood. Unmodified 111In-SB3 showed higher uptake in PC-3 xenografts (8.8 ± 3.0%ID/g) vs. 111In-SB4 (3.1 ± 1.1%ID/g) at 4 h pi. PA-coinjection improved tumor uptake, with 111In-SB3 still showing superior tumor targeting (38.3 ± 7.9%ID/g vs. 7.4 ± 0.3%ID/g for 111In-SB4). Conclusions: Replacement of Gly11 by dAla11 improved in vivo stability, however, at the cost of GRPR-affinity and cell uptake, eventually translating into inferior tumor uptake of 111In-SB4 vs. unmodified 111In-SB3. On the other hand, in-situ NEP-inhibition turned out to be a more efficient and direct strategy to optimize the in vivo profile of 111In-SB3, and potentially other peptide radiotracers.

Radiometal-Dependent Biological Profile of the Radiolabeled Gastrin-Releasing Peptide Receptor Antagonist SB3 in Cancer Theranostics: Metabolic and Biodistribution Patterns Defined by Neprilysin.

Recent advances in oncology involve the use of diagnostic/therapeutic radionuclide-carrier pairs that target cancer cells, offering exciting opportunities for personalized patient treatment. Theranostic gastrin-releasing peptide receptor (GRPR)-directed radiopeptides have been proposed for the management of GRPR-expressing prostate and breast cancers. We have recently introduced the PET tracer 68Ga-SB3 (SB3, DOTA- p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), a receptor-radioantagonist that enables the visualization of GRPR-positive lesions in humans. Aiming to fully assess the theranostic potential of SB3, we herein report on the impact of switching 68Ga to 111In/177Lu-label on the biological properties of resulting radiopeptides. Notably, the bioavailability of 111In/177Lu-SB3 in mice drastically deteriorated compared with metabolically robust 68Ga-SB3, and as a result led to poorer 111In/177Lu-SB3 uptake in GRPR-positive PC-3 xenografts. The peptide cleavage sites were identified by chromatographic comparison of blood samples from mice intravenously receiving 111In/177Lu-SB3 with each of newly synthesized 111In/177Lu-SB3-fragments. Coinjection of the radioconjugates with the neprilysin (NEP)-inhibitor phosphoramidon led to full stabilization of 111In/177Lu-SB3 in peripheral mouse blood and resulted in markedly enhanced radiolabel uptake in the PC-3 tumors. In conclusion, in situ NEP-inhibition led to indistinguishable 68Ga/111In/177Lu-SB3 profiles in mice emphasizing the theranostic prospects of SB3 for clinical use.

PRRT genomic signature in blood for prediction of 177Lu-octreotate efficacy.

BACKGROUND: Peptide receptor radionuclide therapy (PRRT) utilizes somatostatin receptor (SSR) overexpression on neuroendocrine tumors (NET) to deliver targeted radiotherapy. Intensity of uptake at imaging is considered related to efficacy but has low sensitivity. A pretreatment strategy to determine individual PRRT response remains a key unmet need. NET transcript expression in blood integrated with tumor grade provides a PRRT predictive quotient (PPQ) which stratifies PRRT "responders" from "non-responders". This study clinically validates the utility of the PPQ in NETs. METHODS: The development and validation of the PPQ was undertaken in three independent 177Lu-PRRT treated cohorts. Specificity was tested in two separate somatostatin analog-treated cohorts. Prognostic value of the marker was defined in a cohort of untreated patients. The developmental cohort included lung and gastroenteropancreatic [GEP] NETs (n = 72) from IRST Meldola, Italy. The majority were GEP (71%) and low grade (86% G1-G2). Prospective validation cohorts were from Zentralklinik Bad Berka, Germany (n = 44), and Erasmus Medical Center, Rotterdam, Netherlands (n = 42). Each cohort included predominantly well differentiated, low grade (86-95%) lung and GEP-NETs. The non-PRRT comparator cohorts included SSA cohort I, n = 28 (100% low grade, 100% GEP-NET); SSA cohort II, n = 51 (98% low grade; 76% GEP-NET); and an untreated cohort, n = 44 (64% low grade; 91% GEP-NET). Baseline evaluations included clinical information (disease status, grade, SSR) and biomarker (CgA). NET blood gene transcripts (n = 8: growth factor signaling and metabolism) were measured pre-therapy and integrated with tumor Ki67 using a logistic regression model. This provided a binary output: "predicted responder" (PPQ+); "predicted non-responder" (PPQ-). Treatment response was evaluated using RECIST criteria [Responder (stable, partial and complete response) vs Non-Responder)]. Sample measurement and analyses were blinded to study outcome. Statistical evaluation included Kaplan-Meier survival and standard test evaluation analyses. RESULTS: In the developmental cohort, 56% responded to PRRT. The PPQ predicted 100% of responders and 84% of non-responders (accuracy: 93%). In the two validation cohorts (response: 64-79%), the PPQ was 95% accurate (Bad Berka: PPQ + =97%, PPQ- = 93%; Rotterdam: PPQ + =94%, PPQ- = 100%). Overall, the median PFS was not reached in PPQ+ vs PPQ- (10-14 months; HR: 18-77, p < 0.0001). In the comparator cohorts, the predictor (PPQ) was 47-50% accurate for SSA-treatment and 50% as a prognostic. No differences in PFS were respectively noted (PPQ+: 10-12 months vs. PPQ-: 9-15 months). CONCLUSION: The PPQ derived from circulating NET specific genes and tumor grade prior to the initiation of therapy is a highly specific predictor of the efficacy of PRRT with an accuracy of 95%.

Preclinical and first clinical experience with the gastrin-releasing peptide receptor-antagonist [68Ga]SB3 and PET/CT.

PURPOSE: Gastrin-releasing peptide receptors (GRPR) represent attractive targets for tumor diagnosis and therapy because of their overexpression in major human cancers. Internalizing GRPR agonists were initially proposed for prolonged lesion retention, but a shift of paradigm to GRPR antagonists has recently been made. Surprisingly, radioantagonists, such as [(99m)Tc]DB1 ((99m)Tc-N4'-DPhe(6),Leu-NHEt(13)]BBN(6-13)), displayed better pharmacokinetics than radioagonists, in addition to their higher inherent biosafety. We introduce here [(68)Ga]SB3, a [(99m)Tc]DB1 mimic-carrying, instead of the (99m)Tc-binding tetraamine, the chelator DOTA for labeling with the PET radiometal (68)Ga. METHODS: Competition binding assays of SB3 and [(nat)Ga]SB3 were conducted against [(125)I-Tyr(4)]BBN in PC-3 cell membranes. Blood samples collected 5 min postinjection (pi) of the [(67)Ga]SB3 surrogate in mice were analyzed using high-performance liquid chromatography (HPLC) for degradation products. Likewise, biodistribution was performed after injection of [(67)Ga]SB3 (37 kBq, 100 µL, 10 pmol peptide) in severe combined immunodeficiency (SCID) mice bearing PC-3 xenografts. Eventually, [(68)Ga]SB3 (283 ± 91 MBq, 23 ± 7 nmol) was injected into 17 patients with breast (8) and prostate (9) cancer. All patients had disseminated disease and had received previous therapies. PET/CT fusion images were acquired 60-115 min pi. RESULTS: SB3 and [(nat)Ga]SB3 bound to the human GRPR with high affinity (IC50: 4.6 ± 0.5 nM and 1.5 ± 0.3 nM, respectively). [(67)Ga]SB3 displayed good in vivo stability (>85 % intact at 5 min pi). [(67)Ga]SB3 showed high, GRPR-specific and prolonged retention in PC-3 xenografts (33.1 ± 3.9%ID/g at 1 h pi - 27.0 ± 0.9%ID/g at 24 h pi), but much faster clearance from the GRPR-rich pancreas (≈160%ID/g at 1 h pi to <17%ID/g at 24 h pi) in mice. In patients, [(68)Ga]SB3 elicited no adverse effects and clearly visualized cancer lesions. Thus, 4 out of 8 (50 %) breast cancer and 5 out of 9 (55 %) prostate cancer patients showed pathological uptake on PET/CT with [(68)Ga]SB3. CONCLUSION: [(67)Ga]SB3 showed excellent pharmacokinetics in PC-3 tumor-bearing mice, while [(68)Ga]SB3 PET/CT visualized lesions in about 50 % of patients with advanced and metastasized prostate and breast cancer. We expect imaging with [(68)Ga]SB3 to be superior in patients with primary breast or prostate cancer.

Subacute haematotoxicity after PRRT with (177)Lu-DOTA-octreotate: prognostic factors, incidence and course.

PURPOSE: In peptide receptor radionuclide therapy (PRRT), the bone marrow (BM) is one of the dose-limiting organs. The accepted dose limit for BM is 2 Gy, adopted from (131)I treatment. We investigated the incidence and duration of haematological toxicity and its risk factors in patients treated with PRRT with (177)Lu-DOTA(0)-Tyr(3)-octreotate ((177)Lu-DOTATATE). Also, absorbed BM dose estimates were evaluated and compared with the accepted 2 Gy dose limit. METHODS: The incidence and duration of grade 3 or 4 haematological toxicity (according to CTCAE v3.0) and risk factors were analysed. Mean BM dose per unit (gigabecquerels) of administered radioactivity was calculated and the correlations between doses to the BM and haematological risk factors were determined. RESULTS: Haematological toxicity (grade 3/4) occurred in 34 (11 %) of 320 patients. In 15 of the 34 patients, this lasted more than 6 months or blood transfusions were required. Risk factors significantly associated with haematological toxicity were: poor renal function, white blood cell (WBC) count <4.0 × 10(9)/l, age over 70 years, extensive tumour mass and high tumour uptake on the OctreoScan. Previous chemotherapy was not associated. The mean BM dose per administered activity in 23 evaluable patients was 67 ± 7 mGy/GBq, resulting in a mean BM dose of 2 Gy in patients who received four cycles of 7.4 GBq (177)Lu-DOTATATE. Significant correlations between (cumulative) BM dose and platelet and WBC counts were found in a selected group of patients. CONCLUSION: The incidence of subacute haematological toxicity after PRRT with (177)Lu-DOTATATE is acceptable (11 %). Patients with impaired renal function, low WBC count, extensive tumour mass, high tumour uptake on the OctreoScan and/or advanced age are more likely to develop grade 3/4 haematological toxicity. The BM dose limit of 2 Gy, adopted from (131)I, seems not to be valid for PRRT with (177)Lu-DOTATATE.

Nephrotoxicity after PRRT with (177)Lu-DOTA-octreotate.

PURPOSE: After peptide receptor radionuclide therapy (PRRT), renal toxicity may occur, particular in PRRT with (90)Y-labelled somatostatin analogues. Risk factors have been identified for increased probability of developing renal toxicity after PRRT, including hypertension, diabetes and age. We investigated the renal function over time, the incidence of nephrotoxicity and associated risk factors in patients treated with PRRT with [(177)Lu-DOTA(0),Tyr(3)]-Octreotate ((177)Lu-Octreotate). Also, radiation dose to the kidneys was evaluated and compared with the accepted dose limits in external beam radiotherapy and PRRT with (90)Y-radiolabelled somatostatin analogues. METHODS: The annual decrease in creatinine clearance (CLR) was determined in 209 Dutch patients and the incidence of grade 3 or 4 renal toxicity (according to CTCAE v4.03) was evaluated in 323 patients. Risk factors were analysed using a nonlinear mixed effects regression model. Also, radiation doses to the kidneys were calculated and their association with high annual decrease in renal function were analysed. RESULTS: Of the 323 patients, 3 (1 %) developed (subacute) renal toxicity grade 2 (increase in serum creatinine >1.5 - 3.0 times baseline or upper limit of normal). No subacute grade 3 or 4 nephrotoxicity was observed. The estimated average baseline CLR (± SD) was 108 ± 5 ml/min and the estimated average annual decrease in CLR (± SD) was 3.4 ± 0.4 %. None of the risk factors (hypertension, diabetes, high cumulative injected activity, radiation dose to the kidneys and CTCAE grade) at baseline had a significant effect on renal function over time. The mean absorbed kidney dose in 228 patients was 20.1 ± 4.9 Gy. CONCLUSION: Nephrotoxicity in patients treated with (177)Lu-octreotate was low. No (sub)acute grade 3 or 4 renal toxicity occurred and none of the patients had an annual decrease in renal function of >20 %. No risk factors for renal toxicity could be identified. Our data support the idea that the radiation dose threshold, adopted from external beam radiotherapy and PRRT with (90)Y-labelled somatostatin analogues, does not seem valid for PRRT with (177)Lu-octreotate.

Long-term tolerability of PRRT in 807 patients with neuroendocrine tumours: the value and limitations of clinical factors.

PURPOSE: Peptide receptor radionuclide therapy (PRRT) with (90)Y and (177)Lu provides objective responses in neuroendocrine tumours, and is well tolerated with moderate toxicity. We aimed to identify clinical parameters predictive of long-term renal and haematological toxicity (myelodysplastic syndrome and acute leukaemia). METHODS: Of 807 patients studied at IEO-Milan (1997-2013), 793 (98 %) received (177)Lu (278, 34.4 %), (90)Y (358, 44.4 %) or (177)Lu and (90)Y combined (157. 19.5 %), and 14 (2 %) received combinations of PRRT and other agents. Follow-up was 30 months (1-180 months). The parameters evaluated included renal risk factors, bone marrow toxicity and PRRT features. Data analysis included multiple regression, random forest feature selection, and recursive partitioning and regression trees. RESULTS: Treatment with (90)Y and (90)Y + (177)Lu was more likely to result in nephrotoxicity than treatment with (177)Lu alone (33.6 %, 25.5 % and 13.4 % of patients, respectively; p < 0.0001). Nephrotoxicity (any grade), transient and persistent, occurred in 279 patients (34.6 %) and was severe (grade 3 + 4) in 12 (1.5 %). In only 20-27 % of any nephrotoxicity was the disease modelled by risk factors and codependent associations (p < 0.0001). Hypertension and haemoglobin toxicity were the most relevant factors. Persistent toxicity occurred in 197 patients (24.3 %). In only 22-34 % of affected patients was the disease modelled by the clinical data (p < 0.0001). Hypertension (regression coefficient 0.14, p < 0.0001) and haemoglobin toxicity (regression coefficient 0.21, p < 0.0001) were pertinent factors. Persistent toxicity was associated with shorter PRRT duration from the first to the last cycle (mean 387 vs. 658 days, p < 0.004). Myelodysplastic syndrome occurred in 2.35 % of patients (modelled by the clinical data in 30 %, p < 0.0001). Platelet toxicity grade (2.05 ± 1.2 vs. 0.58 ± 0.8, p < 0.0001) and longer PRRT duration (22.6 ± 24 vs. 15.5 ± 9 months, p = 0.01) were relevant. Acute leukaemia occurred in 1.1 % of patients (modelled by the clinical data in 18 %, p < 0.0001). CONCLUSION: Identified risk factors provide a limited (<30 %) risk estimate even with target tissue dosimetry. These data strongly suggest the existence of unidentified individual susceptibilities to radiation-associated disease.

Hypocalcaemia after treatment with [177Lu-DOTA 0,Tyr3]octreotate.

PURPOSE: The aim of this study was to explore the possible mechanisms involved in an observed decline in serum calcium levels in patients with a neuroendocrine tumour (NET) treated with [(177)Lu-DOTA(0),Tyr(3)]octreotate ((177)Lu-octreotate). METHODS: In 47 patients with NET who were normocalcaemic at baseline, serum calcium, albumin, creatinine, alkaline phosphatase, gamma glutamyl transpeptidase, magnesium, phosphate and 25-hydroxyvitamin D were prospectively analysed at baseline and up to 6 months after treatment. Parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3, type 1 aminoterminal propeptide of procollagen, bone-specific alkaline phosphatase, carboxyterminal crosslinking telopeptide of bone collagen, collagen type I crosslinked N-telopeptide, and creatinine and calcium in 24-h urine samples, were evaluated at baseline and at 3 and 6 months. Another 153 patients with NET were included in a retrospective study to estimate the occurrence of hypocalcaemia in a larger patient group. RESULTS: In the prospectively included patients, the mean serum calcium level decreased significantly after treatment (2.31 ± 0.01 to 2.26 ± 0.02 mmol/l, p = 0.02). Eight patients (17%) showed a marked decrease in serum calcium levels with a nadir of ≤ 2.10 mmol/l. In five patients (11%), calcium substitution therapy was prescribed. PTH increased significantly (5.9 ± 0.6 to 6.7 ± 0.8 pmol/l, p = 0.02), presumably in response to the decreasing serum calcium levels. 25-Hydroxyvitamin D remained stable after treatment. Creatinine levels increased significantly (73 ± 3 to 77 ± 3 µmol/l, p = 0.01), but not enough to explain the hypocalcaemia. Phosphate levels remained unaffected. In the retrospectively analysed patients, the mean serum calcium level decreased significantly from 2.33 ± 0.01 at baseline to a nadir of 2.24 ± 0.01 mmol/l at 18 months after treatment (p < 0.001). Of the 153 patients, 33 (22%) showed a serum calcium nadir of ≤ 2.10 mmol/l, and 11 (7%) received calcium substitution therapy. CONCLUSION: The mean serum calcium level decreased significantly after treatment with (177)Lu-octreotate, resulting in mild hypocalcaemia in about 20% of patients. We excluded several potential causes of this hypocalcaemia, so the cause remains unknown. Serum calcium levels should be monitored after peptide receptor radionuclide therapy, and calcium substitution therapy should be initiated if appropriate.

Treatment of gastroenteropancreatic neuroendocrine tumors with peptide receptor radionuclide therapy.

The primary treatment of gastroenteropancreatic neuroendocrine tumors (GEPNETs) is surgery with curative intent or debulking of the tumor mass. In case of metastatic disease, cytoreductive options are limited. A relatively new therapeutic modality, peptide receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogs, is currently available in a number of mostly European centers. Complete and partial responses obtained after treatment with [90Y-DOTA0,Tyr3]octreotide are in the same range as after treatment with [177Lu-DOTA0,Tyr3]octreotate (i.e. 10-30%). However, significant nephrotoxicity has been observed after treatment with [90Y-DOTA0,Tyr3]octreotide. Options to improve PRRT may include combinations of radioactive labeled somatostatin analogs, intra-arterial administration, and the use of radiosensitizing drugs combined with PRRT. Other therapeutic applications of PRRT may include additional therapy cycles in patients with progressive disease after benefit from initial therapy, PRRT in adjuvant or neoadjuvant setting, or PRRT combined with new targeted therapies, such as sunitinib or everolimus. Randomized clinical trials comparing PRRT with other treatment modalities, or comparing various radioactive labeled somatostatin analogs should be undertaken to determine the best treatment options and treatment sequelae for patients with GEPNETs.

Toward Stability Enhancement of NTS1R-Targeted Radioligands: Structural Interventions on [99mTc]Tc-DT1.

The neurotensin subtype 1 receptor (NTS1R) is overexpressed in a number of human tumors, thereby representing a valid target for cancer theranostics with radiolabeled neurotensin (NT) analogs like [99mTc]Tc-DT1 (DT1, N4-Gly7-NT(8-13)). Thus far, the fast degradation of intravenously injected NT-radioligands by neprilysin (NEP) and angiotensin-converting enzyme (ACE) has compromised their clinical applicability. Aiming at metabolic stability enhancements, we herein introduce (i) DT7 ([DAsn14]DT1) and (ii) DT8 ([ß-Homoleucine13]DT1), modified at the C-terminus, along with (iii) DT9 ([(palmitoyl)Lys7]DT1), carrying an albumin-binding domain (ABD) at Lys7. The biological profiles of the new [99mTc]Tc-radioligands were compared with [99mTc]Tc-DT1, using NTS1R-expressing AsPC-1 cells and mice models without or during NEP/ACE inhibition. The radioligands showed enhanced in vivo stability vs. [99mTc]Tc-DT1, with [99mTc]Tc-DT9 displaying full resistance to both peptidases. Furthermore, [99mTc]Tc-DT9 achieved the highest cell internalization and tumor uptake even without NEP/ACE-inhibition but with unfavorably high background radioactivity levels. Hence, unlike C-terminal modification, the introduction of a pendant ABD group in the linker turned out to be the most promising strategy toward metabolic stability, cell uptake, and tumor accumulation of [99mTc]Tc-DT1 mimics. To improve the observed suboptimal pharmacokinetics of [99mTc]Tc-DT9, the replacement of palmitoyl on Lys7 by other ABD groups is currently being pursued.

Somatostatin analogs for the treatment of neuroendocrine tumors.

Somatostatin is an important regulator of endocrine and exocrine secretion, affecting the release of many hormones. The effects of somatostatin are mediated through its interaction with one of five somatostatin receptors. Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) express multiple somatostatin receptors, making them excellent potential therapeutic targets. Many trials have shown that treatment with somatostatin analogs is associated with disease stabilization and prolonged survival. More recently, somatostatin analogs have been shown to have antiproliferative effects, thus broadening the scope of their uses. In this review, we update the current data on the treatment of GEP-NETs with somatostatin analogs, with particular emphasis on the results of the PROMID study. In addition, we discuss the current state of knowledge of novel therapies against GEP-NETs, including the use of somatostatin analogs with broader receptor binding profiles, chimeric somatostatin-dopamine molecules, combinations of somatostatin analogs with other active chemotherapy agents, and peptide receptor-targeted radionuclide therapy.

Imaging of activated macrophages in experimental osteoarthritis using folate-targeted animal single-photon-emission computed tomography/computed tomography.

OBJECTIVE: Evaluation of macrophage activation may provide essential information about the etiology and progression rate of osteoarthritis (OA). Activated macrophages abundantly express folate receptor ß (FRß), which can be targeted using radioactive-labeled folic acid. This study was undertaken to investigate whether macrophage activation can be monitored in small animal models of OA using a folate radiotracer and to determine whether macrophage activation differs in different models of OA with different OA progression. METHODS: Two rat models of OA were used: the mono-iodoacetate (MIA) model, which is a fast-progressing biochemically induced model, and the anterior cruciate ligament transection (ACLT) model, which induces OA at a slower pace. Images were obtained using high-resolution small animal single-photon-emission computed tomography/computed tomography. The specificity of the technique was tested by eradicating macrophages using clodronate-laden liposomes and blockade of FRß by cold folic acid. RESULTS: The MIA model had high initial macrophage activation, with a peak after 2 weeks which disappeared after 8 weeks. The ACLT model showed less activation but was still active 12 weeks after induction. The technique allowed monitoring of the disease process over time, in which late stages of the disease showed less macrophage activation than early stages, especially in the fast-progressing MIA model of OA. CONCLUSION: Our findings indicate that macrophage activation in experimental OA can clearly be demonstrated and monitored by the folate radiotracer. The high resolution, high sensitivity, and high specificity of the technique allow clear localization of macrophage activity in a disease model that is not known for abundant macrophage involvement.