FAP imaging in rare cancer entities-first clinical experience in a broad spectrum of malignancies.

PURPOSE: 68 Ga-FAPI (fibroblast activation protein inhibitor) is a rapidly evolving and highly promising radiotracer for PET/CT imaging, presenting excellent results in a variety of tumor entities, particularly in epithelial carcinomas. This retrospective analysis sought to evaluate the potential and impact of FAPI-PET/CT in rare cancer diseases with respect to improvement in staging and therapy, based on tracer uptake in normal organs and tumors. MATERIAL AND METHODS: Fifty-five patients with rare tumor entities, defined by a prevalence of 1 person out of 2000 or less, received a 68 Ga-FAPI-PET/CT scan. Fourteen women and 41 men (median age 60) were included within the following subgroups: cancer of unknown primary (n = 10), head and neck cancer (n = 13), gastrointestinal and biliary-pancreatic cancer (n = 17), urinary tract cancer (n = 4), neuroendocrine cancer (n = 4), and others (n = 7). Tracer uptake was quantified by standardized uptake values SUVmax and SUVmean and the tumor-to-background ratio (TBR) was determined (SUVmax tumor/SUVmean organ). RESULTS: In 20 out of 55 patients, the primary tumor was identified and 31 patients presented metastases (n = 88), characterized by a high mean SUVmax in primary (10.1) and metastatic lesions (7.6). The highest uptake was observed in liver metastases (n = 6) with a mean SUVmax of 9.8 and a high TBR of 8.7, closely followed by peritoneal carcinomatosis (n = 16) presenting a mean SUVmax of 9.8 and an excellent TBR of 29.6. In terms of the included subgroups, the highest uptake regarding mean SUVmax was determined in gastrointestinal and biliary-pancreatic cancer with 9.8 followed closely by urinary tract cancer with 9.5 and head and neck cancer (9.1). CONCLUSION: Due to excellent tumor visualization and, thereby, sharp contrasts in terms of high TBRs in primary and metastatic lesions in different rare malignancies, 68 Ga-FAPI-PET/CT crystallizes as a powerful and valuable imaging tool, particularly with respect to epithelial carcinomas, and therefore an enhancement to standard diagnostics imaging methodologies. The realization of further and prospective studies is of large importance to confirm the potential of FAP imaging in oncology.

Coating of PLA-nanoparticles with cyclic, arginine-rich cell penetrating peptides enables oral delivery of liraglutide.

Until today, the oral delivery of peptide drugs is hampered due to their instability in the gastrointestinal tract and low mucosal penetration. To overcome these hurdles, PLA (polylactide acid)-nanoparticles were coated with a cyclic, polyarginine-rich, cell penetrating peptide (cyclic R9-CPP). These surface-modified nanoparticles showed a size and polydispersity index comparable to standard PLA-nanoparticles. The zeta potential showed a significant increase indicating successful CPP-coupling to the surface of the nanoparticles. Cryo-EM micrographs confirmed the appropriate size and morphology of the modified nanoparticles. A high encapsulation efficiency of liraglutide could be achieved. In vitro tests using Caco-2 cells showed high viability indicating the tolerability of this novel formulation. A strongly enhanced mucosal binding and penetration was demonstrated by a Caco-2 binding and uptake assay. In Wistar rats, the novel nanoparticles showed a substantial, 4.5-fold increase in the oral bioavailability of liraglutide revealing great potential for the oral delivery of peptide drugs.

F-18 labelled PSMA-1007: biodistribution, radiation dosimetry and histopathological validation of tumor lesions in prostate cancer patients.

PURPOSE: The prostate-specific membrane antigen (PSMA) targeted positron-emitting-tomography (PET) tracer 68Ga-PSMA-11 shows great promise in the detection of prostate cancer. However, 68Ga has several shortcomings as a radiolabel including short half-life and non-ideal energies, and this has motivated consideration of 18F-labelled analogs. 18F-PSMA-1007 was selected among several 18F-PSMA-ligand candidate compounds because it demonstrated high labelling yields, outstanding tumor uptake and fast, non-urinary background clearance. Here, we describe the properties of 18F-PSMA-1007 in human volunteers and patients. METHODS: Radiation dosimetry of 18F-PSMA-1007 was determined in three healthy volunteers who underwent whole-body PET-scans and concomitant blood and urine sampling. Following this, ten patients with high-risk prostate cancer underwent 18F-PSMA-1007 PET/CT (1 h and 3 h p.i.) and normal organ biodistribution and tumor uptakes were examined. Eight patients underwent prostatectomy with extended pelvic lymphadenectomy. Uptake in intra-prostatic lesions and lymph node metastases were correlated with final histopathology, including PSMA immunostaining. RESULTS: With an effective dose of approximately 4.4-5.5 mSv per 200-250 MBq examination, 18F-PSMA-1007 behaves similar to other PSMA-PET agents as well as to other 18F-labelled PET-tracers. In comparison to other PSMA-targeting PET-tracers, 18F-PSMA-1007 has reduced urinary clearance enabling excellent assessment of the prostate. Similar to 18F-DCFPyL and with slightly slower clearance kinetics than PSMA-11, favorable tumor-to-background ratios are observed 2-3 h after injection. In eight patients, diagnostic findings were successfully validated by histopathology. 18F-PSMA-1007 PET/CT detected 18 of 19 lymph node metastases in the pelvis, including nodes as small as 1 mm in diameter. CONCLUSION: 18F-PSMA-1007 performs at least comparably to 68Ga-PSMA-11, but its longer half-life combined with its superior energy characteristics and non-urinary excretion overcomes some practical limitations of 68Ga-labelled PSMA-targeted tracers.

(68)Ga-PSMA-11 dynamic PET/CT imaging in biochemical relapse of prostate cancer.

OBJECTIVES: We aim to investigate the pharmacokinetics and distribution of the recently clinically introduced radioligand (68)Ga-PSMA-11 in men with recurrent prostate cancer (PC) by means of dynamic and whole-body PET/CT. The correlation between PSA levels and (68)Ga-PSMA-11 PET parameters is also investigated. METHODS: 31 patients with biochemical failure after primary PC treatment with curative intent (median age 71.0 years) were enrolled in the analysis. The median PSA value was 2.0 ng/mL (range = 0.1 - 130.0 ng/mL) and the median Gleason score was 7 (range = 5 - 9). 8/31 (25.8 %) of the included patients had a PSA value < 0.5 ng/ml. All patients underwent dynamic PET/CT (dPET/CT) scanning (60 min) of the pelvis and lower abdomen as well as whole-body PET/CT with (68)Ga-PSMA-11. dPET/CT assessment was based on qualitative evaluation, SUV calculation, and quantitative analysis based on a two-tissue compartment model and a non-compartmental approach leading to the extraction of fractal dimension (FD). RESULTS: 22/31 patients (71.0 %) were (68)Ga-PSMA-11-positive, while 9/31 (29.0 %) patients were (68)Ga-PSMA-11-negative. The median PSA value in the (68)Ga-PSMA-11-positive group was significantly higher (median = 2.35 ng/mL; range = 0.19 - 130.0 ng/mL) than in the (68)Ga-PSMA-11-negative group (median value: 0.34 ng/mL; range = 0.10 - 4.20 ng/mL). A total of 76 lesions were semi-quantitatively evaluated. PC recurrence-associated lesions demonstrated a mean SUVaverage = 12.4 (median = 9.0; range = 2.2 - 84.5) and mean SUVmax = 18.8 (median = 14.1; range = 3.1 - 120.3). Dynamic PET/CT studies of the pelvis revealed the following mean values for the PC recurrence-suspicious lesions: K1 = 0.26, k3 = 0.30, influx = 0.14 and FD = 1.24. Time-activity curves derived from PC-recurrence indicative lesions revealed an increasing (68)Ga-PSMA-11 accumulation during dynamic PET acquisition. Correlation analysis revealed a moderate, but significant, correlation between PSA levels and the number of lesions detected on (68)Ga-PSMA-11 PET/CT (r = 0.54) and between PSA levels and SUVaverage (r = 0.48) or SUVmax (r = 0.44). CONCLUSIONS: Ga-PSMA-11 PET/CT demonstrated an overall detection rate of 71.0 % 60 min p.i. of the radiotracer in a mixed patient population with respect to PSA levels and including patients with very low PSA values. Higher PSA values were associated with a higher detection rate. The tracer uptake in PC-recurrence-indicative lesions is increasing during the 60 minutes of dynamic PET acquisition.

Intra-individual comparison of (68)Ga-PSMA-11-PET/CT and multi-parametric MR for imaging of primary prostate cancer.

PURPOSE: Multi-parametric magnetic resonance imaging (MP-MRI) is currently the most comprehensive work up for non-invasive primary tumor staging of prostate cancer (PCa). Prostate-specific membrane antigen (PSMA)-Positron emission tomography-computed tomography (PET/CT) is presented to be a highly promising new technique for N- and M-staging in recurrent PCa-patients. The actual investigation analyses the potential of (68)Ga-PSMA11-PET/CT to assess the extent of primary prostate cancer by intra-individual comparison to MP-MRI. METHODS: In a retrospective study, ten patients with primary PCa underwent MP-MRI and PSMA-PET/CT for initial staging. All tumors were proven histopathological by biopsy. Image analysis was done in a quantitative (SUVmax) and qualitative (blinded read) fashion based on PI-RADS. The PI-RADS schema was then translated into a 3D-matrix and the euclidian distance of this coordinate system was used to quantify the extend of agreement. RESULTS: Both MP-MRI and PSMA-PET/CT presented a good allocation of the PCa, which was also in concordance to the tumor location validated in eight-segment resolution by biopsy. An Isocontour of 50 % SUVmax in PSMA-PET resulted in visually concordant tumor extension in comparison to MP-MRI (T2w and DWI). For 89.4 % of sections containing a tumor according to MP-MRI, the tumor was also identified in total or near-total agreement (euclidian distance ≤1) by PSMA-PET. Vice versa for 96.8 % of the sections identified as tumor bearing by PSMA-PET the tumor was also found in total or near-total agreement by MP-MRI. CONCLUSIONS: PSMA-PET/CT and MP-MRI correlated well with regard to tumor allocation in patients with a high pre-test probability for large tumors. Further research will be needed to evaluate its value in challenging situation such as prostatitis or after repeated negative biopsies.

²¹³Bi-DOTATOC receptor-targeted alpha-radionuclide therapy induces remission in neuroendocrine tumours refractory to beta radiation: a first-in-human experience.

PURPOSE: Radiopeptide therapy using a somatostatin analogue labelled with a beta emitter such as (90)Y/(177)Lu-DOTATOC is a new therapeutic option in neuroendocrine cancer. Alternative treatments for patients with refractory disease are rare. Here we report the first-in-human experience with (213)Bi-DOTATOC targeted alpha therapy (TAT) in patients pretreated with beta emitters. METHODS: Seven patients with progressive advanced neuroendocrine liver metastases refractory to treatment with (90)Y/(177)Lu-DOTATOC were treated with an intraarterial infusion of (213)Bi-DOTATOC, and one patient with bone marrow carcinosis was treated with a systemic infusion of (213)Bi-DOTATOC. Haematological, kidney and endocrine toxicities were assessed according to CTCAE criteria. Radiological response was assessed with contrast-enhanced MRI and (68)Ga-DOTATOC-PET/CT. More than 2 years of follow-up were available in seven patients. RESULTS: The biodistribution of (213)Bi-DOTATOC was evaluable with 440 keV gamma emission scans, and demonstrated specific tumour binding. Enduring responses were observed in all treated patients. Chronic kidney toxicity was moderate. Acute haematotoxicity was even less pronounced than with the preceding beta therapies. CONCLUSION: TAT can induce remission of tumours refractory to beta radiation with favourable acute and mid-term toxicity at therapeutic effective doses.

Benzamides as melanotropic carriers for radioisotopes, metals, cytotoxic agents and as enzyme inhibitors.

Benzamide derivatives are known as antipsychotic and antiemetic drugs. Owing to its neurotropic characteristic this class of compounds was found useful for imaging melanoma and melanoma metastases. [(123I)]BZA (N-(2-diethylaminoethyl)-4-[(123I)]iodobenzamide) was the first example which was clinically applied as an imaging agent demonstrating high tumor uptake. This finding initiated research efforts to further improve the affinity and pharmacological properties of this agent. In order to optimize the use of these molecules with respect to costs and wide spread distribution, (99m)Tc labeled benzamides have been developed. Indeed, several (99m)Tc complexes were found suitable for melanoma imaging; however, they were less eligible than radioiodinated benzamides. Besides their use as radiotracers benzamides have been evaluated for magnetic resonance imaging. Molecular imaging with paramagnetic metal contrast agents for magnetic resonance tomography (MRT) is hampered by the inferior sensitivity of MRT. Biochemical trapping was thought to overcome this problem using the polyamine transporter of melanoma cells. One of the neutral, DTPA based Gd complexes comprising 2-(diethylamino)ethylamine and bis-(2-aminoethyl)amine in the side chain led to intracellular uptake values well above the MRI detection limit. An overview about benzamides used for molecular imaging and as transporters for cytostatic agents as well as inhibitors for histone deacetylases concludes this review, demonstrating that benzamide derivatives represent a versatile class of compounds leading to novel imaging and therapeutic agents.

Antibody-dendrimer conjugates: the number, not the size of the dendrimers, determines the immunoreactivity.

Radioimmunotherapy using antibodies with favorable tumor targeting properties and high binding affinity is increasingly applied in cancer therapy. The potential of this valuable cancer treatment modality could be further improved by increasing the specific activity of the labeled proteins. This can be done either by coupling a large number of chelators which leads to a decreased immunoreactivity or by conjugating a small number of multimeric chelators. In order to systematically investigate the influence of conjugations on immunoreactivity with respect to size and number of the conjugates, the anti-EGFR antibody hMAb425 was reacted with PAMAM dendrimers of different size containing up to 128 chelating agents per conjugation site. An improved dendrimer synthesis protocol was established to obtain compounds of high homogeneity suitable for the formation of defined protein conjugates. The quantitative derivatization of the PAMAM dendrimers with DOTA moieties and the characterization of the products by isotopic dilution titration using (111)In/(nat)In are shown. The DOTA-containing dendrimers were conjugated with high efficiency to hMAb425 by applying Sulfo-SMCC as cross-linking agent and a 10- to 25-fold excess of the thiol-containing dendrimers. The determination of the immunoreactivities of the antibody-dendrimer conjugates by FACS analysis revealed a median retained immunoreactivity of 62.3% for 1.7 derivatization sites per antibody molecule, 55.4% for 2.8, 27.9% for 5.3, and 17.1% for 10.0 derivatization sites per antibody but no significant differences in immunoreactivity for different dendrimer sizes. These results show that the dendrimer size does not influence the immunoreactivity of the derivatized antibody significantly over a wide molecular weight range, whereas the number of derivatization sites has a crucial effect.

Improved syntheses and applicability of different DOTA building blocks for multiply derivatized scaffolds.

DOTA (1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid), which forms extremely stable complexes with a large number of metal ions, is one of the most important and most commonly used chelators for in vivo applications such as cancer diagnosis and therapy. However, many of the published synthesis protocols for DOTA derivatives are complicated and give the products in low yields. Here we report improved synthesis routes for tris-tBu-DOTA, tris-benzyl-DOTA, and thiol-DOTA, and also describe the synthesis of the novel compound tris-4-nitro-benzyl-DOTA. In addition, we determined the applicability of the DOTA derivatives tris-tBu-DOTA, thiol-DOTA, tris-benzyl-DOTA, tris-4-nitrobenzyl-DOTA, tris-allyl-DOTA, DOTA-PFP-ester, and DOTA-PNP-ester for multimerization reactions using amino functionalized PAMAM dendrimers of different sizes. Thiol-DOTA was found to be the best compound for efficient reactions with dendritic scaffolds generating highly homogeneous DOTA-multimers. This DOTA derivative could be quantitatively conjugated to a 128-mer dendrimer.

[Comparison of diagnostic accuracy of (18)F-FDG PET, (123)I-IMT- and (99m)Tc-MIBI SPECT: evaluation of tumour progression in irradiated low grade astrocytomas]. / Vergleich der diagnostischen Genauigkeit von (18)F-FDG PET, (123)I-IMT- und (99m)Tc-MIBI SPECT zur Ermittlung des Progressionsstatus bestrahlter niedriggradiger Astrozytome.

AIM: To evaluates the diagnostic accuracy of the SPECT-tracers 3-(123)I-alpha-methyl-L-tyrosine (IMT) and (99m)Tc(I)- hexakis(2-methoxyisobutylisonitrile) (MIBI) as well as the PET-tracer 2-(18)F-2-deoxyglucose (FDG) for detecting tumour progression in irradiated low grade astrocytomas (LGA). PATIENTS, METHODS: We examined 91 patients (56 males; 35 females; 44.7 +/- 11.5 years), initially suffering from histologically proven LGAs (mean WHO grade II) and treated by stereotactic radiotherapy (59.0 +/- 4.6 Gy). On average 21.9 +/- 11.2 months after radiotherapy, patients presented new Gd-DTPA enhancing lesions on MRI, which did not allow a differentiation between progressive tumour (PT) and non-PT (nPT) at this point of time. PET scans (n = 82) were acquired 45 min after injection of 208 +/- 32 MBq FDG. SPECT scans started 10 min after injection of 269 +/- 73 MBq IMT (n = 68) and 15 min after injection of 706 +/- 63 MBq MIBI (n = 34). Lesions were classified as PT and nPT based on prospective follow-up (clinically, MRI) for 17.2 +/- 9.9 months after PET/SPECT. Lesion-to-normal ratios (L/N) were calculated using contra lateraly mirrored reference regions for the SPECT examinations and reference regions in the contra lateral grey (GM) and white matter (WM) for FDG PET. Ratios were evaluated by Receiver Operating Characteristic (ROC) analysis. RESULTS: In the patient groups nPT and PT, L/N ratios for FDG (GS) were 0.6 +/- 0.3 vs. 1.2 +/- 0.5 (p = 0.003), for FDG (WS) 1.2 +/- 0.4 vs. 2.6 +/- 0.4 (p < 0.001), for IMT 1.1 +/- 0.1 vs. 1.8 +/- 0.4 (p < 0.001) and for MIBI 1.6 +/- 0.7 vs. 2.6 +/- 2.2 (p = 0.554). Areas under the non-parametric ROC-curves were: 0.738 +/- 0.059 for FDG (GS), 0.790 +/- 0.057 for FDG (WS), 0.937 +/- 0.037 for IMT and 0.564 +/- 0.105 for MIBI. CONCLUSION: MIBI-SPECT examinations resulted in a low accuracy and especially in a poor sensitivity even at modest specificity values. A satisfying diagnostic accuracy was reached with FDG PET. Using WM as reference region for FDG PET, a slightly higher AUC as compared to GM was calculated. IMT yielded the best ROC characteristics and the highest diagnostic accuracy for differentiating between PT and nPT in irradiated LGA.

Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors.

Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-XL conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.

A liposomal formulation for the oral application of the investigational hepatitis B drug Myrcludex B.

The aim of this study was the development of a liposomal formulation containing specific tetraether lipids for the oral administration of the investigational hepatitis B peptide drug Myrcludex B. For this purpose, tetraether lipids were extracted from the extremophilic archaeon Sulfolobus acidocaldarius and purified in order to obtain the desired glycerylcaldityltetraether lipids (GCTE). Myrcludex B was synthesized by solid-phase synthesis and incorporated into liposomes containing 5mol% of GCTE. These liposomes showed a size, polydispersity index and zeta potential comparable to the standard liposomes. Cryo-EM micrographs of both liposomal formulations displayed low lamellarity, the prerequisite for high drug loading capacity. Long term storage of the GCTE-liposomes was achieved by freeze-drying using 100-500mM sucrose or trehalose as lyoprotectors. The lyophilized product showed high stability with a recovery rate of 82.7±1.6% of intact Myrcludex B observed after storage for 3months at -20°C as compared to a recovery rate of 83.3±1.3% directly after the freeze-drying process. In vivo, the GCTE-liposomal formulation led to substantial enhancement of the liver uptake of iodine-131-labeled Myrcludex B in Wistar rats. 3h after oral application, approximately 7% of the initial dose (corresponding to a 3.5-fold increase compared to the free peptide) could be detected in the liver. In summary, the GCTE-liposomes enabled efficient oral administration of Myrcludex B and provided long term storage by freeze-drying.

Comparison of 68Ga-DOTATOC biodistribution in patients with and without spleenectomy.

AIM: Ga-68 labeled somatostatin analogues such as 68Ga-DOTA0-Phe1-Tyr3-octrotide (DOTATOC) as PET tracers, have significantly improved the imaging of somatostatin receptors (SSTRs) expressing tumors. Due to unspecific parenchymal binding and the expression of SSTRs on leukocytes in the spleen this is the organ with the highest non-tumor uptake of DOTATOC. Therefore, we investigated the potential changes of normal tissue distribution and tumor concentration in patients with neuroendocrine tumors (NETs) with or without spleenectomy. METHODS: Out of 420 patients with pancreatic NET undergoing 68GA-DOTATOC PET/CT eleven patients with and eleven patients without spleenectomy were derived and matched in regard to tumor histology, tumor load, age and gender. The SUV(max) of liver metastases as well as of the following normal tissues was determined: pituitary gland, thyroid gland, liver parenchyma, kidneys and suprarenal glands. RESULTS: SUV(max) values with and without spleenectomy were: in the liver metastasis (19.17 ± 6.05 versus 37.67 ± 16.31), in the thyroid gland (2.56 ± 1.33 versus 2.66 ± 0.94), in the pituitary gland (4.08 ± 1.79 versus 4.92 ± 1.93) in suprarenal glands (7.18 ± 3.33 versus 9.73 ± 3.46 on the left side and 7.32 ± 3.03 versus 11.19 ± 5.72 on the right side), in the kidneys (8.1 3 ± 4.26 on the left side and 8.11 ± 4.16 on the right side versus 8.62 ± 2.17 on the left side and 9.79 ± 2.18 on the right side) and in normal liver tissue (5.74 ± 1.55 versus 6.22 ± 1.95). The difference was statistically significant (Wilcoxon test P<0.05) in tumor lesions, adrenal and kidney tissue. CONCLUSION: Spleenectomy must be considered as a relevant factor when reporting the outcome of SSTR targeted diagnostics and therapies.

SUV of [68Ga]DOTATOC-PET/CT Predicts Response Probability of PRRT in Neuroendocrine Tumors.

PURPOSE: The goal of our study was to quantify the expression of the somatostatin receptors (SSTR2) using the maximum standardized uptake value (SUVmax) of [(68)Ga]DOTA(0)-Phe(1)-Tyr(3)-octreotide (DOTATOC) positron emission tomography (PET)-computed tomography (CT) in liver metastases of patients with neuroendocrine tumors (NETs) prior to peptide receptor radiation therapy (PRRT) and compare the initial tumor uptake with the final treatment outcome. PROCEDURES: SSTR2 expression of the 60 liver metastases in 30 NET patients was assessed at baseline and after PRRT by measuring SUVmax, tumor to spleen ratio (T/S ratio), and tumor to liver ratio (T/L ratio). Based on morphological changes and tumor size measured at baseline and follow-up contrast-enhanced CT (after three cycles of PRRT), lesions were divided into two groups by the following: (i) responding (n = 40) and (ii) non-responding (n = 20). RESULTS: Statistically significant differences were observed in the mean SUVmax for non-responding vs. responding lesions at baseline (18.00 ± 3.59 vs. 33.55 ± 4.62, p < 0.05) and for the mean T/S ratio (1.20 ± 0.37 vs. 1.90 ± 0.45, p < 0.05) and the mean T/L ratio (3.15 ± 0.53 vs. 4.97 ± 0.62, p < 0.05). Using the receiver operating characteristic curves, SUVmax was found a better metric than both T/L ratio and T/S ratio (area under the curve (AUC) of SUVmax 0.87; T/L ratio 0.78; T/S ratio 0.73) as a stratification criterion. Using a threshold value of >16.4 for SUVmax, the sensitivity and specificity in predicting responding lesions were 95 and 60 %, respectively. CONCLUSION: We propose a SUVmax cutoff of >16.4 from [(68)Ga]DOTATOC-PET-CT to select patients for PRRT. A T/L ratio >2.2 might present a scanner-independent criterion that enables the translation of our results to other institutions. However, the robustness of this arbitrary unit still needs to be evaluated with different PET scanners.

Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: structure-affinity relationships, metabolic fate, and intracellular localization.

Several radioiodinated N-(dialkylaminoalkyl)benzamides have been used for planar scintigraphy and single-photon emission computed tomography (SPECT) of melanoma metastases. In a quest for improved melanoma uptake and tissue selectivity, structure-activity studies for N-(2-diethylaminoethyl)benzamides with variation of phenyl substituents were performed using C57Bl/6 mice bearing B16 melanoma. Compounds 2 (4-amino-5-bromo-N-(2-diethylaminoethyl)-3-[(131)I]iodo-2-methoxybenz amide) and 6 (4-acetamido-N-(2-diethylaminoethyl)-5-[(131)I]iodo-2-methoxybenzamid e) showed at 6 h post iv injection, for example, melanoma uptake of 16.6 and 23.2% ID/g, respectively (mean values, n = 3). Uptake was 3-5 times higher (P < 0.01) than observed with benzamides known from the literature and was probably facilitated by the relatively slow urinary excretion of 2 or 6. In contrast, analogues lacking either the MeO, Ac, AcNH, or Br substituents exhibited reduced tumor uptake and high urinary excretion of radioactivity in various benzamide metabolites. Uptake of radioiodinated benzamides in B16 melanoma is not mediated by a specific mechanism such as sigma-receptor binding. 2 and 6 exhibited similar melanoma uptake values but quite different sigma(1)-receptor affinities of K(i) = 0.278 +/- 0.018 and 5.19 +/- 0.40 microM, respectively. Uptake studies with IMBA (N-(2-diethylaminoethyl)-3-[(131)I]iodo-4-methoxybenzamide) or BZA (N-(2-diethylaminoethyl)-4-[(131)I]iodobenzamide) showed that with increasing dose of unlabeled compound the measured uptake of label was unchanged (IMBA) or even enhanced (BZA) while receptor binding of label decreased. Differential and equilibrium density-gradient centrifugation revealed that most of the radioactivity from labeled IMBA was associated with fractions containing melanin granules. Thus, structure-activity studies indicate that blood clearance rates and metabolic stability are the main determinants for benzamide uptake in melanoma. The high uptake and slow clearance of 6 offer considerable potential for melanoma imaging in patients, and this compound may also prove to be useful for radionuclide therapy.

Investigation of a potential scintigraphic marker of apoptosis: radioiodinated Z-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone.

The imaging of apoptosis represents an attractive diagnostic goal in the area of tumor therapy, degenerative diseases and organ transplantation. Since caspases play a key role during the early period of the intracellular signal cascade of cells undergoing apoptosis we considered benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone [Z-VAD-fmk], a pan-caspase inhibitor, as a potential apoptosis imaging agent. Applying the Tl(TFA)(3)/[131I]iodide method Z-VAD-fmk was successfully labeled at the benzyloxycarbonyl protecting group. The success of radioiodination, however, depended on the presence of carrier iodide resulting in specific radioactivities of 2.6 GBq/micromol and the formation of a mixture of the 2- and 4-iodophenyl derivative (61%) which could not be separated by HPLC. Uptake measurements were performed with Morris hepatoma cells (MH3924Atk8) which showed expression of the Herpes Simplex Virus thymidine kinase (HSVtk) gene. Apoptosis was induced by treatment of the cells with 25 microM ganciclovir. The TUNEL assay revealed 1.3 +/-0.3 and 23 +/-1.1% apoptotic cells immediately and 24 h after therapy, respectively. A two-fold increase of [131I]IZ-VAD-fmk uptake was found at the end of treatment with the HSVtk/suicide system which constantly remained elevated for the following 4 hours. The slow cellular influx and lack of uptake saturation of [131I]IZ-VAD-fmk are evidence for simple diffusion as transport mechanism. In addition, the absolute cellular uptake of [131I]IZ-VAD-fmk was found to be low. This quality was related to the rather high lipophilicity of [131I]IZ-VAD-fmk causing unspecific binding to macromolecules in the medium. Instead of using an inhibitor, synthetic caspase substrates are currently investigated which may accumulate in the apoptotic cell by metabolic trapping thereby enhancing the imaging signal.

A new precursor for the radiosynthesis of [18F]FLT.

In order to improve the [18F]FLT production for nuclear medical purposes, the syntheses and labeling results obtained with six new thymidine derivatives involving an alternative protection group strategy are described. The syntheses of the FLT-labeling precursors were performed using the following protection groups at the 5'-O-position: trityl (Tr) and 4,4'-dimethoxytrityl (DMTr). Formation of an electrophilic center at the 3'-carbon was achieved with methylsulfonyl, p-toluenesulfonyl and 4-nitrobenzenesulfonyl groups. The major difference to previous accomplishments rested upon the 3-N-Boc-protection of the FLT-labeling precursors avoiding the deprotection with ceric ammonium nitrate (CAN). With CAN, a precipitate was formed which was found to interact unfavourably with synthesis automation. Here, deprotection resulted in homogeneous solutions which could immediately be loaded on HPLC. The radiosyntheses were performed with high doses of [18F]fluoride to obtain realistic results for routine production of the clinically interesting radiopharmaceutical, [18F]FLT. It was shown that the nosylated precursors were more favorable for radiofluorination than the mesylated or tosylated derivatives. A positive effect on the radiochemical yield was found with DMTr in comparison to Tr. Best results were obtained using 3-N-Boc-1-[5-O-(4,4'-dimethoxytrityl)-3-O-nosyl-2-deoxy-beta-D-lyxofuranosyl]thymine yielding 1.7 GBq (19.8% EOB) whithin 85 minutes.

Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignansand squalene.

The aim of this study was to evaluate the phenolic antioxidant and squalene content in a range of olive and seed oils. A mean of 290 +/- 38 (SEM) mg squalene/100 g was detected. However, while there was a weak significant difference between extra virgin (424 +/- 21 mg/kg) and refined virgin (340 +/- 31 mg/100 g; P<0.05) olive oils, highly significant differences were evident between extra virgin olive oils (P<0.0001) refined virgin olive oils (P<0.0001) and seed oils (24 +/- 5 mg/100 g). While seed oils were devoid, on average, the olive oils contained 196 +/- 19 mg/kg total phenolics as judged by HPLC analysis, but the value for extra virgin (232 +/- 15 mg/kg) was significantly higher than that of refined virgin olive oil (62 +/- 12 mg/kg; P<0.0001). Appreciable quantities of simple phenols (hydroxytyrosol and tyrosol) were detected in olive oils, with significant differences between extravirgin (41.87 +/- 6.17) and refined virgin olive oils (4.72 +/- 215; P<0.01). The major linked phenols were secoiridoids and lignans. Although extra virgin contained higher concentrations of secoiridoids (27.72 +/- 6.84) than refined olive oils (9.30 +/- 3.81) this difference was not significant. On the other hand, the concentration of lignans was significantly higher (P<0.001) in extra virgin (41.53 +/- 3.93) compared to refined virgin olive oils (7.29 +/- 2.56). All classes of phenolics were shown to be potent antioxidants. In future epidemiologic studies, both the nature and source of olive oil consumed should be differentiated in ascertaining cancer risk.

Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes.

Because olives represent an important component of the Mediterranean diet, it is necessary to establish unequivocal identification and quantitation of the major potential antioxidant phenolic compounds they contain. The major phenolic antioxidants in two types of brined olives were isolated and purified by semi-preparative high performance liquid chromatography. Structural analysis was conducted using UV spectrophotometry, mass spectrometry and nuclear magnetic resonance spectroscopy. In particular, completely assigned 1H and 13C NMR data are presented and errors in literature data are corrected. The data show that tyrosol, hydroxytyrosol, 3-(3, 4-dihydroxyphenyl) propanoic acid (dihydrocaffeic acid), dihydro-p-coumaric acid (phloretic acid), the phenylpropanoid glucosides acteoside (verbascoside) and isoacteoside, along with the flavonoids luteolin and apigenin are major components of the phenolic fraction of brined black olives. Brined green olives contain only hydroxytyrosol and traces of other minor phenolics. Brined olives contain even higher concentrations of phenolic antioxidants than olive oil and may, therefore, be more important modulators of cancer chemopreventive activity.

How do in-vitro release profiles of nanosuspensions from Alzet® pumps correspond to the in-vivo situation? A case study on radiolabeled fenofibrate.

In research and development sufficiently high and constant plasma levels of drug candidates are often requested, but simple solutions of hydrophobic drugs delivered from the commonly used micro-osmotic pumps cannot meet these demands. Nanosuspensions released from implanted osmotic devices can be a strategy to overcome this challenge but little is known about their pharmacokinetic behavior after subcutaneous application. In the current study, four different nanosuspension formulations containing iodinated fenofibrate were prepared, physicochemically characterized and investigated concerning their in-vitro release kinetics from osmotic pumps. One nanosuspension of lower viscosity exhibited thereby an unexpectedly first order release kinetics, whereas the higher viscous counterpart was released in the expected zero-order manner. To assess the relation of the in-vitro release kinetics to the in-vivo fate of nanosuspensions, various [(131)I] iodinated fenofibrate formulations were subcutaneously applied to mice. The biodistribution was followed by means of γ-scintigraphy and γ-scintillation. Two different nanosuspensions released from osmotic pumps were compared to bolus injections of a nanosuspension and an organic drug solution. The distribution and elimination of the bolus injected drug solution were almost completed within 48h. In contrast, a long lasting (>1week) depot at the injection site was formed by the bolus injected nanosuspension. Ex vivo examination of the organs showed a sustained, but exponential decrease of the radiolabel concentration. More constant drug levels in the organs were achieved within the nanosuspensions released from osmotic pumps. The organ levels of [(131)I] labeled fenofibrate were found to be more constant in case of the pump with the higher viscous nanosuspension in contrast to the lower viscous counterpart. However, the very different release profiles of the lower and higher viscous nanosuspension observed in-vitro were not observed in-vivo, as both pumps showed zero order release. In conclusion, nanosuspensions of poorly soluble compounds released from subcutaneously implanted osmotic pumps can be a suitable approach in pharmacokinetic studies. Although the in-vivo release of nanosuspensions differed in the expected release profile from the in-vitro test results, these in-vitro release tests present a valuable tool for the pre-selection of suitable nanosuspension candidates.