Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607

Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites.

Treatment with (90)Y- and (177)Lu-DOTATOC in patients with metastatic neuroendocrine tumors

BACKGROUND: Treatment with (90)Y- or (177)Lu-DOTATOC has recently been introduced in the palliative treatment of somatostatin receptor-expressing neuroendocrine tumors (NETs). The aim of the study was to present clinical experience with (90)Y- and (177)Lu-DOTATOC therapy in the management of NET. METHODS: To prove suitability for treatment each patient underwent scanning with (111)In-DTPAOC or (68)Ga-DOTATOC positron emission tomography/computed tomography. All patients received [(90)Y-DOTATOC] as initial treatment. In case of disease relapse the treatment was repeated. To avoid side effects of repeated [(90)Y] applications, a switch to [(177)Lu-DOTATOC] was carried out. Clinical, biochemical, and radioimaging responses were documented. RESULTS: Twenty patients with metastatic nonresectable NETs (15 pancreas NETs, 2 midgut NETs, 1 gastrinoma, 1 paraganglioma, 1 NET of unknown primary origin) were included. In 8 patients the treatment was repeated more than once (mean, 3 times; range, 2-5 times). After [(90)Y] treatment moderate toxicity was observed in 8 patients. No serious adverse events were documentable. After restaging, a partial remission was found in 5 patients, stable disease in 11 patients, and tumor progression in 4 patients. CONCLUSIONS: Peptide receptor-targeted radionuclide therapy is a promising, safe, and feasible approach in the palliative therapy of patients with NET.

Novel 111In-labelled bombesin analogues for molecular imaging of prostate tumours

PURPOSE: It has been shown that some primary human tumours and their metastases, including prostate and breast tumours, overexpress gastrin-releasing peptide (GRP) receptors. Bombesin (BN) is a neuropeptide with a high affinity for these GRP receptors. We demonstrated successful scintigraphic visualisation of BN receptor-positive tumours in preclinical studies using the radiolabelled BN analogue [(111)In-DTPA-Pro(1),Tyr(4)]BN. However, the receptor affinity as well as the serum stability of this analogue leave room for improvement. Therefore new (111)In-labelled BN analogues were synthesised and evaluated in vitro and in vivo. METHODS AND RESULTS: The receptor affinity of the new BN analogues was tested on human GRP receptor-expressing prostate tumour xenografts and rat colon sections. Analogues with high receptor affinity (low nM range) were selected for further evaluation. Incubation in vitro of GRP receptor-expressing rat CA20948 and human PC3 tumour cells with the (111)In-labelled analogues resulted in rapid receptor-mediated uptake and internalisation. The BN analogue with the best receptor affinity and in vitro internalisation characteristics, Cmp 3 ([(111)In-DTPA-ACMpip(5),Tha(6),betaAla(11),Tha(13),Nle(14)]BN(5-14)), was tested in vivo in biodistribution studies using rats bearing GRP receptor-expressing CA20948 tumours, and nude mice bearing human PC3 xenografts. Injection of (111)In-labelled Cmp 3 in these animals showed high, receptor-mediated uptake in receptor-positive organs and tumours which could be visualised using planar gamma camera and microSPECT/CT imaging. CONCLUSION: With their enhanced receptor affinity and their rapid receptor-mediated internalisation in vitro and in vivo, the new BN analogues, and especially Cmp 3, are promising candidates for use in diagnostic molecular imaging and targeted radionuclide therapy of GRP receptor-expressing cancers.

PEG spacers of different length influence the biological profile of bombesin-based radiolabeled antagonists

INTRODUCTION The gastrin-releasing peptide receptor (GRPR) was shown to be expressed with high density on several types of cancers. Radiolabeled peptides for imaging and targeted radionuclide therapy have been developed. In this study, we evaluated the potential of statine-based bombesin antagonists, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) through oligoethyleneglycol spacers, labeled with (177)Lu and we determined the effect of polyethyleneglycol (PEG) spacer length on in vitro and in vivo properties. METHODS The bombesin antagonists were synthesized on solid phase using Fmoc chemistry; the spacers Fmoc-dPEGx-OH (x=2, 4, 6 and 12) and the DOTA(tBu)3 were coupled using a standard procedure. The peptides were labeled with (177)Lu and evaluated in vitro (lipophilicity, serum stability, internalization and binding affinity assays). Biodistribution studies were performed in PC-3 tumor-bearing nude mice. RESULTS The solid-phase synthesis was straightforward with an overall yield ranging from 30% to 35% based on the first Fmoc cleavage. The hydrophilicity increased with spacer length (logD: -1.95 vs -2.22 of PEG2 and PEG12 analogs, respectively). There is a tendency of increased serum stability by increasing the spacer length (T1/2=246±4 and 584±20 for PEG2 and PEG6 analogs, respectively) which seems to reverse with the PEG12 analog. The IC50 values are similar with the only significant difference of the PEG12 analog. The (177)Lu-labeled PEG4 and PEG6 conjugates showed similar pharmacokinetic with high tumor uptake and excellent tumor-to-kidney ratios (7.8 and 9.7 at 4h for the PEG4 and PEG6 derivatives, respectively). The pancreas uptake was relatively high at 1h but it shows fast washout (0.46%±0.02% IA/g and 0.29%±0.08% IA/g already at 4h). CONCLUSION Among all the studied analogs the PEG4 and PEG6 showed significantly better properties. The very high tumor-to-non-target organ ratios, in particular tumor-to-kidney ratios, already at early time point will be important in regard to safety concerning kidney toxicity.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin

UNLABELLED The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

PET of somatostatin receptor-positive tumors using 64Cu- and 68Ga-somatostatin antagonists: the chelate makes the difference

Somatostatin-based radiolabeled peptides have been successfully introduced into the clinic for targeted imaging and radionuclide therapy of somatostatin receptor (sst)-positive tumors, especially of subtype 2 (sst2). The clinically used peptides are exclusively agonists. Recently, we showed that radiolabeled antagonists may be preferable to agonists because they showed better pharmacokinetics, including higher tumor uptake. Factors determining the performance of radioantagonists have only scarcely been studied. Here, we report on the development and evaluation of four (64)Cu or (68)Ga radioantagonists for PET of sst2-positive tumors.

Comparison of the binding and internalization properties of 12 DOTA-coupled and ¹¹¹In-labelled CCK2/gastrin receptor binding peptides: a collaborative project under COST Action BM0607

Specific overexpression of cholecystokinin 2 (CCK2)/gastrin receptors has been demonstrated in several tumours of neuroendocrine origin. In some of these cancer types, such as medullary thyroid cancer (MTC), a sensitive diagnostic modality is still unavailable and therapeutic options for inoperable lesions are needed. Peptide receptor radionuclide therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CCK2R-targeted radiopharmaceuticals have been described in recent years. As part of the European Union COST Action BM0607 we studied the in vitro and in vivo characteristics of 12 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R binding peptides. In the present study, we analysed binding and internalization characteristics. Stability, biodistribution and imaging studies have been performed in parallel by other centres involved in the project.

Alpha- versus beta-particle radiopeptide therapy in a human prostate cancer model (213Bi-DOTA-PESIN and 213Bi-AMBA versus 177Lu-DOTA-PESIN)

Recurrent prostate cancer presents a challenge to conventional treatment, particularly so to address micrometastatic and small-volume disease. Use of α-radionuclide therapy is considered as a highly effective treatment in such applications due to the shorter range and exquisite cytotoxicity of α-particles as compared with β-particles. (213)Bi is considered an α-emitter with high clinical potential, due to its short half-life (45.6 minutes) being well matched for use in peptide-receptor radionuclide α-therapy; however, there is limited knowledge available within this context of use. In this study, two novel (213)Bi-labeled peptides, DOTA-PEG(4)-bombesin (DOTA-PESIN) and DO3A-CH(2)CO-8-aminooctanoyl-Q-W-A-V-G-H-L-M-NH(2) (AMBA), were compared with (177)Lu (β-emitter)-labeled DOTA-PESIN in a human androgen-independent prostate carcinoma xenograft model (PC-3 tumor). Animals were injected with (177)Lu-DOTA-PESIN, (213)Bi-DOTA-PESIN, or (213)Bi-AMBA to determine the maximum tolerated dose (MTD), biodistribution, and dosimetry of each agent; controls were left untreated or were given nonradioactive (175)Lu-DOTA-PESIN. The MTD of (213)Bi-DOTA-PESIN and (213)Bi-AMBA was 25 MBq (0.68 mCi) whereas (177)Lu-DOTA-PESIN showed an MTD of 112 MBq (3 mCi). At these dose levels, (213)Bi-DOTA-PESIN and (213)Bi-AMBA were significantly more effective than (177)Lu-DOTA-PESIN. At the same time, (177)Lu-DOTA-PESIN showed minimal, (213)Bi-DOTA-PESIN slight, and (213)Bi-AMBA marked kidney damage 20 to 30 weeks posttreatment. These preclinical data indicate that α-therapy with (213)Bi-DOTA-PESIN or (213)Bi-AMBA is more efficacious than β-therapy. Furthermore, (213)Bi-DOTA-PESIN has a better safety profile than (213)Bi-AMBA, and represents a possible new approach for use in peptide-receptor radionuclide α-therapy treating recurrent prostate cancer.

Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL

Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.

Up-regulation of somatostatin receptor density on rat CA20948 tumors escaped from low dose [(177)Lu-DOTA(0),Tyr(3)]octreotate therapy

AIM: Peptide receptor radionuclide therapy using the somatostatin analogue [(177)Lu-DOTA(0),Tyr(3)]octreotate is a convincing treatment modality for metastasized neuroendocrine tumors. Therapeutic doses are administered in 4 cycles with 6-10 week intervals. A high somatostatin receptor density on tumor cells is a prerequisite at every administration to enable effective therapy. In this study, the density of the somatostatin receptor subtype 2 (sst2) was investigated in the rat CA20948 pancreatic tumor model after low dose [(177)Lu-DOTA(0), Tyr(3)]octreotate administration resulting in approximately 20 Gy tumor radiation absorbed dose, whereas 60 Gy is needed to induce complete tumor regression in these and the majority of tumors. METHODS: Sixteen days after inoculation of the CA20948 tumor, male Lewis rats were injected with 185 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate to initiate a decline in tumor size. Approximately 40 days after injection, tumors re-grew progressively after initial response. Quantification of sst2 expression was performed using in vitro autoradiography on frozen sections of three groups: control (not-treated) tumors, tumors in regression and tumors in re-growth. Histology and proliferation were determined using HE- and anti-Ki-67-staining. RESULTS: The sst2 expression on CA20948 tumor cells decreased significantly after therapy to 5% of control level. However, tumors escaping from therapy showed an up-regulated sst2 level of 2-5 times higher sst2 density compared to control tumors. CONCLUSION: After a suboptimal therapeutic dose of [(177)Lu-DOTA(0),Tyr(3)]octreotate, escape of tumors is likely to occur. Since these cells show an up-regulated sst2 receptor density, a next therapeutic administration of radiolabelled sst2 analogue can be expected to be highly effective.

Towards tailored radiopeptide therapy.

PURPOSE Somatostatin receptor-targeted radiopeptide therapy is commonly performed using single radioisotopes. We evaluated the benefits and harms of combining radioisotopes in radiopeptide therapy in patients with neuroendocrine tumor. METHODS Using multivariable-adjusted survival analyses and competing risk analyses we evaluated outcomes in patients with neuroendocrine tumor receiving (90)Y-DOTATOC, (177)Lu-DOTATOC or their combination. RESULTS (90)Y-DOTATOC plus (177)Lu-DOTATOC treatment was associated with longer survival than (90)Y-DOTATOC (66.1 vs. 47.5 months; n = 1,358; p < 0.001) or (177)Lu-DOTATOC alone (66.1 vs. 45.5 months; n = 390; p < 0.001). (177)Lu-DOTATOC was associated with longer survival than (90)Y-DOTATOC in patients with solitary lesions (HR 0.3, range 0.1 - 0.7; n = 153; p = 0.005), extrahepatic metastases (HR 0.5, range 0.3 - 0.9; n = 256; p = 0.029) and metastases with low uptake (HR 0.1, range 0.05 - 0.4; n = 113; p = 0.001). (90)Y-DOTATOC induced higher hematotoxicity rates than combined treatment (9.5% vs. 4.0%, p = 0.005) or (177)Lu-DOTATOC (9.5 vs. 1.4%, p = 0.002). Renal toxicity was similar among the treatments. CONCLUSIONS Using (90)Y and (177)Lu might facilitate tailoring radiopeptide therapy and improve survival in patients with neuroendocrine tumors.

Comparative biodistribution of 12 ¹¹¹In-labelled gastrin/CCK2 receptor-targeting peptides

Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12 (111)In-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides.

Hormographiella aspergillata keratomycosis in a dog

A 4-year-old, female, Border Collie was presented to the University of Bern Veterinary Teaching Hospital, because of a corneal lesion of 10 days duration. The axial cornea presented a whitish fluorescein-positive plaque with irregular margins. A diagnosis of keratomycosis was made based on cytology. Medical therapy with local broad-spectrum antibiotic and fluconazole was instituted. After 1 week of treatment, the improvement was deemed unsatisfactory. Therefore, a lamellar keratectomy and conjunctival pedicle flap were performed. After surgery, the cornea healed uneventfully. Histology confirmed the diagnosis of keratomycosis. The fungus could not be grown in culture and a precise etiological diagnosis could only be obtained with genetic identification of the fungus. A PCR technique was used to amplify the fungal genome from the cornea. Hormographiella aspergillata, the asexual reproductive form of the basidiomycete Coprinopsis cinerea, was identified. As advised in human medicine, we encourage the use of this molecular technique to obtain an early species diagnosis, allowing targeted medical therapy.

Promises of cyclotron-produced 44Sc as a diagnostic match for trivalent β--emitters: in vitro and in vivo study of a 44Sc-DOTA-folate conjugate

In recent years, implementation of 68Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of 44Sc (half-life = 3.97 h, average β+ energy [Eβ+av] = 632 keV) as a valuable alternative to 68Ga (half-life = 68 min, Eβ+av = 830 keV) for imaging and dosimetry before 177Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced 44Sc and its in vitro and in vivo comparison with the 177Lu-labeled pendant. Methods: 44Sc was produced via the 44Ca(p,n)44Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 μA, 30 min) using an enriched 44Ca target (10 mg 44CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of 44Sc-cm09 was tested in human plasma. 44Sc-cm09 was investigated in vitro using folate receptor–positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice Results: Under the given irradiation conditions, 44Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of 44Sc from 44Ca targets allowed formulation of up to 300 MBq of 44Sc in a volume of 200–400 μL of ammonium acetate/HCl solution (1 M, pH 3.5–4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, 44Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor–specific binding to KB tumor cells. PET/CT images of mice injected with 44Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with 44Sc and 177Lu revealed almost identical pharmacokinetics. Conclusion: This study presents a high-yield production and efficient separation method of 44Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of 44Sc for PET imaging. Thus, 44Sc is a valid alternative to 68Ga for imaging and dosimetry before 177Lu-radionuclide tumor therapy.

Differential uptake of (68)Ga-DOTATOC and (68)Ga-DOTATATE in PET/CT of gastroenteropancreatic neuroendocrine tumors

PURPOSE Abundant expression of somatostatin receptors (sst) is a characteristic of neuroendocrine tumors (NET). Thus, radiolabeled somatostatin analogs have emerged as important tools for both in vivo diagnosis and therapy of NET. The two compounds most often used in functional imaging with positron emission tomography (PET) are (68)Ga-DOTATATE and (68)Ga-DOTATOC. Both analogs share a quite similar sst binding profile. However, the in vitro affinity of (68)Ga-DOTATATE in binding the sst subtype 2 (sst2) is approximately tenfold higher than that of (68)Ga-DOTATOC. This difference may affect their efficiency in detection of NET lesions, as sst2 is the predominant receptor subtype on gastroenteropancreatic NET. We thus compared the diagnostic value of PET/CT with both radiolabeled somatostatin analogs ((68)Ga-DOTATATE and (68)Ga-DOTATOC) in the same patients with gastroenteropancreatic NET. PATIENTS AND METHODS Twenty-seven patients with metastatic gastroenteropancreatic NET underwent (68)Ga-DOTATOC and (68)Ga-DOTATATE PET/CT as part of the workup before prospective peptide receptor radionuclide therapy (PRRT). The performance of both imaging methods was analyzed and compared for detection of individual lesions per patient and for eight defined body regions. A region was regarded as positive if at least one lesion was detected in that region. In addition, radiopeptide uptake in terms of the maximal standardized uptake value (SUV(max)) was compared for concordant lesions and renal parenchyma. RESULTS Fifty-one regions were found positive with both (68)Ga-DOTATATE and (68)Ga-DOTATOC. Overall, however, significantly fewer lesions were detected with (68)Ga-DOTATATE in comparison with (68)Ga-DOTATOC (174 versus 179, p < 0.05). Mean (68)Ga-DOTATATE SUV(max) across all lesions was significantly lower compared with (68)Ga-DOTATOC (16.9 ± 6.8 versus 22.1 ± 12.0, p < 0.01). Mean SUV(max) for renal parenchyma was not significantly different between (68)Ga-DOTATATE and (68)Ga-DOTATOC (12.6 ± 2.6 versus 12.6 ± 2.7). CONCLUSIONS (68)Ga-DOTATOC and (68)Ga-DOTATATE possess similar diagnostic accuracy for detection of gastroenteropancreatic NET lesions (with a potential advantage of (68)Ga-DOTATOC) despite their evident difference in affinity for sst2. Quite unexpectedly, maximal uptake of (68)Ga-DOTATOC tended to be higher than its (68)Ga-DOTATATE counterpart. However, tumor uptake shows high inter- and intraindividual variance with unpredictable preference of one radiopeptide. Thus, our data encourage the application of different sst ligands to enable personalized imaging and therapy of gastroenteropancreatic NET with optimal targeting of tumor receptors.