Renal uptake of different radiolabelled peptides is mediated by megalin: SPECT and biodistribution studies in megalin-deficient mice.

PURPOSE: Radiolabelled peptides used for peptide receptor radionuclide therapy are excreted mainly via the kidneys and are partly reabsorbed and retained in the proximal tubular cells. The resulting high renal radiation dose can cause nephrotoxicity, limiting the maximum activity dose and the effectiveness of peptide receptor radionuclide therapy. The mechanisms of kidney reabsorption of these peptides are incompletely understood, but the scavenger receptor megalin has been shown to play a role in the reabsorption of (111)In-octreotide. In this study, the role of megalin in the renal reabsorption of various relevant radiolabelled peptides was investigated. METHODS: Groups of kidney-specific megalin-deficient mice and wild-type mice were injected with (111)In-labelled somatostatin, exendin, neurotensin or minigastrin analogues. Single photon emission computed tomographic (SPECT) images of the kidneys were acquired and analysed quantitatively, or the animals were killed 3 h after injection and the activity concentration in the kidneys was measured. RESULTS: Megalin-deficient mice showed significantly lower uptake of all studied radiolabelled peptides in the kidneys, ranging from 22% ((111)In-octreotide) to 65% ((111)In-exendin) of uptake in wild-type kidneys. Quantitative analysis of renal uptake by SPECT and ex vivo measurements showed a very good correlation. CONCLUSION: Megalin is involved in the renal reabsorption of radiolabelled octreotide, octreotate, exendin, neurotensin and minigastrin. This knowledge may help in the design of strategies to reduce this reabsorption and the resulting nephrotoxicity in peptide receptor radionuclide therapy, enabling more effective therapy. Small-animal SPECT is an accurate tool, allowing in vivo quantification of renal uptake and serial measurements in individual mice.

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.

PURPOSE: 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. METHODS: Determination of IC(50) values was performed using autoradiography, with DOTA-peptides displacing (125)I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using (111)In-labelled peptides. The rat AR42J cell line and the human A431-CCK2R transfected cell line were utilized for in vitro experiments; dissociation constants (K(d)) and apparent number of binding sites (B(max)) were determined. Internalization was determined in receptor-expressing cells by incubating with tracer amounts of peptide at 37 and 4°C for different times up to 120 min. Surface-bound peptide was then stripped either by acid wash or subsequent incubation with 1 µM unlabelled peptide at 4°C. RESULTS: All peptides showed high receptor affinity with IC(50) values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with K(d) values in the 10(-9)-10(-8) M range. B(max) values estimated in A431-CCK2R cells ranged from 0.6 to 2.2 × 10(6) per cell. All peptides showed high levels of internalization when incubated at 37°C. CONCLUSION: All DOTA-conjugated peptides showed high receptor binding and internalization properties and appear suitable for further characterization, as described in other articles of this issue.

Androgen-regulated gastrin-releasing peptide receptor expression in androgen-dependent human prostate tumor xenografts.

Human prostate cancer (PC) overexpresses the gastrin-releasing peptide receptor (GRPR). Radiolabeled GRPR-targeting analogs of bombesin (BN) have successfully been introduced as potential tracers for visualization and treatment of GRPR-overexpressing tumors. A previous study showed GRPR-mediated binding of radiolabeled BN analogs in androgen-dependent but not in androgen-independent xenografts representing the more advanced stages of PC. We have further investigated the effect of androgen modulation on GRPR-expression in three androgen-dependent human PC-bearing xenografts: PC295, PC310 and PC82 using the androgen-independent PC3-model as a reference. Effects of androgen regulation on GRPR expression were initially studied on tumors obtained from our biorepository of xenograft tissues performing reverse transcriptase polymerase chain reaction (RT-PCR) and autoradiography ((125)I-universal-BN). A prospective biodistribution study ((111)In-MP2653) and subsequent autoradiography ((125)I-GRP and (111)In-MP2248) was than performed in castrated and testosterone resupplemented tumor-bearing mice. For all androgen-dependent xenografts, tumor uptake and binding decreased drastically after 7 days of castration. Resupplementation of testosterone to castrated animals restored GRPR expression extensively. Similar findings were concluded from the initial autoradiography and RT-PCR studies. Results from RT-PCR, for which human specific primers are used, indicate that variations in GRPR expression can be ascribed to mRNA downregulation and not to castration-induced reduction in the epithelial fraction of the xenograft tumor tissue. In conclusion, expression of human GRPR in androgen-dependent PC xenografts is reduced by androgen ablation and is reversed by restoring the hormonal status of the animals. This knowledge suggests that hormonal therapy may affect GRPR expression in PC tissue making GRPR-based imaging and therapy especially suitable for non-hormonally treated PC patients.

Kidney protection during peptide receptor radionuclide therapy with somatostatin analogues.

This review focuses on the present status of kidney protection during peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues. This treatment modality for somatostatin receptor-positive tumours is limited by renal reabsorption and retention of radiolabelled peptides resulting in dose-limiting high kidney radiation doses. Radiation nephropathy has been described in several patients. Studies on the mechanism and localization demonstrate that renal uptake of radiolabelled somatostatin analogues largely depends on the megalin/cubulin system in the proximal tubule cells. Thus methods are needed that interfere with this reabsorption pathway to achieve kidney protection. Such methods include coadministration of basic amino acids, the bovine gelatin-containing solution Gelofusine or albumin fragments. Amino acids are already commonly used in the clinical setting during PRRT. Other compounds that interfere with renal reabsorption capacity (maleic acid and colchicine) are not suitable for clinical use because of potential toxicity. The safe limit for the renal radiation dose during PRRT is not exactly known. Dosimetry studies applying the principle of the biological equivalent dose (correcting for the effect of dose fractionation) suggest that a dose of about 37 Gy is the threshold for development of kidney toxicity. This threshold is lower when risk factors for development of renal damage exist: age over 60 years, hypertension, diabetes mellitus and previous chemotherapy. A still experimental pathway for kidney protection is mitigation of radiation effects, possibly achievable by cotreatment with amifostine (Ethylol), a radiation protector, or with blockers of the renin-angiotensin-aldosterone system. Future perspectives on improving kidney protection during PRRT include combinations of agents to reduce renal retention of radiolabelled peptides, eventually together with mitigating medicines. Moreover, new somatostatin analogues with lower renal retention may be developed. Furthermore, knowledge on kidney protection from radiolabelled somatostatin analogues may be expanded to other peptides.

A standardised study to compare prostate cancer targeting efficacy of five radiolabelled bombesin analogues.

PURPOSE: Prostate-specific antigen (PSA)-based screening for prostate cancer (PC) has dramatically increased early diagnosis. Current imaging techniques are not optimal to stage early PC adequately. A promising alternative to PC imaging is peptide-based scintigraphy using radiolabelled bombesin (BN) analogues that bind to gastrin-releasing peptide receptors (GRPR) being overexpressed in PC. When labelled to appropriate radionuclides BN targeting of GRPRs may also provide applications for peptide radionuclide receptor therapy (PRRT). Assessment studies under identical experimental conditions allowing a reliable comparison of the potential of such analogues are lacking. This study was performed to evaluate and directly compare five promising radiolabelled BN analogues for their targeting efficacy for PC under standardised conditions. METHODS: The BN agonists [(111)In]DOTA-PESIN, [(111)In]AMBA, [(111)In]MP2346 and [(111)In]MP2653 and one antagonist [(99m)Tc]Demobesin-1 were evaluated in GRPR-overexpressing human PC-3 tumour-bearing mice to determine peptide stability in vivo, biodistribution and GRPR targeting potential by animal SPECT/CT imaging and ex vivo autoradiography. RESULTS: HPLC analysis of blood showed intact Demobesin-1 at 5 and 15 min after injection (64.1 +/- 1.6% and 41.0 +/- 01%, respectively) being much less for the other compounds. AMBA, the second most stable analogue, showed 36.1 +/- 2.7% and 9.8 +/- 1.1% intact peptide after 5 and 15 min. PC-3 tumour uptake at 1 h was comparable for Demobesin-1, AMBA, PESIN and MP2346 (3.0 +/- 0.4, 2.7 +/- 0.5, 2.3 +/- 0.5 and 2.1 +/- 0.9%ID/g, respectively), but very low for MP2653 (0.9 +/- 0.2%ID/g). In addition, MP2346 showed undesirably high uptake in the kidneys (7.9 +/- 1.9%ID/g) being significantly less for the other analogues. AMBA, MP2346 and PESIN revealed favourable increases in tumour to blood ratios over time while changes in tumour to kidney and pancreas ratios for Demobesin-1 from 1 to 24 h after injection were significantly better than for the other analogues. All analogues visualised PC-3 tumours by SPECT/CT and autoradiography. CONCLUSION: In the present study the BN antagonist Demobesin-1 was the best performing analogue showing superior in vivo stability, highest tumour uptake and retention while pancreatic and renal clearance were rapid. PESIN and AMBA were the best GRP agonists with sufficient in vivo stabilities as well as high tumour uptake and retention. Based on these results all three analogues deserve further evaluation for clinical use in PC patients.

Dose-response effect of Gelofusine on renal uptake and retention of radiolabelled octreotate in rats with CA20948 tumours.

PURPOSE: Peptide receptor radionuclide therapy using ß-emitting radiolabelled somatostatin analogues like DOTA,Tyr3-octreotate shows beneficial results in patients suffering from somatostatin receptor overexpressing tumours. However, after high-dose therapy partial renal reabsorption of radiopeptides may lead to nephrotoxicity. Co-infusion of lysine/arginine lowers renal retention of these radiopeptides without affecting tumour uptake. Recently co-administration of Gelofusine has been described to have a comparable kidney-protecting effect in rats. In the present study optimal dosing of Gelofusine co-administration was studied in tumour-bearing rats. METHODS: Doses of 40, 80, 120 or 160 mg/kg Gelofusine were co-injected with 15 µg DOTA,Tyr3-octreotate, labelled with 3 MBq 111In for biodistribution (24 h post-injection, n = 4 per group) and with 60 MBq 111In for microSPECT imaging experiments at 3, 24 and 48 h post-injection. An additional group of rats received 80 mg/kg Gelofusine plus 400 mg/kg lysine co-injection. Biodistribution studies were performed both in older (475 g) and younger (300 g) rats, the latter bearing CA20948 tumours. RESULTS: Co-injection of 40 mg/kg Gelofusine resulted in 40-50% reduction of renal uptake and retention of 111In-DOTA,Tyr3-octreotate, whereas higher doses further increased the reduction to 50-60% in both groups of rats. Combining Gelofusine and lysine caused 70% reduction of renal uptake. The uptake of radiolabelled octreotate both in somatostatin receptor-expressing normal tissues and tumours was not affected by Gelofusine co-injection. CONCLUSION: In rats co-injection of 80 mg/kg Gelofusine resulted in maximum reduction of renal retention of 111In-DOTA,Tyr3-octreotate, which was further improved when combined with lysine. Tumour uptake of radiolabelled octreotate was not affected, resulting in an increased tumour to kidney ratio.

"Click-to-chelate": in vitro and in vivo comparison of a 99mTc(CO)3-labeled N(tau)-histidine folate derivative with its isostructural, clicked 1,2,3-triazole analogue.

A side-by-side comparison of the synthesis, radiolabeling, and in vitro and in vivo characterization of two new and isostructural (99m)Tc-tricarbonyl folic acid radiotracers comprising either a N(tau)-functionalized histidine (His) chelator or a 1,4-bifunctionalized 1,2,3-triazole His analogue is described. The 1,2,3-triazole-containing folic acid derivative was synthesized in approximately 80% yield by a short reaction sequence including application of click chemistry (the Cu(I)-catalyzed cycloaddition of azides and terminal alkynes). The synthesis of the ligand system and the functionalization of the folic acid derivative were accomplished simultaneously, which prompted us to call this approach "click-to-chelate". In comparison, the reported regioselective synthesis of the N(tau)-His compound provided the final product in only very low yields (<1%). While the efficiency of the syntheses differs considerably, the two isostructural folate derivatives exhibit virtually identical properties with respect to Tc-99m radiolabeling and in vitro and in vivo characteristics as shown by experiments performed with FR-positive KB cells and xenografted mice bearing folate receptor overexpressing tumors. We have demonstrated herein for the first time that a ligand system known to be an excellent chelator for the stable complexation of the organometallic core [M(CO)3] (+) (M = Tc-99m, Re) can be replaced by an isostructural 1,2,3-triazole analogue without influencing the characteristics of the radiometal conjugate. The "click-to-chelate" strategy provides a highly efficient and convenient entry to metal conjugates suitable for diagnostic and potentially therapeutic applications. The described procedures should be readily applicable to any azide-functionalized (bio)molecule and, thus, are likely to represent the method of choice for the future development of radiopharmaceuticals radiolabeled with the organometallic precursors [M(CO)3(H2O)3] (+) (M = (99m)Tc, (188)Re).

Expression of the SST receptor 2 in uveal melanoma is not a prognostic marker.

INTRODUCTION: Uveal melanoma (UM) cells and neurohormone-producing cells both originate from the neural crest. Somatostatin receptors subtype 2 (SSTR2) are over-expressed in several tumors, often from neuroendocrine origin, and synthetic antagonists like octreotide and octreotate are being used as diagnostic or therapeutic agents. We investigated the SSTR2 expression in UM, and determined whether this expression was related to prognosis of the disease. MATERIALS AND METHODS: UM cell lines and fresh primary UM samples were tested for SSTR2 expression by autoradiography (AR) using 125I-Tyr3-octreotate. Furthermore, UM cell lines were analyzed for SSTR2 mRNA expression with quantitative real-time RT-PCR. RESULTS: Using AR, cell-surface SSTR2 expression was demonstrated in two UM metastatic cell lines, but no expression was detected in three cell lines derived from primary UM. However, all primary and metastatic UM cell lines showed mRNA expression levels for SSTR2 using quantitative real-time RT-PCR. Only three of 14 primary UM demonstrated moderate SSTR2 expression, and this expression was not significantly associated with tumor-free survival or any tested prognostic factor. CONCLUSIONS: Based on the rare and low expression of SSTR2 found in primary UM specimens and in UM cell lines, we conclude that SSTR2 is not widely expressed in UM. Furthermore, SSTR2 expression was not associated with tumor-free survival and prognostic factors. Therefore SSTR2 is not suited as prognostic marker or therapeutic target in UM.

Radiation dose distribution in human kidneys by octreotides in peptide receptor radionuclide therapy.

UNLABELLED: Ex vivo autoradiographs of healthy kidney tissue from patients who received (111)In-DTPA-octreotide (DTPA is diethylenetriaminepentaacetic acid) before nephrectomy showed very heterogeneous radioactivity patterns in the kidneys. The consequences of the reported inhomogeneities have been evaluated for radionuclide therapy with (90)Y- DOTA-Tyr(3)-octreotide (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), (177)Lu-DOTA-Tyr(3)-octreotate, and (111)In-DTPA-octreotide by calculating dose distributions and dose-volume histograms (DVHs) for the kidneys. METHODS: Monte Carlo radiation transport calculations were performed by using the MCNP code version 5. The autoradiography data were used in a 2-dimensional model of the kidney tissue sections. A voxel structure inside the MIRD Pamphlet 19 multiregion kidney model was developed to generate a 3-dimensional representation of the autoradiographs. Dose distributions were calculated for the beta-emitter (90)Y, the low-energy electron and gamma-emitter (111)In, and the beta- and gamma-emitter (177)Lu. Isodose curves were generated for the 2-dimensional kidney sections and DVHs for the 3-dimensional kidney model. RESULTS: The isodose curves for the high-energy beta-emitter (90)Y did not show a sign of the inhomogeneous activity distribution, apart from the cortex-medulla boundaries. Both (111)In and (177)Lu isodose curves follow the autoradiographic activity distribution exactly. The 2 gamma-rays from (111)In give higher doses to the low-radioactivity regions in the kidney sections. The DVHs show that the inhomogeneous activity distribution creates considerable volumes within the kidney and within the cortex with lower doses than the average kidney dose, together with volumes receiving much higher doses. This effect is most profound for (177)Lu, but also (111)In shows this heterogeneity in the dose distribution. CONCLUSION: Kidney dosimetry for radionuclide therapy can be based on average MIRD-based dose models for high-energy beta-emitters (such as (90)Y). In contrast, low-energy beta-emitters (such as (177)Lu) and Auger-electron-emitting radionuclides (such as (111)In) produce dose distributions in the kidneys that are very dependent on the activity distribution pattern in the kidney or renal cortex. Complication probability models for renal tissue damage after radionuclide therapy with these latter nuclides need to be developed, as the existing models based on average dose to the kidney or cortex from external beam therapy experience are most probably not valid.

Androgen-dependent expression of the gastrin-releasing peptide receptor in human prostate tumor xenografts.

UNLABELLED: Human prostate cancers (PC) overexpress gastrin-releasing peptide (GRP) receptors. This observation suggests that GRP receptors may be used as new visualization and treatment modalities for these tumors. Radiolabeled GRP receptor-targeting analogs of GRP and bombesin (BN) have successfully been developed for these purposes. Expression of GRP receptors in human prostate tumors is, however, primarily evaluated in early stages of tumor development and information on expression in the more progressive prostate tumors is uncertain. To evaluate GRP receptor expression in all stages of PC, we investigated GRP receptor expression using a panel of 12 established human PC xenograft models representing the different stages of human PC and the effect of antiandrogen treatment (castration). METHODS: Human PC xenografts were grown in male nude mice, and GRP receptor density in the tumors was evaluated using displacement receptor autoradiography with the universal BN receptor analog (125)I-[D-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14) and the BN analog (111)In-[DTPA-Pro(1),Tyr(4)]BN (DTPA is diethylenetriaminepentaacetic acid) before and after castration. RESULTS: Autoradiography showed high-density GRP receptor expression in the androgen-dependent tumors (3/12 models), whereas only very low receptor expression was found in the androgen-responsive and -independent tumors (9/12 models). Castration resulted in GRP receptor downregulation (11%-36% of initial values) in the 3 androgen-dependent tumors. CONCLUSION: High GRP receptor density was only observed in androgen-dependent PC xenografts, indicating high GRP receptor expression in the early, androgen-dependent, stages of prostate tumor development and not in later stages. In addition, castration strongly reduced GRP receptor expression in androgen-dependent tumors, indicating that GRP receptor expression in human PC is androgen-regulated.

Renal uptake and retention of radiolabeled somatostatin, bombesin, neurotensin, minigastrin and CCK analogues: species and gender differences.

INTRODUCTION: During therapy with radiolabeled peptides, the kidney is most often the critical organ. Newly developed peptides are evaluated preclinically in different animal models before their application in humans. In this study, the renal retention of several radiolabeled peptides was compared in male and female rats and mice. METHODS: After intravenous injection of radiolabeled peptides [somatostatin, cholecystokinin (CCK), minigastrin, bombesin and neurotensin analogues], renal uptake was determined in both male and female Lewis rats and C57Bl mice. In addition, ex vivo autoradiography of renal sections was performed to localize accumulated radioactivity. RESULTS: An equal distribution pattern of renal radioactivity was found for all peptides: high accumulation in the cortex, lower accumulation in the outer medulla and no radioactivity in the inner medulla of the kidneys. In both male rats and mice, an increasing renal uptake was found: [(111)In-DTPA]CCK8<[(111)In-DTPA-Pro(1),Tyr(4)]bombesin approximately [(111)In-DTPA]neurotensin<[(111)In-DTPA]octreotide<<[(111)In-DTPA]MG0. Renal uptake of [(111)In-DTPA]octreotide in rats showed no gender difference, and renal radioactivity was about constant over time. In mice, however, renal uptake in females was significantly higher than that in males and decreased rapidly over time in both genders. Moreover, renal radioactivity in female mice injected with [(111)In-DTPA]octreotide showed a different localization pattern. CONCLUSIONS: Regarding the renal uptake of different radiolabeled peptides, both species showed the same ranking order. Similar to findings in patients, rats showed comparable and constant renal retention of radioactivity in both genders, in contrast to mice. Therefore, rats appear to be the more favorable species for the study of the renal retention of radioactivity.

From outside to inside? Dose-dependent renal tubular damage after high-dose peptide receptor radionuclide therapy in rats measured with in vivo (99m)Tc-DMSA-SPECT and molecular imaging.

UNLABELLED: In peptide receptor radionuclide therapy (PRRT), the dose-limiting organ is, most often, the kidney. However, the precise mechanism as well as the exact localization of kidney damage during PRRT have not been fully elucidated. We studied renal damage in rats after therapy with different amounts of [(177)Lu-DOTA(0), Tyr(3)]octreotate and investigated (99m)Tc-DMSA (dimercaptosuccinic acid) as a tool to quantify renal damage after PRRT. EXPERIMENTAL DESIGN: Twenty-nine (29) rats were divided into 3 groups and injected with either 0, 278, or 555 MBq [(177)Lu-DOTA(0), Tyr(3) ]octreotate, leading to approximately 0, 46, and 92 Gy to the renal cortex. More than 100 days after therapy, kidney damage was investigated using (99m)Tc-DMSA single-photon emission computed tomography (SPECT) autoradiography, histology, and blood analyses. RESULTS: In vivo SPECT with (99m)Tc-DMSA resulted in high-resolution (<1.6-mm) images. The (99m)Tc-DMSA uptake in the rat kidneys was inversely related with the earlier injected activity of [(177)Lu-DOTA(0), Tyr(3)]octreotate and correlated inversely with serum creatinine values. Renal ex vivo autoradiograms showed a dose-dependent distribution pattern of (99m)Tc-DMSA. (99m)Tc-DMSA SPECT could distinguish between the rats that were injected with 278 or 555 MBq [(177)Lu-DOTA(0), Tyr(3) ]octreotate, whereas histologic damage grading of the kidneys was nearly identical for these 2 groups. Histologic analyses indicated that lower amounts of injected radioactivity caused damage mainly in the proximal tubules, whereas as well the distal tubules were damaged after high-dose radioactivity. CONCLUSIONS: Renal damage in rats after PRRT appeared to start in a dose-dependent manner in the proximal tubules and continued to the more distal tubules with increasing amounts of injected activity. In vivo SPECT measurement of (99m)Tc-DMSA uptake was highly accurate to grade renal tubular damage after PRRT.

Diagnostic versus therapeutic doses of [(177)Lu-DOTA-Tyr(3)]-octreotate: uptake and dosimetry in somatostatin receptor-positive tumors and normal organs.

AIM: The aim of this study was to investigate the influence of a diagnostic versus therapeutic dose of [(177)Lu-DOTA-Tyr(3)]octreotate on the uptake, effects, and dosimetry in somatostatin receptor subtype 2(sst2)-positive tumors and normal organs in a rat tumor model. MATERIALS AND METHODS: Lewis rats bearing rat pancreatic CA20948-tumor grafts were injected intravenously with different amounts of radioactivity and peptide of [(177)Lu-DOTA-Tyr(3)]octreotate: 3 MBq/0.5 microg (group A), 3 MBq/15 microg (group B), 300 MBq/15 microg (group C), and 555 MBq/15 microg (group D). Biodistribution studies were performed at several time points between 3 and 13 days post injection. Ex vivo and in vitro autoradiography was performed with frozen tumor sections. RESULTS: Normal sst2-positive tissues showed a significantly higher uptake of radioactivity [%IA/g] when a low peptide amount was injected. On the other hand, the radioactivity concentration [%IA/g] in sst2-negative tissues and organs (blood, muscles, kidney, and liver) were comparable (groups A and B), independent of the injected peptide amount. Initially, this held true for the tumors as well. Yet, over time, we found a decrease in the radioactivity concentration in the tumors of groups A and B, because of tumor growth. On the other hand, therapeutic amounts of radioactivity (groups C and D) resulted in a significant reduction of tumor size, where radioactivity concentration remained higher than in groups A and B, despite the use of the high peptide amounts. Ex vivo autoradiograms of tumor sections confirmed these results. In vitro autoradiography performed on adjacent tumor sections revealed a reduced density of sst2 in tumors from animals that received a therapeutic radioactivity dose. Ki67-antibody immunohistochemistry showed an absence of proliferating tumor cells after therapy. CONCLUSIONS: The differences in radioactivity retention in tumors after diagnostic or therapeutic doses, depending on a change in tumor kinetics, have to be taken into account when calculating tumor-absorbed radiation doses.

Breast cancer imaging using radiolabelled somatostatin analogues.

Imaging and therapy using radiolabelled somatostatin analogues are methods successfully used in patients with somatostatin receptor (SSTR)-expressing neuroendocrine tumours. Since these techniques were first introduced, many improvements have been made. SSTR expression has also been reported on breast cancer (BC). Currently mammography, magnetic resonance imaging and ultrasound are the most frequent methods used for BC imaging. Since SSTR expression on BC was demonstrated, clinical studies examining the feasibility of visualizing primary BC using SSTR radioligands have been performed. However, to date SSTR-mediated nuclear imaging is not used clinically in BC patients. The aim of this review is to assess whether recent improvements made within nuclear medicine may enable SSTR-mediated imaging to play a role in BC management. For this we critically analysed results of past studies and discussed the potential of the improvements made within nuclear medicine on SSTR-mediated nuclear imaging of BC. Seven databases were searched for publications on BC imaging with SSTR radioligands. The papers found were analysed by 3 individual observers to identify whether the studies met the pre-set inclusion criteria defined as studies in which nuclear imaging using radiolabelled SST analogues was performed in patients with breast lesions. Twenty-four papers were selected for this review including studies on SSTR-mediated nuclear imaging in BC, neuroendocrine BC and other breast lesions. The analysed studies were heterogeneous with respect to the imaging method, imaging protocol, patient groups and the radiolabelled SST analogues used. Despite the fact that the analysed studies were heterogeneous, sensitivity for primary BC ranged from 36-100%. In a subset of the studies LN lesions were visualized, but sensitivity was lower compared to that for primary tumours. A part of the studies included benign lesions and specificity ranged from 22-100%. Furthermore, false negatives and false positives were reported. In the majority of the studies scan outcome was not associated with BC subtype.

Localization of Radiolabeled Somatostatin Analogs in the Spleen.

Radiolabeled somatostatin (SST) analogs, used to visualize and treat SST receptor (SSTR)-expressing neuroendocrine tumors, also accumulate in the spleen. There is a high interpatient variation and no significant radiation-induced splenic toxicity; however, an absorbed dose-related reduction in spleen size was detected. However, the exact localization of radioactivity and the role of SST receptors in splenic retention are unknown. Therefore, we performed ex vivo micro-SPECT of the isolated spleen from a patient with a pancreatic neoplasm after 1 GBq (27 mCi) Lu-DOTATATE administration, followed by autoradiography and immunohistochemistry. Ex vivo autoradiography demonstrated convincingly that most radioactivity accumulated in red pulp.

Megalin is essential for renal proximal tubule reabsorption of (111)In-DTPA-octreotide.

UNLABELLED: Radiolabeled somatostatin analogs have been shown to be important radiopharmaceuticals for tumor diagnosis and radionuclide therapy. The kidney has appeared to be the critical organ during radionuclide therapy because of peptide reabsorption and retention in the proximal tubules after glomerular filtration. The molecular mechanism of renal reabsorption of these analogs has not been clarified. A possible receptor candidate is megalin, a multiligand scavenger receptor in the renal proximal tubules. The objective of this study was to investigate the role of megalin in tubular reabsorption of radiolabeled somatostatin analogs by using kidney-specific megalin-deficient mice versus mice with normal renal megalin expression. [(111)In-Diethylenetriaminepentaacetic acid (DTPA)]octreotide was used as a practical model of peptide. METHODS: Renal uptake of [(111)In-DTPA]octreotide was determined by animal SPECT scintigraphy at different time points after injection of the tracer and by measurement of radioactivity after isolation of the organs. Furthermore, ex vivo autoradiography of renal sections revealed the zonal distribution of radioactivity in the megalin-deficient and megalin-expressing kidneys. RESULTS: SPECT scintigraphy of [(111)In-DTPA]octreotide at 3 and 24 h after injection clearly showed lower renal radioactivity in megalin-deficient kidneys than in megalin-expressing kidneys, both in male and in female mice, in accordance with counts obtained after isolation of the organ (70%-85% reduction of uptake in the megalin-deficient kidneys, P < 0.001). Renal uptake of [(111)In-DTPA]octreotide was significantly higher in female than in male kidneys (P < 0.001). Ex vivo autoradiograms clearly showed that renal radioactivity was not homogeneously distributed in the megalin-expressing kidneys but localized in the renal cortex. Quantification of the autoradiogram data confirmed the reduced radioactivity in the renal cortex of megalin-deficient kidneys. CONCLUSION: This study revealed the molecular mechanism of [(111)In-DTPA]octreotide uptake in renal proximal tubules involving the receptor megalin. Identification of megalin may be crucial for further research into strategies to reduce renal uptake.

Clinical Relevance of Targeting the Gastrin-Releasing Peptide Receptor, Somatostatin Receptor 2, or Chemokine C-X-C Motif Receptor 4 in Breast Cancer for Imaging and Therapy.

UNLABELLED: Imaging and therapy using radioligands targeting receptors overexpressed on tumor cells is successfully applied in neuroendocrine tumor patients. Because expression of the gastrin-releasing peptide receptor (GRPR), somatostatin receptor 2 (SSTR2), and chemokine C-X-C motif receptor 4 (CXCR4) has been demonstrated in breast cancer, targeting these receptors using radioligands might offer new imaging and therapeutic opportunities for breast cancer patients. The aim of this study was to correlate messenger RNA (mRNA) expression of GRPR, SSTR2, and CXCR4 with clinicopathologic and biologic factors, and with prognosis and prediction to therapy response, in order to identify specific breast cancer patient groups suited for the application of radioligands targeting these receptors. METHODS: First, we studied GRPR and SSTR2 expression in 13 clinical breast cancer specimens by in vitro autoradiography and correlated this with corresponding mRNA levels to investigate whether mRNA levels reliably represent cell surface expression. Next, GRPR, SSTR2, and CXCR4 mRNA levels were measured by quantitative reverse transcriptase polymerase chain reaction in 915 primary breast cancer tissues and correlated with known clinicopathologic and biologic factors, disease-free survival, distant metastasis-free survival, and overall survival (DFS, MFS, and OS, respectively). In 224 adjuvant hormonal treatment-naïve estrogen receptor (ER, ESR1)-positive patients who received tamoxifen as first-line therapy for recurrent or metastatic disease, the expression levels of the receptors were correlated with progression-free survival. RESULTS: Our results showed a significant positive correlation between GRPR and SSTR2 expression analyzed by in vitro autoradiography and by quantitative reverse transcriptase polymerase chain reaction (Spearman's rank correlation coefficient [Rs]=0.94, P<0.001, and Rs=0.73, P=0.0042, respectively). Furthermore, high GRPR and SSTR2 mRNA levels were observed more frequently in ESR1-positive specimens, whereas high CXCR4 expression was associated with ESR1-negative specimens. Also, high mRNA expression of CXCR4 was associated with a prolonged DFS, MFS, and OS (multivariate hazard ratio MFS=0.76 [95% confidence interval, 0.64-0.90], P=0.001), whereas high mRNA levels of GRPR were associated with a prolonged progression-free survival after the start of first-line tamoxifen treatment (multivariate hazard ratio=0.68 [95% confidence interval, 0.48-0.97], P=0.031). CONCLUSION: Our data indicate that imaging and therapy using GRPR or SSTR2 radioligands might especially be beneficial for ESR1-positive breast cancer and CXCR4 radioligands for ESR1-negative breast cancer.

In vitro and in vivo application of radiolabeled gastrin-releasing peptide receptor ligands in breast cancer.

UNLABELLED: Breast cancer (BC) consists of multiple subtypes defined by various molecular characteristics, for instance, estrogen receptor (ER) expression. Methods for visualizing BC include mammography, MR imaging, ultrasound, and nuclear medicine-based methods such as (99m)Tc-sestamibi and (18)F-FDG PET, unfortunately all lacking specificity. Peptide receptor scintigraphy and peptide receptor radionuclide therapy are successfully applied for imaging and therapy of somatostatin receptor-expressing neuroendocrine tumors using somatostatin receptor radioligands. On the basis of a similar rationale, radioligands targeting the gastrin-releasing peptide receptor (GRP-R) might offer a specific method for imaging and therapy of BC. The aim of this study was to explore the application of GRP-R radioligands for imaging and therapy of BC by introducing valid preclinical in vitro and in vivo models. METHODS: GRP-R expression of 50 clinical BC specimens and the correlation with ER expression was studied by in vitro autoradiography with the GRP-R agonist (111)In-AMBA. GRP-R expression was also analyzed in 9 BC cell lines applying (111)In-AMBA internalization assays and quantitative reverse transcriptase polymerase chain reaction. In vitro cytotoxicity of (177)Lu-AMBA was determined on the GRP-R-expressing BC cell line T47D. SPECT/CT imaging and biodistribution were studied in mice with subcutaneous and orthotopic ER-positive T47D and MCF7 xenografts after injection of the GRP-R antagonist (111)In-JMV4168. RESULTS: Most of the human BC specimens (96%) and BC cell lines (6/9) were found to express GRP-R. GRP-R tumor expression was positively (P = 0.026, χ(2)(4) = 12,911) correlated with ER expression in the human BC specimens. Treatment of T47D cells with 10(-7) M/50 MBq of (177)Lu-AMBA resulted in 80% reduction of cells in vitro. Furthermore, subcutaneous and orthotopic tumors from both BC cell lines were successfully visualized in vivo by SPECT/CT using (111)In-JMV4168; T47D tumors exhibited a higher uptake than MCF7 xenografts. CONCLUSION: Targeting GRP-R-expressing BC tumors using GRP-R radioligands is promising for nuclear imaging and therapy, especially in ER-positive BC patients.

Peptide receptor radionuclide therapy (PRRT) with [(177)Lu-DOTA(0),Tyr(3)]octreotate in combination with RAD001 treatment: further investigations on tumor metastasis and response in the rat pancreatic CA20948 tumor model.

BACKGROUND: Previously, we reported on the unexpected development of distant metastases in the subcutaneous rat pancreas CA20948 tumor model after 4.5 weeks of treatment with RAD001-only or in combination with [(177)Lu-DOTA(0),Tyr(3)]octreotate ((177)Lu-DOTATATE) (Cancer Res. 73:12-8, 2013). Moreover, the combination therapy was less effective compared to (177)Lu-DOTATATE-only. In the current study, we address the following questions: (1) Why was the combination therapy less effective? Is (177)Lu-DOTATATE tumor uptake affected by pretreatment with RAD001? (2) Could sudden cessation of RAD001 therapy cause the development of distant metastases? (3) Is (177)Lu-DOTATATE an effective treatment option for these metastases? METHODS: Lewis rats (HanHsd or SsNHsd substrain with a slight difference in immune response) bearing subcutaneous CA20948 tumors were treated with either 125 or 275 MBq (177)Lu-DOTATATE, RAD001, or their combination. RAD001 was given twice a week for 4.5 or 12 weeks, whereas (177)Lu-DOTATATE was given as a single injection. When combined, RAD001 was started either 3 days prior to or 3 days post administration of (177)Lu-DOTATATE. SPECT/CT was performed to quantify (177)Lu-DOTATATE tumor uptake. Where indicated, primary tumors were surgically removed when tumor size is >6,000 mm(3) to enable monitoring for possible metastasis. If metastases were suspected, an (111)In-DTPA-octreotide SPECT/CT scan was performed. Seven rats with metastases were treated with 400 MBq (177)Lu-DOTATATE. RESULTS: Lu-DOTATATE tumor uptake was not significantly affected by RAD001 pretreatment. The occurrence of metastases after RAD001 treatment was not dose dependent in the dose range tested, nor was it related to the duration of RAD001 treatment. In the experiment in which the LEW/SsNsd substrain was used, only 12.5% of RAD001-treated rats showed complete response (CR), compared to 50% tumor regression in the control group. Re-treatment with a high dose of (177)Lu-DOTATATE resulted in CR in only two out of seven animals. CONCLUSION: Less effective anti-tumor effects after the combination of RAD001 + (177)Lu-DOTATATE could not be explained by reduced (177)Lu-DOTATATE tumor uptake after RAD001. Our current data support RAD001-induced immune suppression as the reason for this observation. No evidence was found that cessation of RAD001 treatment caused development of metastases. Metastases appeared to be less sensitive to (177)Lu-DOTATATE treatment than primary tumors.

In vivo enzyme inhibition improves the targeting of [177Lu]DOTA-GRP(13-27) in GRPR-positive tumors in mice.

INTRODUCTION: Gastrin-releasing peptide receptors (GRPR) and GRP-derived analogs have attracted attention due to high receptor expression in frequently occurring human neoplasia. The authors recently synthesized a series of GRPR-affine peptide analogs based on the 27-mer GRP and derivatized with the DOTA chelator at the N-terminus for (111)In-labeling. In this study, the authors evaluated the most promising from these series, DOTA-GRP(13-27), after radiolabeling with (177)Lu for future therapeutic applications. In addition, to improve in vivo stability of the peptide against in vivo degradation by the protease neutral endopeptidase (NEP), the authors coinjected [(177)Lu]DOTA-GRP(13-27) with the potent NEP inhibitor phosphoramidon (PA). The authors also aimed at reducing renal uptake by coadministration of lysine. METHODS: In vivo stability studies were performed in Swiss albino mice. Biodistribution studies were conducted in NMRI nu/nu mice bearing prostate cancer (PC)-3 xenografts. Ex vivo autoradiography was performed using frozen sections from PC-3 xenografts and kidneys. RESULTS AND DISCUSSION: Coadministration of PA significantly increased the percentage of intact radiopeptide in the mouse circulation. From biodistribution and ex vivo autoradiography studies, coadministration of both lysine and PA with [(177)Lu]DOTA-GRP(13-27) appeared to induce a clear improvement of tumor uptake as well as lower levels of renal radioactivity, causing a promising ninefold increase in tumor/kidney ratios.