Bone marrow dosimetry in peptide receptor radionuclide therapy with [177Lu-DOTA(0),Tyr(3)]octreotate.

PURPOSE: Adequate dosimetry is mandatory for effective and safe peptide receptor radionuclide therapy (PRRT). Besides the kidneys, the bone marrow is a potentially dose-limiting organ. The radiation dose to the bone marrow is usually calculated according to the MIRD scheme, where the accumulated activity in the bone marrow is calculated from the accumulated radioactivity of the radiopharmaceutical in the blood. This may underestimate the absorbed dose since stem cells express somatostatin receptors. We verified the blood-based method by comparing the activity in the blood with the radioactivity in bone marrow aspirates. Also, we evaluated the absorbed cross-dose from the source organs (liver, spleen, kidneys and blood), tumours and the so-called "remainder of the body" to the bone marrow. METHODS: Bone marrow aspirates were drawn in 15 patients after treatment with [(177)Lu-DOTA(0),Tyr(3)]octreotate. Radioactivity in the bone marrow was compared with radioactivity in the blood drawn simultaneously. The nucleated cell fraction was isolated from the bone marrow aspirate and radioactivity was measured. The absorbed dose to the bone marrow was calculated. The results were correlated to the change in platelet counts 6 weeks after treatment. RESULTS: A strong linear correlation and high agreement between the measured radioactivities in the bone marrow aspirates and in the blood was found (r=0.914, p<0.001). No correlation between the calculated absorbed dose in the bone marrow and the change in platelets was found. There was a considerable contribution from other organs and the remainder of the body to the bone marrow absorbed dose. CONCLUSION: (1) After PRRT with [(177)Lu-DOTA(0),Tyr(3)]octreotate, the radioactivity concentration in the bone marrow is identical to that in the blood; (2) There is no significant binding of the radiopharmaceutical to bone marrow precursor stem cells; (3) The contribution of the cross dose from source organs and tumours to the bone marrow dose is significant; and (4) There is considerable variation in bone marrow absorbed dose between patients. These findings imply that for individual dose optimization, individual calculation of the bone marrow absorbed dose is necessary.

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.

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.

Five Stabilized 111In-labeled neurotensin analogs in nude mice bearing HT29 tumors.

Neurotensin (NT) receptors are overexpressed in different human tumors, such as human ductal pancreatic adenocarcinoma. New stable neurotensin analogs with high receptor affinity have been synthesized by replacing arginine residues with lysine and arginine derivatives. The aim of this study was to explore the biodistribution, tumor uptake, kidney localization, and stability characteristics of these new analogs in order to develop new diagnostic tools for exocrine pancreatic cancer. Four (111)In-labeled DTPA-chelated NT analogs and one (111)In-labeled DOTA-chelated NT analog were evaluated in NMRI nude mice bearing NT receptor-positive HT29 tumors. Experiments with a coinjection of unlabeled NT or lysine were performed to investigate receptor-mediated uptake and kidney protection, respectively. In addition, the in vivo serum stability of the most promising analog was analyzed. In the biodistribution study in mice, at 4 hours postinjection, a low percentage of the injected dose per gram (%ID/g) of tissue for all compounds was found in NT receptor-negative organs, such as the blood, spleen, pancreas, liver, muscle, and femur. A high uptake was found in the colon, intestine, kidneys, and in implanted HT29 tumors. The coinjection of excess unlabeled neurotensin significantly reduced tumor uptake, showing tumor uptake to be receptor-mediated. To a lesser extent, this was also observed for the colon, but not for other tissues. We concluded that DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH and the DOTA-linked counterpart have the most favorable biodistribution properties regarding tumor uptake.

Anticancer activity of targeted proapoptotic peptides.

UNLABELLED: Tumor-induced angiogenesis can be targeted by RGD (Arg-Gly-Asp) peptides, which bind to alpha(v)beta(3)-receptors upregulated on angiogenic endothelial cells. RGD-containing peptides are capable of inducing apoptosis through direct activation of procaspase-3 to caspase-3 in cells. Additionally, tumor cells overexpressing somatostatin receptors can be targeted by somatostatin analogs. Radiolabeled somatostatin analogs are successfully used to image and treat such tumors via receptor-targeted scintigraphy and therapy. We combined these 2 peptides, RGD and somatostatin, to synthesize a new hybrid peptide, RGD-diethylenetriaminepentaacetic acid (DTPA)-octreotate (c(Arg-Gly-Asp-D-Tyr-Asp)-Lys(DTPA)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr). An earlier study showed that tumor-bearing rats had high receptor-specific uptake of RGD-(111)In-DTPA-octreotate in somatostatin receptor subtype 2-positive tissues and tumors. Furthermore, RGD-(111)In-DTPA-octreotate showed a pronounced tumoricidal effect, which is probably the result of increased apoptosis, as is shown by an increased caspase-3 activity after incubation with (111)In-labeled RGD-DTPA-octreotate in comparison with the 2 monopeptides (111)In-DTPA-RGD and (111)In-DTPA-Tyr(3)-octreotate. In this study, we evaluated the biodistributions of RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate and investigated the caspase-3 activation of the unlabeled compound RGD-DTPA-octreotate in vitro. METHODS: Biodistribution studies on tumor-bearing rats were performed with RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate. The apoptotic activity, by activation of caspase-3 with RGD-DTPA-octreotate and RGD-octreotate, was examined using colorimetric and immunocytochemical assays. RESULTS: The radiolabeled compound, RGD-(111)In-DTPA-octreotate, showed high uptake and retention in the rats in which rat pancreatic CA20948 tumor had been implanted. A major drawback was high renal uptake. In vitro, the unlabeled peptide RGD-DTPA-octreotate induced a significant increase in caspase-3 levels in various cell lines in comparison with RGD and Tyr(3)-octreotate (P < 0.01). Caspase-3 activation was time dependent. To alter the elimination route, we examined the biodistribution of radioiodinated RGD-octreotate without DTPA [c(Arg-Gly-Asp-D-Tyr-Asp)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr], as a model of unlabeled RGD-octreotate, in tumor-bearing rats. (125)I-RGD-octreotate showed a much lower renal uptake than did RGD-(111)In-DTPA-octreotate. Furthermore, the affinity of RGD-octreotate increased in comparison with RGD-DTPA-octreotate (values of 1.4 x 10(-8) mol/L vs. 9.4 x 10(-8) mol/L, respectively, for inhibitory concentration of 50%). Finally, RGD-octreotate was still able to activate caspase-3, as was indicated with immunocytochemistry. CONCLUSION: Because of the high renal uptake, RGD-(111)In-DTPA-octreotate is unsuitable for radionuclide therapy. However, the unlabeled peptides, RGD-DTPA-octreotate and RGD-octreotate, also induced an increase in caspase-3 levels, indicating the therapeutic potential of this compound. Thus, the development of hybrid molecules can become a new approach in the treatment of cancer.

Combination radionuclide therapy using 177Lu- and 90Y-labeled somatostatin analogs.

UNLABELLED: Peptide receptor-targeted radionuclide therapy of somatostatin receptor-expressing tumors is a promising application of radiolabeled somatostatin analogs. Suitable radionuclides are (90)Y, a pure, high-energy beta-emitter (2.27 MeV), and (177)Lu, a medium-energy beta-emitter (0.5 MeV) with a low-abundance gamma. METHODS: Lewis rats, each bearing both a small (approximately 0.5 cm(2)) and a large (7-9 cm(2)) somatostatin receptor-positive rat pancreatic CA20948 tumor in their flanks, were used. We investigated the radiotherapeutic effects of [(90)Y-tetraazacyclododecanetetraacetic acid (DOTA),Tyr(3)]octreotide, [(90)Y-DOTA,Tyr(3)]octreotate, [(177)Lu-DOTA,Tyr(3)]octreotate, and the combination of (90)Y- and (177)Lu-labeled analogs at the same tumor radiation dose (60 Gy). RESULTS: Radiotherapeutic effects of the (90)Y- and (177)Lu-labeled analogs were found in the rat tumor model. In these animals bearing tumors of different sizes, the antitumor effects of the combination of 50% (177)Lu- plus 50% (90)Y-analogs were superior to those in animals treated with either (90)Y- or (177)Lu- analog alone. In smaller tumors, the (90)Y radiation energy was not completely absorbed in the tumor, whereas in larger tumors the increased number of clonogenic tumor cells at the fixed level of absorbed dose may account for the failure of (177)Lu alone to go completely into remission. CONCLUSION: This study shows the superior antitumor effects of the combination of (177)Lu- and (90)Y-somatostatin analogs when compared with either (90)Y- or (177)Lu-analog alone in animals bearing tumors of various sizes.

Uptake of [111In-DTPA0]octreotide in the rat kidney is inhibited by colchicine and not by fructose.

UNLABELLED: The high renal uptake of radiolabeled somatostatin analogs is dose limiting. Lowering this uptake permits higher radioactivity doses and, thus, tumor doses to be administered. We tested the effects of the microtubule drug colchicine on renal uptake of [(111)In-DTPA(0)]octreotide. Also, the effects of fructose were tested. METHODS: Organ radioactivity 24 h after injection of [(111)In-DTPA(0)]octreotide was determined in rats. RESULTS: Coinjection of 1 mg of colchicine per kilogram did not influence renal uptake of [(111)In-DTPA(0)]octreotide, whereas this dose administered 5 h before [(111)In-DTPA(0)]octreotide resulted in significant renal uptake reduction (63%). D-Lysine plus colchicine reduced the uptake by 76% (P < 0.01 vs. D-lysine alone). Liver and blood radioactivity levels were significantly elevated by colchicine. Fructose did not affect the biodistribution of [(111)In-DTPA(0)]octreotide. CONCLUSION: Renal uptake of [(111)In-DTPA(0)]octreotide is dependent on microtubule function in rats. The addition of colchicine to amino acid protocols may permit administration of higher doses, improving the therapeutic window of peptide receptor radionuclide therapy.

Peptide receptor radionuclide therapy in vitro using [111In-DTPA0]octreotide.

UNLABELLED: Peptide receptor radionuclide therapy (PRRT) using [(111)In-DTPA(0)]octreotide (where DTPA is diethylenetriaminepentaacetic acid) is feasible because, besides gamma-radiation, (111)In emits both therapeutic Auger and internal conversion electrons having a tissue penetration of 0.02-10 and 200-500 micro m, respectively. The aim of this study was to investigate the therapeutic effects of [(111)In-DTPA(0)]octreotide in a single-cell model including the effects of incubation time, radiation dose, and specific activity of [(111)In-DTPA(0)]octreotide. Finally, we discriminated between the effects of the Auger electrons and internal conversion electrons in PRRT. METHODS: An in vitro, colony-forming assay to study cell survival after PRRT using the sst subtype 2-positive rat pancreatic tumor cell line CA20948 was developed. RESULTS: In this in vitro system [(111)In-DTPA(0)]octreotide can control tumor growth to 0% survival, and the effects were dependent on incubation time, radiation dose, and specific activity used. Similar concentrations of (111)In-DTPA, which is not internalized into sst-positive tumor cells like [(111)In-DTPA(0)]octreotide, did not influence tumor survival. Excess unlabeled octreotide (10(-6) mol/L) could decrease tumor cell survival to 60% of control; the addition of radiolabeled peptide ([(111)In-DTPA(0)]octreotide [10(-9) mol/L] + 10(-6) mol/L octreotide) did not further decrease survival. CONCLUSION: These in vitro studies show that the therapeutic effect of (111)In is dependent on internalization, enabling the Auger electrons with their very short particle range to reach the nucleus. Our results also indicate that the PRRT effects were receptor mediated.

Increased cell death after therapy with an Arg-Gly-Asp-linked somatostatin analog.

UNLABELLED: Receptor-targeted scintigraphy and radionuclide therapy with radiolabeled somatostatin analogs are successfully applied for somatostatin receptor-positive tumors. The synergistic effects of an apoptosis-inducing factor, for example, the Arg-Gly-Asp (RGD) motif, can increase the radiotherapeutic efficacy of these peptides. Hence, the tumoricidal effects of the hybrid peptide RGD-diethylaminetriaminepentaacetic acid (DTPA)-Tyr3-octreotate (cyclic[c](Arg-Gly-Asp-D-Tyr-Asp)-Lys(DTPA)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr), hereafter referred to as RGD-DTPA-octreotate, were evaluated in comparison with those of RGD (c(Arg-Gly-Asp-D-Tyr-Asp)) and Tyr3-octreotate (D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr). METHODS: The therapeutic effects of RGD-111In-DTPA-octreotate, 111In-DTPA-RGD, and 111In-DTPA-Tyr3-octreotate were investigated with various cell lines by use of a colony-forming assay, and caspase-3 activity was also determined. RESULTS: Tumoricidal effects were found with 111In-DTPA-RGD, 111In-DTPA-Tyr3-octreotate, and RGD-111In-DTPA-octreotate, in order from least effective to most effective. Also, the largest increase in caspase-3 levels was found with RGD-111In-DTPA-octreotate. CONCLUSION: RGD-111In-DTPA-octreotate has more pronounced tumoricidal effects than 111In-DTPA-RGD and 111In-DTPA-Tyr3-octreotate, because of increased apoptosis, as indicated by increased caspase-3 activity.

Preclinical comparison of (111)In-labeled DTPA- or DOTA-bombesin analogs for receptor-targeted scintigraphy and radionuclide therapy.

UNLABELLED: The 14-amino-acid peptide bombesin (BN) has a high affinity for the gastrin-releasing peptide (GRP) receptor that is expressed by a variety of tumors. Recently, high densities of GRP receptors were identified by in vitro receptor autoradiography in human prostate and breast carcinomas using [(125)I-Tyr(4)]BN as radioligand. Radiometal-labeled diethylenetriaminepentaacetic acid (DTPA)-BN derivatives are potentially useful radioligands for receptor-targeted scintigraphy and radiotherapy of GRP receptor-expressing tumors. METHODS: [DTPA-Pro(1),Tyr(4)]BN (A), [DOTA-Pro(1),Tyr(4)]BN (B), [DTPA-epsilon-Lys(3),Tyr(4)]BN (C), and [DOTA-epsilon-Lys(3),Tyr(4)]BN (D) (where DOTA is dodecanetetraacetic acid) were synthesized and studied for competition with binding of [(125)I-Tyr(4)]BN to the GRP receptor. The (111)In-labeled BN analogs were studied in vitro for binding and internalization by GRP receptor-expressing CA20948 and AR42J pancreatic tumor cells as well as in vivo for tissue distribution in rats. Specific tissue binding was tested by coinjection of 0.1 mg [Tyr(4)]BN. RESULTS: All BN analogs competitively inhibited the binding of [(125)I-Tyr(4)]BN to the GRP receptor with 50% inhibitory concentration values in the range of 2-9 nmol/L. All (111)In-labeled analogs showed high and specific time- and temperature-dependent binding and internalization by CA20948 and AR42J cells. In in vivo studies, high and specific binding was found in GRP receptor-positive tissues such as pancreas (0.90, 1.2, 0.54, and 0.79 percentage injected dose per gram for A-D, respectively). In a rat model, the AR42J tumor could clearly be visualized by scintigraphy using [(111)In-DTPA-Pro(1),Tyr(4)]BN as the radioligand. Although [(111)In-DOTA-Pro(1),Tyr(4)]BN showed the highest uptake of radioactivity in GRP receptor-positive tissues as well as higher target-to-blood ratios, [(111)In-DTPA-Pro(1),Tyr(4)]BN was easier to handle and is more practical to use. Therefore, we decided to start phase I studies with this DTPA-conjugated radioligand. CONCLUSION: [(111)In-DTPA-Pro(1),Tyr(4)]BN is a promising radioligand for scintigraphy of GRP receptor-expressing tumors. We are currently performing a phase I study on patients with invasive prostate carcinoma.

Norharman and alcohol-dependency in male Wistar rats.

We examined the effects of ethanol ingestion to rats on levels of the beta-carboline norharman in plasma, brain and liver at the end of ethanol ingestion and 10 h after withdrawal. We also investigated the effect of exogenously administered norharman on the behavioural signs of alcohol withdrawal. Ethanol was given by a liquid diet for 21 days. Norharman plasma levels in alcohol fed rats were significantly elevated compared to both control rats and to rats 10 h after withdrawal. Norharman levels in brains and livers showed a similar pattern. The capacity of the livers of both alcohol-dependent and withdrawal rats to catabolise norharman was significantly reduced compared to control rats. Norharman injected intraperitoneally (6.3 mg/kg) attenuated the behavioural signs of alcohol withdrawal significantly. The mechanism behind the increased norharman levels in alcohol-dependent rats may be inhibition of the synthesis and/or activity of liver enzyme(s) responsible for the breakdown of norharman.

Reduction of skeletal accumulation of radioactivity by co-injection of DTPA in [90Y-DOTA0,Tyr3]octreotide solutions containing free 90Y3+.

Peptide receptor-targeted radionuclide therapy is nowadays being performed with radiolabeled DOTA-conjugated peptides, such as [90Y-DOTA0,Tyr3]octreotide (also known as OctreoTher or 90Y-DOTATOC). The incorporation of 90Y3+ is typically > or = 99%, however, since a total patient dose can be as high as 26 GBq or 700 mCi the amount of free 90Y3+ (= non-DOTA-incorporated) can be substantial. Free 90Y3+ accumulates in bone with undesired radiation of bone marrow as a consequence. 90Y-DTPA is excreted rapidly via the kidneys. Incorporation of free 90Y3+ into 90Y-DTPA might prevent this fraction from being accumulated into bone, therefore we have investigated: the biodistribution in rats of 90YCl3, [90Y-DOTA0,Tyr3]octreotide, and 90Y-DTPA; possibilities to complex 10% of free 90Y3+ in a [90Y-DOTA0,Tyr3]octreotide containing solution into 90Y-DTPA prior to intravenous injection; and effects of 10% free 90Y3+ in [90Y-DOTA0,Tyr3]octreotide solution, in the presence and in the absence of excess DTPA, on the biodistribution of in rats. The following results are presented: 90YCl3 showed high skeletal uptake (i.e., 1% ID (injected dose) per gram femur, with main localization in the epiphyseal plates) and a 24 h total body retention of 74% ID; 90Y-DTPA had rapid renal clearance, and 24 h total body retention of < 5% ID; added free 90Y3+ in [90Y-DOTA0,Tyr3]octreotide solution could rapidly be incorporated into 90Y-DTPA at room temperature; and accumulation of 90Y3+ in femur, blood, and liver was related to the amount of free 90Y3+, whereas these accumulations could be prevented by the addition of DTPA. In conclusion, the addition of excess DTPA to [90Y-DOTA0,Tyr3]octreotide with incomplete 90Y-incorporation is recommended.

Tyr3-octreotide and Tyr3-octreotate radiolabeled with 177Lu or 90Y: peptide receptor radionuclide therapy results in vitro.

Somatostatin analogs promising for peptide receptor scintigraphy (PRS) and peptide receptor radionuclide therapy (PRRT) are D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr(ol) (Tyr 3-octreotide) and D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr (tyr3-octreotate). For radiotherapeutic applications these peptides are being labeled with the beta(-) particle emitters 177Lu or 90Y. We evaluated the therapeutic effects of these analogs chelated with tetra-azacyclododecatatro-acetic acid (DOTA) and labeled with 90Y or 177Lu in an in vitro colony-forming assay using the rat pancreatic tumor cell line CA20948. Furthermore, we investigated the effects of incubation time, radiation dose, and specific activity of [177Lu-DOTA]-D-Phe1-c (Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr (177Lu-octreotate). 177Lu-octreotate could reduce tumor growth to 100% cell kill and effects were dependent on radiation dose, incubation time, and specific activity used. Similar concentrations of 177Lu-DOTA, which is not bound to the cells, had a less pronounced effect on the tumor cell survival. Both tyr3-octreotide and tyr3-octreotate labeled with either 177Lu or 90Y, using DOTA as chelator, were able to control tumor growth in a dose-dependent manner. In all concentrations used radiolabeled tyr3-octreotate had a higher tumor kill compared to radiolabeled tyr3-octreotide, labeled with 177Lu or 90Y. This is in accordance with the higher affinity of tyr3-octreotate for the subtype 2 (sst2)-receptor compared to tyr3-octreotide, leading to a higher amount of cell-associated radioactivity, resulting in a significantly higher tumor radiation dose. In conclusion, tyr3-octreotate labeled with 177Lu or 90Y is the most promising analog for PRRT.

Long-term toxicity of [(177)Lu-DOTA (0),Tyr (3)]octreotate in rats.

PURPOSE AND METHODS: Studies on peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues have shown promising results with regard to tumour control. The efficacy of PRRT is limited by uptake and retention in the proximal tubules of the kidney, which might lead to radiation nephropathy. We investigated the long-term renal toxicity after different doses of [(177)Lu-DOTA(0),Tyr(3)]octreotate and the effects of dose fractionation and lysine co-injection in two tumour-bearing rat models. RESULTS: Significant renal toxicity was detected beyond 100 days after start of treatment as shown by elevated serum creatinine and proteinuria. Microscopically, tubules were strongly dilated with flat epithelium, containing protein cylinders. Creatinine levels rose significantly after 555 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate, but were significantly lower after 278 MBq (single injection) or two weekly doses of 278 MBq. Renal damage scores were maximal after 555 MBq and significantly lower in the 278 and 2x278 MBq groups. Three doses of 185 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate with intervals of a day, a week or a month significantly influenced serum creatinine (469+/-18, 134+/-70 and 65+/-15 micromol/l, respectively; p<0.001). Renal histological damage scores were not significantly influenced by dose fractionation. Lysine co-administration with three weekly treatments of 185 MBq significantly lowered serum creatinine and proteinuria. CONCLUSION: Injection of high doses of [(177)Lu-DOTA(0),Tyr(3)]octreotate resulted in severe renal damage in rats as indicated by proteinuria, elevated serum creatinine and histological damage. This damage was dose dependent and became overt between 100 and 200 days after treatment. Dose fractionation had significant beneficial effects on kidney function. Also, lysine co-injection successfully prevented functional damage.

[(99m)Tc]Demotate 2 in the detection of sst(2)-positive tumours: a preclinical comparison with [(111)In]DOTA-tate.

PURPOSE: The aim of this study was to evaluate [(99m)Tc]Demotate 2 ([(99m)Tc-N(4) (0-1),Asp(0),Tyr(3)]octreotate) as a candidate for in vivo imaging of sst(2)-positive tumours and to compare it with [(111)In]DOTA-tate ([(111)In-DOTA(0),Tyr(3)]octreotate). METHODS: Labelling of Demotate 2 with (99m)Tc was performed at room temperature using SnCl(2) as reductant in the presence of citrate at alkaline pH. Radiochemical analysis involved ITLC and HPLC methods. Peptide conjugate affinities for sst(2) were determined by receptor autoradiography on rat brain cortex sections using [DOTA(0),(125)I-Tyr(3)]octreotate as the radioligand. The affinity profile of Demotate 2 for human sst(1)-sst(5) was studied by receptor autoradiography in cell preparations using the universal somatostatin radioligand [(125)I][Leu(8),(D: )Trp(22),Tyr(25)]somatostatin-28. The internalisation rates of [(99m)Tc]Demotate 2 and [(111)In]DOTA-tate were compared in sst(2)-positive and -negative control cell lines. Biodistribution of radiopeptides was studied in male Lewis rats bearing CA20948 tumours. RESULTS: Peptide conjugates showed selectivity and a high affinity binding for sst(2) (Demotate 2 IC(50)=3.2 nM and DOTA-tate IC(50)=5.4 nM). [(99m)Tc]Demotate 2, like [(111)In]DOTA-tate, internalised rapidly in all sst(2)-positive cells tested, but not in sst(2)-negative control cells. After injection in CA20948 tumour-bearing rats both radiopeptides showed high and specific uptake in the sst(2)-positive organs and in the implanted tumour and rapid excretion from non-target tissues via the kidneys. CONCLUSION: [(99m)Tc]Demotate 2, similarly to the known sst(2)-targeting agent [(111)In]DOTA-tate, showed promising biological qualities for application in the scintigraphy of sst(2)-positive tumours.

Radiolabelling DOTA-peptides with 68Ga.

PURPOSE: A new field of interest is the application of 68Ga-labelled DOTA-conjugated peptides for positron emission tomography (PET). The commercially available or house-made generators require time-consuming and tedious handling of the eluate. Radiolabelling at high specific activities without further purification is not possible, while high specific activities are necessary for peptides that potentially display pharmacological side-effects. Here we present the practical aspects and the results of radiolabelling DOTA-peptides with a TiO2-based commercially available 68Ge/68Ga generator. METHODS: Reaction kinetics and parameters influencing the incorporation of the radionuclide at the highest achievable specific activity were investigated. Since high finger doses were anticipated during handling of the high beta-energy emitter 68Ga, finger dosimetric measurements were performed during radiolabelling and in vivo administration. RESULTS: Fractionated elution of the generator revealed that 80% of the radioactivity was recovered in 1 ml. Bi- and trivalent ionic contaminants that compete for the incorporation of the radionuclide were below 50 nM; thus further tedious and time-consuming purification was avoided. Radiolabelling was performed at pH 3.5-4. Plastic shielding (> or =7-mm wall thickness) around the syringe during administration effectively eliminated the positrons. In rats 68GaCl3 had slow clearance from blood, while 68Ga-EDTA was rapidly cleared via the kidneys. Uptake of 68Ga-DOTATOC in somatostatin receptor-positive tissues was high, with no significant difference between 1 and 4 h post injection. CONCLUSION: DOTA-peptides for PET imaging can be labelled with 68Ga up to specific activities of 1 GBq per nmol within 20 min, enabling the clinical application of peptides that display potential pharmacological side-effects.

Localisation and mechanism of renal retention of radiolabelled somatostatin analogues.

PURPOSE: Radiolabelled somatostatin analogues, such as octreotide and octreotate, are used for tumour scintigraphy and radionuclide therapy. The kidney is the most important critical organ during such therapy owing to the reabsorption and retention of radiolabelled peptides. The aim of this study was to investigate in a rat model both the localisation and the mechanism of renal uptake after intravenous injection of radiolabelled somatostatin analogues. The multi-ligand megalin/cubilin receptor complex, responsible for reabsorption of many peptides and proteins in the kidney, is an interesting candidate for renal endocytosis of these peptide analogues. METHODS: For localisation studies, ex vivo autoradiography and micro-autoradiography of rat kidneys were performed 1-24 h after injection of radiolabelled somatostatin analogues and compared with the renal anti-megalin immunohistochemical staining pattern. To confirm a role of megalin in the mechanism of renal retention of [111In-DTPA]octreotide, the effects of three inhibitory substances were explored in rats. RESULTS: Renal ex vivo autoradiography showed high cortical radioactivity and lower radioactivity in the outer medulla. The distribution of cortical radioactivity was inhomogeneous. Micro-autoradiography indicated that radioactivity was only retained in the proximal tubules. The anti-megalin immunohistochemical staining pattern showed a strong similarity with the renal [111In-DTPA]octreotide ex vivo autoradiograms. Biodistribution studies showed that co-injection of positively charged D-lysine reduced renal uptake to 60% of control. Sodium maleate reduced renal [111In-DTPA]octreotide uptake to 15% of control. Finally, cisplatin pre-treatment of rats reduced kidney uptake to 70% of control. CONCLUSION: Renal retention of [111In-DTPA]octreotide is confined to proximal tubules in the rat kidney, in which megalin-mediated endocytosis may play an important part.

Anti-tumor effect and increased survival after treatment with [177Lu-DOTA0,Tyr3]octreotate in a rat liver micrometastases model.

Peptide receptor scintigraphy with [(111)In-DTPA(0)]octreotide (a stabilized radiolabeled somatostatin (SS) analogue, OctreoScan) is widely used for the visualization and staging of somatostatin receptor-positive tumors. The application of likewise somatostatin analogues as vehicle for the deliverance of radionuclides to somatostatin receptor-positive targets are now in use for peptide receptor-targeted radionuclide therapy (PRRT). Currently preclinical and clinical investigation are ongoing trying to find the optimal combination of radionuclide and ligand. The anti-tumoral effects of such combinations, like [90Y-DOTA degrees, Tyr(3)]octreotide and [(177)Lu-DOTA degrees, Tyr(3)]octreotate, on SSR-positive solid tumors have been reported. In this study we present the anti-tumor effects of (177)Lu-DOTA-tate on: a) a single SSR-positive cell model and b) on a SSR-positive tumor in a rat liver micrometastatic model, mimicking disseminated disease. (177)Lu-DOTA-tate showed anti-tumoral effects in both cases and significant survival in the PRRT-treated rats. (177)Lu-DOTA-tate is a very promising new treatment modality for SSR-positive tumors, including disseminated disease.

DOTA-NOC, a high-affinity ligand of somatostatin receptor subtypes 2, 3 and 5 for labelling with various radiometals.

Earlier studies have shown that modification of the octapeptide octreotide in positions 3 and 8 may result in compounds with increased somatostatin receptor affinity that, if radiolabelled, display improved uptake in somatostatin receptor-positive tumours. The aim of a recent research study in our laboratory was to employ the parallel peptide synthesis approach by further exchanging the amino acid in position 3 of octreotide and coupling the macrocyclic chelator DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to these peptides for labelling with radiometals like gallium-67 or -68, indium-111, yttrium-90 and lutetium-177. The purpose was to find radiopeptides with an improved somatostatin receptor binding profile in order to extend the spectrum of targeted tumours. A first peptide, [111In,90Y-DOTA]-1-Nal3-octreotide (111In,90Y-DOTA-NOC), was isolated which showed an improved profile. InIII-DOTA-NOC exhibited the following IC50 values (nM) when studied in competition with [125I][Leu8, d-Trp22, Tyr25]somatostatin-28 (values for YIII-DOTA-NOC are shown in parentheses): sstr2, 2.9 +/- 0.1 (3.3 +/- 0.2); sstr3, 8 +/- 2 (26 +/- 1.9); sstr5, 11.2 +/- 3.5 (10.4 +/- 1.6). Affinity towards sstr1 and 4 was very low or absent. InIII-DOTA-NOC is superior to all somatostatin-based radiopeptides having this particular type of binding profile, including DOTA-lanreotide, and has three to four times higher binding affinity to sstr2 than InIII,YIII-DOTA-Tyr3-octreotide (InIII,YIII-DOTA-TOC). In addition, [111In]DOTA-NOC showed a specific and high rate of internalization into AR4-2J rat pancreatic tumour cells which, after 4 h, was about two times higher than that of [111In]DOTA-TOC and three times higher than that of [111In]DOTA-octreotide ([111In]DOTA-OC). The internalized radiopeptides were externalized intact upon 2 h of internalization followed by an acid wash. After 2-3 h of externalization a plateau is reached, indicating a steady-state situation explained by reactivation of the receptors followed by re-endocytosis. Biodistribution studies in CA 20948 tumour-bearing rats showed rapid clearance from all sstr-negative tissues except the kidneys. At 4 h the uptake of [111In]DOTA-NOC in the tumour and sstr-positive tissues, such as adrenals, stomach and pancreas, was three to four times higher than that of [111In]DOTA-TOC. Differential blocking studies indicate that this is at least partially due to the uptake mediated by sstr3 and sstr5. These very promising preclinical data justify the use of this new radiopeptide for imaging and potentially internal radiotherapy studies in patients.

The addition of DTPA to [177Lu-DOTA0,Tyr3]octreotate prior to administration reduces rat skeleton uptake of radioactivity.

Peptide receptor-targeted radionuclide therapy is nowadays also being performed with DOTA-conjugated peptides, such as [DOTA(0),Tyr(3)]octreotate, labelled with radionuclides like (177)Lu. The incorporation of (177)Lu is typically >/=99.5%; however, since a total patient dose can be as high as 800 mCi, the amount of free (177)Lu(3+) (= non-DOTA-incorporated) can be substantial. Free (177)Lu(3+) accumulates in bone with unwanted irradiation of bone marrow as a consequence. (177)Lu-DTPA is reported to be stable in serum in vitro, and in vivo it has rapid renal excretion. Transforming free Lu(3+) to Lu-DTPA might reroute this fraction from accumulation in bone to renal clearance. We therefore investigated: (a) the biodistribution in rats of (177)LuCl(3), [(177)Lu-DOTA(0),Tyr(3)]octreotate and (177)Lu-DTPA; (b) the possibilities of complexing the free (177)Lu(3+) in [(177)Lu-DOTA(0),Tyr(3)]octreotate to (177)Lu-DTPA prior to intravenous injection; and (c) the effects of free (177)Lu(3+) in [(177)Lu-DOTA(0),Tyr(3)]octreotate, in the presence and absence of DTPA, on the biodistribution in rats. (177)LuCl(3) had high skeletal uptake (i.e. 5% ID per gram femur, with localization mainly in the epiphyseal plates) and a 24-h total body retention of 80% injected dose (ID). [(177)Lu-DOTA(0),Tyr(3)]octreotate had high and specific uptake in somatostatin receptor-positive tissues, and 24-h total body retention of 19% ID. (177)Lu-DTPA had rapid renal clearance, and 24-h total body retention of 4% ID. Free (177)Lu(3+) in [(177)Lu-DOTA(0),Tyr(3)]octreotate could be complexed to (177)Lu-DTPA. Accumulation of (177)Lu in femur, blood, liver and spleen showed a dose relation to the amount of free (177)Lu(3+), while these accumulations could be normalized by the addition of DTPA. After labelling [DOTA(0),Tyr(3)]octreotate with (177)Lu the addition of DTPA prior to intravenous administration of [(177)Lu-DOTA(0),Tyr(3)]octreotate is strongly recommended.