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.

Evaluation of a radiolabelled cyclic DTPA-RGD analogue for tumour imaging and radionuclide therapy.

Tumours depend on sufficient blood supply for their growth. They are able to promote new blood vessel formation (neoangiogenesis) via angiogenic factors. Inhibition of this process results in tumour involution or necrosis. RGD (Arg-Gly-Asp) peptides are described to antagonise neoangiogenesis, e.g., by binding to alpha(v)beta(3) receptors on blood vessels. In order to visualise neoangiogenesis in tumours in vitro and in vivo, we introduced and tested an RGD analogue [c(Arg-Gly-Asp-D-Tyr-Lys)], coupled to the chelator diethyleletriamepentaacetic acid (DTPA). This analogue can be radiolabelled with both (111)In and (125)I. In autoradiography and immunohistochemistry studies, the (125)I-labelled analogue appeared to bind specifically and with high affinity to alpha(v)beta(3) receptors on neovascular blood vessel sections of different major human cancers, like prostate and breast cancer, which express these receptors. This radioiodinated radiopharmaceutical also bound to and internalised in human carcinoid Bon cells and rat pancreatic CA20948 tumour cells. Internalisation was receptor-specific and appeared to be time and temperature dependent. In vivo in rats, we investigated administration of different peptide amounts (0.1, 0.5, and 100 microg). The best amount of the radiolabelled analogue to be administered to rats appeared to be 0.1 microg/rat, as uptake decreased with increasing peptide amount. We also found receptor-specific accumulation of the (111)In-labelled analogue in the transplantable pancreatic tumour CA20948. The introduction of the DTPA group in this peptide resulted in renal clearance of the radiopharmaceutical, in contrast to the non-DTPA-conjugated compound that is cleared predominantly via the liver. (111)In emits Auger and conversion electrons besides gamma radiation, therefore, this radiopharmaceutical is suitable not only for tumour scintigraphy but also has potential for radionuclide therapy of major human cancers as well. Moreover, after coupling to the chelator DOTA, the analogue could be radiolabelled in a stable way with beta-emitters, e.g., (90)Y and (177)Lu, enlarging its potential. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 186-198 (2000).

Therapy of neuroendocrine tumors with radiolabeled somatostatin-analogues.

UNLABELLED: Peptide receptor scintigraphy with the radioactive somatostatin-analogue [111In-DTPA0]octreotide (DTPA = diethylenetriaminepentaacetic acid) is a sensitive and specific technique to show in vivo the presence and abundance of somatostatin receptors on various tumors. With this technique primary tumors and metastases of neuroendocrine cancers as well as of many other cancer types can be localised. A new application is the use of peptide receptor radionuclide therapy, administrating high doses of 111In- or 90Y-labeled octreotide-analogues. PRECLINICAL: We investigated the radiotherapeutic effect of 90Y- and 111In-labeled [DOTA0,Tyr3]octreotide (DOTA = tetraazacyclododecanetetraacetic acid) or [111In-DTPA0]octreotide in Lewis rats bearing the somatostatin receptor-positive rat pancreatic tumor CA20948 in A) the flank or B) in the liver. PATIENTS: Thirty end-stage patients with mostly neuroendocrine progressing tumors were treated with [111In-DTPA0]octreotide, up to a maximal cumulative patient dose of about 74 GBq, in a phase 1 trial. PRECLINICAL RESULTS: A) Flank model: at least two 111MBq injections of [111In-DOTA0,Tyr3]octreotide were needed to reach tumor response, in 40% of the animals complete tumor remission was found after a follow-up period of 10 months. One or two injections of [90Y-DOTA0,Tyr3] octreotide yielded transient stable disease. B) Liver model: we found that peptide receptor radionuclide therapy is only effective if somatostatin receptors are present on the tumors, and is therefore receptor-mediated. High radioactive doses of 370 MBq [111In-DTPA0]octreotide or 93 MBq [90Y-DOTA0,Tyr3]octreotide can inhibit the growth of somatostatin receptor-positive metastases. CLINICAL RESULTS: There were no major clinical side effects after up to 2 years treatment, except that a transient decline in platelet counts and lymphocyte subsets can occur. Promising beneficial effects on clinical symptoms, hormone production and tumor proliferation were found. Of the 21 patients with progressive disease at baseline and who received a cumulative dose of more than 20 GBq [111In-DTPA0]octreotide, 8 patients showed stabilisation of disease and 6 other patients a reduction in size of tumors. There is a tendency towards better results in patients whose tumors have a higher accumulation of the radioligand. CONCLUSION: Radionuclide therapy with octreotide-derivatives is feasible, both with 111In and 90Y as radionuclides.

Inhibition of uptake of thyroid hormone into rat hepatocytes by preincubation with N-bromoacetyl-3,3',5-triiodothyronine.

To investigate whether affinity coupling of N-bromoacetyl-T3 (BrAcT3) to the T3 membrane carrier results in an inhibition of transport of T3 into the cell, rat hepatocytes in monolayer were incubated for 2 h at 21 C with 1.3 mumol/liter BrAcT3 in medium without protein. After extensive washing, cells were incubated during 20 h at 37 C with [125I]T3 in medium with 0.5% BSA, and products in supernatants were analyzed by LH-20 column chromatography. In addition the apparent affinity constant (Km) and maximal uptake velocity (Vmax) of the high affinity uptake process were estimated using 1 min incubations of hepatocytes with various concentrations of T3. In control experiments (i.e. without BrAcT3 affinity coupling) about 57% of the added T3 was cleared from the medium and further metabolized, 85% of the cleared T3 reappeared in the medium as I-, 15% as conjugates. Addition of propylthiouracil during the 20 h incubation with T3 strongly inhibited deiodination, without a change in T3 clearance. Because T3 is sulfated before deiodination, a concomitant rise in conjugates was observed. Addition of ouabain to control cells during the 20 h incubation with T3 strongly inhibited uptake, with a parallel decrease in I- and conjugate formation. After affinity coupling of BrAcT3, T3 clearance was inhibited (by 30% P less than 0.001). Since I- production was more depressed (by 73%) than T3 clearance, with some rise in conjugate formation (P less than 0.001), inhibition of deiodinase by BrAcT3 also took place. The effects of BrAcT3 and ouabain on uptake of T3 appeared to be additive as were the effects of propylthiouracil and BrAcT3 on deiodination. After affinity coupling of BrAcT3, the Km of T3 uptake did not change significantly; however Vmax was 54% lower (P less than 0.025) indicating a noncompetitive inhibition of the transport system. Preincubation of the cells with N-acetyl-T3 does not alter the characteristics of uptake of T3 by rat hepatocytes as compared to controls, indicating that no binding of this compound occurs. It is concluded that preincubation of hepatocytes with BrAcT3 diminished I- formation from T3; 50% of this inhibition is due to decreased membrane transport and 50% by reduction of deiodination. Inhibition of membrane transport by BrAcT3 is substantiated by a 54% lower Vmax without a significant change in Km as compared to control. The effect of transport of thyroid hormone on metabolism stresses the importance of the membrane carrier in the translocation process.

Active transport of iodothyronines into human cultured fibroblasts.

Thyroid hormone uptake into human cultured fibroblasts was studied using 2-min incubations with labeled iodothyronines. The results indicate the presence of an active T4 uptake process with two saturable sites with apparent Km values of 1.9 and 141 nM, respectively, and an active T3 uptake process with two saturable sites with Km values of 29 and 650 nM. The uptake of both hormones was energy dependent, i.e. inhibited by KCN or by incubation of the cells in the absence of glucose. By analogy with similar findings in rat hepatocytes we postulate that the high affinity systems represent active transport of thyroid hormone into the cell. Preincubation of the cells with 2 mM ouabain resulted in a decrease in the uptake of both T3 and T4, suggesting that a sodium gradient is necessary for transport. Similar to that in rat hepatocytes, uptake of T3 was inhibited by high concentrations of T4, and uptake of T4 was inhibited by high concentrations of T3. These data indicate that regulation of thyroid hormone uptake at the level of the plasma membrane may be operative in humans.

Specific binding sites for L-triiodothyronine in rat liver and kidney cytosol.

Binding of 125I-labelled triiodothyronine (T3) to rat liver and kidney cytosol was examined. In both organs a very similar binding protein was detected. The apparent equilibrium association constants amounted to 4.8 X 10(7) M-1 for the liver protein and to 1.8 X 10(7) M-1 for the kidney protein. In the two organs a high capacity for T3 binding was observed: maximal binding capacities of respectively 4.3 and 33.2 pmol per mg total cytosol protein. Displacement studies using thyroid hormone analogues showed that the binding exhibited similar specificity.