Bert F. Bernard

Yttrium-90 and indium-111 labelling, receptor binding and biodistribution of [DOTA0,d-Phe1,Tyr3]octreotide, a promising somatostatin analogue for radionuclide therapy

In vitro octreotide receptor binding of [111In-DOTA0,d-Phe1,Tyr3]octreotide (111In-DOTATOC) and the in vivo metabolism of90Y or111In-labelled DOTATOC were investigated in rats in comparison with [111In-DTPA0]octreotide [111In-DTPAOC).111In-DOTATOC was found to have an affinity similar to octreotide itself for the octreotide receptor in rat cerebral cortex microsomes. Twenty-four hours after injection of90Y or111In-labelled DOTATOC, uptake of radioactivity in the octreotide receptor-expressing tissues pancreas, pituitary, adrenals and tumour was a factor of 2–6 that after injection of111In-DTPAOC. Uptake of labelled DOTATOC in pituitary, pancreas, adrenals and tumour was almost completely blocked by pretreatment with 0.5 mg unlabelled octreotide, indicating specific binding to the octreotide receptors. These findings strongly indicate that90Y-DOTATOC is a promising radiopharmaceutical for radiotherapy and that111In-DOTATOC is of potential value for diagnosis of patients with octreotide receptor-positive lesions, such as most neuroendocrine tumours.

[177LU-DOTA0, TYR3] OCTREOTATE FOR SOMATOSTATIN RECEPTOR-TARGETED RADIONUCLIDE THERAPY

Receptor‐targeted scintigraphy using radiolabeled somatostatin analogs such as octreotate is being used with great success to demonstrate the in vivo presence of somatostatin receptors on various tumors. A new and promising application for these analogs is radionuclide therapy. Radionuclides suitable for this application include the Auger electron‐emitter 111In and the β‐emitters 90Y (high energy) and 177Lu (low energy). We investigated [DOTA0,Tyr3]octreotate, labeled with the lanthanide 177Lu, in biodistribution and radionuclide therapy experiments using male Lewis rats bearing the somatostatin receptor‐positive rat CA20948 pancreatic tumor. Biodistribution studies in Lewis rats showed the highest uptake in the rat pancreatic CA20948 tumor and sst2‐positive organs, which include the adrenals, pituitary and pancreas, of [177Lu‐DOTA0,Tyr3]octreotate in comparison with 88Y‐ and 111In‐labeled analogs. Kidney uptake of [177Lu‐DOTA0,Tyr3]octreotate could be reduced by approximately 40% by co‐injection of 400 mg/kg D‐lysine. In radionuclide therapy studies, a 100% cure rate was achieved in the groups of rats bearing small (≤1 cm2) CA20948 tumors after 2 doses of 277.5 MBq or after a single dose of 555 MBq [177Lu‐DOTA0,Tyr3]octreotate. A cure rate of 75% was achieved after a single administration of 277.5 MBq. In rats bearing larger (≥1 cm2) tumors, 40% and 50% cure rates were achieved in the groups that received 1 or 2 277.5 MBq injections of [177Lu‐DOTA0,Tyr3]octreotate, respectively. After therapy with [177Lu‐DOTA0,Tyr3]octreotide in rats bearing small tumors, these data were 40% cure after 1 injection with 277.5 MBq and 60% cure after 2 repeated injections. In conclusion, [177Lu‐DOTA0,Tyr3]octreotate has demonstrated excellent results in radionuclide therapy studies in rats, especially in animals bearing smaller tumors. This candidate molecule shows great promise for radionuclide therapy in patients with sst2‐expressing tumors.

Internalization of radiolabelled [DTPA0]octreotide and [DOTA0,Tyr3]octreotide: peptides for somatostatin receptor-targeted scintigraphy and radionuclide therapy.

We compared the internalization of [90Y-DOTA0,Tyr3]octreotide and [111In-DOTA0,Tyr3]octreotide with that of [125I-Tyr3]octreotide and [111In-DTPA0]octreotide in the subtype 2 somatostatin receptor (sst2)-positive rat pancreatic tumour cell lines CA20948 and AR42J and in the somatostatin receptor-negative human anaplastic thyroid tumour cell line ARO. We demonstrated that [111In-DTPA0]octreotide, [90Y-DOTA0,Tyr3]octreotide and [111In-DOTA0,Tyr3]octreotide are internalized by a receptor-specific, time- and temperature-dependent process. The amount of [90Y-DOTA0,Tyr3]octreotide internalized was higher than that of [111In-DOTA0,Tyr3]octreotide and [111In-DTPA0]octreotide.

Characteristics of active transport of thyroid hormone into rat hepatocytes

Thyroid hormone uptake into primary cultured rat hepatocytes was studied using 1-min incubations with radio-iodine-labelled iodothyronines. (1) Uptake of thyroxine indicates two saturable sites with apparent Km values of 1.2 nM and 1.0 microM, and non-saturable uptake. Similar kinetics of triiodothyronine uptake have been observed. (2) The high-affinity systems of both hormones are energy-dependent (i.e., inhibited by KCN and oligomycin). It is postulated that these systems represent active transport of thyroid hormone into the cell. (3) Analysis of mutual inhibition by the substrates for the triiodothyronine and thyroxine transport systems indicates that triiodothyronine and thyroxine cross the cell membrane via separate transport systems. (4) Preincubation with ouabain resulted in a decrease in uptake of both triiodothyronine and thyroxine, suggesting that a sodium gradient is essential for this transport.

Pre-clinical comparison of [DTPA0] octreotide, [DTPA0,Tyr3] octreotide and [DOTA0,Tyr3] octreotide as carriers for somatostatin receptor-targeted scintigraphy and radionuclide therapy

We have evaluated the potential usefulness of radiolabelled [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide as radiopharmaceuticals for somatostatin receptor–targeted scintigraphy and radiotherapy. In vitro somatostatin receptor binding and in vivo metabolism in rats of the compounds were investigated in comparison with [111In‐DTPA0] octreotide. Comparing different peptide–chelator constructs, [DTPA0,Tyr3]octreotide and [DOTA0, Tyr3]octreotide were found to have a higher affinity than [DTPA0]octreotide for subtype 2 somatostatin receptors (sst2) in mouse AtT20 pituitary tumour cell membranes (all IC50 values obtained were in the low nanomolar range). In vivo studies in CA20948 tumor‐bearing Lewis rats revealed a significantly higher uptake of both 111In‐labelled [DOTA0,Tyr3]octreotide and [DTPA0,Tyr3]octreotide in sst2‐expressing tissues than after injection of [111In‐DTPA0]octreotide, showing that substitution of Tyr for Phe at position 3 in octreotide results in an increased affinity for its receptor and in a higher target tissue uptake. Uptake of 111In‐labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide in pituitary, pancreas, adrenals and tumour was decreased to less than 7% of control by pre‐treatment with 0.5 mg unlabelled octreotide/rat, indicating specific binding to sst2. Comparing different radionuclides, [90Y‐DOTA0,Tyr3]octreotide had the highest uptake in sst2‐positive organs, followed by the [111In‐DOTA0,Tyr3]octreotide, whereas [DOTA0, 125I‐Try3]octreotide uptake was low compared to that of the other radiopharmaceuticals, when measured 24 hr after injection. Renal uptake of 111In‐labelled [DTPA0]octreotide, [DTPA0, Tyr3]octreotide and [DOTA0,Tyr3]octreotide was reduced over 50% by an i.v. injection of 400 mg/kg d‐lysine, whereas radioactivity in blood, pancreas and adrenals was not affected. Int. J. Cancer 75:406–411, 1998.

Decreased transport of thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) into rat hepatocytes in primary culture due to a decrease of cellular ATP content and various drugs.

T4, the main secretory product of the thyroid gland, is deiodinated and conjugated in peripheral tissues [ 1,2]. Phenolic ring deiodination of T4 accounts for -80% of the total body production of Ts, the most biologically active iodothyronine [3]. The remainder is produced by the thyroid gland. The other main product of peripheral deiodination is rT3, which is biologically inactive [4]. Initiation of biological activity by Ts occurs after binding to nuclear receptors [5]. At least 70% of the liver nuclear bound Ts is derived from the extracellular compartment and the remainder from local, intracellular deiodination of T4 [6]. The liver, which contains -30% of the total T4 pool and 80% of all intracellularly located T4, is an important organ for the production of thyroid hormone metabolites [7]. Studies related to membranal transport of iodothyronines into hepatic cells are important since they may increase our understanding of regulatory mechanisms involved in the ultimate delivery of thyroid hormone to intracellular active sites like metabolizing enzymes and receptors. fluorescent T3 in cultured iibroblasts [14,15]. We report here on a difference in ATPdependency of the uptake of T3 on the one hand and that of T4 and rTa on the other by rat hepatocytes in primary culture. T4 and rTa showed the most remarkable diminution of transport by small decreases in cellular ATP content. In addition, effects of propranolol, X-ray contrast agents, amiodarone and cytoskeleton-disrupting agents have been studied. The results indicate that besides changes in T4 deiodination [ 1,2] and sulfoconjugation [ 16,171, decrease in cellular uptake of T4 by tissues secondary to decreased cellular ATP concentrations or to the effects of some compounds may be a contributing factor to the clinical condition known as low T3 syndrome. A preliminary account of this work has been published [ 181.

Localisation and mechanism of renal retention of radiolabelled somatostatin analogues

PurposeRadiolabelled 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. ResultsRenal 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.

Evaluation of radiolabelled bombesin analogues for receptor‐targeted scintigraphy and radiotherapy

The 14‐aminoacid peptide bombesin (BN) has a high affinity for the gastrin‐releasing peptide receptor which is expressed by a variety of tumours. Thus, radiometal‐labelled DTPA‐BN derivatives are potentially useful radioligands for receptor‐targeted scintigraphy and radiotherapy of BN receptor‐expressing tumours. A number of such DTPA‐BN analogues, [DTPA‐D‐Tyr6]BN(6–13)NHEt (Et=ethyl), [DTPA‐Tyr5,D‐Phe6]BN(5–13)NHEt, [DTPA‐D‐Phe6,Leu13ΨPhe14]‐BN(6–14), [DTPA‐Tyr5,D‐Phe6,Leu13ΨPhe14]BN(5–14), [DTPA‐Pro1,Tyr4]BN and [DTPA‐Pro1,Tyr4,Nle14]BN, were synthesized and studied for their binding characteristics to the BN receptor on 7315b rat pituitary tumour cell membranes in competition with [125I‐Tyr4]BN. The effects of the BN analogues were determined on basal and BN‐stimulated prolactin secretion by 7315b cells to distinguish between their agonistic and antagonistic characterisitics. Internalization of selected 111In‐labelled BN analogues was studied using the BN receptor‐positive 7315b pituitary tumour and the CA20948 and AR42J exocrine pancreas tumour cell lines. The tissue distribution of these 111In‐labelled BN analogues was investigated in 7315b tumour‐bearing rats. Two DTPA‐conjugated analogues, the antagonist [DTPA‐Tyr5,D‐Phe6]BN(5–13)NHEt and the agonist [DTPA‐Pro1,Tyr4]BN showed the highest affinity for the BN receptor on 7315b cell membranes. Despite similar affinity for the BN receptor, the 111In‐labelled agonist, but not the antagonist, was internalized by the BN receptor‐positive tumour cells. Consonant with this observation, the agonist [111In‐DTPA‐Pro1,Tyr4]BN showed much higher specific uptake in BN receptor‐positive tissues and tumour than the antagonist [111In‐DTPA‐Tyr5,D‐Phe6]BN(5–13)NHEt, with concordant target to background ratios. We conclude that [111In‐DTPA‐Pro1,Tyr4]BN has promising characteristics for applications in nuclear medicine. Int. J. Cancer 81:658–665, 1999.

Pre‐clinical evaluation of [111In‐DTPA‐Pro1, Tyr4]bombesin, a new radioligand for bombesin‐receptor scintigraphy

Bombesin (BN) is a 14‐amino‐acid neuropeptide with a high affinity for the gastrin‐releasing peptide receptor. This receptor has been found to be expressed in a variety of tumours, including lung, breast, prostate and pancreas. A newly synthesized BN analogue, [DTPA‐Pro1,Tyr4]BN, was shown to be a high‐affinity BN‐receptor (BNR) agonist, stimulating prolactin secretion from 7315b cells with an IC50 of 8 nM. The 111In‐labelled analogue was found to bind with high affinity to rat BNR in vitro and in vivo. The radioligand is internalized by BNR‐expressing cells, in contrast to DTPA‐conjugated BN antagonists. Therefore, we further studied the biodistribution of i.v. injected [111In‐DTPA‐Pro1,Tyr4]BN in rats. High and specific uptake was found in tissues of the gastrointestinal tract, notably pancreas. Uptake of radioactivity was blocked by pre‐ or co‐injection of 100 μg [Tyr4]BN, but not when this was administered 30 min after the radioligand. This suggests BNR‐mediated internalization of the radioligand within 30 min. The percentage injected dose (ID) taken up by BNR‐positive tissues was a bell‐shaped function of the amount (0.01–0.1 μg) of injected ligand. Next to the pancreas, highest uptake was observed in the kidneys, which was not blocked by excess [Tyr4]BN. Dynamic gamma camera studies showed rapid clearance of radioactivity from the blood compartment. Urinary excretion amounted to about 35% ID after 1 hr and to 70% ID after 24 hr, with a total body retention of 10% ID. Specific uptake was found in the BNR‐positive CA20948 pancreas tumour and CC531 colon carcinoma in tumour‐bearing rats. The CA20948 tumour, inoculated in the hindleg, was also visualized scintigraphically. [111In‐DTPA‐Pro1, Tyr4]BN appears to be a promising radioligand for scintigraphy of BNR‐expressing tumours. Int. J. Cancer 83:657–663, 1999

Evaluation in vitro and in rats of161Tb-DTPA-octreotide, a somatostatin analogue with potential for intraoperative scanning and radiotherapy

The characteristics of terbium-161 diethylene triamine penta-acetic acid (DTPA) labelled octreotide with respect to specific binding to somatostatin (octreotide) receptors on rat brain cortex membranes, biological activity, uptake and excretion by isolated perfused rat livers and metabolism in vivo in normal and tumour-bearing rats were determined and compared to those of indium-111 DTPA-octreotide. The results of the binding studies demonstrate that161Tb-DTPA-octreotide is a high-affinity radioligand for somatostatin receptors, with an affinity comparable to that of111In-DTPA-octreotide. Rat growth hormone secretion inhibition experiments showed that161Tb-DTPA-octreotide has a similar potency to111In-DTPA-octreotide.161Tb-DTPA-octreotide appeared to be taken up even less by the isolated perfused rat liver than111In-DTPA-octreotide, as almost no tracer disappeared from the perfusion medium. Furthermore, hardly any radioactivity was found in the liver, and excretion into the bile was negligible. The biodistribution studies showed that for octreotide receptor-positive organs, such as pancreas and adrenals, uptake of161Tb-DTPA-octreotide is lower then that of111In-DTPA-octreotide. However, as the clearance from the blood of the former compound is faster than that of the latter, the tissue/blood ratio is higher in the case of161Tb-DTPA-octreotide than with111In-DTPA-octreotide. Furthermore, these studies demonstrated that the uptake of161Tb-DTPA-octreotide by the renal tubular cells after glomerular filtration can be reduced by administration of lysine or sodium maleate. Increase in urine production before and during the experiment had no effect on the kidney uptake of161Tb-DTPA-octreotide. Finally, it appeared that a maximal labelling efficiency of161Tb-DTPA-octreotide is essential, as with decreasing efficiency the uptake in the octreotide receptor-positive organs decreased, whereas non-specific uptake in the other organs was increased. It is concluded that, on the basis of the favourable physical characteristics of161Tb combined with the in vitro and in vivo studies performed with161Tb-DTPA-octreotide, the latter is a promising radiopharmaceutical for both intraoperative scanning and radiotherapy. Studies in patients need to be performed now to see whether161Tb-DTPA-octreotide can indeed open new therapeutic applications for patients bearing octreotide receptor-positive tumours.