Wout A.P. Breeman

Somatostatin receptor scintigraphy with [111In-DTPA-d-Phe1]- and [123I-Tyr3]-octreotide: the Rotterdam experience with more than 1000 patients

Various tumours, classically specified as either neuroendocrine or non-neuroendocrine, contain high numbers of somatostatin receptors, which enable in vivo localization of the primary tumour and its metastases by scintigraphy with the radiolabelled somatostatin analogue octreotide. In addition granulomas and autoimmune processes can be visualized because of local accumulation of somatostatin receptor-positive activated mononuclear leucocytes. In many instances a positive scintigram predicts a favourable response to treatment with octreotide. It is tempting to speculate that octreotide labelled with an appropriate radionuclide might be used in cancer therapy. The successful application of radiolabelled octreotide in scintigraphy indicates the possible usefulness of other radiolabelled peptides, either native peptides or derivatives of these, in, for example, nuclear oncology. The small size of these peptides, e.g. bombesin and substance P, is of the utmost importance for a relatively fast blood clearance, thus leading to low background radioactivity. In this way peptides are powerful alternatives to (fragments of) monoclonal antibodies, the application of which to scintigraphic localization of specific cell surface antigen-bearing tumours is plagued by slow blood clearance and, hence, high background levels.

LOCALISATION OF ENDOCRINE-RELATED TUMOURS WITH RADIOIODINATED ANALOGUE OF SOMATOSTATIN

Various endocrine-related tumours contain large numbers of high-affinity somatostatin receptors. 123I-labelled tyr-3-octreotide (tyr-3-SMS 201-995, a synthetic derivative of somatostatin) was used to localise such tumours in vivo with a gamma-camera. Positive scans were obtained for two meningiomas, two gastrinomas, and one carcinoid; negative scans were obtained for one insulinoma (in which unlabelled octreotide had no effect on insulin levels), one phaeochromocytoma, one adrenal carcinoma (octreotide had no effect on cortisol levels), and three medullary thyroid carcinomas (octreotide had no effect on calcitonin levels). Thus radioiodinated tyr-3-octreotide can label somatostatin receptors in endocrine-related tumours in vivo and can therefore be used for tumour localisation.

[111In-DTPA-D-Phe1]-octreotide, a potential radiopharmaceutical for imaging of somatostatin receptor-positive tumors: synthesis, radiolabeling and in vitro validation.

Somatostatin receptor-positive human tumors can be detected using radioiodinated analogues of somatostatin, both in vitro and in vivo. [123I-Tyr3]-octreotide has been successfully used in the visualization of somatostatin receptor-positive tumors by gamma camera scintigraphy, but this radiopharmaceutical has some major drawbacks, which can be overcome with other radionuclides such as 111In. As starting material for a potentially convenient radiopharmaceutical, a diethylenetriaminopentaacetic acid (DTPA) conjugated derivative of octreotide (SMS 201-995) was prepared. This peptide, [DTPA-D-Phe1]-octreotide (SDZ 215-811) binds more than 95% of added 111In in an easy, single-step labeling procedure without necessity of further purification. The specific somatostatin-like biologic effect of these analogues was proven by the inhibition of growth hormone secretion by cultured rat pituitary cells in a dose-dependent fashion by octreotide, [DTPA-D-Phe1]-octreotide and non-radioactive [115In-DTPA-D-Phe1]-octreotide. The binding of [111In-DTPA-D-Phe1]-octreotide to rat brain cortex membranes proved to be displaced similarly by natural somatostatin as well as by octreotide, suggesting specific binding of [111In-DTPA-D-Phe1]-octreotide to somatostatin receptors. The binding of the indium-labeled compound showed a somewhat lower affinity when compared with the iodinated [Tyr3]-octreotide, but indium-labeled [DTPA-D-Phe1]-octreotide still binds with nanomolar affinity. In conjunction with in vivo studies, these results suggest that [111In-DTPA-D-Phe1]-octreotide is a promising radiopharmaceutical for scintigraphic imaging of somatostatin receptor-positive tumors.

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.

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.

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.

Intraoperative nuclear guidance in benign hyperparathyroidism and parathyroid cancer.

The success of parathyroid surgery is determined by the identification and removal of all hyperactive parathyroid tissue. Ectopic location of parathyroid tumours and fibrosis due to previous operations can cause failure of parathyroidectomy. Parathyroid tumours accumulate and retain 2-methoxyisobutylisonitrile (MIBI) labelled with technetium-99m. This study assesses the value of intra-operative localization of parathyroid tumours using a hand-held gamma detector in patients with hyperparathyroidism and parathyroid cancer. Twenty patients undergoing their first operations for hyperparathyroidism, 15 patients undergoing reoperations for either persistent or recurrent hyperparathyroidism and two patients with parathyroid cancer were studied. Radioactivity in the neck and the mediastinum was recorded by a gamma detector after administration of 370 MBq99mTc-MIBI. Surgical findings and postoperative serum levels of calcium were documented. The sensitivity of the gamma detector in identifying parathyroid tumours was 90.5% in first parathyroidectomies, 88.9% in reoperations for either persistent or recurrent hyperparathyroidism and 100% in parathyroid cancer. One false-positive result was due to a thyroid nodule. Hypercalcaemia ceased in all but one patient postoperatively. It is concluded that employment of the gamma detector is to be advocated in first parathyroidectomies when a parathyroid tumour cannot be discovered, in reoperations for either persistent or recurrent hyperparathyroidism and in surgery for parathyroid cancer.