Internalization of the radioiodinated somatostatin analog [125I-Tyr3]octreotide by mouse and human pituitary tumor cells: increase by unlabeled octreotide

Recently, we developed a technique that allows the in vivo visualization in man of somatostatin receptor-positive neuroendocrine tumors after i.v. injection of [125I-Tyr3]octreotide or [111In-DTPA-D-Phe1]octreotide. Radiotherapy of such tumors using somatostatin analogs coupled to alpha- or beta-emitting radionuclides has been proposed as an application for radiolabeled somatostatin analogs. To develop this concept further, it is of importance to know whether the above-mentioned radiolabeled somatostatin analogs are internalized by the tumor cells, and whether it might be possible to manipulate the degree of internalization. In the present study we investigated the internalization of a stable somatostatin analog, [125I-Tyr3]octreotide, by mouse AtT20/D16V pituitary tumor cells and primary cultures of human GH-secreting pituitary tumor cells. Treatment of the cells with low pH was used to distinguish between membrane-bound (acid-releasable) and internalize (acid-resistant) radioligand. [125I-Tyr3]octreotide showed a time-dependent increasing accumulation in AtT20 cells; after 4 h of incubation, values up to 6-8% of the dose of radioligand added were obtained. Binding and internalization of [125I-Tyr3]octreotide were temperature dependent and inhibited by pertussis toxin. Inhibitors of lysosomal degradation did not increase the amount of internalized radioligand. After 4 h of incubation, 88% of the radioactivity present in the cells was still peptide bound, suggesting a low intracellular breakdown of this radioligand. Six of seven human GH-secreting adenoma cell cultures also internalized [125I-Tyr3]octreotide (variation between 0.24-4.98% of the dose radioligand added). Displacement of binding and internalization of [125I-Tyr3]octreotide by unlabeled octreotide showed a bell-shaped curve in AtT20 cells. At low concentrations (0.1 and 1 nM), binding and internalization were increased, whereas at higher concentrations, saturation occurred. In contrast to this, binding of [125I-Tyr3]octreotide to a broken cell preparation of AtT20 cells was displaced in a dose-dependent manner by unlabeled octreotide, with an IC50 of 0.1 nM. Similar observations were made in the human GH-secreting adenoma cell cultures. In conclusion, a high amount of [125I-Tyr3]octreotide is internalized in a specific-, time-, temperature-, and pertussis toxin-sensitive GTP-binding protein-dependent manner by mouse AtT20 and human GH-secreting pituitary tumor cells. In the presence of a low concentration of unlabeled octreotide, a rapid increase in the amount of [125I-Tyr3]octreotide internalized by AtT20 cells and by the majority of the human GH-secreting adenoma cell cultures was found.(ABSTRACT TRUNCATED AT 400 WORDS

Iodine-131 labelled octreotide: Not an option for somatostatin receptor therapy

Gamma-emitting radiopeptides are useful for scintigraphy of tumours on the basis of receptor binding. Likewise, β-emitting radiopeptides may be used in radionuclide therapy of such tumours. As iodine-131 suggested to be suitable for this purpose, experiments were performed using three somatostatin analogues, in which the effects of coupling of a therapeutic dose of 131I to such peptides were investigated. This study deals with the radioiodination of very small amounts of peptide on a therapeutic scale, the required purification procedures after radioiodination, and the influence of high beta fluxes from 131I. On a peptide during radioiodination and purification. Based on the regularly used therapeutic doses of 131I in cancer treatment and our previous experience with [111In-DTPA-D-Phe1]-octreotide, it was assumed that a minimal effective therapeutic dose of 3.7 GBq 131I has to be coupled to a maximum of ≃ 100 μg peptide, representing only a slight excess of peptide over 131I. This contrasts with non-peptide radiopharmaceuticals in which high compound to radionuclide ratios are usually used. Labelling at low peptide to radionuclide ratios (low labelling yields) results in the formation of di-iodinated compounds, whereas at high peptide to radionuclide ratios (high labelling yields) mono-iodinated products of low specific activity are formed. Thus, after radioiodination the desired mono-iodinated peptide has to be separated from unreacted iodide, and from di-iodinated and unreacted peptide, as both compounds compete for the receptors. Possible radiolysis of the peptide during labelling and separation steps were investigated by irradiating 30 μg unlabelled peptide with 370 MBq 131I in a small volume. The peptide composition of the incubation mixtures was investigated by high-performance liquid chromatography after irradiation for 30 min to 24 h. The results showed that the peptide was degraded with a half-life of less than 1 h. During the preparation of a real therapeutic dose (at much higher β-flux) the peptide will be degraded even faster during the various steps required. In conclusion, intact mono-iodinated 131I-labelled somatostatin analogues for peptide receptor therapy will be difficult to obtain

Maintaining radiochemical purity of [177Lu]Lu-DOTA-PSMA-617 for PRRT by reducing radiolysis

[177Lu]Lu-DOTA-PSMA-617 for PRRT is subject to radiolysis and therefore loses receptor affinity. This will be detrimental for treatment efficacy. In this study optimal quencher(s) (combinations) are determined to maintain radiochemical purity with a downscaled model. Downscaled model in terms of activity, but at similar concentrations. DOTA-PSMA-617 was labeled with [177Lu]LuCl3 with different molar- and volume activities. Either methionine, ethanol or both showed superior effects on the stabilizing radiochemical purity of [177Lu]Lu-DOTA-PSMA-617. As a consequence, radiochemical purity of [177Lu]Lu-DOTA-PSMA-617 could be maintained by the addition of methionine and/or ethanol and downscaled model was proven and complementary

Semi-automated system for concentrating 68Ga-eluate to obtain high molar and volume concentration of 68Ga-Radiopharmaca for preclinical applications

Introduction: 68Ga-radiopharmaceuticals are common in the field of Nuclear Medicine to visualize receptor-mediated processes. In contrast to straightforward labeling procedures for clinical applications, preclinical in vitro and in vivo applications are hampered for reasons like e.g. volume restriction, activity concentration, molar activity and osmolality. Therefore, we developed a semi-automatic system specifically to overcome these problems. A difficulty appeared unexpectedly, as intrinsic trace metals derived from eluate (Zn, Fe and Cu) are concentrated as well in amounts that influence radiochemical yield and thus lower molar activity. Methods: To purify Gallium-68 and to reduce the high elution volume of a 68Ga-generator, a NaCl-based method using a column containing PS-H+ was implemented in a low volume PEEK system. Influence on reducing osmolality, acidity and the amount of PS-H+ resin (15–50 mg) was investigated. [68Ga]Ga was desorbed from the PS-H+ resin with acidified 2-5 M NaCl (containing 0.05 M of HCl) and 68Ga-activity was collected. DOTA-TATE was used as a peptide model. All buffers and additives used for labeling were mixed with Chelex 100 (~1 g/50 mL) for >144 h and eventually filtered using a 0.22 μm filter (Millipore). Quantification of metals was performed after labeling by HPLC (UV). Results: Gallium-68 activity could be desorbed from PS-H+ cation column with 3 M NaCl, and >60% (120–180 MBq) of [68Ga]Ga was collected in 99% (ITLC), and a radiochemical purity of >95% (HPLC). Conclusion: With the here described concentration system and metal purification technique, a low activity containing 68Ga-generator can be used to label DOTA-peptide in preclinical applicable amounts >60 MBq/nmol (40–60 MBq/0.1 mL) and within 20 min

Influence of tumour size on the efficacy of targeted alpha therapy with 213Bi-[DOTA0,Tyr3]-octreotate

BACKGROUND: Targeted alpha therapy has been postulated to have great potential for the treatment of small clusters of tumour cells as well as small metastases. (213)Bismuth, an α-emitter with a half-life of 46 min, has shown to be effective in preclinical as well as in clinical applications. In this study, we evaluated whether (213)Bi-[DOTA(0), Tyr(3)]-octreotate ((213)Bi-DOTATATE), a (213)Bi-labelled somatostatin analogue with high affinity for somatostatin receptor subtype 2 (SSTR(2)), is suitable for the treatment of larger neuroendocrine tumours overexpressing SSTR(2) in comparison to its effectiveness for smaller tumours. We performed a preclinical targeted radionuclide therapy study with (213)Bi-DOTATATE in animals bearing tumours of different sizes (50 and 200 mm(3)) using two tumour models: H69 (human small cell lung carcinoma) and CA20948 (rat pancreatic tumour). METHODS: Pharmacokinetics was determined for calculation of dosimetry in organs and tumours. H69- or CA20948-xenografted mice with tumour volumes of approximately 120 mm(3) were euthanized at 10, 30, 60 and 120 min post injection of a single dose of (213)Bi-DOTATATE (1.5–4.8 MBq). To investigate the therapeutic efficacy of (213)Bi-DOTATATE, xenografted H69 and CA20948 tumour-bearing mice with tumour sizes of 50 and 200 mm(3) were administered daily with a therapeutic dose of (213)Bi-DOTATATE (0.3 nmol, 2–4 MBq) for three consecutive days. The animals were followed for 90 days after treatment. At day 90, mice were injected with 25 MBq (99m)Tc-DMSA and imaged by SPECT/CT to investigate possible renal dysfunction due to (213)Bi-DOTATATE treatment. RESULTS: Higher tumour uptakes were found in CA20948 tumour-bearing animals compared to those in H69 tumour-bearing mice with the highest tumour uptake of 19.6 ± 6.6 %IA/g in CA20948 tumour-bearing animals, while for H69 tumour-bearing mice, the highest tumour uptake was found to be 9.8 ± 2.4 %IA/g. Nevertheless, as the anti-tumour effect was more pronounced in H69 tumour-bearing mice, the survival rate was higher. Furthermore, in the small tumour groups, no regrowth of tumour was found in two H69 tumour-bearing mice and in one of the CA20948 tumour-bearing mice. No renal dysfunction was observed in (213)Bi-DOTATATE-treated mice after the doses were applied. CONCLUSIONS: (213)Bi-DOTATATE demonstrated a great therapeutic effect in both small and larger tumour lesions. Higher probability for stable disease was found in animals with small tumours. (213)Bi-DOTATATE was effective in different neuroendocrine (H69 and CA20948) tumour models with overexpression of SSTR(2) in mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-016-0162-2) contains supplementary material, which is available to authorized users

Therapeutic application of CCK2R-targeting PP-F11: influence of particle range, activity and peptide amount

Background: Targeted radionuclide therapy with high-energy beta-emitters is generally considered suboptimal to cure small tumours (90Y, 177Lu or 213Bi, accounting for the radionuclide specific activities (SAs), the tumour absorbed doses and tumour (radio) biology. Methods: Tumour uptake of 111In-PP-F11 was determined in nude mice bearing CCK2 receptor-transfected A431 xenografts at 1 and 4 h post-injection for escalating peptide masses of 0.03 to 15 nmol/mouse. The absorbed tumour dose was estimated, assuming comparable biodistributions of the 90Y, 177Lu or 213Bi radiolabelled peptides. The linear-quadratic (LQ) model was used to calculate the tumour control probabilities (TCP) as a function of tumour mass and growth. Results: Practically achievable maximum SAs for PP-F11 labelled with 90Y and 177Lu were 400 MBq 90Y/nmol and 120 MBq177Lu/nmol. Both the large elution volume from the 220 MBq 225Ac generator used and reaction kinetics diminished the maximum achieved 213Bi SA in practice: 40 MBq 213Bi/nmol. Tumour uptakes decreased rapidly with increasing peptide amounts, following a logarithmic curve with ED50 = 0.5 nmol. At 0.03 nmol peptide, the (300 mg) tumour dose was 9 Gy after 12 MBq 90Y-PP-F11, and for 111In and 177Lu, this was 1 Gy. A curative dose of 60 Gy could be achieved with a single administration of 111 MBq 90Y labelled to 0.28 nmol PP-F11 or with 4 × 17 MBq 213Bi (0.41 nmol) when its α-radiation relative biological effectiveness (RBE) was assumed to be 3.4. Repeated dosing is preferable to avoid complete tumour receptor saturation. Tumours larger than 200 mg are curable with 90Y-PP-F11; the other radionuclides perform better in smaller tumours. Furthermore, 177Lu is not optimal for curing fast-growing tumours. Conclusions: Receptor saturation, specific radiopharmaceutical activities and absorbed doses in the tumour together favour therapy with the CCK2 receptor-binding peptide PP-F11 labelled with 90Y, despite its longer β-particle range in tissue, certainly for tumours larger than 300 mg. The predicted TCPs are of theoretical nature and need to be compared with the outcome of targeted radionuclide experiments

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

Radioiodinated somatostatin analogue RC-160: preparation, biological activity, in vivo application in rats and comparison with [123I-Tyr3]octreotide

We have evaluated the potential usefulness of the radioiodinated octapeptide RC-160, a somatostatin analogue, which might serve as a radiopharmaceutical for the in vivo detection of somatostatin receptor-positive tumours. For this purpose, iodine-123 and iodine-125 labelled RC-160 was tested for biological activity and applied in vivo in rats bearing the transplantable rat pancreatic tumour CA20948, which expresses somatostatin receptors. Our group has recently described the in vivo visualization of such tumours in rats and in humans with the radioiodinated somatostatin analogue [Tyr3]octreotide. Like [123I-Tyr3]octreotide, 123I-RC-160 showed uptake in and specific binding in vivo to somatostatin receptor-positive organs and tumours. However, blood radioactivity (background) was higher, resulting in a lower tumour to blood (background) ratio. We therefore conclude that in this animal model 123I-RC-160 has no advantage over [123I-Tyr3]octreotide as a radiopharmaceutical for the in vivo use as a somatostatin receptor imager, although, like [123I-Tyr3]octreotide, 123I-RC-160 shows specific binding to different somatostatin receptor-positive organs. Recently differences were reported in affinity between somatostatin and its analogues for somatostatin receptors expressed in different human cancers, like those of the breast, ovary, exocrine pancreas, prostate and colon. Therefore 123I-RC-160 might be of interest for future use in humans as a radiopharmaceutical for imaging octreotide receptor-negative tumours