Bismuth chelate as a contrast agent for X-ray computed tomography.

BACKGROUNDS: Due to the unexpected side effects of the iodinated contrast agents, novel contrast agents for X-ray computed tomography (CT) imaging are urgently needed. Nanoparticles made by heavy metal elements are often employed, such as gold and bismuth. These nanoparticles have the advantages of long in vivo circulation time and tumor targeted ability. However, due to the long residence time in vivo, these nanoparticles may bring unexpected toxicity and, the preparation methods of these nanoparticles are complicated and time-consuming. METHODS: In this investigation, a small molecular bismuth chelate using diethylenetriaminepentaacetic acid (DPTA) as the chelating agent was proposed to be an ideal CT contrast agent. RESULTS: The preparation method is easy and cost-effective. Moreover, the bismuth agent show better CT imaging for kidney than iohexol in the aspect of improved CT values. Up to 500 µM, the bismuth agent show negligible toxicity to L02 cells and negligible hemolysis. And, the bismuth agent did not induce detectable morphology changes to the main organs of the mice after intravenously repeated administration at a high dose of 250 mg/kg. The pharmacokinetics of the bismuth agent follows the first-order elimination kinetics and, it has a short half-life time of 0.602 h. The rapid clearance from the body promised its excellent biocompatibility. CONCLUSIONS: This bismuth agent may serve as a potential candidate for developing novel contrast agent for CT imaging in clinical applications.

Improved Modeling of Plutonium-DTPA Decorporation.

Individuals with significant intakes of plutonium (Pu) are typically treated with chelating agents, such as the trisodium salt form of calcium diethylenetriaminepentaacetate (CaNa3-DTPA, referred to hereafter as Ca-DTPA). Currently, there is no recommended approach for simultaneously modeling plutonium biokinetics during and after chelation therapy. In this study, an improved modeling system for plutonium decorporation was developed. The system comprises three individual model structures describing, separately, the distinct biokinetic behaviors of systemic plutonium, intravenously injected Ca-DTPA and in vivo-formed Pu-DTPA chelate. The system was linked to ICRP Publication 100, "Human Alimentary Tract Model for Radiological Protection" and NCRP Report 156, Development of a Biokinetic Model for Radionuclide-Contaminated Wounds and Procedures for Their Assessment, Dosimetry and Treatment." Urine bioassay and chelation treatment data from an occupationally-exposed individual were used for model development. Chelation was assumed to occur in the blood, soft tissues, liver and skeleton. The coordinated network for radiation dosimetry approach to decorporation modeling was applied using a chelation constant describing the secondorder, time-dependent kinetics of the in vivo chelation reaction. When using the proposed system of models for plutonium decorporation, a significant improvement of the goodness-of-fit to the urinary excretion data was observed and more accurate predictions of postmortem plutonium retention in the skeleton, liver and wound site were achieved.

Cuidado em saúde em adultos com hipertensão arterial autorreferida no Brasil segundo dados da Pesquisa Nacional de Saúde, 2013 / Health care in adults with self-reported hypertension in Brazil according to the National Health Survey, 2013

RESUMO: Objetivo: Descrever indicadores de cuidado em saúde em adultos com hipertensão arterial autorreferida no Brasil, segundo características sociodemográficas. Métodos: Foram utilizados dados da Pesquisa Nacional de Saúde 2013, estudo transversal de base populacional, referentes ao cuidado em saúde com a hipertensão arterial autorreferida quanto ao uso de serviços de saúde. As prevalências e seus intervalos de confiança de 95% (IC95%) foram calculados segundo sexo, idade, raça/cor e escolaridade, representativos para Brasil e grandes regiões. Resultados: A hipertensão arterial foi referida por 21,4% (IC95% 20,8 - 22,0) dos entrevistados, sendo maior em mulheres e em pessoas sem instrução e com ensino fundamental incompleto. Dentre estes, 45,9% (IC95% 44,0 - 47,7) referiram ter recebido assistência médica pela última vez em uma Unidade Básica de Saúde; 81,4% (IC95% 80,1 - 82,7) referiram tomar medicamentos para a hipertensão; e 92,0% (IC95% 91,2 - 92,8) referiram ter realizado todos os exames complementares que foram solicitados. Conclusão: É importante conhecer a cobertura e o acesso aos serviços de saúde para o cuidado aos indivíduos com hipertensão arterial, de modo a avançar na qualidade da assistência prestada e reduzir as desigualdades identificadas.

ABSTRACT: Objective: To describe health care indicators in adults with self-reported hypertension in Brazil, according to socio-demographic characteristics. Methods: Data from the National Health Survey 2013, a cross-sectional population-based study, about health care of self-reported hypertension and health services were used. Prevalence and 95% confidence intervals (95%CI) were calculated for sex, age, race/color skin and schooling levels, representing Brazil and major regions. Results: Hypertension was reported by 21.4% (95%CI 20.8 - 22.0) of respondents, being higher in women and in people without instruction and incomplete middle school. Among these, 45.9% (95%CI 44.0 - 47.7) reported having received medical care for the last time in a basic health unit; 81.4% (95%CI 80.1 - 82.7) reported taking medication for high blood pressure; and 92.0% (95%CI 91.2 - 92.8) reported having taken all requested complementary examinations. Conclusion: It is important to know the coverage and access to health services for the care of patients with hypertension, in order to improve care quality and reduce identified inequalities.

Development of the Plutonium-DTPA Biokinetic Model.

Estimating radionuclide intakes from bioassays following chelation treatment presents a challenge to the dosimetrist due to the observed excretion enhancement of the particular radionuclide of concern where no standard biokinetic model exists. This document provides a Pu-DTPA biokinetic model that may be used for making such determination for plutonium intakes. The Pu-DTPA biokinetic model is intended to supplement the standard recommended biokinetic models. The model was used to evaluate several chelation strategies that resulted in providing recommendations for effective treatment. These recommendations supported early treatment for soluble particle inhalations and an initial 3-day series of DTPA treatments for wounds. Several late chelation strategies were also compared where reduced treatment frequencies proved to be as effective as multiple treatments. The Pu-DTPA biokinetic model can be used to assist in estimating initial intakes of transuranic radionuclides and for studying the effects of different treatment strategies.

Preclinical evaluation of monoclonal antibody 14C5 for targeting pancreatic cancer.

The use of radiolabeled antibodies that are able to target primary tumors as well as metastatic tumor sites with minimal reactivity to normal tissues is a promising approach for treating pancreatic cancer. In this study, the integrin alpha(v)beta(5) is studied as a target for the diagnosis of and potential therapy for human pancreatic cancer by using the radiolabeled murine monoclonal antibody (mAb) 14C5. Biopsy specimens from human pancreatic tumors were examined for the expression of the integrin alpha(v)beta(5). The pancreatic tumor cell line Capan-1 was used to test the in vitro targeting potency of mAb 14C5 labeled with 125/131-iodine and 111-indium. Internalization, retention, and metabolism were investigated in cellular radioimmunoassays. Biodistribution and tumor-targeting characteristics were studied in Capan-1 xenografts. All tumor sections were positive for the integrin alpha(v)beta(5), with an extensive positive staining of the stroma. Saturation binding experiments showed high affinity with comparable K(d)s. In vitro internalization experiments showed a longer intracellular retention of (111)In-p-benzyl isothiocyanate-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA)-14C5 in comparison to (125)I-14C5 and (111)In-p-isothiocyanatobenzyl diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA)-14C5. In vivo radioisotope tumor uptake was maximum at 48-72 hours, with the uptake of (111)In-p-SCN-Bz-DOTA-14C5 (35.84 +/- 8.64 percentage of injected dose per g [%ID/g]) being 3.9- and 2.2-folds higher than (131)I-14C5 (12.16 +/- 1.03%ID/g) and (111)In-p-SCN-Bz-DTPA-14C5 (14.30 +/- 3.76%ID/g), respectively. Planar gamma imaging with mAb 14C5 indicated clear localization of the pancreatic tumors versus minimal normal tissue uptake. mAb 14C5 is a promising new antibody for targeting the integrin alpha(v)beta(5) for the diagnosis of and potential therapy for pancreatic cancer.

Radioimmunotherapy prevents local recurrence of colonic cancer in an experimental model.

BACKGROUND: Radioimmunotherapy (RIT) is suitable for the treatment of microscopic residual disease and might therefore have an adjuvant role after colonic cancer surgery. METHODS: An anastomosis was constructed in male Wag/Rij rats after intraluminal injection of 2 x 10(6) CC531 tumour cells. The biodistribution of (111)In-labelled MG1 monoclonal antibody was assessed after intraperitoneal administration. The therapeutic efficacy of (177)Lu-labelled MG1 (74 MBq per rat), administered on the day of surgery (D0, n = 13) or 5 days later (D5, n = 13), was compared with that of carrier only (n = 13). The primary endpoint was perianastomotic tumour growth 28 days after surgery. RESULTS: (111)In-labelled MG1 preferentially accumulated in perianastomotic CC531 tumours. RIT resulted in a transient reduction in bodyweight in both treatment groups compared with controls, but there were no other signs of clinical discomfort. No macroscopic or microscopic perianastomotic tumour growth was found in eight of 11 animals in the D0 group and 11 of 13 in the D5 group, whereas 11 of 13 controls had macroscopic tumour (P = 0.011 and P = 0.001 respectively). CONCLUSION: This study suggests that RIT may be an effective adjuvant treatment for preventing local recurrence after resection of colonic cancer.

Pre-clinical evaluation of [111In]-benzyl-DOTA-Z(HER2:342), a potential agent for imaging of HER2 expression in malignant tumors.

Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma BT474 and ovarian carcinoma SKOV-3 cell lines. Biodistribution of 111In-benzyl-DOTA-Z(HER2:342) was performed in nude mice bearing LS174T xenografts and compared directly with the biodistribution of 111In-benzyl-DTPA-Z(HER2:342). In vivo, 111In-benzyl-DOTA-Z(HER2:342) demonstrated quick clearance from blood and non-specific organs except the kidneys. Four hours post injection (pi), the tumor uptake of 111In-benzyl-DOTA-Z(HER2:342) (4.4+/-1.0% IA/g) was specific and the tumor-to-blood ratio was 23. The use of benzyl-DTPA provided higher tumor-to-blood and tumor-to-liver ratios. gamma-camera imaging showed clear visualization of HER2-expressing xenografts using 111In-benzyl-DOTA-Z(HER2:342). 111In-benzyl-DOTA-Z(HER2:342) has a potential for imaging of HER2 expression in malignant tumors.

Preclinical pharmacokinetic, biodistribution, toxicology, and dosimetry studies of 111In-DTPA-human epidermal growth factor: an auger electron-emitting radiotherapeutic agent for epidermal growth factor receptor-positive breast cancer.

UNLABELLED: Our objective was to evaluate the pharmacokinetics, normal tissue distribution, radiation dosimetry, and toxicology of human epidermal growth factor (hEGF) labeled with (111)In ((111)In-diethylenetriaminepentaacetic acid [DTPA]-hEGF) in mice and rabbits. METHODS: (111)In-DTPA-hEGF (3.6 MBq; 1.3 or 13 microg) was administered intravenously to BALB/c mice. The blood concentration-time data were fitted to a 3-compartment model. Acute toxicity was studied with female BALB/c mice at 42 times the maximum planned human dose (MBq/kg) or with New Zealand White rabbits at 1 times the maximum planned human dose (MBq/kg) for a phase I clinical trial. Toxicity was evaluated by monitoring body weight, by determination of hematology and clinical biochemistry parameters, and by morphologic examination of tissues. Radiation dosimetry projections in humans were estimated on the basis of the residence times in mice by use of the OLINDA version 1.0 computer program. RESULTS: The largest amounts of radioactivity were taken up by the liver (41.3 +/- 7.8 [mean +/- SD] percentage injected dose [%ID] at 1 h after injection and decreasing to 4.9 +/- 0.3 %ID at 72 h after injection) and kidneys (18.6 +/- 0.8 %ID at 1 h and decreasing to 4.5 +/- 0.2 %ID at 72 h after injection). (111)In-DTPA-hEGF was cleared rapidly from the blood, with a half-life at alpha-phase of 2.7-6.2 min and a half-life at beta-phase of 24.0-36.3 min. The half-life of the long terminal phase could not be accurately determined. The volume of distribution of the central compartment was 340-375 mL/kg, and the volume of distribution at steady state was 430-685 mL/kg. There was no significant difference in the ratio of body weight at 15 d to pretreatment weight for mice administered (111)In-DTPA-hEGF (1.02 +/- 0.01) and mice administered unlabeled DTPA-hEGF (1.01 +/- 0.01). Erythrocyte, leukocyte, and platelet counts and serum alanine aminotransferase and creatinine levels remained in the normal ranges. No morphologic changes were observed by light microscopy in any of 19 tissues sampled. Minor morphologic changes in the liver were observed by electron microscopy. The projected whole-body dose in humans was 0.19 mSv.MBq(-1). The projected doses to the liver, kidneys, and lower large intestine were 0.76, 1.82, and 1.12 mSv.MBq(-1), respectively. CONCLUSION: (111)In-DTPA-hEGF was safely administered to mice and rabbits at multiples of the maximum dose planned for a phase I trial in breast cancer patients.

111In-labelled somatostatin analogues in a rat tumour model: somatostatin receptor status and effects of peptide receptor radionuclide therapy.

PURPOSE: Peptide receptor scintigraphy with the radioactive somatostatin analogue 111In-DTPA-octreotide is a sensitive and specific technique to show in vivo the presence of somatostatin receptors on various tumours. Since 111In emits not only gamma rays but also therapeutic Auger and internal conversion electrons with a medium to short tissue penetration (0.02-10 microm and 200-550 microm, respectively), 111In-DTPA-octreotide is also being used for peptide receptor radionuclide therapy (PRRT). In this study we investigated the therapeutic effects of 111In-DTPA-octreotide in tumours of various sizes. Regrowth of a tumour despite PRRT with 111In-DTPA-octreotide may be due to the lack of crossfire from 111In, whereby any possible receptor-negative tumour cell can multiply. We therefore also investigated the somatostatin receptor status of the tumour before and after PRRT. METHODS: The radiotherapeutic effects of different doses of 111In-DTPA-octreotide in vivo were investigated in Lewis rats bearing small (< or = 1 cm2) or large (> or = 8 cm2) somatostatin receptor-positive rat pancreatic CA20948 tumours expressing the somatostatin receptor subtype 2 (sst2). In addition, the somatostatin receptor density on the tumour after injection of a therapeutic labelled somatostatin analogue was investigated when the tumour was either declining in size or regrowing after an initial reduction in size. To initiate a partial response of the tumour (so that regrowth would follow) and not a complete response, a relatively low dose was administered. RESULTS: Impressive radiotherapeutic effects of 111In-labelled octreotide were observed in this rat tumour model. Complete responses (up to 50%) were found in the animals bearing small (< or 1 cm2) tumours after at least three injections of 111 MBq or a single injection of 370 MBq 111In-DTPA-octreotide, leading to a dose of 6.3-7.8 mGy/MBq (1-10 g tumour). In the rats bearing the larger (> or = 8 cm2) tumours, the effects were much less pronounced and only partial responses were achieved in these groups. Clear sst2 expression was found in the control as well as in the treated tumours. A significantly higher tumour receptor density (p<0.001) was found when the tumours regrew after an initial decline in size after low-dose PRRT in comparison with the untreated tumours. CONCLUSION: Therapy with 111In-labelled somatostatin analogues is feasible but should preferably start as early as possible during tumour development. One might also consider the use of radiolabelled somatostatin analogues in an adjuvant setting after surgery of somatostatin receptor-positive tumours in order to eradicate occult metastases. We showed that PRRT led to an increase in the density of somatostatin receptors when the tumours regrew after an initial decline in size because of PRRT. Upregulation of the somatostatin receptor may lead to higher uptake of radiolabelled peptides in therapeutic applications, which would probably make repeated injections of radiolabelled peptides more effective.

[177Lu]Bz-DTPA-EGF: Preclinical characterization of a potential radionuclide targeting agent against glioma.

Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.

Antitumor effects and normal tissue toxicity of 111In-labeled epidermal growth factor administered to athymic mice bearing epidermal growth factor receptor-positive human breast cancer xenografts.

UNLABELLED: The epidermal growth factor receptor (EGFR) is an attractive target for the design of radiotherapeutic agents for breast cancer because it is present on almost all estrogen receptor-negative, hormone-resistant tumors with a poor prognosis. In this study, we describe the antitumor effects and normal tissue toxicity of the novel Auger electron-emitting radiopharmaceutical (111)In-labeled diethylenetriaminepentaacetic acid-human epidermal growth factor ((111)In-DTPA-hEGF) administered to athymic mice bearing EGFR-positive human breast cancer xenografts. METHODS: Mice bearing subcutaneous MDA-MB-468 or MCF-7 human breast cancer xenografts were treated with 5 weekly doses of (111)In-DTPA-hEGF (total, 27.7-92.5 MBq or 5-17 micro g). Treatment was commenced 6 wk after tumor cell implantation (established tumors) or 1 wk after implantation (nonestablished tumors). Antitumor effects were assessed by use of the slope of the tumor growth curve. Normal tissue toxicity was assessed by use of plasma alanine transaminase and creatinine levels, hematologic indices (leukocytes, platelets, erythrocytes, and hemoglobin), histopathologic examination of the liver and kidneys, and changes in body weight. The uptake of (111)In-DTPA-hEGF in tumors of different sizes (<5-200 mm(3)) was investigated, and microdosimetry estimates were calculated. RESULTS: (111)In-DTPA-hEGF exhibited strong antitumor effects against established MDA-MB-468 xenografts, decreasing their growth rate 3-fold compared with that in normal saline-treated mice (slopes, 0.0225 and 0.0737 d(-1), respectively; P = 0.002). The antitumor effects of (111)In-DTPA-hEGF were much more profound in mice with small, nonestablished MDA-MB-468 tumors, which regressed, than in saline-treated mice (slopes, -0.009 and 0.0297 d(-1), respectively; P < 0.001). The growth of MCF-7 xenografts, with a 100-fold-lower level of EGFR expression, was modestly inhibited by (111)In-DTPA-hEGF compared with that in saline-treated mice (slopes, 0.0250 and 0.0488 d(-1), respectively; P = 0.051). There was a 1.4- to 2-fold decrease in leukocyte and platelet counts with (111)In-DTPA-hEGF treatment, but these counts remained in the normal ranges. There was no change in other biochemical or hematologic parameters or body weight. There was no evidence of morphologic damage to the liver or kidneys. A strong inverse relationship was observed between radiopharmaceutical uptake and tumor size, with small tumors (<5 mm(3)) accumulating >30% of the injected dose (%ID) per gram, compared with 5 %ID/g for tumors measuring 6-30 mm(3). Exceptionally high uptake (>80 %ID/g) was achieved in tumors measuring 1-2 mm(3). Microdosimetry estimates indicated that the nucleus of an MDA-MB-468 cell would receive 90-1,400 cGy, depending on the level of radiopharmaceutical uptake. CONCLUSION: (111)In-DTPA-hEGF exhibited strong antitumor effects against MDA-MB-468 breast cancer xenografts overexpressing EGFR. The highest tumor localization, radiation-absorbed doses, and growth inhibition were achieved for small, nonestablished tumors, suggesting that the radiopharmaceutical may be most valuable for the treatment of small-volume metastatic breast cancer or occult micrometastases in an adjuvant setting.

Preclinical studies of [(99m)Tc]DTPA-mannosyl-dextran .

We report the preclinical testing of a synthetic receptor-binding macromolecule, [(99m)Tc]DTPA-mannosyl-dextran (36 kDa, 8 DTPA and 55 mannosyl units per dextran, K(D) = 0.12 nM), for sentinel node detection. Nonclinical safety studies included cardiac pharmacology safety studies, acute toxicology and pathology studies at 50 and 500 times the scaled human dose in both rats and rabbits after foot pad administration, and perivascular irritation studies in rabbits following intra-muscular administration at 100 and 1000 times the scaled human dose. Biodistribution studies in rabbits at 15 m, 1 h, and 3 h indicated that [(99m)Tc]DTPA-mannosyl-dextran cleared the hind foot pad with a biological half-life of 2.21 +/- 0.27 h. Other than mild hepatocyte hypertrophy in rabbits, no abnormalities in toxicology or pathology were found. Intravenous administration had no effect on survival, any clinical observations, electrocardiograms, or blood pressures. Intramuscular injection had no effect on survival, clinical observations, injection site observations, or injection site histopathology. The estimated absorbed radiation dose to the affected breast was 0.15 mGy/MBq and the effective dose was 1.06 x 10(-2) mSv/MBq. This preclinical study demonstrates that [(99m)Tc]DTPA-mannosyl-dextran has no toxicities and has an acceptable biodistribution and radiation dose.

Evaluation of folate conjugate uptake and transport by the choroid plexus of mice.

PURPOSE: Because the choroid plexus (CP) is enriched in cell surface folate receptors, an investigation was initiated to evaluate whether folate receptor-mediated transcytosis might be exploited to deliver folate conjugates into the brain. METHODS: Balb/c mice were injected with radioactive and fluorescent conjugates of folate to measure and image their uptake by the CP. RESULTS: Retention of a radioactive folate conjugate, folate-diethylenetriaminepentaacetic acid (DTPA)-111In, into the brain of balb/c mice was observed, although repeated injections or prolonged release via an osmotic pump of the compound did not result in increased brain uptake. Uptake of an 125I-labeled anti-folate receptor antibody into the brain was very low, and no competition was observed with unlabeled antibody. Imaging of brain thin-sections and whole brain tissue from a mouse injected with folate-fluorescein revealed strong fluorescence in the CP, but virtually no where else in the brain. CONCLUSIONS: Both fluorescence and radioimaging results demonstrate specific uptake of small molecular weight folate conjugates into CP cells of the murine brain, but no significant transport of the molecules across the CSF. Furthermore, no uptake of larger folate-linked proteins by choroid plexus cells is observed, suggesting folate conjugate size may strongly influence access to CP folate receptors.

Improved kinetic stability of DTPA- dGlu as compared with conventional monofunctional DTPA in chelating indium and yttrium: preclinical and initial clinical evaluation of radiometal labelled minigastrin derivatives.

The development of monofunctional DTPA derivatives has been a major breakthrough in the labelling of proteins or peptides with a variety of radiometals. Although this methodology is simple and useful for indium-111 labelling, the stability of these conjugates is too low for most therapeutic nuclides. Cyclic chelators, such as DOTA, have shown excellent kinetic stability with a variety of radiometals, but the labelling procedure is more difficult, requiring ultra-pure reagents and a heating step that sometimes endangers the biomolecule's integrity. The aim of this work was twofold: (a) to develop a novel, open chain chelator which can be easily labelled with various radiometals, displaying higher kinetic stability than monofunctional DTPA, and (b) to evaluate this chelator in vitro and in vivo when conjugated to a CCK-B receptor ligand as a detection modality for receptor-(over-)expressing tumours. DTPA derivatives of Leu(1)- and dGlu(1)-minigastrin were synthesised. All conjugates could be labelled with (111)In or (88/90)Y at high specific activities (8.5-44.4 GBq/micro mol) and with high radiochemical purity. Serum stability testing was performed, and the labelled conjugates were compared concerning their stability against DTPA challenge. The biodistribution of the radiolabelled Leu(1)- and dGlu(1)-minigastrin derivatives was studied in tumour-bearing nude mice, in one healthy human volunteer and in three patients with metastatic medullary thyroid carcinoma. The transchelation of all tested radiometals to serum proteins was significantly slower with the DTPA-Glu conjugates as compared with their Leu analogues (e.g. transchelation t(1/2) of DTPA- dGlu(1)-minigastrin vs its Leu(1) analogue at 37 degrees C in human serum for (111)In: 239 h vs 91 h; for (90)Y: 130 h vs 53 h). In animals, all labelled CCK-B receptor ligands showed fast and specific uptake in CCK-B-receptor-positive tissues, such as the stomach and tumour, as well as a fast renal clearance pattern. However, DTPA-Leu(1)-minigastrin showed higher background activity in the whole body and those organs known to accumulate the respective free radiometal (e.g. (88)Y-DTPA-Leu(1)-minigastrin had bone uptake of 22%ID/g as compared to only 1.2%ID/g with its dGlu(1) analogue). In humans, fast tumour and stomach uptake was observed for both (111)In-labelled compounds, but DTPA- dGlu(1)-minigastrin lacked the liver, spleen and bone marrow uptake observed with its Leu(1) analogue. In conclusion, anionic amino acid derivatives of DTPA may display improved metabolic stability as compared with monofunctional DTPA conjugates. DTPA- dGlu(1)-minigastrin is preferred to "monofunctional" DTPA-Leu(1)-minigastrin for diagnostic application with (111)In for the in vivo detection of CCK-B receptor-expressing tissues.

Stabilised 111In-labelled DTPA- and DOTA-conjugated neurotensin analogues for imaging and therapy of exocrine pancreatic cancer.

Neurotensin (NT) receptors are overexpressed in exocrine pancreatic cancer and Ewing's sarcoma. The potential utility of native NT in cancer diagnosis and therapy is, however, limited by its rapid degradation in vivo. Therefore, NT analogues were synthesised with modified lysine and arginine derivatives to enhance stability and coupled either to DTPA, to enable high specific activity labelling with indium-111 for imaging, or to DOTA, to enable high specific activity labelling with beta-emitting radionuclides, such as lutetium-177 and yttrium-90. Based on serum stability (4 h incubation at 37 degrees C in human serum) and receptor binding affinity, the five most promising analogues were selected and further evaluated in in vitro internalisation studies in human colorectal adenocarcinoma HT29 cells, which overexpress NT receptors. All five NT analogues bound with high affinity to NT receptors on human exocrine pancreatic tumour sections. The analogues could be labelled with (111)In to a high specific activity. The (111)In-labelled compounds were found to be very stable in serum. Incubation of HT29 cells with the (111)In-labelled analogues at 37 degrees C showed rapid receptor-mediated uptake and internalisation. The most promising analogue, peptide 2530 [DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH] was further tested in vivo in a biodistribution study using HT29 tumour-bearing nude mice. The results of this study showed low percentages of injected dose per gram tissue of this (111)In-labelled 2530 analogue in receptor-negative organs like blood, spleen, pancreas, liver, muscle and femur. Good uptake was found in the receptor-positive HT29 tumour and high uptake was present in the kidneys. Co-injection of excess unlabelled NT significantly reduced tumour uptake, showing that tumour uptake is a receptor-mediated process. With their enhanced stability, maintained high receptor affinity and rapid receptor-mediated internalisation, the (111)In-labelled DTPA- and DOTA-conjugated NT analogues are excellent candidates for imaging and therapy of exocrine pancreatic cancer, peptide 2530 being the most promising analogue.

Albumin-based drug delivery as novel therapeutic approach for rheumatoid arthritis.

We reported recently that albumin is a suitable drug carrier for targeted delivery of methotrexate (MTX) to tumors. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumors and are metabolized by malignant cells. MTX, covalently coupled to human serum albumin (MTX-HSA) for cancer treatment, is currently being evaluated in phase II clinical trials. Because synovium of patients with rheumatoid arthritis (RA) shares various features observed also in tumors, albumin-based drug targeting of inflamed joints might be an attractive therapeutic approach. Therefore, the pharmacokinetics of albumin and MTX in a mouse model of arthritis was examined. Additionally, uptake of albumin by synovial fibroblasts of RA patients and the efficacy of MTX and MTX-HSA in arthritic mice were studied. The results show that when compared with MTX, significantly higher amounts of albumin accumulate in inflamed paws, and significantly lower amounts of albumin are found in the liver and the kidneys. The protein is metabolized by human synovial fibroblasts in vitro and in vivo. MTX-HSA was significantly more effective in suppression of the onset of arthritis in mice than was MTX. In conclusion, albumin appears to be a suitable drug carrier in RA, most likely due to effects on synovial fibroblasts, which might increase therapeutic efficacy and reduce side effects of MTX.

Positron emission tomography shows that intrathecal leptin reaches the hypothalamus in baboons.

Human obesity may be caused by a resistance to circulating leptin. Evidence from rodents and humans suggests that a major component of this resistance is an impairment in the ability of the blood-brain barrier (BBB) to transport leptin from the blood to the brain. One potential way to bypass the BBB is by administering leptin into the intrathecal (i.t.) space. To be effective, i.t. leptin would have to move caudally from the site of injection, enter the cranium, and reach the hypothalamic arcuate nucleus at the base of the pituitary fossa. However, many substances, especially small, lipid-soluble molecules, do not diffuse far from the site of i.t. injection but are resorbed back into blood. To determine whether i.t. leptin can move caudally, we injected leptin conjugated to diethylenetriaminepentaacetic acid (DTPA) and labeled with (68)Ga (G-Ob) into the lumbar space of three baboons. We also studied unconjugated DTPA labeled with (68)Ga, which did not move up the spinal cord but rapidly appeared in blood after i.t. injection. In contrast, G-Ob steadily moved toward the cranium and had reached the hypothalamus 91 and 139 min after i.t. injection in two baboons. We estimated the concentration of leptin in the hypothalamic region to be at least 8 ng/ml, which is about 40 times higher than cerebrospinal fluid levels in normal weight humans and about 4 times higher than the highest level ever recorded after the peripheral administration of leptin. In a third baboon, the leptin neither moved caudally nor appeared in the blood. We conclude that leptin administered i.t. can reach the hypothalamus in therapeutic concentrations, although there is considerable individual variation.

Pre-clinical evaluation of [(111)In-DTPA-Pro(1), Tyr(4)]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-Pro(1),Tyr(4)]BN, was shown to be a high-affinity BN-receptor (BNR) agonist, stimulating prolactin secretion from 7315b cells with an IC(50) of 8 nM. The (111)In-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 [(111)In-DTPA-Pro(1),Tyr(4)]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 microgram [Tyr(4)]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 microgram) of injected ligand. Next to the pancreas, highest uptake was observed in the kidneys, which was not blocked by excess [Tyr(4)]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. [(111)In-DTPA-Pro(1), Tyr(4)]BN appears to be a promising radioligand for scintigraphy of BNR-expressing tumours.

Three-step radioimmunotherapy with yttrium-90 biotin: dosimetry and pharmacokinetics in cancer patients.

A three-step avidin-biotin approach has been applied as a pretargeting system in radioimmunotherapy (RIT) as an alternative to conventional RIT with directly labelled monoclonal antibodies (MoAbs). Although dosimetric and toxicity studies following conventional RIT have been reported, these aspects have not previously been evaluated in a three-step RIT protocol. This report presents the results of pharmacokinetic and dosimetric studies performed in 24 patients with different tumours. Special consideration was given to the dose delivered to the red marrow and to the haematological toxicity. The possible additive dose to red marrow due to the release of unbound yttrium-90 was investigated. The protocol consisted in the injection of biotinylated MoAbs (first step) followed 1 day later by the combined administration of avidin and streptavidin (second step). After 24 h, biotin radiolabelled with 1.85-2.97 GBq/m2 of 90Y was injected (third step). Two different chelating agents, DTPA and DOTA, coupled to biotin, were used in these studies. Indium-111 biotin was used as a tracer of 90Y to follow the biodistribution during therapy. Serial blood samples and complete urine collection were obtained over 3 days. Whole-body and single-photon emission tomography images were acquired at 1, 16, 24 and 40 h after injection. The sequence of images was used to extrapolate 90Y-biotin time-activity curves. Numerical fitting and compartmental modelling were used to calculate the residence time values (tau) for critical organs and tumour, and results were compared; the absorbed doses were estimated using the MIRDOSE3.1 software. The residence times obtained by the numerical and compartmental models showed no relevant differences (<10%); the compartmental model seemed to be more appropriate, giving a more accurate representation of the exchange between organs. The mean value for the tau in blood was 2.0+/-1.1 h; the mean urinary excretion in the first 24 h was 82.5%+/-10.8%. Without considering any contribution of free 90Y, kidneys, liver, bladder and red marrow mean absorbed doses were 1.62+/-1.14, 0.27+/-0.23, 3.61+/-0.70 and 0. 11+/-0.05 mGy/MBq, respectively; the effective dose was 0.32+/-0.06 mSv/MBq, while the dose to the tumour ranged from 0.62 to 15.05 mGy/MBq. The amount of free 90Y released after the injection proved to be negligible in the case of 90Y-DOTA-biotin, but noteworthy in the case of 90Y-DTPA-biotin (mean value: 5.6%+/-2.5% of injected dose), giving an additive dose to red marrow of 0.18+/-0.08 mGy per MBq of injected 90Y-DTPA-biotin. Small fractions of free 90Y originating from incomplete radiolabelling can contribute significantly to the red marrow dose (3.26 mGy per MBq of free 90Y) and may explain some of the high levels of haematological toxicity observed. These results indicate that pretargeted three-step RIT allows the administraton of high 90Y activities capable of delivering a high dose to the tumour and sparing red marrow and other normal organs. Although 90Y-biotin clears rapidly from circulation, the use of DOTA-biotin conjugate for a stable chelation of 90Y is strongly recommended, considering that small amounts of free 90Y contribute significantly in increasing the red marrow dose.

Evaluation of the in vitro stability of gadolinium (III) polyoxometalates.

The gadolinium chelates of lacunary polyoxometalates were evaluated for in vitro stability against rat serum, diethylenetriaminepentaacetic acid (DTPA), endogenous metal cations, and DTPA-doped rat serum. The chelates dissociated rapidly in rat serum. Challenges by DTPA gave relatively slower dissociation rates, whereas challenges by endogenous metal cations (Fe(III), Zn(II), and Cu(II)) occurred at a rate comparable to the serum challenge, suggesting the instability in serum is due to a transmetalation mechanism. Challenges by DTPA-doped serum gave slower rates of dissociation than in native serum, verifying the transmetalation mechanism.