Detection of abscisic-acid-binding proteins in the microsomal protein fraction of Arabidopsis thaliana with abscisic-acid-protein conjugates used as affinity probes

Radioiodinated iododeoxyuridine (IUdR) is a novel, cycle-specific agent that has potential for the treatment of residual malignant glioma after surgery. As only cells in S-phase incorporate IUdR into DNA, a major limitation to this therapy is likely to be proliferative heterogeneity of the tumour cell population. Using a clonogenic end point, we have compared the toxicities of three radioiodoanalogues of IUdR--[123I]IUdR, [125I]IUdR and [131I]IUdR--to the human glioma cell line UVW, cultured as monolayers in the exponential and the plateau phase of growth and as multicellular spheroids. Monolayers treated in the exponential growth phase were most efficiently sterilized by [125I]IUdR (concentration resulting in 37% survival (C37) = 2.36 kBq ml(-1)), while [123I]IUdR and [131I]IUdR were less effective eradicators of clonogens (C37 = 9.75 and 18.9 kBq ml(-1) respectively). Plateau-phase monolayer cultures were marginally more susceptible to treatment with [123I]IUdR and [125I]IUdR (40% clonogenic survival) than [131I]IUdR (60% clonogenic survival). In cells derived from glioma spheroids, both [125I]IUdR and [123I]IUdR were again more effective than [131I]IUdR at concentrations up to and including 20 kBq ml(-1). However, the survival curve for [131I]IUdR crossed the curves for the other agents, resulting in lower survival for [131I]IUdR than [123I]IUdR and [125I]IUdR at concentrations of 40 kBq ml(-1) and higher, the clonogenic survival values at 100 kBq ml(-1) were 13%, 45% and 28% respectively. It was concluded that IUdR incorporating the Auger electron emitters 123I and 125I killed only cells that were in S-phase during the period of incubation with the radiopharmaceutical, whereas the superior toxicity to clonogenic cells in spheroids of [131I]IUdR at higher concentration was due to cross-fire beta-irradiation. These findings suggest that [131I]IUdR or combinations of [131I]IUdR and [123I]IUdR or [125I]IUdR may be more effective than Auger electron emitters alone for the treatment of residual glioma, if proliferative heterogeneity exists.     

Targeted radiotherapy of multicell neuroblastoma spheroids with high specific activity [125I]meta-iodobenzylguanidine

PURPOSE: Iodine-125 induces cell death by a mechanism similar to that of high linear energy transfer (high-LET) radiation. This study investigates the cytotoxicity of high-specific-activity [125I]meta-iodobenzylguanidine (125I-mIBG) in human SK-N-MC neuroblastoma cells grown as three-dimensional multicellular spheroids. MATERIALS AND METHODS: Spheroids were incubated with high-specific-activity 125I-mIBG (6 mCi/microg, 1000 times that of the conventional specific activity used for autoradiography). Cytotoxicity was assessed by fluorescence viability markers and confocal microscopy for intact spheroids, fluorescence-activated cell sorting and clonogenic assay, and clonogenic assays for dispersed whole spheroids. Distribution of radioactive mIBG was determined by quantitative light-microscope autoradiography of spheroid cryostat sections. Dose estimation was based on temporal knowledge of the retained radioactivity inside spheroids, and of the radiolabel's emission characteristics. Findings were compared with those of spheroids treated under the same conditions with 131I-mIBG, cold mIBG, and free iodine-125. RESULTS: 125I-mIBG exerted significant cell killing. Complete spheroids were eradicated when they were treated with 500 microCi of 125I-mIBG, while those treated with 500 microCi or 1000 microCi of 131I-mIBG were not. The observed difference in cytotoxicity between treatments with 125I- and 131I-mIBG could not be accounted for by the absorbed dose of spheroid alone. The peripheral, proliferating cell layer of the spheroids remained viable at the moderate radioactivity of 100 microCi for both isotopes. Cytotoxicity induced by 125I-mIBG was quantitatively comparable by the peripheral rim thickness to that of 131I-mIBG at the dose of 100 microCi. The peripheral rim thickness decreased most significantly in the first 17 hours after initial treatment. There was no statistical decrease in the rim thickness identified afterwards for the second, third, and fourth days of incubation. CONCLUSION: The cytotoxic effect of high-specific-activity 125I-mIBG appears to be comparable to, if not more efficient than that of conventionally used 131I-mIBG at the same level of total radioactivity. 125I-mIBG may improve the therapeutic index over that of 131I-mIBG in the clinical management of metastatic neuroblastoma due to the short range of Auger electrons.     

No-carrier-added synthesis of meta-[131I]iodobenzylguanidine

No-carrier-added meta-[131I]iodobenzylguanidine ([131I]MIBG) was prepared starting with two different metallated precursors. Attempted preparation of 3-(tri-n-butylstannyl)benzylguanidine was not successful. An alternate two-step strategy using 3-(tri-n-butylstannyl)benzylamine could be used to prepare radio-iodinated [131I]MIBG in an overall radiochemical yield of 30-33%. Synthesis of [131I]MIBG via the radioiododesilylation of 3-trimethylsilylbenzylguanidine was also investigated. Yields were dependent on temperature, precursor concentration, solvent and nature of the oxidant. Radiochemical yields of 90% were obtained in 5 min at room temperature using either N-chlorosuccinimide or hydrogen peroxide in trifluoroacetic acid as oxidants. The percentage of specific binding in vitro of no-carrier-added MIBG to SK-N-SH neuroblastoma cells remained constant over a 2 log activity range, while the binding of MIBG prepared by isotopic exchange dropped by a factor of seven. In normal mice, heart and adrenal uptake of no-carrier-added [131I]MIBG was found to be higher than that of [131I]MIBG prepared by isotopic exchange.     

Radiochemical purity of iodine-131 labelled metaiodobenzylguanidine infusion fluids: a report from clinical practice

Iodine-131 labelled metaiodobenzylguanidine ([131I]MIBG) has a diagnostic and therapeutic role in the management of neural crest tumours, particularly neuroblastoma, malignant phaeochromocytoma and paraganglioma. With therapeutic amounts of [131I]MIBG it is essential that the amount of free [131I]iodide, the most important impurity, is known. In clinical practice the percentage of free [131I]iodide seen in a [131I]MIBG infusion concentrate increased from 2.2% +/- 0.67% to 3.6% +/- 0.39% (mean +/- SD; n = 23) 1 day after production. At the time of use the percentage of free [131I]iodide was always below our upper limit of acceptance of 5%. Since 5% of free [131I]iodide is within practical reach in our environment, a higher percentage at the time of preadministration quality control is not accepted in the Netherlands Cancer Institute.     

Peripheral pain mechanisms

A promising new treatment for glioma involves Auger electron emitters such as 125I or 123I conjugated to deoxyuridine (IUdR). However, the presence in tumour deposits of non-proliferating cells with clonogenic potential poses a major limitation to this cycle-specific therapy. We have used multicellular tumour spheroids derived from the human glioma cell line UVW to study [125I]IUdR-targeted radiotherapy in aggregates containing cells in different proliferative states. Autoradiographic identification of labelled cells indicated that nuclear incorporation of [125I]IUdR decreased markedly with increasing size of spheroid. IUdR incorporation was maximal in the surface layer of cells and decreased with depth within spheroids. Radiopharmaceutical uptake corresponded closely to the regions of cell cycling as indicated by staining for the nuclear antigen Ki67. The uptake of drug was enhanced by increasing the duration of incubation from 52 h to 104 h. These observations suggest that significant sparing of non-cycling malignant cells would result from treatment delivered as a single injection of radiolabelled IUdR. To achieve maximal therapeutic effect. IUdR should be administered by multiple injections, by slow release from biodegradable implants or by slow-pump delivery.     

Acute liver necrosis induced by iodine-131-MIBG in the treatment of metastatic carcinoid tumors

Iodine-131-metaiodobenzylguanidine (MIBG) is used in the treatment of carcinoid tumors. Temporary palliation with complete subjective symptomatic response has been reported in these patients. This treatment is usually well tolerated and side-effects are generally limited to nausea, mild hepatic toxicity with spontaneous recovery and temporary myelosuppression. Our case report shows that repeated treatment with [131I]MIBG in a patient with extensive carcinoid liver metastasis may cause severe hepatic toxicity leading to death. Factors such as concomitant use of 5-fluorouracil and the progressive nature of the disease may have contributed to this event.     

Iodine-131-metaiodobenzylguanidine dosimetry in cancer therapy: risk versus benefit

In the treatment of neural crest tumors, such as pheochromocytoma, with[131I]MIBG, bone marrow toxicity limits the amount of administered activity and, thus, a therapeutically useful tumor dose. METHODS: We calculated tumor doses in a series of diagnostic studies with [123I]MIBG using accurate quantification of SPECT and planar scintigraphy. By extrapolating diagnostic results to therapeutic activities of [131I]MIBG, we could compare the results with whole-body doses from a series of therapies. RESULTS: The tumor dose was DT = 2.2 mGy MBq(-1) (median value of 27 measurements, range 0.04 < or = DT < or = 20 mGy MBq(-1) and the whole-body dose in a series of 16 patients undergoing 50 therapies was DWB = 0.12 +/- 0.04 mGy MBq(-1) (mean +/- s.d.). The therapeutic ratio varied between 130 to below 10 in some patients. CONCLUSION: The results were compared with published data. We found clearly skewed distribution of tumor doses, with a majority of tumors receiving only a few mGy per MBq administered activity. In some patients, however, doses did reach 20 mGy MBq(-1).     

Synthesis of 4'-epi-iodo-4'-deoxy-daunorubicin, a potential cancer radiotherapeutic agent

We have prepared 4'-epi-iodo-4'-deoxy-daunorubicin (IDDNR)(1), a doxorubicin analog, via a 5-step synthesis involving a protected daunorubicin triflate derivative (4). This triflate derivative will allow the facile and regiospecific nucleophilic preparation of I-125 or Br-80 m labelled analogs of IDDNR. Auger electron-emitting I-125- or Br-80 m-labelled analogs of IDDNR may have potential as cancer radiotherapeutic agents.     

Therapeutic efficacy and dose-limiting toxicity of Auger-electron vs. beta emitters in radioimmunotherapy with internalizing antibodies: evaluation of 125I- vs. 131I-labeled CO17-1A in a human colorectal cancer model

Recent clinical results suggest that higher anti-tumor efficacy may be achieved with internalizing monoclonal antibodies (MAbs) at lower toxicity when labeled with Auger-electron, as compared to conventional beta-emitters. The aim of our study was to compare the toxicity and anti-tumor efficacy of the 125I-labeled internalizing MAb, CO17-1A, with its 131I-labeled form in a human colon cancer model in nude mice. Biodistribution studies were performed in nude mice bearing s.c. human colon cancer xenografts. For therapy, the mice were injected either with unlabeled 125I- or 131I-labeled C017-1A at equitoxic doses. Control groups were left untreated, were given a radiolabeled isotype-matched irrelevant antibody or a tumor-specific, but noninternalizing antibody. The maximum tolerated activities (MTD) of 131I-and 125I-CO17-1A without artificial support were 300 microCi and 3 mCi, respectively. Myelotoxicity was dose-limiting; bone marrow transplantation allowed for an increase of the MTD to 400 microCi of 131I-17-1A, whereas the MTD of 125I-17-1A with bone marrow support had not been reached at 5 mCi. Whereas no significant therapeutic effects were seen with unlabeled C017-1A, tumor growth was retarded with 131I-CO17-1A. With the 125I-label, however, therapeutic results were clearly superior. In contrast, no significant difference was observed in the therapeutic efficacy of the 131I- vs. 125I-labeled, noninternalizing antibodies. Our data indicate a superiority of Auger-electron emitters, such as 125I, as compared to therapy with conventional beta-emitters with internalizing antibodies. The lower toxicity of Auger emitters may be due to the short path length of their low-energy electrons, which can reach the nuclear DNA only if the antibody is internalized (as is the case in antigen-expressing tumor tissue, but not in the stem cells of the red marrow).     

Immunohistochemical analysis of intratumoral heterogeneity of [131I]cG250 antibody uptake in primary renal cell carcinomas

In previous studies, highly heterogeneous uptake of 131I-labelled chimeric monoclonal antibody G250 ([131I]cG250) in primary renal cell carcinomas has been observed (intratumoral differences > factor 100). In this study, we investigated a possible correlation between intratumoral antibody uptake and four immunohistochemically determined parameters: G250 antigen expression, blood vessel density, neovascularization and percentage of viable tumour cells. Whole tumour slices of four different tumours were cut into 1-cm3 cubes, and in each cube the [131I]cG250 uptake was determined. The correlation between [131I]cG250 uptake and each individual parameter was determined in a multiple regression analysis. Additionally, the data were reanalysed after introducing arbitrary cut-off values for each parameter. If a sample showed expression of a parameter above the introduced threshold value, this sample fulfilled one condition. Subsequently, the Pearson correlation coefficients were calculated from [131I]cG250 uptake and the number of fulfilled conditions (0-3). All tumour samples with high [131I]cG250 uptake [> 0.1% of the injected dose per gram (ID g(-1))] showed high antigen expression (> 50%). However, not all samples with high antigen expression displayed high uptake. A statistically significant correlation between [131I]cG250 uptake and antigen expression was found (beta = 0.44, 0.69 and 0.74) in three out of four tumours analysed. Of the other determined parameters, no consistent correlation with [131I]cG250 uptake was found; only the percentage of viable tumour cells correlated significantly in two out of four tumours (beta = 0.80 and 0.26). Calculation of the Pearson correlation coefficients showed a statistically significant correlation between [131I]cG250 uptake and an increased number of fulfilled conditions in all tumours, indicating that each of the individual parameters contribute to the uptake of [131I]cG250. These observations indicate that high antigen expression is a prerequisite for high antibody uptake. However, regional differences in antibody uptake within a tumour cannot be explained by antigen expression alone.     

I-123 MIBG scintigraphy in adults. A report of clinical experience

Thirty-one adult patients with clinical findings suggestive of pheochromocytoma were studied with I-123 MIBG. All patients had images obtained at 24 and 48 hours. Five patients had abnormal uptake proved to be because of I-123 MIBG avid tumors. The remaining 26 patients had no proven tumors and showed physiologic uptake in various organs. The 24-hour images were of high quality. In all cases, the 48-hour images contributed no significant additional information. Only in 1 patient did the 48-hour image add some certainty. Physiologic uptake was seen in the salivary glands, liver, G.I. tract, and urinary bladder in all patients (100%). Uptake was also observed in the lung and heart (90%), normal adrenal glands (32%), thyroid (29%), spleen (23%) uterus (13%), and neck muscles (6%). The authors' experience indicates that I-123 MIBG gives superior images compared to the previously used I-131 MIBG, that the optimum imaging time for adults is 18-24 hours, and that normal distribution patterns including uterine and neck muscle uptake should be familiar to physicians interpreting the studies.     

Rate of insufficient samples for fine-needle aspiration for nonpalpable breast lesions in a multicenter clinical trial: The Radiologic Diagnostic Oncology Group 5 Study. The RDOG5 investigators

Metaiodobenzylguanidine (MIBG) was developed 18 yr ago for scintigraphic imaging of the adrenomedullary tumors pheochromocytoma and neuroblastoma. Many studies have shown the usefulness of this agent for the management of patients with neuroblastoma or pheochromocytoma, and the 131I-labeled form was recently approved by the Food and Drug Administration for use in the U.S. This article summarizes our current concepts on the diagnostic use of MIBG in children. The radioisotopes available for labeling of MIBG and related compounds, the dosimetry, metabolism and mechanisms of uptake and retention are discussed. Our protocols for imaging both 131I-MIBG and 123I-MIBG, along with the normal distribution of these compounds, are reviewed. The use of MIBG for the management of neuroblastoma, and comparisons with other radiotracers available for imaging neuroblastomas are also addressed.     

Offspring of xenogeneically-reconstituted scid/scid mice are capable of a primary xenogeneic immune response to DNP-KLH

The distribution of [125I]SRIF-28 ([Leu8,D-Trp22,125I-Tyr25]somatostatin-28), [125I]204-090 ([Tyr3]octreotide) and [125I]CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) labelled recognition sites was studied by autoradiography in rat brain at embryonic day 18 (E 18) and postnatal day 5 (P 5). These results were compared with mRNA expression of somatostatin receptors SSTR1-5 (named sst1-5 now) as studied by in situ hybridization. [125I]SRIF-28, [125I]204-090 and [125I]CGP 23996 binding displayed different although partially overlapping distributions, and showed an increase between E 18 and P 5, which was less marked for [125I]204-090 binding. -125I-204-090 binding and sst2 receptor mRNA were similarly distributed, whereas [125I]CGP 23996 binding did not correlate with any single somatostatin receptor mRNA. The data suggest that most SRIF receptor subtypes in rat brain are present before birth, but evolve differently.     

Treatment of neuroblastoma stage 4 with 131I-meta-iodo-benzylguanidine, high-dose chemotherapy and immunotherapy. A pilot study

Disseminated neuroblastoma after infancy has a prognosis of approximately 10-20% with conventional therapy. We investigated the role of high-dose chemotherapy (HDCT) with peripheral blood stem cell (PBSC) rescue in combination with 131I-metaiodobenzylguanidine ([131I-m]IBG). 11 children with neuroblastoma stage 4 were pretreated within the German Neuroblastoma Trial NB90 and included in a high-dose concept for consolidation. Remission was documented by ultrasound, CT, NMR, or [123I-m]IBG scanning. HDCT was a combination of melphalan (180 mg/m2), carboplatin (1,500 mg/m2) and etoposide (40 mg/kg). All children were treated by [131I-m]IBG (0.58 GBq/kg) prior to high-dose treatment. All 11 children were additionally treated with antiGD2 murine- or chimeric-antibody (ch14.18). 4 children had no change to their remission status but three achieved a complete response (from a partial response to first line) and one a partial response (from no response to first line). The other 3 children progressed, 2 dying of their disease. Using Kaplan-Meier analysis, the probability of progression-free survival was 0.70 +/- 0.15 with a median observation time of 19 months. 9/11 children are alive, 8 without progression or relapse, whilst 2 have died of their disease. The combination of mIBG plus high-dose chemotherapy with PBSC support supplemented by immunotherapy with antiGD2 antibody appears to be a feasible and effective treatment regimen for disseminated neuroblastoma in this limited series. Larger numbers of patients should be treated to confirm these results.     

The synthesis, radioiodination and preliminary biological study of the new carboxylic derivatives of dithizone

Synthesis, characteristics and radioiodination of the new carboxylic derivatives of dithizone are described in this paper. We have applied the carboxy dithizones for preparation of radioactive compounds by coupling with [131I]-histamine. Preliminary biological studies of the new radiodithizone were done in rats after two different application routs: peripheral i.v. injection and direct injection to splenic artery. Biodistribution of the carboxy dithizone-[131I]-histamine conjugate (i.v. injection) was quite different than that for free [131I]-histamine. However, uptake of activity in pancreas was low (0.81% g-1 of tissue). Direct application of the conjugate to splenic artery resulted in high activity retention in pancreas after 30 and 45 min post injection (respectively 8.8 and 12.4% g-1 of tissue) indicating potential usefulness of the new radiodithizone for in vivo monitoring of pancreas.     

I-131 localization in acute megakaryocytic leukemia

A case of acute megakaryocytic leukemia in a 13-month-old infant was encountered in whom an abnormal bone scan was associated with abnormal I-131 MIBG uptake in the femoral bone marrow. The normal platelet will take up MIBG and thus, uptake of this radiopharmeceutical by the malignant megakaryocyte, a platelet precursor, may be caused by the same mechanism.     

Construction of PCR-ligated long flanking homology cassettes for use in the functional analysis of six unknown open reading frames from the left and right arms of Saccharomyces cerevisiae chromosome XV

To examine the capacity of chemical protectors to mitigate damage caused by chronic irradiation by incorporated radionuclides in vitro, cells must be maintained in the presence of the protector during the course of the irradiation. Such long exposures to chemical protectors at concentrations high enough to afford protection usually results in extreme chemotoxicity. To overcome this problem, experimental conditions were developed to allow Chinese hamster V79 cells to be maintained in 5% DMSO for prolonged periods (up to 72 h) with no observable chemotoxicity. Under these conditions, the capacity of DMSO to protect against damage to V79 cells caused by unbound 32P and 3H2O and DNA-incorporated (131)IdU, [3H]dThd and 125IdU was examined. The dose modification factors for 32P, 3H2O, (131)IdU, [3H]dThd and 125IdU were 2.6+/-0.5, 2.3+/-0.3, 1.0+/-0.1, 1.16+/-0.07 and 1.07+/-0.02, respectively. These results show that 5% DMSO is capable of protecting cultured V79 cells against lethal damage caused by beta particles emitted by unbound 32P and 3H2O, whereas little or no protection is afforded against damage caused by beta particles emitted by DNA-incorporated (131)I and 3H or low-energy Auger electrons emitted by DNA-incorporated 125I.     

Human biodistribution and dosimetry of iodine-123-fluoroalkyl analogs of beta-CIT

Two new N-omega-fluoroalkyl analogs of [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([123I]beta-CIT), the fluoroethyl and fluoropropyl compounds ([123I]FE-CIT and [123I]FP-CIT, respectively), have been shown to have faster kinetics and better selectivity for the dopamine transporter than [123I]beta-CIT. We examined the organ biodistribution and radiation safety of these two compounds in six healthy volunteers who received an injection with each of the two compounds 2 weeks apart. Data were obtained on the Strichman 860 whole-body scanner. Transmission scans were obtained in all subjects prior to the injection of the radiotracer with a line source and used to derive organ-specific attenuation correction factors. Whole-body planar images were acquired every hour for the first 6 h, and at 24 h. Attenuation-corrected regional conjugate counts were converted into units of activity using a calibration factor obtained for each subject by dividing whole-body conjugate decay-corrected counts from the first acquisition by the injected activity. Radiation dose estimates were on average higher for [123I]CIT-FE than for [123I]CIT-FP, with the lower large intestine receiving the highest exposure: 0.15+/-13% mGy/MBq (mean +/-COV) and 0.12+/-14% mGy/MBq for [123I]FE-CIT and [123I]FP-CIT, respectively, followed by the upper large intestine and the spleen.     

Cell death induced by a 131I-labeled monoclonal antibody in ovarian cancer multicell spheroids

Treatment of OVCAR-3 spheroids with 131I-OC125 monoclonal antibody produced a decrease in spheroid volume and a concomitant rise in necrotic cell number. No increase in apoptotic cell number was observed during incubation of spheroids with the labeled antibody. Necrosis began early, reaching a maximum after 3 Gy of accumulated dose delivered at a dose rate of 1.8 cGy/h. Higher accumulated doses induced necrosis for longer incubation times. Thus, dose rate and time are both determinants of ultimate radiation effects when spheroids are incubated with labeled antibodies, although dose rate is the most important factor.