Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular-scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS).

Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 ((10)B) atoms to individual tumour cells. Cell killing results from the (10)B (n, α)(7) Li neutron capture and fission reactions that occur if a sufficient number of (10)B atoms are localized in the tumour cells. Intranuclear (10)B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of (10)B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular-scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high-grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols.

Microdosimetric study for interpretation of outcomes from boron neutron capture therapy clinical trials.

Boron neutron capture therapy is a brachyradiotherapy utilizing the (10)B(n,alpha)(7)Li reaction that has been used to treat glioblastoma multiforme (GBM), melanoma and colon carcinoma liver metastases. GBM clinical trials resulted in modestly improved life expectancies compared with conventional therapies. Early results trials focused on malignant melanoma and colon carcinoma provide dramatically better results. Macrodosimetry cannot explain these apparent differences. The dichotomy can only be understood using microdosimetry techniques. A computer program has been created to provide an improved tissue model. This model permits the dose in each cell's cytoplasm, nucleus, and the interstitium to be calculated for ellipsoidal cells placed in either random or ordered locations. The nuclei can be centered or eccentric. The new model provides insight into the micro level for differences in the trials. The differences arise from the tissue's cellular geometry and the effects of neighboring cells. These results help to explain the observed clinical outcomes.

The microdosimetry of boron neutron capture therapy in a randomised ellipsoidal cell geometry.

Two reactions deliver the majority of local dose in boron neutron capture therapy. The ionised particles (protons, alpha particles and lithium nuclei) produced in the two reactions, 10B(n,alpha,gamma)7Li and 14N(n,p)14O, have short ranges that are less than -14 microm (which is on the order of the diameter of a typical human cell). The ionised particles are heavy and are in the 2+ charge state in the case of the boron reactions. These heavy 2+ ions will do significant damage to molecules near their tracks. Thus, the distribution of nitrogen and, in particular, of boron determines the spatial characteristics of the radiation field. Since the distribution of nitrogen is nearly homogeneous in the brain and is not easily altered for the purpose of radiotherapy, the spatial variation in the radiation dose is due mainly to the spatial distribution of boron. This implies that the spatial distribution of boron determines the microscopic energy deposition and therefore the spatial characteristics of the microscopic dose. The microscopic dose from the (n,alpha) and (n,p) reactions has been examined in detail and, as averred, the proton dose is relatively homogeneous except for statistical variability. The statistical variability in essence adds a false spatial variability that would not be seen if a large number of histories were performed. Since the majority of spatial variability occurs in the boron distribution, the (n,p) reaction can be suppressed to better understand the spatial distribution effects on the microscopic dose. Programs have been written in FORTRAN using Monte Carlo techniques to model ellipsoidal cells that are either randomly sized and located in the region of interest or are arranged in a face centred cubic array and are identical except for the location of the nuclei, which may be random. It is shown that closely packed prolate ellipsoidal cells with a large eccentricity in one dimension will receive a larger nuclear dose than cells that are more sparsely packed. This demonstrates that the boron content of a cell and its nucleus can have a significant impact upon the dose to neighbouring cells. The local boron distribution in a region of interest can be shown to affect the macrodosimetric dose, with possible implications for clinical outcomes.

Synthesis and evaluation of radioiodinated (E)-18-iodo-17-octadecenoic acid as a model iodoalkenyl fatty acid for myocardial imaging.

125I-labeled (E)-18-iodo-17-octadecenoic acid (13) has been prepared and evaluated in rats to determine the myocardial uptake and retention and degree of in vivo deiodination of this model iodovinyl-substituted fatty acid, which contains no structural perturbation to inhibit metabolism. This new agent was prepared by NaI-chloramine-T treatment of (17-carbomethoxyheptadec-1-en-1-yl)boronic acid (11) prepared by catecholborane treatment of methyl 17-octadecynoate (10), followed by basic hydrolysis to the free acid (13). The pivotal substrate, 17-octadecynoic acid (9), was prepared by two new routes. The 125I-labeled acid 13 showed high myocardial uptake (1 h, 1.90-2.28% dose/g) with 45% washout after 2 h but lower heart/blood ratios in comparison to analogues containing the tellurium heteroatom. Deiodination was low for the first 2 h after injection (2 h, 61% dose/g). Excellent myocardial images were obtained in a dog with the 123I-labeled agent.

Synthesis and evaluation of radioiodinated 2-(2(RS)-aminopropyl)-5- iodothiophenes as brain imaging agents.

Methods have been developed for the preparation of 2-(2(RS)-aminopropyl)-5-iodothiophenes. The syntheses and physical properties of 2-(2(RS)-aminopropyl)-5-iodothiophene and N-isopropyl-2-(2(RS)-aminopropyl)-5-iodothiophene are described. The radioiodinated agents are of interest because of the high expected uptake and prolonged brain retention that may result from binding to high-capacity, relatively nonspecific amine binding sites. Radioiodine was introduced into the 5-position of 2-(2(RS)-aminopropyl)-5-iodothiophene and N-isopropyl-2-(2(RS)-aminopropyl)-5-iodothiophene by radioiodination of the corresponding 5-boronic acid or 5-(trimethylstannyl) derivatives. Tissue distribution studies in rats with 2-(2(RS)-aminopropyl)-5-[125I]iodothiophene showed high brain uptake (5 min, 2.77% dose/g; 30 min, 2.51% dose/g) and good brain/blood (B/B) ratios (5 min, 6/1; 30 min 3.8/1. A comparison of the brain uptake of the N-isopropyl derivative with the 2(RS)-aminopropyl analogue demonstrated higher initial brain uptake and brain to blood ratios (5 min, 3.2% dose/g; 10.3/1) but more rapid washout (30 min, 1.37% dose; 2.8/1). These data suggest that radiolabeled 2-(2(RS)-aminopropyl)-5-iodothiophenes are potentially useful agents for cerebral perfusion imaging by single-photon-emission computerized tomography (SPECT).

Synthesis and characterization of radioiodinated N-(3-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropanes: potential dopamine reuptake site imaging agents.

Methods have been developed for the preparation of radioiodinated N-substituted 2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropanes. The syntheses, physical properties, radiolabeling, and characterization of the pharmacological properties of N-(3(Z)-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropane (12) and N-(3(E)-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropane (13) are described. 2 beta-Carbomethoxy-3 beta-(p-substituted-phenyl)tropanes are potent ligands for the dopamine transporter. The radioiodinated derivatives are of interest because of the high uptake and prolonged striatal retention that may result from specific binding to low-capacity, high-affinity, dopamine reuptake sites. Radioiodine was introduced into the 3Z and 3E-position of N-(3-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropane by iododemetalation of the corresponding 3-(tri-n-butylstannyl) derivatives. Competition binding data of various dopamine reuptake ligands with rat striatal tissue preparation for either [125I]-12 or [125I]-13 exhibited the following order of potency: E-13 > Z-12 > GBR 12909 >> mazindol >>> (-)-cocaine. Tissue distribution studies in rats showed that the E-13 was the best analogue. E-13 showed high striatal uptake (60 min, 1.23% dose/g; 120 min, 0.61% dose/g) and high striatal to cerebellum ratios (60 min, 15.9/1; 120 min, 16.5/1). These studies indicate that iodine-123-labeled E-13 is a potentially useful agent for imaging the dopamine reuptake sites by single-photon-emission computerized tomography.

Effect of tellurium position on the myocardial uptake of radioiodinated 18-iodotellura-17-octadecenoic acid analogues.

The effect of tellurium (Te) position on myocardial specificity and retention of fatty acids in which radioiodide is stabilized as a trans-(E)-vinyl iodide has been evaluated in rats. Five analogues of 18-iodo-17-octadecenoic acid (ICH = CH-R-Te-R'-COOH) with Te at positions 5, 7, 9, 11, and 13 were prepared by coupling of a trans-diiodoalkene (ICH = CH-R-I) with the requisite sodium [(alkoxycarbonyl)alkyl]telluride substrate (NaTe-R'-COOR"; R" = Me or Et), followed by basic hydrolysis. By varying R and R', a series of analogues with a chain length of 18 carbon atoms was prepared. The telluride substrates were generated in situ by NaBH4 reduction of the corresponding ditellurides, and the diiodoalkenes were prepared by sodium iodide-chloramine-T treatment of the corresponding vinylboronic acids [(HO)2BCH = CH-R-I)]. The vinylboronic acids were prepared by treatment of the terminal acetylenes (HC identical to C-R-I), synthesized from commercially available materials, with catecholborane. All new compounds were analyzed by TLC, NMR, MS, and elemental analyses. The 125I analogues [(E)-125ICH = CH-R-Te-R'-COOH] were prepared in the same manner and evaluated in rats (four per group). Heart uptake and retention were dependent upon the Te position. The analogue with Te at position 5 showed the most pronounced (5-min values) heart uptake (3.7-4.1 dose/g), myocardial retention, and heart/blood ratios (37:1) and is a candidate for radiolabeling with 123I and further evaluation as a myocardial imaging agent.

Myocardial imaging agents: synthesis, characterization, and evaluation of (Z)- and (Z,E)-18-[82Br]bromo-5-tellura-17-octadecenoic acids.

The effects of replacement of radioiodide with radiobromide on the biological properties of a model vinyl halide substituted tellurium fatty acid have been evaluated. The use of a facile radiobromination method involving the reaction of [82Br]Br2 with vinylmercuric bromide substrates has been investigated. Unexpectedly, both the cis- and trans-vinyl bromide products are formed as confirmed by chromatographic and spectral studies. With use of this technique, (E,Z)-1-[82Br]bromo-13-iodo-1-tridecene was prepared and used as the substrate to prepare (E,Z)-18-[82Br]bromo-5-tellura-17-octadecenoic acid (13-E,Z), which was evaluated in rats. Both [82Br]-13-E,Z and [82Br]-13-Z, prepared by an established procedure using N-chorosuccinimide-Br- reaction with the trans-boronovinyl substrates, showed tissue distribution properties similar to those of (E)-18-[125I]iodo-5-tellura-17-octadecenoic acid. These results demonstrate that replacement of I with Br and also the stereochemistry about the olefinic bond do not drastically affect heart uptake and retention.

New myocardial imaging agents: stabilization of radioiodine as a terminal vinyl iodide moiety on tellurium fatty acids.

To determine the myocardial uptake and retention properties of radioiodinated tellurium fatty acids, we prepared two new tellurium fatty acids in which iodine-125 has been chemically stabilized by attachment as a trans-vinyl iodide (I-CH = CH-R-Te-R'-COOH) and evaluated them in rats. Fabrication of 18-iodo-13-tellura-17-octadecenoic acid was accomplished by coupling 1,5-diiodo-1-pentene with sodium 12-(methoxycarbonyl)-n-dodecan-1-yl telluride. The [5-125I]-1,5-diiodo-1-pentene was prepared by an organoborane technique involving 125I+ treatment of 5-iodo-1-penten-1-ylboronic acid [I(CH2)3CH = CHB(OH)2]. The absolute heart uptake of this agent was moderate (1.47-2.52% dose/g after 60 min), but the heart/blood ratios were low (approximately 2.6:1). Only marginal in vivo deiodination occurred, since the thyroid uptake was low (15-18% dose/g after 60 min). The effect of tellurium in position 13 was unexpected. To determine if the low heart specificity and low heart/blood ratios were dependent upon the position of the tellurium, we prepared an analogue with the same chain length, 18-[125I]iodo-7-tellura-17-octadecenoic acid, in the same manner by reaction of [11-125I]-1,11-diiodo-1-undecene with sodium 6-(methoxycarbonyl)-n-hexan-1-yl telluride. This agent showed pronounced heart uptake (2.47-3.94% dose/g after 60 min) and prolonged retention (1.76-3.14% dose/g after 4 h) in rats. Furthermore, the heart/blood ratios remained high for several hours (13:1 after 60 min; 9:1 after 4 h). Iodine-123 labeled 18-iodo-7-tellura-17-octadecenoic acid is an attractive new compound for evaluation as a myocardial imaging agent.

Rapid and mild syntheses of radioiodine-labeled radiopharmaceuticals.

Radioiodine-labeled pharmaceuticals have been used extensively in diagnostic nuclear medicine. We have developed a rapid and mild method for incorporating radioiodine into functionally substituted molecules. The new process involves the reaction of the radioiodide ion with organoboranes in the presence of gentle oxidizing reagents. The radioiodide is utilized nearly quantitatively in a matter of seconds. The radiochemical yields are excellent, and parallel those obtained when iodine monochloride is reacted with organoboranes.

Evaluation of fluorine-18-BPA-fructose for boron neutron capture treatment planning.

UNLABELLED: Boron neutron capture therapy (BNCT) using 4-[10B]boronophenylalanine-fructose (BPA-Fr) is in Phase II clinical trials to validate BNCT as a treatment for glioblastoma multiforme and melanoma. Successful BNCT depends on knowledge of the distribution of boron-containing agents in both tumor and normal tissue as currently determined by chemical confirmation of boron deposition in surgically removed malignant tissue before BNCT. METHODS: We used PET to noninvasively obtain in vivo information on the pharmacokinetics of the 18F-labeled analog of BPA-Fr in two patients with glioblastoma multiforme. Time-activity curves generated from the bolus injection of 18F-BPA-Fr were coinvolved to simulate a continuous infusion used for BNCT therapy. RESULTS: Distribution of 18F-BPA-Fr by PET was found to be consistent with tumor as identified by MR imaging. The 18F-BPA-Fr tumor-to-normal brain uptake ratio was 1.9 in Patient 1 and 3.1 in Patient 2 at 52 min after injection. The 18F-BPA-Fr uptake ratio in glioblastoma paralleled that of nonlabeled BPA-Fr seen in patients as previously determined by boron analysis of human glioblastoma tissue obtained from pre-BNCT surgical biopsy. CONCLUSION: Knowledge of the biodistribution of BPA-Fr enables pre-BNCT calculation of expected tissue dosimetry for a selected dose of BPA-Fr at a specific neutron exposure. Fluorine-18-BPA-Fr PET is capable of providing in vivo BPA-Fr biodistribution data that may prove valuable for patient selection and pre-BNCT treatment planning.

Uptake and washout of borocaptate sodium and borono-phenylalanine in cultured melanoma cells: a multi-nuclear NMR study.

The cellular uptake and washout of the two principal boron neutron capture therapy (BNCT) agents, borocaptate sodium (BSH) and borono-phenylalanine (BPA), were monitored on-line, noninvasively, using nuclear magnetic resonance (NMR) spectroscopy. The uptake and washout of inorganic borate (B(i)) was also followed for comparison. M2R mouse melanoma cells grown on polystyrene microspheres were perfused inside the NMR sample tube. (11)B NMR was used to detect the presence of B(i), BSH and BPA, and (19)F NMR was applied to detect fluorinated BPA ((19)F-BPA). The results revealed chemical modifications of BSH due to spontaneous formation of the borocaptate dimer, BSSB, in the culture medium. BPA readily formed a complex with glucose contained in the culture medium but was also converted in the cells to a yet unidentified compound in a reaction that probably involves the hydrolysis of BPA and the release of B(i). The cellular accumulation ratio for BPA was significantly higher than 1 and was also significantly higher than that for BSH. On the other hand, the cellular retention time observed for BSH was much longer than for BPA, indicating a strong trapping of BSH in cells.

High resolution computed tomography and MRI for monitoring lung tumor growth in mice undergoing radioimmunotherapy: correlation with histology.

A model lung tumor system has been developed in mice for the evaluation of vascular targeted radioimmunotherapy. In this model, EMT-6 mammary carcinoma tumors growing in the lung are treated with 213Bi, an alpha particle emitter, which is targeted to lung blood vessels using a monoclonal antibody. Smaller tumors (< 100 microm in diameter) are cured, but larger tumors undergo a period of regression and then regrow and ultimately prove lethal. The goal of this work was to determine if external imaging with MRI or CT could be used routinely to monitor the growth/ regression of lung tumors in live mice. To attempt to evaluate individual tumors in vivo, animals were initially imaged with magnetic resonance imaging (MRI). High resolution MRI images could be obtained only after sacrifice when lungs were not moving. In contrast, high resolution computed tomography (CT) produced evaluable images from anesthetized animals. Serial CT images (up to 5/animal) were collected over a 17 day period of tumor growth and treatment. When tumored animals became moribund, animals were sacrificed and lungs were inflated with fixative, embedded in paraffin, and then sectioned serially to compare the detection of tumors by high resolution CT with detection by histology. CT proved most useful in detecting lung tumors located in the hilar area and least useful in detecting serosal surface and anterior lobe tumor foci. Overall, CT images of live animals revealed tumors in approximately 2/3 of cases detected in histologic serial sections when relatively few tumors were present per lung. Detection of lesions and their resolution post therapy were complicated due to residual hemorrhagic, regressing tumor nodules and the development of lung edema both of which appeared as high density areas in the CT scans. We conclude that the microCT method used could identify some lung tumors as small as 100 microm in diameter; however, no concrete evaluation of therapy induced regression of the tumors could be made with CT analyses alone.

Synthesis and evaluation of a new series of 17alpha-[(123)I]iodovinyl estradiols.

A series of 17alpha-substituted estradiols was synthesized in which the stereochemical characteristics of carbons 20 and 21 were modified. It was found that the (Z)-isomer demonstrated more favorable receptor binding affinity than the corresponding (E)-isomer.

Gadolinium-labeled liposomes containing amphiphilic Gd-DTPA derivatives of varying chain length: targeted MRI contrast enhancement agents for the liver.

Paramagnetic liposomal contrast agents were synthesized and utilized for selective augmentation of T1 MR imaging of the livers of normal Balb/c mice. Amphiphilic gadolinium complexes, which mimic phospholipids, were incorporated into the lamella of small unilamellar liposomes (SUV) such that they become an integral part of its surface. The amphiphilic complexing agents consisted of DTPA reagents in which a pair of alkyl chains of varying lengths are attached via amide linkages. The in vivo lifetimes of the amphiphilic agents were found to be dependent on the chain length of the alkyl groups.

In vivo boron-11 MRI and MRS using (B24H22S2)4- in the rat.

In vivo boron-11 magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) were performed on a rat that had been infused with a potential boron neutron capture therapy agent, Na4B24H22S2, using methods for detecting nuclei with a short T2 relaxation time. MRI and MRS were also performed on a euthanized rat that had been similarly infused in vivo. Boron-11 spectral intensities decreased in the living rat over a 25-h period. The results demonstrate the capability of MRI and MRS to noninvasively monitor the distribution and excretion of boron agents in vivo.

The syntheses and in vivo biodistribution of novel boronated unnatural amino acids.

A series of boronated, unnatural amino acids were prepared and their biodistribution determined in melanoma bearing mice. The unnatural amino acids were prepared utilizing recently developed borylation. The majority of the syntheses utilize metal catalyzed additions of diboron agents to unsaturated carbonyl compounds. Biodistribution studies in mice bearing melanoma tumors indicated that all the boronated amino acids were taken up by the melanoma tumors. The data for the cyclic five-membered ring analogue, 1-amino-3-boronocyclopentanecarboxylic acid, was most striking, exhibiting a nearly 22:1 ratio of boron concentration for tumor to brain at the 2 h time point, dropping to 7.3 after 6 h. The tumor to blood and tumor to skin ratios were also quite high. It is important to note that all of the amino acids were synthesized as racemic and diastereomeric mixtures. Thus there is a high probability that a single enantiomer of 1-amino-3-boronocyclopentanecarboxylic acid might exhibit far higher selectivity.

Biological evaluation of boronated unnatural amino acids as new boron carriers.

There is a pressing need for new and more efficient boron delivery agents to tumor cells for use in boron neutron capture therapy (BNCT). A class of boronated unnatural cyclic amino acids has demonstrated a remarkable selectivity toward tumors in animal and cell culture models, far superior to currently used agents in clinical BNCT. One of these amino acids, 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC), has shown a tumor to blood ratio of 8 and a tumor to normal brain ratio of nearly 21 in a melanoma bearing mouse model. This work represents further biological characterization of this compound for tumor targeting in an EMT6 murine mammary carcinoma mouse model and a T98G human glioblastoma cell line. Female BALB/c mice bearing EMT6 tumors were injected with the fructose complex form of racemic mixtures of cis and trans isomers of ABCPC in identical concentrations. Boron concentrations were measured in the tumor, blood, brain, skin, and liver tissues at 1, 3, and 5 h post-injection. These observations revealed a remarkable difference in racemic mixtures of cis and trans isomers in tumor targeting by boron. This implies that further separation of the L and D forms of this compound may enhance tumor targeting to an even higher degree than that provided by the racemic mixtures. Since the uptake measurements were made in homogenized tumor and normal tissues, little is known about the subcellular location of the boron arising from the various isomeric forms of the amino acid. To study subcellular delivery of boron from ABCPC in T98G human glioblastoma cells, we employed secondary ion mass spectrometry (SIMS) based technique of ion microscopy, which is capable of quantitatively imaging isotopic (elemental) gradients in cells and tissues at 500 nm spatial resolution. The T98G cells were exposed to the nutrient medium containing 100 ppm boron equivalent of a mixture of both L and D isomers of ABCPC in the form of a fructose complex for 1 h. Following this treatment, the cells were fast frozen, freeze-fractured, and freeze-dried for SIMS analysis. Within an hour of exposure, ABCPC provided partitioning of intracellular to extracellular boron of 3/1. SIMS imaging revealed that boron from ABCPC was distributed throughout the cell, including the nucleus. This level of boron delivery within an hour of exposure is superior to p-boronophenylalanine (BPA) and sodium borocaptate (BSH), which have been previously studied by SIMS in the same cell line. These encouraging observations provide compelling support for further isomeric separations of ABCPC into the D and L forms for enhanced tumor targeting and continued testing of these compounds as new boron carriers in BNCT.