Michael S. Makar

Neuropathology of ablation of rat gliosarcomas and contiguous brain tissues using a microplanar beam of synchrotron-wiggler-generated X rays

Adult‐rat‐brain tissues display an unusually high resistance to necrosis when serially irradiated with parallel, thin slices of a microplanar (i.e., microscopically thin and macroscopically broad) beam of synchrotron‐wiggler‐generated, approx. 35–120 keV (median approx. 50 keV) Gd‐filtered X rays at skin‐entrance absorbed doses of 312 to 5000 Gy per slice. Such microplanar beams were used to irradiate young adult rats bearing right frontocerebral 9L gliosarcomas (approx. 4 mm diameter), through a volume of tissue containing the tumor and contiguous brain tissue, either in a single array or in 2 orthogonally crossed arrays of tissue slices. Each array included 101 parallel microplanar slices, 100 μm center‐to‐center distance, each slice being approx. 25 μm wide and 12 mm high, with skin‐entrance absorbed doses of 312.5 Gy or 625 Gy per slice. Compared with unirradiated controls with a median survival time of 20 days after tumor initiation, the median survival time was extended in irradiated rats by 139 days (625 Gy, crossed arrays), 96 days (312.Gy, crossed arrays) or 24 days (625 Gy, single array). The tumors disappeared in 22 of the 36 irradiated rats, 4/ 11 even after unidirectional microbeam irradiation. The extent and severity of radiation damage to the normal brain in rats with or without tumor was graded histopathologically. Correlation of those grades with radiation doses shows that loss of tissue structure was confined to beam‐crossing regions and that only minor damage was done to zones of the brain irradiated unidirectionally. Int. J. Cancer 78:654–660, 1998.

Accumulation of Boron in Malignant and Normal Cells Incubated In Vitro with Boronophenylalanine, Mercaptoborane or Boric Acid

The short (< 10 microns) ranges of alpha and 7Li particles produced during boron neutron capture therapy (BNCT) make the partitioning of the boronated drug within and without the cell of critical importance. The evaluation of the potential usefulness of a boron-containing substance for BNCT requires information about its intracellular accumulation. In the present report, an in vitro method is described for direct measurement of intracellular boron based on rapid centrifugation of cells through a layer of mineral oil and silicon oil to strip away extracellular growth medium. The intracellular concentrations of boronophenylalanine (BPA), mercaptoborane (BSH) and horic acid in malignant cells and in normal cells have been compared. The accumulation ratio is defined as the ratio of the intracellular to the extracellular boron concentration. Boric acid showed an accumulation ratio of 1 while the ratios for BSH and BPA were dependent on cell type and tended to be greater for BPA than for BSH in malignant but not in normal cells.

Synthesis, toxicity and biodistribution of two 5,15-di[3,5-(nido-carboranylmethyl)phenyl]porphyrins in EMT-6 tumor bearing mice

The total synthesis of a 5,15-di[3,5-(o-carboranylmethyl)phenyl]porphyrin 5, its zinc(II) complex 6, and the corresponding nido-carboranylporphyrins 7 and 8 are reported. The molecular structures of porphyrin 6 and of potassium nido-carborane were obtained and are described. The biodistribution of nido-carboranylporphyrins 7 and 8 in BALB/c mice bearing EMT-6 mammary tumors are presented and compared. Both compounds are effective tumor localizers and delivered therapeutic concentrations of boron to tumors (mean+/-standard deviation): 32.5+/-7.1 and 54.3+/-14 microg/g for 7 and 8, respectively, 2 days after the last of 3 injections of a total boron dose of 23 mg/kg body weight. The zinc(II) complex 8 was found to deliver 1.2-1.7 times higher amounts of boron to tumors than 7, with lower tumor-to-blood boron concentration ratios (9.8/1 and 4.7/1 for 7 and 8, respectively, 2 days after injections). The tumor-to-brain boron concentration ratios were >100/1 for both porphyrins 2 days after administration. Both nido-carboranylporphyrins 7 and 8 were well-tolerated at the concentrations used (75 and 78 mg/kg body weight, respectively) and no morbidity or mortality were observed in these studies.

In vitro determination of toxicity, binding, retention, subcellular distribution and biological efficacy of the boron neutron capture agent DAC-1

In boron neutron capture therapy (BNCT), 10B is delivered selectively to the tumour cells and the nuclide then forms high-LET radiation (4He2+ and 7Li3+) upon neutron capture. Today much research is focused on development of a variety of boron compounds aimed for BNCT. The compounds must be thoroughly analysed in preclinical tests regarding basic characteristics such as binding and subcellular distribution to enable accurate estimations of dose-modifying factors. DAC-1,2-[2-(3-amino-propyl)-1,2-dicarba-closo-dodecaboran (12)-1-yl-methoxy]- 1,3-propanediol was synthesized at our laboratories and the human colon carcinoma cells LS-174T were used as an in vitro model. The boron compound showed a remarkable intracellular accumulation, 20-100 times higher than the boron content in the culture medium, in cultured cells and was not removed by extensive washes. Approximately half of the boron taken up also remained within the cells for at least 4 days. The DAC-1 compound alone was not toxic at boron concentrations below 2.5 micrograms B/g. The intracellular distribution of the boron compound was investigated by subcellular fractionation experiments and low pH treatments. It is possible that DAC-1 binds to some intracellular molecules or to membranes connected with organelles in the cytoplasm or even to the inside of the outer cell membrane. Another possibility is that the compound, due to the somewhat lipophilic properties, is embedded in the membranes. Thermal neutron irradiations were carried out at the Brookhaven Medical Research Reactor (BMRR). At a survival level of 0.1, DAC-1 + thermal neutrons were about 10.5 times more effective in cell inactivation than the thermal neutrons alone. Monte Carlo calculations gave a mean value of the 10B-dependent specific energy, the dose, of 0.22 Gy. The total physical dose during irradiation of DAC-1-containing cells with a neutron fluence of 0.18 x 10(12) n/cm2 was 0.39 Gy. The dose-modifying factor, at survival level 0.1, when comparing irradiation with thermal neutrons with and without DAC-1 was 3.4, while the dose-modifying factor when comparing neutron irradiations of cells with DAC-1 and irradiation of the cells with 60Co-gamma was 7.3. The results are encouraging and in vivo tests of tissue distributions and tumour uptake should now be carried out.

Uptake, toxicity and radiation effects of the boron compounds DAAC-1 and DAC-1 in cultured human glioma cells

PURPOSE To study the uptake, toxicity and radiation effects in vitro of a diol-amino acid-carborane (DAAC-1) and make comparisons with the previously studied diol-amine-carborane (DAC-1). MATERIALS AND METHODS Toxicity and radiation effects were studied with clonogenic survival, uptake by measuring the cellular boron content and the subcellular distribution was investigated after organelle separation with centrifugation. The studied cell line was human glioma U343. RESULTS DAAC-1 showed an accumulation of 1-1.5 times, compared with the culture medium, and was non-toxic up to 47 microg boron/ml. The accumulation of DAC-1 was about 90 times, but toxic effects were detectable already at the concentration 5 microg boron/ml. None of the compounds was localized in the cell nucleus. Following irradiation with thermal neutrons, DAC-1 was about 2.5 times more effective than DAAC-1 and about 4.9 times more effective than neutrons alone, at the survival level 0.2. The dose modifying factors, when compared with the neutron beam alone, were for both DAAC-1 and DAC-1 about 1.5 and about 5 when compared with 60Co-gamma-radiation. CONCLUSIONS DAAC-1 was less toxic than DAC-1 but gave less accumulation of boron. Both substances gave significant boron-dependent cell inactivation when the test cells were exposed to thermal neutrons.

Enhancement of the radiation response of EMT-6 tumours by a copper octabromotetracarboranylphenylporphyrin

OBJECTIVE The carborane-containing porphyrin, copper (II) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(3-[1,2-dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin (CuTCPBr), was investigated as a potential radiation enhancing agent for X-ray radiotherapy (XRT) in a subcutaneously implanted EMT-6 murine carcinoma. METHOD The biodistribution and toxicological profile of this porphyrin has been shown to be favourable for another bimodal radiotherapy technique, boron neutron-capture therapy. For the XRT studies, CuTCPBr was formulated in either 9% Cremophor (BASF Corporation, Ludwigschafen, Germany) EL and 18% propylene glycol (9% CRM) or a revised formulation comprising 1% Cremophor ELP, 2% Tween 80 (JT Baker, Mansfield, MA), 5% ethanol and 2.2% PEG 400 (CTEP formulation), which would be more clinically acceptable than the original 9% CRM formulation. Using the 9% CRM formulation of CuTCPBr, doses of 100, 210 or 400 mg kg(-1) of body weight were used in combination with single doses of 25-35 Gy 100 kVp X-rays. RESULTS While doses of 100 mg kg(-1) and 210 mg kg(-1) did not result in any significant enhancement of tumour response, the 400 mg kg(-1) dose did. A dose modification factor of 1.20±0.10 was obtained based on the comparison of doses that produced a 50% local tumour control probability. With the CTEP formulation of CuTCPBr, doses of 83 and 170 mg kg(-1) produced significant radiation enhancement, with dose modification factors based on the TCP(50) of 1.29±0.15 and 1.84±0.24, respectively. CONCLUSION CuTCPBr significantly enhanced the efficacy of XRT in the treatment of EMT-6 carcinomas in mice. The CTEP formulation showed a marked improvement, with over 9% CRM being associated with higher dose modification factors. Moreover, the radiation response in the skin was not enhanced.

Cell Survival Following in Vitro Irradiation at Depth in a Lucite Phantom as a Measure of Epithermal Beam RBE

The radiation field produced in tissue during BNCT consists of a mixture of components with differing linear energy transfer (LET) characteristics. In addition to the high-LET products of the 10B(n,α)7Li reaction, the interaction of the neutron beam with the nuclei of elements in tissue will deliver an unavoidable, non-specific background dose, from a mixture of high- and low-LET radiation components, to both tumor and normal tissue. Thermal neutron capture by hydrogen releases a gamma ray through the 1H(n,γ)2H reaction. The capture of thermal neutrons by nitrogen in tissue, the 14N(n,p)14C reaction, releases a high-LET proton with an energy of 590 keV. Contaminating fast neutrons (those with kinetic energies >10keV) in the epithermal neutron beam produce high-LET recoil protons through collisions with hydrogen nuclei (1H(n,n′)p reaction) in tissue. Because the energies of the nitrogen capture proton and the fast neutron recoil proton tend to be in the same range, the biological effects of these high-LET components of the dose are most conveniently measured as a combined “proton dose”. This high-LET “proton dose” must be multiplied by an experimentally determined factor for relative biological effectiveness (RBE) in order to express the total BNCT dose in photon-equivalent units.

BSMel: An Aromatic Amino Acid Analog of High Boron Content, Easily Prepared from B-10 Enriched and Stereochemically Pure Precursors

In the familiar format of BNCT for cancer, the cells of a malignant tumor are sensitized to thermal neutrons by systemic administration of a B-10 containing compound that is selectively taken up by malignant cells. The boron-loaded tumor cells are then killed by neutron irradiation within a defined target volume. Effective BNCT requires a minimum of about 30μg 10B/g in the targeted malignant cells. To produce a useful therapeutic gain, there should be a differential of three or four fold between the boron concentration in malignant cells compared to that in the “normal” cells. This demanding pattern of boron delivery must be accomplished by a compound that has no serious toxicity to any vital tissue, either within the treatment volume or elsewhere in the body. Given these daunting requirements, it is remarkable that two compounds, BSH and L-Bpa, are currently in clinical trials, aspiring to the cure of glioblastoma multiforme. These gratifying results are hard won by many people over more than three decades. Hopefully, a careful choice of therapeutic targets, along with diligent attention to the literature of metabolite analog design, will allow new BNCT applications using new boron delivery molecules to reach clinical trial in a shorter time frame.

Dose-Response Analysis for Boron Neutron Capture Therapy of the B16 Murine Melanoma Using p-Boronophenylalanine

Boron Neutron Capture Therapy (BNCT) of a well-pigmented B16 melanoma implanted subcutaneously in the mouse thigh has been carried out at the Brookhaven Medical Research Reactor (BMRR) using the synthetic amino acid p-boronophenylalanine (BPA) as the boron delivery agent. The response of the B16 melanoma to BNCT was compared with the response to 250 kVp x-rays using both tumor growth delay and an in vivo/in vitro assay that measures clonogenic survival. These experiments allow a comparison of tumor growth delay, log cell kill and damage to normal tissues produced by BNCT or photon irradiation.