Fazlul Alam

Synthesis of a carboranyl nucleoside for potential use in neutron capture therapy of cancer

Abstract The reaction of 2′,3′-O-(dibutylstannylene)uridine (1) with 3-bromopropyne yielded 2′,(3′)-O-(3-propynyl)uridine (2 and 3). The mixture of 2 and 3 on acetylation followed by chromatographic purification yielded 3′,5′-di-O-acetyl-2′-O-(3-propynyl) uridine (4). Reaction of 4 with bis(acetonitrile)decaborane produced 3′,5′-di-O-acetyl-2′-O-(o-carboran-l-ylmethyl)uridine (5). Deacetylation of 5 gave 2′-O-(o-carboran-l-ylmethyl) uridine (6), a boronated nucleoside for potential use in neutron capture therapy of cancer.

Boronation of antibodies with mercaptoundecahydro-closo-dodecaborate(2-) anion for potential use in boron neutron capture therapy

The anionic polyhedral borane derivative, mercaptoundecahydro-closo-dodecaborate(2-), has been evaluated as a boronating agent for antibodies. The objective of these studies was the selective delivery of boron to neoplasms for neutron capture therapy. Incubation of a large excess of this anion with the polyclonal antibody antithymocyte globulin (ATG) resulted in the incorporation of 9-13 mol of the anion per mol of antibody. The extent of boron incorporation into the protein was measured either by tritium-labeled B12H11SH2- or by direct boron determination with neutron activation analysis. The nature of the covalent linkage of the anion to the antibody appeared to involve the formation of a new disulfide bond by a thiol-disulfide exchange. The number of boron atoms incorporated into antibodies by this method appeared to be inadequate for neutron capture therapy. However, such boronated antibodies may have potential for the detection of molecules of biologic interest by means of electron energy loss spectroscopy.

Delivery of Boron-10 for Neutron Capture Therapy by Means of Monoclonal Antibody - Starburst Dendrimer Immunoconjugates

The use of monoclonal antibodies (MoAbs) for the delivery of radionuclides, drugs and toxins for therapeutic purposes has been the subject of intensive investigation over the past decade. A few investigators, including ourselves, have focused on the possible use of MoAbs directed against tumor associated antigens (TAA) for targeting boron-10 to tumors1,2. Using a high molecular weight macromolecule, poly-DL-lysine and a methyl isocyanato-polyhedral borane, Na(CH3)3 NB10H8NCO, we have prepared a boronated polylysine (BPL) containing 23% boron by weight and having > 1700 boron atoms per polymeric unit3. This boronated macromolecule was then attached to MoAbs utilizing two heterobifunctional reagents N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP), which was used to introduce potential sulfhydryl groups into proteins, and sulfo mmaleimidobenzoyl-N-hydroxysuccinimide ester (sMBS), which was used to introduce maleimido groups on MoAbs4. The resulting immunoconjugates retained a high degree of in vitro immunoreactivity but had lost their in vivo tumor localizing properties5. It became apparent that an alternative approach was required to produce boron containing immunoconjugates that would retain both their immunoreactivity and in vivo tumor localizing properties6. The purpose of the present report is to describe our most recent efforts to prepare boron containing immunoconjugates using a neutrally charged precision macromolecule consisting of repetitive polyamido amino groups(PAMAM) arranged in a starburst pattern (“starburst” dendrimers)7.

Boron-10 concentration measurements using the solid-state nuclear track detector CR-39 and automatic image analysis

Calibration curves are determined for measuring the concentration of /sup 10/B in the blood of rats using an autoradiographic procedure, with the polycarbonate solid-state nuclear track detector CR-39 and an image analysis system for automatic track counting. The calibration curves indicate that for the etch procedure used, the nitrogen concentration in the blood is an important interfering input for /sup 10/B concentration measurements. By discriminating against small racks, the sensitivity to the blood nitrogen concentration can be reduced to the point that a variation in the nitrogen weight fraction of 0.01 g N/g blood causes a variation in the predicted /sup 10/B concentration of 0.3 {mu}g /sup 10/B/ml blood.

Boron Compounds for Neutron Capture Therapy

There have been two main approaches to the development of boron compounds for neutron capture therapy (BNCT). One has involved the synthesis of boronated analogues of organic structures which possess a high degree of selectivity for neoplastic cells. These include amino acids, nucleic acid precusors, porphyrins and promazines. The second approach has emphasized the use and incorporation of boron compounds into monoclonal antibodies targeted against tumor associated antigens. There have been several important requirements in achieving the use of antibodies for BNCT. First, the conjugation of boron to monoclonal antibodies must occur with significant retention of the antibody’s immunoreactivity. Second, sufficient numbers of boron atoms have to be incorporated and at least 103 boron atoms per protein molecule is necessary if a goal of 109 boron atoms per tumor cell is to be attained. Third, separation of the boron-containing antibody from the unconjugated species and from the boron entity used in the conjugation is essential. Finally, the boron-loaded antibody must have the ability for targeting all the tumor cells, under in vivo conditions with a high degree of selectivity. Research at The Ohio State University on the incorporation of boron-containing polymers into monoclonal antibodies has already been described1. The work presented herein outlines the synthesis of boronated analogues of promazines and phthalocyanines, structures which have a demonstrated proclivity for certain neoplasms. The tissue distribution data in tumor-bearing animals for certain of these compounds are presented.

Tumor Targeting Agents for Neutron Capture Therapy

The development of compounds for use as delivery agents in the treatment of cancer by neutron capture therapy has largely focused on the use of boron-10 even though there are other nuclides having higher capture cross section values for thermal neutrons. The major reasons are: (1) it is nonradioactive and comprises approximately 20% of naturally occurring boron; (2) boron-10 enriched materials are readily available commercially; (3) the products of the fission reaction are largely high LET recoiling 7Li and 4He (alpha) nuclei; (4) the pathlength of these particles is such that they are confined to a radius approximating the diameter of a single cell; and (5) the chemistry of boron is such that it may be incorporated into a multitude of different stable chemical structures.

Carboranyl Precursors of Nucleic Acids--Potential DNA Probes for BNCT

It has been determined that the biological effectiveness of BNCT will be maximized if the capture reaction were to occur in the cell nucleus in comparison with the cytoplasm, on cell membrane or in extracellular spaces.1–2 This has been the basis for the ongoing synthesis of boron compounds which are chemically similar to the building blocks of the nucleic acids. Initially, the approach concentrated on the preparation of purine and pyrimidine bases which contain boron.3–7 The rationale was that such structures might emulate the naturally-occurring purine and pyrimidine bases and become more selectively incorporated into tumor cell nuclei in comparison to normal cells due to the higher proliferative rates of the former. Of the different boron compounds which were initially synthesized, many were unstable, toxic or failed to become incorporated into nucleic acids. One structure which resembled more closely its natural counterpart was 5-dihydroxyboryluracil.8 Its synthesis encouraged Schinazi and Prusoff to prepare the first boron-containing pyrimidine nucleoside.9

Pre-clinical studies on boron neutron capture therapy.

The present report provides an overview of the multidisciplinary research effort on BNCT that currently is in progress at The Ohio State University. Areas under investigation include the preparation of boron containing monoclonal antibodies, the synthesis of boron containing derivatives of promazines and phathalocyanines, the development of a rat model for the treatment of glioblastoma by means of BNCT, the design of an accelerator-based neutron irradiation facility, and 10B concentration measurements using alpha track autoradiographic methods. Progress in each of these areas is described and the direction of future research is indicated.

Determination of Boron Concentration by Means of Direct Current Plasma - Atomic Emission Spectroscopy

The accurate measurement of total boron content in biological samples with a sensitivity in the ppm range is essential for evaluating the potential usefulness of various tumor localizing boron-containing compounds for Boron Neutron Capture Therapy (BNCT)1. Among the procedures that have been used are spectrophotometric analyses involving various complexing agents2–4. These methods are time consuming and require that the boron compounds can be oxidized to boric acid. Relatively low sensitivity and interference from various contaminants further limit their usefulness. Recently, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) has been shown to be sensitive enough for the detection of microgram quantities of boron in biological samples5,6, although the sensitivity is adversely affected by high concentrations of inorganic salts in the samples7. Since alkaline fusion is not suitable for use with ICP8, samples were digested by exposure to either perchloric acid5,6 or nitric acid6. There may be a danger of explosion with the former, and with the latter, it may be necessary to decompose the tissues in teflon-lined digestion bombs. Both procedures, therefore, have their limitations. The objectives of the present study were to determine whether DCPAES could be used to quantify boron in a variety of chemical compounds including polyhedral boranes and carboranes, to improve the procedure for the digestion of tissue samples, to determine if this method could be used to quantify cellular uptake of boron, and finally to define the limits of boron detection in biologic samples by means of this method.