Dianne M. Adams

Boron Neutron Capture Therapy of Brain Tumors: Biodistribution, Pharmacokinetics, and Radiation Dosimetry of Sodium Borocaptate in Patients with Gliomas

OBJECTIVEThe purpose of this study was to obtain tumor and normal brain tissue biodistribution data and pharmacokinetic profiles for sodium borocaptate (Na2B12H11SH) (BSH), a drug that has been used clinically in Europe and Japan for boron neutron capture therapy of brain tumors. The study was performed with a group of 25 patients who had preoperative diagnoses of either glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) and were candidates for debulking surgery. Nineteen of these patients were subsequently shown to have histopathologically confirmed diagnoses of GBM or AA, and they constituted the study population. METHODSBSH (non-10 B-enriched) was infused intravenously, in a 1-hour period, at doses of 15, 25, and 50 mg boron/kg body weight (corresponding to 26.5, 44.1, and 88.2 mg BSH/kg body weight, respectively) to groups of 3, 3, and 13 patients, respectively. Multiple samples of tumor tissue, brain tissue around the tumors, and normal brain tissue were obtained at either 3 to 7 or 13 to 15 hours after infusion. Blood samples for pharmacokinetic studies were obtained at times up to 120 hours after termination of the infusion. Sixteen of the patients underwent surgery at the Beijing Neurosurgical Institute and three at The Ohio State University, where all tissue samples were subsequently analyzed for boron content by direct current plasma-atomic emission spectroscopy. RESULTSBlood boron values peaked at the end of the infusion and then decreased triexponentially during the 120-hour sampling period. At 6 hours after termination of the infusion, these values had decreased to 20.8, 29.1, and 62.6 &mgr;g/ml for boron doses of 15, 25, and 50 mg/kg body weight, respectively. For a boron dose of 50 mg/kg body weight, the maximum (mean ± standard deviation) solid tumor boron values at 3 to 7 hours after infusion were 17.1 ± 5.8 and 17.3 ± 10.1 &mgr;g/g for GBMs and AAs, respectively, and the mean tumor value averaged across all samples was 11.9 &mgr;g/g for both GBMs and AAs. In contrast, the mean normal brain tissue values, averaged across all samples, were 4.6 ± 5.1 and 5.5 ± 3.9 &mgr;g/g and the tumor/normal brain tissue ratios were 3.8 and 3.2 for patients with GBMs and AAs, respectively. The large standard deviations indicated significant heterogeneity in uptake in both tumor and normal brain tissue. Regions histopathologically classified either as a mixture of tumor and normal brain tissue or as infiltrating tumor exhibited slightly lower boron concentrations than those designated as solid tumor. After a dose of 50 mg/kg body weight, boron concentrations in blood decreased from 104 &mgr;g/ml at 2 hours to 63 &mgr;g/ml at 6 hours and concentrations in skin and muscle were 43.1 and 39.2 &mgr;g/g, respectively, during the 3- to 7-hour sampling period. CONCLUSIONWhen tumor, blood, and normal tissue boron concentrations were taken into account, the most favorable tumor uptake data were obtained with a boron dose of 25 mg/kg body weight, 3 to 7 hours after termination of the infusion. Although blood boron levels were high, normal brain tissue boron levels were almost always lower than tumor levels. However, tumor boron concentrations were less than those necessary for boron neutron capture therapy, and there was significant intratumoral and interpatient variability in the uptake of BSH, which would make estimation of the radiation dose delivered to the tumor very difficult. It is unlikely that intravenous administration of a single dose of BSH would result in therapeutically useful levels of boron. However, combining BSH with boronophenylalanine, the other compound that has been used clinically, and optimizing their delivery could increase tumor boron uptake and potentially improve the efficacy of boron neutron capture therapy.

Folate Receptor-Mediated Liposomal Delivery of a Lipophilic Boron Agent to Tumor Cells in Vitro for Neutron Capture Therapy

AbstractPurpose. This study was aimed at the in vitro evaluations of folate receptor (FR)-targeted liposomes as carriers for a lipophilic boron agent, K[nido-7-CH3(CH2)15-7,8-C2B9H11], in FR-overexpressing tumor cells for neutron capture therapy. Methods. Large unilamellar vesicles (∼200 nm in diameter) were prepared with the composition of egg PC/chol/K[nido-7-CH3(CH2)15-7,8-C2B9H11] (2:2:1, mol/mol), with an additional 0.5 mol % of folate-PEG-DSPE or PEG-DSPE added for the FR-targeted or nontargeted liposomal formulations, respectively. Results. Boron-containing, FR-targeted liposomes readily bound to KB cells, an FR-overexpressing cell line, and were internalized via FR-mediated endocytosis. The boron uptake in cells treated with these liposomes was approximately 10 times greater compared with those treated with control liposomes. In contrast, FR-targeted and nontargeted liposomes showed no difference in boron delivery efficiency in F98 cells, which do not express the FR. The subcellular distribution of the boron compound in KB cells treated with the FR-targeted liposomes was investigated by cellular fractionation experiments, which showed that most of the boron compound was found in either the cytosol/endosomal or cell membrane fractions, indicating efficient internalization of the liposomal boron. Conclusion. FR-targeted liposomes incorporating the lipophilic boron agent, K[nido-7-CH3(CH2)15-7,8-C2B9H11], into its bilayer were capable of specific receptor binding and receptor-mediated endocytosis in cultured KB cells. Such liposomes warrant further investigations for use in neutron capture therapy.

Evaluation of systemically administered radiolabeled epidermal growth factor as a brain tumor targeting agent

We have previously reported a method for labeling epidermal growth factor (EGF) with technetium-99m and have shown that 99mTc-EGF localized in EGF receptor (R) positive intracerebral C6EGFR rat gliomas following intratumoral (i.t.) injection of the radioligand. In the present study, we have evaluated the potential use of 99mTc-EGF as a tumor targeting agent after systemic administration to Fischer rats bearing intracerebral implants of C6EGFR gliomas. Radiolocalization was determined following intravenous (i.v.) or intracarotid (i.c.) injection with or without hyperosmotic mannitol induced disruption of the blood–brain barrier (BBB-D). As determined by γ-scintillation counting, 4 h after i.c. injection of 99mTc-EGF, 0.34% of the injected dose per gram (% ID/g) was localized in C6EGFR tumors, which expressed 105–106 EGFR sites per cell, compared to 0.07% ID/g in animals bearing C6 wildtype gliomas, which do not express EGFR. The corresponding tumor to brain ratios were 5.6 and 1.6, respectively. Tumors could be visualized by external γ-scintigraphy in rats bearing C6EGFR but not C6 wildtype gliomas, thereby establishing that radiolocalization was dependent upon receptor expression. Intracarotid administration of 99mTc-EGF significantly increased tumor uptake compared to i.v. injection (0.34 vs 0.14% ID/g, p<0.04). BBB-D disruption, followed by i.c. injection of 99mTc-EGF, however, did not significantly enhance tumor uptake compared to i.c. injection without BBB-D (0.45% vs 0.34% ID/g, p>0.1). The uptake of 99mTc-EGF was ∼4–9% ID/g in the liver and 12–20% ID/g in the kidneys after i.c. or i.v. administration. External γ-scintigraphy of regions of interest over the liver and kidneys revealed that ∼70–80% of the whole body radioactivity accumulated in these organs, and only 0.47–0.83% in the tumor following i.v. or i.c. administration of 99mTc-EGF. Our study has demonstrated that EGF can be used as a specific targeting agent for EGFR (+) rat brain tumors. However, it is unlikely that systemic injection of EGF-based bioconjugates can deliver sufficient amounts of the ligand to brain tumors for therapeutic purposes and direct delivery by means of either intratumoral injection or a variant of it such as convection enhanced delivery will be required.

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.

In Vivo Distribution of Boronated Monoclonal Antibodies and Starburst Dendrimers

Monoclonal antibodies (MoAbs) are theoretically among the most specific of all tumor targeting agents. In the span of ten years a voluminous literature has developed on their potential use for the diagnosis and treatment of cancer. Drugs, radionuclides, and toxins have been linked to MoAbs, but, unfortunately, their therapeutic efficacy has been limited due to a variety of problems1. Attempts to use MoAbs to target 10B to tumors for BNCT have been unsuccessful for many of these same reasons. In order to sustain a lethal 10B (n,α)Li7 reaction at the cellular level, approximately ≈109 10B atoms must be delivered to each tumor cell2. This requirement has led to the use of macromolecules such as boronated poly-D,L-lysine (BPL) or boronated starburst dendrimers (BSD), which can be linked to MoAbs by means of heterobifunctional reagents3,4. The purpose of the present study was to evaluate the in vivo distribution of a MoAb directed against the murine B16 melanoma5 that has been boronated using a starburst dendrimer, and to compare this to the distribution patterns of native MoAb and unmodified dendrimers of varying molecular weights.

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.

Epidermal growth factor (EGF) as a potential targeting agent for delivery of boron to malignant gliomas

Epidermal growth factor (EGF) is a single-chain, 53-residue polypeptide that binds to a transmembrane glycoprotein with tyrosine kinase activity1 After binding, the receptor-ligand complex is promptly internalized and then degraded in lysosomes. The EGF receptor (EGFR) gene is homologous to the c-erb B oncogene2 and is often amplified in human glioblastomas. Studies by Bigner et al. 3 revealed that in a series of 33 human glioma biopsies, 15 showed amplification of of the EGFR gene. Similiar or even higher frequencies of amplification have been observed by others4,5, and this often is associated with an increase in cell surface receptor expression6,7. The distribution of EGFR in high grade gliomas is variable, which probably reflects the cellular heterogenity of these tumors. However, in 25 of 42 human gliomas studied by Torp et al. 6, more than 50% of the tumor cells bound biotinylated EGF. Attempts have been made to use EGFRs as targets for radioimmunotherapy, and in one series of patients with recurrent glioblastomas, 50% showed some evidence of a clinical response with no significant toxicity8,9, although there was no followup report indicating improved survival. Nevertheless, the EGFR still remains one of the more attractive targets on high grade gliomas10. Targeting the EGFR by means of boronated EGF may be easier than using boronated monoclonal antibodies (MoAbs) directed against EGFR since EGF has a relatively low molecular weight (~6 kD) compared to MoAbs, and it should be possible to construct relatively small conjugates, which would diffuse more rapidly into poorly vascularized regions of the tumor. The rapid internalization and degradation of EGF-EGFR complexes, as well as their high affinity constant, are further advantages, if the boron persists intracellulary. The purpose of the present study was to develop methodology for the boronation of EGF and to study its in vitro affinity for glioma cells.

A Rat Model for the Treatment of Melanoma Metastatic to the Brain by Means of Neutron Capture Therapy

Melanoma metastatic to the brain is a serious clinical problem for which there currently is no satisfactory treatment1,2. Boron neutron capture therapy (BNCT) has been shown by Mishima et al. to be clinically effective in the treatment of cutaneous melanoma using 10B-enriched boronophenylalaine (BPA) as the capture agent3. Similarly, Coderre et al. have demonstrated efficacy of BNCT in the treatment of the Harding-Passey melanoma in BALB/c mice4. Our previous experience in the treatment of a rat glioma by means of BNCT5 has lead us to develop a rat model for the treatment of melanoma metastatic to the brain. We have employed a human melanoma cell line, MRA 27, which when implanted intracerebrally into immunologically deficient nude rats, grows progressively and ultimately kills the host. Survival time is dependent upon the size of the initial tumor inoculum, and death occurs as a result of the expanding intracranial mass. Although human melanoma metastatic to the brain may be multicentric6, the biological behavior of intracerebrally implanted MRA 27 simulates it in a number of ways, and therefore provides a good model for the human tumor. The purpose of the present study was to evaluate the efficacy of BNCT for the treatment of intracerebrally implanted MRA 27 using BPA as the capture agent and to compare its efficacy with external beam gamma irradiation.

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.

Distinct and Non-Cross-Reactive Epitopes are Recognized on B16 Melanoma by LAK Cells and Anti-B16 Monoclonal Antibodies

Abstract Two rat anti-B16 melanoma monoclonal antibodies (MoAb), designated IB16–6 and IB16–8, recognize an epitope expressed with high density on the surface of B16 parental cells and B16-F1, F10, F10FLR, and BL6 sublines. The purpose of this study was to define by means of cytolytic and clonogenic assays whether these MoAbs reacted with the same or distinct determinants as those recognized on B16 targets by lymphokine-activated killer (LAK) cells. Using 125I-labeled antibody and Scatchard analysis, the affinity constant (KA ) of IB16–6 was determined to range from 5.6 to 9.4 × 105 liter/M and the number of receptor sites per B16 cell was 4.8 × 104 to 2.5 × 105. The effects of anti-B16 MoAb on LAK activity were determined by either preincubating 51Cr-labeled B16 target cells with varying concentrations of MoAb, followed by the cytolytic assay, or exposing unlabeled B16 cells to MoAb, and then carrying out a 10-day clonogenic assay. Over a wide range of antibody concentrations, IB16–6 and IB16–8 had minimal effects on LAK activity, and even at MoAb concentrations up to 1 mg there were no changes in target cell sensitivity or colony-forming ability. Enzymatic treatment of B16 melanoma cells with either trypsin or pronase completely removed the epitope recognized by MoAb IB16–6 but did not alter B16 sensitivity to LAK cells. These observations indicate that the LAK recognition unit was distinct from the epitope reactive with MoAb IB16–6 and that the B16 determinant(s) recognized by LAK cells is resistant to proteolytic enzymes. The molecular structure of each of these remains to be determined.