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Several neuroendocrine tumors are known to express both the somatostatin receptor subtype 2 (SSTR2) and the norepinephrine transporter (NET), and radiopharmaceuticals directed toward both these targets such as MIBG and octreotide derivatives are routinely used in the clinic. To investigate the possibility of targeting both NET and SSTR2 conjointly, a conjugate of radioiodinated MIBG and octreotate was synthesized. Attempts to synthesize the radioiodinated target compound (MIBG-octreotate; [ (131)I] 12a) from a tin precursor were futile; however, it could be accomplished from a bromo precursor by exchange radioiodination in 3-36% ( n = 10) radiochemical yields. The total uptake of [ (131)I] 12a in SK-N-SH human neuroblastoma cells transfected to express SSTR2 (SK-N-SHsst2) was similar to that for [ (125)I]MIBG at all time points (34.9 +/- 2.4% vs 43.8 +/- 1.2% at 4 h; p < 0.05), while it was substantially lower (5.4 +/- 0.3% vs 35.9 +/- 1.2%) in the SH-SY5Y cell line, a subclone of SK-N-SH line that is known to express SSTR2. The NET blocker desipramine reduced the uptake of [ (131)I] 12a only to a small extent, further suggesting a limited role of NET in its binding and accumulation. Uptake of [ (131)I] 12a in SK-N-SHsst2 cells was 8-10-fold higher ( p < 0.05) than that of [ (125)I]I-Gluc-TOCA, an octreotide analogue, at all time points over a 4 h period and was reduced to about 20% by 10 muM octreotide demonstrating that the uptake of [ (131)I] 12a in this cell line is predominantly mediated by SSTR2. The intracellularly trapped radioactivity in SK-N-SHsst2 cells was substantially higher for [ (131)I] 12a compared to that for [ (125)I]OIBG-octreotate, an isomeric congener of 12a. Because MIBG has more specific NET-mediated uptake than OIBG, this suggests at least a partial role for NET-mediated uptake of [ (131)I] 12a in this cell line. While further refinement in the structure of the conjugate-probably interposition of a flexible and/or cleavable linker between the MIBG and octreotate moieties-may be necessary to make it a substrate/ligand for both NET and SSTR2, this conjugate is demonstrated to be much superior than I-Gluc-TOCA with respect to the uptake in SSTR2-expressing cells.

Background: Somatostatin analogues labelled with radiometals or radiohalogens are useful for the imaging and treatment of somatostatin receptor-containing tumours. In this study, the procedures for the radioiodination of glucose-Tyr3-octreotate (gluc-Tyr3-tate) and radiolabelling of DOTA-Tyr3-octreotate (DOTA-Tyr3-tate) with 111In, 177Lu and 125I were compared and their metabolism in rats was analyzed. The usefulness of high performance liquid chromatography (HPLC) analysis and instant thin-layer chromatography on silica gel (ITLC-SG) for both radiochemical purity determination and analysis of metabolism in urine was investigated., Materials and Methods: For labelling with radiometals, the formation of a complex with the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) functionality of the peptide was employed. Radioiodination was performed by the chloramime-T method. The radiochemical purity of radiolabelled peptides and the analyses of rat urine were determined by HPLC and/or ITLC-SG methods. Male Wistar rats were used in the elimination studies., Results: DOTA-Tyr3-tate was simply radiolabelled with radiometals with high yield and high radiochemical purity. Stopping of the reaction was a critical step for radioiodination, therefore labelling of gluc-Tyr3-tate and DOTA-Tyr3-tate with 125I was not so simple and the reaction product had to be purified by preparative HPLC analysis. Whereas 111In-DOTA-Tyr3-tate and 177Lu-DOTA-Tyr3-tate were eliminated in rat urine in a practically unchanged form, a significant proportion of metabolites was observed with radioiodinated peptides, particularly at longer time intervals., Conclusion: Labelling of DOTA-Tyr3-tate with radiometals is simple and the radiochemical purity of prepared compounds is very high, while iodination of the peptides demands purification of the product by preparative HPLC. The analysis of rat urine showed that excretion of radioiodinated peptides included a significant proportion of metabolites.

An attractive feature of targeted radionuclide therapy is the ability to select radionuclides and targeting vehicles with characteristics that are best suited for a particular clinical application. One combination that has been receiving increasing attention is the use of monoclonal antibodies (mAbs) specifically reactive to receptors and antigens that are expressed in tumor cells to selectively deliver the alpha-particle-emitting radiohalogen astatine-211 (211At) to malignant cell populations. Promising results have been obtained in preclinical models with multiple 211At-labeled mAbs; however, translation of the concept to the clinic has been slow. Impediments to this process include limited radionuclide availability, the need for suitable radiochemistry methods operant at high activity levels and lack of data concerning the toxicity of alpha-particle emitters in humans. Nonetheless, two clinical trials have been initiated to date with 211At-labeled mAbs, and others are planned for the near future.

Unlabelled: Two characteristics of alpha-particles that enhance their potential for targeted radiotherapy are their high energy and approximately cellular range. Unfortunately, these properties also can have negative consequences, confounding the production of clinically relevant levels of radiopharmaceutical because of radiolytic effects. The purpose of this study was to evaluate the effect of radiation dose on the astatine species present before initiation of a labeling reaction and the potential role of these molecules in the efficiency of N-succinimidyl 3-(211)At-astatobenzoate (SAB) synthesis. The ranges of radiation dose evaluated were selected to reflect those that might be encountered in SAB synthesis for the preparation of clinical doses of (211)At-labeled radiopharmaceuticals., Methods: The distribution of astatine species present in methanol, and the yields for the synthesis of SAB from N-succinimidyl 3-(tri-n-butylstannyl)benzoate as a function of radiation dose, were determined by high-performance liquid chromatography. Radiation doses in the range of 500-12,000 Gy were evaluated using different (211)At time-activity combinations, and the effect of acetic acid, a normal component of astatodestannylation reactions, also was studied. Finally, the effect of the reducing agent sodium sulfite also was evaluated to characterize the nature of the species produced by radiolysis., Results: At radiation doses below 1,000 Gy, high-performance liquid chromatography analysis indicated that more than 90% of the (211)At was present in methanol as a single species, At(1), whereas at higher doses, a second peak, At(2), emerged. At(1) decreased and At(2) increased in a radiation dose-dependent fashion, with At(2) becoming the predominant species at about 3,000 Gy. At(2) was identified as a reduced form of astatine, presumably astatide, which could not be efficiently oxidized to a species suitable for electrophilic astatination. In methanol/acetic acid, more than 95% of the astatine was present as At(2) even at doses below 1,400 Gy., Conclusion: The emergence of a reduced form of astatine, At(2), at higher radiation doses is consistent with the decline in SAB yields observed under these conditions. Alteration of the chemical form of the astatine by radiolysis could account for the declining yields noted in the preparation of clinical-level (211)At-labeled radiopharmaceuticals and when the labeling chemistry is initiated hours after (211)At production.

meta-[(211)At]Astatobenzylguanidine ([(211)At]MABG), an analogue of meta-iodobenzylguanidine (MIBG) labeled with the alpha-emitter (211)At, targets the norepinephrine transporter. Because MABG has been shown to have excellent characteristics in preclinical studies, it has been considered to be a promising targeted radiotherapeutic for the treatment of tumors such as micrometastatic neuroblastoma that overexpress the norepinephrine transporter. To facilitate clinical evaluation of this agent, a convenient method for the high level synthesis of [(211)At]MABG that is adaptable for kit formulation has been developed. A tin precursor anchored to a solid-support was treated with a methanolic solution of (211)At in the presence of a mixture of H(2)O(2)/HOAc as the oxidant; [(211)At]MABG was isolated by simple solid-phase extraction. By using C-18 solid-phase extraction, the radiochemical yield from 25 batches was 63+/-13%; however, loss of radioactivity during evaporation of the methanolic solution was a problem. This difficulty was avoided by use of a cation exchange resin cartridge for isolation of [(211)At]MABG, which resulted in radiochemical yields of 63+/-9% in a shorter duration of synthesis. The radiochemical purity was more than 90% and no chemical impurity has been detected. The final doses were sterile and apyrogenic. These results demonstrate that [(211)At]MABG can be prepared via a kit method at radioactivity levels anticipated for initiation of clinical studies.

Adults with primary malignant glioma have an unacceptably poor outcome. Most of these tumors recur at or adjacent to the site of origin, which indicates that failure to eradicate local tumor growth is a major factor contributing to poor outcome. Therefore, locoregional therapies may improve local control and overall outcome for malignant glioma patients. Malignant gliomas selectively express several factors that are not present on normal CNS tissue. Regional administration of radiolabeled monoclonal antibodies targeting tumor-specific antigens expressed by malignant gliomas offers an innovative therapeutic strategy that has recently demonstrated encouraging antitumor activity and acceptable toxicity in clinical trials at a number of centers. Most studies have utilized monoclonal antibodies against tenascin-C, an extracellular matrix glycoprotein ubiquitously expressed by malignant gliomas. This review summarizes clinical trials performed using radiolabeled antitenascin-C monoclonal antibodies for malignant glioma patients to date and highlights future plans to further develop this therapeutic strategy.

Introduction: The neurohistological findings in patients treated with targeted beta emitters such as (131)I are poorly described. We report a histopathologic analysis from patients treated with combined external beam therapy and a brachytherapy consisting of a (131)I-labeled monoclonal antibody (mAb) injected into surgically created resection cavities during brain tumor resections., Methods: Directed tissue samples of the cavity walls were obtained because of suspected tumor recurrence from 28 patients. Samples and clinical follow-up were evaluated on all patients (Group A) based on total radiation dose received and a subset of these (n=18; Group B, proximal therapy subset) who had received external beam therapy within

Indirect effects may contribute to the efficacy of radiotherapy by sterilizing malignant cells that are not directly irradiated. However, little is known of the influence of indirect effects in targeted radionuclide treatment. We compared gamma-radiation-induced bystander effects with those resulting from exposure to three radiohaloanalogues of meta-iodoben-zylguanidine (MIBG): [(131)I]MIBG (low linear energy transfer (LET) alpha-emitter), [(123)I]MIBG (high LET Auger electron emitter), and meta-[(211)At]astatobenzylguanidine ([(211)At]MABG) (high LET alpha-emitter). Cells exposed to media from gamma-irradiated cells exhibited a dose-dependent reduction in survival fraction at low dosage and a plateau in cell kill at > 2 Gy. Cells treated with media from [(131)I]MIBG demonstrated a dose-response relationship with respect to clonogenic cell death and no annihilation of this effect at high radiopharmaceutical dosage. In contrast, cells receiving media from cultures treated with [(211)At]MABG or [(123)I]MIBG exhibited dose-dependent toxicity at low dose but elimination of cytotoxicity with increasing radiation dose (i.e. U-shaped survival curves). Therefore radionuclides emitting high LET radiation may elicit toxic or protective effects on neighboring untargeted cells at low and high dose respectively. We conclude that radiopharmaceutical-induced bystander effects may depend on LET and be distinct from those elicited by conventional radiotherapy.

Introduction: When labeled with iodine-131, the antitenascin monoclonal antibody (mAb) 81C6 has shown promise as a targeted radiotherapeutic in patients with brain tumors. Because of its more favorable gamma-ray properties, lutetium-177 might be a better low-energy beta-emitter for this type of therapy., Materials and Methods: Chimeric 81C6 (ch81C6) was labeled with (177)Lu using the acyclic 1B4M ligand and the macrocyclic ligands NHS-DOTA and MeO-DOTA and evaluated for binding to tenascin. Three paired-label tissue distribution experiments were performed in normal mice receiving one of the (177)Lu-labeled immunoconjugates plus (125)I-labeled ch81C6 labeled using Iodogen. Paired-label experiments in athymic mice bearing subcutaneous D54 MG human glioma xenografts were done to directly compare the biodistribution of ch81C6-1B4M-(177)Lu and (125)I-labeled ch81C6, and ch81C6-MeO-DOTA-(177)Lu and (125)I-labeled ch81C6. Similar comparisons were done using murine (mu) instead of ch81C6. The primary parameter utilized for evaluation was the (177)Lu/(125)I uptake ratio in each tissue., Results: In the studies performed in normal mice, the NHS-DOTA ligand yielded the highest (177)Lu/(125)I uptake ratios in tissues indicative of loss of label from the chelate; for this reason, only 1B4M and MeO-DOTA were evaluated further. The (177)Lu/(125)I ratio in bone increased gradually with time for the chimeric conjugates; however, there were no significant differences between ch81C6-1B4M-DTPA-(177)Lu and ch81C6-MeO-DOTA-(177)Lu. In contrast, mu81C6-1B4M-DTPA-(177)Lu and mu81C6-MeO-DOTA-(177)Lu showed a more dramatic increase in the (177)Lu/(125)I ratio in bone - from 2.4+/-0.3 and 1.7+/-0.2 at Day 1 to 8.5+/-1.1 and 4.2+/-0.5 at Day 7, respectively., Conclusion: With these antitenascin constructs, the nature of the mAb had a profound influence on the relative degree of loss of (177)Lu from these immunoconjugates. MeO-DOTA shows promise as a bifunctional chelate for labeling 81C6 mAbs with (177)Lu.

This protocol describes a detailed procedure for the synthesis of N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB), an agent useful in the radio-iodination of proteins, including monoclonal Abs, and peptides that undergo internalization after receptor or antigen binding. In this procedure, the tin precursor N-succinimidyl 4-[N1,N2-bis(tert-butyloxycarbonyl)guanidinomethyl]-3-(trimethylstannyl)benzoate (Boc-SGMTB, 3) was first radio-iodinated to [*I]Boc-SGMIB, a derivative of [*I]SGMIB with the guanidine function protected with Boc groups. Treatment of [*I]Boc-SGMIB with trifluoroacetic acid delivered the final product. The total time for the synthesis and purification of [*I]Boc-SGMIB and its subsequent de-protection is approximately 140 min.

ELPs are genetically engineered, thermally responsive polypeptides that preferentially accumulate in solid tumors subjected to focused, mild hyperthermia. In this paper, we report the biodegradation, pharmacokinetics, tumor localization, and tumor spatial distribution of (14)C-labeled ELPs that were radiolabeled during their biosynthesis in Escheriehia coli. The in vitro degradation rate of a thermally responsive (14)C-labeled ELP1 ([(14)C] ELP1) with a molecular weight of 59.4 kDa, upon exposure to murine serum, was 2.49 wt.%/day. The apparent in vivo degradation rate of ELP1 after intravenous injection of nude mice was 2.46 wt.%/day and its terminal half-life was 8.7 h. The tumor accumulation and spatial distribution of intravenously administered ELP1 and a control ELP that was designed to remain soluble in heated tumors (ELP2) were examined in both heated (41.5 degrees C) and unheated tumors. ELP1 accumulated at a significantly higher concentration in heated tumors than ELP1 in unheated tumors and ELP2 in heated tumors. Quantitative autoradiography of tumor sections provided similar tumor accumulation results as the whole tumor analysis but, in addition, showed that ELP1 had a more homogeneous distribution in heated tumors and a greater concentration in the tumor center than either control treatment.

Intra-articular drug delivery is the preferred standard for targeting pharmacologic treatment directly to joints to reduce undesirable side effects associated with systemic drug delivery. In this study, a biologically based drug delivery vehicle was designed for intra-articular drug delivery using elastin-like polypeptides (ELPs), a biopolymer composed of repeating pentapeptides that undergo a phase transition to form aggregates above their transition temperature. The ELP drug delivery vehicle was designed to aggregate upon intra-articular injection at 37 degrees C, and form a drug 'depot' that could slowly disaggregate and be cleared from the joint space over time. We evaluated the in vivo biodistribution and joint half-life of radiolabeled ELPs, with and without the ability to aggregate, at physiological temperatures encountered after intra-articular injection in a rat knee. Biodistribution studies revealed that the aggregating ELP had a 25-fold longer half-life in the injected joint than a similar molecular weight protein that remained soluble and did not aggregate. These results suggest that the intra-articular joint delivery of ELP-based fusion proteins may be a viable strategy for the prolonged release of disease-modifying protein drugs for osteoarthritis and other arthritides.

We report a method to incorporate a stable isotope (13C) and a radioactive isotope (14C) into a recombinant polypeptide during Escherichia coli culture in M9 minimal medium supplemented with universally labeled 13C- or 14C-labeled glucose. We chose a thermally responsive elastin-like polypeptide (ELP) as a model polypeptide for this study because of its utility in various biotechnology applications such as drug delivery and tissue engineering. High cell densities were obtained by step-wise adaptation of E. coli to M9 medium in addition to supplementing the medium with trace elements that facilitated growth of E. coli. Furthermore, an optimal concentration of isopropyl-beta-d-thiogalactopyranoside was determined for induction of ELP expression to achieve high yield (mg/L culture) of the ELP. The incorporation of carbon isotopes was stoichiometrically related to the ratio of labeled glucose to unlabeled glucose in the culture medium. The isotope-labeled variants retained the physicochemical properties of the unlabeled ELP, specifically its temperature dependent aggregation behavior. As an example of the utility of this method, the in vitro stability of 14C-labeled ELP in PBS and mouse serum was conveniently quantified by SDS-PAGE and autoradiography. In addition, the in vivo stability of the 14C-labeled ELP in plasma was determined along with its plasma pharmacokinetics.

Novel methods are needed for the radiohalogenation of cell-internalizing proteins and peptides because rapid loss of label occurs after lysosomal processing when these molecules are labeled using conventional radioiodination methodologies. We have developed a radiolabeled prosthetic group that contains multiple negatively charged D-amino acids to facilitate trapping of the radioactivity in the cell after proteolysis of the labeled protein. N(epsilon)-(3-[(125)I]iodobenzoyl)-Lys(5)-N(alpha)-maleimido-Gly(1)-GEEEK ([(125)I]IB-Mal-D-GEEEK) was synthesized via iododestannylation in 90.3 +/- 3.9% radiochemical yields. This radioiodinated agent was conjugated to iminothiolane-treated L8A4, an anti-epidermal growth factor receptor variant III (EGFRvIII) specific monoclonal antibody (mAb) in 54.3 +/- 17.7% conjugation yields. In vitro assays with the EGFRvIII-expressing U87MGDeltaEGFR glioma cell line demonstrated that the internalized radioactivity for the [(125)I]IB-Mal-D-GEEEK-L8A4 conjugate increased from 14.1% at 1 h to 44.7% at 24 h and was about 15-fold higher than that of directly radioiodinated L8A4 at 24 h. A commensurately increased tumor uptake in vivo in athymic mice bearing subcutaneous U87MGDeltaEGFR xenografts (52.6 +/- 14.3% injected dose per gram versus 17.4 +/- 3.5% ID/g at 72 h) also was observed. These results suggest that [(125)I]IB-Mal-d-GEEEK is a promising reagent for the radioiodination of internalizing mAbs.

Unlabelled: Results from animal experiments have shown that human IgG2/mouse chimeric antitenascin 81C6 (ch81C6) monoclonal antibody exhibited higher tumor accumulation and enhanced stability compared with its murine parent. Our objective was to determine the effect of these differences on the maximum tolerated dose (MTD), pharmacokinetics, dosimetry, and antitumor activity of (131)I-ch81C6 administered into the surgically created resection cavity (SCRC) of malignant glioma patients., Methods: In this phase I trial, eligible patients received a single injection of (131)I-ch81C6 administered through a Rickham catheter into the SCRC. Patients were stratified as newly diagnosed and untreated (stratum A), newly diagnosed after external beam radiotherapy (XRT) (stratum B), and recurrent (stratum C). (131)I-ch81C6 was administered either before (stratum A) or after (stratum B) conventional XRT for newly diagnosed patients. In addition, chemotherapy was prescribed for all patients after (131)I-ch81C6 administration. Dose escalation was performed independently for each stratum. Patients were observed for toxicity and response until death or progressive disease., Results: We treated 47 patients with (131)I-ch81C6 doses up to 4.44 GBq (120 mCi), including 35 with newly diagnosed tumors (strata A and B) and 12 with recurrent disease (stratum C). Dose-limiting hematologic toxicity defined the MTD to be 2.96 GBq (80 mCi) for all patients, regardless of treatment strata. Neurologic dose-limiting toxicity developed in 3 patients; however, none required further surgery to debulk radiation necrosis. Median survival was 88.6 wk and 65.0 wk for newly diagnosed and recurrent patients, respectively., Conclusion: The MTD of (131)I-ch81C6 is 2.96 GBq (80 mCi) because of dose-limiting hematologic toxicity. Although encouraging survival was observed, (131)I-ch81C6 was associated with greater hematologic toxicity, probably due to the enhanced stability of the IgG2 construct, than previously observed with (131)I-murine 81C6.

Unlabelled: Recent studies have shown that indirect effects of ionizing radiation may contribute significantly to the effectiveness of radiotherapy by sterilizing malignant cells that are not directly hit by the radiation. However, there have been few investigations of the importance of indirect effects in targeted radionuclide treatment. Our purpose was to compare the induction of bystander effects by external beam gamma-radiation with those resultant from exposure to 3 radiohaloanalogs of metaiodobenzylguanidine (MIBG): (131)I-MIBG (low-linear-energy-transfer [LET] beta-emitter), (123)I-MIBG (potentially high-LET Auger electron emitter), and meta-(211)At-astatobenzylguanidine ((211)At-MABG) (high-LET alpha-emitter)., Methods: Two human tumor cell lines-UVW (glioma) and EJ138 (transitional cell carcinoma of bladder)-were transfected with the noradrenaline transporter (NAT) gene to enable active uptake of MIBG. Medium from cells that accumulated the radiopharmaceuticals or were treated with external beam radiation was transferred to cells that had not been exposed to radioactivity, and clonogenic survival was determined in donor and recipient cultures., Results: Over the dose range 0-9 Gy of external beam radiation of donor cells, 2 Gy caused 30%-40% clonogenic cell kill in recipient cultures. This potency was maintained but not increased by higher dosage. In contrast, no corresponding saturation of bystander cell kill was observed after treatment with a range of activity concentrations of (131)I-MIBG, which resulted in up to 97% death of donor cells. Cellular uptake of (123)I-MIBG and (211)At-MABG induced increasing recipient cell kill up to levels that resulted in direct kill of 35%-70% of clonogens. Thereafter, the administration of higher activity concentrations of these high-LET emitters was inversely related to the kill of recipient cells. Over the range of activity concentrations examined, neither direct nor indirect kill was observed in cultures of cells not expressing the NAT and, thus, incapable of active uptake of MIBG., Conclusion: Potent toxins are generated specifically by cells that concentrate radiohalogenated MIBG. These may be LET dependent and distinct from those elicited by conventional radiotherapy.

Introduction: Radioimmunotherapy with anti-HER2 monoclonal antibodies (mAbs) such as trastuzumab is a promising strategy for treating HER2-positive breast and ovarian carcinoma patients. The objective of this study was to determine the cytotoxic effectiveness of trastuzumab labeled with the 7.2-h half-life alpha-particle emitter 211At., Methods: Experiments were performed on SKBr-3, BT-474 and the transfected MCF7/HER2-18 human breast carcinoma cell lines. Intrinsic radiosensitivity was determined after exposure to external beam irradiation. The cytotoxicity of 211At-labeled trastuzumab was measured by clonogenic assays. The distribution of HER2 receptor expression on the cell lines was measured using fluorescence-activated cell sorting. A pharmacokinetic (PK)/microdosimetric model was established to assess the effects of specific activity (SA), HER2 receptor expression and absorbed dose on survival fraction (SF)., Results: With external beam irradiation, the 2-Gy SF for BT-474, SKBr-3 and MCF7/HER2-18 cells was 0.78, 0.53 and 0.64 Gy, respectively. Heterogeneous HER2 expression was observed, with a subpopulation of cells lacking measurable receptor (14.5%, SKBr-3; 0.34%, MCF-7/HER2; 1.73%, BT-474). When plotted as a function of activity concentration, SF curves were biphasic and inversely proportional to SA; however, when the model was applied and absorbed doses calculated, the SF curve was monoexponential independent of SA. Thus, the PK model was able to demonstrate the effects of competition between cold and labeled mAb. These studies showed that the relative biological effectiveness of 211At-labeled trastuzaumab was about 10 times higher than that of external beam therapy., Conclusion: These in vitro studies showed that 211At-labeled trastuzumab mAb is an effective cytotoxic agent for the treatment of HER2-positive tumor cells. The SA of the labeled mAb and the homogeneity of HER2 receptor expression are important variables influencing the efficiency of cell killing.

Purpose: An inverse correlation has been established between tumor levels of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) and a positive outcome after alkylator chemotherapy. Quantitative imaging of AGT could provide important information for patient-specific cancer treatment. Several radiolabeled analogues of O6-benzylguanine (BG), a potent AGT inactivator, have been developed and shown to be capable of labeling pure AGT protein. Herein, two of these analogues--O6-3-[*I]iodobenzylguanine ([*I]IBG) and O6-3-[*I]iodobenzyl-2'-deoxyguanosine ([*I]IBdG)--were further evaluated in two murine xenograft models. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, also was investigated as an alternative agent., Methods: Several biodistribution studies of radioiodinated IBG and IBdG were performed in TE-671 human rhabdomyosarcoma and DAOY human medulloblastoma murine xenograft models. Mice were treated with BG or its nucleoside analogue dBG to deplete the tumor AGT content. The effect of unlabeled IBG and that of 7,8-benzoflavone (BF), an inhibitor of the cytochrome P-450 isozyme CYP1A2, on the tumor uptake of the tracers was determined. The uptake of (AcO)2-[131I]IBdG along with that of [125I]IBdG in DAOY cells in vitro was determined in the presence and absence of a nucleoside transporter inhibitor, dipyridamole., Results: Pretreatment of mice either with BG or dBG failed to reduce tumor levels of [*I]IBG or [*I]IBdG even though such treatments completely depleted tumor AGT content. Treatment of mice with BF increased tumor uptake of [125I]IBG by 56%; however, differentiation of tumors with and without AGT still was not possible. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, had a higher uptake in vitro in DAOY tumor cells. However, its uptake, like that of [125I]IBdG, was blocked by dipyridamole., Conclusions: Taken together, these results suggest that labeled agents that are more specific for cellular AGT and that are more metabolically stable are needed.

A procedure for the synthesis of N-succinimidyl 3-iodobenzoate labeled with any iodine isotope ([*I]SIB), which is an agent used in the radioiodination of proteins and peptides, from its tin precursor N-succinimidyl 3-(tri-n-butylstannyl)benzoate (STB) is described. Also included are protocols for the synthesis of an unlabeled standard of SIB and the tin precursor. Radioiododestannylation of STB using tert-butylhydroperoxide as the oxidant gives [*I]SIB in 80% radiochemical yields. The total time for the synthesis of [*I]SIB from STB is approximately 95 min. Use of [*I]SIB yields radioiodinated proteins that are considerably more stable in vivo than those radioiodinated by the direct electrophilic method.

Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-[(3-tri-n-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 +/- 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 +/- 4.9% and 46.8 +/- 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 +/- 0.8% of input dose compared to 9.67 +/- 0.43% for N(alpha)-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 +/- 4.0% ID/g, 18.8 +/- 7.7% ID/g, and 0.9 +/- 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 +/- 1.2% ID/g, 4.7 +/- 1.4% ID/g, and 0.8 +/- 0.5% ID/g. Both in vitro and in vivo catabolism studies did not suggest the severance of the lys0 along with its appendages from the peptide. Taken together, although GIBLO maintained affinity to SSTR2, its tumor uptake both in vitro and in vivo was substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be important.

This protocol describes the step-by-step procedure for the synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB), an agent widely used for labeling proteins and peptides with the positron-emitting radionuclide 18F. The protocols for the synthesis of unlabeled SFB and the quaternary salt precursor 4-formyl-N,N,N-trimethyl benzenaminium trifluoromethane sulfonate also are described. For the [18F]SFB synthesis, the quaternary salt is first converted to 4-[18F]fluorobenzaldehyde. Oxidation of the latter provides 4-[18F]fluorobenzoic acid, which is converted to [18F]SFB by treatment with N,N-disuccinimidyl carbonate. Using this method, [18F]SFB can be synthesized in decay-corrected radiochemical yields of 30%-35% and a specific radioactivity of 11-12 GBq micromol(-1). The total synthesis and purification time required is about 80 min, starting from delivery of the [18F]fluoride. [18F]SFB remains an optimal reagent for labeling proteins and peptides with 18F because of good conjugation yields and metabolic stability.

Introduction: MR1-1 is a single-chain Fv (scFv) fragment that binds with high affinity to epidermal growth factor receptor variant III, which is overexpressed on gliomas and other tumors but is not present on normal tissues. The objective of this study was to evaluate four different methods for labeling MR1-1 scFv that had been previously investigated for the radioiodinating of an intact anti-epidermal growth factor receptor variant III (anti-EGFRvIII) monoclonal antibody (mAb) L8A4., Methods: The MR1-1 scFv was labeled with (125)I/(131)I using the Iodogen method, and was also radiohalogenated with acylation agents bearing substituents that were positively charged--N-succinimidyl-3-[*I]iodo-5-pyridine carboxylate and N-succinimidyl-4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB)--and negatively charged--N-succinimidyl-3-[*I]iodo-4-phosphonomethylbenzoate ([*I]SIPMB). In vitro internalization assays were performed with the U87MGDeltaEGFR cell line, and the tissue distribution of the radioiodinated scFv fragments was evaluated in athymic mice bearing subcutaneous U87MGDeltaEGFR xenografts., Results and Conclusion: As seen previously with the anti-EGFRvIII IgG mAb, retention of radioiodine activity in U87MGDeltaEGFR cells in the internalization assay was labeling method dependent, with SGMIB and SIPMB yielding the most prolonged retention. However, unlike the case with the intact mAb, the results of the internalization assays were not predictive of in vivo tumor localization capacity of the labeled scFv. Renal activity was dependent on the nature of the labeling method. With MR1-1 labeled using SIPMB, kidney uptake was highest and most prolonged; catabolism studies indicated that this uptake primarily was in the form of epsilon-N-3-[*I]iodo-4-phosphonomethylbenzoyl lysine.

Purpose: To assess the efficacy and toxicity of intraresection cavity iodine-131-labeled murine antitenascin monoclonal antibody 81C6 (131I-m81C6) among recurrent malignant brain tumor patients., Patients and Methods: In this phase II trial, 100 mCi of 131I-m81C6 was injected directly into the surgically created resection cavity (SCRC) of 43 patients with recurrent malignant glioma (glioblastoma multiforme [GBM], n = 33; anaplastic astrocytoma [AA], n = 6; anaplastic oligodendroglioma [AO], n = 2; gliosarcoma [GS], n = 1; and metastatic adenocarcinoma, n = 1) followed by chemotherapy., Results: With a median follow-up of 172 weeks, 63% and 59% of patients with GBM/GS and AA/AO tumors were alive at 1 year. Median overall survival for patients with GBM/GS and AA/AO tumors was 64 and 99 weeks, respectively. Ten patients (23%) developed acute hematologic toxicity. Five patients (12%) developed acute reversible neurotoxicity. One patient (2%) developed irreversible neurotoxicity. No patients required reoperation for radionecrosis., Conclusion: In this single-institution phase II study, administration of 100 mCi of 131I-m81C6 to recurrent malignant glioma patients followed by chemotherapy is associated with a median survival that is greater than that of historical controls treated with surgery plus iodine-125 brachytherapy. Furthermore, toxicity was acceptable. Administration of a fixed millicurie dose resulted in a wide range of absorbed radiation doses to the SCRC. We are now conducting a phase II trial, approved by the US Food and Drug Administration, using patient-specific 131I-m81C6 dosing, to deliver 44 Gy to the SCRC followed by standardized chemotherapy. A phase III multicenter trial with patient-specific dosing is planned.

Targeted radiotherapy using radiolabelled meta-iodobenzylguanidine (MIBG) is a promising treatment option for bladder cancer, restricting the effects of radiotherapy to malignant cells thereby increasing efficacy and decreasing morbidity of radiotherapy. We investigated the efficacy of a combined gene therapy and targeted radiotherapy approach for bladder cancer using radiolabelled MIBG. The effectiveness of alternative radiohalogens and alternative preparations of radiolabelled MIBG for this therapeutic strategy were compared. Bladder cancer cells, EJ138, were transfected with a gene encoding the noradrenaline transporter (NAT) under the control of a tumour specific telomerase promoter, enabling them to actively take up radiolabelled MIBG. This resulted in tumour-specific cell kill. Uptake and retention of radioactivity in cells transfected with the NAT gene were compared with that obtained in cells transfected with the sodium iodide symporter (NIS) gene. Substantially greater uptake and longer retention of radioactivity in NAT-transfected cells was observed. Carrier-added (c.a.) [131I]MIBG, no-carrier added (n.c.a.) [131I]MIBG, and [211At]-labelled benzylguanidine (i.e. [211At] meta-astatobenzylguanidine (MABG)) were compared with respect to efficiency of induction of cell kill. N.c.a[(131)I]MIBG was more cytotoxic than c.a.[131I]MIBG. However, the alpha-emitter [211At]MABG was, by three orders of magnitude, more effective in causing tumour cell kill than the beta-emitter [131I]MIBG. We conclude that NAT gene transfer combined with the administration of n.c.a.[131I]MIBG or [211At]MABG, is a promising novel treatment approach for bladder cancer therapy.

Unlabelled: A variety of promising targeted radiotherapeutics labeled with alpha-emitters have been developed. Clinical investigation of these radiopharmaceuticals requires the production of high activity levels, which can be hindered by alpha-particle-mediated radiolytic effects on labeling chemistry. The purpose of this study was to investigate the effects of radiation dose on the synthesis of N-succinimidyl 3-(211)At-astatobenzoate (SAB), a compound used in our clinical trials for labeling antibodies with alpha-particle-emitting (211)At., Methods: Yields for the synthesis of SAB as a function of the radiation dose received by the reaction medium were determined. The variables studied included the radiohalogenation precursors N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) and N-succinimidyl 3-(trimethylstannyl)benzoate (MeSTB); the solvents chloroform, benzene, and methanol; and the addition of acetic acid and the oxidant N-chlorosuccinimide. The (211)At product spectra were determined from high-performance liquid chromatograms and then plotted against radiation dose., Results: SAB production declined rapidly with increasing dose, consistent with the documented radiolytic decomposition of BuSTB and MeSTB in chloroform. Even though these tin precursors were not appreciably degraded in benzene, SAB could not be produced in this solvent; instead, highly lipophilic (211)At-labeled species were generated in nearly quantitative yields. Although a dose-dependent decline in SAB yield also was observed in methanol, both in the presence and in the absence of an oxidant, the results were better than those obtained with the other solvents. An unexpected observation was that SAB could be obtained at a yield of greater than 30% when the reaction was run in methanol without the addition of acetic acid or an oxidant; these 2 components previously were considered essential for astatodestannylation., Conclusion: Radiolytic factors can play an important role in the synthesis of clinical-level activities of (211)At-labeled radiopharmaceuticals, necessitating the development of reaction conditions different from those that are used successfully at lower activity levels.

The development of O(6)-(3-[(125)I]iodobenzyl)-2'-deoxyguanosine ([(125)I]IBdG), the glycosylated analogue of the O(6)-3-iodobenzylguanine (IBG), as an agent for the in vivo mapping of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) is described. Synthesis of its tin precursor, O(6)-3-trimethylstannylbenzyl-2'-deoxyguanosine (TBdG) was achieved in four steps from deoxyguanosine. Radioiodination of TBdG in a single step gave [(125)I]IBdG in 70-85% isolated radiochemical yield. [(125)I]IBdG bound specifically to pure AGT with an IC(50) of 7.1 microM. From paired-label assays, [(125)I]IBdG showed a 2- to 3-fold higher cellular uptake than [(131)I]IBG in DAOY medulloblastoma, TE-671 rhabdomyosarcoma, SK-Mel-28 melanoma, and HT-29 colon carcinoma human cell lines. Uptake of both labeled compounds in these cell lines decreased with increasing concentrations of unlabeled O(6)-benzylguanine (BG) when BG was present in the medium during incubation with the labeled compounds. Compared to BG, unlabeled IBdG diminished the uptake of [(125)I]IBdG and [(131)I]IBG in DAOY cells more efficiently (IC(50)<1 microM vs >10 microM for BG). There was no significant change in cell-bound activity of [(125)I]IBdG and [(131)I]IBG when BG was removed from the incubation medium before incubating cells with the tracers, suggesting that only a very small portion of radioactivity taken up by the cells is AGT bound. This was corroborated by gel-electrophoresis performed on extracts from cells treated with varying amounts of BG and then incubated with [(125)I]IBdG in the presence of BG. No radiolabeled AGT band was discernable by phosphor-imaging, signifying low cellular AGT binding of the radiotracer. In contrast, when cell extracts were prepared from BG pre-treated cells and aliquots were incubated with [(125)I]IBdG subsequently, the intensity of radiolabeled AGT band decreased linearly as a function of BG concentration. This suggests that the low level of [(125)I]IBdG that binds to AGT does so in a concentration dependent manner. These data suggest that IBdG is transported across the cell membrane to a higher degree than IBG. However, to be a practical tracer for quantifying cellular AGT, considerable localization of such derivatives need to occur within the cell nucleus where AGT is present predominantly.

Unlabelled: The objective was to perform dosimetry and evaluate dose-response relationships in newly diagnosed patients with malignant brain tumors treated with direct injections of (131)I-labeled anti-tenascin murine 81C6 monoclonal antibody (mAb) into surgically created resection cavities (SCRCs) followed by conventional external-beam radiotherapy and chemotherapy., Methods: Absorbed doses to the 2-cm-thick shell, measured from the margins of the resection cavity interface, were estimated for 33 patients with primary brain tumors. MRI/SPECT registrations were used to assess the distribution of the radiolabeled mAb in brain parenchyma. Results from biopsies obtained from 15 patients were classified as tumor, radionecrosis, or tumor and radionecrosis, and these were correlated with absorbed dose and dose rate. Also, MRI/PET registrations were used to assess radiographic progression among patients., Results: This therapeutic strategy yielded a median survival of 86 and 79 wk for all patients and glioblastoma multiforme (GBM) patients, respectively. The average SCRC residence time of (131)I-mu81C6 mAb was 76 h (range, 34-169 h). The average absorbed dose to the 2-cm cavity margins was 48 Gy (range, 25-116 Gy) for all patients and 51 Gy (range, 27-116 Gy) for GBM patients. In MRI/SPECT registrations, we observed a preferential distribution of (131)I-mu81C6 mAb through regions of vasogenic edema. An analysis of the relationship between the absorbed dose and dose rate and the first biopsy results yielded a most favorable absorbed dose of 44 Gy. A correlation between decreased survival and irreversible neurotoxicity was noted. A comparative analysis, in terms of median survival, was performed with previous brachytherapy clinical studies, which showed a proportional relationship between the average boost absorbed dose and the median survival., Conclusion: This study shows that (131)I-mu81C6 mAb increases the median survival of GBM patients. An optimal absorbed dose of 44 Gy to the 2-cm cavity margins is suggested to reduce the incidence of neurologic toxicity. Further clinical studies are warranted to determine the effectiveness of (131)I-mu81C6 mAb based on a target dose of 44 Gy rather than a fixed administered activity.

Unlabelled: The high energy and short range of alpha-particles make them attractive for targeted radiotherapy. However, these properties can be problematic when the production of high activity levels of alpha-particle-emitting radiotherapeutics is required. For example, difficulties were encountered in the production of N-succinimidyl 3-[211At]-astatobenzoate (SAB), when 370-MBq doses of 211At-labeled antibody were required. The purpose of this study was to investigate a potential cause of this behavior--radiolytic degradation of the radiohalogenation precursor., Methods: Both N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) and N-succinimidyl 3-trimethylstannylbenzoate (MeSTB) were incubated with various 211At time-activity combinations such that the radiation dose received by the reaction medium ranged from about 0 to 20,000 Gy. Studies were performed using chloroform, methanol, and benzene as the solvent, and both at neutral pH and at a pH of approximately 5.5, as used in SAB synthesis. The fraction of tin precursor remaining and the generation of unlabeled byproducts were determined from high-performance liquid chromatograms and then plotted against radiation dose., Results: Extensive radiolytic decomposition of BuSTB and MeSTB was observed in chloroform, with 50% degradation taking place even at doses below 500 Gy. Formation of a byproduct, most likely N-succinimidyl 3-chlorobenzoate, increased with radiation dose. A greater degree of stability was seen in both methanol and benzene, with more than 85% of the precursor remaining at 3,500 Gy. No cold byproducts were observed with either solvent., Conclusion: The nature of the solvent profoundly influences the ability to synthesize high activity levels of SAB and possibly other 211At-labeled radiopharmaceuticals.

Radioiodinated meta-iodobenzylguanidine (MIBG) is used in the diagnosis and therapy of various neuroendocrine tumors. As a part of our efforts to develop an MIBG analogue with improved characteristics for these applications, a synthesis of 3-[131I]iodo-4-methylbenzylguanidine ([131I]MeIBG) was developed. Unlabeled MeIBG and the tin precursor, N, N'-(bis-tert-butyloxycarbonyl)-N-(4-methyl-3-trimethylstannylbenzyl) guanidine were synthesized in two steps from 3-iodo-4-methylbenzylalcohol. Radioiodinated MeIBG was synthesized at a no-carrier-added level by the iododestannylation of the tin precursor in about 85% radiochemical yield. The accumulation of [131I]MeIBG (38.9+/-3.0% of input counts) by human neuroblastoma SK-N-SH cells in vitro was 87% that of [125I]MIBG (44.5+/-3.0%) and a number of Uptake-1 inhibiting conditions reduced the uptake of both tracers in this cell line to a similar degree suggesting that introduction of a methyl substituent at the 4-position of MIBG did not adversely affect its biological characteristics.

Clinical application of local hyperthermia as a means for modulating drug and macro-molecular tumour uptake have been slow to develop, due in part to the difficulty in designing and comparing heating protocols. The thermal isodose formula developed by Sapareto and Dewey is used in cytotoxicity and radiosensitization hyperthermia protocols to compare different time/temperature combinations; however, its relevance to other end-points has not been evaluated. The current study was undertaken to determine whether heating protocols of different time and temperature, but predicted to be thermally equivalent by this formula, had similar effects on the tumour and normal tissue distribution of radiolabelled tumour-specific (anti-tenascin 81C6) and non-specific (anti-dansyl TPS3.2) monoclonal antibodies (mAbs). Two thermally equivalent heating protocols, 4 h at 41.8 degrees C and 45 min at 43 degrees C, were compared in mice with subcutaneous D54 MG human glioma xenografts. A 4-fold increase in xenograft localization of 81C6 mAb was achieved relative to that in non-heated control groups with both heating protocols. Both hyperthermia protocols also resulted in improved tumour:normal tissue ratios. However, differences in absolute tumour and normal tissue uptake were seen, suggesting that the thermal isodose formula has limited usefulness in the design and comparison of hyperthermia protocols for enhancing the tumour uptake of radiolabelled mAbs.

Improving drug and macromolecular delivery of anti-cancer agents to tumours results in greater efficacy without increased toxicity. The current study was undertaken to assess the effects of the timing of injection of tumour specific and non-specific monoclonal antibodies (mAbs) relative to a hyperthermia treatment on tumour and normal tissue uptake. Using a local hyperthermia protocol of 45 min at 43 degrees C, uptake in tumour and normal tissues was measured at 1, 4, 12, 24, 48 and 72 h after injection. An anti-tenascin chimeric mAb, ch81C6, served as the specific mAb in a D-54 MG glioma xenograft mouse model. The chimeric mAb chTPS3.2 served as the control. A five-to-eight-fold increase in uptake of the tumour-targeted mAb was achieved in the heated tumours when compared with the non-heated tumours at 1 h. Differences in absolute tumour uptake of the specific mAb between the mice injected prior to hyperthermia and mice injected post-hyperthermia were seen only at 1 and 12 h. The median uptakes in the tumours of mice injected pre-heat were 25%ID/g at 1 h and 43.5%ID/g at 12 h, while in the animals injected post-hyperthermia the median uptakes were 45.5%ID/g and 80.2%ID/g, respectively. Blood levels of both the specific and non-specific mAbs were consistently higher over the initial 12 h period in the mice injected post-hyperthermia. Normal tissue uptake was also increased at most time points in the mice injected post-hyperthermia. The clinical importance of the differences in specific mAb uptake in tumour detected statistically at 1 and 12 h is questionable, given the highly variable nature of mAb uptake in vivo. Tumour targeting mAbs administered in combination with heat may be injected either prior to or immediately following hyperthermia treatment, with the expectation that levels of uptake in tumour will be relatively equivalent. Absolute normal tissue levels will be higher in patients receiving the mAb post-hyperthermia.

Treatment of malignant brain tumors with conventional approaches is largely unsuccessful because curative doses generally cannot be delivered without excessive toxicity to normal brain. Radioimmunotherapy is emerging as an attractive alternative for glioma therapy because of the potential for more selectively irradiating tumor cells while sparing normal tissues. Several institutions are engaged in phase I and phase II trials investigating the therapeutic potential of monoclonal antibodies (mAbs) labeled with the beta-emitters (131)I and (90)Y and the alpha-emitter (211)At in patients with recurrent and newly diagnosed brain tumors. The current status of these trials will be discussed with regard to efficacy, toxicity, and future directions.

Derivatives of the somatostatin analogues octreotide and octreotate labeled with radioiosotopes are used in the diagnosis and therapy of somatostatin receptor (SSTR)-positive tumors. A method has been devised to synthesize {N-(4-guanidinomethyl-3-iodobenzoyl)-Phe1-octreotate (GMIBO). Receptor binding assay and scatchard analysis yielded a Kd of 4.83 +/- 0.19 nM for this peptide. Derivatives of this peptide labeled with radioiodine ([*I]GMIBO) and the alpha-particle-emitting radiohalogen 211At N-(3-[211At]astato-4-guanidinomethylbenzoyl)-Phe1-octreotate; [211At]AGMBO} were prepared in a single step from a tin precursor in radiochemical yields of 30-35% and 15-20%, respectively. Paired-label internalization assays performed with the SSTR-positive D341 Med human medulloblastoma cell line demonstrated that [125I]GMIBO and [211At]AGMBO were specifically internalized 20-40% more than Nalpha-(1-deoxy-D-fructosyl)-[131I]I-Tyr3-octreotate ([131I]I-Glu-TOCA), the radioiodinated octreotide derivative previously shown to exhibit maximum internalization in this cell line. Uptake of [131I]GMIBO in D341 Med subcutaneous xenografts in a murine model (8.34 +/- 1.82 versus 8.10 +/- 2.23% ID/g at 1h) and SSTR-expressing normal tissues was comparable to that of [125I]I-Glu-TOCA and was shown to be specific. However, the uptake of [131I]GMIBO also was substantially higher in liver (16.9 +/- 3.15 versus 1.39 +/- 0.45% ID/g at 1 h) and in kidneys (44.33 +/- 6.47 versus 3.44 +/- 0.68% ID/g at 1h) compared to that of [125I]I-Glu-TOCA. These data suggest that these novel peptide conjugates retain their specificity for SSTR both in vitro and in vivo; however, because of their higher accumulation in normal tissues they would be best applied in settings amenable to loco-regional administration such as medulloblastoma neoplastic meningitis.

An important consideration in the development of effective strategies for radioimmunotherapy is the nature of the radiation emitted by the radionuclide. Radionuclides decaying by the emission of alpha-particles offer the possibility of matching the cell specific reactivity of monoclonal antibodies with radiation with a range of only a few cell diameters. Furthermore, alpha-particles have important biological advantages compared with external beam radiation and beta-particles including a higher biological effectiveness, which is nearly independent of oxygen concentration, dose rate and cell cycle position. In this review, the clinical settings most likely to benefit from alpha-particle radioimmunotherapy will be discussed. The current status of preclinical and clinical research with antibodies labeled with 3 promising alpha-particle emitting radionuclides - (213)Bi, (225)Ac, and (211)At - also will be summarized.

Purpose: As a part of our efforts to develop a meta-iodobenzylguanidine (MIBG) analogue with improved characteristics for the diagnosis and treatment of neuroendocrine tumours, 3-[131I]iodo-4-methyl-benzylguanidine ([131I]MeIBG) has been developed. The purpose of this study was to evaluate [131I]MeIBG in vitro using the uptake-1 positive SK-N-SH neuroblastoma cell line and in vivo in normal mice and mice bearing human neuroblastoma xenografts., Methods: The ability of SK-N-SH human neuroblastoma cells to retain [131I]MeIBG in vitro over a period of 4 days, in comparison to [125I]MIBG, was determined by a paired-label assay. Paired-label biodistributions of [131I]MeIBG and [125I]MIBG were performed in normal mice as well as in athymic mice bearing SK-N-SH and IMR-32 human neuroblastoma xenografts., Results: Retention of [131I]MeIBG by SK-N-SH cells in vitro was increased by factors of 1.2, 1.5, 2.0, 2.5 and 3.1 compared with [125I]MIBG at 8, 24, 48, 72 and 96 h, respectively. In normal mice, the uptake of [131I]MeIBG in the heart was similar to that of [125I]MIBG at 1 and 4 h; in contrast, myocardial uptake of [131I]MeIBG was 1.6-fold higher than that of [125I]MIBG (p<0.05) at 24 h. When mice were pre-treated with the uptake-1 inhibitor desipramine (DMI), the heart uptake of both tracers was reduced to about half that in untreated controls at 1 h post injection (p<0.05). The hepatic uptake of [131I]MeIBG was two- to threefold lower than that of [125I]MIBG. On the other hand, blood levels of [131I]MeIBG were substantially higher (up to sixfold), especially at early time points. Uptake of [131I]MeIBG in heart and tumour at 1 h in the murine SK-N-SH model was specific and comparable to that of [125I]MIBG. However, [131I]MeIBG uptake was 1.6- to 1.7-fold lower than that of [125I]MIBG over 4-48 h. While the uptake of both tracers in IMR32 xenografts was similar, it was not uptake-1 mediated., Conclusion: Introduction of a methyl group at the 4-position of MIBG seems to be advantageous in terms of higher tumour retention in vitro and lower hepatic uptake in vivo. However, the slower blood clearance of MeIBG may be problematic for some applications.

Monoclonal antibodies such as L8A4, reactive with the epidermal growth factor receptor variant III, internalize after receptor binding resulting in proteolytic degradation by lysosomes. Labeling internalizing mAbs requires the use of methodologies that result in the trapping of labeled catabolites in tumor cells after intracellular processing. Herein we have investigated the potential utility of N-succinimidyl-3-[131I]iodo-4-phosphonomethylbenzoate ([131I]SIPMB), an acylation agent that couples the corresponding negatively charged acid [131I]IPMBA to the protein, for this purpose. Biodistribution studies demonstrated that [131I]IPMBA cleared rapidly from normal tissues and exhibited thyroid levels < or =0.1% injected dose, consistent with a low degree of dehalogenation. Biodistribution experiments in athymic mice bearing subcutaneous D-256 human glioma xenografts were performed to compare L8A4 labeled using [131I]SIPMB to L8A4 labeled with 125I using both the analogous positively charged acylation agent N-succinimidyl-4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) and Iodogen. Tumor uptake of [131I]SIPMB-L8A4 (41.9+/-3.5% ID/g) was nearly threefold that of L8A4 labeled using Iodogen (14.0+/-1.1% ID/g) after 2 days, and tumor to tissue ratios remained uniformly high throughout with [131I]SIPMB-L8A4. Thyroid uptake increased for the Iodogen labeled mAb (3.55+/-0.36 %ID at 5 days) whereas that of [131I]SIPMB labeled mAb remained low (0.21+/-0.04% ID at 5 days). In the second biodistribution, L8A4 labeled using [131I]SIPMB and [125I]SGMIB showed no difference in normal tissue uptake and had nearly identical tumor uptake ([131I]SIPMB, 41.8+/-14.2% ID/g; [125I]SGMIB, 41.6+/-15.8% ID/g, at 4 days). These results suggest that [131I]SIPMB may be a viable acylation agent for the radioiodination of internalizing mAbs.

Conventional treatment of brain tumours includes surgical, radiotherapeutic and chemotherapeutic modalities. Nonetheless, the outcome of patients with brain tumours, in particular glioblastoma, remains poor. Immunotherapy with armed or unarmed monoclonal antibodies targeting tumour-specific antigens has emerged in the last two decades as a novel potential adjuvant treatment for all types of neoplasia. Many challenges to its implementation as a safe and viable therapy for brain tumours still need to be addressed; nevertheless, results from ongoing Phase I/II clinical trials are encouraging, as disease stabilisation and patient survival prolongation have been observed. Advances in preclinical and clinical research indicate that treatment of brain tumours with monoclonal antibodies can be increasingly adjusted to the characteristics of the targeted tumour and its environment. This aspect relies on the careful selection of the target antigen and corresponding specific monoclonal antibody, and antibody format (size, class, affinity), conjugation to the appropriate toxin or radioactive isotope (half-life, range), and proper compartmental administration.

Background: A fluorine substituted derivative of meta-iodobenzylguanidine (MIBG), 4-fluoro-3-iodobenzylguanidine (FIBG), is retained in SK-N-SH human neuroblastoma cells in vitro to a higher degree than the MIBG., Method: To investigate whether the higher retention of FIBG is due to differences in the catabolic degradation of the two tracers, in vitro paired-label studies were performed using SK-N-SH cells., Results: No detectable amount of benzyl amines, benzoic acids or hippuran derivatives, potential catabolites of these tracers, were seen in either case. Even after 48 h, the cell culture supernatants contained exclusively intact I-MIBG and I-FIBG. In contrast, in some cases, HPLC analysis of cell lysates indicated the presence of a very polar compound(s) as the predominant species with smaller quantities of intact tracers. The per cent total radioactivity in the lysate at each time point that was associated with intact I-FIBG was (average [range]) 25.4% [20.3-30.5], 22.5% [19.3-25.6], and 18.8% [14.3-23.3], at 0 h, 24 h and 48 h, respectively. The corresponding values for I-MIBG were 24.3% [21.0-27.5], 19.1% [11.7-26.5] and 17.4% [14.6-20.1]. No significant amount of activity was associated with high molecular weight species for either halobenzylguanidine, indicating that protein binding was not a major factor.

We report a phase 1 study of pharmacokinetics, dosimetry, toxicity, and response of (131)I anti-tenascin chimeric 81C6 for the treatment of lymphoma. Nine patients received a dosimetric dose of 370 MBq (10 mCi). Three patients received an administered activity of 1480 MBq (40 mCi), and 2 developed hematologic toxicity that required stem cell infusion. Six patients received an administered activity of 1110 MBq (30 mCi), and 2 developed toxicity that required stem cell infusion. The clearance of whole-body activity was monoexponential with a mean effective half-life of 110 hours (range, 90-136 hours) and a mean effective whole-body residence time of 159 hours (range, 130-196 hours). There was rapid uptake within the viscera; however, tumor uptake was slower. Activity in normal viscera decreased proportional to the whole body; however, tumor sites presented a slow clearance (T(1/2), 86-191 hours). The mean absorbed dose to whole-body was 67 cGy (range, 51-89 hours), whereas the dose to tumor sites was 963 cGy (range, 363-1517 cGy). Despite lack of a "blocking" antibody, 1 of 9 patients attained a complete remission and 1 a partial remission. These data demonstrate this radiopharmaceutical to be an encouraging agent for the treatment of lymphoma particularly if methods to protect the normal viscera are developed.

Background: Targeted radiotherapy achieves malignant cell-specific concentration of radiation dosage by tumour-affinic molecules conjugated to radioactive atoms. Combining gene therapy with targeted radiotherapy is attractive because the associated cross-fire irradiation of the latter induces biological bystander effects upon neighbouring cells overcoming low gene transfer efficiency., Methods: We sought to maximise the tumour specificity and efficacy of noradrenaline transporter (NAT) gene transfer combined with treatment using the radiopharmaceutical meta-[(131)I]iodobenzylguanidine ([(131)I]MIBG). Cell-kill was achieved by treatment with the beta-decay particle emitter [(131)I]MIBG or the alpha-particle emitter [(211)At]MABG. We utilised our novel transfected mosaic spheroid model (TMS) to determine whether this treatment strategy could result in sterilisation of spheroids containing only a small proportion of NAT-expressing cells., Results: The concentrations of [(131)I]MIBG and [(211)At]MABG required to reduce to 0.1% the survival of clonogens derived from the TMS composed of 100% of NAT gene-transfected cells were 1.5 and 0.004 MBq/ml (RSV promoter), 8.5 and 0.0075 MBq/ml (hTR promoter), and 9.0 and 0.008 MBq/ml (hTERT promoter), respectively. The concentrations of radiopharmaceutical required to reduce to 0.1% the survival of clonogens derived from 5% RSV/NAT and 5% hTERT/NAT TMS were 14 and 23 MBq/ml, respectively, for treatment with [(131)I]MIBG and 0.018 and 0.028 MBq/ml, respectively, for treatment with [(211)At]MABG., Conclusions: These results indicate that the telomerase promoters have the capacity to drive the expression of the NAT. The potency of [(211)At]MABG is approximately three orders of magnitude greater than that of [(131)I]MIBG. Spheroids composed of only 5% of cells expressing NAT under the control of the RSV or hTERT promoter were sterilised by radiopharmaceutical treatment. This observation is indicative of bystander cell-kill.

The utility of external beam radiotherapy for the treatment of malignant brain tumours is compromised by the need to avoid excessive radiation damage to normal CNS tissues. This review describes the current status of targeted radiotherapy, an alternative strategy for brain tumour treatment that offers the exciting prospect of increasing the specificity of tumour cell irradiation.

We have obtained encouraging responses in recent Phase I studies evaluating (131)I-labeled human/murine chimeric 81C6 anti-tenascin monoclonal antibody (ch81C6) administered into surgically-created tumor resection cavities in brain tumor patients. However, because the blood clearance is slow, hematologic toxicity has been higher than seen with murine 81C6 (mu81C6). In the current study, a series of paired-label experiments were performed in athymic mice bearing subcutaneous D-245 MG human glioma xenografts to compare the biodistribution of the fragment ch81C6 F(ab')(2) labeled using Iodogen to a) intact ch81C6, b) mu81C6, and c) ch81C6 F(ab')(2) labeled using N-succinimidyl 3-[(131)I]iodobenzoate. Tumor retention of radioiodine activity for the F(ab')(2) fragment was comparable to that for intact ch81C6 for the first 24 h and to that for mu81C6 for the first 48 h; as expected, blood and other normal tissue levels declined faster for ch81C6 F(ab')(2.) Radiation dosimetry calculations suggest that (131)I-labeled ch81C6 F(ab')(2) may warrant further evaluation as a targeted radiotherapeutic for the treatment of brain tumors.

Radioiodinated meta-iodobenzylguanidine (MIBG) is used in the diagnosis and therapy of various neuroendocrine tumors. To investigate whether an additional guanidine function in the structure of MIBG will yield analogues that may potentially enhance tumor-to-target ratios, two derivatives-one with a guanidine moiety and another with a guanidinomethyl group at the 4-position of MIBG-were prepared. In the absence of any uptake-1 inhibiting conditions, the uptake of 4-guanidinomethyl-3-[(131)I]iodobenzylguanidine ([(131)I]GMIBG) by SK-N-SH cells in vitro was 1.7+/-0.1% of input counts, compared to a value of 40.3+/-1.4% for [(125)I[MIBG suggesting that guanidinomethyl group at the 4-position negated the biological properties of MIBG. On the other hand, 4-guanidino-3-[(131)I]iodobenzylguanidine ([(131)I]GIBG) had an uptake (5.6+/-0.3%) that was 12-13% that of [(125)I]MIBG (46.1+/-2.7%), and the ratio of uptake by control over DMI-treated (nonspecific) cultures was higher for [(131)I]GIBG (20.9+/-0.3) than [(125)I]MIBG itself (15.0+/-2.7). The exocytosis of [(131)I]GIBG and [(125)I]MIBG from SK-N-SH cells was similar. The uptake of [(131)I]GIBG in the mouse target tissues, heart and adrenals, as well as in a number of other tissues was about half that of [(125)I]MIBG. These results suggest that substitution of guanidine functions, especially a guanidinomethyl group, in MIBG structure may not be advantageous.

O(6)-Benzylguanine derivatives with suitable radionuclides attached to the benzyl ring are potentially useful in the noninvasive imaging of the DNA repair protein, alkylguanine-DNA alkyltransferase (AGT). Previously, O(6)-3-[(131)I]iodobenzylguanine ([(131)I]IBG) was prepared using a two-step approach; we now report its synthesis in a single step by the radioiododestannylation of O(6)-3-(trimethylstannyl)benzylguanine in 85-95% radiochemical yield. The in vitro specific uptake of [(131)I]IBG in DAOY human medulloblastoma cells, in TE-671 human rhabdomyosarcoma cells and a CHO cell line transfected to express AGT was linear (r(2) = 0.9-1.0) as a function of cell density. After intravenous injection of [(131)I]IBG in athymic mice bearing TE-671 xenografts, tumor uptake was 1.38 +/- 0.34% ID/g at 0.5 h and declined at 2 and 4 h. Preadministration of O(6)-(3-iodobenzyl)guanine (IBG) at 0.5 h increased uptake not only in tumor but also in several normal tissues. Notable exceptions were thyroid (p < 0.05), lung (p <0.05) and stomach. After intratumoral injection of [(131)I]IBG in the same xenograft model, the uptake in tumors that were depleted of AGT by BG treatment (165.8 +/- 27.5% ID/g) was about 60% of that in control mice (272.4 +/- 48.2% ID/g; p < 0.05).

A major obstacle to successful cancer chemotherapy is drug resistance. Multidrug resistance (MDR) is often seen with chemotherapeutic agents such as anthracycline derivatives, vinca alkaloids and taxanes. Multiple aspects of cellular biochemistry have been implicated in the MDR process. Cellular mechanisms of resistance are due to the presence of efflux pumps, P-glycoprotein (P-gp) and multiple resistance-associated protein (MRP), which belong to the ATP-binding cassette (ABC) family of transporters. Another form of drug resistance is involved in the chemotherapy of cancers with alkylating agents such as nitrosourea derivatives and nitrogen mustards. The cytotoxicity of these agents is primarily due to alkylation of the DNA guanine residues at their O6-position, which leads, via a cascade of events, to DNA strand breaks. The DNA repair protein, alkylguanine-DNA alkyl transferase (AGT) removes the alkyl groups from the lesions stoichiometrically to a cysteine in its active site. This process is irreversible and results in the degradation of the protein and its recovery is entirely from de novo synthesis. Noninvasive methodologies for monitoring the transport activity of these efflux pumps and determining tumor content of AGT could serve as critical tools for optimizing chemotherapeutic protocols on a patient-specific basis and gaining an understanding of the dynamics of resistance in living patients. In this review, we will describe the efforts made to date to synthesize radioactive probes of chemotherapy resistance and their use to quantitate these transporters and DNA repair protein by radionuclide imaging.

Purpose: The monoclonal antibody (MAb) trastuzumab (Herceptin) effectively treats HER2-overexpressing extracerebral breast neoplasms. Delivery of such macromolecule therapeutic agents to intracerebral metastases, however, is limited by the tight junctions characteristic of the cerebral vasculature. Direct intracerebral microinfusion (ICM) is a technique that bypasses this blood-brain barrier and allows for a greater delivery of drugs directly into intracerebral tumors., Experimental Design: A human breast cancer cell line transfected to overexpress HER2, MCF-7/HER2-18, was transplanted into the cerebrum of athymic rats. Saline, trastuzumab, or an isotype-matched control MAb was delivered systemically or by ICM to assess toxicity and efficacy., Results: No clinical or histological toxicity related to trastuzumab was evident under any of the conditions studied. Delivery of trastuzumab (2 mg/kg) i.p. led to a median survival of 26.5 days, whereas treatment with trastuzumab (2 mg/kg) by ICM increased the median survival by 96% to 52 days, with two of nine rats surviving >120 days (P = 0.009). Treatment with an isotype-matched control MAb (16 mg/kg) resulted in a median survival of 21 days, which did not differ significantly from the survival of rats treated by ICM with saline (16 days; P = 0.42). Treatment by ICM with trastuzumab (16 mg/kg) led to a median survival of 45 days, with 2 of 10 rats surviving >120 days. These results represent 181% and 114% increases in median survival over the saline and MAb controls, respectively (P < 0.001)., Conclusion: ICM of trastuzumab is safe and superior to systemic delivery as therapy for HER2-overexpressing intracerebral neoplasms in an athymic rat model.

Unlabelled: The sodium/iodide symporter (NIS) has been identified as an attractive target for cancer therapy. The efficacy of (131)I-iodide for NIS-expressing tumor therapy may be limited by a combination of poor cellular retention and unfavorable physical characteristics (long physical half-life and low linear-energy-transfer [LET] radiative emissions). On the other hand, (211)At-astatide is also transported by NIS and offers several therapeutic advantages over (131)I-iodide due to its physical characteristics (short half-life, high LET alpha-particle emissions). The objective of this study was to directly compare the radiotoxicity of both radionuclides using a NIS-transfected cultured cell model., Methods: Cytotoxicity was determined by colony-forming assays. Also, a first-order pharmacokinetic model was used to simulate the closed compartmental system between the medium and cells. Experimental data were then fitted to this model and used to estimate the transfer coefficients between medium and cells, k(m)(c), and between cells and medium, k(c)(m). Using the pharmacokinetic model, the cumulated activity concentrations in the medium and cells were calculated. Monte Carlo transport methods were then used to assess absorbed doses from (131)I and (211)At., Results: (211)At-Astatide was significantly more cytotoxic than (131)I-iodide in this closed compartmental system. For (211)At-astatide, absorbed doses per unit administered activity were 54- to 65-fold higher than for (131)I-iodide. Both NIS-expressing and control cells showed increased sensitivity to (211)At over (131)I, with significantly lower D(0) (absorbed dose required to reduce the survival fraction to e(-1)) and SF(2) (2-Gy survival fraction) values, highlighting the higher intrinsic cytotoxicity of alpha-particles. However, NIS-independent (nonspecific) binding of (211)At-astatide was higher than that of (131)I-iodide, therefore, yielding a lower absorbed dose ratio between NIS-transfected and -nontransfected cells., Conclusion: Treatment of NIS-expressing cells with (211)At-astatide resulted in higher absorbed doses and increased cytotoxicity per unit administered activity than that observed with (131)I-iodide. These results suggest that (211)At-astatide may be a promising treatment strategy for the therapy of NIS-expressing tumors.