Conformational insights into the lesion and sequence effects for arylamine-induced translesion DNA synthesis: 19F NMR, surface plasmon resonance, and primer kinetic studies.

Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3'-next flanking base (G*CT vs G*CA; G*, FABP, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl; FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair ( Jain , V. , Hilton , B. , Lin , B. , Patnaik , S. , Liang , F. , Darian , E. , Zou , Y. , Mackerell , A. D. , Jr. , and Cho , B. P. ( 2013 ) Nucleic Acids Res. , 41 , 869 - 880 ). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo(-)) and Dpo4. We obtained dynamic (19)F NMR spectra for two 19-mer modified templates during primer elongation: G*CA [d(5'-CTTACCATCG*CAACCATTC-3')] and G*CT [d(5'-CTTACCATCG*CTACCATTC-3')]. We found that lesion stacking is favored in the G*CT sequence compared to the G*CA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and G*CA > G*CT. Primer extension was stalled at (n) and near (n - 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct's chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo(-) vs Dpo4). Steady-state kinetics analysis with Kf-exo(-) revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis.

Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site.

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase ß (pol ß) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol ß. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,ß)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol ß, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol ß binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

SIB-DOTA: a trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies.

A major drawback of internalizing monoclonal antibodies (mAbs) radioiodinated with direct electrophilic approaches is that tumor retention of radioactivity is compromised by the rapid washout of iodo-tyrosine, the primary labeled catabolite for mAbs labeled via this strategy. In our continuing efforts to develop more versatile residualizing labels that could overcome this problem, we have designed SIB-DOTA, a prosthetic labeling template that combines the features of the prototypical, dehalogenation-resistant N-succinimidyl 3-iodobenzoate (SIB) with DOTA, a useful macrocyclic chelator for labeling with radiometals. Herein we describe the synthesis of the unlabeled standard of this prosthetic moiety, its protected tin precursor, and radioiodinated SIB-DOTA. An anti-EGFRvIII-reactive mAb, L8A4 was radiolabeled with [(131)I]SIB-DOTA in 27.1±6.2% (n=2) conjugation yields and its targeting properties to the same mAb labeled with [(125)I]SGMIB both in vitro and in vivo using U87MG·ΔEGFR cells and xenografts were compared. In vitro paired-label internalization assays showed that the intracellular radioactivity from [(131)I]SIB-DOTA-L8A4 was 21.4±0.5% and 26.2±1.1% of initially bound radioactivity at 16 and 24h, respectively. In comparison, these values for [(125)I]SGMIB-L8A4 were 16.7±0.5% and 14.9±1.1%. Similarly, the SIB-DOTA prosthetic group provided better tumor targeting in vivo than SGMIB over 8 d period. These results suggest that SIB-DOTA warrants further evaluation as a residualizing agent for labeling internalizing mAbs including those targeted to EGFRvIII.

A case of curative-intent hepatectomy for colon cancer metastatic to the scapula and liver.

Colon cancer is the second leading cause of cancer death in the United States. Patients with colon cancer metastatic to liver and bone are deemed non-curable and have a poor prognosis. We present a case of recurrent colon cancer with synchronous hepatic and bony metastases treated with radiation, chemotherapy, and curative-intent hepatectomy. The patient is alive and free of disease recurrence, off chemotherapy, more than 2 years post-hepatectomy.

Meta-iodobenzylguanidine and analogues: chemistry and biology.

Meta-iodobenzylguanidine (MIBG) is a structural analogue of the neurotransmitter norepinephrine (NE) and is taken up by cells rich in sympathetic neurons by an active uptake process mediated by the NE transporter, which is referred to as uptake-1. It is a valuable agent in the diagnosis of myocardial abnormalities as well as that of several neuroendocrine tumors such as neuroblastoma, pheochromocytoma and carcinoid tumors. MIBG labeled with (131)I also is used extensively in the therapy of several neuroendocrine tumors. Over the years, a substantial amount of work has been undertaken to improve its clinical utility. Currently, radio-iodinated MIBG used in the clinic is prepared by an exchange radio-iodination method and, thus, is of low specific activity. For possible better targeting and to ward off pharmacological effects, its preparation at a no-carrier-added level both by solution-phase and solid-phase synthesis has been developed. For potential use in the treatment of micrometastatic diseases, synthesis of an analogue labeled with the a emitter (211)At was devised. Development of analogues labeled with positron emitting radionuclides such as (124)I, (18)F, and (76)Br has been reported. Further, efforts have been put in to improve its pharmacokinetic properties by structural modifications. Various aspects of these developments are reviewed herein.

TMPRSS2-ERG fusion, a common genomic alteration in prostate cancer activates C-MYC and abrogates prostate epithelial differentiation.

The high prevalence of TMPRSS2-ERG rearrangements ( approximately 60%) in prostate cancer (CaP) leads to androgenic induction of the ETS-related gene (ERG) expression. However, the biological functions of ERG overexpression in CaP remain to be understood. ERG knockdown in TMPRSS2-ERG expressing CaP cells induced striking morphological changes and inhibited cell growth both in cell culture and SCID mice. Evaluation of the transcriptome and specific gene promoters in ERG siRNA-treated cells and investigation of gene expression signatures of human prostate tumors revealed ERG-mediated activation of C-MYC oncogene and the repression of prostate epithelial differentiation genes (PSA and SLC45A3/Prostein). Taken together, these data combining cell culture and animal models and human prostate tumors reveal that ERG overexpression in prostate tumor cells may contribute to the neoplastic process by activating C-MYC and by abrogating prostate epithelial differentiation as indicated by prostate epithelial specific markers.

Synthesis and evaluation of glycosylated octreotate analogues labeled with radioiodine and 211At via a tin precursor.

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.

No-carrier-added synthesis of a 4-methyl-substituted meta-iodobenzylguanidine analogue.

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.

Imaging drug resistance with radiolabeled molecules.

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.

A validated LC method for imatinib mesylate.

An isocratic reversed-phase liquid chromatography method with UV detection has been developed for the purity evaluation of imatinib mesylate in bulk drug. The method is selective and is capable of detecting all process intermediates and other related compounds, which may be present at trace levels in the drug substance. The method was validated on a Symmetry Shield RP18 analytical column (150 x 4.6 mm, 5 microm), mobile phase consisting of 30 mM sodium octane sulphonic acid in 10 mM aqueous KH2PO4 (pH 2.5 with H3PO4): MeOH in the ratio of 42:58 v/v. The flow rate was set at 1.0 ml/min and the column was maintained at room temperature. The injection volume was set to 10 microl and the detector was set at a wavelength of 237 nm. The method was validated in terms of system precision, method precision, linearity, accuracy, limit of detection and limit of quantification.

Synthesis of ring- and side-chain-substituted m-iodobenzylguanidine analogues.

With the goal of developing MIBG analogues with improved targeting properties especially for oncologic applications, several radioiodinated ring- and side-chain-substituted MIBG analogues were synthesized. Except for 3-[(131)I]iodo-4-nitrobenzylguanidine and N-hydroxy-3-[(131)I]iodobenzylguanidine, the radioiodinated analogues were prepared at no-carrier-added levels from their respective tin precursors. The radiochemical yields generally were in the range of 70-90% except for 3-amino-5-[(131)I]iodobenzylguanidine for which a radiochemical yield of about 40% was obtained. While the silicon precursor N(1),N(2)-bis(tert-butyloxycarbonyl)-N(1)-(4-nitro-3-trimethylsilylbenzyl)guanidine did not yield 3-[(131)I]iodo-4-nitrobenzylguanidine, its deprotected derivative, N(1)-(4-nitro-3-trimethylsilylbenzyl)guanidine was radioiodinated in a modest yield of 20% providing 3-[(131)I]iodo-4-nitrobenzylguanidine. Exchange radioiodination of 3-iodo-4-nitrobenzylguanidine gave 3-[(131)I]iodo-4-nitrobenzylguanidine in 80% radiochemical yield. No-carrier-added [(131)I]NHIBG was prepared from its silicon precursor N(1)-hydroxy-N(3)-(3-trimethylsilylbenzyl)guanidine in 85% radiochemical yield.

Biological evaluation of ring- and side-chain-substituted m-iodobenzylguanidine analogues.

A number of ring- and side-chain-substituted m-iodobenzylguanidine analogues were evaluated for their lipophilicity, in vitro stability, uptake by SK-N-SH human neuroblastoma cells in vitro, and biodistribution in normal mice. As expected, the lipophilicity of m-iodobenzylguanidine increased when a halogen was introduced onto the ring and decreased with the addition of polar hydroxyl, amino, and nitro substitutents. Most of the derivatives showed reasonable stability up to 24 h in PBS at 37 degrees C. While N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine and 3,4-dihydroxy-5-[(131)I]iodobenzylguanidine generated a more nonpolar product in addition to the free iodide, 3-[(131)I]iodo-4-nitrobenzylguanidine decomposed to a product more polar than the parent compound. The specific uptake of 4-chloro-3-[(131)I]iodobenzylguanidine, 3-[(131)I]iodo-4-nitrobenzylguanidine, and N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine by SK-N-SH human neuroblastoma cells in vitro, relative to that of m-[(125)I]iodobenzylguanidine, was 117 +/- 10%, 50 +/- 4%, and 12 +/- 2%, respectively. The specific uptake of the known m-iodobenzylguanidine analogues 4-hydroxy-3-[(131)I]iodobenzylguanidine and 4-amino-3-[(131)I]iodobenzylguanidine was 80 +/- 4% and 66 +/- 4%, respectively. None of the other m-iodobenzylguanidine derivatives showed any significant specific uptake by SK-N-SH cells. Heart uptake of 4-chloro-3-[(131)I]iodobenzylguanidine in normal mice was higher than that of m-[(125)I]iodobenzylguanidine at later time points (11 +/- 1% ID/g versus 3 +/- 1% ID/g at 24 h; p < 0.05) while uptake of 3-[(131)I]iodo-4-nitrobenzylguanidine and of N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine in the heart was lower than that of m-iodobenzylguanidine at all time points. In accordance with the in vitro results, none of the other novel m-iodobenzylguanidine derivatives showed any significant myocardial or adrenal uptake in vivo.

A polar substituent-containing acylation agent for the radioiodination of internalizing monoclonal antibodies: N-succinimidyl 4-guanidinomethyl-3-[131I]iodobenzoate ([131I]SGMIB).

The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies.

Radiolabeled guanine derivatives for the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase: 6-(4-[(18)F]Fluoro-benzyloxy)-9H-purin-2-ylamine and 6-(3-[(131)I]Iodo-benzyloxy)-9H-purin-2-ylamine.

Two radiolabeled analogues of 6-benzyloxy-9H-purin-2-ylamine (O(6)-benzylguanine; BG) potentially useful in the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase (AGT) were synthesized. Fluorine-18 labeling of the known 6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine (FBG; 6) was accomplished by the condensation of 4-[(18)F]fluorobenzyl alcohol with 2-aminopurin-6-yltrimethylammonium chloride (4) or 2-amino-6-chloropurine in average decay-corrected radiochemical yields of 40 and 25%, respectively. Unlabeled 6-(3-iodo-benzyloxy)-9H-purin-2-ylamine (IBG; 7) was prepared from 4 and 3-iodobenzyl alcohol. Radioiodination of 9, prepared from 7 in two steps, and subsequent deprotection gave [(131)I]7 in about 70% overall radiochemical yield. The IC(50) values for the inactivation of AGT from CHO cells transfected with pCMV-AGT were 15 nM for IBG and 50 nM for FBG. The binding of [(18)F]6 and [(131)I]7 to purified AGT was specific and saturable with both exhibiting similar IC(50) values (5-6 microM).

Radioiodination and astatination of octreotide by conjugation labeling.

Octreotide was coupled to 3-iodobenzoyl and 3-iodonicotinoyl moieties to obtain [N-(3-iodobenzoyl)-D-Phe(1)]octreotide (IBO) and [N-(3-iodonicotinoyl)-D-Phe(1)]octreotide (INO), respectively. The IC(50) values for the binding of IBO and INO to CA20948 rat pancreatic tumor membranes were 0.90 and 0.13 nM, respectively, compared with 0.35 nM for octreotide itself. Starting from N-succinimidyl 3-[(131)I]iodobenzoate and N-succinimidyl 5-[(131)I]iodopyridine-3- carboxylate, [(131)I]IBO and [(131)I]INO were prepared in overall radiochemical yields of 35%-50%. Likewise, ¿N-(3-[(211)At]astatobenzoyl)-D-Phe(1)¿octreotide ([(211)At]ABO) was prepared in similar yield from N-succinimidyl 3-[(211)At]astatobenzoate. In vitro assays with AR42J rat pancreatic tumor cells demonstrated a higher retention of cell-internalized radioiodine activity for [(131)I]INO compared with [(125)I]IBO. Tissue distribution studies with both conjugates revealed low levels of activity in the thyroid suggesting that dehalogenation of these peptides was minimal.

Astatine-211-labeled radiotherapeutics: an emerging approach to targeted alpha-particle radiotherapy.

Targeted radiotherapy or endoradiotherapy is an appealing approach to cancer treatment because of the potential for delivering curative doses of radiation to tumor while sparing normal tissues. Radionuclides that decay by the emission of alpha-particles such as the heavy halogen astatine-211 (211At) offer the exciting prospect of combining cell-specific molecular targets with radiation having a range in tissue of only a few cell diameters. Herein, the radiobiological advantages of alpha-particle targeted radiotherapy will be reviewed, and the rationale for using 211At for this purpose will be described. The chemistry of astatine is similar to that of iodine; however, there are important differences which make the synthesis and evaluation of 211At-labeled compounds more challenging. Perhaps the most successful approach that has been developed involves the astatodemetallation of tin, silicon or mercury precursors. Astatine-211 labeled agents that have been investigated for targeted radiotherapy include [211At]astatide, 211At- labeled particulates, 211At-labeled naphthoquinone derivatives, 211At-labeled methylene blue, 211At-labeled DNA precursors, meta-[211At]astatobenzylguanidine, 211At-labeled biotin conjugates, 211At-labeled bisphosphonates, and 211At-labeled antibodies and antibody fragments. The status of these 211At-labeled compounds will be discussed in terms of their labeling chemistry, cytotoxicity in cell culture, as well as their tissue distribution and therapeutic efficacy in animal models of human cancers. Finally, an update on the status of the first clinical trial with an 211At-labeled targeted therapeutic, 211At-labeled chimeric anti-tenascin antibody 81C6, will be provided.

Iodopyridine-for-iodobenzene substitution for use with low molecular weight radiopharmaceuticals: application to m-iodobenzylguanidine.

Substituting a pyridine ring for a benzene ring in the acylation agent N-succinimidyl 3-iodobenzoate has resulted in a useful approach for the radiohalogenation of monoclonal antibodies, peptides, and labeled biotin conjugates. It was hypothesized that such a substitution in m-iodobenzylguanidine (MIBG), a radiotracer used in the detection and treatment of neuroendocrine tumors, might result in an analogue with more rapid normal tissue clearance, thereby facilitating its use for tumor therapy. For the preparation of this analogue, 3-guanidinomethyl-5-iodopyridine (GMIP; 9b), the silicon precursor 4 was synthesized starting from 5-bromonicotinic acid. Attempts to convert 4 to 9b under various conditions were not successful. Radioiodinated 9b could be prepared by the iododestannylation of the tin precursor 8 in 65-70% radiochemical yield. A number of in vitro, in vivo, and ex vivo studies showed that pyridine-for-benzene substitution in MIBG yielded a compound that no longer was taken up by the uptake-1 pathway.

Survival and DNA damage in Chinese hamster V79 cells exposed to alpha particles emitted by DNA-incorporated astatine-211.

Asynchronous Chinese hamster V79 lung fibroblasts were incubated at 37 degrees C for 30 min with the thymidine analog 5-[211At]astato-2'-deoxyuridine (211AtdU, exposure from DNA-incorporated activity) or with [211At]astatide (211At-, exposure from extracellular activity), and DNA-incorporated activity was determined. The 211AtdU content in cellular DNA increased as a function of extracellular concentration. Incorporation of 211At- was less than 1% of that of 211AtdU. After exposure, cells were frozen in the presence of 10% DMSO. One month later, survival was determined by the colony-forming assay, and DNA double-strand breaks (DSBs) were measured by the neutral elution method (pH 9.6). The survival curve for 211AtdU was biphasic (D37 = 2.8 decays per cell), reflecting killing of 211At-DNA-labeled cells and of unlabeled cells irradiated by 211At in neighboring labeled cells. The toxicity of 211At- decaying outside the cell (30-min exposure) was negligible. Analysis of the survival curve produced a D0 of 1.3 decays/cell for 211At-labeled cells. The yield of DSBs from the decay of DNA-incorporated 211At was compared with that from DNA-incorporated 125I. Each decay of 211At produced at least 10 times the number of DSBs as that obtained per 125I decay. The extreme radiotoxicity of DNA-incorporated 211AtdU seems to be associated with considerable damage to the mammalian cell genome.

Preparation of 5-[131I]iodo- and 5-[211At]astato-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil by a halodestannylation reaction.

To circumvent the in vivo instability of 5-iodo-2'-deoxyuridine (IUdR), a 2'-fluorine-substituted analogue, 5-iodo-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (FIAU) recently has been introduced. To facilitate the preparation of radioiodinated FIAU as well as its astatinated analogue, a tin precursor, 5-trimethylstannyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)ura cil (FTAU) was synthesized. Both [125/131I]FIAU and 5-[211At]astato-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (FAAU) were prepared from FTAU in more than 85% radiochemical yield under mild conditions. The in vitro serum stability of both fluorine-substituted derivatives was higher than that of the corresponding unsubstituted parents. The enhanced stability of fluorinated derivatives was even more apparent in whole blood. The uptake of [125I]FIAU in D-247 MG human glioma cells in vitro was 20-fold higher than that of [125I]IUdR over an activity concentration range of 5-100 kBq/mL; the uptake of FAAU was not significantly different from that of 5-[211At]astato-2'-deoxyuridine (AUdR). Accumulation of radioiodine in mouse thyroid in vivo with [131I]FIAU was fivefold lower than [125I]IUdR, indicating that the former was less susceptible to deiodination. The tissue uptake of FAAU was similar to that reported for AUdR.

Toxicity to neuroblastoma cells and spheroids of benzylguanidine conjugated to radionuclides with short-range emissions.

Radiolabelled meta-iodobenzylguanidine (MIBG) is selectively taken up by tumours of neuroendocrine origin, where its cellular localization is believed to be cytoplasmic. The radiopharmaceutical [131I]MIBG is now widely used in the treatment of neuroblastoma, but other radioconjugates of benzylguanidine have been little studied. We have investigated the cytotoxic efficacy of beta, alpha and Auger electron-emitting radioconjugates in treating neuroblastoma cells grown in monolayer or spheroid culture. Using a no-carrier-added synthesis route, we produced 123I-, 125I-, 131I- and 211At-labelled benzylguanidines and compared their in vitro toxicity to the neuroblastoma cell line SK-N-BE(2c) grown in monolayer and spheroid culture. The Auger electron-emitting conjugates ([123I]MIBG and [125I]MIBG) and the alpha-emitting conjugate ([211At]MABG) were highly toxic to monolayers and small spheroids, whereas the beta-emitting conjugate [131I]MIBG was relatively ineffective. The Auger emitters were more effective than expected if the cellular localization of MIBG is cytoplasmic. As dosimetrically predicted however, [211At]MABG was found to be extremely potent in terms of both concentration of radioactivity and number of atoms ml(-1) administered. In contrast, the Auger electron emitters were ineffective in the treatment of larger spheroids, while the beta emitter showed greater efficacy. These findings suggest that short-range emitters would be well suited to the treatment of circulating tumour cells or small clumps, whereas beta emitters would be superior in the treatment of subclinical metastases or macroscopic tumours. These experimental results provide support for a clinical strategy of combinations ('cocktails') of radioconjugates in targeted radiotherapy.