Mechanism of the palladium-catalyzed hydrothiolation of alkynes to thioethers: a DFT study.

The mechanisms of the palladium-catalyzed hydrothiolation of alkynes with thiols were investigated using density functional theory at the B3LYP/6-31G(d, p) (SDD for Pd) level. Solvent effects on these reactions were explored using the polarizable continuum model (PCM) for the solvent tetrahydrofuran (THF). Markovnikov-type vinyl sulfides or cis-configured anti-Markovnikov-type products were formed by three possible pathways. Our calculation results suggested the following: (1) the first step of the cycle is a proton-transfer process from thiols onto the palladium atom to form a palladium-thiolate intermediate. The palladium-thiolate species is attacked on alkynes to obtain an elimination product, liberating the catalyst. (2) The higher activation energies for the alkyne into the palladium-thiolate bond indicate that this step is the rate-determining step. The Markovnikov-type vinyl sulfide product is favored. However, for the aromatic alkyne, the cis-configured anti-Markovnikov-type product is favored. (3) The activation energy would reduce when thiols are substituted with an aromatic group. Our calculated results are consistent with the experimental observations of Frech and colleagues for the palladium-catalyzed hydrothiolation of alkynes to thiols.

Bystander effect in tumor cells produced by Iodine-125 labeled human lymphocytes.

PURPOSE: To investigate the ability of human lymphocytes labeled with DNA-incorporated (125)I to exert an inhibitory (antiproliferative) bystander effect on co-cultured human colon adenocarcinoma LS174T cells in vitro. MATERIALS AND METHODS: Human peripheral blood lymphocytes were stimulated to synthesize DNA in the presence of phytohemagglutinin (PHA) and labeled with 5-[(125)I]iodo-2'-deoxyuridine. Human colon adenocarcinoma LS174T cells were co-cultured with the (125)I-labeled lymphocytes in various ratios for 5 days and the proliferation of the LS174T cells was assessed. Further, the supernatant media from these co-cultures were: (i) Transferred to LS174T cells and their proliferation measured after 5 days, (ii) used to assess the clonogenic survival of LS174T cells, and (iii) screened for factors that suppress growth. RESULTS: A significant reduction in the proliferation of LS174T cells was observed when co-cultured either with (125)I-labeled lymphocytes (56 ± 3.5%) or the supernatant media (52.5 ± 1.3%) obtained from these co-cultures. Clonogenic survival of LS174T cells grown in the supernatant media corroborated the decrease in tumor cell growth. CONCLUSION: The observed reduction in the proliferation of LS174T cells in presence of (125)I-labeled lymphocytes or media obtained from such co-cultures can be attributed to an inhibitory (antiproliferative) bystander effect, probably mediated by factor(s) released from the dying (125)I-labeled lymphocytes.

Effect of distance between decaying (125)I and DNA on Auger-electron induced double-strand break yield.

PURPOSE: To determine the possible effects of (125)I-to-DNA distance on the magnitude and mechanism of Auger-electron induced-double-strand break (DSB) production. MATERIALS AND METHODS: We have synthesized a series of (125)I-labeled Hoechst (H) derivatives ((125)IE-H, (125)IB-H, (125)I-C(8)-H and (125)I-C(12)-H). While all four molecules share a common DNA minor groove binding bis-benzimidazole motif, they are designed to position (125)I at varying distances from the DNA helix. Each Hoechst derivative was incubated at 4°C in phosphate buffered saline (PBS) together with supercoiled (SC) (3)H-pUC19 plasmid DNA (ratio 3:1) ± the •OH scavenger dimethyl sulfoxide (DMSO) (0.2 M). Aliquots were analyzed on agarose gels over time and DSB yields per decay of (125)I atom were determined. Docking of the iodinated compounds on a DNA molecule was carried out to determine the distance between the iodine atom and the central axis of DNA. RESULTS: In the absence of DMSO, the results show that the DSB yields decrease monotonically as the (125)I atom is distanced - by 10.5 Å to 13.9 Å - from the DNA helix ((125)IEH: 0.52 ± 0.01; (125)IB-H: 0.24 ± 0.03; (125)I-C(8)-H: 0.18 ± 0.02; (125)I-C(12)-H: 0.10 ± 0.00). In the presence of DMSO, DSB yields for (125)IEH (0.49 ± 0.02) and (125)IB-H (0.26 ± 0.04) remain largely unchanged indicating that DSB are entirely produced by direct effects. Strikingly, (125)I-C(8)-H or (125)I-C(12)-H, did not produce detectable DSB in the presence of DMSO under similar conditions suggesting when (125)I atom is positioned > 12 Å from the DNA, DSB are entirely produced by indirect effects. CONCLUSION: These results suggest that at a critical distance between the (125)I atom and the DNA helix, DSB production switches from an 'all' direct to an 'all' indirect mechanism, the latter situation being comparable to the decay of (125)I free in solution. These experimental findings were correlated with theoretical expectations based on microdosimetry.

Computational and biological evaluation of quinazolinone prodrug for targeting pancreatic cancer.

Our concept of enzyme-mediated cancer imaging and therapy aims to use radiolabeled compounds to target hydrolases over-expressed on the extracellular surface of solid tumors. A data mining approach identified extracellular sulfatase 1 (SULF1) as an enzyme expressed on the surface of pancreatic cancer cells. We designed, synthesized, and characterized 2-(2'-sulfooxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-S)) as well as its radioiodinated form ((125) IQ(2-S)) as a prodrug with potential for hydrolysis by SULF1. IQ(2-S) was successfully docked in silico into three enzymes - homolog of SULF1, alkaline phosphatase, and prostatic acid phosphatase. The incubation of (125) IQ(2-S) and (125) IQ(2-P) with the three enzymes in solution confirms the docking results and enzyme selectivity for the analogs. The hydrolysis of both radioactive compounds produces the water-insoluble, fluorescent product 2-(2'-hydroxyphenyl)-6-[(125) I]iodo-4-(3H)-quinazolinone ((125) IQ(2-OH)). The in vitro incubation of (127) IQ(2-S) and (127) IQ(2-P) with pancreatic, ovarian, and prostate cancer cells expressing studied hydrolases also results in their hydrolysis and the precipitation of (127) IQ(2-OH) fluorescent crystals on the cell surface. To our knowledge, these findings are the first to report the targeting of a radioactive substrate to SULF1 and that this prodrug may be potentially useful in the imaging ((123) I/(124) I/(131) I) and radiotherapy ((131) I) of pancreatic cancer.

Human placental alkaline phosphatase-mediated hydrolysis correlates tightly with the electrostatic contribution from tail group.

Human placental alkaline phosphatase has been identified as a hydrolase that is significantly overexpressed on the surface of various solid tumor cells, and is therefore a suitable prodrug design target for non-invasive cancer imaging and therapy. Structure-based prediction of enzymatic activities is essential for rational prodrug design. We have been probing the catalytic proficiency--(k(cat) /K(M) )/k(w)--of placental alkaline phosphatase toward several widely diverse substrate structures experimentally and correlating these results to in silico predictions that are based on the free energy estimates obtained from docking of each substrate structure with placental alkaline phosphatase. We have found that electrostatic contribution from the tail group is the most crucial factor to determine the catalytic efficiencies of the substrates. The electrostatic contribution and the total binding energy of the tail group are well correlated with catalytic efficiencies (R² = 0.79 and 0.89, respectively). However, hydrophobic contribution from the tail group does not correlate with the catalytic efficiencies (negative correlation, R² = 0.27). This supports the prior hypothesis stating that alkaline phosphatase-mediated differential hydrolysis of its substrates is attributable to the differential interactions with the tail group, determined by the electrostatic contributions from the non-bridging oxygen atoms. Calculation of the electrostatic potentials within the active site of human placental alkaline phosphatase also suggests that the local positive electrostatic environment may account for its capability to distinguish various substrates. Our study is likely to have immediate implications in the design of prodrugs against human placental alkaline phosphatase and other esterases overexpressed by human tumor cells.

DNA double-strand breaks induced by decay of (123)I-labeled Hoechst 33342: role of DNA topology.

PURPOSE: To determine double-strand-break (DSB) yields produced by decay of minor-groove-bound (123)I-labeled Hoechst 33342 ((123)IEH) in supercoiled (SC) and linear (L) forms of pUC19 DNA, to compare strand-break efficiency of (123)IEH with that of (125)IEH, and to examine the role of DNA topology in DSB induction by these Auger electron emitters. MATERIALS AND METHODS: Tritium-labeled SC and L pUC19 DNA were incubated with (123)IEH (0-10.9 MBq) at 4 degrees C. After (123)I had completely decayed (10 days), samples were analyzed on agarose gel, and single-strand-break (SSB) and DSB yields were measured. RESULTS: Each (123)I decay in SC DNA produces a DSB yield of 0.18 +/- 0.01. On the basis of DSB yields for (125)IEH (0.52 +/- 0.02 for SC and 1.62 +/- 0.07 for L, reported previously) and dosimetric expectations, a DSB yield of approximately 0.5 (3 x 0.18) per (123)I decay is expected for L DNA. However, no DSB are observed for the L form, even after approximately 2 x 10(11) decays of (123)I per microg DNA, whereas a similar number of (125)I decays produces DSB in approximately 40% of L DNA. CONCLUSION: (123)IEH-induced DSB yield for SC but not L DNA is consistent with the dosimetric expectations for Auger electron emitters. These studies highlight the role of DNA topology in DSB production by Auger emitters and underscore the failure of current theoretical dosimetric methods per se to predict the magnitude of DSB.

Novel prodrugs for targeting diagnostic and therapeutic radionuclides to solid tumors.

Most cancer therapeutics (chemo, radiation, antibody-based, anti-angiogenic) are at best partially and/or temporarily effective. In general, the causes for failure can be summarized as: (i) poor diffusion and/or nonuniform distribution of drug/prodrug molecules in solid tumors; (ii) high drug concentration and retention in normal tissues (leading to side effects); (iii) requirement for plasma-membrane permeability and/or internalization of drug/prodrug molecules; (iv) low uptake of drug by tumor; (v) lack of retention of drug within tumor (most have gradient-driven reversible binding); and (vi) multidrug resistance. We are developing an innovative technology that aims to surmount these problems by actively concentrating and permanently entrapping radioimaging and radiotherapeutic prodrugs specifically within solid tumors. The approach will enable noninvasive sensing (imaging) and effective therapy of solid tumors, allowing tumor detection, diagnosis, and treatment to be closely coupled (personalized medicine).

DMSO increases radioiodination yield of radiopharmaceuticals.

A high-yield radioiodination method for various types of molecules is described. The approach employs DMSO as precursor solvent, a reaction ratio of 2-5 precursor molecules per iodine atom, 5-10 microg oxidant, and a 10-25 microl reaction volume. The solution is vortexed at room temperature for 1-5 min and progress of the reaction is assessed by HPLC. Radioiodinated products are obtained in > or = 95% yield and meet the requirements for radiotracer imaging, biodistribution studies, and molecular and cellular biology research.

Computational modeling and experimental evaluation of a novel prodrug for targeting the extracellular space of prostate tumors.

We are developing a noninvasive approach for targeting imaging and therapeutic radionuclides to prostate cancer. Our method, Enzyme-Mediated Cancer Imaging and Therapy (EMCIT), aims to use enzyme-dependent, site-specific, in vivo precipitation of a radioactive molecule within the extracellular space of solid tumors. Advanced methods for data mining of the literature, protein databases, and knowledge bases (IT. Omics LSGraph and Ingenuity Systems) identified prostatic acid phosphatase (PAP) as an enzyme overexpressed in prostate cancer and secreted in the extracellular space. Using AutoDock 3.0 software, the prodrug ammonium 2-(2'-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-P)) was docked in silico into the X-ray structure of PAP. The data indicate that IQ(2-P) docked into the PAP active site with a calculated inhibition constant (K(i)) more favorable than that of the PAP inhibitor alpha-benzylaminobenzylphosphonic acid. When (125)IQ(2-P), the radioiodinated form of the water-soluble prodrug, was incubated with PAP, rapid hydrolysis of the compound was observed as exemplified by formation of the water-insoluble 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone ((125)IQ(2-OH)). Similarly, the incubation of IQ(2-P) with human LNCaP, PC-3, and 22Rv1 prostate tumor cells resulted in the formation of large fluorescent IQ(2-OH) crystals. No hydrolysis was seen in the presence of normal human cells. Autoradiography of tumor cells incubated with (125)IQ(2-P) showed accumulation of radioactive grains ((125)IQ(2-OH)) around the cells. We anticipate that the EMCIT approach will enable the active in vivo entrapment of radioimaging and radiotherapeutic compounds within the extracellular spaces of primary prostate tumors and their metastases.

Using Hoechst 33342 to target radioactivity to the cell nucleus.

We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated (125I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with 125I-H33342 at 37 degrees C and accumulating 125I decays at -90 degrees C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (gamma-H2AX) formation. 125I-H33342 decay leads to a monoexponential decrease in cell survival with a D0 of 122 125I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 gamma-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against gamma radiation. We conclude that cell killing by 125I-H33342 and the induction of gamma-H2AX foci are highly correlated.

Evaluation of chemical, physical, and biologic properties of tumor-targeting radioiodinated quinazolinone derivative.

Our group is developing a novel technology, enzyme-mediated cancer imaging and therapy (EMCIT), that aims to entrap radioiodinated compounds within solid tumors for noninvasive tumor detection and therapy. In this approach, a water-soluble, radioiodinated prodrug is hydrolyzed in vivo to a highly water-insoluble compound by an enzyme overexpressed extracellularly by tumor cells. We have synthesized and characterized the water-soluble prodrug, 2-(2'-phosphoryloxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]5, which is readily hydrolyzed by alkaline phosphatase, an enzyme expressed by many tumor cell lines, to a water-insoluble drug, 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]1. In the course of our study, we discovered that ammonium 2-(2'-phosphoryloxyphenyl)-6-tributylstannyl-4-(3H)-quinazolinone, an intermediate in the radioiodination of the prodrug, exists as two isomers (3 and 4) whose radioiodination leads, respectively, to [(125)I]6 and [(125)I]5. These prodrugs have different in vitro and in vivo biologic activities. Compound 6 is not hydrolyzed by alkaline phosphatase (ALP), whereas 5 is highly soluble (mg/mL) in aqueous solution and is rapidly dephosphorylated in the presence of ALP to 1, a water-insoluble molecule (ng/mL). Mouse biodistribution studies indicate that [(125)I]6 has high uptake in kidney and liver and [(125)I]5 has very low uptake in all normal organs. Compounds 3 and 6 are converted, respectively, to 4 and 5 after incubation in DMSO. The stability of 5 in human serum is high. The minimum ALP concentration needed to hydrolyze 5 is much greater than the ALP level in the blood of patients with cancer, and the latter should not affect the pharmacokinetics of the compound. Incubation of 5 with viable human and mouse tumor-cell lines--but not with normal human cells and mouse tissues--leads to its hydrolysis and the formation of large crystals of 1. We expect that 5 will also be hydrolyzed in vivo by tumor cells that express phosphatase activity extracellularly and anticipate the specific precipitation of radioiodinated 1 within tumor cell clusters. This should lead to high tumor-to-normal-tissue ratios and enable imaging (SPECT/PET) and radionuclide therapy of solid tumors.

In silico design, synthesis, and biological evaluation of radioiodinated quinazolinone derivatives for alkaline phosphatase-mediated cancer diagnosis and therapy.

As part of the development of enzyme-mediated cancer imaging and therapy, a novel technology to entrap water-insoluble radioactive molecules within solid tumors, we show that a water-soluble, radioactive quinazolinone prodrug, ammonium 2-(2'-phosphoryloxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ(2-P)), is hydrolyzed by alkaline phosphatase to a water-insoluble, radiolabeled drug, 2-(2'-hydroxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ(2-OH)). Biodistribution data suggest the existence of two isoforms of the prodrug (IQ(2-P(I)) and IQ(2-P)), and this has been confirmed by their synthesis and characterization. Structural differences of the two isoforms have been examined using in silico molecular modeling techniques and docking methods to describe the interaction/binding between the isoforms and human placental alkaline phosphatase (PLAP), a tumor cell, membrane-associated, hydrolytic enzyme whose structure is known by X-ray crystallographic determination. Docking data show that IQ(2-P), but not IQ(2-P(I)), fits the active binding site of PLAP favorably and interacts with the catalytic amino acid Ser(92), which plays an important role in the hydrolytic process. The binding free energies (DeltaG(binding)) of the isoforms to PLAP predict that IQ(2-P) will be the better substrate for PLAP. The in vitro incubation of the isoforms with PLAP leads to the rapid hydrolysis of IQ(2-P) only and confirms the in silico expectations. Fluorescence microscopy shows that in vitro incubation of IQ(2-P) with mouse and human tumor cells causes the extracellular, alkaline phosphatase-mediated hydrolysis of the molecule and precipitation of fluorescent crystals of IQ(2-OH). No hydrolysis is seen in the presence of normal mouse and human cells. Furthermore, the intratumoral injection of 125IQ(2-P) into alkaline phosphatase-expressing solid human tumors grown s.c. in nude rats results in efficient hydrolysis of the compound and retention of approximately 70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) does not produce any measurable hydrolysis (approximately 1%) or retention of radioactivity at the injected site. These studies support the enzyme-mediated cancer imaging and therapy technology and show the potential of such quinazolinone derivatives in the in vivo radiodetection (123I/124I) and therapy (131I) of solid tumors.

Induction of apoptosis in human tumor cells after exposure to Auger electrons: comparison with gamma-ray exposure.

To clarify the contribution of apoptosis to cell death in four human solid tumor cell lines, clonogenic cell survival (indicator of radiosensitivity) and induction of caspase-3 (CASP-3)/caspase-3-like proteases (CASP-3LP) and the production of DNA fragmentation (markers for apoptosis) were studied in RKO, LS174T, MCF7 and TE671 cells exposed to DNA-incorporated Auger-electron-emitting (125)I (5-[(125)I]iodo-2'-deoxyuridine) or gamma-radiation. Clonogenic survival was assessed by colony-forming assay, CASP-3/CASP-3LP induction with a fluorogenic substrate and DNA fragmentation by ligation-mediated polymerase chain reaction. For (125)I, log dose-survival curves had no shoulder [high-linear-energy-transfer (LET)-like] and decreased exponentially at different rates in various cell lines. Induction of CASP-3/CASP-3LP in radiosensitive RKO and LS174T cells was threefold greater than that in radioresistant TE671 and MCF7 cells. Nucleosomal laddering in (125)I-radiosensitive cell lines was dose-dependent, and no laddering was detected in radioresistant lines. For gamma-radiation, the survival curve for LS174T cells was monoexponential and that for the other lines exhibited a distinct shoulder (low-LET-like). The most radiosensitive cell line, LS174T, showed the highest induction of CASP-3/CASP-3LP, and the most radioresistant line, TE671, showed the lowest induction. Although DNA laddering was not detectable in TE671 cells, it was observed in other lines, being most prominent in LS174T cells. We conclude that apoptosis initiated by DNA-incorporated (125)I is dose-dependent, correlates with cell radiosensitivity and takes place through a CASP-3-mediated pathway, whereas that after gamma-irradiation probably occurs via a CASP-3-independent pathway and/or a CASP-3-mediated pathway and does not correlate with cell radiosensitivity.

Inhibitory and stimulatory bystander effects are differentially induced by Iodine-125 and Iodine-123.

The bystander effect, originating from cells irradiated in vitro, describes responses of surrounding cells not targeted by the radiation. Previously we demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated (125)I inhibits growth of co-injected LS174T cells (inhibitory bystander effect; Proc. Natl. Acad. Sci. USA 99, 13765-13770, 2002). We have repeated these studies using cells exposed to lethal doses of (123)I, an Auger electron emitter whose emission spectrum is identical to that of (125)I, and report herein that the decay of (123)I within tumor cell DNA stimulates the proliferation of neighboring unlabeled tumor cells growing subcutaneously in nude mice (stimulatory bystander effect). Similar inhibitory bystander effects ((125)I) and stimulatory bystander effects ((123)I) are obtained in vitro. Moreover, supernatants from cultures with (125)I-labeled cells are positive for tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), and those from cultures with (123)I-labeled cells are positive for angiogenin. These findings call for the re-evaluation of current dosimetric approaches for the estimation of dose-response relationships in individuals after radiopharmaceutical administration or radiocontamination and demonstrate a need to adjust all "calculated" dose estimates by a dose modification factor (DMF), a radionuclide-specific constant that factors in hitherto not-so-well recognized biophysical processes.

Determination of active components in Cynanchum chinense R. Br. by capillary electrophoresis.

A method for the determination of 7-O-alpha-l-rhamnopyranosyl-kaempferol-3-O-beta-d-glucopyranoside (GL) and 7-O-alpha-l-rhamnopyranosyl-kaempferol-3-O-alpha-l-rhamnopyranoside (RH) in the traditional Chinese herb Cynanchum chinense R. Br. by capillary electrophoresis has been developed. With botate buffer (30 mmol/L, pH 9.50) as running buffer and an applied voltage of 20 kV, the compounds were completely separated within 6 min and detected at UV 254 nm. The correlation coefficients of the calibration curves for GL and RH were 0.9990 and 0.9992, respectively, over the concentration ranges (15.0-1000.0 and 12.0-1000.0 microg/mL), and the recoveries were from 91.4 to 107.1%.

5-[123I/125I]iodo-2'-deoxyuridine in metastatic lung cancer: radiopharmaceutical formulation affects targeting.

UNLABELLED: This study assesses targeting of lung metastases in mice with the radioiodinated thymidine analog 5-[(123)I/(125)I]iodo-2'-deoxyuridine ((123)I-IUdR/(125)I-IUdR), formulated with varying amounts of tributyltin precursor and injected intravenously. METHODS: Six- to 8-wk-old C57BL/6 mice were injected intravenously with B16F10 melanoma cells. Two weeks later, when lung tumors were established, the animals were injected intravenously with (125)I-IUdR synthesized using 1, 35, 100, 150, 200, or 250 microg 5-tributylstannyl-2'-deoxyuridine (SnUdR) in the presence of an oxidant. Nontumor-bearing mice were also injected with these formulations and served as control animals. Twenty-four hours later, the animals were killed, and the radioactivity associated with the lungs and other tissues was measured in a gamma-counter. The percentage injected dose per gram tissue (%ID/g) and tumor-to-nontumor ratios (T/NT ratios) were calculated. Phosphor imaging was done on lungs from tumor-bearing and nontumor-bearing mice injected with (125)I-IUdR formulated with each tin precursor concentration. Scintigraphy was also performed 3 and 24 h after intravenous injection of (123)I-IUdR. RESULTS: The %ID/g (125)I-IUdR was higher in lungs of tumor-bearing animals than in lungs of control animals. Although the increase in SnUdR present led to a small but statistically significant decrease in the radioactive content of normal lungs, a 3-fold increase was observed in the lungs of tumor-bearing animals with radiopharmaceutical formulated with 100 microg SnUdR (5 microg per mouse). This enhancement in radioactive uptake by the lungs led to approximately 14-fold increases in T/NT ratios. Phosphor imaging ((125)I-IUdR) of lungs as well as scintigraphy ((123)I-IUdR) of whole animals substantiated these findings. CONCLUSION: The formulation for the synthesis of radio-IUdR that leads to the highest %ID/g in tumor and the best T/NT ratio has been identified. Further studies are required to determine the factors responsible for specific enhancement in IUdR tumor uptake.

Activation of diverse pathways to apoptosis by (125)IdUrd and gamma-photon exposure.

PURPOSE: To delineate the mechanisms underlying induction of apoptosis in malignant cells irradiated by DNA-incorporated iodine-125 or gamma-photons. MATERIALS AND METHODS: Human tumor cells (RKO, LS174T, TE671, and MCF7) were irradiated by DNA-incorporated 5-[125I]iodo-2'-deoxyuridine (125IdUrd) or by gamma-photons. Clonogenic survival was determined by the colony-forming assay. Caspase-3 induction was measured with a fluorogenic substrate assay, and DNA fragmentation was determined by ligation-mediated polymerase chain reaction. DNA arrays were used to assess the expression of the B-cell lymphoma/leukaemia-2 (Bcl-2) family and related genes in RKO cells and in caspase-3-gene-defective MCF7 cells. RESULTS: After 125IdUrd or y-photon exposure, the highest induction of caspase-3 was observed in the radiation-sensitive cell lines (RKO and LS174T). DNA fragmentation was prominent in the radiosensitive cells and undetectable in TE671 (125IdUrd and gamma-photons) and MCF7 (125IdUrd only) cells. Exposure of RKO and MCF7 cells to 125I decay led to up-regulation of several pro-apoptotic and antiapoptotic Bcl-2 family genes whereas y-irradiation produced minimal activation. CONCLUSIONS: Apoptosis generated by a DNA-incorporated Auger electron emitter is induced through the mitochondrial/caspase-3-mediated pathway and correlates with cellular radiosensitivity. Apoptosis caused by y-radiation can be signaled without activation of Bcl-2 family genes, and DNA fragmentation occurs with or without caspase-3 activation.

Therapeutic potential of 5-[125I]iodo-2'-deoxyuridine and methotrexate in the treatment of advanced neoplastic meningitis.

PURPOSE: To assess the therapeutic potential of methotrexate (MTX) and 5-[1251]liodo-2'-deoxyuridine (125IdUrd) administered sequentially in rats bearing advanced (ten-day-old) intrathecal (i.t.) TE671 human rhabdomyosarcoma tumours. MATERIALS AND METHODS: Nude rats were injected with TE671 cells through an i.t. placed catheter. Ten days later, the animals were injected i.t. over a 12-day period with (i) saline daily, (ii) MTX every other day, (iii) 125IdUrd every other day, or (iv) MTX and 125IdUrd on alternating days. Onset of paralysis was determined as a function of time, and the medians for onset (M), percentage of cells killed (% kill), and log cell kill were calculated. RESULTS: The data show that (i) injection of MTX leads to a moderate delay in the onset of paralysis (M(MTX) = 29 d versus Msaline = 20 d), (ii) administration of 125IdUrd is more effective (M(IdUrd) = 36 d), and (iii) sequential administration of MTX- 125IdUrd further increases the therapeutic efficacy of 125IdUrd (M(MTX)-IdUrd = 47 d). CONCLUSIONS: Intrathecal injection of MTX-(125)IdUrd is efficacious in the therapy of advanced intrathecal tumours.

Synthesis and biologic evaluation of a radioiodinated quinazolinone derivative for enzyme-mediated insolubilization therapy.

We have developed a new strategy that aims to concentrate therapeutic radionuclides within solid tumors. This approach, which we have named EMIT (enzyme-mediated insolubilization therapy), is a method for enzyme-dependent, site-specific, in vivo precipitation of a radioactive molecule (from a water-soluble precursor) within the extracellular space of solid tumors. The prodrug, ammonium 2-(2'-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone, labeled with iodine-125 ((125)IPD) and its authentic compound labeled with iodine-127 (IPD) have been synthesized, purified, and characterized. The alkaline phosphatase (ALP)-mediated conversion of these water-soluble nonfluorescent prodrugs to the water-insoluble fluorescent species, iodine-125-labeled 2-(2'-hydroxyphenyl)-6-iodo-4-(3H)-quinazolinone ((125)ID) and its iodine-127-labeled derivative (ID), has been demonstrated in vitro. Biodistribution studies in mice indicate that both (125)IPD and (125)ID are minimally retained by most tissues and organs. In addition, following its intravenous injection in mice, (125)IPD is localized in ALP-rich regions and converted to (125)ID, which remains indefinitely within the tissues where it is produced. We believe that EMIT is a strategy that will lead to the active and specific concentration and entrapment of therapeutic radionuclides within solid tumors, the consequent protracted irradiation of tumor cells within the range of the emitted particles, and the effective therapy of solid tumors.