As part of the development of enzyme-mediated cancer imaging and therapy, a novel technology to entrap waterinsoluble radioactive molecules within solid tumors, we show that a water-soluble, radioactive quinazolinone prodrug, ammonium 2-(2 ¶-phosphoryloxyphenyl)-6-[ 125 I]iodo-4-(3H)-quinazolinone ( 125 IQ 2-P ), is hydrolyzed by alkaline phosphatase to a water-insoluble, radiolabeled drug, 2-(2 ¶-hydroxyphenyl)-6-[ 125 I]iodo-4-(3H)-quinazolinone ( 125 IQ 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 (#G 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 phosphatasemediated 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 125 IQ 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 f70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) does not produce any measurable hydrolysis (f1%) 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 ( 123 I/ 124 I) and therapy ( 131 I) of solid tumors.

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.

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.

Purpose To determine the possible effects of 125I-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 125I-labeled Hoechst (H) derivatives (125IE–H, 125IB-H, 125I-C8-H and 125I-C12-H). While all four molecules share a common DNA minor groove binding bis-benzimidazole motif, they are designed to position 125I at varying distances from the DNA helix. Each Hoechst derivative was incubated at 4 °C in phosphate buffered saline (PBS) together with supercoiled (SC) 3H-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 125I 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 125I atom is distanced – by 10.5 Å to 13.9 Å – from the DNA helix (125IEH: 0.52 ± 0.01; 125IB– H: 0.24 ± 0.03; 125I-C8–H:0.18 ± 0.02; 125I-C12–H: 0.10 ± 0.00). In the presence of DMSO, DSB yields for 125IEH (0.49 ± 0.02) and 125IB-H (0.26 ± 0.04) remain largely unchanged indicating that DSB are entirely produced by direct effects. Strikingly, 125I-C8–H or 125I-C12 –H, did not produce detectable DSB in the presence of DMSO under similar conditions suggesting when 125I 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 125I 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 125I free in solution. These experimental findings were correlated with theoretical expectations based on microdosimetry.

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 A-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. [Cancer Res 2007;67(5):2197-205]

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.

Intrathecal injection of MTX-(125)IdUrd is efficacious in the therapy of advanced intrathecal tumours.

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 and 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 γ -radiation. Clonogenic survival was assessed by the colony-forming assay, caspase-3 induction with a fluorogenic substrate, and DNA fragmentation by ligation-mediated PCR. For 125 I, the log dosesurvival curves had no shoulder (high-LET-like) and decreased exponentially at different rates in various cell lines. Induction of caspase-3 in radiosensitive RKO and LS174T cells was 3-fold greater than 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 γ -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, and the most radioresistant line, TE671, the lowest. Although DNA laddering was not detectable in TE671 cells, it was observed in the other lines, being most prominent in LS174T cells. We conclude that apoptosis initiated by DNA-incorporated 125 I is dose-dependent, correlates with radiation sensitivity, and takes place through the CASP-3-mediated pathway, whereas that after γ -irradiation probably occurs via CASP-3-independent and/or CASP-3-mediated pathways and does not correlate with cell radiosensitivity.