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 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.

Enzyme-mediated cancer imaging and therapy (EMCIT) is a novel approach in which radioactive water-soluble molecules are precipitated in vivo following their hydrolysis by extracellular enzymes overexpressed by cancer cells. AutoDock 3.0 was used to model the interaction-binding between a series of iodinated quinazolinone derivatives and human placental alkaline phosphatase (PLAP, crystal structure in the Protein Data Bank) and to assess the effects of structural modification of the derivatives. Ammonium 2-(2',4'-diphosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ2-P,4-P), having the most favorable calculated inhibition constant, was synthesized and characterized. Concentration-dependent, PLAP-mediated conversion of IQ2-P,4-P (4)/125IQ2-P,4-P (6) to water-insoluble 2-(2',4'-dihydroxyphenyl)-6-[127I/125I]iodo-4-(3H)-quinazolinone (127IQ2-OH,4-OH (2)/125IQ2-OH,4-OH (7)) was observed in solution. Autoradiography indicated that 6 is hydrolyzed by human cancer cells and the resulting 7 precipitates on exterior cell surfaces. Biodistribution studies in mice demonstrated that 6 is minimally retained by normal tissues. The findings support the validity of the EMCIT approach.