Research Article
Computational Modeling and Experimental Evaluation of a Novel Prodrug for Targeting the Extracellular Space of Prostate Tumors 1
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Pavel Pospisil, Ketai Wang, Ayman F. Al Aowad, Lakshmanan K. Iyer, 1 1 S. James Adelstein, and Amin I. Kassis
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1 Department of Radiology, Harvard Medical School, Boston, Massachusetts and 2Bauer Center for Genomics Research, Harvard University, Cambridge, Massachusetts
Abstract 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 (IQ2-P) was docked in silico into the X-ray structure of PAP. The data indicate that IQ2-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 125IQ2-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-[125I]iodo-4-(3H)-quinazolinone (125IQ2-OH). Similarly, the incubation of IQ2-P with human LNCaP, PC-3, and 22Rv1 prostate tumor cells resulted in the formation of large fluorescent IQ2-OH crystals. No hydrolysis was seen in the presence of normal human cells. Autoradiography of tumor cells incubated with 125IQ2-P showed accumulation of radioactive grains (125IQ2-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]
Introduction Prostate cancer is the most frequently diagnosed malignancy in men (33%) and the second leading cause of cancer death in the United States (10%).3 The American Cancer Society estimates that 234,300 new cases of invasive prostate cancer will be identified in the United States this year. A substantial proportion of these people will develop metastatic disease at some point, and f30,000 will die. Routine diagnosis is based on prostate-specific antigen
Requests for reprints: Amin I. Kassis, Department of Radiology, Harvard Medical School, Armenise Building, Room D2-137, 200 Longwood Avenue, Boston, MA 02115. Phone: 617-432-7777; Fax: 617-432-2419; E-mail:
[email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3309
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determination in blood or discovery of a palpable mass within the prostate. Despite current therapies, the outcome is often lymph node spread, bone metastases, and death. The development of technologies that enable noninvasive determination of this disease and therapeutic intervention at an early stage is clearly to be desired. Such technologies would be designed to detect prostate cancer, move meaningful intervention to an earlier point in its progression, prevent the development of metastatic disease, and minimize patient inconvenience and incapacitation. Toward these objectives, we present a novel approach, Enzyme-Mediated Cancer Imaging and Therapy (EMCIT; refs. 1–5), which potentially enables the active entrapment of a radioisotopically labeled compound within the extracellular spaces of primary prostate tumors and their metastases (Fig. 1). This noninvasive technique is based on the rapid uptake of radioactive molecules and their enzyme-dependent, site-specific, in vivo precipitation within solid tumors (in contrast to minimal uptake in normal tissues). The compound can be labeled with an isotope having decay characteristics suitable for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging (e.g., 123I and 124I) or for therapy (e.g., 131I). The prototype for this approach was first developed for alkaline phosphatase (ALP; EC 3.1.3.1), a hydrolase with monophosphoesteric activity that is overexpressed on the plasma membranes of many tumor cell types (1, 2, 6, 7). A suitable substrate, the prodrug ammonium 2-(2¶-phosphoryloxyphenyl)-6-iodo-4-(3H)quinazolinone (127IQ2-P), and its radioiodinated analogue (125IQ2-P) were synthesized, purified, and characterized (1, 5), and their ALP-mediated hydrolysis to the water-insoluble, fluorescent 2-(2¶hydroxyphenyl)-6-iodo-4-(3H)-quinazolinone derivatives (127IQ2-OH and 125IQ2-OH) was shown (1, 3, 5). These studies also indicated that (a) IQ2-P is a highly water-soluble molecule (mg/mL) that is stable in human serum and readily dephosphorylated by ALP to the water-insoluble IQ2-OH derivative; (b) the in vitro incubation of 127 IQ2-P/125IQ2-P derivatives with several ALP-expressing human and mouse tumor cell lines results in the efficient and rapid formation of the corresponding water-insoluble derivatives 127IQ2-OH/ 125 IQ2-OH; and (c) the intratumoral injection of 125IQ2-P into ALP-expressing solid human tumors grown in rats leads to the efficient hydrolysis of the compound and the retention of f70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) leads to little measurable hydrolysis (f1%) and lack of retention of radioactivity at injected sites. In addition, we observed that the pharmacokinetic properties of IQ2-P in mice were not consistent (5). Subsequently, we recognized that the
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http://www.cancer.org
Cancer Res 2007; 67: (5). March 1, 2007
Cancer Research
synthesis of its stannylated quinazolinone precursor (SnQ2-P) produces a mixture of two compounds, SnQ2-P and SnQ2-P(I) (f1:1 ratio), whose radioiodination leads to the formation of 125 IQ2-P and its cyclic isoform 125IQ2-P(I), respectively. Both iodinated molecules have been docked onto ALP, and the data indicate that the calculated binding energies for 125IQ2-P are more favorable than for its cyclic isoform (3). Furthermore, it has been shown that pure 125 IQ2-P can be prepared following an overnight incubation of the tin precursor mixture in DMSO and that, on its i.v. injection into mice, there is minimal retention (9 (49), it follows that the rate of hydrolysis for phosphorylated substrates within the interstitial fluid is likely to be much higher in PAP-expressing tumors. In addition, the relative ALP-IQ2-P and PAP-IQ2-P binding energies and predicted K i and IC50 values are lower for PAP (see table within Fig. 2), supporting the view that prostate cancer (i.e., PAP-expressing tumors) may be a better candidate than ALP-expressing tumors for the EMCIT approach. In conclusion, using advanced computational data-mining and modeling methods, we have identified PAP as a suitable target for the EMCIT technology being developed in our laboratories. The water-soluble prodrug IQ2-P has been docked in silico into the crystal structure of PAP. The iodinated PAP substrate (125IQ2-P and 127IQ2-P) has been synthesized. The incubation of 125IQ2-P with PAP in solution leads to the formation of its dephosphorylated
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analogue 125IQ2-OH. Similarly, the in vitro incubation of IQ2-P with several human prostate tumor cell lines (but not normal cells) results in the hydrolysis of this water-soluble, nonfluorescent prodrug and the formation of water-insoluble, fluorescent IQ2-OH crystals, many of which are attached to these prostate cancer cells. It is our hope that these quinazolinone-based radiopharmaceuticals will eventually be developed into a novel, noninvasive method for imaging (123I-SPECT and 124I-PET) and treating (131I) prostate tumors and their metastases. In addition, the proposed EMCIT approach may (a) function as a prognostic marker for the noninvasive sensing of precancerous, cancerous, and metastatic signatures of prostate cancers in individual patients; (b) move meaningful intervention to a much earlier point in cancer progression; (c) provide a technique for evaluating the early response of individual tumors to therapy, thus facilitating selection of effective treatment by allowing rapid identification of ineffective treatments whose side effects might not be balanced by expected benefits; and (d) allow detection, diagnosis, staging, and treatment to be closely coupled.
Acknowledgments Received 9/7/2006; revised 11/1/2006; accepted 12/15/2006. Grant support: U.S. Department of Defense grant W81XWH-06-1-0043 (A.I. Kassis). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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