Targeting Sigma Receptor-Binding Benzamides As in Vivo Diagnostic and Therapeutic Agents for Human Prostate Tumors1

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[CANCER RESEARCH 59, 4578–4583, September 15, 1999] Targeting Sigma Receptor-binding Benzamides as in Vivo Diagnostic and Therapeutic Agents for Human Prostate Tumors1

Christy S. John,2 Bertold J. Vilner, Brian C. Geyer, Terry Moody, and Wayne D. Bowen Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, Washington, DC 20037 [C. S. J., B. C. G.]; Unit on Receptor Biochemistry and Pharmacology, Laboratory of Medicinal Chemistry, National Institutes of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892 [B. J. V., W. D. B.]; and Cell and Cancer Biology Department, Medicine Branch, National Cancer Institute, Rockville, Maryland 20850 [T. M.]

ABSTRACT currently limited by routine diagnostic modalities, such as magnetic resonance imaging, computed tomography, and ultrasound. The skel- Sigma receptors are known to be expressed in a variety of human etal metastases are routinely diagnosed with radionuclide skeletal tumor cells, including breast, neural, and melanoma tumors. A very high imaging. However, the soft tissue metastases and involvement of density (1.0–1.5 million receptors/cell) of sigma receptors was also reported in a human androgen-dependent prostate tumor cell line (LNCaP). pelvic lymph nodes cannot be accurately assessed with current tech- In this study, we show that a very high density of sigma receptors is also niques. Therefore, there is a need for a reliable noninvasive diagnostic expressed in an androgen-independent human prostate tumor cell line procedure to determine the lymphatic and soft tissue spread of pros- (DU-145). Pharmacological binding studies using the sigma-1-selective tate neoplasm. Accurate, early detection of prostate tumor and its ؍ ؉ 3 ligand [ H]( )-pentazocine showed a high-affinity binding (Kd 5.80 nM, metastases would improve patient management and outcome of ther- 111 3 ؍ Bmax 1800 fmol/mg protein). Similarly, binding studies with [ H]1,3-di- apy. Recently, the In-labeled murine antibody 7E11-C5.3-GYK o-tolylguanidine in the presence of dextrallorphan also showed a high- (conjugated to diethylenetriaminepentacetic acid linker chelator), -fmol/mg protein). Radioio 1930 ؍ nM, B 15.71 ؍ affinity binding (K d max which binds to prostate-specific antigen, was clinically studied and -dinated benzamide N-[2-(1؅-piperidinyl)ethyl]-3-[125I]iodo-4-methoxy approved by the Food and Drug Administration under the commercial benzamide ([125I]PIMBA) was also shown to bind DU-145 cells in a dose-dependent manner. Three different radioiodinated benzamides, name “ProstaScint” for clinical staging and management of patients -125I]PIMBA, 4-[125I]iodo-N-[2-(1؅-piperidinyl)ethyl]benzamide, and with prostate tumors (2–4). However, this product has several limi] 2-[125I]-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide, were screened for tations, such as slow clearance of antibody from the plasma, high their potential to image human prostate tumors in nude mice bearing uptake in liver and intestine, production of human antimouse antibod- human prostate cells (DU-145) xenografts. All three compounds showed a ies in some patients, the need for repeat patient imaging up to 5 days fast clearance from the blood pool and a high uptake and retention in the postinjection, and the high cost of the drug. Radiolabeled small tumor. Therapeutic potential of nonradioactive PIMBA was studied using molecule(s) peptide or nonpeptide certainly would be preferred be- in vitro colonogenic assays. A dose-dependent inhibition of cell colony cause of their rapid clearance from the blood pool and normal organs. formation was found in two different human prostate cells. These results Sigma receptors are nonopiate, nondopaminergic, membrane- demonstrate the potential use of sigma receptor binding ligands in non- bound proteins that possess high affinity for haloperidol and various invasive diagnostic imaging of prostate cancer and its treatment. other neuroleptics (5). It is known that there are at least two sigma receptors subtypes, termed sigma-1 and sigma-2 (6). Sigma-1 sites INTRODUCTION can be selectively labeled by [3H](ϩ)-pentazocine, whereas DTG3 is Prostate cancer is the male malignancy with the highest incidence a non-subtype-selective ligand for labeling both sigma-1 and sigma-2 3 rate in the Western hemisphere. It is the second leading cause of death sites (7, 8). Sigma-2 sites can be labeled with the use of [ H]DTG in in men in the United States, with the number of new incidences this the presence of dextrallorphan, which masks the labeling of sigma-1 year expected to be ϳ184,500. The total number of expected deaths sites (8). Sigma receptors are present not only in central nervous from prostate cancer this year is ϳ39,200. The current methods of system but also in other tissues, such as the liver, kidneys, lungs, diagnosis include digital rectal examination and prostate-specific an- gonads, and ovaries (9, 10). The endogenous sigma-ligand(s) are not tigen blood test. The effective treatment of prostate cancer requires known; however, progesterone has been suggested to be a candidate early detection and accurate staging of the disease. Generally, at the (11). The pharmacological significance of sigma receptor-binding clinical presentation, the majority of patients have disease that has sites remain elusive due to lack of functional and structural informa- extended beyond the prostate, i.e., local or distant metastases. Many tion. However, recently, the sigma-l binding site, a Mr 30,000 protein early-stage prostate tumors require androgen for survival, and thera- from guinea pig liver, was purified and cloned. The amino acid pies that are designed to interfere with steroid metabolism have been sequence of this protein showed no homology to any known mam- effective. Most prostate cancer patients, however, show relapse of malian proteins, but a partial homological resemblance with a fungal disease and acquire a more aggressive androgen-independent meta- protein involved in sterol synthesis was observed (12). static disease (1). There is no currently available curative therapy; On the basis of in vitro pharmacological binding studies with therefore, the development of new diagnostic imaging agents for tritiated ligands, we recently discovered that sigma receptors are accurate staging and new cytotoxic therapies is of utmost importance. expressed in high densities (1.0–1.5 million receptors/cell) on andro- The diagnosis of soft tissue primary or metastatic prostate carci- gen-dependent (LNCaP) human prostate tumor cells (13). Therefore, noma or even its residual/recurrent lesions after radical prostectomy is we reasoned that the compounds binding sigma receptors with a modest to high affinity could be potentially used for in vivo labeling Received 1/29/99; accepted 7/16/99. of such receptors, thereby enabling noninvasive imaging of human The costs of publication of this article were defrayed in part by the payment of page prostate tumor sites. In addition, sigma sites could be an attractive charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Cancer Institute Grant CA 58496 and by 3 The abbreviations used are: DTG, 1,3-di-o-tolylguanidine; PIMBA, N-[2-(1Ј-piper- Research Corporation Technologies (Tucson, AZ). idinyl)ethyl]-3-iodo-4-methoxybenzamide; HPLC, high-performance liquid chromatogra- 2 To whom requests for reprints should be addressed, at 2300 I Street NW, 530 Ross phy; 2-[125I]BP, 2-[125I]-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide; 4-[125I]PAB, Hall, Department of Biochemistry and Molecular Biology, The George Washington 4-[125I]iodo-N-[2-(1Ј-piperidinyl)ethyl]benzamide; %ID, percentage injected dose; 2-IBP, University Medical Center, Washington, DC 20037. Phone: (202) 994-5031; Fax: (202) N-(N-benzylpiperidin-4-yl)-2-iodobenzamide; 4-IPAB, N-[2-(1Ј-piperidinyl)ethyl]4-iodo- 994-8974; E-mail: [email protected]. benzamide. 4578

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1999 American Association for Cancer Research. SIGMA RECEPTOR-BINDING BENZAMIDES FOR PROSTATE CANCER target not only for diagnostic imaging but also for therapeutic inter- residue was dissolved in methanol (400 ␮l) and injected into HPLC fitted with vention. Sigma receptors are also expressed in a variety of other a reverse-phase C18 column and eluted with methanol-Tris buffer (10 mM,pH human tumors, such as malignant melanoma (13, 14), non-small cell 5.5; 80:20, v/v). The retention time at a flow rate of 1.0 ml/min was ϳ9 min. lung carcinoma (15), breast (16, 17), and tumors of neural origin (18). The fractions containing the desired compound were pooled together and Pharmacological binding studies with membrane preparations from cospotted on TLC along with authentic nonradioactive and developed in CHCl :methanol (90:10). The R of nonradioactive and [125I]PIMBA was biopsied tumors tissue and the corresponding normal tissues have 3 f found to be 0.85 in the above solvent system. The chemical synthesis and indicated that sigma receptors are overexpressed with respect to structures of all three benzamides are shown in Fig. 1. normal tissue (19, 20). Sigma receptor-binding ligands have also shown the inhibition of Cell Culture proliferation in mammary adenocarcinoma (MCF-7 and MDA-MB- 231), colon carcinoma cells (LIM l2l5 and WIDr), melanoma cells, Human prostate tumor cells DU-145, PC-3 and LNCaP were purchased and neural tumor cells in culture (21, 22). These results have impli- from American Type Culture Collection (Manassas, VA) and cultured in serum cated that sigma binding sites may play an important role in cell supplemented DMEM containing 10% heat-inactivated fetal bovine serum growth, differentiation, and cell proliferation as well. Here, we eval- (Life Technologies, Inc.) at 37°C. The cells were adherent and split 1:5 ratio uated the noninvasive imaging potential of three radioiodinated ben- using trypsin-EDTA (Life Technologies, Inc.). zamides in a nude mouse model hosting prostate tumor xenografts. The therapeutic potential of one of the sigma-binding ligands was also Pharmacology evaluated in three different prostate cell colonogenic assays. In Vitro Sigma-1 Binding Assay. Guinea pig brain membranes (300–500 ␮g of protein) were incubated with 3 nM [3H](ϩ)-pentazocine (51.7 Ci/mmol) in 0.5 ml of 50 mM Tris-HCl (pH 8.0) for 120 min at 25°C. PIMBA was added MATERIALS AND METHODS Ϫ Ϫ in concentrations ranging from 10 4 to 10 12 M. Assays were terminated by Reagents for organic syntheses were purchased from Aldrich Chemical Co. the addition of 5 ml ice-cold 10 mM Tris-HCl (pH 8.0) and filtered through (Milwaukee, WI) and used without further purification. HPLC-grade solvents glass fiber filters using a Brandel cell harvester (Gaithersburg, MD). Filters were purchased from Fisher Scientific Company and used without further were then washed twice with 5 ml of ice-cold 10 mM Tris-HCl (pH 8.0). The distillation. Proton NMR spectra were recorded on a Bruker 300 AM spec- nonspecific binding was determined in the presence of 10 ␮M haloperidol. The trometer. NMR peak patterns were described by the following designations: s, filters were soaked in 0.5% polyethyleneimine for at least 30 min at 25°C prior singlet; b, broad; d, doublet; t, triplet; q, quartet; m, multiplet; and arom, to use. Scintillation counting was carried out in Ecoscint (National Diagnos- aromatic protons. Chemical shifts were expressed as ppm using CDCl3 as tics, Manville, NJ) after an overnight extraction of counts. Protein concentra- internal standard. TLC was performed on Analtech uniplate silica gel GF plates tion was determined by the Lowry method. ␮ ϫ ␮ (250 m, 10 20 cm) and developed with CHCl3:methanol (90:10). Radio- In Vitro Sigma-2 Binding Assay. Rat liver homogenates (150–200 gof TLC was performed using a radiochromatogram scanner (Packard model protein) were incubated with 3 nM [3H]DTG (39.4 Ci/mmol) in the presence of 7220/21). Na125I was obtained from Amersham (Arlington Heights, IL). Ra- 1 ␮M dextrallorphan (to mask sigma-1 sites). The procedure was same as dioactivity was measured using a Capintec radioisotope calibrator CRC-4. above. IC50s were determined using the computerized iterative curve-fitting 3 [ H]DTG (35.4 Ci/mmol) was bought from DuPont-NEN (Boston, MA). program GraphPad InPlot4 (GraphPad Software, San Diego, CA). Ki was 3 [ H](ϩ)-pentazocine (51.7 Ci/mmol) was synthesized as described previously calculated from IC50s using Cheng-Prusoff equation. (8). Dextrallorphan was provided by F. I. Carroll (Research Triangle Institute, Scatchard Analysis of Binding of Tritiated Sigma Ligands in Human Research Triangle Park, NC). Haloperidol, Tris-HCl, and polyethyleneimine Prostate Tumor (DU-145) Cell Membranes. Crude membranes from DU- were purchased from Sigma Chemical Co. (St. Louis, MO). 145 cells were prepared as follows: homogenization of cells (Potter-Elvehjem homogenizer with Teflon pestle) was carried out in ice-cold 10 mM Tris-HCl Chemical Synthesis (pH 7.4). The homogenate was centrifuged at 31,000 ϫ g for 15 min at 4°C, and the pellet was resuspended in ice-cold 10 mM Tris-HCl (pH 7.4) to a Preparation of PIMBA (1). PIMBA was prepared as described previously protein concentration of 15–20 mg/ml, as determined by method of Lowry (23). using BSA as standard. Binding assay with [3H]DTG and [3H](ϩ)-pentazocine -Synthesis of N-[2-(1؅-Piperidinyl)ethyl]-4-methoxy-3-trimethyl stannyl were carried out as described previously, using 200–250 ␮g of membrane benzamide (2). A mixture of PIMBA (1; 500 mg, 1.3 mmol), tetrakis(triph- protein per tube in a final volume of 250 ␮l. Scatchard plots of both radioli- enylphosphine)-palladium(0) (150 mg, 0.13 mmol, 10% molar equivalent), gands were carried out using a combination of radiolabeled and unlabeled bis(trimethylstannyl) (510 mg, 1.5 mmol), triethylamine (50 ml), and THF (50 ligand to achieve a concentration range of 1–400 nM for [3H]DTG and ml) was heated at reflux overnight. The reaction mixture was evaporated to 0.1–1000 nM for [3H](ϩ)-pentazocine. dryness in vacuum. The residue was dissolved in ethylacetate and eluted on a Competition Binding Studies of [125I]PIMBA in Human Prostate Can- silica gel column loaded with hexanes. The column was first eluted with cer Cells (DU-145). The affinity of compounds tested for sites labeled by hexanes (50 ml) and then EtOAc:NEt (9:1) to give a light yellow oil. 1H NMR 3 radiolabeled [125I]PIMBA was determined by cell binding assays. The in vitro (CDCl ) yielded: 0.266 (s, 9H, Me ); 1.42–1.47 (m, 2H, CH ); 1.56–1.59 (m, 3 3 2 binding assays were carried out with DU-145 prostate tumor whole cells as 4H, CH ); 2.42–2.55 (m, 6H, CH ); 3.46–3.49 (s, 2H, NCH ); 3.81 (s, [3H], 2 2 2 follows. The medium from T75 tissue culture flasks were decanted, and cells OMe); and 6.80–7.79 (m, 4 H, NH and arom). were scraped using a cell scraper and suspended in RPMI 1640 (1 ϫ mod) with Radiochemical Synthesis of [125I]PIMBA (3) glutamine medium (Cellgro, Herndon, VA). The cells suspension was centrifuged for 5–6 min on a Sorvall RT6000B refrigerated centifuge at 2000 rpm [125I]PIMBA was prepared using the following method. An ethanolic so- for 5 min. The medium was decanted and the cells were suspended again in lution of N-[2-(1Ј-piperidinyl)ethyl]-4-methoxy-3-trimethyl stannylbenzamide RPMI 1640 (1 ϫ mod) with glutamine medium without serum. An aliquot of (2; 1 mg/ml) was prepared. To 50 ␮l of this solution was added Na125I (0.5–1.0 the cell (50,000–100,000 cells) suspension was incubated with the radiophar- mCi, 3–5 ml) in 0.1 N NaOH, followed by the addition of 0.05 N HCl (100 ␮l) maceutical (0.05 nM) and the increasing concentrations (1.0 nM to 100 ␮M)of to adjust to pH 4.0–5.5. A freshly prepared solution (100 ␮l) of chloramine-T competing sigma ligands. The total volume was kept constant at 1.0 ml with (1 mg/ml) was added to the above mixture, and the solution was incubated at the medium, and the suspension was incubated at 37°C for 1 h. At the end of room temperature for 15 min. After this time, 200 ␮l of sodium metabisulfite this period, the cell suspension was filtered through a Brandel cell harvester (3 mg/ml) were added, and the solution was incubated for an additional 5 min. (Brandel, Gaithersburg, MD) and washed twice. The activity associated with Finally, a saturated solution of sodium bicarbonate (500 ␮l) was added to the cells on filters was counted using PackardGamma counter. The data were reaction vial, and the radioactivity was extracted with chloroform (2 ϫ 1 ml). analyzed by GraphPad InPlot 4 program (GraphPad Software) using nonlinear The organic layer was separated and evaporated with a stream of argon. The regress analysis. 4579

The base layer consisted of 3 ml of 0.5% agarose in SIT medium containing 5% fetal bovine serum in six-well plates. The top layer consisted of 3 ml of SIT medium in 0.3% agarose, benzamides, and 3 ϫ 104 single viable cells. For each cell line and benzamide concentration, triplicate wells were plated. After 2 weeks, 1 ml of 0.1% p-iodonitrotetrazolium violet was added, and after 16 h at 37°C, the plates were screened for colony formation; the number of colonies that were Ͼ100 ␮m in diameter were counted using an Omnicon image analysis system.

RESULTS AND DISCUSSION Our previous in vitro pharmacological binding studies with tritiated ligands showed that sigma receptors are expressed in high densities (1.0–1.5 million receptors/cell) on androgen-dependent (LNCaP) human prostate tumor cells (13). Here, we investigated the expression of tritiated sigma-binding ligand in another androgen-independent human prostate tumor (DU-145) cell line. Pharmacological binding studies using the sigma-1-selective ligand [3H](ϩ)-pentazocine showed a high-affinity binding in human prostate tumor cells (DU- ϭ ϭ 145, an androgen-independent cell line; Kd 5.80 nM, Bmax 1800 fmol/mg protein). Similarly, binding studies with [3H]DTG in the presence of dextrallorphan also showed a high-affinity binding ϭ ϭ (Kd 15.71 nM, Bmax 1930 fmol/mg protein). A representative example of Scatchard analysis is shown in Fig. 2. 2-IBP and radioiodinated 2-[125I]BP were synthesized using a procedure described in the literature (24). Similarly, 4-IPAB and radioiodinated 4-[125I]PAB 125 Fig. 1. Radiochemical synthesis of [ I]PIMBA and chemical structures of were synthesized as described previously (14). The synthesis of non- 4-[125I]PAB and 2-[125I]BP. radioactive PIMBA involved the condensation of 4-methoxybenzoyl chloride with 2(1-piperidine)ethylamine in the presence of triethyl- Biodistribution Studies in Nude Mice Bearing Human Prostate Tumor amine. The resulting benzamide was iodinated using electrophilic (DU-145) Xenograft thallation (23). Pharmacological characterization of 2-IBP and BALB/c nude mice (18–25 g) rats were injected with a suspension of 4-IPAB have been reported previously (14, 16) Receptor binding ϳ5.0–9.0 million DU-145 cells in 0.2–0.3 ml of saline in the right flank of affinity for PIMBA was carried out for sigma-1 receptor sites in animals. The animals were housed in a pathogen-free temperature-controlled guinea pig brain membranes using [3H](ϩ)-pentazocine. Its affinity isolation room, and the diet consisted of autoclaved rodent chow and auto- for sigma-2 sites was determined in rat liver membranes using claved water given ad libitum. After ϳ3 weeks, palpable tumors were observed [3H]DTG in the presence of dextrallorphan. PIMBA exhibited a high in ϳ90% of injected animals. The animals were anesthetized with ketamine/ ϭ affinity for sigma-1 sites (Ki 11.8 nM) and a modest affinity for xylazine and injected i.v. with [125I]PIMBA or 2-[125I]BP or 4-[125I]PAB sigma-2 sites (K ϭ 206 nM; Table 4). Similarly, 4-IPAB and 2-IBP (7–12 ␮Ci) in 0.3 ml of saline containing 10% ethanol solution. At 1, 6, and i also displayed a high affinity for sigma-1 receptor subtype, showing 24 h postinjection, blood samples were drawn by cardiac puncture, and the nude mice were sacrificed thereafter by cardiectomy while under ketamine/ Kis of 2.57 and 1.64 nM, respectively. 2-IBP was also found to have ϭ xylazine anesthesia. The organs of interest and the tumors were then excised, a high affinity for sigma-2 sites (Ki 29.6 nM) whereas 4-IPAB had blotted with tissue paper, and weighed, and the radioactivity was counted. The a modest affinity for sigma-2 subtypes (205 nM). Thus, all three %ID per organ was determined by comparison of the tissue radioactivity with radioligands evaluated for their potential to image prostate cancer in suitably diluted, known quantity aliquots of the injected dose. The results of this study had a high affinity for sigma-1 receptor subtype (1–15 nM). biodistribution studies are summarized in Tables 1–3. 2-IBP had a high affinity for sigma-2 sites as well. To synthesize radioiodinated [125I]PIMBA with a high specific In Vitro Growth Assays activity, we obtained a trimethylstannyl precursor for radioiodinations Growth assays in vitro were conducted using three different human prostate in high yields by a palladium catalyzed deiodostannylation reaction of cancer cell lines (DU-145, LNCaP, and PC-3) and the agarose cloning system. PIMBA with bis(trimethyl stannane). The radioiodinated compound

Table 1 Tissue distribution of 4-[125I]PAB in BALB/c nude mice hosting human prostate (DU-145) tumor xenografta Organ 1 h 6 h 12 h 24 h Blood 0.96 Ϯ 0.21 0.12 Ϯ 0.01 0.08 Ϯ 0.01 0.03 Ϯ 0.01 Heart 2.05 Ϯ 0.26 0.10 Ϯ 0.01 0.04 Ϯ 0.00 0.02 Ϯ 0.01 Liver 8.95 Ϯ 2.15 0.80 Ϯ 0.21 0.34 Ϯ 0.10 0.18 Ϯ 0.02 Lung 4.02 Ϯ 1.01 0.29 Ϯ 0.08 0.12 Ϯ 0.02 0.06 Ϯ 0.02 Kidney 1.91 Ϯ 0.36 0.41 Ϯ 0.06 0.07 Ϯ 0.01 0.06 Ϯ 0.01 Spleen 0.35 Ϯ 0.12 0.21 Ϯ 0.03 0.01 Ϯ 0.00 0.01 Ϯ 0.00 Stomach 1.48 Ϯ 0.32 0.36 Ϯ 0.10 0.07 Ϯ 0.00 0.07 Ϯ 0.02 Small intestine 13.25 Ϯ 3.61 0.62 Ϯ 0.12 0.12 Ϯ 0.01 0.06 Ϯ 0.01 Large intestine 1.94 Ϯ 0.35 4.39 Ϯ 0.44 0.18 Ϯ 0.02 0.15 Ϯ 0.04 Brain 1.90 Ϯ 0.14 0.09 Ϯ 0.02 0.01 Ϯ 0.00 0.00 Muscle 0.55 Ϯ 0.15 0.03 Ϯ 0.01 0.00 0.00 Tumor 2.23 Ϯ 0.45 0.83 Ϯ 0.18 0.50 Ϯ 0.11 0.28 Ϯ 0.09 Tumor/blood 3 14 Tumor/muscle 6 57 a Values are mean % ID/g Ϯ SD (n ϭ 4). 4580

Table 2 Tissue distribution of [125I]PIMBA, in BALB/c nude mice hosting human prostate tumor cells. This finding has significant implications for a prostate (DU-145) tumor xenograft diagnostic and therapeutic application of cancer for various sigma Organ 1 h 6 h 12 h receptor binding ligands. Similar findings were also reported for Blood 0.27 Ϯ 0.08 0.01 Ϯ 0.01 0.00 somatostatin receptors that are expressed on a variety of human Heart 0.41 Ϯ 0.10 0.01 Ϯ 0.00 0.00 Ϯ Ϯ tumors also (25). Multiple binding sites were reported for the binding Liver 1.12 0.22 0.29 0.09 0.10 123 Lung 0.74 Ϯ 0.18 0.06 Ϯ 0.01 0.00 of I-labeled octreotide against unlabeled peptides in primary car- Kidney 1.23 Ϯ 0.20 0.16 Ϯ 0.05 0.01 cinoid tumors. The expression of sigma-1 and sigma-2 sites was Spleen 1.10 Ϯ 0.11 0.08 Ϯ 0.02 0.00 3 ϩ Stomach 1.60 Ϯ 0.21 0.14 Ϯ 0.03 0.06 further confirmed by Scatchard analysis using [ H]( )-pentazocine, a Small intestine 3.10 Ϯ 0.87 0.19 Ϯ 0.04 0.05 highly selective sigma-1 ligand. Kd for sigma-1 site was found to be Ϯ Ϯ Large intestine 0.85 0.18 0.79 0.20 0.13 5.80 Ϯ 1.3 nM (r ϭ 0.966), and B was 1800 Ϯ 117 fmol/mg of Brain 0.15 Ϯ 0.05 0.01 Ϯ 0.00 0.00 max Muscle 0.21 Ϯ 0.04 0.02 Ϯ 0.00 0.00 protein. A representative Scatchard plot is shown in Fig. 1. Similarly, Ϯ Ϯ 3 Tumor 1.34 0.14 0.71 0.13 0.21 the dissociation constant (Kd) for sigma-2 binding site using [ H]DTG Tumor/blood 5 70 in the presence of dextrallorphan was found to be 15.71 Ϯ 3.6 nM.A Tumor/muscle 6 70 high density of sigma-2 sites was found (B ϭ 1930 Ϯ 176 fmol/mg a Values are mean % ID/g Ϯ SD (n ϭ 4). max protein). The total amount of sigma-1 and sigma-2 binding sites (ϳ3700 fmol/mg of protein) translates to ϳ1.8 million sigma recep- was prepared by electrophilic iododestannylation reaction in the pres- tors/cell. This number is one logarithmic scale higher than somatosta- ence of chloramine-T as the oxidizing agent in very high yields tin and VIP receptors expressed on human tumors (26). Therefore, (85–94%; Fig. 1). The specific activity of [125I]PIMBA ranged from sigma receptors are very attractive targets for diagnostic/therapeutic 1850 to 2030 (Ci/mmol), as determined by HPLC. Similarly, radio- oncological applications. iodinated 4-[125I]PAB and 2-[125I]BP were also prepared in high yield The potential for noninvasive imaging of prostate tumors and its and with high specific activities, as reported previously (14, 24). metastases was studied for three radioiodinated benzamides in We had earlier reported the binding characteristics of 2-[125I]BP in BALB/c nude mice hosting human prostate tumor cell line (DU-145). DU-145 prostate tumor cells and showed that the binding curve was All three ligands showed a fast clearance from the blood pool, curvilinear, indicating binding to the multiple sites (24). Here, we indicating a low plasma binding. (Tables 1–3). As expected, all three investigated the in vitro binding characteristics of [125I]PIMBA to radioiodinated benzamides showed hepatic and bowel uptakes. There DU-145 cells. A high degree of specific binding was observed in the were, however, remarkable differences in the liver uptake and reten- cells that were in proliferating stages of cell cycle (2–4 days tion of the three compounds. 2-[125I]BP and 4-[125I]PAB had a high postsplit). Four different classes of sigma ligands were used for uptake (ϳ8%ID/g) at 1 h postinjection, and [125I]PIMBA had a very competition studies, i.e., three benzamides (PIMBA, 4-IBP, and low liver uptake (1.12%ID/g). Similarly, lung and kidney uptake of 4-IPAB), one arylethylenediamine (BD1008), one butyrophenone radioactivity was also high for 2-[125I]BP and 4-[125I]PAB but rela- (haloperidol), and a benzenesulfonamide (N-[2-(1Ј-piperidinyl)- tively low for [125I]PIMBA. Stomach and bowel activity (total, ethyl]4-iodobenzenesulfonamide). The inhibition of specific binding 35%ID/g) was very high for 2-[125I]BP, presumably due to its higher of [125I]PIMBA was found to be dose dependent for all six sigma lipophilicity due to the presence of benzyl group. [125I]PIMBA receptor-binding agents studied. Kis for various competing ligands are showed much lower amounts of radioactivity in stomach and bowel listed in Table 5. The rank order of potency for various sigma receptor (total, 5% ID/g). This uptake may be attributed in part to the lipophi- binding ligands for competition at sites labeled by [125I]PIMBA was found to be: (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrro- lidinyl)ethylamine (BD1008) Ͼ PIMBA Ͼ haloperidol Ͼ 4-IPAB and Table 4 Receptor binding data for iodobenzamidesa Ј N- [2-(1 -piperidinyl)ethyl]4-iodobenzenesulfonamide. It is apparent Sigma-1 (Ki,nM) guinea 3 ϩ from the plots that the binding data fits a two-site model better than a pig brain, [ H]( )- Sigma-2 (Ki,nM) rat liver, Compound pentazocine [3H]DTG ϩ dextrallorphan one-site model. Fig. 3 shows representative examples of nonlinear PIMBA 11.82 Ϯ 0.68 206 Ϯ 11 regression fit for both one-site and two-site fits. This finding is not 4-IPAB 2.57 Ϯ 0.70 205 Ϯ 67 surprising in the light of our previous results in DU-145 cells and also 2-IBP 1.64 Ϯ 0.15 29.6 Ϯ 0.49 a in various breast cancer cells (T47D and MCF-7), whereby curvilinear Twelve concentrations of unlabeled test ligand ranging from 0.05 to 10,000 nM were plots were obtained when various radioiodinated benzamides were incubated with guinea pig brain membranes and [3H](ϩ)-pentazocine (sigma-1 receptors) or with rat liver membranes and [3H]DTG in the presence of 1 ␮M dextrallorphan competed against sigma ligands. These results indicated that radioio- (sigma-2 receptors) as described in “Materials and Methods.” Values are the averages of dinated ligand [125I]PIMBA binds to multiple sites present on human two to three experiments Ϯ SE. Each experiment was carried out in duplicate.

Table 3 Tissue distribution of 2-[125I]BP, in BALB/c nude mice hosting human prostate (DU-145) tumor xenografta Organ 1 h 6 h 12 h 24 h Blood 0.52 Ϯ 0.19 0.12 Ϯ 0.09 0.02 Ϯ 0.01 0.01 Ϯ 0.01 Heart 2.43 Ϯ 0.68 0.15 Ϯ 0.09 0.003 Ϯ 0.00 0.00 Ϯ 0.01 Liver 7.87 Ϯ 1.92 0.47 Ϯ 0.25 0.28 Ϯ 0.10 0.05 Ϯ 0.01 Lung 4.41 Ϯ 1.66 0.28 Ϯ 0.20 0.01 Ϯ 0.02 0.03 Ϯ 0.02 Kidney 4.89 Ϯ 1.83 0.37 Ϯ 0.25 0.03 Ϯ 0.01 0.03 Ϯ 0.01 Spleen 7.29 Ϯ 2.63 0.28 Ϯ 0.24 0.01 Ϯ 0.00 0.01 Ϯ 0.00 Stomach 15.74 Ϯ 7.73 2.10 Ϯ 1.98 0.23 Ϯ 0.00 0.07 Ϯ 0.02 Small intestine 12.97 Ϯ 3.68 0.78 Ϯ 0.52 0.26 Ϯ 0.01 0.03 Ϯ 0.01 Large intestine 8.84 Ϯ 5.88 1.68 Ϯ 1.10 0.45 Ϯ 0.02 0.08 Ϯ 0.04 Brain 2.32 Ϯ 0.48 0.13 Ϯ 0.03 0.01 Ϯ 0.00 0.00 Muscle 1.43 Ϯ 0.53 0.09 Ϯ 0.03 0.03 0.01 Tumor 6.63 Ϯ 0.45 2.01 Ϯ 0.18 0.83 Ϯ 0.11 0.35 Ϯ 0.09 Tumor/blood 13 16 41 35 Tumor/muscle 6 20 28 35 a Values are mean % ID/g Ϯ SD (n ϭ 4). 4581

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1999 American Association for Cancer Research. SIGMA RECEPTOR-BINDING BENZAMIDES FOR PROSTATE CANCER licity of drug, receptor-bound fraction, and also nonspecific binding. The uptake of activity in tumor was observed to be very high for 2-[125I]BP (6.63%ID/g) at 1 h postinjection and modest uptake for 4-[125I]PAB and [125I]PIMBA was observed 2.2 and 1.34%ID/g, respectively. The higher uptake of 2-[125I]BP at one and six h postinjection may be due to higher high affinity of 2-IBP for sigma-2 subtypes (29 nM), as compared with low affinities of 4-IPAB and PIMBA for sigma-2 subtypes. Tumor:muscle ratios were high for all three compounds at 1 h postinjection; however, at 6 h postinjection, tumor:muscle and tumor:blood ratios were very high for [125I]P- IMBA. Therefore, of the three compounds studied, [125I]PIMBA appeared to be most promising. Another important factor in imaging prostate carcinoma is the excretion of radioactivity from the bladder. There was a significant release of radioactivity in the bladder for 2-[125I]BP, followed by [125I]PIMBA and 4-[125I]PAB. These results indicated that tumor uptake and retention of three radioiodinated benzamides studied here look promising and tumor to blood and tumor to muscle ratios are high enough at early time points for visualization of prostate tumors. Our recent studies in vitro cell binding studies using radioiodinated sigma receptor ligand, 4-[125I]iodo-N-[2-(1Ј-piper-idinyl)ethyl]benzamide, 4-[125I]PAB showed that 4-[125I]PAB had a higher specific binding in breast cancer cells (T47D and MCF-7) and also in human prostate tumor

Fig. 3. Competition binding profiles. One-site (A) and two-site (B) fits for [125I]P- IMBA in DU-145 against various sigma binding ligands.

Table 6 Human prostate cancer cell line clonal growtha Cell line colony no.

Addition DU-145 LNCaP PC-3 None 52 Ϯ 944Ϯ 14 91 Ϯ 31 PIMBA, 10 ␮M 10 Ϯ 48Ϯ 624Ϯ 12 a Values are means Ϯ SD of three determinations.

Table 7 Dose-dependent inhibition of colony formation of human prostate tumorsa Addition DU-145 PC-3 None 573 Ϯ 105 1077 Ϯ 27 PIMBA, 0.1 ␮M 487 Ϯ 43 787 Ϯ 183 PIMBA, 1 ␮M 335 Ϯ 104 550 Ϯ 100 PIMBA, 10 ␮M 89 Ϯ 20 485 Ϯ 131 Values are means Ϯ SD of three determinations.

3 3 Fig. 2. Scatchard plot for binding of [ H](ϩ)-pentazocine (A) and [ H]DTG plus 1 ␮M (DU-145) cells when the cells were in logarithmic-phase growth than dextrallorphan (B) in human prostate (DU-145) tumor cells. those at quiescent stages. The stimulation of mitosis with insulin or Ϯ 125 125 Table 5 Inhibition constants (for one-site fit, mean K1, SE for [ I]PIMBA binding a 10% serum resulted in higher binding of sigma ligand 4-[ I]PAB. in human prostate (DU-145) cancer cells These results imply that sigma binding ligands could be useful for non Compound IC50 (nM) invasive scintigraphic imaging of proliferating tumors and also in BD1008 0.6 Ϯ 0.2 monitoring the progress of chemotherapy (28). Furthermore, support PIMBA 5.2 Ϯ 1.5 Haloperidol 15 Ϯ 3 of imaging proliferating tumors was shown in vitro binding studies by 4-IPAB 81 Ϯ 15 others (29). We have recently designed several radioiodinated ligands 4-IPBSb 100 Ϯ 24 that bind to sigma receptors in tumor cell cultures in vitro and in a Confirmation of sigma-like pharmacological profiles of [125I]PIMBA in DU-145 several animal models possessing human tumor xenografts. Further intact cells. The values are average of two experiments Ϯ SE carried out in triplicate as indicated in the text. studies in patients with metastatic prostate carcinoma are required to b 4-IPBS, N-[2-(1Ј-piperidinyl)ethyl]4-iodobenzenesulfonamide. confirm the validity of these preclinical results. 4582

On the basis of these pharmacokinetic findings and the kinetics of 10. Wolfe, S. A., Culp, S. G., and DeSouza, E. B. Sigma-receptors in endocrine organs: tumor uptake and retention, we decided to study the antiproliferative identification, characterization, and autoradiographic localization in rat pituitary, adrenals, testis, and ovary. Endocrinology, 124: 1160–1172, 1989. activity of PIMBA in different prostate tumor cell lines. Sigma ago- 11. Su, T-P., London, E. D., and Jaffe, J. H. Steroid binding at sigma receptors suggests nists have been shown to induce apoptosis in a variety of human a link between endocrine, nervous, and immune systems. Science (Washington DC), neural tumor (SK-N-SH) cells (30). In this study, the antiproliferative/ 240: 219–221, 1988. 12. Hanner, M., Moebius, F. F., Flandorfer, A., Knaus, H-G., Striessnig, J., Kempner, E., antineoplastic activity of PIMBA was determined in various human and Glossman, H. Purification, molecular cloning, and expression of the mammalian prostate tumor cell colonogenic assays. The results indicated that there sigma-1 binding site. Proc. Natl. Acad. Sci. USA, 93: 8072–8077, 1996. was a significant decrease in the number of colonies formed, when 13. Vilner, B. J., John, C. S., and Bowen, W. D. Sigma-1 and sigma-2 receptors are ␮ expressed in a wide variety of human and rodent tumor cell lines. Cancer Res., 55: treated with 10 M PIMBA, for all three prostate cell lines (androgen- 408–413, 1995. dependent and androgen-independent) as compared with the control. 14. John, C. S., Bowen, W. D., Saga, T., Kinuya, S., Vilner, B. J., Baumgold, J., Paik, DU-145 and LNCaP seemed to exhibit the maximum response (80% C. H., Reba, R. C., Neumann, R. D., Varma, V. M., and McAfee, J. G. A malignant melanoma imaging agent: synthesis, characterization, in vitro binding and biodistri- colonies death) in vitro assays (Table 6). Furthermore, a dose esca- bution of iodine-125-(2-piperidinylaminoethyl) 4-iodobenzamide. J. Nucl. Med., 34: lation study was performed with PIMBA on DU-145 and PC-3 cells. 2169–2175, 1993. A dose-dependent inhibition of colony growth formation was ob- 15. John, C. S., Bowen, W. D., Varma, V. M., McAfee, J. G., and Moody, T. W. Sigma receptors are expressed in human non-small cell lung carcinoma. Life Sci., 56: served (Table 7). On the basis of acute i.v. toxicity in Sprague Dawley 2385–2392, 1995. 4 rats and rabbits for IPAB, LD50 was found to be 110 mg/kg. It is 16. John, C. S., Vilner, B. J., and Bowen, W. D. Synthesis and characterization of concluded from these results that the in vivo toxicity of the iodoben- [125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide, a new sigma receptor radiophar- maceutical: high affinity binding to MCF-7 breast tumor cells. J. Med. Chem., 37: zamides evaluated may be very low, whereas in vitro cytotoxicity is 1737–1739, 1994. modest. Because a significant cytotoxicity was observed only at 17. John, C. S., Vilner, B. J., Gulden, M. E., Efange, S. M. N., Langason, R. B., Moody, higher concentrations, the cytotoxic effect may or may not be medi- T. W., and Bowen, W. D. Synthesis and pharmacological characterization of 4-[125I]- N-(N-benzyl piperidin-4-yl)-4-iodobenzamide: a high-affinity sigma receptor ligand ated by the sigma receptor. These results suggest that sigma receptor for potential imaging of breast cancer. Cancer Res., 55: 3022–3027, 1995. binding ligands would not be only useful for in vivo receptor diag- 18. Vilner, B. J., and Bowen, W. D. Characterization of sigma-like binding sites of nostic imaging or targeted radiotherapy but may also play an impor- NB41A3, S-20Y, and N1E-115 neuroblastomas, C6 glioma, and NG108–15 neuro- tant role in targeting various malignancies as chemotherapeutics. blastoma-glioma hybrid cells: further evidence for sigma-2 receptors. In: J-M. Ka- menka and E. F. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1999 American Association for Cancer Research. Targeting Sigma Receptor-binding Benzamides as in Vivo Diagnostic and Therapeutic Agents for Human Prostate Tumors

Christy S. John, Bertold J. Vilner, Brian C. Geyer, et al.

Cancer Res 1999;59:4578-4583.

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