Shawn Hillier

Preclinical Evaluation of Novel Glutamate-Urea-Lysine Analogues That Target Prostate-Specific Membrane Antigen as Molecular Imaging Pharmaceuticals for Prostate Cancer

Prostate-specific membrane antigen (PSMA) is expressed in normal human prostate epithelium and is highly up-regulated in prostate cancer. We previously reported a series of novel small molecule inhibitors targeting PSMA. Two compounds, MIP-1072, (S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)pentyl)ureido)pentanedioic acid, and MIP-1095, (S)-2-(3-((S)-1carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioic acid, were selected for further evaluation. MIP-1072 and MIP-1095 potently inhibited the glutamate carboxypeptidase activity of PSMA (K(i) = 4.6 +/- 1.6 nmol/L and 0.24 +/- 0.14 nmol/L, respectively) and, when radiolabeled with (123)I, exhibited high affinity for PSMA on human prostate cancer LNCaP cells (K(d) = 3.8 +/- 1.3 nmol/L and 0.81 +/- 0.39 nmol/L, respectively). The association of [(123)I]MIP-1072 and [(123)I]MIP-1095 with PSMA was specific; there was no binding to human prostate cancer PC3 cells, which lack PSMA, and binding was abolished by coincubation with a structurally unrelated NAALADase inhibitor, 2-(phosphonomethyl)pentanedioic acid (PMPA). [(123)I]MIP-1072 and [(123)I]MIP-1095 internalized into LNCaP cells at 37 degrees C. Tissue distribution studies in mice showed 17.3 +/- 6.3% (at 1 hour) and 34.3 +/- 12.7% (at 4 hours) injected dose per gram of LNCaP xenograft tissue, for [(123)I]MIP-1072 and [(123)I]MIP-1095, respectively. [(123)I]MIP-1095 exhibited greater tumor uptake but slower washout from blood and nontarget tissues compared with [(123)I]MIP-1072. Specific binding to PSMA in vivo was shown by competition with PMPA in LNCaP xenografts, and the absence of uptake in PC3 xenografts. The uptake of [(123)I]MIP-1072 and [(123)I]MIP-1095 in tumor-bearing mice was corroborated by single-photon emission computed tomography/computed tomography (SPECT/CT) imaging. PSMA-specific radiopharmaceuticals should provide a novel molecular targeting option for the detection and staging of prostate cancer.

99mTc-Labeled Small-Molecule Inhibitors of Prostate-Specific Membrane Antigen for Molecular Imaging of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and small-molecule radiopharmaceuticals targeting PSMA rapidly detect the location and extent of disease. Here we evaluated preclinically 4 novel 99mTc-labeled small-molecule inhibitors of PSMA with the potential for clinical translation for molecular imaging of prostate cancer in humans. Methods: Four PSMA inhibitors derived from the glutamate-urea-glutamate or glutamate-urea-lysine pharmacophores conjugated to CIM or TIM chelators were radiolabeled with 99mTc and evaluated in vitro and in vivo. Results: High-affinity, saturable binding to PSMA on LNCaP cells was observed with Kd values of 0.64 ± 0.46 nM for 99mTc-MIP-1427, 1.07 ± 0.89 nM for 99mTc-MIP-1404, 1.75 ± 0.32 nM for 99mTc-MIP-1428, and 4.35 ± 0.35 nM for 99mTc-MIP-1405. 99mTc-labeled PSMA inhibitors did not bind human prostate cancer PC3 cells, which lack PSMA, demonstrating specificity, and binding was abolished with 2-(phosphonomethyl)pentanedioic acid (PMPA), a structurally unrelated PSMA inhibitor. 99mTc-labeled PSMA inhibitors were shown to internalize at 37°C. Uptake in LNCaP xenografts ranged from 9.3% to 12.4% injected dose per gram at 1 h after injection and from 7.2% to 11.0% at 4 h, with tumor-to-blood ratios ranging from 29:1 to 550:1 and tumor–to–skeletal muscle ratios ranging from 31:1 to 157:1 at 4 h. 99mTc-MIP-1404 exhibited the best combination of high tumor uptake and rapid clearance from kidney and nontarget tissues. 99mTc-MIP-1404 specifically bound to PSMA in vivo as demonstrated by the absence of uptake in PC3 xenografts and by competition with PMPA. SPECT/CT imaging corroborated the tissue distribution results, demonstrating uptake only in PSMA-expressing kidney and tumor tissue and clearance through the urinary bladder. Conclusion: These 99mTc-labeled radiopharmaceuticals targeting PSMA may provide a SPECT molecular imaging option to assist in the initial diagnosis of prostate cancer and the management of patient care by monitoring disease progression.

123I-MIP-1072, a Small-Molecule Inhibitor of Prostate-Specific Membrane Antigen, Is Effective at Monitoring Tumor Response to Taxane Therapy

Because traditional endpoints in oncology trials are not always applicable for metastatic prostate cancer, better ways of following response to treatment are needed. Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed in normal human prostate epithelium and is upregulated in prostate cancer. (S)-2-(3-((S)-1-carboxy-5-((4-123I-iodobenzyl)amino)pentyl)ureido)pentanedioic acid, 123I-MIP-1072, targets PSMA and was evaluated for monitoring the growth of PSMA-positive LNCaP cells in vitro and as xenografts after paclitaxel therapy. Methods: LNCaP and 22Rv1 cells were treated with paclitaxel (0–100 nM) for 48 h, after which binding of 123I-MIP-1072 was examined. Cell number was determined by MTS assay, and PSMA expression was analyzed by Western blotting. LNCaP xenograft–bearing mice were treated with paclitaxel (6.25 mg/kg) for 3.5 cycles of 5 d on and 2 d off. Tissue distribution of 123I-MIP-1072 was determined on days 2 and 23 from the start of paclitaxel treatment. Results: Paclitaxel (10–100 nM) inhibited LNCaP and 22Rv1 cell growth after 48 h, and binding of 123I-MIP-1072 was proportional to cell number. Western blot analysis verified there was no paclitaxel-dependent change in PSMA expression. Treatment of LNCaP xenografts with paclitaxel resulted in a decrease in tumor volume (−21%), compared with an increase in the untreated xenografts (+205%) by day 23. Tumor uptake of 123I-MIP-1072 was proportional to changes in tumor mass: decreased by paclitaxel treatment and increased in untreated mice. Conclusion: Treatment of LNCaP cells or xenograft tumors with paclitaxel resulted in growth inhibition, which was detected with 123I-MIP-1072. The high specificity of 123I-MIP-1072 for prostate cancer may allow monitoring of tumor progression in patients before, during, and after chemotherapy.

Novel Polar Single Amino Acid Chelates for Technetium-99m Tricarbonyl-Based Radiopharmaceuticals with Enhanced Renal Clearance: Application to Octreotide

Single amino acid chelate (SAAC) systems for the incorporation of the M(CO)(3) moiety (M = Tc/Re) have been successfully incorporated into novel synthetic strategies for radiopharmaceuticals and evaluated in a variety of biological applications. However, the lipophilicity of the first generation Tc(CO)(3)-dipyridyl complexes has resulted in substantial hepatobiliary uptake when either examined as lysine derivatives or integrated into biologically active small molecules and peptides. Here we designed, synthesized, and evaluated novel SAAC systems that have been chemically modified to promote overall Tc(CO)(3)L(3) complex hydrophilicity with the intent of enhancing renal clearance. A series of lysine derived SAAC systems containing functionalized polar imidazole rings and/or carboxylic acids were synthesized via reductive alkylation of the epsilon amino group of lysine. The SAAC systems were radiolabeled with (99m)Tc, purified, and evaluated for radiochemical stability, lipophilicity, and tissue distribution in rats. The log P values of the (99m)Tc complexes were determined experimentally and ranged from -0.91 to -2.33. The resulting complexes were stable (>90%) for at least 24 h. Tissue distribution in normal rats of the lead (99m)Tc complexes demonstrated decreased liver (<1 %ID/g) and gastrointestinal clearance (<1.5%ID/g) and increased kidney clearance (>15 %ID/g) at 2 h after injection compared to the dipyridyl lysine complex (DpK). One of the new SAAC ligands, [(99m)Tc]bis-carboxymethylimidazole lysine, was conjugated to the N-terminus of Tyr-3 octreotide and evaluated for localization in nude mice bearing AR42J xenografts to examine tissue distribution, tumor uptake and retention, clearance, and route of excretion for comparison to (111)In-DOTA-Tyr-3-octreotide and (99m)Tc-DpK-Tyr-3-octreotide. (99m)Tc-bis-(carboxymethylimidazole)-lysine-Tyr-3-octreotide exhibited significantly less liver uptake and gastrointestinal clearance compared to (99m)Tc-DpK-Tyr-3-octreotide while maintaining tumor uptake in the same mouse model. These novel chelators demonstrate that lipophilicity can be controlled and organ distribution significantly altered, opening up broad application of these novel SAAC systems for radiopharmaceutical design.

Synthesis and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel (99m)Tc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC(50) values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC(50)=4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({(99m)Tc(CO)(3)}(+)) to afford the {(99m)Tc(CO)(3)}(+) complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {(99m)Tc(CO)(3)}(+) radiolabeled PSMA inhibitors.

Comprehensive Radiolabeling, Stability, and Tissue Distribution Studies of Technetium-99m Single Amino Acid Chelates (SAAC)

Technetium tricarbonyl chemistry has been a subject of interest in radiopharmaceutical development over the past decade. Despite the extensive work done on developing chelates for Tc(I), a rigorous investigation of the impact of changing donor groups and labeling conditions on radiochemical yields and/or distribution has been lacking. This information is crucially important if these platforms are going to be used to develop molecular imaging probes. Previous studies on the coordination chemistry of the {M(CO)(3)}(+) core have established alkylamine, aromatic nitrogen heterocycles, and carboxylate donors as effective chelating ligands. These observations led to the design of tridentate ligands derived from the amino acid lysine. Such amino acid analogues provide a tridentate donor set for chelation to the metal and an amino acid functionality for conjugation to biomolecules. We recently developed a family of single amino acid chelates (SAAC) that serve this function and can be readily incorporated into peptides via solid-phase synthesis techniques. As part of these continuing studies, we report here on the radiolabeling with technetium-99m ((99m)Tc) and stability of a series of SAAC analogues of lysine. The complexes studied include cationic, neutral, and anionic complexes. The results of tissue distribution studies with these novel complexes in normal rats demonstrate a range of distribution in kidney, liver, and intestines.

Comparison of High-Specific-Activity Ultratrace 123/131I-MIBG and Carrier-Added 123/131I-MIBG on Efficacy, Pharmacokinetics, and Tissue Distribution

Metaiodobenzylguanidine (MIBG) is an enzymatically stable synthetic analog of norepinephrine that when radiolabled with diagnostic ((123)I) or therapeutic ((131)I) isotopes has been shown to concentrate highly in sympathetically innervated tissues such as the heart and neuroendocrine tumors that possesses high levels of norepinephrine transporter (NET). As the transport of MIBG by NET is a saturable event, the specific activity of the preparation may have dramatic effects on both the efficacy and safety of the radiodiagnostic/radiotherapeutic. Using a solid labeling approach (Ultratrace), noncarrier-added radiolabeled MIBG can be efficiently produced. In this study, specific activities of >1200 mCi/micromol for (123)I and >1600 mCi/micromol for (131)I have been achieved. A series of studies were performed to assess the impact of cold carrier MIBG on the tissue distribution of (123/131)I-MIBG in the conscious rat and on cardiovascular parameters in the conscious instrumented dog. The present series of studies demonstrated that the carrier-free Ultratrace MIBG radiolabeled with either (123)I or (131)I exhibited similar tissue distribution to the carrier-added radiolabeled MIBG in all nontarget tissues. In tissues that express NETs, the higher the specific activity of the preparation the greater will be the radiopharmaceutical uptake. This was reflected by greater efficacy in the mouse neuroblastoma SK-N-BE(2c) xenograft model and less appreciable cardiovascular side-effects in dogs when the high-specific-activity radiopharmaceutical was used. The increased uptake and retention of Ultratrace (123/131)I-MIBG may translate into a superior diagnostic and therapeutic potential. Lastly, care must be taken when administering therapeutic doses of the current carrier-added (131)I-MIBG because of its potential to cause adverse cardiovascular side-effects, nausea, and vomiting.

Preclinical evaluation of an 131I-labeled benzamide for targeted radiotherapy of metastatic melanoma

Radiolabeled benzamides are attractive candidates for targeted radiotherapy of metastatic melanoma as they bind melanin and exhibit high tumor uptake and retention. One such benzamide, N-(2-diethylamino-ethyl)-4-(4-fluoro-benzamido)-5-iodo-2-methoxy-benzamide (MIP-1145), was evaluated for its ability to distinguish melanin-expressing from amelanotic human melanoma cells, and to specifically localize to melanin-containing tumor xenografts. The binding of [(131)I]MIP-1145 to melanoma cells in vitro was melanin dependent, increased over time, and insensitive to mild acid treatment, indicating that it was retained within cells. Cold carrier MIP-1145 did not reduce the binding, consistent with the high capacity of melanin binding of benzamides. In human melanoma xenografts, [(131)I]MIP-1145 exhibited diffuse tissue distribution and washout from all tissues except melanin-expressing tumors. Tumor uptake of 8.82% injected dose per gram (ID/g) was seen at 4 hours postinjection and remained at 5.91% ID/g at 24 hours, with tumor/blood ratios of 25.2 and 197, respectively. Single photon emission computed tomography imaging was consistent with tissue distribution results. The administration of [(131)I]MIP-1145 at 25 MBq or 2.5 GBq/m(2) in single or multiple doses significantly reduced SK-MEL-3 tumor growth, with multiple doses resulting in tumor regression and a durable response for over 125 days. To estimate human dosimetry, gamma camera imaging and pharmacokinetic analysis was performed in cynomolgus monkeys. The melanin-specific binding of [(131)I]MIP-1145 combined with prolonged tumor retention, the ability to significantly inhibit tumor growth, and acceptable projected human dosimetry suggest that it may be effective as a radiotherapeutic pharmaceutical for treating patients with metastatic malignant melanoma.

Molecular imaging of human ACE-1 expression in transgenic rats.

OBJECTIVES The aim of this study was to develop a molecular imaging strategy that can monitor myocardial angiotensin-converting enzyme (ACE)-1 upregulation as a function of progressive heart failure. BACKGROUND High-affinity technetium-99m-labeled lisinopril (Tc-Lis) has been shown to specifically localize in tissues that express ACE in vivo, such as the lungs. Whether Tc-Lis can also detect upregulation of ACE in the heart, by external in vivo imaging, has not been established. METHODS Twenty-one ACE-1 over-expressing transgenic (Tg) and 18 wild-type control rats were imaged using in vivo micro single-positron emission computed tomography (SPECT)-computed tomography (CT) at 10, 30, 60, and 120 min after Tc-Lis injection. A subgroup of rats received nonradiolabeled (cold) lisinopril before the Tc-Lis injection to evaluate nonspecific binding. After imaging, the rat myocardium was explanted, ex vivo images were acquired, and percent injected dose per gram gamma-well was counted, followed by an assessment of enzyme-linked immunosorbent assay-verified ACE activity and messenger ribonucleic acid expression. RESULTS On micro SPECT-CT, myocardial ACE-1 uptake was best visualized in Tg rats at 120 min after Tc-Lis injection. The quantitative uptake of Tc-Lis in the myocardium was 5-fold higher in mutant Tg than in control rats at each time point after tracer injection. The percent injected dose per gram uptake was 0.74 ± 0.13 in Tg myocardium at 30 min and was reduced substantially to 0.034 ± 0.003% when pre-treated with cold lisinopril (p = 0.029). Enzyme activity assay showed a >30-fold higher level of ACE-1 activity in the myocardium of Tg rats than in controls. The ACE-1 messenger ribonucleic acid was quantified, and lisinopril was found to have no effect on ACE-1 gene expression. CONCLUSIONS The Tc-Lis binds specifically to ACE, and the activity can be localized in Tg rat hearts that over-express human ACE-1 with a signal intensity that is sufficiently high to allow external imaging. Such a molecular imaging strategy may help identify susceptibility to heart failure and may allow optimization of pharmacologic intervention.

Synthesis, cytotoxicity, and insight into the mode of action of Re(CO) 3 thymidine complexes

Nucleoside analogues are extensively used in the treatment of cancer and viral diseases. The antiproliferative properties of organorhenium(I) complexes, however, have been scarcely explored to date. Herein we present the syntheses, characterization, and in vitro evaluation of ReI(CO)3 core complexes of thymidine and uridine. For the binding of the ReI(CO)3 core, a tridentate dipicolylamine metal chelate was introduced at positions C5′, C2′, N3, and C5 with spacers of various lengths. The corresponding organometallic thymidine complexes were fully characterized by IR and NMR spectroscopy and mass spectrometry. Their cytotoxicity was assessed against the A549 lung carcinoma cell line. Toxicity is dependent on the site and mode of conjugation as well as on the nature and the length of the tether. Moderate toxicity was observed for conjugates carrying the rhenium moiety at position C5′ or N3 (IC50=124–160 μM). No toxicity was observed for complexes modified at C2′ or C5. Complex 53, with a dodecylene spacer at C5′, exhibits remarkable toxicity and is more potent than cisplatin, with an IC50 value of 6.0 μM. To the best of our knowledge, this is the first report of the antiproliferative properties of [M(CO)3]+1–nucleoside conjugates. In competitive inhibition experiments with A549 cell lysates and purified recombinant human thymidine kinase 1 (hTK‐1), enzyme inhibition was observed for complexes modified at either N3 or C5′, but our results suggest that the toxicity cannot be attributed solely to interaction with hTK‐1.