Prostate-specific membrane antigen (PSMA) is expressed in normal human prostate epithelium and is highly upregulated 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)-1-carboxy-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 (Ki = 4.6 ± 1.6 and 0.24 ± 0.14 nM, respectively), and when radiolabeled with 123I exhibited high affinity for PSMA on human prostate cancer LNCaP cells (Kd = 3.8 ± 1.3 and 0.81 ± 0.39 nM, respectively). The association of [123I]MIP-1072 and [123I]MIP-1095 with PSMA was specific; there was no binding to human prostate cancer PC3 cells, which lack PSMA, and binding was abolished by co-incubation with a structurally unrelated NAALADase inhibitor, 2-(phosphonomethyl)pentanedioic acid (PMPA). [123I]MIP-1072 and [123I]MIP-1095 internalized into LNCaP cells at 37 °C. Tissue distribution studies in mice demonstrated 17.3 ± 6.3 (at 1 hr) and 34.3 ± 12.7 (at 4 hr) % injected dose per gram of tissue, for [123I]MIP-1072 and [123I]MIP-1095, respectively. [123I]MIP-1095 exhibited greater tumor uptake but slower washout from blood and non-target tissues compared to [123I]MIP-1072. Specific binding to PSMA in vivo was demonstrated by competition with PMPA in LNCaP xenografts, and the absence of uptake in PC3 xenografts. The uptake of [123I]MIP-1072 and [123I]MIP-1095 in tumor bearing mice was corroborated by SPECT/CT imaging. PSMA-specific radiopharmaceuticals should provide a novel molecular targeting option for the detection and staging of prostate cancer.
Prostate specific membrane antigen (PSMA) is a validated molecular marker for prostate cancer. A series of glutamate-urea (Glu-urea-X) heterodimeric inhibitors of PSMA were designed and synthesized where X = epsilon-N-(o-I, m-I, p-I, p-Br, o-Cl, m-Cl, p-Cl, p-F, H)-benzyl-Lys and epsilon-(p-I, p-Br, p-Cl, p-F, H)-phenylureido-Lys. The affinities for PSMA were determined by screening in a competitive binding assay. PSMA binding of the benzyllysine series was significantly affected by the nature of the halogen substituent (IC(50) values, Cl < I = Br << F = H) and the ring position of the halogen atom (IC(50) values, p-I < o-I << m-I). The halogen atom had little affect on the binding affinity in the para substituted phenylureido-Lys series. Two lead iodine compounds were radiolabeled with (123)I and (131)I and demonstrated specific PSMA binding on human prostate cancer cells, warranting evaluation as radioligands for the detection, staging, and monitoring of prostate cancer.
New classes of physiologically responsive magnetic resonance (MR) contrast agents are being developed that are activated by enzymes, secondary messengers, pH, and temperature. To this end, we have prepared a new class of enzyme-activated MR contrast agents using a self-immolative mechanism and investigated the properties of these agents using novel in vitro assays. We have synthesized in nine steps a Gd(III) agent 1 that is activated by the oncologically significant beta-glucuronidase. 1 consists of Gd(III)DO3A (DO3A = 1,4,7-tricarboxymethylene-1,4,7,10-tetraazacyclododecane) bearing a pendant beta-glucuronic acid moiety connected by a self-immolative linker to the macrocycle. LC-MS analysis reveals that 1 is enzymatically processed as predicted by bovine liver beta-glucuronidase, generating 2-aminoethylGdDO3A, 2. Compound 2 was prepared independently in a four-step synthetic procedure. Complex 1 displays a decrease in relaxivity upon titration with bicarbonate anion. The relaxivity increases when 1 is converted to 2 in a buffer mimicking in vivo anion concentrations (Parker, D. In Crown Compounds: Towards Future Applications; Cooper, S. R., Ed.; VCH: New York, 1992; pp 51-67) by 17%, while the relaxivity decreases by 27% for the same experiment in human blood serum. Hydrolytic kinetics catalyzed by bovine liver beta-glucuronidase at interstitial pH = 7.4 fit the Michaelis-Menten model with k cat/Km = 74.9 +/- 10.9 M(-1) s(-1). Monitoring of bulk water proton T1 during incubation with enzyme shows an increase in T1 that mirrors results obtained through the relaxivity measurements of compounds 1 and 2.
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.
We report a mechanistic investigation of an isomeric series of β-galactosidase-activated magnetic resonance contrast agents. Our strategy focuses on the synthesis of macrocyclic caged-complexes that coordinatively saturate a chelated lanthanide. Enzyme cleavage of the complex results in an open coordination site available for water that creates a detectable MR contrast agent. The complexes consist of a DO3A Gd(III) chelator modified with a galactopyranose at the N-10 position of the macrocycle. We observed significant differences in relaxometric properties and coordination geometry that can be correlated to subtle variations of the linker between the macrocycle and the galactopyranose. After synthesis and purification of the R, S, and racemic mixtures of complexes 1 and 3 and measurement of the hydration number, water residence lifetime, and longitudinal relaxation rates, we propose mechanisms for water exclusion from the lanthanide in the precleavage state. While the stereochemistry of the linker does not influence the agents' properties, the mechanism of water exclusion for each isomer is significantly influenced by the position of modification. Data for one series with a methyl group substituted on the sugar-macrocycle linker at the R-position suggests a steric mechanism where the galactopyranose sugar blocks water from the Gd(III) center. In contrast, our observations for a second series with methyl substitution at the β position of the sugar-macrocycle linker are consistent with a mechanism in which a bidentate anion occupies two available coordination sites of Gd(III) in the precleavage state.
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