Preclinical evaluation of novel glutamate-urea-lysine analogues that target prostate-specific membrane antigen as molecular imaging pharmaceuticals for prostate cancer

Abstract

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.

Figure 1

A. Binding of [¹²³I]MIP-1072 or [¹²³I]MIP-1095 to LNCaP and PC3 cells. Cells were incubated for 1 hr with each compound in the absence or presence of unlabeled compound or PMPA. B. Saturation binding analysis of [¹²³I]MIP-1072 and [¹²³I]MIP-1095. LNCaP cells were incubated at 4 °C for 1 hr with 30–300,000 pM [¹²³I]MIP-1072 or [¹²³I]MIP-1095. The K_d and B_max were determined by non-linear regression analysis. C. LNCaP cellular internalization of [¹²³I]MIP-1072 and [¹²³I]MIP-1095. LNCaP cells were incubated with 100 nM radiolabeled compound for the indicated time, washed, and treated with a mild acid buffer to separate cell surface bound (dashed lines) from total bound material (solid lines). The results are representative of two independent experiments.

Figure 2

Selective targeting of PSMA in vivo with [¹²³I]-MIP-1072 and [¹²³I]-MIP-1095. Radiolabeled compound was injected into mice bearing LNCaP xenografts and imaged by SPECT/CT at 4 hr (top) or mice bearing PC3 PIP (PSMA +) or PC3 flu (PSMA −) xenografts and imaged by SPECT/CT at 2 hr (bottom). Each mouse was injected with approximately 1 mCi of radiolabeled compound at a specific activity >1000 mCi/µmol.

Figure 3

Specific binding of [¹²³I]MIP-1072 (A) and [¹²³I]MIP-1095 (B) to PSMA in vivo. Radiolabeled compound (2 µCi/mouse at >1000 mCi/µmol) was injected alone (LNCaP tumor , PC3 tumor ) or co-injected with 50 mg/kg PMPA (LNCaP tumor , or PC3 tumor ) via the tail vein. Data are expressed as %ID/g.