Discovery of a new class of integrin antibodies for fibrosis.

Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.

99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer.

UNLABELLED: 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 (99m)Tc-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 (99m)Tc 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 (99m)Tc-MIP-1427, 1.07 ± 0.89 nM for (99m)Tc-MIP-1404, 1.75 ± 0.32 nM for (99m)Tc-MIP-1428, and 4.35 ± 0.35 nM for (99m)Tc-MIP-1405. (99m)Tc-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. (99m)Tc-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. (99m)Tc-MIP-1404 exhibited the best combination of high tumor uptake and rapid clearance from kidney and nontarget tissues. (99m)Tc-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 (99m)Tc-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.

UNLABELLED: 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-(123)I-iodobenzyl)amino)pentyl)ureido)pentanedioic acid, (123)I-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 (123)I-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 (123)I-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 (123)I-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 (123)I-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 (123)I-MIP-1072. The high specificity of (123)I-MIP-1072 for prostate cancer may allow monitoring of tumor progression in patients before, during, and after chemotherapy.

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.

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.

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.

DNA adducts formed by a novel antitumor agent 11beta-dichloro in vitro and in vivo.

The multifunctional molecule 11beta-dichloro consists of a ligand for the androgen receptor linked to a bifunctional alkylating group, permitting it to create DNA adducts that bind the androgen receptor. We propose that binding of the androgen receptor to 11beta-DNA adducts acts to both shield damaged sites from repair and disrupt the expression of genes essential for growth and survival. We investigated the formation 11beta-DNA adducts in tumor xenograft and nontumor tissues in mice. Using [14C]-11beta-dichloro, we show that the molecule remains intact in blood and is widely distributed in mouse tissues after i.p. injection. Covalent 11beta-guanine adducts identified in DNA that had been allowed to react with 11beta-dichloro in vitro were also found in DNA isolated from cells in culture treated with 11beta-dichloro as well as in DNA isolated from liver and tumor tissues of mice treated with the compound. We used accelerator mass spectrometry to determine the levels of [14C]-11beta-DNA adducts in LNCaP cells treated in culture as well as in liver tissue and LNCaP xenograft tumors in treated mice. The level of DNA adducts in tumor tissue was found to be similar to that found in LNCaP cells in culture treated with 2.5 micromol/L 11beta-dichloro. Our results indicate that 11beta-dichloro has sufficient stability to enter the circulation, penetrate tissues, and form DNA adducts that are capable of binding the androgen receptor in target tissues in vivo. These data suggest the involvement of our novel mechanisms in the antitumor effects of 11beta-dichloro.

Disruption of gene expression and induction of apoptosis in prostate cancer cells by a DNA-damaging agent tethered to an androgen receptor ligand.

The goal of our work was the design of DNA-damaging agents that disrupt both DNA repair and signaling pathways in prostate tumor cells. A DNA alkylator (N,N-bis-2-chloroethyl aniline) was linked to a steroid ligand (17beta-hyroxy-estra-Delta(4(5),9(10))-3-one) to produce a complex molecule (11beta-dichloro) that forms DNA adducts that bind the androgen receptor (AR). We speculated that DNA adducts in an AR-DNA adduct complex would be camouflaged from DNA repair proteins that would otherwise remove the adducts in prostate cancer cells. Furthermore, transcription dependent on the AR would be antagonized by AR redistribution to sites distant from AR-driven promoters. The anticancer potential of 11beta-dichloro was demonstrated against prostate cancer cells in vitro and in vivo. 11beta-dichloro induces a unique pattern of gene disruption, induces apoptosis in apoptosis-resistant cells, and shows promising anticancer activity in animals.

A rationally designed genotoxin that selectively destroys estrogen receptor-positive breast cancer cells.

We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is proposed that the association of such proteins with damaged sites in DNA can compromise protein function and/or DNA repair resulting in increased toxicity. We describe the synthesis of a bifunctional compound consisting of an aniline mustard linked to the 7alpha position of estradiol. This novel compound can form covalent DNA adducts that have high affinity for the estrogen receptor. Breast cancer cells that express high levels of the estrogen receptor showed increased sensitivity to the cytotoxic effects of the new compound.